WO2015038426A1 - Self-immolative linkers containing mandelic acid derivatives, drug-ligand conjugates for targeted therapies and uses thereof - Google Patents

Self-immolative linkers containing mandelic acid derivatives, drug-ligand conjugates for targeted therapies and uses thereof Download PDF

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WO2015038426A1
WO2015038426A1 PCT/US2014/054236 US2014054236W WO2015038426A1 WO 2015038426 A1 WO2015038426 A1 WO 2015038426A1 US 2014054236 W US2014054236 W US 2014054236W WO 2015038426 A1 WO2015038426 A1 WO 2015038426A1
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drug
alkyl
compound
fragment
reaction
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PCT/US2014/054236
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French (fr)
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Aranapakam M. Venkatesan
Roger A. Smith
Nitin K. Damle
Raman Kuman BAKSHI
Arjan Viram ODEDRA
Srinivas Kumar
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Asana Biosciences, Llc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment

Definitions

  • Targeted anti-neoplastic therapies are desired, in order to improve therapeutic outcome and to reduce toxicity of the delivered therapeutic.
  • One approach to targeted antineoplastic therapies is the use of highly cytotoxic drugs linked to a targeting molecule that is highly specific for cell surface receptors on tumor or other neoplastic cells.
  • de Groot et al propose activatable prodrugs having the organization "specifier-spacer-drug", which are converted by enzymatic cleavage to provide a [spacer-drug] that subsequently undergoes spontaneous spacer elimination to release the drug.
  • a chemical means for conjugating drugs to ligands is through a spacer or linker that is a "self-immolative linker".
  • self-immolative linkers include PABC or PAB (para-aminobenzyloxycarbonyl linkers. See, e.g., Feng et al, US Patent No. 7,375,078.
  • This PAB linker unit may be referred to as an electronic cascade spacer.
  • the amide bond linking the carboxyl terminus of a peptide unit and the para-aminobenzyl of PAB may act as a substrate and proteolytically cleavable. Upon cleavage, the aromatic amine becomes electron-donating and initiates an electronic cascade that leads to the expulsion of the leaving group, which releases the free drug after elimination of carbon dioxide. See, e.g., US Patent No. 7,375,078, citing (de Groot et al, (2001) J Organic Chemistry, 66(26): 8815-8830). See, also, de Groot et al, US Patent 7,705,045.
  • the '078 patent describes limitations of the PAB type linker; for example, that certain PAB -containing conjugates may not be suitable substrates for some cleaving enzymes or may cleave too slowly to achieve efficacy.
  • the '078 patent proposes alternative heterocyclic self-immolative linkers and conjugates. Still other self-immolative linkers are described in US Patent No. 7,091,186 and WO
  • the present invention describes a novel protease-cleavable self-immolative drug-linker system capable of releasing a variety of drugs.
  • the invention provides a cleavable para-amino mandelic acid (PAMA) derived linker which is useful for forming a conjugate between a drug and targeting moiety, having the structure IX:
  • PAMA para-amino mandelic acid
  • L is a di-, tri- or tetra- amino acid chain
  • Z is an optional amine blocking group
  • X is a conjugatable group, such as an OH or NH 2 , or in the context of a therapeutic and targeting conjugate is bound directly or indirectly to a drug fragment or prodrug fragment
  • Wi and W 2 are independently N or CR 2 , and W is absent or present, provided that when W is absent, W 3 is independently NR 3 , O or S, and when W is present, W and W 3 are
  • R 1 is a conjugatable group, for example having a formula selected from the group consisting of -CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) admirOCH 2 CHR 8 -, C C 6 alkyl-, and -(CH 2 CH 2 OCH 2 CH 2 0) n -, wherein n is 1 to 8, R 6 is H, C C 6 alkyl, or C 2 -C 3 hydroxyalkyl; R 7 is C C 6 alkyl, C C 3 hydroxyalkyl, or -(CH 2 ) 2 NH(Ci-C 3 alkyl) 2 ; R 8 is H, C C 3 alkyl or C C 3 hydroxyalkyl, or in the context of a therapeutic and drug targeting conjugate is bound to the targeting moiety via this R 1 moiety; and R 2 is H, C 1 -C6 alkyl, C 1 -C6
  • R 2 group shown in the structure IX above indicates that one or more R 2 groups may be attached to the ring. This same R 2 group is described explicitly in the definition for W, W 2 and W 3 .
  • the invention provides a therapeutic drug and targeting conjugate characterized by formula (I):
  • L is a di-, tri- or tetra- amino acid chain
  • X is (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR 4 - which is the point of attachment to -OC(O)-, (ii) X is -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR 4 - or - O- as the point of attachment to -N(H)C(0)-; (iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or (iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR 4 - which is part of the drug fragment or prodrug fragment; p is 0 or 1; m is 1 to 6, with the proviso that when m is 1, p is 0; LG is a ligand which targets
  • B-LG is ; wherein said LG has at least one thiol moiety or at least one amino moiety which forms the point of attachment to B; p" is 1 to 6; Wi and W 2 are independently N or CR 2 , and W is absent or present, provided that when W is absent, W 3 is independently NR 3 , O or S, and when W is present, W and W 3 are independently N or CR 2 , provided that at least one of Wi, W 2 , and W 3 is CR 2 ; R 1 is -CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) deliberatelyOCH 2 CHR 8 -, C C 6 alkyl-,
  • R 2 is H, C C 6 alkyl, C C 6 alkoxy, halogen, C 1 -C6 fluoroalkyl, or cyano
  • R 3 is H, C 1 -C6 alkyl, C 2 -C6 hydroxyalkyl or C 2 -C6
  • R 4 is H, C 1 -C6 alkyl, C 1 -C6 hydroxyalkyl or C 1 -C6 perfluoroalkyl
  • R 6 is H, Ci-C 6 alkyl, or C 2 -C 3 hydroxyalkyl
  • R 7 is Ci-C 6 alkyl, C 1 -C3 hydroxyalkyl, or - (CH 2 ) 2 NH(Ci-C 3 alkyl) 2
  • R 8 is H, C C 3 alkyl or C C 3 hydroxyalkyl
  • R 9 and R 10 are independently H or C 1 -C6 alkyl
  • R 12 , R 13 and R 14 are independently selected from H, C 1 -C6 alkyl, C 2 -C 3 hydroxyalkyl
  • Z is an optional amine blocking group.
  • X is (i) or (iii) and the drug fragment or prodrug fragment Y is a DNA damaging agent having the structure of formula (III) or (IV), respectively, below:
  • Minor Groove Binder means a DNA minor groove binding group which is a substituted lH-indole-2-carbonyl group as described herein.
  • the invention provides novel ligands for use in the present targeting and therapeutic conjugates, such as the anti-HERl-scFvFc of SEQ ID NO: ABl and the anti- HER2-scFvFc of SEQ ID NO: AA2.
  • the invention provides a method of delivering at least one therapeutically active drug, said method comprising administering a conjugate comprising a targeting ligand and at least one PAMA linker system bearing a therapeutic drug or prodrug fragment, wherein the therapeutically active drug or prodrug fragment is released from the conjugate following cleavage of the peptidic substrate moiety incorporated within the PAMA linker by a protease, for example a lysosomal protease.
  • the targeting ligand is also therapeutically active.
  • the drug or prodrug is selected from the group consisting of a DNA damaging agent, a microtubule disrupting agent, and a cytotoxic protein or polypeptide.
  • the invention provides a method of treating a disease or disorder associated with the presence of a specific cell surface molecule on cells of a subject, comprising administering to the subject a therapeutically effective amount of a conjugate of the invention comprising at least one ligand that specifically binds to the cell surface molecule.
  • the disease or disorder is a neoplastic disease such as cancer.
  • the invention provides a method for producing a therapeutic drug and targeting conjugate, comprising the steps of providing a cleavable para-amino mandelic acid (PAMA) derived linker having the structure IX:
  • PAMA para-amino mandelic acid
  • L is a di-, tri- or tetra- amino acid chain
  • Z is an optional amine blocking group
  • X is a conjugatable group, such as an OH or NH 2 , which can be bound directly or indirectly to a drug fragment or prodrug fragment
  • Wi and W 2 are independently N or CR 2 , and W is absent or present, provided that when W is absent, W 3 is independently NR 3 , O or S, and when W is present, W and W 3 are independently N or CR 2 , provided that at least one of Wi, W 2 , and W 3 is CR 2
  • R 1 is a conjugatable group, for example having a formula selected from
  • R 6 is H, C C 6 alkyl, or C 2 -C 3 hydroxyalkyl
  • R 7 is C C 6 alkyl, C C 3 hydroxyalkyl, or -(CH 2 ) 2 NH(Ci-C 3 alkyl) 2
  • R 8 is H, C C 3 alkyl or C C 3 hydroxyalkyl, which can be bound to a targeting moiety
  • kit that comprises a conjugate of the invention.
  • the conjugate may be in the presence or absence of one or more of pharmaceutically acceptable carriers or excipients.
  • the kit may optionally contain instructions for administering the conjugate to a subject, e.g., for use in an anti-neoplastic regimen.
  • a synthetic process for preparing a tert-butyl (4- hydroxynaphthalen-2-yl)carbamate comprises (i) admixing 1,3- dihydroxynapthalene and diphenylmethylamine in an aromatic solvent such as toluene solution; (ii) heating the toluene solution at 80°C to 125°C for about 4 to about 8 hours; (iii) combining the reaction mass with palladium hydroxide, di-tert butyl carbonate, and dioxane:water; (iv) shaking the reaction mixture of (c) at 60-80 psi hydrogen pressure for about 24 to 48 hours; and (v) filtering the reaction mixture of (d) and concentrating to yield crude tert-butyl (4-hydroxynaphthalen-2-yl)carbamate.
  • the process further comprises filtering and concentrating the tert-butyl (4-hydroxynaphthalen-2-yl)carbamate product of step (v) to column chromatography on silica gel using ethyl acetate-hexane (1 :9) as eluent.
  • a synthetic process for preparing a compound 29a comprises (a) combining an alcohol having the structure of compound 26a and cesium chloride in a dimethylformide solution, wherein the compound
  • 26a has the structure: wherein Z is amine blocking group and L is an amino acid selected from Val-Cit or Gly-Gly-Phe-Gly; cooling the reaction mixture to about 0°C and adding trichloroacetonitrile; allowing the reaction mixture to warm to room temperature with stirring; pouring the reaction mixture over water and extracting with ethylacetate; washing the combined organic layers with water and brine, followed by separating the organic layer, drying, concentrating, and purifying crude product by silica gel column chromatography using 10% methanol in dichloromethane as eluent to yield the trichloracetimidate product which has the structure 27a:
  • Compound 34a is useful in synthesis of a drug and targeting conjugate as described herein.
  • the process comprises (a) combining carbonyldiimidazole and 3 (5-(2- (dimethylamino)ethoxy)-6-methoxy-lH-indole-2-carboxylic acid; DMMI) (Scheme 2) (or the carboxylic acid compound of another DNA minor groove binding group which is a substituted lH-indole-2-carbonyl group) in a DMF solution and stirring for about 2 hours at ambient temperature; (b) combining the reaction mixture with a stirred DMF solution of amine 29a and sodium carbonate, wherein Z is a blocking group and L is a di-peptide, tri- peptide, or tetra-peptide; (c) removal of solid following reaction and neutralizing remaining solution with formic acid.
  • FIG. 1 is a line graph illustrating self-immolation and release of HMC (7-hydroxy-4- methyl-2H-chromen-2-one, or 7-hydroxy-4-methyl-coumarin, which serves as an illustrative surrogate hydroxy-linked drug) from the compound "Cbz-Val-Cit-PAMA-(methyl-
  • FIG. 2 is a line graph illustrating self-immolation and release of HMC from the compound "Cbz-Val-Cit-PAMA-(amide)-HMC":
  • FIG. 3 is a line graph illustrating self-immolation and release of HMC from the compound "Cbz-Val-Cit-PAMA-(PEG-amide)-HMC":
  • PABE-HMC was used as reference compound.
  • FIG. 4 is a line graph illustrating self-immolation and release of AMC (7-amino-4- methyl-2H-chromen-2-one, or 7-amino-4-methyl-coumarin, which serves as an illustrative surrogate amine-linked drug) from the compound "Cbz-Val-Cit-PAMA-(PEG-amide)-
  • Cathepsin B The reference compound was the compound "Cbz-Val-Cit-PABC-AMC":
  • FIG. 6A is a line graph illustrating self-immolation and release of the cytotoxic
  • FIG. 6B is a line graph illustrating self-immolation and release of CBI-DMMI from the compou "Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-Pro-CBI-DMMI”:
  • FIG. 6C is a line graph illustrating self-immolation and release of CBI-DMMI from the compou "Cbz-Val-Cit-PAMA-(Boc-PEG4-amide)-Pro-CBI-DMMI”:
  • FIG. 7 is a line graph illustrating self-immolation and release/generation of a cytotoxic drug (Compound A) from anti-HER2 ADC-v2 [Cbz-Val-Cit-PAMA-(anti-HER2 scFv-Fc-succinimido-butanoyl-PEG3-amide)-(Compound 91); Example 39] in the presence and absence of Cathepsin B.
  • FIGs. 8A and 8B provide the results of an LC-ESI-MS analysis of the anti-5T4 scADC of Example 37. The analysis was performed as described in Example 37.
  • FIG. 8A provides the ESI-MS charge states of scADC peak pre-reduced completely with DTT (dithiothreitol) to generate monomeric scADC.
  • FIG. 8B provides the deconvoluted mass spectrum showing relative abundance of non-conjugated (54669.6 Da) and singly conjugated (56000.7 Da) monomer of the scADC described in (A).
  • a para-amino mandelic acid (PAMA) derived linker is provided herein which is useful in a conjugate containing both a drug fragment or prodrug fragment and a targeting moiety (herein also called a "ligan "), said linker having the structure IX:
  • Wi and W 2 are independently N or CR 2 , and W is absent or present, provided that when W is absent, W 3 is independently NR 3 , O or S, and when W is present, W and W 3 are independently N or CR 2 , provided that at least one of Wi, W 2 , and W 3 is CR 2 ;
  • L is a di-, trior tetra- amino acid chain;
  • Z is an optional amine blocking group;
  • X is bound directly or indirectly to a drug fragment or prodrug fragment;
  • R 1 is bound to a targeting moiety via a group having a formula selected from -CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) n OCH 2 CHR 8 -, C C 6 alkyl- or -(CH 2 CH 2 OCH 2 CH 2 0) n -, wherein n is 1 to 8, R 6 is H, C C 6 alkyl, or C 2 -C 3 hydroxyalkyl;
  • R 7 is C C 6 alkyl
  • a drug and targeting conjugate which comprises the cleavable PAMA linker described herein, wherein each of W, Wi, W 2 and W 3 is CR 2 , R 1 is - CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) n OCH 2 CHR 8 -, and R 2 is H.
  • the conjugate or linker fragment A illustrated below serves as a substrate for one or more proteases following intracellular uptake of the conjugate by a targeted cell.
  • These fragments are designed to be cleaved by enzymes within the cathepsin family (e.g., cathepsins A, B, C, D, E, F, G, H, K, LI, L2, O, W, or Z), including particularly cathepsin B and cathepsin L following uptake of the conjugate of the invention into target cells of the targeted cell population.
  • the cathepsins are found primarily in lysosomes, but at least cathepsin K has been found to have extracellular activity.
  • Cathepsins are typically associated with a variety of cancers, and thus in one embodiment cleavage of the conjugate of the invention is designed for targeted cancers cells.
  • the protease substrate can be targeted to other cell types by designing it such that the protease substrate is cleavable for example by other cathepsins or other lysosomal enzymes, thus permitting the targeting-drug conjugates described herein to be designed and used in treatment of and therapies for conditions other than cancer including, e.g., certain viral infections, certain bacterial infections, COPD, chronic periodontitis, and pancreatitis.
  • fragment A illustrated below binds to the protease active site, such that the proteolytic activity results in cleavage of the amide bond between Z-L and the aryl or heteroaryl amine as indicated by the dashed line in B.
  • L refers to a di-, tri- or tetra-peptidyl group, i.e., a peptide composed of two, three or four amino acid residues.
  • the peptide L is optionally bonded to group Z, is linked to a drug moiety through the para-amino mandelic acid (PAMA) derived self-immolative linker in structure (IX), and forms part of the enzyme-cleavable substrate.
  • PAMA para-amino mandelic acid
  • Suitable peptides for L include, without limitation, Val-Cit, Gly-Gly-Phe-Gly, Phe-Lys, Val- Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp- Cit, Phe-Ala, Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.
  • Z refers to an optional amine blocking group attached to the N- terminus of the peptide residue L.
  • Z may be selected from C 1 -C6 acyl, optionally substituted aroyl, optionally substituted heteroaroyl, (aryl)alkyl-carbonyl, C 3 -C 6 cycloalkyl-carbonyl, C3-C6 heterocycloalkyl-carbonyl, (alkoxy)carbonyl (e.g., t-butoxycarbonyl known as BOC), (aryloxy)carbonyl, (heteroaryloxy)carbonyl, (aryl alkoxy)carbonyl (e.g., benzyloxycarbonyl or carbobenzyloxy, known as Cbz; and 9-Fluorenylmethyloxycarbonyl known as FMOC), (heteroaryl alkoxy)carbonyl, C 3 -C6 (cycloalkoxy)carbonyl, C 3 -C6
  • R 11 is H, C 1 -C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocycloalkyl, (aryl)alkyl, or (heteroaryl)alkyl.
  • Z groups include acetyl, pyrroloyl, and t- butylcarbonyl.
  • Suitable examples of groups Z-L are: Cbz- Val-Cit, Cbz-Phe-Cit, Cbz-Phe-Ala, Cbz- Phe-Lys, Cbz-Val-Lys, Cbz-Phe-Arg, Cbz-Val-Arg, Gly-Phe-Leu-Gly, and Gly-Gly-Phe- Gly.
  • groups Z-L are: Cbz- Val-Cit, Cbz-Phe-Cit, Cbz-Phe-Ala, Cbz- Phe-Lys, Cbz-Val-Lys, Cbz-Phe-Arg, Cbz-Val-Arg, Gly-Phe-Leu-Gly, and Gly-Gly-Phe- Gly.
  • the group Z-L forms the part of the substrate which binds to the protease "S3-S 2 -S 1 " binding pockets or subsites, using the terminology as described by I. Schechter and A. Berger in Biochemical and Biophysical Research Communications, 27, 157-162 (1967).
  • the group Z-L may be considered as the "P 3 -P 2 -Pi" positions of the protease substrate.
  • Di-, tri- and tetra-peptidyl fragments L that are suitable as substrate fragments "P 2 -Pi", “P3- P 2 -P 1 ", and “P4-P3-P 2 -P 1 " for proteases such as Cathepsin-B, -L and -S are well known in the art, for example as described by P. Ruzza et al., J. Peptide Science, 12, 455-461 (2006); and Y. Choe et al., J. Biol. Chem., 281, 12824-12832 (2006).
  • the compound B Upon binding to and cleavage by a protease, the compound B is converted to intermediate compound C which spontaneously undergoes a rearrangement or "self- immolation" to form compound D and eliminate fragment X.
  • X is -OC(0)Y
  • the reaction continues, to eliminate CO2 and the drug Y-H.
  • X is -N(H)C(0)Y
  • the reaction continues, to eliminate HNCO and the drug Y-H.
  • the drug Y-H is formed as indicated in the scheme below.
  • the drug Y-H is formed also as
  • a therapeutic drug and targeting conjugate which is of formula (I):
  • Wi and W 2 are independently N or CR 2 , and W is absent or present, provided that when W is absent, W 3 is independently NR 3 , O or S, and when W is present, W and W 3 are independently N or CR 2 , provided that at least one of Wi, W 2 , and W 3 is CR 2 ;
  • R 1 is - CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) n OCH 2 CHR 8 -, C C 6 alkyl-, or -(CH 2 CH 2 OCH 2 CH 2 0) n -, wherein n is 1 to 8;
  • LG is a ligand which specificall
  • B-LG is ; wherein said LG has at least one thiol moiety or at least one amino moiety which forms a point of attachment to B;
  • L is a di-, tri- or tetra- amino acid chain;
  • X is (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR 4 - which is the point of attachment to -OC(O)-, (ii) -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -O- or -NR 4 - as the point of attachment to -N(H)C(0)-;
  • each of W, Wi, W 2 and W 3 is CR 2
  • R 1 is -CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) administratOCH 2 CHR 8 -
  • R 2 is H
  • formula IA has the structure:
  • L is selected from the group consisting of (a) Val-Cit or (b) Gly-Gly-Phe-Gly;
  • X is -OC(0)Y, wherein Y is a drug or prodrug having a - NR 4 - as the point of attachment; or a drug or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug or prodrug.
  • R 1 is -
  • Z is an amine blocking group selected from the group consisting of Ci-C 6 acyl, optionally substituted aroyl, optionally substituted heteroaroyl, (aryl)alkyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C 3 -C6 heterocycloalkyl-carbonyl, (alkoxy)carbonyl, (aryloxy)carbonyl, (heteroaryloxy)carbonyl, (aryl alkoxy)carbonyl, (heteroaryl alkoxy)carbonyl, C 3 -C6 (cycloalkoxy)carbonyl, C 3 -C6 (heterocycloalkoxy)carbonyl, and R n NH-CO-.
  • R 11 is H, C 1 -C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocycloalkyl, (aryl)alkyl, or (heteroaryl)alkyl.
  • W is CR 2
  • R 2 is H
  • each of Wi , W 2 , and W 3 are
  • R 1 is -CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) n OCH 2 CHR 8 - and formula IB has the structure:
  • W is absent, W 1 is CR 2 , R 2 is H, R 1 is - CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) n OCH 2 CHR 8 - and the formula ICi or ICii has the structure:
  • linker Various uses of the linker, the conjugate, functional fragments thereof, and compositions and methods employing the same are provided herein.
  • the invention provides a synthetic process for preparing a tert-butyl (4- hydroxynaphthalen-2-yl)carbamate (compound 6 in Scheme 1).
  • the process comprises (i) admixing 1,3-dihydroxynaphthalene and diphenyl methylamine in a toluene solution and (ii) heating the toluene solution at about 80°C to about 125°C for about 4 to about 8 hours, about 5 to about 7 hours, or about 4 hours, about 5 hours, about 6 hours, about 7 hours or about 8 hours.
  • the heating may be about 90°C to about 110°C, or about 100°C.
  • the reaction mixture is combined with palladium hydroxide, di-tert butyl carbonate, and dioxane: water.
  • the ratio of dioxane to water is about 4 parts dioxane to about 1 part water (volume/volume). However, higher or lower amounts may be selected.
  • this combination is mixed at about 60 psi to about 80 psi hydrogen gas pressure for about 24 to 48 hours. However, these pressures and times may be adjusted as needed.
  • the reaction mixture is filtered and concentrated to yield crude tert- butyl (4-hydroxynaphthalen-2-yl)carbamate.
  • N-Boc Pro-CBI (12 in Scheme 1) and Pro-CBI (2 in Scheme 1) is illustrated, e.g., in Scheme 1.
  • the invention provides a synthetic process for preparing compound
  • this reaction is performed at a temperature from about 0 °C to about room temperature (about 20°C to about 25°C) for about 2 to about 6 hours. In one embodiment, the reaction is proceeds for about 4 hours. Following this, the reaction mixture is cooled to about -10°C to about 5°C, or 0°C before or at the time of adding trichloroacetonitrile and the reaction mixture may be allowed to warm to room temperature with stirring. The reaction mixture is then poured over water and extracted with ethylacetate.
  • the combined organic layers are washed and the crude product is purified.
  • washing is performed with water and brine, followed by separating the organic layer, drying, concentrating, and purifying crude product by silica gel column chromatography.
  • the eluent is 10% methanol in
  • This compound is useful in synthesis of a drug and targeting conjugate comprising a therapeutic drug associated with a cell specific targeting moiety via a linker which is specifically cleavable by a lysosomal protease.
  • the synthetic process involves combining carbonyldiimidazole and an optionally substituted Indole-2- carboylic acid derivative in a DMF solution and stirring for about 2 hours at ambient temperature.
  • the compound is 3 ("DMMI") as described in Scheme 2.
  • MGB minor groove binders
  • the reaction mixture is combined with a stirred DMF solution of amine 29a and an inorganic base such as sodium carbonate or potassium carbonate, wherein amine 29a has the structure:
  • Z is an N-blocking group and L is a di-peptide, tri-peptide, or tetra-peptide fragment.
  • the solid is filtered and removed and the remaining solution is neutralized with an acid such as formic acid or acetic acid.
  • the DMF is removed
  • a synthetic process for preparing compound of structural formula 40a is described.
  • This compound is useful in synthesis of a drug and targeting conjugate comprising a therapeutic drug associated with a cell specific targeting moiety via a linker which is specifically cleavable by a lysosomal protease.
  • the synthetic process involves the t-Boc protected compound of formula 34a was deprotected using an inorganic acid dissolved in an organic solvent such as ethylacetate.
  • the deprotected amino derivative was dissolved in an aprotic organic solvent such as DMF and reacted with succinate ester 27 in the presence of an organic or an inorganic base such as sodium or
  • cleavable group is intended to mean a moiety that is cleaved in vivo by the biological environment.
  • substrate also refers to the structural fragment that binds to the active site of the enzyme and is then cleaved by that enzyme.
  • the cleavable groups are selected so that activation occurs at the desired site of action, which can be a site in or near the target cells (e.g., carcinoma cells) or tissues such as at the site of therapeutic action or marker activity.
  • target cells e.g., carcinoma cells
  • exemplary enzymatically cleavable groups include natural amino acids or peptide sequences that end with a natural amino acid, and are attached at their carboxyl terminus to the linker.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs, N, N-dialkylated amino acid, and amino acid mimetic that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acid mimetic refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.
  • unnatural amino acid is intended to represent the "D" stereochemical form of the twenty naturally occurring amino acids.
  • the term “unnatural amino acid” includes homologues of the natural amino acids, and synthetically modified forms of the natural amino acids. Such synthetically modified forms include, but are not limited to, amino acids having -CH 2 - chains shortened or lengthened by up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprised halogenated groups, preferably halogenated alkyl and aryl groups.
  • heteroatom refers to oxygen, sulfur or nitrogen.
  • the number of carbon atoms present in a given group is designated "Cx- Cy", where x and y are the lower and upper limits, respectively.
  • a group designated as “C 1 -C6” contains from 1 to 6 carbon atoms.
  • the carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like. Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming from left to right the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent
  • arylalkyloxycarbonyl refers to the group (C6-C14 aryl)-(Ci-C6 alkyl)-O-C(O)-. Terms not defined herein have the meaning commonly attributed to them by those skilled in the art.
  • Ci-C 8 acyl- refers to a group having a straight, branched, or cyclic configuration or a combination thereof, attached to the parent structure through a carbonyl functionality. Such groups may be saturated or unsaturated, aliphatic or aromatic, and carbocyclic or heterocyclic. Examples of a Ci-C 8 acyl- group include acetyl-, benzoyl-, nicotinoyl-, propionyl-, isobutyryl-, oxalyl-, and the like. Lower-acyl refers to acyl groups containing one to four carbons.
  • An acyl group can be unsubstituted or substituted with one or more of halogen, NH 2 , (C C 6 alkyl)amino-, di(C C 6 alkyl)amino-, (Ci-C 6 alkyl)C(0)N(Ci-C 3 alkyl)-, CN, hydroxyl, C 1 -C6 alkoxy, C 1 -C6 alkyl, C6-C14 aryl, C 1 -C9 heteroaryl, or C3-C8 cycloalkyl.
  • halogen NH 2 , (C C 6 alkyl)amino-, di(C C 6 alkyl)amino-, (Ci-C 6 alkyl)C(0)N(Ci-C 3 alkyl)-, CN, hydroxyl, C 1 -C6 alkoxy, C 1 -C6 alkyl, C6-C14 aryl, C 1 -C9 heteroaryl, or C3-C
  • Alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, for example, a C 1 -C 12 alkyl group may have from 1 to 12 (inclusive) carbon atoms in it.
  • Examples of C 1 -C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec -butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.
  • Ci-Cs alkyl groups include, but are not limited to, methyl, propyl, pentyl, hexyl, heptyl, 3-methylhex-l-yl, 2,3-dimethylpent- 2-yl, 3-ethylpent-l-yl, octyl, 2-methylhept-2-yl, 2,3-dimethylhex-l-yl, and 2,3,3- trimethylpent-l-yl.
  • An alkyl group can be unsubstituted or substituted with one or more of halogen, NH 2 , (C C 6 alkyl)NH, (Ci-C 6 alkyl)(Ci-C 6 alkyl)N-, -N(C C 3 alkyl)C(0)(C C 6 alkyl), -NHC(0)(C C 6 alkyl), -NHC(0)H, -C(0)NH 2 , -(alkyl)amido-, -C(0)N(C C 6 alkyl)(C C 6 alkyl), CN, hydroxyl, C 6 alkoxy, C C 6 alkyl, -C(0)OH, -C(0)0(C C alkyl), -C(0)(C C 6 alkyl), C 6 -C 14 aryl, C C 9 heteroaryl, C 3 -C 8 cycloalkyl, d-C 6 haloalkyl, C C 6 aminoalkyl-, -OC(0)(Ci-
  • Alkoxy- refers to the group R-O- where R is an alkyl group, as defined above.
  • Exemplary C 1 -C6 alkoxy- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1-propoxy, n-butoxy and t-butoxy.
  • An alkoxy group can be unsubstituted or substituted with one or more of halogen, hydroxyl, C 1 -C6 alkoxy, NH 2 , (C 1 -C6 alkyl)amino-, di(Ci-C6 alkyl)amino-, (C C 6 alkyl)C(0)N(C C 3 alkyl)-, (alkyl)carboxyamido-, HC(0)NH-, H 2 NC(0)-, (C C 6 alkyl)NHC(0)-, di(C C 6 alkyl)NC(O)-, CN, C0 2 H, (C C 6
  • Aryl refers to an aromatic hydrocarbon group.
  • Examples of a C6-C14 aryl group include, but are not limited to, phenyl, a-naphthyl, ⁇ -naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenanaphthyl.
  • Examples of a C - Cioaryl group include, but are not limited to, phenyl, a-naphthyl, ⁇ -naphthyl, biphenyl, and tetrahydronaphthyl.
  • An aryl group can be unsubstituted or substituted with one or more of C 1 -C6 alkyl, halogen, haloalkyl, hydroxyl, hydroxyl(Ci-C6 alkyl)-, NH 2 , aminoalkyl-, dialkylamino-, -COOH, -C(0)0-(C C 6 alkyl), -OC(0)(C C 6 alkyl), N-alkylamido-, - C(0)NH 2 , (C C 6 alkyl)amido-, N0 2 , (aryl)alkyl, C C 6 alkoxy, C 6 -C 10 aryloxyl, C 2 -C 10 heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carboxyamido-, (aryl)alkyl
  • Spirocyclic refers to a 3, 4, 5, 6 or 7 membered cyclic compound optionally having one or two heteroatoms optionally having 0 to 2 double bonds and containing a sp 3 -hybridized carbon, wherein the 2 nd cyclic system contains the same sp 3 - hybridized carbon atom.
  • the following examples denote some of the examples of spirocyclic system.
  • (Aryl)alkyl refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with an aryl group as defined above.
  • ( C i4 Aryl) alkyl- moieties include benzyl, benzhydryl, 1-phenylethyl, 2-phenylethyl, 3- phenylpropyl, 2-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl and the like.
  • An (aryl)alkyl group can be unsubstituted or substituted with one or more of halogen, CN, NH 2 , hydroxyl, (C C 6 alkyl)amino-, di(Ci_C 6 alkyl)amino-, (Ci-C 6 alkyl)C(0)N(C C 3 alkyl)-, (C C 6 alkyl)carboxyamido-, HC(0)NH-, H 2 NC(0)-, (C C 6 alkyl)NHC(O)-, di(C C 6 alkyl)NC(O)-, C C 6 alkoxy, C C 6 alkyl, C0 2 H, (C C 6 alkoxy)carbonyl-, (C C 6 alkyl)C(O)-, C 6 -Ci 4 aryl, Ci-Cg heteroaryl, C 3 -C 8 cycloalkyl, Ci-C 6 haloalkyl, amino(Ci-C 6 alkyl)-, (
  • (Alkoxy)carbonyl- refers to the group alkyl-O-C(O)-.
  • Exemplary (d- C 6 alkoxy)carbonyl- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1- propoxy, n-butoxy and t-butoxy.
  • An (alkoxy)carbonyl group can be unsubstituted or substituted with one or more of halogen, hydroxyl, NH 2 , (Ci-C 6 alkyl)amino-, di(Ci-C 6 alkyl)amino-, (C C 6 alkyl)C(0)N(C C 3 alkyl)-, (alkyl)carboxyamido-, HC(0)NH-, H 2 NC(0)-, (C C 6 alkyl)NHC(0)-, di(C C 6 alkyl)NC(O)-, CN, C C 6 alkoxy, C0 2 H, (C C 6 alkoxy)carbonyl-, (Ci-C 6 alkyl)C(0)-, C6-C14 aryl, Ci-Cg heteroaryl, C3-C8 cycloalkyl, C 1 -C6 haloalkyl, amino(Ci-C6 alkyl)-, (C 1 -C6 alkyl)carbox
  • (Alkyl)amido- refers to a -C(0)NH- group in which the nitrogen atom of said group is attached to a Ci-C 6 alkyl group, as defined above.
  • Representative examples of a (Cr C 6 alkyl)amido- group include, but are not limited to, -C(0)NHCH 3 , -C(0)NHCH 2 CH 3 , - C(0)NHCH 2 CH 2 CH 3 , -C(0)NHCH 2 CH 2 CH 2 CH 3 , -C(0)NHCH 2 CH 2 CH 2 CH 2 CH 3 , - C(0)NHCH(CH 3 ) 2 , -C(0)NHCH 2 CH(CH 3 ) 2 , -C(0)NHCH(CH 3 )CH 2 CH 3 , -C(0)NH-C(CH 3 ) 3 and -C(0)NHCH 2 C(CH 3 ) 3 .
  • (Alkyl)amino- refers to an -NH group, the nitrogen atom of said group being attached to a alkyl group, as defined above.
  • Representative examples of an (C 1 -C6 alkyl)amino- group include, but are not limited to CH 3 NH-, CH 3 CH 2 NH-, CH 3 CH 2 CH 2 NH-, CH 3 CH 2 CH 2 CH 2 NH-, (CH 3 ) 2 CHNH-, (CH 3 ) 2 CHCH 2 NH-, CH 3 CH 2 CH(CH 3 )NH- and
  • An (alkyl)amino group can be unsubstituted or substituted with one or more of halogen, NH 2 , (C C 6 alkyl)amino-, di(C C 6 alkyl)amino-, (Ci-C 6 alkyl)C(0)N(Ci-C 3 alkyl)-, (Ci-C 6 alkyl)carboxyamido-, HC(0)NH-, H 2 NC(0)-, (C C 6 alkyl)NHC(O)-, di(C C 6 alkyl)NC(O)-, CN, hydroxyl, C C 6 alkoxy, C C 6 alkyl, C0 2 H, (C C 6 alkoxy)carbonyl-, (Ci-C 6 alkyl)C(O)-, C 6 -Ci 4 aryl, C C 9 heteroaryl, C 3 -C 8 cycloalkyl, C C 6 haloalkyl, amino(
  • Aminoalkyl- refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with -NH 2 ; one or both H of the NH 2 may be replaced by a substituent.
  • Alkylcarboxyl- refers to an alkyl group, defined above that is attached to the parent structure through the oxygen atom of a carboxyl (C(O)-O-) functionality.
  • Examples of (d- C 6 alkyl)carboxyl- include acetoxy, propionoxy, propylcarboxyl, and isopentylcarboxyl.
  • (Alkyl)carboxyamido- refers to a -NHC(O)- group in which the carbonyl carbon atom of said group is attached to a C 1 -C6 alkyl group, as defined above.
  • Representative examples of a (Ci-C 6 alkyl)carboxyamido- group include, but are not limited to, - NHC(0)CH 3 , -NHC(0)CH 2 CH 3 , -NHC(0)CH 2 CH 2 CH 3 , -NHC(0)CH 2 CH 2 CH 2 CH3, - NHC(0)CH 2 CH 2 CH 2 CH 2 CH 3 , -NHC(0)CH(CH 3 ) 2 , -NHC(0)CH 2 CH(CH 3 ) 2 , - NHC(0)CH(CH 3 )CH 2 CH 3 , -NHC(0)-C(CH 3 ) 3 and -NHC(0)CH 2 C(CH 3 ) 3 .
  • (Aryl)amino refers to a radical of formula (aryl)-NH-, wherein aryl is as defined above.
  • (Aryl)oxy refers to the group Ar-O- where Ar is an aryl group, as defined above.
  • Cycloalkyl refers to a non-aromatic, saturated, monocyclic, bicyclic or polycyclic hydrocarbon ring system.
  • Representative examples of a C 3 -Ci 2 cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cycloheptyl, cyclooctyl, decahydronaphthalen-l-yl, octahydro-lH-inden-2-yl, decahydro-lH-benzo[7]annulen-2-yl, and dodecahydros-indacen- 4-yl.
  • C 3 -Cio cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
  • C 3 - Cg cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and octahydropentalen-2-yl.
  • a cycloalkyl can be unsubstituted or substituted with one or more of halogen, NH 2 , (C 1 -C6 alkyl)NH, (C 1 -C6 alkyl)(C C 6 alkyl)N-, -N(C C 3 alkyl)C(0)(C C 6 alkyl), -NHC(0)(C C 6 alkyl), -NHC(0)H, -C(0)NH 2 , -C(0)NH(Ci-C 6 alkyl), -C(0)N(Ci-C 6 alkyl)(Ci-C 6 alkyl), CN, hydroxyl, C 6 alkoxy, Ci-C 6 alkyl, -C0 2 H, -C(0)0(C C 6 alkyl), -C(0)(C C 6 alkyl), C 6 -Ci 4 aryl, C C 9 heteroaryl, C 3 -C 8 cycloalkyl, C C 6 haloalkyl, C C 6 amino
  • Halo or "halogen” refers to -F, -CI, -Br and -I.
  • C 1 -C6 Haloalkyl- refers to a i-C alkyl group, as defined above, wherein one or more of the Ci-C 6 alkyl group's hydrogen atoms has been replaced with -F, -CI, -Br, or -I.
  • Each substitution can be independently selected from -F, -CI, -Br, or -I.
  • Representative examples of an C 1 -C6 haloalkyl- group include, but are not limited to, -CH 2 F, -CC1 3 , -CF 3 ,
  • CH 2 CF 3 -CH 2 C1, -CH 2 CH 2 Br, -CH 2 CH 2 I, -CH 2 CH 2 CH 2 F, -CH 2 CH 2 CH 2 C1, - CH 2 CH 2 CH 2 Br, -CH 2 CH 2 CH 2 CH 2 I, -CH 2 CH 2 CH 2 CH 2 CH 2 Br, -CH 2 CH 2 CH 2 CH 2 I, -
  • Heteroaryl refers to a monocyclic, bicyclic, or polycyclic aromatic ring system containing at least one ring atom selected from the heteroatoms oxygen, sulfur and nitrogen.
  • C 1 -C9 heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4- triazole, 1 -methyl- 1,2,4-triazole, lH-tetrazole, 1-methyltetrazole, benzoxazole,
  • Bicyclic C 1 -C9 heteroaryl groups include those where a phenyl, pyridine, pyrimidine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring having one or two nitrogen atoms in the ring, one nitrogen atom together with either one oxygen or one sulfur atom in the ring, or one O or S ring atom.
  • Examples of monocyclic C 1 -C4 heteroaryl groups include 2H-tetrazole, 3H-l,2,4-triazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, imidazole, and pyrrole.
  • a heteroaryl group can be unsubstituted or substituted with one or more of C 1 -C6 alkyl, halogen, haloalkyl, hydroxyl, CN, hydroxyl(Ci-C 6 alkyl)-, NH 2 , aminoalkyl-, dialkylamino-, -C0 2 H, -C(0)0(C C 6 alkyl), -OC(0)(C C 6 alkyl), N-alkylamido-, - C(0)NH 2 , (Ci-C 6 alkyl)carboxyamido-, N0 2 , (aryl)alkyl, Ci-C 6 alkoxy, C 6 -Ci 0 aryloxyl, C 2 - Cio heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carbox
  • Heterocycle or “heterocyclyl” refers to monocyclic, bicyclic and polycyclic groups in which at least one ring atom is a heteroatom.
  • a heterocycle may be saturated or partially saturated.
  • Exemplary Ci-Cgheterocyclyl- groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran,
  • Ci heterocyclyl- radicals would include but are not limited to oxaziranyl, diaziridinyl, and diazirinyl
  • C 2 heterocyclyl- radicals include but are not limited to aziridinyl, oxiranyl, and diazetidinyl
  • C 9 heterocyclyl- radicals include but are not limited to azecanyl, tetrahydroquinolinyl, and perhydroisoquinolinyl.
  • a heterocyclyl group can be unsubstituted or substituted with one or more of C 1 -C6 alkyl, halogen, haloalkyl, hydroxyl, hydroxyl(C C 6 alkyl)-, -NH 2 , aminoalkyl-, dialkylamino-, -C0 2 H, -C(0)0-(C C 6 alkyl), -OC(0)(C C 6 alkyl), N-alkylamido-, -C(0)NH 2 , (C C 6 alkyl)amido-, or N0 2 .
  • Heterocyclyl(alkyl)- refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with a heterocycle group as defined above.
  • Heterocyclyl(Ci-C6 alkyl)- moieties include 1-piperazinylethyl, 4- morpholinylpropyl, 6-piperazinylhexyl, and the like.
  • a heterocyclyl(alkyl) group can be unsubstituted or substituted with one or more of halogen, NH 2 , (C 1 -C6 alkyl)amino-, di(Ci-C6 alkyl)amino-, (C C 6 alkyl)C(0)N(C C 3 alkyl)-, (C C 6 alkyl)carboxyamido-, HC(0)NH-, H 2 NC(0)-, (Ci-C 6 alkyl)NHC(O)-, di(C C 6 alkyl)NC(0)-, CN, hydroxyl, C C 6 alkoxy, C C6 alkyl, C0 2 H, (C 1 -C6 alkoxy)carbonyl-, (Ci-C 6 alkyl)C(0)-, 4- to 7-membered monocyclic heterocycle, C6-C14 aryl, Ci-Cgheteroaryl, or C3-C8 cycloalkyl.
  • halogen
  • Hydroxylalkyl- refers to a alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with hydroxyl groups.
  • Examples of d- C 6 hydroxylalkyl- moieties include, for example, -CH 2 OH, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH, - CH 2 CH(OH)CH 2 OH, -CH 2 CH(OH)CH 3 , -CH(CH 3 )CH 2 OH and higher homologs.
  • “Monocyclic heterocyclyl” refers to monocyclic groups in which at least one ring atom is a heteroatom.
  • a heterocycle may be saturated or partially saturated.
  • Exemplary monocyclic Ci-Cgheterocyclyl- groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3, 6-tetrahydropyridine-l-yl, tetrahydropyran, pyran, thiane, thiine, piperazine, oxazine, 5,6-dihydro-4H-l,3-oxazin-2-yl, 4-methyl-3,4- dihydro-2H-l,4-benzoxazin-7-yl, thiazine, dithiane, and di
  • Ciheterocyclyl- radicals would include but are not limited to oxaziranyl, diaziridinyl, and diazirinyl
  • C 2 heterocyclyl- radicals include but are not limited to aziridinyl, oxiranyl, and diazetidinyl
  • Cgheterocyclyl- radicals include but are not limited to azecanyl.
  • a heterocyclyl group can be unsubstituted or substituted with one or more of Ci-C 6 alkyl, halogen, haloalkyl, hydroxyl, hydroxyl(Ci-C6 alkyl)-, NH 2 , aminoalkyl-, dialkylamino-, -CO 2 H, - C(0)0(Ci-C 6 alkyl), -OC(0)(Ci-C 6 alkyl), N-alkylamido-, -C(0)NH 2 , (C C 6 alkyl)amido-, or N0 2 .
  • Perfluoroalkyl- refers to alkyl group, defined above, having two or more fluorine atoms. Examples of a Ci-C 6 perfluoroalkyl- group include CF 3 , CH 2 CF 3 , CF 2 CF 3 and CH(CF 3 ) 2 .
  • optionally substituted means that at least one hydrogen atom of the optionally substituted group has been substituted with one or more of halogen, NH 2 , -NH(Ci-C 6 alkyl), -N(C C 6 alkyl)(C C 6 alkyl), -N(Ci-C 3 alkyl)C(0)(Ci-C 6 alkyl), - NHC(0)(C C 6 alkyl), -NHC(0)H, -C(0)NH 2 , -C(0)NH(C C 6 alkyl), -C(0)N(C C 6 alkyl)(C C 6 alkyl), CN , hydroxyl, C C 6 alkoxy, d-Ce alkyl, -C0 2 H, -C(0)0(C C 6 alkyl), - C(0)(Ci-C 6 alkyl), C 6 -Ci 4 aryl , C C 9 heteroaryl, or C 3 -C 8 cycloalkyl.
  • the aryl and the heteroaryl moieties are optionally substituted with optionally substituted C 1 -C6 alkyl, C 1 -C6 alkyl-amino, -O-C 1 -C6 alkyl, -O-optionally substituted C6-C 10 aryl, -O-optionally substituted C 1 -C6 heteroaryl, -O-optionally substituted C 3 -C6 heterocyclyl, -mono and dialkyl amino, halogen, CN, -C0 2 H, -COO-C 1 -C6 alkyl, -CONH 2j - CO-dialkylamino, -S0 2 NH 2j -optionally substituted C6-C 10 aryl, -optionally substituted d- C 10 heteroaryl, -optionally substituted C 2 -C6 heterocyclyl, or -O-C 1 -C6 alkyl-amino.
  • the drug or prodrug which is delivered inside the cells via a conjugate according to formula (I) may be selected with a view to the desired physiologic effect, e.g., therapeutic, cytotoxic, immunomodulatory, or the like, and the disease or condition which is being treated.
  • cytotoxic means toxic to cells or a selected cell population (e.g., cancer cells).
  • the toxic effect may result in cell death and/or lysis.
  • the toxic effect may be a sublethal destructive effect on the cell, e.g., slowing or arresting cell growth.
  • the drug or prodrug may be selected from a group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, amongst others.
  • drug refers to an amino acid-based molecule or a small molecule chemical compound which is biologically active and provides a desired physiological effect following administration to a subject in need thereof (e.g., an active pharmaceutical ingredient).
  • prodrug refers to a precursor of an active drug, that is, a compound that can be transformed to an active drug. Typically such a prodrug is subject to processing in vivo, which converts the drug to a physiologically active form. In some instances, a prodrug may itself have a desired physiologic effect.
  • the drug or prodrug (Y-H) when present as a part of the conjugate, is bound to the conjugate in the form of a drug fragment or a prodrug fragment (Y).
  • Y drug fragment or a prodrug fragment
  • the reaction continues, to eliminate CO 2 and the drug Y-H.
  • the reaction continues, to eliminate HNCO and the drug Y-H.
  • the drug Y-H is formed as indicated in the scheme below.
  • the drug Y-H is formed as indicated in the scheme below.
  • suitable drug or prodrug may be selected from amongst DNA damaging agents such as DNA alkylating agents and DNA strand breaking agents (e.g., calicheamicin, duocarmycins) and microtubule disrupting agents, such as microtubule depolymerizing agents (e.g., auristatins, maytansinoids) and microtubule polymerizing agents (e.g., taxanes).
  • DNA damaging agents such as DNA alkylating agents and DNA strand breaking agents (e.g., calicheamicin, duocarmycins)
  • microtubule disrupting agents such as microtubule depolymerizing agents (e.g., auristatins, maytansinoids) and microtubule polymerizing agents (e.g., taxanes).
  • DNA damaging agents such as DNA alkylating agents and DNA strand breaking agents (e.g., calicheamicin, duocarmycins)
  • microtubule disrupting agents such as microtubul
  • suitable DNA damaging drugs or prodrugs may include DNA minor groove binders such as duocarmycins and calicheamicins.
  • Duocarmycins includes duocarmycin SA and related analogs and prodrug forms, such as the
  • CBI cyclopropa[c]benzo[e]indol-4-one
  • Pro-CBI- DMMI cyclopropa[c]benzo[e]indol-4-one (CBI) analog CBI-DMMI and the pro-drug form Pro-CBI- DMMI, and related compounds identified herein.
  • Calicheamicins includes related enediynes, e.g., esperamicin.
  • Still other drugs or prodrugs which function as DNA damaging drugs or prodrugs includes lexitropsins.
  • the drug or prodrug selected may be an anti-tubulin agent.
  • anti-tubulin agents include, auristatins, taxanes and vinca alkyloids.
  • Auristatins includes dolastatin and other cytotoxic or cytostatic agents of the auristatin class, such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and auristatin F phenylenediamine (AFP).
  • MMAE monomethylauristatin E
  • MMAF monomethylauristatin F
  • AFP auristatin F phenylenediamine
  • Taxanes includes, for example, paclitaxel, docetaxel, tesetaxel and cabazitaxel.
  • Vinca alkaloids includes, e.g., vincristine, vinblastine, vindesine, and vinorelbine.
  • antitubulin agents include, for example, tubulysins, T67 (Tularik), baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine,
  • anti -tubulin agents includes maytansinoids; maytansinoids includes maytansine, DM-1 and DM-4 (ImmunoGen).
  • Still other classes of compounds or compounds with these or other cytotoxic modes of action may be selected, including, e.g., mitomycin C, mitomycin A, daunorubicin, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, aminopterin, bleomycin, 9-amino camptothecin, l-(chloromethyl)-2,3-dihydro-lH-benzo[e]indol-5-ol, pyrrolobenzodiazepine (PBD) polyamide and dimers thereof.
  • mitomycin C mitomycin A
  • daunorubicin doxorubicin
  • morpholino-doxorubicin morpholino-doxorubicin
  • cyanomorpholino-doxorubicin aminopterin
  • 9-amino camptothecin l-(chloromethyl)-2,3-dihydro-lH-benzo[e
  • cytotoxic agents include, for example, puromycins, CC-1065, SN-38, topotecan, rhizoxin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, discodermolide, eleutherobin, and mitoxantrone.
  • these drugs or prodrugs may contain amine groups or hydroxy groups which form the point of attachment to X as defined in formula (I).
  • amine-containing drugs examples include, e.g., monomethylauristatin E (MMAE), monomethyl Auristatin F (MMAF), epirubicin, doxorubicin, or the duocarmycin analog AS- 1-145.
  • hydroxy-containing drugs or prodrugs include, e.g., pro-duocarmycin SA, duocarmycin C2, carelsin, epirubin or doxorubicin.
  • the drug fragment or prodrug fragment is a DNA damaging agent that comprises a minor groove binder, which is an optionally-substituted lH-indole-2-carbonyl group incorporating a spiroheterocyclic amine, which is bound to a duocarmycin analog fragment such as CBI or Pro-CBI.
  • the minor groove binder non-covalently binds in the minor groove of double-stranded DNA, and the duocarmycin analog fragment such as CBI is understood by those in the art to covalently bind to DNA, thereby damaging the DNA.
  • the DNA damaging agent has the structure of formula (III) or (IV), below: N—
  • minor groove binder is a fragment that binds to and/or within the minor groove of double stranded deoxyribonucleic acid (DNA).
  • suitable minor groove binders include the following compounds (A)-(L) below:
  • the minor groove binders (B) to (L) and their related carboxylic acids were heretofore unknown, and can be used in a variety of indications, in addition to the conjugates of formula (I). Methods for generating these compounds are provided in the synthetic schemes described herein.
  • the drug fragment or prodrug fragment is a DNA damaging agent having the structure of formula (III) or (IV), wherein the minor groove binder has the following structure:
  • Q is N(CH 3 ) 2 or is selected from the following groups:
  • spirocyclic amines are commercially available, e.g., from commercial vendors such as Synthonix Inc, 2713 Connector Drive, Wake Forest, NC 27587, or they can be readily synthesized by methods known to those skilled in the art.
  • the drug is selected from the group consisting of maytansine, which is characterized by the structure of formula (V):
  • the drug is monomethylauristatin F (VI) or monomethylauristatin E (VII):
  • a "ligand” is any molecule that specifically binds or complexes with (herein also referred to as "targeting") a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population.
  • targeting a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population.
  • the cell is permissive for uptake of the ligand or ligand-drug-conjugate, which is then internalized into the cell.
  • a ligand that "specifically binds or complexes with” or “targets” a cell surface molecule means a ligand (which can be part of a conjugate of the invention) that preferentially associates with a cell surface molecule via intermolecular forces.
  • the ligand can preferentially associate with the cell surface molecule with a Kd of less than about 50 nM, less than about 5 nM, or less than 500 pM.
  • Techniques for measuring binding affinity of a ligand to a cell surface molecule are well-known; for example, one suitable technique, namely surface plasmon resonance (SPR), is described in Example 37.
  • the drug fragment or prodrug fragment Y is released as the drug or prodrug Y-H by a cascade of chemical rearrangements or reactions.
  • this cleavage occurs intracellular ly following uptake by the cells of the targeted cell population.
  • the ligand is used for targeting and has no detectable therapeutic effect as separate from the drug which it delivers.
  • the ligand functions both as a targeting moiety and as a therapeutic or immunomodulatory agent (e.g., to enhance the activity of the active drug or prodrug).
  • p" is 1 to 6.
  • a single ligand may be bound to multiple drug-linker fragments, i.e., 2, 3, 4, 5 or 6 drug-linker fragments.
  • the drug-linker fragments may be the same or they may be different from one another.
  • a single ligand-drug-conjugate contains 2 drug-linker fragments.
  • the ligand may be a polypeptide or protein.
  • the ligand is a cytokine.
  • One suitable class of cytokines includes interleukins.
  • suitable interleukins (IL) include, e.g., IL2, IL6 and IL-12.
  • the ligand is an immunoglobulin.
  • an immunoglobulin may be a full-length antibody or a functional fragment of an antibody.
  • functional fragment of an antibody it is meant herein that a sufficient portion of an antibody is provided that the immunoglobulin effectively binds or complexes with the cell surface molecule for its target cell population.
  • An immunoglobulin may be purified, generated recombinantly, generated synthetically, or combinations thereof, using techniques known to those of skill in the art. While immunoglobulins within or derived from IgG antibodies are particularly well-suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g., IgG, IgA, IgM, IgD and IgE.
  • immunoglobulins are well-suited as sources of the immunoglobulins.
  • immunoglobulins from other mammalian origins e.g., murine or other rodent, or rabbit origin
  • suitable immunoglobulins include, without limitation, a monoclonal antibody, a chimeric antibody, a humanized antibody, an immunoadhesin, and a truncated version of any of the immunoglobulins including, e.g., truncated versions such as F(Ab) 2 , Minibody, FAb, Single-domain Ab, scFv, tandem/bis-scFv, F(ab) 3 , scFv-Fc (or scFvFc), IgG CH, dsFv, diabody, triabody, or tetrabody.
  • Ab indicates antibody; bis-scFv indicates bispecific scFv; dsFc indicates disulfite Fc; Fab indicates antigen-binding fraction of immunoglobulin; Fc indicates crystallizable fraction of immunoglobulins; Fv indicates variable fragments of immunoglobulin; IgG indicates immunoglobulin G; and scFv indicates single-chain Fv.
  • scFv-Fc single chain Fv-Fc antibody
  • Fab' or F(ab') 2 fragment examples of suitable single chain Fv-Fc antibody (scFv-Fc, or scFvFc) or a functional Fab' or F(ab') 2 fragment are identified below in the experimental section. Such antibodies are not a limitation on the present invention.
  • the immunoglobulin selected may be one that can recognize a tumor associated antigen.
  • These immunoglobulins can be derived from human, murine or rabbit in origin.
  • Some suitable monoclonal antibodies include BR96 mAb (Trail, P.A., et al., "Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates", Science, 1993, 261, 212-215); (mAb against the Her2neu antigen such as Herceptin in advanced Breast cancer” Cancer Treat Rev. 26, 287-90, 2000); mAbs against CD40 antigen, such as S2C6 mAb (Francisco, J.
  • immunoglobulins include, without limitation, trastuzamab; panitumumab; brentuximab; gemtuzumab; and inotuzumab.
  • immunoglobulins include the anti-5T4 antibody and the anti-HER-1 and anti-HER2 antibodies described herein.
  • the term “5T4 antigen-binding portion” refers to a polypeptide sequence capable of selectively binding to a 5T4 antigen.
  • the 5T4 antigen-binding portion generally comprises a single chain scFv-Fc form engineered from an anti-5T4 antibody.
  • a single-chain variable fragment is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a linker peptide, and further connected to an Fc region comprising a hinge region and CH2 and CH3 regions of an antibody (any such combinations of antibody portions with each other or with other peptide sequences is sometimes referred to herein as an "immunofusion" molecule).
  • the scFv section may be C-terminally linked to the N-terminus of the Fc section by a linker peptide.
  • At least a portion of the 5T4 antigen-binding portion of the immunofusion molecules may originate from a murine source.
  • a murine source For example, one may obtain an immunofusion molecule by expressing a polynucleotide engineered to encode at least a murine anti-5T4 scFv region having the polypeptide sequence according to SEQ ID NO: A.
  • at least a portion of the 5T4-antigen binding portion may be generated to be chimeric or humanized according to well-known methods. See, Borras et al., J. Biol. Chem. 2010 Mar 19;285(12):9054-66.
  • an immunofusion molecule having a 5T4- antigen binding portion with a humanized scFv portion by expressing a polynucleotide engineered to encode at least the polypeptide sequence according to SEQ ID NO: B.
  • the Fv portion of the 5T4 antigen-binding portion may be engineered by well-known molecular biology techniques to comprise one or more amino acid substitutions in the VH region.
  • the Fc portion of the 5T4 antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge, CH2 and CH3 regions of an anti-5T4 antibody. For example, it is possible to engineer a
  • polynucleotide to encode at least an Fc portion having the polypeptide sequence according to SEQ ID NO: C.
  • a polynucleotide encoding a peptide wherein the single chain Fv and Fc regions are linked together may encode at least a chimeric 5T4 antigen-binding portion of an antibody- drug conjugate molecule having the polypeptide sequence according to SEQ ID NO: D or may encode a humanized 5T4 antigen-binding portion having the polypeptide sequence according to SEQ ID NOs: E or F.
  • a polypeptide linker such as one having the polypeptide sequence ASTC (SEQ ID NO: Y) or ASTX (SEQ ID NO: Z) (where "X” refers to any amino acid or a direct peptide bond between the adjacent amino acids), may fuse the C-terminus of ScFv portion to the N- terminus of the Fc portion of the 5T4 antigen-binding portion.
  • X refers to any amino acid or a direct peptide bond between the adjacent amino acids
  • any amino acid substitution, insertion, or deletion or use of a peptidomimetic does not substantially reduce the affinity or specificity of the 5T4 antigen- binding portion.
  • An immunofusion molecule having an amino acid substitution, insertion, or deletion or a peptidomimetic in the 5T4 antigen-binding portion preferably retains greater than 75%, preferably greater than 80%, preferably greater than 85%, preferably greater than 90%, or preferably greater than 95% of affinity or specificity for binding the 5T4 antigen compared to an antibody-drug conjugate molecule with an unmodified 5T4-antigen binding portion.
  • antibodies may be produced recombinantly, synthetically, or by other suitable method known in the art.
  • Such methods and constructs utilize the nucleic acid sequences encoding the polypeptides and peptide sequences identified herein.
  • such methods and constructs for antibody production utilize sequences which are naturally or artificially modified, e.g., natural variants or codon optimized variants of the SEQ ID NOs provided herein (e.g., A).
  • a variety of codon optimization schema are known in the art. See, e.g., UpGeneTM and OptimizerTM, which are web-based optimization methods.
  • the term "anti-HERl antigen-binding portion” refers to a immunoglobulin polypeptide sequence capable of selectively binding to a HERl antigen.
  • the HERl antigen-binding portion generally comprises a single chain scFv-Fc form engineered from an anti-HERl antibody.
  • a single- chain variable fragment (scFvFc) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a linker peptide, and further connected to an Fc region comprising a hinge region and CH 2 and CH 3 regions of an IgG antibody.
  • VH variable heavy
  • VL light chains
  • the scFv section may be C-terminally linked to the N-terminus of the Fc section by a linker peptide.
  • At least a portion of the HERl antigen-binding immunofusion molecules may originate from a murine source.
  • a murine source For example, one may obtain an immunofusion molecule by expressing a polynucleotide engineered to encode at least a murine anti-HERl scFv region having the polypeptide sequence according to SEQ ID NO: AB2 or AC2.
  • at least a portion of the HERl-antigen binding portion may be generated to be humanized or fully human according to well-known methods. See, Borras et al., J. Biol. Chem. 2010 Mar 19;285(12):9054-66.
  • an immunofusion molecule having a HERl- antigen binding portion with a humanized or fully human scFv portion by expressing a polynucleotide engineered to encode at least the polypeptide sequence according to SEQ ID NO: AB2 or AC2, respectively.
  • the Fv portion of the HERl antigen-binding portion may be engineered by well-known molecular biology techniques to comprise one or more amino acid substitutions in the VH region.
  • the Fc portion of the HERl antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge, CH2 and CH3 regions of an anti-HERl antibody.
  • a polynucleotide encoding a peptide wherein the single chain Fv and Fc regions are linked together may encode at least a chimeric HERl antigen-binding portion of an antibody-drug conjugate molecule having the polypeptide sequence according to SEQ ID NO: AB2 or AC2 or may encode a humanized HERl antigen-binding portion having the polypeptide sequence according to SEQ ID NOs: AB1 or AC1, respectively.
  • a polypeptide linker such as one having the polypeptide sequence ASTC (SEQ ID NO: Y) or ASTX (SEQ ID NO: Z) (where "X” refers to any amino acid or a direct peptide bond between the adjacent amino acids), may fuse the C -terminus of scFv portion to the N- terminus of the Fc portion of the HERl antigen-binding portion.
  • X refers to any amino acid or a direct peptide bond between the adjacent amino acids
  • an immunofusion molecule having a peptide linker according to SEQ ID NO: Y benefits from the presence of a cysteine residue that can be preferentially conjugated in a site-specific manner.
  • any amino acid substitution, insertion, or deletion or use of a peptidomimetic does not substantially reduce the affinity or specificity of the HERl antigen- binding portion.
  • An immunofusion molecule having an amino acid substitution, insertion, or deletion or a peptidomimetic in the HERl antigen-binding portion preferably retains greater than 75%, preferably greater than 80%, preferably greater than 85%, preferably greater than 90%, or preferably greater than 95% of affinity and specificity for binding the HERl antigen compared to an antibody-drug conjugate molecule with an unmodified HERl -antigen binding portion.
  • antibodies may be produced recombinantly, synthetically, or by other suitable method known in the art.
  • Such methods and constructs utilize the nucleic acid sequences encoding the polypeptides and peptide sequences identified herein.
  • such methods and constructs for antibody production utilize sequences which are naturally or artificially modified, e.g., natural variants or codon optimized variants of the SEQ ID NOs provided herein (e.g., AB 1).
  • a variety of codon optimization schema are known in the art. See, e.g., UpGeneTM and OptimizerTM, which are web-based optimization methods.
  • the term "anti-HER2 antigen-binding portion” refers to a polypeptide sequence capable of selectively binding to a HER2 antigen.
  • the HER2 antigen-binding portion generally comprises a single chain scFv-Fc form engineered from an anti-HER2 antibody.
  • a single-chain variable fragment (scFvFc) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a linker peptide, and further connected to an Fc region comprising a hinge region and CH2 and CH3 regions of an antibody.
  • VH variable heavy
  • VL light chains
  • the scFv section may be C-terminally linked to the N-terminus of the Fc section by a linker peptide.
  • At least a portion of the HER2 antigen-binding portion of the immunofusion molecules may originate from a murine source.
  • a murine source For example, one may obtain an immunofusion molecule by expressing a polynucleotide engineered to encode at least a murine anti-HER2 scFv region having the polypeptide sequence according to SEQ ID NO: AA2.
  • at least a portion of the HER2-antigen binding portion may be generated to be humanized according to well-known methods. See, Borras et al., J. Biol. Chem. 2010 Mar 19;285(12):9054-66.
  • an immunofusion molecule having a HER2- antigen binding portion with a humanized or fully human scFv portion by expressing a polynucleotide engineered to encode at least the polypeptide sequence according to SEQ ID NO: AA1.
  • the Fv portion of the HER2 antigen-binding immunofusion may be engineered by well-known molecular biology techniques to comprise one or more amino acid substitutions in the VH region.
  • the Fc portion of the HER2 antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge, CH2 and CH3 regions of an anti-HER2 antibody.
  • a polynucleotide encoding a peptide wherein the single chain Fv and Fc regions are linked together may encode at least a humanized HER2 antigen-binding portion of an antibody-drug conjugate molecule having the polypeptide sequence according to SEQ ID NO: AA1 or may encode a fully human HER2 antigen-binding portion having the polypeptide sequence.
  • a polypeptide linker such as one having the polypeptide sequence ASTC (SEQ ID NO: 1
  • any amino acid substitution, insertion, or deletion or use of a peptidomimetic does not substantially reduce the affinity or specificity of the HER2 antigen- binding portion.
  • An immunofusion molecule having an amino acid substitution, insertion, or deletion or a peptidomimetic in the HER2 antigen-binding portion preferably retains greater than 75%, preferably greater than 80%, preferably greater than 85%, preferably greater than 90%, or preferably greater than 95% of affinity or specificity for binding the HER2 antigen compared to an antibody-drug conjugate molecule with an unmodified HER1 -antigen binding portion.
  • antibodies may be produced recombinantly, synthetically, or by other suitable method known in the art.
  • Such methods and constructs utilize the nucleic acid sequences encoding the polypeptides and peptide sequences identified herein.
  • such methods and constructs for antibody production utilize sequences which are naturally or artificially modified, e.g., natural variants or codon optimized variants of the SEQ ID NOs provided herein (e.g., AA1).
  • a variety of codon optimization schema are known in the art. See, e.g., UpGeneTM and OptimizerTM, which are web-based optimization methods.
  • the targeting ligands, the PAMA-based self-immolative linkers and the drug or prodrug fragments described herein can be assembled into the therapeutic drug and targeting conjugate of the invention, for example according to the disclosed techniques and methods.
  • a method for producing a therapeutic drug and targeting conjugate comprising providing a cleavable para-amino mandelic acid (PAMA) derived linker having the structure IX:
  • L is a di-, tri- or terra- amino acid chain.
  • Z is an optional amine blocking group.
  • X is a conjugatable group, such as OH or NH 2 .
  • Wi and W 2 are independently N or CR 2 , and W is absent or present, provided that when W is absent, W 3 is independently NR 3 , O or S, and when W is present, W and W 3 are independently N or CR 2 , provided that at least one of Wi, W 2 , and W 3 is CR 2 .
  • R 2 is H, C C 6 alkyl, C C 6 alkoxy, halogen, C 1 -C6 fluoroalkyl, or cyano.
  • R 1 is a conjugatable group, such as a group having a formula selected from -CONR 6 CHR 7 CH 2 (OCH 2 CH 2 ) n OCH 2 CHR 8 -, C C 6 alkyl-, or -(CH 2 CH 2 OCH 2 CH 2 0) n -, wherein n is 1 to 8, R 6 is H, C C 6 alkyl, or C 2 -C 3 hydroxyalkyl; R 7 is C C 6 alkyl, C C 3 hydroxyalkyl, or -(CH 2 ) 2 NH(Ci-C 3 alkyl) 2 ; R 8 is H, Ci-C 3 alkyl or Ci-C 3 hydroxyalkyl.
  • a drug or prodrug fragment is also provided, and is conjugated to each of the PAMA derived linkers via the X group as described below.
  • X can be (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR 4 - which is the point of attachment to -OC(O)-, (ii) X is -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR 4 - or -O- as the point of attachment to -N(H)C(0)-; (iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or (iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR 4 - which is part of the drug fragment or prodrug fragment.
  • conjugation is not necessarily fixed; for example, the PAMA derived linker can first be conjugated to a ligand, and then to a drug or prodrug fragment, or the order of conjugation can be reversed. Suitable conjugation schemes are discussed herein and certain embodiments are presented in the Examples.
  • more than one thiol or other suitable moiety such as at least one amine moiety of a single LG serves as point of attachment to a B moiety of a drug-linker fragment.
  • a single ligand may have multiple linker conjugate fragments bound thereto through moiety B.
  • particularly suitable ligands (LG) are characterized by containing one or more thiol moieties or one or more amine moieties in their structure that are suitable for conjugation. At least one of these thiol or amine moieties serves as a point of attachment to B.
  • the thiol moiety is the point of attachment, with the attachment achieved via reaction with a maleimide or haloacetamide moiety in the PAMA linker-drug compound to the succinimide moiety or acetamide moiety of the LG to B, which is characterized by the following fragment:
  • a ligand may be modified or engineered to contain at least one thiol moiety suitable for conjugation.
  • a ligand e.g., an immunoglobulin
  • a ligand may be engineered to present a Cys residue for site-specific conjugation.
  • the number of available sulfide bonds available for the conjugation reaction may be controlled.
  • a reduction reaction is utilized to disrupt any possible adducts of the engineered cysteine (Cys) residue with glutathione or cysteines.
  • the ligand prior to the conjugation reaction, the ligand is reduced using dithiothreitol (DTT) in PBS (phosphate buffered saline; 20 mM sodium phosphate, pH 7.2, 150 mM NaCl) at 1 mg/mL to 10 mg/mL protein concentration, preferably about 3 mg/mL protein concentration, for 15 to 90 minutes or preferably about 45 min.
  • DTT dithiothreitol
  • PBS phosphate buffered saline
  • 150 mM NaCl sodium phosphate, pH 7.2
  • the ligand is then subjected to desalting to remove DTT by using a desalting column, for example in 50 mM Tris-Cl, 150 mM NaCl, 250 mM Arginine (pH 8.2) using a Hi-Prep 26/10 Desalting Column (GE healthcare).
  • Buffer-exchanged ligand is then concentrated and stirred slowly for 15 to 60 minutes, preferably for 30 min, to allow oxidative regeneration of the native interchain disulfide bonds.
  • the ligand is then incubated in the presence of the maleimide or haloacetamide linker at 5 to 50 fold molar excess, more preferably at about 15 fold molar excess, for 10 min to 6 h and preferably for about 90 min with slow stirring for conjugation to occur.
  • the conjugated linker - immunoglobulin is desalted to 20 mM PBS containing 10% glycerol.
  • the preceding is exemplary, and other suitable conditions and reagent may be selected which allow a Cys residue to be presented for conjugation.
  • Characterization of the conjugate is carried out by using a variety of analytical methods known to those skilled in the art. For example, LC-ESI-MS analysis of the reduced conjugate (i.e., analysis of the monomer unit) permits determination of the relative amounts of ligand having 0, 1 or more linker-drug groups attached.
  • one or more lysine (Lys) residues in the ligand LG may be employed for conjugation.
  • Methods for conjugation to lysine are well-known to those skilled in the art, e.g., M.P. Brun and L. Gauzy-Lazo, Methods in Molecular Biology 2013, 1045: 173-187; L.R. Milgrom, M.P. Deonarain, Innovations in Pharmaceutical Technology 2011, 38:56-59; W.C. Widdison et al., J. Med. Chem. 2006, 49:4392-4408.
  • a ligand may be modified or engineered to contain at least one amine moiety suitable for conjugation.
  • the product resulting from the ligand conjugation reaction may be filtered and purified using known techniques. In one embodiment, filtration removes any agglomerated conjugates and allows separation and isolation of non-agglomerated conjugates.
  • the reaction product may be a mixture of conjugates, in which p" is 1, 2, 3, 4, 5 or 6.
  • a composition of the invention contains conjugates in which the average p" is 2. Such a composition may additionally contain one or more conjugates in which p" is 1, 3, or 4. Minor amounts of conjugates in which p" is 5 or 6 may be present.
  • Still other compositions will contain a mixture of conjugates in which p" is 1 or 2. Such a mixture may contain approximately equivalent amounts of p" is 1 and p" is 2. Alternatively, such a mixture may contain more than 50% of p" is 2. In still another alternative, such a mixture may contain more than 50% of p" is 1. Still other compositions will contain a mixture of conjugates in with p" is 1, 2 or 3.
  • conjugates of formula (I) as described herein may contain any combination of ligand and drug/prodrug with the self-immolative linker of formula (IX) and defined herein.
  • Illustrative conjugates of formula (I) include, without limitation,
  • LG is ligand, such as an immunoglobulin protein or polypeptide which specifically targets a cell surface antigen and wherein LG contains at least or at least one amine, or at least one thiol group that forms a point of attachment to the succinimide moiety or the acetamide moiety of B; n is 1, 2 or 3; and p" is 1, 2, 3 or 4. In one embodiment, these structures have p" is 2. In still another embodiment, a composition of the invention contains conjugates which have an average p is 2.
  • Scheme 1 provides the preparation of intermediate compound 6 and Pro-CBI- 2 reagent starting from 2,4-dihydroxy naphthalene.
  • 1 ,3-Dihydroxy naphthalene 4 and diphenyl methylamine were heated at elevated temperatures.
  • the reaction was performed in an aromatic solvent such as toluene, xylenes or 1 ,2-dichlorobenzene.
  • the reaction was performed at about 100 to about 150°C.
  • the reaction was performed in a sealed tube. The product was then reacted with an excess of di-tert-butyl dicarbonate and palladium hydroxide to provide compound 6.
  • the reaction was performed in a parr shaker or an autoclave.
  • the reaction was performed at about 60 psi of hydrogen pressure. In a further embodiment, the reaction was performed with about 3 equivalents of di-tert-butyl dicarbonate. In yet another embodiment, the reaction was performed in dioxane: water or THF:water. To compound 6 was successively added benzyl bromide or p-methoxybenzyl bromide, potassium carbonate or in the presence of an inorganic base and tetrabutyl ammonium iodide to provide compound 7. In one embodiment, the reaction was performed in anhydrous DMF or acetone. In another embodiment, the reaction was performed at RT.
  • compound 7 was reacted with p- toluenesulfonic acid.
  • the reaction was performed in THF or dioxane.
  • the reaction was performed at about -20 to about 0°C.
  • N- iodosuccinimide was then added.
  • the addition was performed drop wise.
  • the reaction was complete, the mixture warmed to RT to provide product 8.
  • Compound 8 was reacted with a strong base at reduced temperatures.
  • the strong base was 60% sodium hydride or potassium hydride.
  • the reaction was performed at about 0°C.
  • the reaction was performed in DMF.
  • reaction was performed in an ether such as THF.
  • compound 10 was reacted with ammonium formate and a catalyst at elevated temperatures to afford product 11.
  • the reaction was performed in a mixture of THF and MeOH or dioxane and methanol mixture.
  • the catalyst was 10% Pd/C.
  • the elevated temperatures were about 70 to about 100°C.
  • compound 12 N-Boc-CBI
  • the reaction was performed in toluene. See, Lajiness and Boger, J. Org. Chem. 2011, 76, 583-587, which is herein incorporated by reference.
  • Compound 12 was treated with HC1 in ethyl acetate or in diethyl ether at reduced temperatures to provide compound 2 as the HC1 salt (Pro-CBI). In one embodiment, the reaction was performed at about -78°C to -10°C.
  • Scheme 2 describes a method for the synthesis of compound 3 (DMMI).
  • 3-Hydroxy- 4-methoxybenzaldehyde 5 was reacted with an inorganic base such as potassium carbonate or 1,2-dichloroethane or acetone at elevated temperatures to yield compound 35.
  • the elevated temperature was about 70°C.
  • the reaction was performed in DMF or acetone. Methyl chloroacetate and sodium azide were reacted to provide methyl azedoacetate.
  • the reaction was performed using DMSO or THF. Methyl azedoacetate was then reacted with aldehyde 35 to yield 36.
  • the reaction was performed in methanol or ethanol.
  • reaction was cooled to about -30°C.
  • Azido 36 was then heated to elevated temperatures to yield compound 37.
  • the reaction was performed in high boiling aromatic solvents such as toluene or xylenes.
  • the reaction was performed at reflux.
  • Compound 37 was then reacted with an amine in the presence of sodium carbonate or potassium carbonate at elevated temperatures.
  • the reaction was performed in water or ethanol: water mixture.
  • the amine was dimethyl amine or a spirocyclic amine such as 52, 53, 66, 75, 82, 79, 81 or any spirocyclic amine of general term Q.
  • the amine was 40% aqueous dimethyl amine solution.
  • the elevated temperature was about 100°C.
  • the product was then acidified to provide 3 (DMMI).
  • the acidification was performed in water.
  • the acidification was performed with an acid such as HCI or acetic acid.
  • Coupling of 2 (Pro-CBI) and 3 (DMMI) was accomplished by an EDC coupling reaction to provide 1 (Pro CBI-DMMI) (L. F. Tietze et al. (2008), Chem. Med. Chem. 3: 1946-1955), as outlined in Scheme 3.
  • the coupling was performed using EDC HCI.
  • the coupling was performed in DMF or THF.
  • Scheme 4 describes the synthesis of compound 20a ("Cbz-Val-Cit-PAMA-(methyl carboxylate)-HMC").
  • An optionally substituted 4-nitrobenzaldehyde 13A, zinc iodide and trimethylsilyl cyanide or KCN were reacted to provide compound 14.
  • the reaction was performed in DCM or THF.
  • the optionally substituted benzaldehyde is 4-nitro-benzaldehyde.
  • the reaction was performed at elevated temperatures.
  • To compound 14 was added an acid at elevated temperatures to obtain compound 15.
  • the reaction was performed in acetic acid.
  • the reaction was performed at about 100 to about 120°C.
  • the acid was hydrochloric acid.
  • reaction was performed in MeOH or ethanol. In a further embodiment, the reaction was performed at about 85 to about 100°C.
  • Compound 16 was then reduced using a reducing agent. In one embodiment, the reduction was performed using a catalyst and hydrogen gas under inert conditions. In a further embodiment, the catalyst was Pd/C.
  • Compound 17 was then reacted with Fmoc-Cit-OH or Fmoc-protected appropriate amino acid residues and EEDQ to provide compound 18. In one embodiment, the reaction was performed in DCM:THF or DMF.
  • reaction was performed in DMF or THF.
  • Compound 21 was reacted with Cbz-Val-OSu and an excess of di-isopropyl ethylamine.
  • the reaction was performed in DMF.
  • the reaction was performed at reduced temperatures.
  • the reaction was performed at about 0°C to about RT. Dilution of the mixture
  • Scheme 6 details the preparation of compound 23 from compound 20a.
  • To compound 20a was added an excess of LiOH.H 2 0 or NaOH.
  • the reaction was performed using MeOH:water or THF:MeOH:water.
  • the reaction was performed using about 3 to about 5 equivalents of LiOH.H 2 0.
  • the solution was then acidified with an acid to obtain the acid product.
  • the acid was HC1.
  • the acid product was then reacted with a slight excess of benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), and DIPEA to provide compound 23.
  • the reaction was performed in DMF or THF.
  • the reaction was performed at reduced temperatures.
  • the reaction was performed at 0°C to about RT.
  • the reaction was performed using about 1.2 to about 1.5 equivalents of CH 3 OCH 2 CH 2 OCH 2 CH 2 NH 2 .
  • Scheme 7 describes the synthesis of compound 25 (Cbz-Val-Cit-PAMA-(PEG- amide)-OC(O)-AMC) which was utilized as described herein to demonstrate the function of the PAMA linker for protease-activated self-immolation of a drug surrogate, 7-amino-4- methylcoumarin (AMC).
  • the described PAMA linker is connected to the warhead or the model compound via carbamate linker.
  • the 4-amino group in compound 17 was reacted with Cbz-Val-Cit-OH in the presence of about 5 equivalents of EEDQ to yield compound 20.
  • the reaction was performed in DCM:THF:MeOH.
  • the ester group of compound 20 was then converted to the PEG amide by a two-step process.
  • the ester group was hydrolyzed to the carboxylic acid.
  • the hydrolysis was performed using an inorganic base.
  • the inorganic base was LiOH or NaOH.
  • the carboxylic acid was reacted with NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 3 in presence of PyBOP and DIPEA to yield compound 24.
  • the reaction was performed at reduced temperatures to RT.
  • the reaction was performed at 0°C to RT.
  • Compound 24 was subsequently reacted with the isocyanate derivative of AMC (4-methyl-7-amino-coumarine) to yield compound 25.
  • the reaction was performed in DMF or THF.
  • an iodo acetyl group was introduced to the PAMA based liker/cytotoxic payload components.
  • Compound 102a was prepared to target the thiol group of the cysteine or the -NH 2 group of lysine residues of the antibody.
  • Compound 102a was prepared starting from the amine 101a.
  • the appropriately substituted Z-L-PAMA- (iodoacetyl-PEG-3-amide)-CBI-91 was reacted with compound 103 in a borate buffer: dioxane mixture and an inorganic base to provide compound 102a.
  • the inorganic base was sodium or potassium carbonate.
  • Scheme 9 provides the synthesis of compound 26, which can be used as a synthon to prepare an appropriate ADC.
  • compound 20 was reacted with an excess of an inorganic base such as LiOH.H 2 0 or NaOH.
  • an inorganic base such as LiOH.H 2 0 or NaOH.
  • the reaction was performed in MeOH:water or THF:MeOH:water.
  • the reaction was performed using about 2 equivalents of LiOH.H 2 0.
  • the product was neutralized.
  • the neutralization was performed using acidic resin (for example, Amberlyst® (Rohm Haas)) to provide the acid product.
  • the acid product was the reacted with NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NHB0C, PyBOP, and a slight excess of DIPEA to provide compound 26.
  • the reaction was performed in DMF or THF.
  • the reaction was performed at reduced temperatures.
  • the reaction was performed at 0°C.
  • the reaction was performed using about 1.5 to about 3 equivalents of DIPEA.
  • Scheme 10 depicts the synthesis of compound 29.
  • compound 20 was hydrolyzed by reaction with an excess of LiOH or other inorganic base.
  • the reaction was performed in THF and water.
  • the reaction was performed using about 6 to 10 equivalents of LiOH.
  • the reaction was performed at reduced temperatures.
  • the reaction was performed at 0°C to RT.
  • the mixture was neutralized with an acid to provide the acid product.
  • the acid was citric acid or acetic acid.
  • the acid product was then reacted with NH 2 CH 2 CH 2 O-CH 2 CH 2 O- CH 2 CH 2 NHBoc, PyBOP, and DIPEA to provide compound 28.
  • the reaction was performed in DMF. In another embodiment, the reaction was performed at reduced temperatures. In a further embodiment, the reaction was performed at 0°C to about RT. Compound 28 was then reacted with TFA at reduced temperatures. In one embodiment, the reaction was performed in DCM or dichloroethane. In another embodiment, the reaction was performed at about -10 to about 0°C. To the resulting solution was added succinate ester 27 and an excess of DIPEA to afford compound 29. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed using about 2 to about 5 equivalents of DIPEA.
  • Scheme 11 depicts the synthesis of compound 31 and was prepared similarly to the route described in Scheme 10.
  • compound 20 was reacted with an excess of LiOH.H 2 0 an inorganic base such as NaOH.
  • the reaction was performed in THF and water.
  • the reaction was performed using about 3 to about 5 equivalents of LiOH.H 2 0.
  • the reaction was performed at reduced temperatures.
  • the reaction was performed at 0°C to about RT.
  • the mixture was neutralized with an acid to provide the acid product.
  • the acid was citric acid.
  • the acid product was then reacted with NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NHB0C, PyBOP, and DIPEA to provide compound 30.
  • the reaction was performed in DMF or THF.
  • the reaction was performed at reduced temperatures.
  • the reaction was performed at 0°C to about RT.
  • Compound 30 was then reacted with TFA at reduced temperatures.
  • the reaction was performed in DCM.
  • the reaction was performed at about -10 to about 0°C.
  • succinate ester 27 and DIPEA was added to afford compound 31.
  • the reaction was performed in DMF or THF.
  • the ether formation between the PAMA based linker and the compound 2a was prepared by a novel procedure as outlined in Scheme 12.
  • An appropriately substituted PAMA linker of structure 26 was reacted with an excess of CS 2 CO 3 .
  • the reaction was performed in DMF or THF.
  • the reaction was performed with about 2 to about 5 equivalents of Cs 2 C0 3 .
  • To this mixture at reduced temperatures was then added an excess of CI 3 CN to provide compound 32.
  • this reaction was performed at about 0°C to about RT.
  • the reaction was performed using about 10 to about 15 equivalents of CCI 3 CN.
  • Compound 40 was prepared as described in Scheme 13. Specifically, compound 34 was coupled with DMMI via a CDI mediated reaction as outlined in Scheme 11. Other DNA minor groove binder groups are synthetically introduced in a similar manner. Specifically, DMMI 3 was reacted with a slight excess of CDI at reduced temperatures. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using 1.3 equivalents of CDI. The mixture was then added to amine 34 and an excess of sodium or potassium carbonate to obtain compound 39. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed using about 10 to about 15 equivalents of sodium carbonate.
  • reaction was performed in ethyl acetate. In another embodiment, the reaction was performed at about -10 to about 0°C. The resulting product was then reacted with succinate ester 27 and sodium carbonate to provide compound 40. In one embodiment, the reaction was performed in DMF or THF.
  • reaction was performed in MeOH and water or MeOH:THF:water. In a further embodiment, the reaction was performed using about 2 to about 5 equivalents of LiOH.H 2 0. In another embodiment, the reaction was performed at about 0°C to about RT. The mixture was then neutralized to provide the acid product. In one embodiment, the neutralization was performed using an acidic resin. The acid product was then reacted with
  • reaction was performed in DMF or THF. In a further embodiment, the reaction was performed at about 0°C to about RT. In another embodiment, the reaction was performed using about 1.5 to about 2.5 equivalents of DIPEA.
  • Scheme 15 depicts the synthesis of compound 44 from alcohol 41.
  • To alcohol 41 was added about 2 to about 5 equivalents of CS 2 CO 3 .
  • the mixture was then combined with CCI 3 CN at reduced temperatures to yield compound 42.
  • the reduced temperature was about 0°C to about RT.
  • the reaction was performed in DMF or THF.
  • An excess of compound 42 and Boc-Pro-CBI were then added to a suspension of 4A molecular sieves in dry CH 3 CN.
  • the reaction was performed with about 1.5 to about 2 equivalents of compound 42.
  • the product was reacted with a Lewis acid such as BF 3 .ether at reduced temperatures.
  • the reaction was performed at -10 to about 0°C.
  • Scheme 16 provides a route to compound 46 from compound 3.
  • DMMI compound 3 was added a slight excess of CDI at reduced temperatures.
  • the reaction was performed in DMF or THF In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed with about 1.3 equivalents of CDI.
  • the product was then reacted with amine 44 and an excess of sodium or potassium carbonate.
  • the reaction was performed in DMF.
  • the reaction was performed with about 10 to about 15 equivalents of sodium carbonate.
  • the mixture was then neutralized to obtain compound 45.
  • the neutralization was performed using formic acid.
  • To compound 45 was added HC1 in EtOAc at reduced temperatures. In one embodiment, the reaction was performed in EtOAc. In another embodiment, the reaction was performed at about -10 to about 0°C.
  • succinate ester 27 and sodium carbonate to provide compound 46.
  • the reaction was performed using DMF.
  • Scheme 17 provides the combined synthesis of compounds 52 and 53, both routes beginning with starting material compound 47.
  • compound 47 was reacted with an excess of ethyl cyanoacetate and an excess of NEt 3 in the presence of 4A molecular sieves to provide compound 48.
  • the reaction was performed in DCM.
  • the reaction is performed with about 1.5 to about 2 equivalents of ethyl cyanoacetate.
  • the reaction is performed with about 3 to about 5 equivalents of NEt 3 .
  • Compound 48 was then reacted with KCN at elevated temperatures to provide compound 49.
  • the reaction was performed using ethanol and water.
  • the reaction was performed at about 80 to about 100°C.
  • Compound 49 was then reacted with concentrated HC1 at elevated temperatures to provide diacid 50.
  • the reaction was performed in a sealed tube. In another embodiment, the reaction was performed at about 100°C.
  • To compound 50 was added ⁇ , ⁇ '- dicyclohexylcarbodiimide.
  • the reaction was performed in DMF or THF. 4-Methoxy benzyl amine and an excess of NEt 3 or DIEA were then added. In one embodiment, the reaction was performed using about 2 to about 4 equivalents of NEt 3 .
  • the product was then reacted with Ac 2 0 and sodium acetate at elevated temperatures to provide compound 51. In one embodiment, the reaction was performed at about 100°C.
  • Compound 51 was then utilized to prepare compounds 52 and 53 via separate routes.
  • compound 51 was reacted with palladium hydroxide in the presence of hydrogen to provide amine 52.
  • the reaction was performed in acetic acid: 1,4-dioxane: water.
  • the reaction was performed in 1.5: 1.5: 1 v/v of acetic acid:l,4-dioxane:water.
  • compound 51 was reacted with an excess of eerie ammonium nitrate provide compound 53.
  • the reaction was performed using acetonitrile: water.
  • the reaction was performed using about 2 to about 8 equivalents of eerie ammonium nitrate.
  • Scheme 18 describes the preparation of compound 56 from starting materials 32 and 52.
  • To spirocyclic amine 52 were added compound 32 and an excess of K 2 CO 3 to provide compound 54.
  • the reaction was performed in DMF or THF.
  • the reaction was performed using about 3 to about 8 equivalents of K 2 CO 3 .
  • To compound 54 was added an excess of Lil at elevated temperatures to provide compound 55.
  • the reaction was performed in pyridine.
  • the reaction was performed using 6 equivalents of Lil.
  • the reaction was performed at about 200°C.
  • the reaction was performed in a microwave reactor.
  • To Pro-CBI 2 was added compound 55 and an excess of EDC ' HCl at reduced temperatures to provide compound 56.
  • the reaction was performed in DMF or THF.
  • the reaction was performed using 4 to about 6 equivalents of EDC ' HCl.
  • the reaction was performed at about 0°C to about RT.
  • Scheme 19 depicts a route to compound 59 via compounds 32 and 53 and is similar to the route described in Scheme 18.
  • amine 53 was reacted with compound 32 and an excess of K 2 CO 3 or NaH at elevated temperatures to provide compound 57.
  • the reaction was performed using DMF or THF.
  • the reaction was performed using about 2.5 to about 3.5 equivalents of K 2 CO 3 .
  • the reaction was performed at about 100°C.
  • To derivative 57 was added Lil at elevated temperatures to provide compound 58.
  • the reaction was performed in pyridine.
  • the reaction was performed at about 200°C.
  • the reaction was performed using about 6 to about 10 equivalents of Lil.
  • reaction was performed in a microwave reactor. Finally, Pro-CBI 2 was reacted with compound 58 and excess EDC.HC1 to provide compound 59. In one embodiment, the reaction was performed using DMF. In another embodiment, the reaction was performed at about 0 to about 10°C. In a further embodiment, the reaction was performed using about 3 to about 5 equivalents of EDC.HC1. Scheme 20
  • Scheme 20 provides the synthesis of compound 65 from starting material 32. Specifically, ester 32 was reacted with a slight excess of Cs 2 C0 3 . In one embodiment, the reaction was performed in ethanol and water. In another embodiment, the reaction was performed in a 1 : 1 v/v ethanol to water solution. In a further embodiment, the reaction was performed using about 1.5 to about 3 equivalents of CS 2 CO 3 . The mixture was then acidified to provide compound 60. In one embodiment, the acidification was performed using HC1. In another embodiment, the acidification was performed using 4N HC1. To compound 60 was added benzyl alcohol, DCC, and an excess of DMAP at reduced temperatures to provide compound 61. In one embodiment, the reaction was performed in DCM.
  • the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using about 2.5 to about 3.5 equivalents of DMAP. To amine 53 was added compound 61 and an excess of K 2 CO 3 or NaH at elevated temperatures to provide compound 62. In one embodiment, the reaction was performed using DMF. In another embodiment, the reaction was performed at about 100 to about 120°C. In a further embodiment, the reaction was performed using about 2.5 to about 5 equivalents of K 2 CO 3 . To benzyl ester 62 was added palladium hydroxide under hydrogen to provide amine 63. In one embodiment, the reaction was performed in acetic acid, 1,4-dioxane, and water.
  • the reaction was performed in a 2:2: 1 acetic acid, 1,4-dioxane, and water solution.
  • To amine 63 were added acetic acid and an excess of formaldehyde or para formaldehyde.
  • the reaction was performed in MeOH or ethanol.
  • the reaction was performed using about 2 to about 4 equivalents of formaldehyde.
  • the mixture was then charged with sodium triacetoxy borohydride at reduced temperatures to provide compound 64.
  • the reaction was performed at about 0°C to about RT.
  • compound 64 was reacted with Pro-CBI 2 and an excess of EDC.HC1 at reduced temperatures to provide compound 65.
  • the reaction was performed using DMF.
  • the reaction is performed using about 5 to about 10 equivalents of EDC.HC1.
  • the reaction is performed at about 0°C to about RT.
  • reaction is performed using THF.
  • the reducing agent is LiAlH 4 .
  • reaction is performed using about 4 to about 6 equivalents of the reducing agent.
  • the starting temperature for the reaction is about -10 to about 0°C.
  • the temperature of the reaction is then elevated to about 65 to about 75°C.
  • To spiroamine 66 were added compound 32 and an excess of K 2 CO 3 or NaH at elevated temperatures to provide compound 67.
  • the reaction is performed in DMF or THF.
  • the reaction was performed with about 2 to about 5 equivalents of K 2 CO 3 . In a further embodiment, the reaction was performed at about 80 to about 100°C.
  • To compound 67 was added palladium hydroxide under hydrogen to provide amine 68.
  • the reaction was performed in acetic acid, 1,4- dioxane, and water.
  • the reaction was performed in a 2:2:1 acetic acid, 1,4-dioxane, and water solution.
  • To amine 68 were added acetic acid, an excess of formalin or para formaldehyde and sodium cyanoborohydride or sodium triacetoxy borohydride at reduced temperatures to provide compound 69.
  • the reaction was performed in MeOH or MeOH:THF. In a further embodiment, the reaction was performed at about 0°C to about RT. In another embodiment, the reaction was performed with about 2 to about 5 equivalents of formalin. In still a further embodiment, the reaction was performed in formalin 40% in water. To compound 69 was added to an inorganic base such as NaOH or LiOH.H 2 0 and the mixture acidified to yield compound 70. In one embodiment, the reaction was performed in a MeOH:water or THF:MeOH:water. In another embodiment, the reaction was performed using about 6 to about 10 equivalents of LiOH.H 2 0. In a further embodiment, the acidification was performed using HC1.
  • reaction was performed in DMF. In another embodiment, the reaction was performed using about 5 to about 10 equivalents of EDC.HC1. In a further embodiment, the reaction was performed at about 0°C to about RT.
  • Scheme 22 provides the synthesis of compound 75, which is similar to the routes discussed in Schemes 20 and 21.
  • a reducing agent at reduced to elevated temperatures to provide compound 73 in a manner similar to Scheme 21.
  • the reaction is performed using THF.
  • the reducing agent is LiAlH 4 .
  • the reaction is performed using about 4 to about 6 equivalents of the reducing agent.
  • the starting temperature for the reaction is about 0°C to about RT.
  • the temperature of the reaction is then elevated to about 80 to about 100°C.
  • the mixture was then basified at reduced temperatures to provide compound 73.
  • the basification is performed using NaOH or NH 4 OH.
  • the basification was performed using 10% aqueous NaOH. In a further embodiment, the basification is performed at about 0°C.
  • To triphenylphosphine and diol 73 was added DBAD or DEAD.
  • the reaction was performed at about 0°C to about RT.
  • the reaction was performed in THF.
  • a solution of the product was then washed with an acid and basified to provide spirocyclic 74.
  • the acid was HC1.
  • the reaction was performed in DCM.
  • the aqueous layer was then basified to provide spirocyclic 74.
  • the pH of the aqueous layer adjusted to pH 10.
  • the basification was performed using NaOH.
  • To compound 74 was added palladium hydroxide under hydrogen to provide amine 75.
  • the reaction was performed in acetic acid, 1,4-dioxane, and water.
  • the reaction was performed in a 4:4: 1 acetic acid, 1,4-dioxane, and water solution.
  • Scheme 23 depicts the preparation of compound 82, which route is similar to that described in Scheme 21.
  • amine 66 was reacted with acetic acid, formaldehyde or formaline and sodium cyanoborohydride or sodium triacetoxy borohydride at reduced temperatures to provide product 82a.
  • the reaction was performed in MeOH of THF:MeOH.
  • the reaction was performed using about 2 to about 5 equivalents of formaldehyde.
  • the reaction was performed using about 3 to about 8 equivalents of cyanoborohydride.
  • the reaction was performed at about 0°C to about RT.
  • compound 82a was added palladium hydroxide and hydrogen to provide amine 82.
  • reaction was performed in acetic acid: 1,4-dioxane: water. In another embodiment, the reaction was performed in a 2:2: 1 v/v solution of acetic acid: 1,4-dioxane: water.
  • Scheme 24 describes the synthesis of compound 81.
  • Compound 73 was reacted with palladium hydroxide and hydrogen to provide the corresponding amine.
  • the reaction was performed using acetic acid, 1,4-dioxane, and water.
  • the reaction was performed using a 2:2: 1 v/v solution of acetic acid: 1,4- dioxane: water.
  • the amine was then reacted with Boc anhydride and an excess of triethyl amine or DIEA at reduced temperatures to provide compound 76.
  • the reaction was performed in THF and water.
  • the reaction was performed at about 0°C to about RT.
  • the reaction was performed using about 2 to about 4 equivalents of triethylamine.
  • Compound 76 was reacted with TEA or DIEA, followed by methane sulfonylchloride at reduced temperatures to provide compound 77.
  • the reaction was performed in DCM or THF.
  • the reaction was performed at about -10 to about 0°C.
  • Compound 77 was reacted with Na 2 S.H 2 0 NO at elevated temperatures to provide compound 78.
  • the reaction was performed in DMF or THF. .
  • the reaction was performed at about 105 to about 120°C.
  • Compound 78 was then reacted with HCl at reduced temperatures to provide compound 79.
  • the reaction was performed in ethyl acetate or ethanol. In another embodiment, the reaction was performed using HCl. In a further embodiment, the reaction was performed at about -10 to about 0°C. Compound 78 were added H 2 O 2 solution, water and Na 2 W0 4 .2H 2 0 at elevated temperatures to provide compound 80. In one embodiment, the reaction was performed in THF or THF:MeOH. In another embodiment, the reaction was performed in a sealed tube. In a further embodiment, compound 80 was reacted with the 33% in a water H 2 O 2 solution. In yet another embodiment, the reaction was performed with about 6 to about 10 equivalents of a H 2 O 2 solution.
  • reaction was performed with about 3 to about 6 equivalents of Na 2 W0 4 .2H 2 0. In another embodiment, the reaction was performed at about 65 to about 75°C.
  • compound 80 was reacted with HC1 at reduced temperatures to provide amine 81 as the HC1 salt. In one embodiment, the reaction was performed in ethyl acetate. In another embodiment, the reaction was performed at 0°C to - 10°C.
  • Scheme 25 provides the synthesis of compounds 91-94, all starting from compound 32.
  • To amine 75, 82, 89, or 81 were added compound 32 and K 2 CO 3 at elevated temperatures to provide compounds 83, 84, 85, or 86, respectively.
  • the reaction was performed in DMF or DMSO.
  • the reaction was performed using about 2 to about 4 equivalents of K 2 CO 3 .
  • the reaction was performed at about 80 to about 100°C.
  • Compound 83 was reacted with an inorganic base such as NaOH or LiOH.H 2 0.
  • the reaction was performed in MeOH and water or THF:MeOH:water.
  • the reaction was performed using about 3 to about 5 equivalents of LiOH.H 2 0.
  • the mixture was then acidified with HC1 to provide compound 87 as the HC1 salt.
  • Compound 87 was reacted with Pro-CBI 2 and EDC.HC1 at reduced temperatures to provide product 91.
  • the reaction was performed in DMF.
  • the reaction was performed at about 0°C to about RT.
  • the reaction was performed using EDC.HC1.
  • the coupling of the PAMA based linker to the warhead via an ether linkage can be performed by following the pathway depicted in Scheme 26, using an "Alloc" protecting group.
  • Compound 20 was reacted with LiOH.H 2 0 at reduced temperatures.
  • the reaction was performed in methanol and water or MeOH:THF:water.
  • the reaction was performed in a 10: 1 mixture of methanol: water.
  • the reaction was performed with about 3 to about 5 equivalents of LiOH.H 2 0.
  • the reaction was performed at about 0°C. The product was then neutralized to yield the acid.
  • the neutralization was performed using an acidic resin (e.g., Amberlyst®, available from Sigma-Aldrich).
  • the acid was then reacted with NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NHAII0C, PyBOP, and DIPEA at reduced temperatures to obtain compound 95.
  • the reaction was performed in DMF.
  • the reaction was performed at about 0°C to about RT.
  • Alcohol 95 was reacted with an excess of CS 2 CO 3 , cooled to a reduced temperatures, and CCI 3 CN was added to obtain compound 96.
  • the reaction was performed in DMF.
  • about 2 to about 5 equivalents of CS 2 CO 2 was utilized.
  • the reaction was cooled to about 0°C.
  • trichloroacetimidate 96 and Boc-Pro-CBI 2a were added to a suspension of molecular sieves.
  • the reaction was performed using 2 to about 4 equivalents of trichloroacetimidate 96.
  • the reaction was performed using MeCN.
  • the mixture was then cooled to reduced temperatures and reacted with a Lewis acid such as BF 3 .ether. Additional BF 3 ether was added as needed.
  • the mixture was cooled to about -10°C to about 0°C.
  • the mixture was neutralized using NEt 3 or DIEA to afford compound 97.
  • Scheme 27 provides the synthesis of compound 100 via compound 87.
  • compound 87 was reacted with CDI at reduced temperatures to provide compound 98.
  • the reaction was performed in DMF
  • the reaction was performed using about 5 to about 10 equivalents of CDI.
  • the reaction was performed at about -10 to about 0°C.
  • Compound 98 was then reacted with amine 97 and excess sodium carbonate to afford compound 99.
  • the reaction was performed in DMF.
  • the reaction was performed using about 10 to about 15 equivalents of sodium carbonate.
  • Compound 99 was reacted Pd(PPh 3 ) 4 and 1,3-dimethylbarbituric acid to provide the amine product.
  • the reaction was performed in DCM.
  • the amine product was reacted with an excess of succinate ester 27 and an excess of sodium carbonate to provide compound 100.
  • the reaction was performed in DMF.
  • the reaction was performed with about 2 equivalents of succinate ester 27 and/or sodium carbonate.
  • Scheme 28 provides the synthesis of compound 102. Specifically, amine 101 is reacted with an excess of the succinate ester 103 and an inorganic base such as sodium carbonate to obtain compound 102. In one embodiment, the reaction is performed in a buffer. In another embodiment, the reaction is performed in a borate buffer. In a further embodiment, the reaction was performed in a 0.1 M borate buffer (pH 8.0):dioxane. In yet another embodiment, the reaction is performed with about 2 equivalents of succinate ester.
  • a composition containing a therapeutic drug and targeting conjugate of formula (I), or a mixture thereof, may be prepared.
  • a composition contains a therapeutic drug and targeting conjugate of any of formula (IA), formula (IB), formula (ICi) or formula (ICii). While the following specification will reference formula (I), it will be understood that any of these subgeneric structures, or combinations thereof, may be combined in to a single pharmaceutical composition unless otherwise specified.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a conjugate of formula (I) in a pharmaceutically acceptable carrier optionally with other pharmaceutically inert or inactive ingredients.
  • a composition may contain a mixture of conjugates of formula (I) in which each LG has 1-6 drugs (via 1-6 B-linker-drug fragments) conjugated thereto.
  • Such a composition may contain a mixture of conjugates of formula (I), wherein p" averages 2.
  • Such a composition may contain conjugates of formula (I), wherein a selected percentage of the conjugates is characterized by p" is 1, a selected percentage of the conjugates is characterized by p" is 2, and with a significantly smaller percentage of the conjugates being characterized by p" over 3 or higher. Still other mixtures can be prepared according to the invention.
  • a conjugate of formula (I), or a mixture of conjugate of formula (I) is combined with one or more excipients and/or other therapeutic agents as described below.
  • formulation of a conjugate of formula (I) it will be understood that a mixture of conjugates of formula (I) can be similarly formulated, unless stated otherwise.
  • compositions provided herein comprise an amount of a conjugate of formula (I) that is therapeutically effective.
  • dosage of the conjugate of formula (I) to achieve a therapeutic effect will depend on the formulation, age, weight and sex of the patient and route of delivery. It is also contemplated that the treatment and dosage of the conjugate of formula (I) may be administered in unit dosage form and that one skilled in the art would adjust the unit dosage form accordingly to reflect the relative level of activity. The decision as to the particular dosage to be employed (and the number of times to be administered per day) is within the discretion of the ordinarily-skilled physician, and may be varied by titration of the dosage to the particular circumstances to produce the desired therapeutic effect.
  • the therapeutically effective amount is about 0.001 to 10 mg of ADC protein per kg body weight. In another embodiment, the therapeutically effective amount is less than about 10 mg/kg, about 1 mg/kg, about 0.5 mg/kg, about 0.25 mg/kg, about 0.1 mg/kg, about 100 ⁇ g/kg, about 75 ⁇ g/kg, about 50 ⁇ g/kg, about 25 ⁇ g/kg, about 10 ⁇ g/kg, or about 1 ⁇ g/kg.
  • the therapeutically effective amount of the conjugate of formula (I) can be determined based on clinical studies and individual patient response and depends on the condition treated, the particular conjugate administered, the route of delivery, the age, weight, severity of the patient's symptoms and response pattern of the patient.
  • the therapeutically effective amount may be provided on regular schedule, i.e. , daily, weekly, monthly, or yearly basis or on an irregular schedule with varying
  • the therapeutically effective amount to be administered may vary.
  • the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses.
  • the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses.
  • Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months.
  • the number and frequency of dosages corresponding to a completed course of therapy will be determined according to the recommendations of the relevant regulatory bodies and judgment of a health-care practitioner.
  • the therapeutically effective amounts described herein refer to total amounts administered for a given time period; that is, if more than one different conjugate of formula (I) thereof is administered, the therapeutically effective amounts correspond to the total amount administered.
  • the pharmaceutical compositions containing a conjugate of formula (I) may be formulated neat or with one or more pharmaceutical carriers for administration.
  • the amount of the pharmaceutical carrier(s) is determined by the solubility and chemical nature of the conjugate of formula (I), chosen route of administration and standard pharmacological practice.
  • the pharmaceutical carrier(s) may be solid or liquid and may incorporate both solid and liquid carriers.
  • suitable liquid carriers are known and may be readily selected by one of skill in the art.
  • Such carriers may include, e.g., saline, buffered saline, human albumin, polyethylene glycol, and mixtures thereof.
  • solid carriers and excipients are known to those of skill in the art.
  • the conjugates of formula (I) may be administered by any route, taking into consideration the specific condition for which it has been selected.
  • the conjugates of formula (I) may, be delivered by injection, intravascularly, subcutaneously, intravesically, intramuscularly, intracranially, epidurally, among others.
  • the compositions are formulated for parenteral delivery of a conjugate of formula (I), e.g., intravenous or intraperitoneal delivery.
  • the conjugate of formula (I) may be administered alone, it may also be administered in the presence of one or more pharmaceutical carriers that are physiologically compatible.
  • the carriers may be in liquid form and must be pharmaceutically acceptable.
  • Liquid pharmaceutical compositions are typically sterile solutions or suspensions. When liquid carriers are utilized for parenteral administration, they are desirably sterile liquids. Liquid carriers are typically utilized in preparing solutions.
  • the conjugate of formula (I) is dissolved in a liquid carrier.
  • the conjugate of formula (I) is suspended in a liquid carrier.
  • One of skill in the art of formulations would be able to select a suitable liquid carrier, depending on the parenteral route of administration.
  • conjugates of formula (I) and/or other medication(s) or therapeutic agent(s) may be administered in a single composition.
  • the present invention is not so limited.
  • the conjugates of formula (I) may be administered in one or more separate formulations from other conjugates of formula (I), chemotherapeutic agents, or other agents as is desired.
  • the invention provides a method of delivering a therapeutically active drug, said method comprising administering a therapeutic drug and targeting conjugate of formula (I) as defined herein, wherein said drug fragment or prodrug fragment is converted to a therapeutically active drug or prodrug following cleavage of the di/tri/tetrapeptide substrate within the ligand-drug conjugate by one or more proteases.
  • a "patient” or “subject” is a mammal, e.g. , a human or a veterinary patient or subject, e.g., mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.
  • treating or “treatment” is meant to encompass administering to a subject a therapeutic conjugate of the present invention for the purposes of amelioration of one or more symptoms of a disease or disorder, including palliative care.
  • a “therapeutically effective amount” refers to the minimum amount of the active compound which effects treatment.
  • a conjugate as described herein contains a ligand designed to target a desired cell surface molecule, such as a receptor or a growth factor.
  • a desired cell surface molecule such as a receptor or a growth factor.
  • suitable targets include, e.g., CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70,
  • CD 133 carcinogenic embryonic antigen (CEA), epidermal growth factor receptor- 1 (EGFR or HER1 or erbBl), epidermal growth factor receptor (EGFRviii), human epidermal growth factor receptor-2 (HER2 or erbB2), epidermal growth factor receptor-3 (HER3 or erbB3), MET, insulin-like growth factor receptor 1 (IGFIR), platelet-derived growth factor receptor alpha and beta (PDGFRalpha and PDGFRbeta), EphrinA receptors 1-8 (EphAl-8), EphrinB receptors 1-6 (EphBl-6), folate receptor (FolRalpha), prostate specific membrane antigen (PSMA), MUC-1, MUC-16, high molecular weight melanoma-associated antigen (HMW- MAA) or chondroitin sulfate proteoglycan (CSPG), epithelial cell adhesion molecule (EPCAM), 5T4 oncofetal trophoblast glycoprotein, Ti
  • the invention provides a method of treating a disease or disorder associated with the presence of a specific cell surface molecule on cells of a subject, comprising administering to the subject a therapeutically effective amount of a conjugate of the invention comprising at least one ligand that specifically binds to the cell surface molecule.
  • the disease or disorder is a neoplastic disease such as cancer.
  • such a composition is designed for use in a therapeutic in anti-neoplastic regimen.
  • the ligand is desirably selected to target a neoplastic cell based on the presence of specific cell surface molecules on the neoplastic cell.
  • neoplastic cells include those involved in cancer of the prostate, head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone, lung (small cell or non-small cell), colon, rectum, stomach, bladder, uterus, cervix, breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes, kidney (renal cancer), liver, intestines, pancreas, brain (e.g., glioblastoma), central nervous system, adrenal gland, or skin or a leukemia.
  • said cancer is cancer of the prostate.
  • a cancer patient has at least one solid or liquid tumor.
  • kits e.g., kits, vials with preformulated parental compositions of the invention, or other packages of containing the conjugates or formulated conjugates described herein.
  • the kits may be organized to indicate a single formulation or combination of formulations to be taken at each desired time.
  • the kit contains packaging or a container with the compound of formula (I) formulated for the desired delivery route.
  • the kit contains instructions on dosing and an insert regarding the active agent.
  • the kit may further contain instructions for monitoring circulating levels of product and materials for performing such assays including, e.g. , reagents, well plates, containers, markers or labels, and the like.
  • Such kits are readily packaged in a manner suitable for treatment of a desired indication.
  • Other suitable components to include in such kits will be readily apparent to one of skill in the art, taking into consideration the desired indication and the delivery route.
  • compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration.
  • a package or kit can include the conjugates in each dosage unit (e.g., solution or other unit described above or utilized in drug delivery), and optionally instructions for administering the doses daily, weekly, or monthly, for a predetermined length of time or as prescribed. If varying concentrations of a composition, of the components of the composition, or the relative ratios of the conjugates or agents within a composition over time is desired, a package or kit may contain a sequence of dosage units which provide the desired variability.
  • the package has indicators for each period.
  • the package is a labeled blister package, dial dispenser package, or bottle.
  • the packaging means of a kit may itself be geared for administration, such as a syringe, pipette, eye dropper, or other such apparatus, from which the formulation may be applied to an affected area of the body, injected into a subject, or even applied to and mixed with the other components of the kit.
  • compositions of these kits also may be provided in dried or lyophilized forms.
  • reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in a separate package.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g. , injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g. , injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits also may include, or be packaged with a separate instrument for assisting with the injection/administration or placement of the composition within the body of a subject.
  • a separate instrument may be a syringe, pipette, forcep, measuring spoon, eye dropper or any such medically approved or appropriate delivery means.
  • the para-amino mandelic acid (PAMA) derived linker system was demonstrated to provide protease-activated self-immolative release of both hydroxy- and amine -linked drugs, by multiple methods.
  • HMC 7-hydroxy-4-methylcoumarin
  • AMC 7-amino-4-methylcoumarin
  • fluorescence measurements were used to assay the amount of surrogate prodrug/drug released.
  • Compounds were prepared incorporating variations of the PAMA linker and these surrogate drugs, but without inclusion of a targeting ligand such as an antibody. These compounds were incubated (pH 5.0, 37 °C) in the presence of cathepsin B, which was used as a representative protease, and release of the surrogate drug was measured by fluorescence.
  • Cbz-Val-Cit-PABE-HMC or Cbz-Val-Cit-PABC-AMC was included in the assay.
  • Cbz-Val-Cit-PABC-AMC is an analog of Cbz-Val-Cit-PABC-Doxorubicin, which was reported by Dubowchik and Firestone, Bioorg. Med. Chem. Lett., 8 (1998), 3341-3346; and Dubowchik et. al., Bioconj. Chem., 13 (2002), 855-869.
  • LC -MS/MS was used for detection of the prodrug and/or drug released.
  • Compounds were prepared incorporating variations of the PAMA linker and using Pro CBI-DMMI as a hydroxy-linked prodrug of the cytotoxic duocarmycin analog drug CBI- DMMI.
  • the prepared compounds evaluated using this method did not include a targeting ligand such as an antibody. These compounds were incubated (pH 5.0, 37 °C) in the presence of cathepsin B, and the formation of duocarmycin analog drug CBI-DMMI was determined by LC -MS/MS.
  • the drug linked to PAMA is a prodrug, i.e., Pro CBI- DMMI, which upon its release is converted under the assay conditions to CBI-DMMI.
  • Pro CBI- DMMI a prodrug
  • Multiple compounds were demonstrated to release/generate the duocarmycin analog drug CBI-DMMI under the cathepsin B assay conditions. Results are provided in Figure 6 and 7. These compounds were also found to be stable (i.e., no significant release/formation of CBI- DMMI over time) under the assay conditions in the absence of cathepsin B. This demonstrates that protease cleavage (in this case by cathepsin B) is required to activate the PAMA linker to self-immolate and release the drug.
  • compounds are prepared incorporating variations of the PAMA linker and using N-methyl auristatin E (MMAE) as an amine-linked cytotoxic drug.
  • MMAE N-methyl auristatin E
  • the prepared compounds evaluated using this method do not include a targeting ligand such as an antibody.
  • These compounds are incubated (pH 5.0, 37 °C) in the presence of cathepsin B, and the formation of N-methyl auristatin E drug is determined by LC-MS/MS.
  • antibody drug conjugates were prepared incorporating the PAMA linker and a cytotoxic prodrug or drug, and the drug release was determined by mass spectrometry.
  • an anti-5T4 scFvFc antibody conjugated to a PAMA derived linker incorporating Pro CBI-DMMI was evaluated. Incubation of this anti-5T4 scADC in the presence of cathepsin B (pH 5.0, 37 °C) for two hours resulted in the formation of a new signal in the MALDI mass spectrum corresponding to Pro CBI-DMMI. Under the same conditions in the absence of cathepsin B, there was no signal for the mass of Pro CBI- DMMI.
  • protease cleavage in this case by cathepsin B is achieved on the antibody drug conjugate (ADC), namely anti-5T4 scFvFc incorporating the PAMA linker and Pro CBI-DMMI as drug, and that protease cleavage is required to activate the PAMA linker to self-immolate and release the prodrug/drug.
  • ADC antibody drug conjugate
  • compounds are prepared incorporating variations of the PAMA linker and using N-methyl auristatin E (MMAE) as an amine-linked cytotoxic drug.
  • MMAE N-methyl auristatin E
  • the prepared compounds to be evaluated using this method do not include a targeting ligand such as an antibody.
  • These compounds are incubated (pH 5.0, 37 °C) in the presence of cathepsin B, and the formation of N-methyl auristatin E drug is determined by mass spectrometry.
  • LC-MS/MS was used to measure the prodrug and/or drug released, for an antibody drug conjugate (ADC) incorporating the PAMA linker and a cytotoxic duocarmycin analog prodrug.
  • ADC antibody drug conjugate
  • an anti-HER2 scFvFc antibody conjugated to a PAMA derived linker incorporating duocarmycin prodrug analog compound 91 was evaluated, taking measurements at multiple time points.
  • the para-amino mandelic acid (PAMA) derived linker system was demonstrated to have utility to inhibit tumor cell proliferation and tumor growth, when conjugated with a targeting ligand and a cytotoxic prodrug or drug, for example as an antibody drug conjugate (ADC), in both in vitro and in vivo assays.
  • ADC antibody drug conjugate
  • anti-5T4 scFvFc antibody conjugated to the PAMA linker and Pro CBI-DMMI as the drug was demonstrated in vitro to cause potent inhibition of tumor cell proliferation in a 5T4-overexpressing transfectant of MDA-MB-231 breast carcinoma cells.
  • the in vitro inhibition was found to be ca. 100-fold less potent for native MDA-MB-231 tumor cells that have low expression of the 5T4 antigen.
  • anti-HER2 scFvFc antibody conjugated to the PAMA linker and Pro CBI-DMMI as the drug was demonstrated in vivo to cause strong inhibition of tumor growth in a SKOV3 cervical carcinoma mouse xenograft model, at a dose of 2 mg/kg iv, dosing once per week for three weeks.
  • the para-amino mandelic acid (PAMA) derived linker system was incorporated into conjugate compounds having a targeting ligand (for example, an scFvFc or IgG antibody), and an amine -linked cytotoxic drug (for example, N-methyl auristatin E).
  • a targeting ligand for example, an scFvFc or IgG antibody
  • an amine -linked cytotoxic drug for example, N-methyl auristatin E.
  • Utility of the PAMA-linked conjugate to inhibit tumor cell proliferation was demonstrated in vitro by measuring cytotoxicity in cell assays wherein the tumor cells harbor the antigen that is targeted by the targeting ligand.
  • Utility to inhibit tumor growth in vivo was achieved by measuring tumor growth inhibition in mouse xenograft studies, wherein the tumors are grown from tumor cells that harbor the antigen that is targeted by the targeting ligand.
  • MHz megahertz (frequency)
  • m multiplet
  • q quartet
  • t triplet
  • d doublet
  • s singlet
  • br broad
  • CDC1 3 deutero chloroform
  • DMSO-d 6 deutero dimethyl sulfoxide
  • calcd is calculated, min is minutes, h is hours, g is grams, mol is moles, mmol is millimoles, mL is milliliters
  • °C degree centigrade
  • HPLC High Performance Liquid Chromatography
  • LC-MS Liquid Chromatography-Mass Spectroscopy
  • UPLC Ultra Performance Liquid Chromatography
  • NMR Nuclear Magnetic Resonance
  • TLC thin layer chromatography
  • ESI electrospray ionization
  • EI electron impact ionization
  • LC liquid chromatography
  • HPLC spectra were recorded using Agilent 1100 and 1200 series with Quaternary pump systems with diode array detector (DAD) detection using Atlantis CI 8, HypesilTM BDS column, SunfireTM CI 8 column, XbridgeTM C 18 and Zorbax® C18 (50 mm x 4.6 mm x 5 ⁇ ), (150 mm x 4.6 mm x 3 ⁇ ) & (250 mm x 4.6 mm x 5 ⁇ ) columns.
  • DAD diode array detector
  • LCMS spectra were recorded using Agilent 1100 and 1200 series with Quaternary pump systems with diode array detector (DAD) detection using Atlantis® CI 8, Xterra® C8 and Zorbax® C18 (50 mm x 4.6 mm x 5 ⁇ ) and (150 mm x 4.6 mm x 3 ⁇ ) columns.
  • UPLC spectra were recorded using Waters Acquity® UPLC-SQD systems with diode array detector (DAD) detection using Acquity® BEH C18 and HSS T3 C18 (50 mm x 2.1 mm x 1.7 ⁇ ) & (75 mm x 2.1 mm x 1.8 ⁇ ) columns.
  • a mobile phase of 0.01% of formic acid or TFA with acetonitrile and 0.01% of formic acid or TFA with water with a flow rate of 0.5 mL/min or 1.0 mL/min or 1.5 mL/min at temperature of RT or 40 °C was used.
  • a toluene (250 mL) solution of 1 ,3-dihydroxy naphthalene 4 (50 g, 0.3123 mol) and diphenyl methylamine (69.92 mL, 0.406 mol) was heated at 100°C for 6 h in a sealed tube. After that reaction mass was transfer to a par shaker and were added di-tert-butyl dicarbonate (204 mL, 0.94 mol), palladium hydroxide (17 g; 20% Pd(OH) 2 /C) and dioxane: water (4: 1 , 250 mL). The mixture was shaken at 60 psi hydrogen pressure for 48 h.
  • the amine salt 2 (CBI) was dissolved in THF:water (4: 1 ; 40 mL). Triethyl amine (1.0 eq.) and Boc 2 0 (1.0 eq.) were added successively and mixture was stirred at RT. After 4 h, volatiles were removed under reduced pressure and the crude material was purified by silica gel (60-120 mesh) column chromatography to provide compound 2a.
  • a toluene solution of 1 ,3-dihydroxy naphthalene 4 and diphenyl methylamine was heated at 100°C for 4 to 6 h in a sealed tube. After that reaction mass was transfer to a par shaker and were added di-tert-butyl dicarbonate, palladium hydroxide (20% Pd(OH) 2 /C) and dioxane: water. The mixture was shaken at 60 psi hydrogen pressure for 4 to 6 h.
  • Example 3 describes the synthesis of compound 3 ("DMMI"). To a stirred solution of 3-hydroxy-4-methoxybenzaldehyde 5 in DMF was added potassium carbonate and 1, 2- dichloroethane. The mixture was stirred at 70°C for 16 h. The mixture was cooled down to RT and 1,2-dichloroethane was removed under reduced pressure to yield compound 35.
  • DMMI dimethyl methacrylate
  • the crude acid (0.900 g) was dissolved in DMF (10 mL) and NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 3 (0.571 g, 4.85 mmol), benzotriazol-l -yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (1.68 g, 3.23 mmol), DIPEA (0.86 mL, 4.85 mmol) were added successively at 0°C. The mixture was stirred for 16 h at RT under nitrogen atmosphere. The mixture was diluted with cold water (50 mL), extracted with 10% butanol in ethyl acetate (3 x 50 mL).
  • the crude acid was dissolved in DMF (3.0 mL) and NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NHB0C (0.04 g, 0.16 mol), PyBOP (0.10 g, 0.20 mmol), DIPEA (0.03 mL, 0.27 mmol) were added successively at 0°C.
  • the mixture was stirred for 18 h at RT under nitrogen atmosphere.
  • the mixture was diluted with minimum amount of ice water, and extracted with ethyl acetate (3 x 25 mL). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated. Crude product was purified by column chromatography (silica gel) using DCM/MeOH.
  • the crude acid was dissolved in DMF (5.0 mL) and NH 2 CH 2 CH 2 OCH 2 CH 2 O- CH 2 CH 2 OCH 2 CH 2 NHB0C (0.12 g, 0.41 mol), PyBOP (0.27 g, 0.52 mmol), DIPEA (0.12 mL, 0.69 mmol) were added successively at 0°C.
  • the mixture was stirred for 18 h at RT under nitrogen atmosphere.
  • the mixture was diluted with minimum amount of ice water, and extracted with ethyl acetate (3 x 25 mL). Combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated.
  • the compounds listed in Table 1 incorporate 7-hydroxy-4-methylcoumarin (HMC) as a surrogate for a hydroxy-linked drug, and 7-amino-4-methylcoumarin (AMC) as a surrogate for an amine-linked drug, and variations in the PAMA linker structure. These compounds were assayed using a fluorescence assay method as described below (Example 155) to determine the amount of surrogate prodrug/drug released under different conditions and for variations in the PAMA linker structure of formula (IX). These compounds are also used as synthetic intermediates to prepare a conjugate of formula (I), in which HMC or AMC is incorporated as a surrogate drug/prodrug.
  • HMC 7-hydroxy-4-methylcoumarin
  • AMC 7-amino-4-methylcoumarin
  • the compounds listed in Table 2 and Table 3 are prepared in a similar manner to the Examples described above. These compounds also incorporate 7-hydroxy-4-methylcoumarin (HMC) as a surrogate for a hydroxy-linked drug, and 7-amino-4-methylcoumarin (AMC) as a surrogate for an amine-linked drug, and they further incorporate variations in the PAMA linker structure of formula (IX). These compounds are assayed using the fluorescence assay method as described below (Example 155) to determine the amount of surrogate prodrug/drug released under different conditions and for different variations in the PAMA linker structure of formula (IX). The compounds listed in Table 2 and Table 3 are also used as synthetic intermediates to prepare a conjugate of formula (I), in which HMC or AMC is incorporated as a surrogate drug/prodrug.
  • HMC 7-hydroxy-4-methylcoumarin
  • AMC 7-amino-4-methylcoumarin
  • the crude acid was dissolved in DMF (30 mL) and NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NHB oc (1.8 g, 0.0064 mol), PyBOP (4.2 g, 0.0080 mol), DIPEA (1.85 mL, 0.0107 mol) were added successively at 0°C. The mixture was stirred for 16 h at RT under nitrogen atmosphere. Volatiles were removed under reduced pressure. Crude product was purified by column chromatography (silica gel) using DCM/MeOH.
  • the product was dissolved in Ac 2 0 (70 mL) and sodium acetate (3.83 g, 46.71 mmol) was added. The mixture was heated at 100°C for 3 h. Acetic anhydride was removed under reduced pressure, diluted with water (200 mL) and extracted with ethyl acetate (300 mL x 3). Combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated to provide crude material. The crude product was purified by column chromatography (silica gel) in DCM/MeOH.
  • the aqueous layer was basified to pH 10 using IN NaOH solution and then extracted with ethyl acetate (100 mL x 3). Combined ethyl acetate layer was washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • This compound was prepared from ester 84 using experimental procedure similar to the preparation of compound 87. The acid was used in next step without characterization.
  • the crude acid was dissolved in DMF (30 mL) and NH 2 CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NHAII0C (5.60 g, 20.46 mmol), PyBOP (13.3 g, 25.58 mole), DIPEA (8.75 mL, 51.16 mmol) were added successively at 0°C.
  • the mixture was stirred for 16 h at RT under nitrogen atmosphere.
  • the mixture was diluted with ice water, stirred for 1 h, the solid was filtered off and washed with diethyl ether to obtained compound 95 as a light brown colored solid.
  • Anti-5T4 scFv-Fc engineered to present two Cysteine residues for site-specific conjugation was covalently linked to Cbz-Val-Cit-PAMA-(MB-PEG3-amide)-Pro-CBI- DMMI (compound 40), to form the corresponding anti-5T4 single-chain antibody drug conjugate ("anti-5T4 scADC").
  • Anti-5T4 single chain antibody-Fc fusion protein (“anti-5T4 scFv-Fc") was prepared in CHO DG44 cells, as disclosed in US Provisional Application Number 61/835,858, filed June 17, 2013, herein incorporated by reference.
  • scFv-Fc Prior to the conjugation reaction, reduction of the scFv-Fc was performed with 0.8mM DTT in 20mM sodium phosphate, pH 7.2, 150mM NaCl at 3 mg/mL protein concentration for 45 min to break any possible adducts of the engineered Cys residue with glutathione or cysteines.
  • the reduced scFv-Fc sample was then subjected to desalting in 50mM Tris-Cl, 150 mM NaCl, 250 mM Arginine (pH 8.2) to remove DTT using Hi-Prep 26/10 Desalting (GE Healthcare) column. Buffer exchanged scFv-Fc was then concentrated to 3 mg/mL and stirred slowly for 30 min to allow oxidative regeneration of the native disulfides.
  • scFv-Fc prepared in the above step was incubated in the presence of Cbz- Val-Cit-PAMA-(MB-PEG3-amide)-Pro-CBI-DMMI (compound 40) at 15-fold molar excess for 90 min with slow stirring for conjugation to occur.
  • the conjugated scFv-Fc (“anti-5T4 scADC") was desalted to 20 mM PBS (phosphate buffer saline) containing 10% glycerol.
  • Anti-5T4 scADC was analyzed by SDS-PAGE (reducing and non-reducing), MALDI-MS (Bruker AutoflexTM III), LC-ESI-MS (Bruker HCT Ultra) and SE-HPLC. Sinapinic acid in the presence of 50% acetonitrile and 0.1% TFA was used to formulate samples for MALDI.
  • a Zorbax® 300sb-C3 column was used for reverse phase chromatography.
  • Non-reducing SDS-PAGE of the scADC yielded a band at position corresponding to the dimeric scFv-Fc (anti-5T4 scADC) at -110 kDa.
  • Negligible intensity of the monomeric band at -55 kDa indicated near complete formation of the covalently dimerized protein after air oxidation and conjugation.
  • this sample of anti-5T4 scADC incorporated a distribution of 0, 1 and 2 linker-drug groups per scFvFc antibody, with an average of > 1.6 linker-drug groups per scFvFc antibody. That is, the drug-to-antibody ratio (DAR) for this sample was determined to be ca. 1.6.
  • the structure of this anti-5T4 scADC is comprised as follows:
  • Ab-S- is derived from anti-5T4 scFvFc (i.e., Ab-SH is anti-5T4 scFvFc)
  • Affinity of anti-5T4 scADC to 5T4 was determined by SPR analysis using a BIAcoreTM T200 instrument (GE Healthcare). Briefly, protein A affinity purified 5T4- extracellular domain-human Fc fusion protein extracellular was covalently immobilized on a BIAcoreTM CM5 sensor chip by amine coupling method using reagents and instructions provided by the manufacturer. In the binding study, anti-5T4 scADC was serially diluted to a concentration series and flowed over the immobilized antigen for a fixed period of time, followed by flow of buffer to dissociate the antigen. At the end of the dissociation cycle, regeneration of chip the surface was carried out at low pH.
  • Fluorescence-activated cell sorting (FACS) analysis was carried out to determine cell binding of the anti-5T4 scADC, by using a MDA-MB-231 breast carcinoma cell line that over-expressed 5T4 (generated by stable transfection). Selective binding of the anti-5T4 scADC to the 5T4-high-expressing (transfectant) MDA-MB-231 cells versus the native 5T4-low-expressing MDA-MB-231 cells was observed, in close agreement to observations for the non-conjugated scFv-Fc antibody.
  • FACS Fluorescence-activated cell sorting
  • Cell were routinely cultured as monolayers in nutrient medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin G and 100 ⁇ g/mL streptomycin sulfate at 37 °C under 5% C02 atmosphere. Cells of an exponentially-growing monolayer culture were harvested and plated at a concentration of 4000 cells/well in 96 well cell culture plate and allowed to grow overnight under standard cell growth conditions.
  • an IC 50 value of 0.8 nM was obtained for the anti- 5T4 scADC.
  • Non-conjugated scFv-Fc antibody did not result in any cell growth inhibition of the recombinant MDA-MB-231 cells.
  • Native MDA-MB-231 cells, previously confirmed to have extremely low basal expression of 5T4 were also used in the cell growth inhibition assays.
  • the 5T4-low-expressing cell line was approximately 100-fold less sensitive to growth inhibition by the anti-5T4 scADC.
  • Example 38 Conjugation of anti-HER2 scFv-Fc to a PAMA-Linker-Drug to form an anti-HER2 antibody drug conjugate (anti-HER2 scADC-vl)
  • anti-HER2 scFv-Fc engineered to present two cysteine residues for site-specific conjugation was covalently linked to Cbz-Val-Cit-PAMA- (MB-PEG3-amide)-Pro-CBI-DMMI (compound 40), to form the corresponding anti-HER2 single-chain antibody drug conjugate ("anti-HER2 scADC-vl ").
  • the anti- HER2 scFv-Fc reduced and re-oxidized in the above reaction was treated with 10-fold molar excess of the linker-drug molecule 40 for 90 min to generate the conjugate anti-HER2 scADC-vl , which was then buffer-exchanged into 20 mM PBS, 10% glycerol at pH 7.2 for storage.
  • the anti-HER2 scADC-vl generated was further purified using a Hi-trap Butyl sepharose FF (GE Healthcare) column.
  • the scADC was mixed with equal volume of 50 mM Tris-Cl pH-7.0, 1.5M ammonium sulfate and clarified by filtration prior to loading on to the column. Proteins bound to the column were eluted with a linear gradient of 50 mM Tris-Cl, pH 7. Each collected fraction was analyzed by MALDI mass spectrometry to identify the fractions containing scADC, which were pooled.
  • the pooled scADC was buffer exchanged into 20 mM PBS, 10% glycerol, pH 7.
  • Example 37 LC-ESI mass spectrometric analysis as described in Example 37 detected greater than 65% scADC in the final preparation with 2 linker-drug molecules per the scFv- Fc dimer.
  • BIAcore Surface Plasmon Resonance
  • this sample of anti-HER2 scADC-vl incorporated a distribution of 0, 1 and 2 linker-drug groups per anti-HER2 scFv- Fc antibody, with an average of > 1.3 linker-drug groups per scFv-Fc antibody.
  • the structure of this anti-HER2 scADC-vl is comprised as follows:
  • Athymic male & female nude mice (Hsd: Athymic Nude-FoxnlTM) 5-6 weeks old, weighing 20-22 g were obtained from Harlan, Netherlands. Animals were taken care as per the Regulations of Committee for the Purpose of Control and Supervision of
  • mice were kept under acclimatization in the experimental room for a period of at least 5 days. Animals were individually numbered and the cage cards indicating the experiment, study number, date of tumor implantation, date of randomization, tumor type, mouse strain, gender, and individual mouse number were displayed to corresponding cages. After randomization, group identity, test compound, dosage, schedule and route of administration were added.
  • Cancer cells SKOV-3 (Ovarian) with 70-80% confluent and viability of >90 % were chosen for the study. Cancer cells SKOV-3 (5 X 10 6 cells) were resuspended in 200 of PBS or serum free media containing 50% of matrigel kept in ice. Subcutaneous injection of cells: Nude mice (Hsd: Athymic Nude-FoxnlTM) housed in Individual Ventilated Cages (IVCs) were used. Cancer cell line (SKOV-3) was propagated in the animals by injecting the cancer cells subcutaneously in the flanks or back of the animals. The implanted area was monitored for growth of tumor.
  • the scADC test sample was dissolved in sterile lx PBS, which resulted in a clear solution and was administered intravenously via tail vein.
  • the test solution was freshly prepared on the days of administration and the dose volume was kept at 5 mL/kg body weight. For each animal group, separate new syringe and needles were used.
  • Clinical signs and body weight Animals were observed individually for visible general clinical signs once every three days during the study period. All the animals were checked for morbidity and mortality. Body weights were measured once every three days during the study period. The % change in body weights of individual mice was calculated.
  • Antitumor activity was evaluated as maximum tumor volume inhibition versus the vehicle (formulation buffer) control group. Data evaluation was performed using statistical software Graph Pad version 5.
  • anti-HER2 scFv-Fc unconjugated antibody
  • anti-HER2 scADC-vl were dosed to the tumor-bearing mice at 2 mg/kg IV, Q7D x 3, i.e., dosing once every seven days for a total of three doses.
  • Anti-HER2 scADC-vl therapy demonstrated significant antitumor activity (inhibition of tumor growth), measured up to Day 33, in comparison to vehicle-treated control animals.
  • Anti-HER2 scFv-Fc antibody provided no significant antitumor activity, in comparison to vehicle-treated control animals.
  • Example 39 Conjugation of anti-HER2 scFv-Fc to a PAMA-Linker-Drug to form an anti-HER2 antibody drug conjugate (anti-HER2 scADC-v2)
  • anti-HER2 scADC-v2 anti-HER2 single-chain antibody drug conjugate
  • This conjugation process included the generation of reduced anti-HER2 scFv-Fc in the presence of 800 ⁇ DTT for 45 min at room temperature in 20 mM sodium phosphate, 150 mM NaCl, pH 7.0 followed by desalting into 50 mM Tris, 50 mM NaCl, 125 mM Arginine, pH 7.0 and air oxidation with slow stirring.
  • the reduced, air-oxidized anti-HER2 scFv-Fc was diluted in conjugation buffer (50 mM Tris, 50 mM NaCl, 125 mM Arginine, pH 7.0) to a final concentration of 0.03 mg/mL.
  • conjugation buffer 50 mM Tris, 50 mM NaCl, 125 mM Arginine, pH 7.0
  • the conjugation reaction was allowed to proceed for 1.5 h at room temperature with slow rocking.
  • the conjugated protein was purified by butyl sepharose chromatography in a manner similar to Example 38.
  • cytotoxicity assay was carried out in 96-well plates; approximately five thousand cells were seeded in 100 ⁇ of medium in each well of the 96-well plates. After 24 h, cells were treated with different concentrations of the anti-HER2 scADC-v2. 96 h after the treatment, media was removed from the wells, and 10 ⁇ of cell titre aqueous reagent was added to 100 ⁇ of media in each well. Plates were incubated for 3 hours and absorbance in each well at 490 nm was read using an ELISA plate reader.
  • IC 50 values were determined after analysis of the drug response curve with four parameter non-linear regression equation using GraphPad Prism® software. Average IC 50 values of 4.0 nM and 40 nM were determined for the anti-HER2 scADC-v2 in NCI-N87 and SKBR3 tumor cell lines, respectively. In comparison, the non-conjugated anti-HER2 scFv- Fc antibody did not result in any cell growth inhibition in these two cell lines.
  • anti-HER2 scADC-v2 was evaluated in HER2-expressing tumor xenograft models, for example SKOV3 tumor xenograft model.
  • the anti-HER2 scADC-v2 was administered at a dose of 3 mg/kg mouse weight, IV, followed by 1 mg/kg IV Q4D x 4.
  • Antitumor activity was assessed based on the observed inhibition of tumor growth or tumor suppression, in comparison to mice treated with vehicle (formulation buffer). Approximately 100% tumor growth inhibition by the anti-HER2 scADC-v2 was observed on Day 18.
  • the non-conjugated anti-HER2 scFv-Fc antibody was also active, but the percent tumor growth inhibition observed on Day 18 was significantly less than that observed for that observed for the anti-HER2 scADC-v2.
  • Example 40 Conjugation of anti-HER2 IgG (trastuzumab) to a PAMA-Linker-Drug, where the drug is the amine-linked cytotoxic drug MMAE, to form an anti-HER2 antibody drug conjugate (anti-HER2 ADC-v3)
  • This compound 103 incorporates the PAMA linker and the amine-linked cytotoxic drug N- methyl auristatin E (MMAE).
  • Compound 104 is a related MMAE-drug/PAMA-linker compound that is also used to prepare antibody-drug conjugates.
  • the drug-linker compound 103 was prepared by the following steps.
  • the reaction mixture was stirred for an additional 12 h.
  • the reaction mixture was poured over ice-cold water (30 mL).
  • the solid that formed was filtered off and washed with water and hexane, dried under reduced pressure, and then purified by column chromatography (silica gel) using dichloromethane/methanol as eluent.
  • the compound 103-c was obtained (0.16 g, 87%) with 62% purity (LCMS); MS calcd. for C 68 Hio 2 N 1 oOi 6 : 1314.7, Found: 1315.6 (M + 1).
  • the crude carboxylic acid was dissolved in DMF (5 mL) and amine 103-f (0.055 g, 0.161 mmol), PyBOP (0.084 g, 0.161mmol) and DIPEA (0.037 mL, 0.215 mmol) were added successively at 0 °C.
  • the reaction mixture was stirred for 3 h at RT.
  • the reaction mixture was diluted with ice water and extracted with ethyl acetate (3 x 25 mL). Combined organic phases were washed with brine solution, dried over anhydrous sodium sulphate, and concentrated under reduced pressure.
  • Compound 104 is a drug-linker compound, related to 103, that can also be used for conjugation to a targeting ligand such as an scFvFc or IgG antibody.
  • anti-HER2 ADC-v3 anti-HER2 IgG antibody drug conjugate
  • PAMA linker PAMA linker
  • MMAE cytotoxic drug
  • Trastuzumab (T-mAb) was obtained from a commercial source and was purified by gel filtration chromatography before conjugation. T-mAb (0.2 mg/mL) was treated with 3 molar equivalents of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) in 0.025 M sodium borate pH 8, 0.025 M NaCl, 1 mM diethylenetriamine penta-acetic acid (DTP A) for 2 h at 37 °C. The mixture was then cooled to 4 °C.
  • TCEP tris-(2-carboxyethyl)phosphine hydrochloride
  • the partially reduced T-mAb was then treated with 6 molar equivalents of drug-linker compound 103 for 1 h at 4°C.
  • the conjugated and unconjugated fractions were purified by Butyl Sepharose chromatography on a Hi-Trap Butyl HP (5 mL) column (GE Healthcare) equilibrated with 50 mM Tris-Cl pH 7.5, 750 mM Ammonium Sulfate. Bound proteins were eluted using a linear gradient of water in 20 column volumes. Each peak eluted from the Butyl Sepharose column was analyzed by SDS- PAGE and LC-ESI-MS to identify those corresponding to conjugated antibody. Purified ADC fractions were pooled and buffer exchanged into sodium phosphate buffer, pH 7.4 150 mM sodium chloride and 5% glycerol.
  • the mass spectrometric data was acquired using Esquire Control software and analyzed by DataAnalysis software. Analysis of conjugated T-mAb LC-ESI-MS data in a manner similar to that described in Example 37 indicated that the sample of conjugated T- mAb consisted of major fractions containing either 2 or 4 conjugation events per antibody molecule giving rise to an approximate average drug to antibody ratio (DAR) of 3.
  • DAR drug to antibody ratio
  • the anti-HER2 ADC-v3 was determined to incorporate a distribution of about 2 to about 4 linker-drug groups per anti-HER2 IgG antibody.
  • the structure of this anti-HER2 ADC-v3 is comprised as follows:
  • R 6 is H
  • Ab-S- is derived from trastuzumab (i.e., Ab-SH is trastuzumab)
  • p ranges from about 2 to about 4 in this sample, and the average p is about 3.
  • ADC-v3 was evaluated in HER2-expressing tumor xenograft models, for example SKOV3 tumor xenograft model. Following a procedure similar to that described in Example 38, the anti-HER2 ADC-v3 was administered at a dose of 3 mg/kg mouse weight, IV, on Day 0, followed by 1 mg/kg IV Q4D x 4 (on Days 4, 8, & 12). Antitumor activity was assessed based on the observed inhibition of tumor growth or tumor suppression, in comparison to mice treated with vehicle (formulation buffer). Approximately 100% tumor growth inhibition by the anti-HER2 ADC-v3 was observed on Day 18. In this SKOV3 model, the non-conjugated Trastuzumab antibody was also highly active.
  • Example 41 Conjugation of anti-HER2 IgG (trastuzumab) to a PAMA-Linker-Drug to form an anti-HER2 antibody drug conjugate (anti-HER2 ADC-v4)
  • anti-HER2 IgG (trastuzumab) was covalently linked to compound 100:
  • anti-HER2 ADC-v4 anti-HER2 antibody drug conjugate
  • Trastuzumab (0.2 mg/mL) was treated with 3 fold molar equivalents of TCEP (tris-(2-carboxyethyl)phosphine hydrochloride) in 0.025 M sodium borate pH 8, 0.025M NaCl, 1 mM DTPA (diethylenetriamine penta-acetic acid) for 2 h at 37 °C. The mixture was then cooled to 4 °C. Partially reduced Trastuzumab from the above reaction was treated with 6 fold molar equivalents of linker-drug 91 for 1 h at 4 °C. The conjugated protein was purified by butyl sepharose chromatography in a manner similar to Example 38.
  • TCEP tris-(2-carboxyethyl)phosphine hydrochloride
  • LC-ESI-mass spectrometric analysis of anti-HER2 scADC-v4 following purification by butyl sepharose chromatography indicated the preparation to be a heterogenous mixture of ADCs with DAR values from about 2 to about 6.
  • this sample of anti-HER2 ADC- v4 incorporated ⁇ 2 to ⁇ 6 linker-drug groups per anti-HER2 Trastuzumab IgG antibody, with an average of 3.7 linker-drug groups per Trastuzumab antibody.
  • the structure of this anti- HER2 ADC-v4 is comprised as follows:
  • anti-HER2 ADC-v4 was evaluated in HER2-expressing tumor xenograft models, for example SKOV3 tumor xenograft model.
  • the anti-HER2 ADC-v4 was administered at a dose of 3 mg/kg mouse weight, IV, on Day 0, followed by 1 mg/kg IV Q4D x 4 (on Days 4, 8, 12).
  • Antitumor activity was assessed based on the observed inhibition of tumor growth or tumor suppression, in comparison to mice treated with vehicle (formulation buffer). Approximately 100% tumor growth inhibition by the anti-HER2 ADC-v4 was observed on Day 18.
  • the non-conjugated Trastuzumab antibody was also highly active.
  • treatment with the anti-HER2 ADC-v4 at 5 mg/kg IV Q4D x 4 caused strong tumor growth inhibition (ca. 80% on Day 21) that was significantly greater than that caused by the non- conjugated Trastuzumab antibody also dosed at 5 mg/kg IV Q4D x 4.
  • the illustrative conjugates of the following Table 6 are prepared in a similar manner.
  • the fluorescence drug release assay was carried out in 96-well black opaque plates (Greiner) in a final volume of 100 ⁇ , in duplicate. Each well contained 25 mM Sodium acetate/1 mM EDTA buffer (pH 5.0), 40 nM activated human liver Cathepsin B (Enzo Life Sciences) and 40 ⁇ test substrate (Trigger-linker molecules).
  • Control incubations included assay buffer without enzyme (buffer blank, pH 5.0) and substrate incubated in assay buffer in the absence of enzyme (substrate blank at pH 5.0). For each time point, substrate blank value was subtracted from the test values to get the final value. RFU for each time point was plotted against time to obtain a time versus RFU curve (progress curve).

Abstract

The invention provides a therapeutic drug and targeting conjugate, pharmaceutical compositions containing these conjugates in pharmaceutical composition, and uses of these conjugates in anti-neoplastic and other therapeutic regimens. Also provided are novel intermediates thereof. The conjugates provide a therapeutic drug fragment or prodrug fragment bound to a targeting moiety via a linker which comprises a substrate cleavable by a protease such as Cathepsin B. The targeting moiety is a ligand which targets a cell surface molecule, such as a cell surface receptor on an anti-neoplastic cell. The ligand may function solely as a targeting moiety or may itself have a therapeutic effect. Following administration of the therapeutic drug and targeting conjugate of formula I and exposure of the conjugate to the protease specific for the substrate, the linker is cleaved and the targeting moiety is separated from the conjugate, which causes the drug fragment or prodrug fragment to convert to the drug or prodrug. The recited conjugates are useful in anti-neoplastic therapies. Also provided are methods of making the therapeutic drug and targeting conjugates and intermediates thereof, and kits comprising the therapeutic drug and targeting conjugates.

Description

SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG - LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of the filing date of U.S. Provisional
Application No. 61/877,439, filed September 13, 2013, entitled Self-Immolative Linkers Containing Mandelic Acid Derivatives, Drug - Ligand Conjugates For Targeted Therapies And Uses Thereof, the disclosure of which is hereby incorporated herein by reference.
BACKGROUND
Targeted anti-neoplastic therapies are desired, in order to improve therapeutic outcome and to reduce toxicity of the delivered therapeutic. One approach to targeted antineoplastic therapies is the use of highly cytotoxic drugs linked to a targeting molecule that is highly specific for cell surface receptors on tumor or other neoplastic cells.
In WO 02/083180, de Groot et al propose activatable prodrugs having the organization "specifier-spacer-drug", which are converted by enzymatic cleavage to provide a [spacer-drug] that subsequently undergoes spontaneous spacer elimination to release the drug. A chemical means for conjugating drugs to ligands is through a spacer or linker that is a "self-immolative linker". Examples of such self-immolative linkers include PABC or PAB (para-aminobenzyloxycarbonyl linkers. See, e.g., Feng et al, US Patent No. 7,375,078.
This PAB linker unit may be referred to as an electronic cascade spacer. The amide bond linking the carboxyl terminus of a peptide unit and the para-aminobenzyl of PAB may act as a substrate and proteolytically cleavable. Upon cleavage, the aromatic amine becomes electron-donating and initiates an electronic cascade that leads to the expulsion of the leaving group, which releases the free drug after elimination of carbon dioxide. See, e.g., US Patent No. 7,375,078, citing (de Groot et al, (2001) J Organic Chemistry, 66(26): 8815-8830). See, also, de Groot et al, US Patent 7,705,045. The '078 patent describes limitations of the PAB type linker; for example, that certain PAB -containing conjugates may not be suitable substrates for some cleaving enzymes or may cleave too slowly to achieve efficacy. The '078 patent proposes alternative heterocyclic self-immolative linkers and conjugates. Still other self-immolative linkers are described in US Patent No. 7,091,186 and WO
2012/113847.
Despite the efforts and progress in this field, some limitations remain and there has been increasing interest in devising alternative enzyme-cleavable drug-linker technologies. The present invention describes a novel protease-cleavable self-immolative drug-linker system capable of releasing a variety of drugs. SUMMARY OF THE INVENTION
The invention provides a cleavable para-amino mandelic acid (PAMA) derived linker which is useful for forming a conjugate between a drug and targeting moiety, having the structure IX:
Figure imgf000004_0001
(IX)
wherein, L is a di-, tri- or tetra- amino acid chain; Z is an optional amine blocking group; X is a conjugatable group, such as an OH or NH2, or in the context of a therapeutic and targeting conjugate is bound directly or indirectly to a drug fragment or prodrug fragment; Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are
independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2; R1 is a conjugatable group, for example having a formula selected from the group consisting of -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, C C6 alkyl-, and -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(Ci-C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl, or in the context of a therapeutic and drug targeting conjugate is bound to the targeting moiety via this R1 moiety; and R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano. It should be recognized that the R2 group shown in the structure IX above, and similarly in other structures elsewhere in this document, indicates that one or more R2 groups may be attached to the ring. This same R2 group is described explicitly in the definition for W, W2 and W3.
In another aspect, the invention provides a therapeutic drug and targeting conjugate characterized by formula (I):
Figure imgf000004_0002
wherein, L is a di-, tri- or tetra- amino acid chain; X is (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- which is the point of attachment to -OC(O)-, (ii) X is -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- or - O- as the point of attachment to -N(H)C(0)-; (iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or (iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR4- which is part of the drug fragment or prodrug fragment; p is 0 or 1; m is 1 to 6, with the proviso that when m is 1, p is 0; LG is a ligand which targets a cell surface molecule, for example a cell
surface receptor or antigen; B-LG is
Figure imgf000005_0001
; wherein said LG has at least one thiol moiety or at least one amino moiety which forms the point of attachment to B; p" is 1 to 6; Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2; R1 is -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, C C6 alkyl-,
or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8; R2 is H, C C6 alkyl, C C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano; R3 is H, C1-C6 alkyl, C2-C6 hydroxyalkyl or C2-C6
perfluoroalkyl; R4 is H, C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 perfluoroalkyl; R6 is H, Ci-C6 alkyl, or C2-C3 hydroxyalkyl; R7 is Ci-C6 alkyl, C1-C3 hydroxyalkyl, or - (CH2)2NH(Ci-C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl; R9 and R10 are independently H or C1-C6 alkyl; R12, R13 and R14 are independently selected from H, C1-C6 alkyl, C2-C3 hydroxyalkyl; and Z is an optional amine blocking group.
In one example, X is (i) or (iii) and the drug fragment or prodrug fragment Y is a DNA damaging agent having the structure of formula (III) or (IV), respectively, below:
Binder
Figure imgf000005_0002
where "Minor Groove Binder" means a DNA minor groove binding group which is a substituted lH-indole-2-carbonyl group as described herein. In one aspect, the invention provides novel ligands for use in the present targeting and therapeutic conjugates, such as the anti-HERl-scFvFc of SEQ ID NO: ABl and the anti- HER2-scFvFc of SEQ ID NO: AA2.
In another aspect, the invention provides a method of delivering at least one therapeutically active drug, said method comprising administering a conjugate comprising a targeting ligand and at least one PAMA linker system bearing a therapeutic drug or prodrug fragment, wherein the therapeutically active drug or prodrug fragment is released from the conjugate following cleavage of the peptidic substrate moiety incorporated within the PAMA linker by a protease, for example a lysosomal protease. Optionally, the targeting ligand is also therapeutically active. In one embodiment, the drug or prodrug is selected from the group consisting of a DNA damaging agent, a microtubule disrupting agent, and a cytotoxic protein or polypeptide.
In yet another aspect, the invention provides a method of treating a disease or disorder associated with the presence of a specific cell surface molecule on cells of a subject, comprising administering to the subject a therapeutically effective amount of a conjugate of the invention comprising at least one ligand that specifically binds to the cell surface molecule. In one embodiment, the disease or disorder is a neoplastic disease such as cancer.
In still another aspect, the invention provides a method for producing a therapeutic drug and targeting conjugate, comprising the steps of providing a cleavable para-amino mandelic acid (PAMA) derived linker having the structure IX:
Figure imgf000006_0001
(IX)
wherein, L is a di-, tri- or tetra- amino acid chain; Z is an optional amine blocking group; X is a conjugatable group, such as an OH or NH2, which can be bound directly or indirectly to a drug fragment or prodrug fragment; Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2; R1 is a conjugatable group, for example having a formula selected from
-CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, C C6 alkyl-, or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(Ci-C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl, which can be bound to a targeting moiety; and R2 is H, Ci-C6 alkyl, Ci-C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano, and conjugating the cleavable para-amino mandelic acid (PAMA) derived linker to a targeting molecule through R1 and to a drug or prodrug fragment through X.
In still another aspect, a kit is provided that comprises a conjugate of the invention.
The conjugate may be in the presence or absence of one or more of pharmaceutically acceptable carriers or excipients. The kit may optionally contain instructions for administering the conjugate to a subject, e.g., for use in an anti-neoplastic regimen.
In still another embodiment, a synthetic process for preparing a tert-butyl (4- hydroxynaphthalen-2-yl)carbamate is provided. The process comprises (i) admixing 1,3- dihydroxynapthalene and diphenylmethylamine in an aromatic solvent such as toluene solution; (ii) heating the toluene solution at 80°C to 125°C for about 4 to about 8 hours; (iii) combining the reaction mass with palladium hydroxide, di-tert butyl carbonate, and dioxane:water; (iv) shaking the reaction mixture of (c) at 60-80 psi hydrogen pressure for about 24 to 48 hours; and (v) filtering the reaction mixture of (d) and concentrating to yield crude tert-butyl (4-hydroxynaphthalen-2-yl)carbamate. The process further comprises filtering and concentrating the tert-butyl (4-hydroxynaphthalen-2-yl)carbamate product of step (v) to column chromatography on silica gel using ethyl acetate-hexane (1 :9) as eluent.
In still a further embodiment, a synthetic process for preparing a compound 29a is provided. This method comprises (a) combining an alcohol having the structure of compound 26a and cesium chloride in a dimethylformide solution, wherein the compound
Figure imgf000007_0001
26a has the structure: wherein Z is amine blocking group and L is an amino acid selected from Val-Cit or Gly-Gly-Phe-Gly; cooling the reaction mixture to about 0°C and adding trichloroacetonitrile; allowing the reaction mixture to warm to room temperature with stirring; pouring the reaction mixture over water and extracting with ethylacetate; washing the combined organic layers with water and brine, followed by separating the organic layer, drying, concentrating, and purifying crude product by silica gel column chromatography using 10% methanol in dichloromethane as eluent to yield the trichloracetimidate product which has the structure 27a:
Figure imgf000008_0001
^7a (b) combining the trichloracetimidate product 27a defined in (a) with Boc-Pro-CBI 2a (Scheme 4) and dry acetonitrile in a suspension of molecular sieves; under cooling conditions at -10 °C to -20 °C in the presence of Lewis acid such as BF3.etherate; neutralizing the reaction mixture; removing volatiles; and purified by silica gel column chromatography using dichloromethane
yield compound of structure 28a:
Figure imgf000008_0002
(c) combining the methanol solution of compound 28a, Boc20, and NEt3 at 0 °C and allowing the reaction to proceed; removing the volatiles and purifying the crude reaction product by purified by silica gel column
yield compound 29a:
Figure imgf000008_0003
29a
a further aspect, a synthetic process for preparing a compound 34a
Figure imgf000008_0004
is provided.
Compound 34a is useful in synthesis of a drug and targeting conjugate as described herein. The process comprises (a) combining carbonyldiimidazole and 3 (5-(2- (dimethylamino)ethoxy)-6-methoxy-lH-indole-2-carboxylic acid; DMMI) (Scheme 2) (or the carboxylic acid compound of another DNA minor groove binding group which is a substituted lH-indole-2-carbonyl group) in a DMF solution and stirring for about 2 hours at ambient temperature; (b) combining the reaction mixture with a stirred DMF solution of amine 29a and sodium carbonate, wherein Z is a blocking group and L is a di-peptide, tri- peptide, or tetra-peptide; (c) removal of solid following reaction and neutralizing remaining solution with formic acid.
Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a line graph illustrating self-immolation and release of HMC (7-hydroxy-4- methyl-2H-chromen-2-one, or 7-hydroxy-4-methyl-coumarin, which serves as an illustrative surrogate hydroxy-linked drug) from the compound "Cbz-Val-Cit-PAMA-(methyl-
carboxylate)-HMC " :
Figure imgf000009_0001
upon treatment with Cathepsin B. Release of HMC after treatment with Cathepsin B was compared to a reference compound ("Cbz-Val-Cit-PABE-HMC") having the structure:
Figure imgf000009_0002
FIG. 2 is a line graph illustrating self-immolation and release of HMC from the compound "Cbz-Val-Cit-PAMA-(amide)-HMC":
Figure imgf000009_0003
upon treatment with Cathepsin B. Cbz-Val-Cit-
PABE-HMC was used as reference compound. FIG. 3 is a line graph illustrating self-immolation and release of HMC from the compound "Cbz-Val-Cit-PAMA-(PEG-amide)-HMC":
Figure imgf000010_0001
upon treatment with Cathepsin B. Cbz-Val-Cit
PABE-HMC was used as reference compound.
FIG. 4 is a line graph illustrating self-immolation and release of AMC (7-amino-4- methyl-2H-chromen-2-one, or 7-amino-4-methyl-coumarin, which serves as an illustrative surrogate amine-linked drug) from the compound "Cbz-Val-Cit-PAMA-(PEG-amide)-
OC(0)-AMC":
Figure imgf000010_0002
upon treatment with
Cathepsin B. The reference compound was the compound "Cbz-Val-Cit-PABC-AMC":
Figure imgf000010_0003
FIG. 5 is a line graph illustrating self-immolation and release of HMC from the compound "Cbz-Val-Cit-PAMA-(MB-PEG3-amide)-HMC" (n=l in the following structure) and the compound "Cbz-Val-Cit-PAMA-(MB-PEG4-amide)-HMC" (n=2 in the following
Figure imgf000010_0004
structure): n " = 1 l ,, 2e- upon treatment with Cathepsin B. "MB" means 4-(maleimido)-butanoyl. The reference compound was Cbz- Val-Cit-PABE-AMC. FIG. 6A is a line graph illustrating self-immolation and release of the cytotoxic
/
\
duocarmycin analog "CBI-DMMI":
Figure imgf000011_0001
from the compound
"Cbz- Val-Cit- -(PEG3 -amide)-Pro-CBI-DMMI" :
Figure imgf000011_0002
upon treatment with Cathepsin B. The amount of CBI-DMMI released in the assay was measured using LC-MS/MS. Cbz-Val-Cit-PAMA-(PEG3-amide)-Pro-CBI-DMMI" was incubated in buffer without Cathepsin B enzyme for comparison.
FIG. 6B is a line graph illustrating self-immolation and release of CBI-DMMI from the compou "Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-Pro-CBI-DMMI":
Figure imgf000011_0003
upon treatment with Cathepsin B. The amount of CBI-DMMI released in the assay was measured using LC-MS/MS. Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-Pro-CBI-DMMI" was incubated in buffer without Cathepsin B enzyme for comparison. FIG. 6C is a line graph illustrating self-immolation and release of CBI-DMMI from the compou "Cbz-Val-Cit-PAMA-(Boc-PEG4-amide)-Pro-CBI-DMMI":
Figure imgf000012_0001
upon treatment with Cathepsin B. The amount of CBI-DMMI released in the assay was measured using LC-MS/MS. Cbz-Val-Cit-PAMA-(Boc-PEG4-amide)-Pro-CBI-DMMI was incubated in buffer without Cathepsin B enzyme for comparison.
FIG. 7 is a line graph illustrating self-immolation and release/generation of a cytotoxic drug (Compound A) from anti-HER2 ADC-v2 [Cbz-Val-Cit-PAMA-(anti-HER2 scFv-Fc-succinimido-butanoyl-PEG3-amide)-(Compound 91); Example 39] in the presence and absence of Cathepsin B.
FIGs. 8A and 8B provide the results of an LC-ESI-MS analysis of the anti-5T4 scADC of Example 37. The analysis was performed as described in Example 37. FIG. 8A provides the ESI-MS charge states of scADC peak pre-reduced completely with DTT (dithiothreitol) to generate monomeric scADC. FIG. 8B provides the deconvoluted mass spectrum showing relative abundance of non-conjugated (54669.6 Da) and singly conjugated (56000.7 Da) monomer of the scADC described in (A).
DETAILED DESCRIPTION OF THE INVENTION
A para-amino mandelic acid (PAMA) derived linker is provided herein which is useful in a conjugate containing both a drug fragment or prodrug fragment and a targeting moiety (herein also called a "ligan "), said linker having the structure IX:
Figure imgf000012_0002
(IX) wherein, Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2; L is a di-, trior tetra- amino acid chain; Z is an optional amine blocking group; X is bound directly or indirectly to a drug fragment or prodrug fragment; R1 is bound to a targeting moiety via a group having a formula selected from -CONR6CHR7CH2(OCH2CH2)nOCH2CHR8-, C C6 alkyl- or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(C C3 alkyl)2; R8 is H, Ci-C3 alkyl or Ci-C3 hydroxyalkyl; and R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano.
In one embodiment, a drug and targeting conjugate is provided which comprises the cleavable PAMA linker described herein, wherein each of W, Wi, W2 and W3 is CR2, R1 is - CONR6CHR7CH2(OCH2CH2)nOCH2CHR8-, and R2 is H.
The conjugate or linker fragment A illustrated below serves as a substrate for one or more proteases following intracellular uptake of the conjugate by a targeted cell. These fragments are designed to be cleaved by enzymes within the cathepsin family (e.g., cathepsins A, B, C, D, E, F, G, H, K, LI, L2, O, W, or Z), including particularly cathepsin B and cathepsin L following uptake of the conjugate of the invention into target cells of the targeted cell population. The cathepsins are found primarily in lysosomes, but at least cathepsin K has been found to have extracellular activity. Cathepsins are typically associated with a variety of cancers, and thus in one embodiment cleavage of the conjugate of the invention is designed for targeted cancers cells. However, the protease substrate can be targeted to other cell types by designing it such that the protease substrate is cleavable for example by other cathepsins or other lysosomal enzymes, thus permitting the targeting-drug conjugates described herein to be designed and used in treatment of and therapies for conditions other than cancer including, e.g., certain viral infections, certain bacterial infections, COPD, chronic periodontitis, and pancreatitis. For example, the fragment A illustrated below binds to the protease active site, such that the proteolytic activity results in cleavage of the amide bond between Z-L and the aryl or heteroaryl amine as indicated by the dashed line in B.
As used herein, the term "L" refers to a di-, tri- or tetra-peptidyl group, i.e., a peptide composed of two, three or four amino acid residues. The peptide L is optionally bonded to group Z, is linked to a drug moiety through the para-amino mandelic acid (PAMA) derived self-immolative linker in structure (IX), and forms part of the enzyme-cleavable substrate. Suitable peptides for L include, without limitation, Val-Cit, Gly-Gly-Phe-Gly, Phe-Lys, Val- Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp- Cit, Phe-Ala, Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.
The moiety "Z" refers to an optional amine blocking group attached to the N- terminus of the peptide residue L. Z may be selected from C1-C6 acyl, optionally substituted aroyl, optionally substituted heteroaroyl, (aryl)alkyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C3-C6 heterocycloalkyl-carbonyl, (alkoxy)carbonyl (e.g., t-butoxycarbonyl known as BOC), (aryloxy)carbonyl, (heteroaryloxy)carbonyl, (aryl alkoxy)carbonyl (e.g., benzyloxycarbonyl or carbobenzyloxy, known as Cbz; and 9-Fluorenylmethyloxycarbonyl known as FMOC), (heteroaryl alkoxy)carbonyl, C3-C6 (cycloalkoxy)carbonyl, C3-C6
(heterocycloalkoxy)carbonyl, and RnNH-CO-,wherein R11 is H, C1-C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, (aryl)alkyl, or (heteroaryl)alkyl. Other examples of Z groups include acetyl, pyrroloyl, and t- butylcarbonyl.
Suitable examples of groups Z-L are: Cbz- Val-Cit, Cbz-Phe-Cit, Cbz-Phe-Ala, Cbz- Phe-Lys, Cbz-Val-Lys, Cbz-Phe-Arg, Cbz-Val-Arg, Gly-Phe-Leu-Gly, and Gly-Gly-Phe- Gly. However, many other Z-L combinations will be apparent from the information provided herein.
For example, in the case where L is a dipeptide fragment, the group Z-L forms the part of the substrate which binds to the protease "S3-S2-S1" binding pockets or subsites, using the terminology as described by I. Schechter and A. Berger in Biochemical and Biophysical Research Communications, 27, 157-162 (1967). Using further Schechter and Berger terminology, in the embodiment where L is a dipeptide fragment, the group Z-L may be considered as the "P3-P2-Pi" positions of the protease substrate.
Figure imgf000014_0001
Di-, tri- and tetra-peptidyl fragments L that are suitable as substrate fragments "P2-Pi", "P3- P2-P1", and "P4-P3-P2-P1" for proteases such as Cathepsin-B, -L and -S are well known in the art, for example as described by P. Ruzza et al., J. Peptide Science, 12, 455-461 (2006); and Y. Choe et al., J. Biol. Chem., 281, 12824-12832 (2006). Other studies describing Cathepsin- B, -L and -S labile peptidyl groups that are suitable for use as group L in the conjugates of the invention, and their application have been reported, for example in prodrug and enzyme inhibitor structures; this includes examples with N-blocking groups (Z) such as
carbobenzyloxy, acetyl, and formyl moieties. For example, see G. M. Dubowchik and R. A. Firestone, Bioorg. Med. Chem. Lett., 8, 3341-3346 (1998); G. M. Dubowchik et al., Bioorg. Med. Chem. Lett., 8, 3347-3352 (1998); G. M. Dubowchik et al., Bioconjugate Chem., 13, 855-869 (2002); D. Bromme et al., Biol. Chem. Hoppe-Seyler, 375, 343-347 (1994); D. H. Pliura et al., Biochem J., 288, 759-762 (1992); A. Krantz et al., Biochemistry, 30, 4678-4687 (1991); R. A. Smith et al., J. Am. Chem. Soc, 110, 4429-4431 (1988); and R. A. Smith et al., Biochem. Biophys. Res. Commun., 155, 1201-1206 (1988). Finally, suitable and preferred peptidyl groups, for use in prodrugs, that are labile to lysosomal proteases were described in a study using solid tumor homogenates rather than selected proteases such as Cathepsin B; see Y. Shiose et al., Bioconjugate Chem., 20, 60-70 (2009).
Upon binding to and cleavage by a protease, the compound B is converted to intermediate compound C which spontaneously undergoes a rearrangement or "self- immolation" to form compound D and eliminate fragment X. In embodiment (i) wherein X is -OC(0)Y, the reaction continues, to eliminate CO2 and the drug Y-H. In embodiment (ii) wherein X is -N(H)C(0)Y, the reaction continues, to eliminate HNCO and the drug Y-H. In embodiment (iii) wherein X is a drug or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug or prodrug, the drug Y-H is formed as indicated in the scheme below. In embodiment (iv) where X is a drug or prodrug fragment Y bound to the a carbon via a -NR4- which is part of the drug or prodrug, the drug Y-H is formed also as
Figure imgf000015_0001
B C D
In one embodiment, making use of the para-amino mandelic acid (PAMA) derived linker defined herein, a therapeutic drug and targeting conjugate is provided which is of formula (I):
Figure imgf000016_0001
wherein, Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2; R1 is - CONR6CHR7CH2(OCH2CH2)nOCH2CHR8-, C C6 alkyl-, or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8; LG is a ligand which specificall
example a cell surface receptor or antigen; B-LG is
Figure imgf000016_0002
; wherein said LG has at least one thiol moiety or at least one amino moiety which forms a point of attachment to B; L is a di-, tri- or tetra- amino acid chain; X is (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- which is the point of attachment to -OC(O)-, (ii) -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -O- or -NR4- as the point of attachment to -N(H)C(0)-; (iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or (iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR4- which is part of the drug fragment or prodrug fragment; p is 0 or 1 ; m is 1 to 6 with the a proviso that, when m is 1, p is 0; p" is 1 to 6; R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, Ci-C6 fluoroalkyl, or cyano; R3 is H, Ci-C6 alkyl, C2-C6 hydroxyalkyl or C2-C6 perfluoroalkyl; R4 is H, C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 perfluoroalkyl; R6 is H, Ci-C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2N(C C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl; R9 and R10 are independently H or Ci-C6 alkyl; R12, R13 and R14 are independently selected from H, C C6 alkyl or C2-C3 hydroxyalkyl; and Z is an amine blocking group.
In still further embodiments, a therapeutic drug and targeting conjugate of formula (IA), formula (IB), formula (ICi) or formula (ICii) are provided. While much of the following description will reference formula (I) for purposes of convenience, it will be understood that the following definitions and uses are applicable to any of these subgeneric structures, or combinations thereof. In one embodiment, each of W, Wi, W2 and W3 is CR2, R1 is -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, R2 is H and formula IA has the structure:
Figure imgf000017_0001
CO-(NH)p-(CH2)m-B- LG
(IA)
P"
In one embodiment of formula (IA), L is selected from the group consisting of (a) Val-Cit or (b) Gly-Gly-Phe-Gly; X is -OC(0)Y, wherein Y is a drug or prodrug having a - NR4- as the point of attachment; or a drug or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug or prodrug. R1 is -
CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, wherein n is 1 to 8. Z is an amine blocking group selected from the group consisting of Ci-C6 acyl, optionally substituted aroyl, optionally substituted heteroaroyl, (aryl)alkyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C3-C6 heterocycloalkyl-carbonyl, (alkoxy)carbonyl, (aryloxy)carbonyl, (heteroaryloxy)carbonyl, (aryl alkoxy)carbonyl, (heteroaryl alkoxy)carbonyl, C3-C6 (cycloalkoxy)carbonyl, C3-C6 (heterocycloalkoxy)carbonyl, and RnNH-CO-. R11 is H, C1-C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, (aryl)alkyl, or (heteroaryl)alkyl.
In another embodiment, W is CR2, R2 is H, each of Wi, W2, and W3 are
independently N or CR2, R1 is -CONR6CHR7CH2(OCH2CH2)nOCH2CHR8- and formula IB has the structure:
Figure imgf000017_0002
CO-(NH)p-(CH2)m-B{ LG
(IB) P"
In still a further embodiment, W is absent, W1 is CR2, R2 is H, R1 is - CONR6CHR7CH2(OCH2CH2)nOCH2CHR8- and the formula ICi or ICii has the structure:
Figure imgf000018_0001
Various uses of the linker, the conjugate, functional fragments thereof, and compositions and methods employing the same are provided herein.
The following definitions are used in connection with the compounds and conjugates of the present invention unless the context indicates otherwise.
Figure imgf000018_0002
In one aspect, the invention provides a synthetic process for preparing a tert-butyl (4- hydroxynaphthalen-2-yl)carbamate (compound 6 in Scheme 1). The process comprises (i) admixing 1,3-dihydroxynaphthalene and diphenyl methylamine in a toluene solution and (ii) heating the toluene solution at about 80°C to about 125°C for about 4 to about 8 hours, about 5 to about 7 hours, or about 4 hours, about 5 hours, about 6 hours, about 7 hours or about 8 hours. The heating may be about 90°C to about 110°C, or about 100°C. Following this heating, the reaction mixture is combined with palladium hydroxide, di-tert butyl carbonate, and dioxane: water. In one example, the ratio of dioxane to water is about 4 parts dioxane to about 1 part water (volume/volume). However, higher or lower amounts may be selected. In one embodiment, this combination is mixed at about 60 psi to about 80 psi hydrogen gas pressure for about 24 to 48 hours. However, these pressures and times may be adjusted as needed. Following this, the reaction mixture is filtered and concentrated to yield crude tert- butyl (4-hydroxynaphthalen-2-yl)carbamate. One example of this synthetic process and its further utility for the preparation of N-Boc Pro-CBI (12 in Scheme 1) and Pro-CBI (2 in Scheme 1) is illustrated, e.g., in Scheme 1.
In another aspect, the invention provides a synthetic process for preparing compound
Figure imgf000019_0001
of formula 29a . The method involves
(a) combining an alcohol having the structure 26a and cesium chloride in an aprotic polar solvent such as dimethylformide, wherein the compound 26a has the structure:
Figure imgf000019_0002
wherein Z is an amine blocking group and L is an amino acid chain, as defined elsewhere in this specification. In one embodiment, the amino acid chain is Val-Cit or Gly-Gly-Phe-Gly. Typically, this reaction is performed at a temperature from about 0 °C to about room temperature (about 20°C to about 25°C) for about 2 to about 6 hours. In one embodiment, the reaction is proceeds for about 4 hours. Following this, the reaction mixture is cooled to about -10°C to about 5°C, or 0°C before or at the time of adding trichloroacetonitrile and the reaction mixture may be allowed to warm to room temperature with stirring. The reaction mixture is then poured over water and extracted with ethylacetate. The combined organic layers are washed and the crude product is purified. In one embodiment, washing is performed with water and brine, followed by separating the organic layer, drying, concentrating, and purifying crude product by silica gel column chromatography. In one embodiment, the eluent is 10% methanol in
dichloromethane. The resulting product is a trichloracetimidate which has the structure 27a:
Figure imgf000020_0001
. This trichloracetimidate product 27a defined
is combined with 2a
Figure imgf000020_0002
(N-Boc-Pro-CBI) and dry acetonitrile in a suspension of molecular sieves and the reaction mixture is cooled to about -10 °C to about - 20 C in the presence of a Lewis acid. Following this, the reaction mixture is neutralized, volatiles removed; and the crude product is purified by silica gel column chromatography using dichloromethane (DCM):MeOH:NH3 solution as eluent to obtain the compound of
Figure imgf000020_0003
structure 28a: . The methanol solution containing compound 28a is combined with Boc20, and NEt3 at 0 °C and the reaction is allowed to proceed. The volatiles are then removed and the crude reaction product is purified. In one embodiment, purification is performed by silica gel column chromatography using DCM:MeOH as eluent to yield compound 29a. However, other purification methods may be utilized. One example of this synthetic process is illustrated, e.g., in Scheme 12 herein. In still a further embodiment, a synthetic process for preparing compound of structural formula 34a is described. This compound is useful in synthesis of a drug and targeting conjugate comprising a therapeutic drug associated with a cell specific targeting moiety via a linker which is specifically cleavable by a lysosomal protease. The synthetic process involves combining carbonyldiimidazole and an optionally substituted Indole-2- carboylic acid derivative in a DMF solution and stirring for about 2 hours at ambient temperature. In one embodiment, the compound is 3 ("DMMI") as described in Scheme 2. However, other minor groove binders (MGB) related to DMMI may be selected. The reaction mixture is combined with a stirred DMF solution of amine 29a and an inorganic base such as sodium carbonate or potassium carbonate, wherein amine 29a has the structure:
BocHN
Figure imgf000021_0001
29a
wherein Z is an N-blocking group and L is a di-peptide, tri-peptide, or tetra-peptide fragment. Following the reaction, the solid is filtered and removed and the remaining solution is neutralized with an acid such as formic acid or acetic acid. The DMF is removed
Figure imgf000021_0002
is obtained.
Similar procedures can be performed with an optionally substituted indole-2- carboxylic acid derivative with the Pro-CBI portion of the molecule, and/or with variation Z, L, and/or variation in the length of the PEG group (different number of -OCH2CH2- fragments).For example, the compound comprising the following formula, where Z-L is Cbz- -Cit, was prepared in a similar manner:
Figure imgf000021_0003
In still a further embodiment, a synthetic process for preparing compound of structural formula 40a is described. This compound is useful in synthesis of a drug and targeting conjugate comprising a therapeutic drug associated with a cell specific targeting moiety via a linker which is specifically cleavable by a lysosomal protease. The synthetic process involves the t-Boc protected compound of formula 34a was deprotected using an inorganic acid dissolved in an organic solvent such as ethylacetate. The deprotected amino derivative was dissolved in an aprotic organic solvent such as DMF and reacted with succinate ester 27 in the presence of an organic or an inorganic base such as sodium or
Figure imgf000022_0001
Similarly compounds corresponding structure 100a was prepared starting from the
.
Figure imgf000022_0002
The term "cleavable group", "cleavable moiety", or "substrate" is intended to mean a moiety that is cleaved in vivo by the biological environment. The term "substrate" also refers to the structural fragment that binds to the active site of the enzyme and is then cleaved by that enzyme. The cleavable groups are selected so that activation occurs at the desired site of action, which can be a site in or near the target cells (e.g., carcinoma cells) or tissues such as at the site of therapeutic action or marker activity. Such cleavage is enzymatic and exemplary enzymatically cleavable groups include natural amino acids or peptide sequences that end with a natural amino acid, and are attached at their carboxyl terminus to the linker.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs, N, N-dialkylated amino acid, and amino acid mimetic that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid. The term "unnatural amino acid" is intended to represent the "D" stereochemical form of the twenty naturally occurring amino acids. The term "unnatural amino acid" includes homologues of the natural amino acids, and synthetically modified forms of the natural amino acids. Such synthetically modified forms include, but are not limited to, amino acids having -CH2- chains shortened or lengthened by up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprised halogenated groups, preferably halogenated alkyl and aryl groups. The term heteroatom refers to oxygen, sulfur or nitrogen.
In general, the number of carbon atoms present in a given group is designated "Cx- Cy", where x and y are the lower and upper limits, respectively. For example, a group designated as "C1-C6" contains from 1 to 6 carbon atoms. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like. Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming from left to right the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent
"arylalkyloxycarbonyl" refers to the group (C6-C14 aryl)-(Ci-C6 alkyl)-O-C(O)-. Terms not defined herein have the meaning commonly attributed to them by those skilled in the art.
"Acyl-" refers to a group having a straight, branched, or cyclic configuration or a combination thereof, attached to the parent structure through a carbonyl functionality. Such groups may be saturated or unsaturated, aliphatic or aromatic, and carbocyclic or heterocyclic. Examples of a Ci-C8acyl- group include acetyl-, benzoyl-, nicotinoyl-, propionyl-, isobutyryl-, oxalyl-, and the like. Lower-acyl refers to acyl groups containing one to four carbons. An acyl group can be unsubstituted or substituted with one or more of halogen, NH2, (C C6 alkyl)amino-, di(C C6 alkyl)amino-, (Ci-C6 alkyl)C(0)N(Ci-C3 alkyl)-, CN, hydroxyl, C1-C6 alkoxy, C1-C6 alkyl, C6-C14 aryl, C1-C9 heteroaryl, or C3-C8 cycloalkyl. "Alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, for example, a C1-C12 alkyl group may have from 1 to 12 (inclusive) carbon atoms in it. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec -butyl, tert-butyl, isopentyl, neopentyl, and isohexyl. Examples of Ci-Cs alkyl groups include, but are not limited to, methyl, propyl, pentyl, hexyl, heptyl, 3-methylhex-l-yl, 2,3-dimethylpent- 2-yl, 3-ethylpent-l-yl, octyl, 2-methylhept-2-yl, 2,3-dimethylhex-l-yl, and 2,3,3- trimethylpent-l-yl. An alkyl group can be unsubstituted or substituted with one or more of halogen, NH2, (C C6 alkyl)NH, (Ci-C6 alkyl)(Ci-C6 alkyl)N-, -N(C C3 alkyl)C(0)(C C6 alkyl), -NHC(0)(C C6 alkyl), -NHC(0)H, -C(0)NH2, -(alkyl)amido-, -C(0)N(C C6 alkyl)(C C6 alkyl), CN, hydroxyl, C C6 alkoxy, C C6 alkyl, -C(0)OH, -C(0)0(C C alkyl), -C(0)(C C6 alkyl), C6-C14 aryl, C C9 heteroaryl, C3-C8 cycloalkyl, d-C6 haloalkyl, C C6 aminoalkyl-, -OC(0)(Ci-C6 alkyl), (alkyl)carboxyamido-, and N02.
"Alkoxy-" refers to the group R-O- where R is an alkyl group, as defined above. Exemplary C1-C6 alkoxy- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1-propoxy, n-butoxy and t-butoxy. An alkoxy group can be unsubstituted or substituted with one or more of halogen, hydroxyl, C1-C6 alkoxy, NH2, (C1-C6 alkyl)amino-, di(Ci-C6 alkyl)amino-, (C C6 alkyl)C(0)N(C C3 alkyl)-, (alkyl)carboxyamido-, HC(0)NH-, H2NC(0)-, (C C6alkyl)NHC(0)-, di(C C6 alkyl)NC(O)-, CN, C02H, (C C6
alkoxy)carbonyl-, (C1-C6 alkyl)C(O)-, C6-C14 aryl, C1-C9 heteroaryl, C3-C8 cycloalkyl, C1-C6 haloalkyl, amino(Ci-C6 alkyl)-, (alkyl)carboxyl- or N02.
Aryl refers to an aromatic hydrocarbon group. Examples of a C6-C14 aryl group include, but are not limited to, phenyl, a-naphthyl, β-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenanaphthyl. Examples of a C - Cioaryl group include, but are not limited to, phenyl, a-naphthyl, β-naphthyl, biphenyl, and tetrahydronaphthyl. An aryl group can be unsubstituted or substituted with one or more of C1-C6 alkyl, halogen, haloalkyl, hydroxyl, hydroxyl(Ci-C6 alkyl)-, NH2, aminoalkyl-, dialkylamino-, -COOH, -C(0)0-(C C6 alkyl), -OC(0)(C C6 alkyl), N-alkylamido-, - C(0)NH2, (C C6 alkyl)amido-, N02, (aryl)alkyl, C C6 alkoxy, C6-C10 aryloxyl, C2-C10 heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carboxyamido-, (aryl)alkyl-, (aryl)amino-, cycloalkenyl-,
di(alkyl)amino-, heteroaryl-, (heteroaryl)alkyl-, heterocyclyl-, heterocyclyl(alkyl)-, (HO-Cr C6 alkyl)NH-, (OH-Ci-C6 alkyl)2N or a spirocyclic substituent as defined before. The term "Spirocyclic" refers to a 3, 4, 5, 6 or 7 membered cyclic compound optionally having one or two heteroatoms optionally having 0 to 2 double bonds and containing a sp3-hybridized carbon, wherein the 2nd cyclic system contains the same sp3- hybridized carbon atom. The following examples denote some of the examples of spirocyclic system.
Figure imgf000025_0001
"(Aryl)alkyl" refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with an aryl group as defined above. ( C i4 Aryl) alkyl- moieties include benzyl, benzhydryl, 1-phenylethyl, 2-phenylethyl, 3- phenylpropyl, 2-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl and the like. An (aryl)alkyl group can be unsubstituted or substituted with one or more of halogen, CN, NH2, hydroxyl, (C C6 alkyl)amino-, di(Ci_C6 alkyl)amino-, (Ci-C6 alkyl)C(0)N(C C3 alkyl)-, (C C6 alkyl)carboxyamido-, HC(0)NH-, H2NC(0)-, (C C6 alkyl)NHC(O)-, di(C C6 alkyl)NC(O)-, C C6 alkoxy, C C6 alkyl, C02H, (C C6 alkoxy)carbonyl-, (C C6 alkyl)C(O)-, C6-Ci4 aryl, Ci-Cg heteroaryl, C3-C8 cycloalkyl, Ci-C6haloalkyl, amino(Ci-C6 alkyl)-, (C1-C6 alkyl)carboxyl-, C1-C6 carboxyamidoalkyl-, or NO2.
"(Alkoxy)carbonyl-" refers to the group alkyl-O-C(O)-. Exemplary (d- C6alkoxy)carbonyl- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1- propoxy, n-butoxy and t-butoxy. An (alkoxy)carbonyl group can be unsubstituted or substituted with one or more of halogen, hydroxyl, NH2, (Ci-C6 alkyl)amino-, di(Ci-C6 alkyl)amino-, (C C6 alkyl)C(0)N(C C3 alkyl)-, (alkyl)carboxyamido-, HC(0)NH-, H2NC(0)-, (C C6alkyl)NHC(0)-, di(C C6 alkyl)NC(O)-, CN, C C6 alkoxy, C02H, (C C6 alkoxy)carbonyl-, (Ci-C6 alkyl)C(0)-, C6-C14 aryl, Ci-Cg heteroaryl, C3-C8 cycloalkyl, C1-C6 haloalkyl, amino(Ci-C6 alkyl)-, (C1-C6 alkyl)carboxyl-, C1-C6 carboxyamidoalkyl-, or N02.
"(Alkyl)amido-" refers to a -C(0)NH- group in which the nitrogen atom of said group is attached to a Ci-C6 alkyl group, as defined above. Representative examples of a (Cr C6 alkyl)amido- group include, but are not limited to, -C(0)NHCH3, -C(0)NHCH2CH3, - C(0)NHCH2CH2CH3, -C(0)NHCH2CH2CH2CH3, -C(0)NHCH2CH2CH2CH2CH3, - C(0)NHCH(CH3)2, -C(0)NHCH2CH(CH3)2, -C(0)NHCH(CH3)CH2CH3, -C(0)NH-C(CH3)3 and -C(0)NHCH2C(CH3)3.
"(Alkyl)amino-" refers to an -NH group, the nitrogen atom of said group being attached to a alkyl group, as defined above. Representative examples of an (C1-C6 alkyl)amino- group include, but are not limited to CH3NH-, CH3CH2NH-, CH3CH2CH2NH-, CH3CH2CH2CH2NH-, (CH3)2CHNH-, (CH3)2CHCH2NH-, CH3CH2CH(CH3)NH- and
(CH3)3CNH-. An (alkyl)amino group can be unsubstituted or substituted with one or more of halogen, NH2, (C C6 alkyl)amino-, di(C C6 alkyl)amino-, (Ci-C6 alkyl)C(0)N(Ci-C3 alkyl)-, (Ci-C6 alkyl)carboxyamido-, HC(0)NH-, H2NC(0)-, (C C6 alkyl)NHC(O)-, di(C C6 alkyl)NC(O)-, CN, hydroxyl, C C6 alkoxy, C C6 alkyl, C02H, (C C6 alkoxy)carbonyl-, (Ci-C6 alkyl)C(O)-, C6-Ci4 aryl, C C9 heteroaryl, C3-C8 cycloalkyl, C C6 haloalkyl, amino(Ci-C6 alkyl)-, (C1-C6 alkyl)carboxyl-, C1-C6 carboxyamidoalkyl-, or N02.
"Aminoalkyl-" refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with -NH2; one or both H of the NH2 may be replaced by a substituent.
"Alkylcarboxyl-" refers to an alkyl group, defined above that is attached to the parent structure through the oxygen atom of a carboxyl (C(O)-O-) functionality. Examples of (d- C6alkyl)carboxyl- include acetoxy, propionoxy, propylcarboxyl, and isopentylcarboxyl.
"(Alkyl)carboxyamido-" refers to a -NHC(O)- group in which the carbonyl carbon atom of said group is attached to a C1-C6 alkyl group, as defined above. Representative examples of a (Ci-C6 alkyl)carboxyamido- group include, but are not limited to, - NHC(0)CH3, -NHC(0)CH2CH3, -NHC(0)CH2CH2CH3, -NHC(0)CH2CH2CH2CH3, - NHC(0)CH2CH2CH2CH2CH3, -NHC(0)CH(CH3)2, -NHC(0)CH2CH(CH3)2, - NHC(0)CH(CH3)CH2CH3, -NHC(0)-C(CH3)3 and -NHC(0)CH2C(CH3)3.
"(Aryl)amino" refers to a radical of formula (aryl)-NH-, wherein aryl is as defined above. "(Aryl)oxy" refers to the group Ar-O- where Ar is an aryl group, as defined above.
"Cycloalkyl" refers to a non-aromatic, saturated, monocyclic, bicyclic or polycyclic hydrocarbon ring system. Representative examples of a C3-Ci2 cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cycloheptyl, cyclooctyl, decahydronaphthalen-l-yl, octahydro-lH-inden-2-yl, decahydro-lH-benzo[7]annulen-2-yl, and dodecahydros-indacen- 4-yl. Representative examples of a C3-Cio cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
decahydronaphthalen-l-yl, and octahydro-lH-inden-2-yl. Representative examples of a C3- Cg cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and octahydropentalen-2-yl. A cycloalkyl can be unsubstituted or substituted with one or more of halogen, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)(C C6 alkyl)N-, -N(C C3 alkyl)C(0)(C C6 alkyl), -NHC(0)(C C6 alkyl), -NHC(0)H, -C(0)NH2, -C(0)NH(Ci-C6 alkyl), -C(0)N(Ci-C6 alkyl)(Ci-C6 alkyl), CN, hydroxyl, C C6 alkoxy, Ci-C6 alkyl, -C02H, -C(0)0(C C6 alkyl), -C(0)(C C6 alkyl), C6-Ci4 aryl, C C9 heteroaryl, C3-C8 cycloalkyl, C C6 haloalkyl, C C6 aminoalkyl-, -OC(0)(C C6 alkyl), C C6 carboxyamido alkyl-, and N02. Additionally, each of any two hydrogen atoms on the same carbon atom of the carbocyclic ring can be replaced by an oxygen atom to form an oxo (=0) substituent.
"Halo" or "halogen" refers to -F, -CI, -Br and -I.
"C1-C6 Haloalkyl-" refers to a i-C alkyl group, as defined above, wherein one or more of the Ci-C6alkyl group's hydrogen atoms has been replaced with -F, -CI, -Br, or -I.
Each substitution can be independently selected from -F, -CI, -Br, or -I. Representative examples of an C1-C6 haloalkyl- group include, but are not limited to, -CH2F, -CC13, -CF3,
CH2CF3, -CH2C1, -CH2CH2Br, -CH2CH2I, -CH2CH2CH2F, -CH2CH2CH2C1, - CH2CH2CH2CH2Br, -CH2CH2 CH2CH2I, -CH2CH2CH2CH2CH2Br, -CH2CH2CH2CH2CH2I, -
CH2CH(Br)CH3, -CH2 CH(C1)CH2CH3, -CH(F)CH2CH3 and -C(CH3)2(CH2C1).
"Heteroaryl" refers to a monocyclic, bicyclic, or polycyclic aromatic ring system containing at least one ring atom selected from the heteroatoms oxygen, sulfur and nitrogen.
Examples of C1-C9 heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4- triazole, 1 -methyl- 1,2,4-triazole, lH-tetrazole, 1-methyltetrazole, benzoxazole,
benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic C1-C9 heteroaryl groups include those where a phenyl, pyridine, pyrimidine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring having one or two nitrogen atoms in the ring, one nitrogen atom together with either one oxygen or one sulfur atom in the ring, or one O or S ring atom. Examples of monocyclic C1-C4 heteroaryl groups include 2H-tetrazole, 3H-l,2,4-triazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, imidazole, and pyrrole. A heteroaryl group can be unsubstituted or substituted with one or more of C1-C6 alkyl, halogen, haloalkyl, hydroxyl, CN, hydroxyl(Ci-C6 alkyl)-, NH2, aminoalkyl-, dialkylamino-, -C02H, -C(0)0(C C6 alkyl), -OC(0)(C C6 alkyl), N-alkylamido-, - C(0)NH2, (Ci-C6 alkyl)carboxyamido-, N02, (aryl)alkyl, Ci-C6 alkoxy, C6-Ci0 aryloxyl, C2- Cio heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carboxyamido-, (aryl)alkyl-, (aryl)amino-, cycloalkenyl-,
di(alkyl)amino-, heteroaryl-, (heteroaryl)alkyl-, heterocyclyl-, heterocyclyl(alkyl)-, (HO-Cr C6 alkyl)NH-, (OH-C1-C6 alkyl)2N or a spirocyclic substituent as defined before.
"Heterocycle" or "heterocyclyl" refers to monocyclic, bicyclic and polycyclic groups in which at least one ring atom is a heteroatom. A heterocycle may be saturated or partially saturated. Exemplary Ci-Cgheterocyclyl- groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran,
dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,6-tetrahydropyridine-l- yl, tetrahydropyran, pyran, thiane, thiine, piperazine, oxazine, 5,6-dihydro-4H-l,3-oxazin-2- yl, 2,5-diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.2]octane, 3,6- diazabicyclo [3.1.1 Jheptane, 3 ,8-diazabicyclo [3.2.1] octane, 6-oxa-3 , 8- diazabicyclo[3.2. l]octane, 7-oxa-2,5-diazabicyclo[2.2.2]octane, 2,7-dioxa-5- azabicyclo[2.2.2]octane, 2-oxa-5-azabicyclo[2.2. l]heptane-5-yl, 2-oxa-5- azabicyclo[2.2.2]octane, 3,6-dioxa-8-azabicyclo[3.2.1]octane, 3-oxa-6- azabicyclo [3.1.1 Jheptane, 3-oxa-8-azabicyclo[3.2.1]octan-8-yl, 5,7-dioxa-2- azabicyclo[2.2.2]octane, 6,8-dioxa-3-azabicyclo[3.2.1]octane, 6-oxa-3- azabicyclo [3.1.1 Jheptane, 8-oxa-3-azabicyclo[3.2.1]octan-3-yl, 2-methyl-2,5- diazabicyclo[2.2.1]heptane-5-yl, l,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl, 3-hydroxy-8- azabicyclo[3.2.1]octan-8-yl-, 7-methyl-3-oxa-7,9-diazabicyclo[3.3.1]nonan-9-yl, 9-oxa-3- azabicyclo [3.3.1 ]nonan-3 -yl, 3 -oxa-9-azabicyclo [3.3.1 ]nonan-9-yl, 3 ,7-dioxa-9- azabicyclo [3.3.1 ]nonan-9-yl, 4-methyl-3 ,4-dihydro-2H- 1 ,4-benzoxazin-7 -yl, thiazine, dithiane, and dioxane. The contemplated heterocycle rings or ring systems have a minimum of 3 members. Therefore, for example, Ci heterocyclyl- radicals would include but are not limited to oxaziranyl, diaziridinyl, and diazirinyl, C2heterocyclyl- radicals include but are not limited to aziridinyl, oxiranyl, and diazetidinyl, C9heterocyclyl- radicals include but are not limited to azecanyl, tetrahydroquinolinyl, and perhydroisoquinolinyl. A heterocyclyl group can be unsubstituted or substituted with one or more of C1-C6 alkyl, halogen, haloalkyl, hydroxyl, hydroxyl(C C6 alkyl)-, -NH2, aminoalkyl-, dialkylamino-, -C02H, -C(0)0-(C C6alkyl), -OC(0)(C C6 alkyl), N-alkylamido-, -C(0)NH2, (C C6 alkyl)amido-, or N02.
"Heterocyclyl(alkyl)-" refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with a heterocycle group as defined above. Heterocyclyl(Ci-C6 alkyl)- moieties include 1-piperazinylethyl, 4- morpholinylpropyl, 6-piperazinylhexyl, and the like. A heterocyclyl(alkyl) group can be unsubstituted or substituted with one or more of halogen, NH2, (C1-C6 alkyl)amino-, di(Ci-C6 alkyl)amino-, (C C6 alkyl)C(0)N(C C3 alkyl)-, (C C6 alkyl)carboxyamido-, HC(0)NH-, H2NC(0)-, (Ci-C6 alkyl)NHC(O)-, di(C C6 alkyl)NC(0)-, CN, hydroxyl, C C6 alkoxy, C C6 alkyl, C02H, (C1-C6 alkoxy)carbonyl-, (Ci-C6 alkyl)C(0)-, 4- to 7-membered monocyclic heterocycle, C6-C14 aryl, Ci-Cgheteroaryl, or C3-C8 cycloalkyl.
"Hydroxylalkyl-" refers to a alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with hydroxyl groups. Examples of d- C6 hydroxylalkyl- moieties include, for example, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, - CH2CH(OH)CH2OH, -CH2CH(OH)CH3, -CH(CH3)CH2OH and higher homologs.
"Monocyclic heterocyclyl" refers to monocyclic groups in which at least one ring atom is a heteroatom. A heterocycle may be saturated or partially saturated. Exemplary monocyclic Ci-Cgheterocyclyl- groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3, 6-tetrahydropyridine-l-yl, tetrahydropyran, pyran, thiane, thiine, piperazine, oxazine, 5,6-dihydro-4H-l,3-oxazin-2-yl, 4-methyl-3,4- dihydro-2H-l,4-benzoxazin-7-yl, thiazine, dithiane, and dioxane. The contemplated heterocycle ring systems have a minimum of 3 members. Therefore, for example,
Ciheterocyclyl- radicals would include but are not limited to oxaziranyl, diaziridinyl, and diazirinyl, C2heterocyclyl- radicals include but are not limited to aziridinyl, oxiranyl, and diazetidinyl, Cgheterocyclyl- radicals include but are not limited to azecanyl. A heterocyclyl group can be unsubstituted or substituted with one or more of Ci-C6 alkyl, halogen, haloalkyl, hydroxyl, hydroxyl(Ci-C6 alkyl)-, NH2, aminoalkyl-, dialkylamino-, -CO2H, - C(0)0(Ci-C6 alkyl), -OC(0)(Ci-C6 alkyl), N-alkylamido-, -C(0)NH2, (C C6 alkyl)amido-, or N02.
"Perfluoroalkyl-" refers to alkyl group, defined above, having two or more fluorine atoms. Examples of a Ci-C6perfluoroalkyl- group include CF3, CH2CF3, CF2CF3 and CH(CF3)2.
The term "optionally substituted" as used herein means that at least one hydrogen atom of the optionally substituted group has been substituted with one or more of halogen, NH2, -NH(Ci-C6 alkyl), -N(C C6 alkyl)(C C6 alkyl), -N(Ci-C3alkyl)C(0)(Ci-C6 alkyl), - NHC(0)(C C6alkyl), -NHC(0)H, -C(0)NH2, -C(0)NH(C C6 alkyl), -C(0)N(C C6 alkyl)(C C6 alkyl), CN , hydroxyl, C C6 alkoxy, d-Ce alkyl, -C02H, -C(0)0(C C6 alkyl), - C(0)(Ci-C6 alkyl), C6-Ci4 aryl, C C9 heteroaryl, or C3-C8 cycloalkyl.
The aryl and the heteroaryl moieties are optionally substituted with optionally substituted C1-C6 alkyl, C1-C6 alkyl-amino, -O-C1-C6 alkyl, -O-optionally substituted C6-C10 aryl, -O-optionally substituted C1-C6 heteroaryl, -O-optionally substituted C3-C6 heterocyclyl, -mono and dialkyl amino, halogen, CN, -C02H, -COO-C1-C6 alkyl, -CONH2j - CO-dialkylamino, -S02NH2j -optionally substituted C6-C10 aryl, -optionally substituted d- C10 heteroaryl, -optionally substituted C2-C6 heterocyclyl, or -O-C1-C6 alkyl-amino.
The drug or prodrug which is delivered inside the cells via a conjugate according to formula (I) may be selected with a view to the desired physiologic effect, e.g., therapeutic, cytotoxic, immunomodulatory, or the like, and the disease or condition which is being treated.
The term "cytotoxic" means toxic to cells or a selected cell population (e.g., cancer cells). The toxic effect may result in cell death and/or lysis. In certain instances, the toxic effect may be a sublethal destructive effect on the cell, e.g., slowing or arresting cell growth.
In order to achieve a cytotoxic effect, the drug or prodrug may be selected from a group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, amongst others.
As used herein, the term "drug" refers to an amino acid-based molecule or a small molecule chemical compound which is biologically active and provides a desired physiological effect following administration to a subject in need thereof (e.g., an active pharmaceutical ingredient). The term "prodrug" refers to a precursor of an active drug, that is, a compound that can be transformed to an active drug. Typically such a prodrug is subject to processing in vivo, which converts the drug to a physiologically active form. In some instances, a prodrug may itself have a desired physiologic effect.
In one embodiment, when present as a part of the conjugate, the drug or prodrug (Y-H) is bound to the conjugate in the form of a drug fragment or a prodrug fragment (Y). For example, as described above with reference to formula (I), in embodiment (i) wherein X is -OC(0)Y, the reaction continues, to eliminate CO2 and the drug Y-H. In embodiment (ii) wherein X is -N(H)C(0)Y, the reaction continues, to eliminate HNCO and the drug Y-H. In embodiment (iii) wherein X is a drug or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug or prodrug, the drug Y-H is formed as indicated in the scheme below. In embodiment (iv) where X is a drug or prodrug fragment Y bound to the a carbon via a -NH- or -NR4- which is part of the drug or prodrug, the drug Y-H is formed
Figure imgf000031_0001
As described above, suitable drug or prodrug may be selected from amongst DNA damaging agents such as DNA alkylating agents and DNA strand breaking agents (e.g., calicheamicin, duocarmycins) and microtubule disrupting agents, such as microtubule depolymerizing agents (e.g., auristatins, maytansinoids) and microtubule polymerizing agents (e.g., taxanes). Still other suitable drugs or prodrugs will be apparent to one of skill in the art in view of the information provided herein.
For example, suitable DNA damaging drugs or prodrugs may include DNA minor groove binders such as duocarmycins and calicheamicins. Duocarmycins includes duocarmycin SA and related analogs and prodrug forms, such as the
cyclopropa[c]benzo[e]indol-4-one (CBI) analog CBI-DMMI and the pro-drug form Pro-CBI- DMMI, and related compounds identified herein. Calicheamicins includes related enediynes, e.g., esperamicin. Still other drugs or prodrugs which function as DNA damaging drugs or prodrugs includes lexitropsins.
The drug or prodrug selected may be an anti-tubulin agent. Examples of anti-tubulin agents include, auristatins, taxanes and vinca alkyloids. Auristatins includes dolastatin and other cytotoxic or cytostatic agents of the auristatin class, such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and auristatin F phenylenediamine (AFP). Taxanes includes, for example, paclitaxel, docetaxel, tesetaxel and cabazitaxel. Vinca alkaloids includes, e.g., vincristine, vinblastine, vindesine, and vinorelbine. Other antitubulin agents include, for example, tubulysins, T67 (Tularik), baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine,
cryptophycins, cemadotin, combretastatins, discodermolide, and eleutherobin. Another group of anti -tubulin agents includes maytansinoids; maytansinoids includes maytansine, DM-1 and DM-4 (ImmunoGen).
Still other classes of compounds or compounds with these or other cytotoxic modes of action may be selected, including, e.g., mitomycin C, mitomycin A, daunorubicin, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, aminopterin, bleomycin, 9-amino camptothecin, l-(chloromethyl)-2,3-dihydro-lH-benzo[e]indol-5-ol, pyrrolobenzodiazepine (PBD) polyamide and dimers thereof. Other suitable cytotoxic agents include, for example, puromycins, CC-1065, SN-38, topotecan, rhizoxin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, discodermolide, eleutherobin, and mitoxantrone.
With reference to formula (I), these drugs or prodrugs may contain amine groups or hydroxy groups which form the point of attachment to X as defined in formula (I).
Examples of amine-containing drugs include, e.g., monomethylauristatin E (MMAE), monomethyl Auristatin F (MMAF), epirubicin, doxorubicin, or the duocarmycin analog AS- 1-145. Examples of hydroxy-containing drugs or prodrugs include, e.g., pro-duocarmycin SA, duocarmycin C2, carelsin, epirubin or doxorubicin.
In one example, the drug fragment or prodrug fragment is a DNA damaging agent that comprises a minor groove binder, which is an optionally-substituted lH-indole-2-carbonyl group incorporating a spiroheterocyclic amine, which is bound to a duocarmycin analog fragment such as CBI or Pro-CBI. The minor groove binder non-covalently binds in the minor groove of double-stranded DNA, and the duocarmycin analog fragment such as CBI is understood by those in the art to covalently bind to DNA, thereby damaging the DNA. In one embodiment, the DNA damaging agent has the structure of formula (III) or (IV), below: N—
Groove Binder
Figure imgf000033_0001
The term "minor groove binder" (MGB) is a fragment that binds to and/or within the minor groove of double stranded deoxyribonucleic acid (DNA). Examples of suitable minor groove binders include the following compounds (A)-(L) below:
Figure imgf000033_0002
(L) In one embodi
Figure imgf000034_0001
The minor groove binders (B) to (L) and their related carboxylic acids were heretofore unknown, and can be used in a variety of indications, in addition to the conjugates of formula (I). Methods for generating these compounds are provided in the synthetic schemes described herein.
In still another embodiment, the drug fragment or prodrug fragment is a DNA damaging agent having the structure of formula (III) or (IV), wherein the minor groove binder has the following structure:
Figure imgf000034_0002
(M)
wherein Q is N(CH3)2 or is selected from the following groups:
Figure imgf000034_0003
Figure imgf000035_0001
i— N COOH HN^^< NH
X
(M17)
M16) (M18)
Figure imgf000035_0002
These spirocyclic amines are commercially available, e.g., from commercial vendors such as Synthonix Inc, 2713 Connector Drive, Wake Forest, NC 27587, or they can be readily synthesized by methods known to those skilled in the art.
In still another example, the drug is selected from the group consisting of maytansine, which is characterized by the structure of formula (V):
Figure imgf000035_0003
(V) In a further example, the drug is monomethylauristatin F (VI) or monomethylauristatin E (VII):
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000038_0001
As used herein, a "ligand" is any molecule that specifically binds or complexes with (herein also referred to as "targeting") a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. In one embodiment, following specific binding or complexing of the ligand with its receptor, the cell is permissive for uptake of the ligand or ligand-drug-conjugate, which is then internalized into the cell.
As used herein, a ligand that "specifically binds or complexes with" or "targets" a cell surface molecule means a ligand (which can be part of a conjugate of the invention) that preferentially associates with a cell surface molecule via intermolecular forces. For example, the ligand can preferentially associate with the cell surface molecule with a Kd of less than about 50 nM, less than about 5 nM, or less than 500 pM. Techniques for measuring binding affinity of a ligand to a cell surface molecule are well-known; for example, one suitable technique, namely surface plasmon resonance (SPR), is described in Example 37.
Without wishing to be bound by theory and with reference to formula (I), it is believed that following uptake by the cell, the fragment of formula I characterized by the
Figure imgf000039_0001
structure: , serves as a substrate for one or more enzymes, e.g., lysosomal proteases. Following cleavage of formula I at the amide between the terminal carbonyl moiety of L and the -NH- of the 4-amino-phenyl moiety or the related amino- heterocyclic moiety, the drug fragment or prodrug fragment Y is released as the drug or prodrug Y-H by a cascade of chemical rearrangements or reactions. Suitably, this cleavage occurs intracellular ly following uptake by the cells of the targeted cell population.
In one embodiment, the ligand is used for targeting and has no detectable therapeutic effect as separate from the drug which it delivers. In another embodiment, the ligand functions both as a targeting moiety and as a therapeutic or immunomodulatory agent (e.g., to enhance the activity of the active drug or prodrug).
With reference to the structure of formula (I), p" is 1 to 6. Thus, a single ligand may be bound to multiple drug-linker fragments, i.e., 2, 3, 4, 5 or 6 drug-linker fragments. Where a single ligand-drug-conjugate contains multiple drug-linker fragments, the drug-linker fragments may be the same or they may be different from one another. In one embodiment, a single ligand-drug-conjugate contains 2 drug-linker fragments.
The ligand may be a polypeptide or protein.
In one example, the ligand is a cytokine. One suitable class of cytokines includes interleukins. Examples of suitable interleukins (IL) include, e.g., IL2, IL6 and IL-12. In another example, the ligand is an immunoglobulin.
As used herein, an immunoglobulin may be a full-length antibody or a functional fragment of an antibody. By "functional fragment of an antibody", it is meant herein that a sufficient portion of an antibody is provided that the immunoglobulin effectively binds or complexes with the cell surface molecule for its target cell population. An immunoglobulin may be purified, generated recombinantly, generated synthetically, or combinations thereof, using techniques known to those of skill in the art. While immunoglobulins within or derived from IgG antibodies are particularly well-suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g., IgG, IgA, IgM, IgD and IgE. Human or humanized immunoglobulins are well-suited as sources of the immunoglobulins. However, immunoglobulins from other mammalian origins, e.g., murine or other rodent, or rabbit origin, may be selected. Examples of suitable immunoglobulins include, without limitation, a monoclonal antibody, a chimeric antibody, a humanized antibody, an immunoadhesin, and a truncated version of any of the immunoglobulins including, e.g., truncated versions such as F(Ab)2, Minibody, FAb, Single-domain Ab, scFv, tandem/bis-scFv, F(ab)3, scFv-Fc (or scFvFc), IgG CH, dsFv, diabody, triabody, or tetrabody.
As used herein, the abbreviation Ab indicates antibody; bis-scFv indicates bispecific scFv; dsFc indicates disulfite Fc; Fab indicates antigen-binding fraction of immunoglobulin; Fc indicates crystallizable fraction of immunoglobulins; Fv indicates variable fragments of immunoglobulin; IgG indicates immunoglobulin G; and scFv indicates single-chain Fv.
Examples of suitable single chain Fv-Fc antibody (scFv-Fc, or scFvFc) or a functional Fab' or F(ab')2 fragment are identified below in the experimental section. Such antibodies are not a limitation on the present invention.
When the drug moiety is a cytotoxic payload, the immunoglobulin selected may be one that can recognize a tumor associated antigen. These immunoglobulins can be derived from human, murine or rabbit in origin. Some suitable monoclonal antibodies include BR96 mAb (Trail, P.A., et al., "Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates", Science, 1993, 261, 212-215); (mAb against the Her2neu antigen such as Herceptin in advanced Breast cancer" Cancer Treat Rev. 26, 287-90, 2000); mAbs against CD40 antigen, such as S2C6 mAb (Francisco, J. A., et al, "Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14" Cancer Res. 2000, 60, 3225-3231); mAbs against the CD30 antigens such as AC 10 mAb (Bowen, M.A., et al, " Functional Effects of CD30 on a large granular lymphoma cell line YT" J. Immunol., 151, 5896-5906, 1993); mAbs against CD27 antigen, such as CD70 (Lens, S.M., et al , "Aberrant expression and reverse signaling of CD70 on malignant B cells", Br. J. Haematology, 1996, 106(2), 491-503; Franke, A.E., et al., "Cell surface receptor targeted therapy of acute myeloid leukemia: a review", Cancer Biother. Radiopharm. 2000, 27, 64-70. Breitling, F., et al, S. Recombinant Antibodies, John Wiley and Sons, NY 1998). Examples of suitable immunoglobulins include, without limitation, trastuzamab; panitumumab; brentuximab; gemtuzumab; and inotuzumab. Examples of other immunoglobulins include the anti-5T4 antibody and the anti-HER-1 and anti-HER2 antibodies described herein.
One desirable anti-5T4 antibody is disclosed in US Provisional Application Number 61/835,858, filed June 17, 2013, herein incorporated by reference. As used herein, the term "5T4 antigen-binding portion" refers to a polypeptide sequence capable of selectively binding to a 5T4 antigen. In exemplary antibody-drug conjugate molecules, the 5T4 antigen-binding portion generally comprises a single chain scFv-Fc form engineered from an anti-5T4 antibody. A single-chain variable fragment (scFv-Fc) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a linker peptide, and further connected to an Fc region comprising a hinge region and CH2 and CH3 regions of an antibody (any such combinations of antibody portions with each other or with other peptide sequences is sometimes referred to herein as an "immunofusion" molecule). Within such a scFvFc molecule, the scFv section may be C-terminally linked to the N-terminus of the Fc section by a linker peptide.
At least a portion of the 5T4 antigen-binding portion of the immunofusion molecules may originate from a murine source. For example, one may obtain an immunofusion molecule by expressing a polynucleotide engineered to encode at least a murine anti-5T4 scFv region having the polypeptide sequence according to SEQ ID NO: A. Additionally, at least a portion of the 5T4-antigen binding portion may be generated to be chimeric or humanized according to well-known methods. See, Borras et al., J. Biol. Chem. 2010 Mar 19;285(12):9054-66. Thus, one may obtain an immunofusion molecule having a 5T4- antigen binding portion with a humanized scFv portion by expressing a polynucleotide engineered to encode at least the polypeptide sequence according to SEQ ID NO: B.
In some examples, the Fv portion of the 5T4 antigen-binding portion may be engineered by well-known molecular biology techniques to comprise one or more amino acid substitutions in the VH region. The Fc portion of the 5T4 antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge, CH2 and CH3 regions of an anti-5T4 antibody. For example, it is possible to engineer a
polynucleotide to encode at least an Fc portion having the polypeptide sequence according to SEQ ID NO: C.
A polynucleotide encoding a peptide wherein the single chain Fv and Fc regions are linked together may encode at least a chimeric 5T4 antigen-binding portion of an antibody- drug conjugate molecule having the polypeptide sequence according to SEQ ID NO: D or may encode a humanized 5T4 antigen-binding portion having the polypeptide sequence according to SEQ ID NOs: E or F.
A polypeptide linker, such as one having the polypeptide sequence ASTC (SEQ ID NO: Y) or ASTX (SEQ ID NO: Z) (where "X" refers to any amino acid or a direct peptide bond between the adjacent amino acids), may fuse the C-terminus of ScFv portion to the N- terminus of the Fc portion of the 5T4 antigen-binding portion. Thus, it is possible to engineer a polynucleotide to encode at least a linker having the polypeptide sequence according to SEQ ID NOs: Y or Z. While either SEQ ID NOs: Y or Z may be used as a linker, an immunofusion molecule having a peptide linker according to SEQ ID NO: Y benefits from site-specific conjugation due to the presence of the cysteine residue.
Preferably, any amino acid substitution, insertion, or deletion or use of a peptidomimetic does not substantially reduce the affinity or specificity of the 5T4 antigen- binding portion. An immunofusion molecule having an amino acid substitution, insertion, or deletion or a peptidomimetic in the 5T4 antigen-binding portion preferably retains greater than 75%, preferably greater than 80%, preferably greater than 85%, preferably greater than 90%, or preferably greater than 95% of affinity or specificity for binding the 5T4 antigen compared to an antibody-drug conjugate molecule with an unmodified 5T4-antigen binding portion.
These antibodies may be produced recombinantly, synthetically, or by other suitable method known in the art. Such methods and constructs utilize the nucleic acid sequences encoding the polypeptides and peptide sequences identified herein. Alternatively, such methods and constructs for antibody production utilize sequences which are naturally or artificially modified, e.g., natural variants or codon optimized variants of the SEQ ID NOs provided herein (e.g., A). A variety of codon optimization schema are known in the art. See, e.g., UpGene™ and Optimizer™, which are web-based optimization methods.
Additionally, a number of commercial institutions perform codon optimization using proprietary schema, e.g., SignGen Laboratories, DNA2.0, OpenX, amongst others.
As used herein, the term "anti-HERl antigen-binding portion" refers to a immunoglobulin polypeptide sequence capable of selectively binding to a HERl antigen. In exemplary antibody-drug conjugate molecules, the HERl antigen-binding portion generally comprises a single chain scFv-Fc form engineered from an anti-HERl antibody. A single- chain variable fragment (scFvFc) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a linker peptide, and further connected to an Fc region comprising a hinge region and CH2 and CH3 regions of an IgG antibody. Within such a scFvFc molecule, the scFv section may be C-terminally linked to the N-terminus of the Fc section by a linker peptide.
At least a portion of the HERl antigen-binding immunofusion molecules may originate from a murine source. For example, one may obtain an immunofusion molecule by expressing a polynucleotide engineered to encode at least a murine anti-HERl scFv region having the polypeptide sequence according to SEQ ID NO: AB2 or AC2. Additionally, at least a portion of the HERl-antigen binding portion may be generated to be humanized or fully human according to well-known methods. See, Borras et al., J. Biol. Chem. 2010 Mar 19;285(12):9054-66. Thus, one may obtain an immunofusion molecule having a HERl- antigen binding portion with a humanized or fully human scFv portion by expressing a polynucleotide engineered to encode at least the polypeptide sequence according to SEQ ID NO: AB2 or AC2, respectively.
In some examples, the Fv portion of the HERl antigen-binding portion may be engineered by well-known molecular biology techniques to comprise one or more amino acid substitutions in the VH region. The Fc portion of the HERl antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge, CH2 and CH3 regions of an anti-HERl antibody. For, example, it is possible to engineer a polynucleotide to encode at least an Fc portion having the polypeptide sequence according to SEQ ID NO: C.
A polynucleotide encoding a peptide wherein the single chain Fv and Fc regions are linked together may encode at least a chimeric HERl antigen-binding portion of an antibody-drug conjugate molecule having the polypeptide sequence according to SEQ ID NO: AB2 or AC2 or may encode a humanized HERl antigen-binding portion having the polypeptide sequence according to SEQ ID NOs: AB1 or AC1, respectively.
A polypeptide linker, such as one having the polypeptide sequence ASTC (SEQ ID NO: Y) or ASTX (SEQ ID NO: Z) (where "X" refers to any amino acid or a direct peptide bond between the adjacent amino acids), may fuse the C -terminus of scFv portion to the N- terminus of the Fc portion of the HERl antigen-binding portion. Thus, it is possible to engineer a polynucleotide to encode at least a linker having the polypeptide sequence according to SEQ ID NOs: Y or Z. While either SEQ ID NOs: Y or Z may be used as a linker, an immunofusion molecule having a peptide linker according to SEQ ID NO: Y benefits from the presence of a cysteine residue that can be preferentially conjugated in a site-specific manner.
Preferably, any amino acid substitution, insertion, or deletion or use of a peptidomimetic does not substantially reduce the affinity or specificity of the HERl antigen- binding portion. An immunofusion molecule having an amino acid substitution, insertion, or deletion or a peptidomimetic in the HERl antigen-binding portion preferably retains greater than 75%, preferably greater than 80%, preferably greater than 85%, preferably greater than 90%, or preferably greater than 95% of affinity and specificity for binding the HERl antigen compared to an antibody-drug conjugate molecule with an unmodified HERl -antigen binding portion.
These antibodies may be produced recombinantly, synthetically, or by other suitable method known in the art. Such methods and constructs utilize the nucleic acid sequences encoding the polypeptides and peptide sequences identified herein. Alternatively, such methods and constructs for antibody production utilize sequences which are naturally or artificially modified, e.g., natural variants or codon optimized variants of the SEQ ID NOs provided herein (e.g., AB 1). A variety of codon optimization schema are known in the art. See, e.g., UpGene™ and Optimizer™, which are web-based optimization methods.
Additionally, a number of commercial institutions perform codon optimization using proprietary schema, e.g., SignGen Laboratories, DNA2.0, OpenX, amongst others.
As used herein, the term "anti-HER2 antigen-binding portion" refers to a polypeptide sequence capable of selectively binding to a HER2 antigen. In exemplary antibody-drug conjugate molecules, the HER2 antigen-binding portion generally comprises a single chain scFv-Fc form engineered from an anti-HER2 antibody. A single-chain variable fragment (scFvFc) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a linker peptide, and further connected to an Fc region comprising a hinge region and CH2 and CH3 regions of an antibody. Within such a scFvFc molecule, the scFv section may be C-terminally linked to the N-terminus of the Fc section by a linker peptide.
At least a portion of the HER2 antigen-binding portion of the immunofusion molecules may originate from a murine source. For example, one may obtain an immunofusion molecule by expressing a polynucleotide engineered to encode at least a murine anti-HER2 scFv region having the polypeptide sequence according to SEQ ID NO: AA2. Additionally, at least a portion of the HER2-antigen binding portion may be generated to be humanized according to well-known methods. See, Borras et al., J. Biol. Chem. 2010 Mar 19;285(12):9054-66. Thus, one may obtain an immunofusion molecule having a HER2- antigen binding portion with a humanized or fully human scFv portion by expressing a polynucleotide engineered to encode at least the polypeptide sequence according to SEQ ID NO: AA1.
In some examples, the Fv portion of the HER2 antigen-binding immunofusion may be engineered by well-known molecular biology techniques to comprise one or more amino acid substitutions in the VH region. The Fc portion of the HER2 antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge, CH2 and CH3 regions of an anti-HER2 antibody. For, example, it is possible to engineer a polynucleotide to encode at least an Fc portion having the polypeptide sequence according to SEQ ID NO: AA1.
A polynucleotide encoding a peptide wherein the single chain Fv and Fc regions are linked together may encode at least a humanized HER2 antigen-binding portion of an antibody-drug conjugate molecule having the polypeptide sequence according to SEQ ID NO: AA1 or may encode a fully human HER2 antigen-binding portion having the polypeptide sequence.
A polypeptide linker, such as one having the polypeptide sequence ASTC (SEQ ID
NO: Y) or ASTX (SEQ ID NO: Z) (where "X" refers to any amino acid or a direct peptide bond between the adjacent amino acids), may fuse the C-terminus of ScFv portion to the N- terminus of the Fc portion of the HER2 antigen-binding portion. Thus, it is possible to engineer a polynucleotide to encode at least a linker having the polypeptide sequence according to SEQ ID NOs: Y or Z. While either SEQ ID NOs: Y or Z may be used as a linker, an immunofusion molecule having a peptide linker according to SEQ ID NO: Y benefits from site-specific conjugation due to the presence of the cysteine residue.
Preferably, any amino acid substitution, insertion, or deletion or use of a peptidomimetic does not substantially reduce the affinity or specificity of the HER2 antigen- binding portion. An immunofusion molecule having an amino acid substitution, insertion, or deletion or a peptidomimetic in the HER2 antigen-binding portion preferably retains greater than 75%, preferably greater than 80%, preferably greater than 85%, preferably greater than 90%, or preferably greater than 95% of affinity or specificity for binding the HER2 antigen compared to an antibody-drug conjugate molecule with an unmodified HER1 -antigen binding portion.
These antibodies may be produced recombinantly, synthetically, or by other suitable method known in the art. Such methods and constructs utilize the nucleic acid sequences encoding the polypeptides and peptide sequences identified herein. Alternatively, such methods and constructs for antibody production utilize sequences which are naturally or artificially modified, e.g., natural variants or codon optimized variants of the SEQ ID NOs provided herein (e.g., AA1). A variety of codon optimization schema are known in the art. See, e.g., UpGene™ and Optimizer™, which are web-based optimization methods.
Additionally, a number of commercial institutions perform codon optimization using proprietary schema, e.g., SignGen Laboratories, DNA2.0, OpenX, amongst others. These and other immunoglobulins, cytokines, or cell surface molecule binding agent may be selected for use as targeting moieties in the conjugates described herein.
The targeting ligands, the PAMA-based self-immolative linkers and the drug or prodrug fragments described herein can be assembled into the therapeutic drug and targeting conjugate of the invention, for example according to the disclosed techniques and methods. Thus, there is provided a method for producing a therapeutic drug and targeting conjugate, comprising providing a cleavable para-amino mandelic acid (PAMA) derived linker having the structure IX:
Figure imgf000046_0001
(IX)
In this structure, L is a di-, tri- or terra- amino acid chain. Z is an optional amine blocking group. X is a conjugatable group, such as OH or NH2. Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2. R2 is H, C C6 alkyl, C C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano. A ligand is then provided, and one or more cleavable para-amino mandelic acid (PAMA) derived linkers can be conjugated to the ligand through R1 as described in more detail below. For example, R1 is a conjugatable group, such as a group having a formula selected from -CONR6CHR7CH2(OCH2CH2)nOCH2CHR8-, C C6 alkyl-, or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(Ci-C3 alkyl)2; R8 is H, Ci-C3 alkyl or Ci-C3 hydroxyalkyl. A drug or prodrug fragment is also provided, and is conjugated to each of the PAMA derived linkers via the X group as described below. For example, once conjugated, and in the context of the full therapeutic and targeting conjugate of the invention, X can be (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- which is the point of attachment to -OC(O)-, (ii) X is -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- or -O- as the point of attachment to -N(H)C(0)-; (iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or (iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR4- which is part of the drug fragment or prodrug fragment. The order of conjugation is not necessarily fixed; for example, the PAMA derived linker can first be conjugated to a ligand, and then to a drug or prodrug fragment, or the order of conjugation can be reversed. Suitable conjugation schemes are discussed herein and certain embodiments are presented in the Examples.
It is also a specific object of the invention to provide a conjugatable para-amino mandelic acid derived linker of Formula IX, where the X and R1 moieties are conjugatable groups as detailed above.
Therapeutic and targeting conjugates of the invention, and methods for producing them, are described below by way of non-limiting example.
With reference to the structure of formula (I) above within the brackets, in one example more than one thiol or other suitable moiety such as at least one amine moiety of a single LG serves as point of attachment to a B moiety of a drug-linker fragment. In other words, a single ligand may have multiple linker conjugate fragments bound thereto through moiety B. Thus, particularly suitable ligands (LG) are characterized by containing one or more thiol moieties or one or more amine moieties in their structure that are suitable for conjugation. At least one of these thiol or amine moieties serves as a point of attachment to B. Typically, the thiol moiety is the point of attachment, with the attachment achieved via reaction with a maleimide or haloacetamide moiety in the PAMA linker-drug compound to the succinimide moiety or acetamide moiety of the LG to B, which is characterized by the following fragment:
Figure imgf000047_0001
Where the ligand does not natively contain an available thiol moiety that is suitable for conjugation, or where disruption of a native thiol moiety would disrupt the desired targeting function of the ligand, a ligand may be modified or engineered to contain at least one thiol moiety suitable for conjugation.
For example, a ligand (e.g., an immunoglobulin) may be engineered to present a Cys residue for site-specific conjugation. Where it is desired to limit the number of drug moieties conjugated to a ligand via a B-fragment of the conjugate, the number of available sulfide bonds available for the conjugation reaction may be controlled. In one aspect, a reduction reaction is utilized to disrupt any possible adducts of the engineered cysteine (Cys) residue with glutathione or cysteines. For example, prior to the conjugation reaction, the ligand is reduced using dithiothreitol (DTT) in PBS (phosphate buffered saline; 20 mM sodium phosphate, pH 7.2, 150 mM NaCl) at 1 mg/mL to 10 mg/mL protein concentration, preferably about 3 mg/mL protein concentration, for 15 to 90 minutes or preferably about 45 min. Following reduction, the ligand is then subjected to desalting to remove DTT by using a desalting column, for example in 50 mM Tris-Cl, 150 mM NaCl, 250 mM Arginine (pH 8.2) using a Hi-Prep 26/10 Desalting Column (GE healthcare). Buffer-exchanged ligand is then concentrated and stirred slowly for 15 to 60 minutes, preferably for 30 min, to allow oxidative regeneration of the native interchain disulfide bonds. The ligand is then incubated in the presence of the maleimide or haloacetamide linker at 5 to 50 fold molar excess, more preferably at about 15 fold molar excess, for 10 min to 6 h and preferably for about 90 min with slow stirring for conjugation to occur. The conjugated linker - immunoglobulin is desalted to 20 mM PBS containing 10% glycerol. However, the preceding is exemplary, and other suitable conditions and reagent may be selected which allow a Cys residue to be presented for conjugation.
Characterization of the conjugate is carried out by using a variety of analytical methods known to those skilled in the art. For example, LC-ESI-MS analysis of the reduced conjugate (i.e., analysis of the monomer unit) permits determination of the relative amounts of ligand having 0, 1 or more linker-drug groups attached.
In still another embodiment, one or more lysine (Lys) residues in the ligand LG may be employed for conjugation. Methods for conjugation to lysine are well-known to those skilled in the art, e.g., M.P. Brun and L. Gauzy-Lazo, Methods in Molecular Biology 2013, 1045: 173-187; L.R. Milgrom, M.P. Deonarain, Innovations in Pharmaceutical Technology 2011, 38:56-59; W.C. Widdison et al., J. Med. Chem. 2006, 49:4392-4408. Where the ligand LG does not natively contain an available amine moiety that is suitable for conjugation, or where disruption of a native amine moiety would disrupt the desired targeting function of the ligand, a ligand may be modified or engineered to contain at least one amine moiety suitable for conjugation.
The product resulting from the ligand conjugation reaction may be filtered and purified using known techniques. In one embodiment, filtration removes any agglomerated conjugates and allows separation and isolation of non-agglomerated conjugates. The reaction product may be a mixture of conjugates, in which p" is 1, 2, 3, 4, 5 or 6. In one embodiment, a composition of the invention contains conjugates in which the average p" is 2. Such a composition may additionally contain one or more conjugates in which p" is 1, 3, or 4. Minor amounts of conjugates in which p" is 5 or 6 may be present. Still other compositions will contain a mixture of conjugates in which p" is 1 or 2. Such a mixture may contain approximately equivalent amounts of p" is 1 and p" is 2. Alternatively, such a mixture may contain more than 50% of p" is 2. In still another alternative, such a mixture may contain more than 50% of p" is 1. Still other compositions will contain a mixture of conjugates in with p" is 1, 2 or 3.
The conjugates of formula (I) as described herein may contain any combination of ligand and drug/prodrug with the self-immolative linker of formula (IX) and defined herein. Illustrative conjugates of formula (I) include, without limitation,
Figure imgf000049_0001
Figure imgf000050_0001

Figure imgf000051_0001

Figure imgf000052_0001
wherein LG is ligand, such as an immunoglobulin protein or polypeptide which specifically targets a cell surface antigen and wherein LG contains at least or at least one amine, or at least one thiol group that forms a point of attachment to the succinimide moiety or the acetamide moiety of B; n is 1, 2 or 3; and p" is 1, 2, 3 or 4. In one embodiment, these structures have p" is 2. In still another embodiment, a composition of the invention contains conjugates which have an average p is 2.
Still other conjugates according to formula (I) are illustrated in the table below, the examples below, and/or or will be apparent to one of skill in the art based upon the description provided herein.
Methods useful for making the compounds discussed herein are set forth in the Examples below and generalized in Schemes 1 to 28. One of skill in the art will recognize that Schemes 1-28 can be adapted to produce the compounds of the invention and pharmaceutically accepted salts thereof according to the present invention. In the reactions described, reactive functional groups, such as hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, may be protected to avoid unwanted reactions. Conventional protecting groups may be used in accordance with standard practice.
The materials needed to synthesize and characterize PAMA-based linkers and conjugates of the invention were prepared by the procedures outlined in Schemes 1 to 28. The cytotoxic payloads 56, 59, 65, 71, 91, 92, 93, and 94 were prepared by reacting the Pro- CBI 2 part of the molecule with the appropriately substituted minor groove binders. Scheme 1
Figure imgf000053_0001
11 N-Boc-CBI (12) Pro-CBI (2)
Scheme 1 provides the preparation of intermediate compound 6 and Pro-CBI- 2 reagent starting from 2,4-dihydroxy naphthalene. 1 ,3-Dihydroxy naphthalene 4 and diphenyl methylamine were heated at elevated temperatures. In one embodiment, the reaction was performed in an aromatic solvent such as toluene, xylenes or 1 ,2-dichlorobenzene. In another embodiment, the reaction was performed at about 100 to about 150°C. In a further embodiment, the reaction was performed in a sealed tube. The product was then reacted with an excess of di-tert-butyl dicarbonate and palladium hydroxide to provide compound 6. In one embodiment, the reaction was performed in a parr shaker or an autoclave. In another embodiment, the reaction was performed at about 60 psi of hydrogen pressure. In a further embodiment, the reaction was performed with about 3 equivalents of di-tert-butyl dicarbonate. In yet another embodiment, the reaction was performed in dioxane: water or THF:water. To compound 6 was successively added benzyl bromide or p-methoxybenzyl bromide, potassium carbonate or in the presence of an inorganic base and tetrabutyl ammonium iodide to provide compound 7. In one embodiment, the reaction was performed in anhydrous DMF or acetone. In another embodiment, the reaction was performed at RT. Under an inert atmosphere and at reduced temperatures, compound 7 was reacted with p- toluenesulfonic acid. In one embodiment, the reaction was performed in THF or dioxane. In another embodiment, the reaction was performed at about -20 to about 0°C. N- iodosuccinimide was then added. In one embodiment, the addition was performed drop wise. After the reaction was complete, the mixture warmed to RT to provide product 8. Compound 8 was reacted with a strong base at reduced temperatures. In one embodiment, the strong base was 60% sodium hydride or potassium hydride. In another embodiment, the reaction was performed at about 0°C. In a further embodiment, the reaction was performed in DMF. To this solution was then added (s)-(+)-glycidal nosylate at the reduced temperature to yield product 9. To compound 9 was added ethyl magnesium bromide to provide compound 10. In one embodiment, the reaction was performed in an ether such as THF. Compound 10 was reacted with ammonium formate and a catalyst at elevated temperatures to afford product 11. In one embodiment, the reaction was performed in a mixture of THF and MeOH or dioxane and methanol mixture. In another embodiment, the catalyst was 10% Pd/C. In a further embodiment, the elevated temperatures were about 70 to about 100°C. To compound 11 was added 1,1 -(azodicarbonyl)dipiperidine followed by tributyl phosphine to provide compound 12 (N-Boc-CBI). In one embodiment, the reaction was performed in toluene. See, Lajiness and Boger, J. Org. Chem. 2011, 76, 583-587, which is herein incorporated by reference. Compound 12 was treated with HC1 in ethyl acetate or in diethyl ether at reduced temperatures to provide compound 2 as the HC1 salt (Pro-CBI). In one embodiment, the reaction was performed at about -78°C to -10°C.
Figure imgf000054_0001
Figure imgf000054_0002
37 DMMI (3)
Scheme 2 describes a method for the synthesis of compound 3 (DMMI). 3-Hydroxy- 4-methoxybenzaldehyde 5 was reacted with an inorganic base such as potassium carbonate or 1,2-dichloroethane or acetone at elevated temperatures to yield compound 35. In one embodiment, the elevated temperature was about 70°C. In another embodiment, the reaction was performed in DMF or acetone. Methyl chloroacetate and sodium azide were reacted to provide methyl azedoacetate. In one embodiment, the reaction was performed using DMSO or THF. Methyl azedoacetate was then reacted with aldehyde 35 to yield 36. In one embodiment, the reaction was performed in methanol or ethanol. After the mixture was cooled, sodium methoxide was added to give product 36. In one embodiment, the reaction was cooled to about -30°C. Azido 36 was then heated to elevated temperatures to yield compound 37. In one embodiment, the reaction was performed in high boiling aromatic solvents such as toluene or xylenes. In another embodiment, the reaction was performed at reflux. Compound 37 was then reacted with an amine in the presence of sodium carbonate or potassium carbonate at elevated temperatures. In one embodiment, the reaction was performed in water or ethanol: water mixture. In another embodiment, the amine was dimethyl amine or a spirocyclic amine such as 52, 53, 66, 75, 82, 79, 81 or any spirocyclic amine of general term Q. In a further embodiment, the amine was 40% aqueous dimethyl amine solution. In one embodiment, the elevated temperature was about 100°C. The product was then acidified to provide 3 (DMMI). In one embodiment, the acidification was performed in water. In another embodiment, the acidification was performed with an acid such as HCI or acetic acid.
Figure imgf000055_0001
Coupling of 2 (Pro-CBI) and 3 (DMMI) was accomplished by an EDC coupling reaction to provide 1 (Pro CBI-DMMI) (L. F. Tietze et al. (2008), Chem. Med. Chem. 3: 1946-1955), as outlined in Scheme 3. Specifically, the coupling was performed using EDC HCI. In one embodiment, the coupling was performed in DMF or THF.
Figure imgf000056_0001
Figure imgf000056_0002
Scheme 4 describes the synthesis of compound 20a ("Cbz-Val-Cit-PAMA-(methyl carboxylate)-HMC"). An optionally substituted 4-nitrobenzaldehyde 13A, zinc iodide and trimethylsilyl cyanide or KCN were reacted to provide compound 14. In one embodiment, the reaction was performed in DCM or THF. In a further embodiment, the optionally substituted benzaldehyde is 4-nitro-benzaldehyde. In another embodiment, the reaction was performed at elevated temperatures. To compound 14 was added an acid at elevated temperatures to obtain compound 15. In one embodiment, the reaction was performed in acetic acid. In another embodiment, the reaction was performed at about 100 to about 120°C. In still another embodiment, the acid was hydrochloric acid. To compound 15 was added an acid at elevated temperatures to provide product 16. In one embodiment, the acid was concentrated H2SO4. In another embodiment, the reaction was performed in MeOH or ethanol. In a further embodiment, the reaction was performed at about 85 to about 100°C. Compound 16 was then reduced using a reducing agent. In one embodiment, the reduction was performed using a catalyst and hydrogen gas under inert conditions. In a further embodiment, the catalyst was Pd/C. Compound 17 was then reacted with Fmoc-Cit-OH or Fmoc-protected appropriate amino acid residues and EEDQ to provide compound 18. In one embodiment, the reaction was performed in DCM:THF or DMF. To a stirred solution of triphenyl phosphine, DBAD (di-tert-butyl azodicarboxylate) was added, followed by the addition of HMC. In one embodiment, the reaction was performed at about 0°C to about RT. Compound 18 was then added to this solution, followed by an organic tertiary amine base such as triethylamine or DIEA to provide product 19. To compound 19 was added piperidine at reduced temperatures. In one embodiment, the reduced temperature was about 0°C. In another embodiment, the reaction was performed in DMF or THF. The product was then reacted with Cbz-Val-OSu and diisopropyl ethylamine. Quenching of the product with water provided compound 20a.
Figure imgf000057_0001
To compound 19 was added concentrated ammonium hydroxide solution or methanolic ammonia to provide compound 21. In one embodiment, the reaction was performed in DMF or THF. Compound 21 was reacted with Cbz-Val-OSu and an excess of di-isopropyl ethylamine. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed at reduced temperatures. In a further embodiment, the reaction was performed at about 0°C to about RT. Dilution of the mixture
Figure imgf000057_0002
Scheme 6 details the preparation of compound 23 from compound 20a. To compound 20a was added an excess of LiOH.H20 or NaOH. In one embodiment, the reaction was performed using MeOH:water or THF:MeOH:water. In another embodiment, the reaction was performed using about 3 to about 5 equivalents of LiOH.H20. The solution was then acidified with an acid to obtain the acid product. In one embodiment, the acid was HC1. The acid product was then reacted with a slight excess of benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), and DIPEA to provide compound 23. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed at reduced temperatures. In a further embodiment, the reaction was performed at 0°C to about RT. In yet another embodiment, the reaction was performed using about 1.2 to about 1.5 equivalents of CH3OCH2CH2OCH2CH2NH2.
Scheme 7
Figure imgf000058_0001
Scheme 7 describes the synthesis of compound 25 (Cbz-Val-Cit-PAMA-(PEG- amide)-OC(O)-AMC) which was utilized as described herein to demonstrate the function of the PAMA linker for protease-activated self-immolation of a drug surrogate, 7-amino-4- methylcoumarin (AMC). In this sequence of reactions, the described PAMA linker is connected to the warhead or the model compound via carbamate linker. The 4-amino group in compound 17 was reacted with Cbz-Val-Cit-OH in the presence of about 5 equivalents of EEDQ to yield compound 20. In one embodiment, the reaction was performed in DCM:THF:MeOH. The ester group of compound 20 was then converted to the PEG amide by a two-step process. First, the ester group was hydrolyzed to the carboxylic acid. In one embodiment, the hydrolysis was performed using an inorganic base. In another embodiment, the inorganic base was LiOH or NaOH. The carboxylic acid was reacted with NH2CH2CH2OCH2CH2OCH3 in presence of PyBOP and DIPEA to yield compound 24. In one embodiment, the reaction was performed at reduced temperatures to RT. In another embodiment, the reaction was performed at 0°C to RT. Compound 24 was subsequently reacted with the isocyanate derivative of AMC (4-methyl-7-amino-coumarine) to yield compound 25. In one embodiment, the reaction was performed in DMF or THF.
Scheme 8
Figure imgf000059_0001
101a
Figure imgf000059_0002
As shown in Scheme 8, an iodo acetyl group was introduced to the PAMA based liker/cytotoxic payload components. Compound 102a was prepared to target the thiol group of the cysteine or the -NH2 group of lysine residues of the antibody. Compound 102a was prepared starting from the amine 101a. The appropriately substituted Z-L-PAMA- (iodoacetyl-PEG-3-amide)-CBI-91 was reacted with compound 103 in a borate buffer: dioxane mixture and an inorganic base to provide compound 102a. In one embodiment, the inorganic base was sodium or potassium carbonate. Scheme 9
Figure imgf000060_0001
Scheme 9 provides the synthesis of compound 26, which can be used as a synthon to prepare an appropriate ADC. Specifically, compound 20 was reacted with an excess of an inorganic base such as LiOH.H20 or NaOH. In one embodiment, the reaction was performed in MeOH:water or THF:MeOH:water. In another embodiment, the reaction was performed using about 2 equivalents of LiOH.H20. After completion, the product was neutralized. In one embodiment, the neutralization was performed using acidic resin (for example, Amberlyst® (Rohm Haas)) to provide the acid product. The acid product was the reacted with NH2CH2CH2OCH2CH2OCH2CH2NHB0C, PyBOP, and a slight excess of DIPEA to provide compound 26. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed at reduced temperatures. In a further embodiment, the reaction was performed at 0°C. In yet another embodiment, the reaction was performed using about 1.5 to about 3 equivalents of DIPEA.
Scheme 10
Figure imgf000060_0002
Scheme 10 depicts the synthesis of compound 29. Initially, compound 20 was hydrolyzed by reaction with an excess of LiOH or other inorganic base. In one embodiment, the reaction was performed in THF and water. In another embodiment, the reaction was performed using about 6 to 10 equivalents of LiOH. In a further embodiment, the reaction was performed at reduced temperatures. In still another embodiment, the reaction was performed at 0°C to RT. After consumption of starting material 20, the mixture was neutralized with an acid to provide the acid product. In one embodiment, the acid was citric acid or acetic acid. The acid product was then reacted with NH2CH2CH2O-CH2CH2O- CH2CH2NHBoc, PyBOP, and DIPEA to provide compound 28. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed at reduced temperatures. In a further embodiment, the reaction was performed at 0°C to about RT. Compound 28 was then reacted with TFA at reduced temperatures. In one embodiment, the reaction was performed in DCM or dichloroethane. In another embodiment, the reaction was performed at about -10 to about 0°C. To the resulting solution was added succinate ester 27 and an excess of DIPEA to afford compound 29. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed using about 2 to about 5 equivalents of DIPEA.
Scheme 11
Figure imgf000061_0001
31
Scheme 11 depicts the synthesis of compound 31 and was prepared similarly to the route described in Scheme 10. Initially, compound 20 was reacted with an excess of LiOH.H20 an inorganic base such as NaOH. In one embodiment, the reaction was performed in THF and water. In another embodiment, the reaction was performed using about 3 to about 5 equivalents of LiOH.H20. In a further embodiment, the reaction was performed at reduced temperatures. In still another embodiment, the reaction was performed at 0°C to about RT. After consumption of starting material 20, the mixture was neutralized with an acid to provide the acid product. In one embodiment, the acid was citric acid. The acid product was then reacted with NH2CH2CH2OCH2CH2OCH2CH2OCH2CH2NHB0C, PyBOP, and DIPEA to provide compound 30. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed at reduced temperatures. In a further embodiment, the reaction was performed at 0°C to about RT. Compound 30 was then reacted with TFA at reduced temperatures. In one embodiment, the reaction was performed in DCM. In another embodiment, the reaction was performed at about -10 to about 0°C. To the resulting TFA salt solution was added succinate ester 27 and DIPEA to afford compound 31. In one embodiment, the reaction was performed in DMF or THF.
Scheme 12
Figure imgf000062_0001
The ether formation between the PAMA based linker and the compound 2a was prepared by a novel procedure as outlined in Scheme 12. An appropriately substituted PAMA linker of structure 26 was reacted with an excess of CS2CO3. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed with about 2 to about 5 equivalents of Cs2C03. To this mixture at reduced temperatures was then added an excess of CI3CN to provide compound 32. In one embodiment, this reaction was performed at about 0°C to about RT. In another embodiment, the reaction was performed using about 10 to about 15 equivalents of CCI3CN. To a suspension of 4 A molecular sieves in CH3CN were added a slight excess of compound 32 and compound 2a (Boc-Pro-CBI). In one embodiment, the reaction was performed using about 1.5 to about 3 equivalents of compound 2a. To this mixture at reduced temperatures was added a Lewis acid such as BF3-efher. In one embodiment, the reaction was performed at about -10 to about 0°C. Additional BF3- ether was added as needed. Neutralization of the mixture provided compound 33. In one embodiment, the neutralization was performed using a tertiary organic base such as NEt3. Finally, compound 33 was added Boc20 and an excess of NEt3 to yield compound 34. In one embodiment, the reaction was performed in MeOH or ethanol. In another embodiment, the reaction was performed using about 2 equivalents of NEt3.
Scheme 13
Figure imgf000063_0001
Compound 40 was prepared as described in Scheme 13. Specifically, compound 34 was coupled with DMMI via a CDI mediated reaction as outlined in Scheme 11. Other DNA minor groove binder groups are synthetically introduced in a similar manner. Specifically, DMMI 3 was reacted with a slight excess of CDI at reduced temperatures. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using 1.3 equivalents of CDI. The mixture was then added to amine 34 and an excess of sodium or potassium carbonate to obtain compound 39. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed using about 10 to about 15 equivalents of sodium carbonate. To compound 39 was added HC1 in EtOAc at reduced temperatures. In one embodiment, the reaction was performed in ethyl acetate. In another embodiment, the reaction was performed at about -10 to about 0°C. The resulting product was then reacted with succinate ester 27 and sodium carbonate to provide compound 40. In one embodiment, the reaction was performed in DMF or THF.
Scheme 14
Figure imgf000064_0001
To compound 20 was added an excess of LiOH.H20 or an inorganic base such as NaOH at reduced temperatures. See, Scheme 14. In one embodiment, the reaction was performed in MeOH and water or MeOH:THF:water. In a further embodiment, the reaction was performed using about 2 to about 5 equivalents of LiOH.H20. In another embodiment, the reaction was performed at about 0°C to about RT. The mixture was then neutralized to provide the acid product. In one embodiment, the neutralization was performed using an acidic resin. The acid product was then reacted with
NH2CH2CH2OCH2CH2OCH2CH2NHB0C, PyBOP, and a slight excess of DIPEA at reduced temperatures to provide compound 41. In one embodiment, the reaction was performed in DMF or THF. In a further embodiment, the reaction was performed at about 0°C to about RT. In another embodiment, the reaction was performed using about 1.5 to about 2.5 equivalents of DIPEA. Scheme 15
Figure imgf000065_0001
Figure imgf000065_0002
Scheme 15 depicts the synthesis of compound 44 from alcohol 41. To alcohol 41 was added about 2 to about 5 equivalents of CS2CO3. The mixture was then combined with CCI3CN at reduced temperatures to yield compound 42. In one embodiment, the reduced temperature was about 0°C to about RT. In another embodiment, the reaction was performed in DMF or THF. An excess of compound 42 and Boc-Pro-CBI were then added to a suspension of 4A molecular sieves in dry CH3CN. In one embodiment, the reaction was performed with about 1.5 to about 2 equivalents of compound 42. The product was reacted with a Lewis acid such as BF3.ether at reduced temperatures. In one embodiment, the reaction was performed at -10 to about 0°C. The mixture was then neutralized to afford compound 43. In one embodiment, the neutralization was performed using an organic tertiary base such as NEt3 Compound 43 was then reacted with an excess of NEt3 or DIEA at reduced temperatures. In one embodiment, the reaction was performed in MeOH or ethanol. In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using about 2 to about 3 equivalents of NEt3. Finally, Boc20 was added to this mixture to yield compound 44. Scheme 16
Figure imgf000066_0001
46
Scheme 16 provides a route to compound 46 from compound 3. To DMMI compound 3 was added a slight excess of CDI at reduced temperatures. In one embodiment, the reaction was performed in DMF or THF In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed with about 1.3 equivalents of CDI. The product was then reacted with amine 44 and an excess of sodium or potassium carbonate. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed with about 10 to about 15 equivalents of sodium carbonate. The mixture was then neutralized to obtain compound 45. In one embodiment, the neutralization was performed using formic acid. To compound 45 was added HC1 in EtOAc at reduced temperatures. In one embodiment, the reaction was performed in EtOAc. In another embodiment, the reaction was performed at about -10 to about 0°C. To the resulting solution was added succinate ester 27 and sodium carbonate to provide compound 46. In one embodiment, the reaction was performed using DMF.
Scheme 17
Figure imgf000067_0001
Scheme 17 provides the combined synthesis of compounds 52 and 53, both routes beginning with starting material compound 47. Specifically, compound 47 was reacted with an excess of ethyl cyanoacetate and an excess of NEt3 in the presence of 4A molecular sieves to provide compound 48. In one embodiment, the reaction was performed in DCM. In another embodiment, the reaction is performed with about 1.5 to about 2 equivalents of ethyl cyanoacetate. In a further embodiment, the reaction is performed with about 3 to about 5 equivalents of NEt3. Compound 48 was then reacted with KCN at elevated temperatures to provide compound 49. In one embodiment, the reaction was performed using ethanol and water. In another embodiment, the reaction was performed at about 80 to about 100°C. Compound 49 was then reacted with concentrated HC1 at elevated temperatures to provide diacid 50. In one embodiment, the reaction was performed in a sealed tube. In another embodiment, the reaction was performed at about 100°C. To compound 50 was added Ν,Ν'- dicyclohexylcarbodiimide. In one embodiment, the reaction was performed in DMF or THF. 4-Methoxy benzyl amine and an excess of NEt3 or DIEA were then added. In one embodiment, the reaction was performed using about 2 to about 4 equivalents of NEt3. The product was then reacted with Ac20 and sodium acetate at elevated temperatures to provide compound 51. In one embodiment, the reaction was performed at about 100°C.
Compound 51 was then utilized to prepare compounds 52 and 53 via separate routes.
In this first route, compound 51 was reacted with palladium hydroxide in the presence of hydrogen to provide amine 52. In one embodiment, the reaction was performed in acetic acid: 1,4-dioxane: water. In another embodiment, the reaction was performed in 1.5: 1.5: 1 v/v of acetic acid:l,4-dioxane:water.
In the second route, compound 51 was reacted with an excess of eerie ammonium nitrate provide compound 53. In one embodiment, the reaction was performed using acetonitrile: water. In another embodiment, the reaction was performed using about 2 to about 8 equivalents of eerie ammonium nitrate.
Scheme 18
Figure imgf000068_0001
Scheme 18 describes the preparation of compound 56 from starting materials 32 and 52. To spirocyclic amine 52 were added compound 32 and an excess of K2CO3 to provide compound 54. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed using about 3 to about 8 equivalents of K2CO3. To compound 54 was added an excess of Lil at elevated temperatures to provide compound 55. In one embodiment, the reaction was performed in pyridine. In another embodiment, the reaction was performed using 6 equivalents of Lil. In a further embodiment, the reaction was performed at about 200°C. In yet another embodiment, the reaction was performed in a microwave reactor. To Pro-CBI 2 was added compound 55 and an excess of EDC'HCl at reduced temperatures to provide compound 56. In one embodiment, the reaction was performed in DMF or THF. In another embodiment, the reaction was performed using 4 to about 6 equivalents of EDC'HCl. In a further embodiment, the reaction was performed at about 0°C to about RT. Scheme 19
Figure imgf000069_0001
Scheme 19 depicts a route to compound 59 via compounds 32 and 53 and is similar to the route described in Scheme 18. Specifically, amine 53 was reacted with compound 32 and an excess of K2CO3 or NaH at elevated temperatures to provide compound 57. In one embodiment, the reaction was performed using DMF or THF. In another embodiment, the reaction was performed using about 2.5 to about 3.5 equivalents of K2CO3. In a further embodiment, the reaction was performed at about 100°C. To derivative 57 was added Lil at elevated temperatures to provide compound 58. In one embodiment, the reaction was performed in pyridine. In another embodiment, the reaction was performed at about 200°C. In a further embodiment, the reaction was performed using about 6 to about 10 equivalents of Lil. In yet another embodiment, the reaction was performed in a microwave reactor. Finally, Pro-CBI 2 was reacted with compound 58 and excess EDC.HC1 to provide compound 59. In one embodiment, the reaction was performed using DMF. In another embodiment, the reaction was performed at about 0 to about 10°C. In a further embodiment, the reaction was performed using about 3 to about 5 equivalents of EDC.HC1. Scheme 20
Figure imgf000070_0001
Scheme 20 provides the synthesis of compound 65 from starting material 32. Specifically, ester 32 was reacted with a slight excess of Cs2C03. In one embodiment, the reaction was performed in ethanol and water. In another embodiment, the reaction was performed in a 1 : 1 v/v ethanol to water solution. In a further embodiment, the reaction was performed using about 1.5 to about 3 equivalents of CS2CO3. The mixture was then acidified to provide compound 60. In one embodiment, the acidification was performed using HC1. In another embodiment, the acidification was performed using 4N HC1. To compound 60 was added benzyl alcohol, DCC, and an excess of DMAP at reduced temperatures to provide compound 61. In one embodiment, the reaction was performed in DCM. In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using about 2.5 to about 3.5 equivalents of DMAP. To amine 53 was added compound 61 and an excess of K2CO3 or NaH at elevated temperatures to provide compound 62. In one embodiment, the reaction was performed using DMF. In another embodiment, the reaction was performed at about 100 to about 120°C. In a further embodiment, the reaction was performed using about 2.5 to about 5 equivalents of K2CO3. To benzyl ester 62 was added palladium hydroxide under hydrogen to provide amine 63. In one embodiment, the reaction was performed in acetic acid, 1,4-dioxane, and water. In a further embodiment, the reaction was performed in a 2:2: 1 acetic acid, 1,4-dioxane, and water solution. To amine 63 were added acetic acid and an excess of formaldehyde or para formaldehyde. In one embodiment, the reaction was performed in MeOH or ethanol. In another embodiment, the reaction was performed using about 2 to about 4 equivalents of formaldehyde. The mixture was then charged with sodium triacetoxy borohydride at reduced temperatures to provide compound 64. In one embodiment, the reaction was performed at about 0°C to about RT. Finally, compound 64 was reacted with Pro-CBI 2 and an excess of EDC.HC1 at reduced temperatures to provide compound 65. In one embodiment, the reaction was performed using DMF. In another embodiment, the reaction is performed using about 5 to about 10 equivalents of EDC.HC1. In a further embodiment, the reaction is performed at about 0°C to about RT.
Scheme 21
Figure imgf000071_0001
To compound 53 was added an excess of a reducing agent at reduced to elevated temperatures to provide compound 66 in a manner similar to Scheme 20 and as outlined in Scheme 21. In one embodiment, the reaction is performed using THF. In another embodiment, the reducing agent is LiAlH4. In a further embodiment, the reaction is performed using about 4 to about 6 equivalents of the reducing agent. In yet another embodiment, the starting temperature for the reaction is about -10 to about 0°C. In still a further embodiment, the temperature of the reaction is then elevated to about 65 to about 75°C. To spiroamine 66 were added compound 32 and an excess of K2CO3 or NaH at elevated temperatures to provide compound 67. In one embodiment, the reaction is performed in DMF or THF. In another embodiment, the reaction was performed with about 2 to about 5 equivalents of K2CO3. In a further embodiment, the reaction was performed at about 80 to about 100°C. To compound 67 was added palladium hydroxide under hydrogen to provide amine 68. In one embodiment, the reaction was performed in acetic acid, 1,4- dioxane, and water. In a further embodiment, the reaction was performed in a 2:2:1 acetic acid, 1,4-dioxane, and water solution. To amine 68 were added acetic acid, an excess of formalin or para formaldehyde and sodium cyanoborohydride or sodium triacetoxy borohydride at reduced temperatures to provide compound 69. In one embodiment, the reaction was performed in MeOH or MeOH:THF. In a further embodiment, the reaction was performed at about 0°C to about RT. In another embodiment, the reaction was performed with about 2 to about 5 equivalents of formalin. In still a further embodiment, the reaction was performed in formalin 40% in water. To compound 69 was added to an inorganic base such as NaOH or LiOH.H20 and the mixture acidified to yield compound 70. In one embodiment, the reaction was performed in a MeOH:water or THF:MeOH:water. In another embodiment, the reaction was performed using about 6 to about 10 equivalents of LiOH.H20. In a further embodiment, the acidification was performed using HC1. To compound 70 were added Pro-CBI 2 and EDC.HC1 at reduced temperatures to provide compound 71. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed using about 5 to about 10 equivalents of EDC.HC1. In a further embodiment, the reaction was performed at about 0°C to about RT.
Scheme 22
Figure imgf000072_0001
72 73 74
75
Scheme 22 provides the synthesis of compound 75, which is similar to the routes discussed in Schemes 20 and 21. To diacid 72 was added an excess of a reducing agent at reduced to elevated temperatures to provide compound 73 in a manner similar to Scheme 21. In one embodiment, the reaction is performed using THF. In another embodiment, the reducing agent is LiAlH4. In a further embodiment, the reaction is performed using about 4 to about 6 equivalents of the reducing agent. In yet another embodiment, the starting temperature for the reaction is about 0°C to about RT. In still a further embodiment, the temperature of the reaction is then elevated to about 80 to about 100°C. The mixture was then basified at reduced temperatures to provide compound 73. In one embodiment, the basification is performed using NaOH or NH4OH. In another embodiment, the basification was performed using 10% aqueous NaOH. In a further embodiment, the basification is performed at about 0°C. To triphenylphosphine and diol 73 was added DBAD or DEAD. In one embodiment, the reaction was performed at about 0°C to about RT. In another embodiment, the reaction was performed in THF. A solution of the product was then washed with an acid and basified to provide spirocyclic 74. In one embodiment, the acid was HC1. In another embodiment, the reaction was performed in DCM. The aqueous layer was then basified to provide spirocyclic 74. In one embodiment, the pH of the aqueous layer adjusted to pH 10. In a further embodiment, the basification was performed using NaOH. To compound 74 was added palladium hydroxide under hydrogen to provide amine 75. In one embodiment, the reaction was performed in acetic acid, 1,4-dioxane, and water. In a further embodiment, the reaction was performed in a 4:4: 1 acetic acid, 1,4-dioxane, and water solution.
Scheme 23
Figure imgf000073_0001
66 82a 82
Scheme 23 depicts the preparation of compound 82, which route is similar to that described in Scheme 21. Specifically, amine 66 was reacted with acetic acid, formaldehyde or formaline and sodium cyanoborohydride or sodium triacetoxy borohydride at reduced temperatures to provide product 82a. In one embodiment, the reaction was performed in MeOH of THF:MeOH. In another embodiment, the reaction was performed using about 2 to about 5 equivalents of formaldehyde. In a further embodiment, the reaction was performed using about 3 to about 8 equivalents of cyanoborohydride. In yet another embodiment, the reaction was performed at about 0°C to about RT. Finally, to compound 82a was added palladium hydroxide and hydrogen to provide amine 82. In one embodiment, the reaction was performed in acetic acid: 1,4-dioxane: water. In another embodiment, the reaction was performed in a 2:2: 1 v/v solution of acetic acid: 1,4-dioxane: water. Scheme 24
Figure imgf000074_0001
Figure imgf000074_0002
80 81
Scheme 24 describes the synthesis of compound 81. Compound 73 was reacted with palladium hydroxide and hydrogen to provide the corresponding amine. In one embodiment, the reaction was performed using acetic acid, 1,4-dioxane, and water. In another embodiment, the reaction was performed using a 2:2: 1 v/v solution of acetic acid: 1,4- dioxane: water. The amine was then reacted with Boc anhydride and an excess of triethyl amine or DIEA at reduced temperatures to provide compound 76. In one embodiment, the reaction was performed in THF and water. In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using about 2 to about 4 equivalents of triethylamine. Compound 76 was reacted with TEA or DIEA, followed by methane sulfonylchloride at reduced temperatures to provide compound 77. In one embodiment, the reaction was performed in DCM or THF. In another embodiment, the reaction was performed at about -10 to about 0°C. Compound 77 was reacted with Na2S.H20 NO at elevated temperatures to provide compound 78. In one embodiment, the reaction was performed in DMF or THF. . In another embodiment, the reaction was performed at about 105 to about 120°C. Compound 78 was then reacted with HCl at reduced temperatures to provide compound 79. In one embodiment, the reaction was performed in ethyl acetate or ethanol. In another embodiment, the reaction was performed using HCl. In a further embodiment, the reaction was performed at about -10 to about 0°C. Compound 78 were added H2O2 solution, water and Na2W04.2H20 at elevated temperatures to provide compound 80. In one embodiment, the reaction was performed in THF or THF:MeOH. In another embodiment, the reaction was performed in a sealed tube. In a further embodiment, compound 80 was reacted with the 33% in a water H2O2 solution. In yet another embodiment, the reaction was performed with about 6 to about 10 equivalents of a H2O2 solution. In still a further embodiment, the reaction was performed with about 3 to about 6 equivalents of Na2W04.2H20. In another embodiment, the reaction was performed at about 65 to about 75°C. Finally, compound 80 was reacted with HC1 at reduced temperatures to provide amine 81 as the HC1 salt. In one embodiment, the reaction was performed in ethyl acetate. In another embodiment, the reaction was performed at 0°C to - 10°C.
Scheme 25
Figure imgf000075_0001
75; X = O 83; X = O
82; X = N-Me 84; X = W-Me
79; X = S 85; X = S
81 ; X = S02 86 X = S02
Figure imgf000075_0002
89; X = S 92: X = W-Me
90; X = S02 93: X = S
94: x = so2
Scheme 25 provides the synthesis of compounds 91-94, all starting from compound 32. To amine 75, 82, 89, or 81 were added compound 32 and K2CO3 at elevated temperatures to provide compounds 83, 84, 85, or 86, respectively. In one embodiment, the reaction was performed in DMF or DMSO. In another embodiment, the reaction was performed using about 2 to about 4 equivalents of K2CO3. In a further embodiment, the reaction was performed at about 80 to about 100°C. Compound 83 was reacted with an inorganic base such as NaOH or LiOH.H20. In one embodiment, the reaction was performed in MeOH and water or THF:MeOH:water. In another embodiment, the reaction was performed using about 3 to about 5 equivalents of LiOH.H20. The mixture was then acidified with HC1 to provide compound 87 as the HC1 salt. Compound 87 was reacted with Pro-CBI 2 and EDC.HC1 at reduced temperatures to provide product 91. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed at about 0°C to about RT. In a further embodiment, the reaction was performed using EDC.HC1.
Scheme 26
Figure imgf000076_0001
The coupling of the PAMA based linker to the warhead via an ether linkage can performed by following the pathway depicted in Scheme 26, using an "Alloc" protecting group. Compound 20 was reacted with LiOH.H20 at reduced temperatures. In one embodiment, the reaction was performed in methanol and water or MeOH:THF:water. In another embodiment, the reaction was performed in a 10: 1 mixture of methanol: water. In a further embodiment, the reaction was performed with about 3 to about 5 equivalents of LiOH.H20. In still another embodiment, the reaction was performed at about 0°C. The product was then neutralized to yield the acid. In one embodiment, the neutralization was performed using an acidic resin (e.g., Amberlyst®, available from Sigma-Aldrich). The acid was then reacted with NH2CH2CH2OCH2CH2OCH2CH2NHAII0C, PyBOP, and DIPEA at reduced temperatures to obtain compound 95. In one embodiment, the reaction was performed in DMF. In a further embodiment, the reaction was performed at about 0°C to about RT. Alcohol 95 was reacted with an excess of CS2CO3, cooled to a reduced temperatures, and CCI3CN was added to obtain compound 96. In one embodiment, the reaction was performed in DMF. In one embodiment, about 2 to about 5 equivalents of CS2CO2 was utilized. In a further embodiment, the reaction was cooled to about 0°C. To a suspension of molecular sieves was added trichloroacetimidate 96 and Boc-Pro-CBI 2a. In one embodiment, the reaction was performed using 2 to about 4 equivalents of trichloroacetimidate 96. In another embodiment, the reaction was performed using MeCN. The mixture was then cooled to reduced temperatures and reacted with a Lewis acid such as BF3.ether. Additional BF3 ether was added as needed. In one embodiment, the mixture was cooled to about -10°C to about 0°C. The mixture was neutralized using NEt3 or DIEA to afford compound 97.
Figure imgf000077_0001
Figure imgf000077_0002
Scheme 27 provides the synthesis of compound 100 via compound 87. Specifically, compound 87 was reacted with CDI at reduced temperatures to provide compound 98. In one embodiment, the reaction was performed in DMF In another embodiment, the reaction was performed using about 5 to about 10 equivalents of CDI. In a further embodiment, the reaction was performed at about -10 to about 0°C. Compound 98 was then reacted with amine 97 and excess sodium carbonate to afford compound 99. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed using about 10 to about 15 equivalents of sodium carbonate. Compound 99 was reacted Pd(PPh3)4 and 1,3-dimethylbarbituric acid to provide the amine product. In one embodiment, the reaction was performed in DCM. The amine product was reacted with an excess of succinate ester 27 and an excess of sodium carbonate to provide compound 100. In one embodiment, the reaction was performed in DMF. In another embodiment, the reaction was performed with about 2 equivalents of succinate ester 27 and/or sodium carbonate.
Scheme 28
Figure imgf000078_0001
Scheme 28 provides the synthesis of compound 102. Specifically, amine 101 is reacted with an excess of the succinate ester 103 and an inorganic base such as sodium carbonate to obtain compound 102. In one embodiment, the reaction is performed in a buffer. In another embodiment, the reaction is performed in a borate buffer. In a further embodiment, the reaction was performed in a 0.1 M borate buffer (pH 8.0):dioxane. In yet another embodiment, the reaction is performed with about 2 equivalents of succinate ester.
A composition containing a therapeutic drug and targeting conjugate of formula (I), or a mixture thereof, may be prepared. In still further embodiments, a composition contains a therapeutic drug and targeting conjugate of any of formula (IA), formula (IB), formula (ICi) or formula (ICii). While the following specification will reference formula (I), it will be understood that any of these subgeneric structures, or combinations thereof, may be combined in to a single pharmaceutical composition unless otherwise specified.
A pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a conjugate of formula (I) in a pharmaceutically acceptable carrier optionally with other pharmaceutically inert or inactive ingredients. In one embodiment, as a result of the conjugation process, a composition may contain a mixture of conjugates of formula (I) in which each LG has 1-6 drugs (via 1-6 B-linker-drug fragments) conjugated thereto.
Suitably, no or substantially no agglomerate conjugates are present, optionally having been removed by filtration or other post-conjugation processing. Such a composition may contain a mixture of conjugates of formula (I), wherein p" averages 2. Such a composition may contain conjugates of formula (I), wherein a selected percentage of the conjugates is characterized by p" is 1, a selected percentage of the conjugates is characterized by p" is 2, and with a significantly smaller percentage of the conjugates being characterized by p" over 3 or higher. Still other mixtures can be prepared according to the invention.
In a further embodiment, a conjugate of formula (I), or a mixture of conjugate of formula (I), is combined with one or more excipients and/or other therapeutic agents as described below. When reference is made to formulation of a conjugate of formula (I), it will be understood that a mixture of conjugates of formula (I) can be similarly formulated, unless stated otherwise.
The pharmaceutical compositions provided herein comprise an amount of a conjugate of formula (I) that is therapeutically effective. Specifically, the dosage of the conjugate of formula (I) to achieve a therapeutic effect will depend on the formulation, age, weight and sex of the patient and route of delivery. It is also contemplated that the treatment and dosage of the conjugate of formula (I) may be administered in unit dosage form and that one skilled in the art would adjust the unit dosage form accordingly to reflect the relative level of activity. The decision as to the particular dosage to be employed (and the number of times to be administered per day) is within the discretion of the ordinarily-skilled physician, and may be varied by titration of the dosage to the particular circumstances to produce the desired therapeutic effect. In one embodiment, the therapeutically effective amount is about 0.001 to 10 mg of ADC protein per kg body weight. In another embodiment, the therapeutically effective amount is less than about 10 mg/kg, about 1 mg/kg, about 0.5 mg/kg, about 0.25 mg/kg, about 0.1 mg/kg, about 100 μg/kg, about 75 μg/kg, about 50 μg/kg, about 25 μg/kg, about 10 μg/kg, or about 1 μg/kg. However, the therapeutically effective amount of the conjugate of formula (I) can be determined based on clinical studies and individual patient response and depends on the condition treated, the particular conjugate administered, the route of delivery, the age, weight, severity of the patient's symptoms and response pattern of the patient.
The therapeutically effective amount may be provided on regular schedule, i.e. , daily, weekly, monthly, or yearly basis or on an irregular schedule with varying
administration days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered may vary. In one embodiment, the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses. In another embodiment, the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The number and frequency of dosages corresponding to a completed course of therapy will be determined according to the recommendations of the relevant regulatory bodies and judgment of a health-care practitioner. The therapeutically effective amounts described herein refer to total amounts administered for a given time period; that is, if more than one different conjugate of formula (I) thereof is administered, the therapeutically effective amounts correspond to the total amount administered.
The pharmaceutical compositions containing a conjugate of formula (I) may be formulated neat or with one or more pharmaceutical carriers for administration. The amount of the pharmaceutical carrier(s) is determined by the solubility and chemical nature of the conjugate of formula (I), chosen route of administration and standard pharmacological practice. The pharmaceutical carrier(s) may be solid or liquid and may incorporate both solid and liquid carriers. A variety of suitable liquid carriers are known and may be readily selected by one of skill in the art. Such carriers may include, e.g., saline, buffered saline, human albumin, polyethylene glycol, and mixtures thereof. Similarly, a variety of solid carriers and excipients are known to those of skill in the art. The conjugates of formula (I) may be administered by any route, taking into consideration the specific condition for which it has been selected. The conjugates of formula (I) may, be delivered by injection, intravascularly, subcutaneously, intravesically, intramuscularly, intracranially, epidurally, among others. In one embodiment, the compositions are formulated for parenteral delivery of a conjugate of formula (I), e.g., intravenous or intraperitoneal delivery.
Although the conjugate of formula (I) may be administered alone, it may also be administered in the presence of one or more pharmaceutical carriers that are physiologically compatible. The carriers may be in liquid form and must be pharmaceutically acceptable. Liquid pharmaceutical compositions are typically sterile solutions or suspensions. When liquid carriers are utilized for parenteral administration, they are desirably sterile liquids. Liquid carriers are typically utilized in preparing solutions. In one embodiment, the conjugate of formula (I) is dissolved in a liquid carrier. In another embodiment, the conjugate of formula (I) is suspended in a liquid carrier. One of skill in the art of formulations would be able to select a suitable liquid carrier, depending on the parenteral route of administration.
The conjugates of formula (I) and/or other medication(s) or therapeutic agent(s) may be administered in a single composition. However, the present invention is not so limited.
In other embodiments, the conjugates of formula (I) may be administered in one or more separate formulations from other conjugates of formula (I), chemotherapeutic agents, or other agents as is desired.
The invention provides a method of delivering a therapeutically active drug, said method comprising administering a therapeutic drug and targeting conjugate of formula (I) as defined herein, wherein said drug fragment or prodrug fragment is converted to a therapeutically active drug or prodrug following cleavage of the di/tri/tetrapeptide substrate within the ligand-drug conjugate by one or more proteases.
A "patient" or "subject" is a mammal, e.g. , a human or a veterinary patient or subject, e.g., mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.
The term "treating" or "treatment" is meant to encompass administering to a subject a therapeutic conjugate of the present invention for the purposes of amelioration of one or more symptoms of a disease or disorder, including palliative care. A "therapeutically effective amount" refers to the minimum amount of the active compound which effects treatment.
In one embodiment, a conjugate as described herein contains a ligand designed to target a desired cell surface molecule, such as a receptor or a growth factor. Examples of suitable targets include, e.g., CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70,
CD 133, carcinogenic embryonic antigen (CEA), epidermal growth factor receptor- 1 (EGFR or HER1 or erbBl), epidermal growth factor receptor (EGFRviii), human epidermal growth factor receptor-2 (HER2 or erbB2), epidermal growth factor receptor-3 (HER3 or erbB3), MET, insulin-like growth factor receptor 1 (IGFIR), platelet-derived growth factor receptor alpha and beta (PDGFRalpha and PDGFRbeta), EphrinA receptors 1-8 (EphAl-8), EphrinB receptors 1-6 (EphBl-6), folate receptor (FolRalpha), prostate specific membrane antigen (PSMA), MUC-1, MUC-16, high molecular weight melanoma-associated antigen (HMW- MAA) or chondroitin sulfate proteoglycan (CSPG), epithelial cell adhesion molecule (EPCAM), 5T4 oncofetal trophoblast glycoprotein, Tie-2, oncofetal antigen and vascular endothelial growth factor receptor-2 (VEGFR2). Still other suitable targets will be apparent to one of skill in the art. Thus, the invention provides a method of treating a disease or disorder associated with the presence of a specific cell surface molecule on cells of a subject, comprising administering to the subject a therapeutically effective amount of a conjugate of the invention comprising at least one ligand that specifically binds to the cell surface molecule. In one embodiment, the disease or disorder is a neoplastic disease such as cancer.
Thus in one embodiment, such a composition is designed for use in a therapeutic in anti-neoplastic regimen. In such an instance, the ligand is desirably selected to target a neoplastic cell based on the presence of specific cell surface molecules on the neoplastic cell. Examples of neoplastic cells include those involved in cancer of the prostate, head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone, lung (small cell or non-small cell), colon, rectum, stomach, bladder, uterus, cervix, breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes, kidney (renal cancer), liver, intestines, pancreas, brain (e.g., glioblastoma), central nervous system, adrenal gland, or skin or a leukemia. In another embodiment, said cancer is cancer of the prostate. In one embodiment, a cancer patient has at least one solid or liquid tumor.
Also provided herein are products, e.g., kits, vials with preformulated parental compositions of the invention, or other packages of containing the conjugates or formulated conjugates described herein. The kits may be organized to indicate a single formulation or combination of formulations to be taken at each desired time.
Suitably, the kit contains packaging or a container with the compound of formula (I) formulated for the desired delivery route. Suitably, the kit contains instructions on dosing and an insert regarding the active agent. Optionally, the kit may further contain instructions for monitoring circulating levels of product and materials for performing such assays including, e.g. , reagents, well plates, containers, markers or labels, and the like. Such kits are readily packaged in a manner suitable for treatment of a desired indication. Other suitable components to include in such kits will be readily apparent to one of skill in the art, taking into consideration the desired indication and the delivery route.
The compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration. For continuous administration, a package or kit can include the conjugates in each dosage unit (e.g., solution or other unit described above or utilized in drug delivery), and optionally instructions for administering the doses daily, weekly, or monthly, for a predetermined length of time or as prescribed. If varying concentrations of a composition, of the components of the composition, or the relative ratios of the conjugates or agents within a composition over time is desired, a package or kit may contain a sequence of dosage units which provide the desired variability.
A number of packages or kits are known in the art for dispensing pharmaceutical agents for periodic oral use. In one embodiment, the package has indicators for each period. In another embodiment, the package is a labeled blister package, dial dispenser package, or bottle.
The packaging means of a kit may itself be geared for administration, such as a syringe, pipette, eye dropper, or other such apparatus, from which the formulation may be applied to an affected area of the body, injected into a subject, or even applied to and mixed with the other components of the kit.
The compositions of these kits also may be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in a separate package.
The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g. , injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of packages and as discussed above, the kits also may include, or be packaged with a separate instrument for assisting with the injection/administration or placement of the composition within the body of a subject. Such an instrument may be a syringe, pipette, forcep, measuring spoon, eye dropper or any such medically approved or appropriate delivery means.
Protease-activated self-immolative release in model systems
The para-amino mandelic acid (PAMA) derived linker system was demonstrated to provide protease-activated self-immolative release of both hydroxy- and amine -linked drugs, by multiple methods.
In one method, 7-hydroxy-4-methylcoumarin (HMC) was used as a surrogate for a hydroxy-linked drug, and 7-amino-4-methylcoumarin (AMC) was used as a surrogate for an amine -linked drug, and fluorescence measurements were used to assay the amount of surrogate prodrug/drug released. Compounds were prepared incorporating variations of the PAMA linker and these surrogate drugs, but without inclusion of a targeting ligand such as an antibody. These compounds were incubated (pH 5.0, 37 °C) in the presence of cathepsin B, which was used as a representative protease, and release of the surrogate drug was measured by fluorescence. Multiple compounds were demonstrated to release the surrogate drug under the cathepsin B assay conditions. As a positive control or reference compound in these assays, Cbz-Val-Cit-PABE-HMC or Cbz-Val-Cit-PABC-AMC was included in the assay. Cbz-Val-Cit-PABC-AMC is an analog of Cbz-Val-Cit-PABC-Doxorubicin, which was reported by Dubowchik and Firestone, Bioorg. Med. Chem. Lett., 8 (1998), 3341-3346; and Dubowchik et. al., Bioconj. Chem., 13 (2002), 855-869. It was reported by Dubowchik and coworkers that the PABC-linker in Cbz-Val-Cit-PABC-Doxorubicin underwent cathepsin-B-activated self-immolation to release Doxorubicin (as measured by HPLC) with a half -life of 4 h. Compounds incorporating the PAMA linker conjugated to a drug surrogate were determined, using the fluorescence assay as described above, to release the surrogate drug at rates that were comparable to or greater than the drug release rates determined for the two reference compounds. Results are provided in Figures 1 to 5. Compounds incorporating the PAMA linker conjugated to a drug surrogate were also incubated under the same conditions but in the absence of cathepsin B, and were found to be stable (i.e., no significant release of surrogate drug over time). This demonstrates that protease cleavage (e.g., by cathepsin B) is required to activate the PAMA linker to self-immolate and release the drug.
In a second method, LC -MS/MS was used for detection of the prodrug and/or drug released. Compounds were prepared incorporating variations of the PAMA linker and using Pro CBI-DMMI as a hydroxy-linked prodrug of the cytotoxic duocarmycin analog drug CBI- DMMI. The prepared compounds evaluated using this method did not include a targeting ligand such as an antibody. These compounds were incubated (pH 5.0, 37 °C) in the presence of cathepsin B, and the formation of duocarmycin analog drug CBI-DMMI was determined by LC -MS/MS. In this case the drug linked to PAMA is a prodrug, i.e., Pro CBI- DMMI, which upon its release is converted under the assay conditions to CBI-DMMI. Multiple compounds were demonstrated to release/generate the duocarmycin analog drug CBI-DMMI under the cathepsin B assay conditions. Results are provided in Figure 6 and 7. These compounds were also found to be stable (i.e., no significant release/formation of CBI- DMMI over time) under the assay conditions in the absence of cathepsin B. This demonstrates that protease cleavage (in this case by cathepsin B) is required to activate the PAMA linker to self-immolate and release the drug.
In a similar manner, using the second method with LC-MS/MS detection, compounds are prepared incorporating variations of the PAMA linker and using N-methyl auristatin E (MMAE) as an amine-linked cytotoxic drug. The prepared compounds evaluated using this method do not include a targeting ligand such as an antibody. These compounds are incubated (pH 5.0, 37 °C) in the presence of cathepsin B, and the formation of N-methyl auristatin E drug is determined by LC-MS/MS.
In a third method, antibody drug conjugates were prepared incorporating the PAMA linker and a cytotoxic prodrug or drug, and the drug release was determined by mass spectrometry. In one example, an anti-5T4 scFvFc antibody conjugated to a PAMA derived linker incorporating Pro CBI-DMMI was evaluated. Incubation of this anti-5T4 scADC in the presence of cathepsin B (pH 5.0, 37 °C) for two hours resulted in the formation of a new signal in the MALDI mass spectrum corresponding to Pro CBI-DMMI. Under the same conditions in the absence of cathepsin B, there was no signal for the mass of Pro CBI- DMMI. This demonstrates that protease cleavage (in this case by cathepsin B) is achieved on the antibody drug conjugate (ADC), namely anti-5T4 scFvFc incorporating the PAMA linker and Pro CBI-DMMI as drug, and that protease cleavage is required to activate the PAMA linker to self-immolate and release the prodrug/drug.
In a similar manner, using the third method with mass spectrometry detection, compounds are prepared incorporating variations of the PAMA linker and using N-methyl auristatin E (MMAE) as an amine-linked cytotoxic drug. The prepared compounds to be evaluated using this method do not include a targeting ligand such as an antibody. These compounds are incubated (pH 5.0, 37 °C) in the presence of cathepsin B, and the formation of N-methyl auristatin E drug is determined by mass spectrometry.
In a fourth method, LC-MS/MS was used to measure the prodrug and/or drug released, for an antibody drug conjugate (ADC) incorporating the PAMA linker and a cytotoxic duocarmycin analog prodrug. In one example, an anti-HER2 scFvFc antibody conjugated to a PAMA derived linker incorporating duocarmycin prodrug analog compound 91 was evaluated, taking measurements at multiple time points. Incubation of this anti-HER2 scADC in the presence of cathepsin B (pH 5.0, 37 °C) resulted in the formation of the CBI analog of compound 91, whereas in the absence of cathepsin B there was no significant formation of the CBI analog of compound 91 during 24 hours of incubation.
Inhibition of tumor cell proliferation and tumor growth
The para-amino mandelic acid (PAMA) derived linker system was demonstrated to have utility to inhibit tumor cell proliferation and tumor growth, when conjugated with a targeting ligand and a cytotoxic prodrug or drug, for example as an antibody drug conjugate (ADC), in both in vitro and in vivo assays. For example, anti-5T4 scFvFc antibody conjugated to the PAMA linker and Pro CBI-DMMI as the drug was demonstrated in vitro to cause potent inhibition of tumor cell proliferation in a 5T4-overexpressing transfectant of MDA-MB-231 breast carcinoma cells. The in vitro inhibition was found to be ca. 100-fold less potent for native MDA-MB-231 tumor cells that have low expression of the 5T4 antigen. As another example, anti-HER2 scFvFc antibody conjugated to the PAMA linker and Pro CBI-DMMI as the drug was demonstrated in vivo to cause strong inhibition of tumor growth in a SKOV3 cervical carcinoma mouse xenograft model, at a dose of 2 mg/kg iv, dosing once per week for three weeks.
In a similar manner, the para-amino mandelic acid (PAMA) derived linker system was incorporated into conjugate compounds having a targeting ligand (for example, an scFvFc or IgG antibody), and an amine -linked cytotoxic drug (for example, N-methyl auristatin E). Utility of the PAMA-linked conjugate to inhibit tumor cell proliferation was demonstrated in vitro by measuring cytotoxicity in cell assays wherein the tumor cells harbor the antigen that is targeted by the targeting ligand. Utility to inhibit tumor growth in vivo was achieved by measuring tumor growth inhibition in mouse xenograft studies, wherein the tumors are grown from tumor cells that harbor the antigen that is targeted by the targeting ligand.
EXAMPLES
All reactions were carried out under dry nitrogen or argon atmosphere unless otherwise specified. Unless otherwise stated, all the raw starting materials, solvents and reagents were purchased from commercial sources (e.g. , Fluoro Chem., GLR Scientific Co., Alfa Aesar, Matrix Scientific, Sonia Industries, Sisco Research Laboratories Pvt. Ltd., Hindustan Platinum, S. D. Fine Chemical Limited, Qualigens Fine chemicals, FChemicals, Apollo Scientific Limited, Sigma Aldrich Chemicals Pvt. Ltd.) and used as such without further purification, or reagents were synthesized by procedures known in the art. Monomethyl-auristatin E (MMAE) was purchased from Concortis Biosystems, San Diego, USA.
The following abbreviations are used and have the indicated definitions: MHz is megahertz (frequency), m is multiplet, q is quartet, t is triplet, d is doublet, s is singlet, br is broad, CDC13 is deutero chloroform, DMSO-d6 is deutero dimethyl sulfoxide, calcd is calculated, min is minutes, h is hours, g is grams, mol is moles, mmol is millimoles, mL is milliliters, N is Normal (concentration), M is molarity (concentration), μΜ is micromolar, °C is degree centigrade, HPLC is High Performance Liquid Chromatography, LC-MS is Liquid Chromatography-Mass Spectroscopy, UPLC is Ultra Performance Liquid Chromatography, NMR is Nuclear Magnetic Resonance, TLC is thin layer chromatography, ESI is electrospray ionization, EI is electron impact ionization, LC is liquid chromatography, MS is mass spectroscopy, LCMS is liquid chromatography-coupled mass spectrometry, Hz is hertz, MHz is megahertz, THF is tetrahydrofuran, MeOH is methanol, EtOH is ethanol, DCM is dichloromethane, DEA is diethylamine, DIPEA is diisopropyl ethylamine, DMA is dimethylacetamide, DMF is Ν,Ν-dimethyl formamide, DMSO is dimethyl sulfoxide, EtOH is ethyl alcohol, EtOAc is ethyl acetate, RT is room temperature, HC1 is hydrogen chloride or hydrochloric acid, TFA is trifluoroacetic acid, EtMgBr is ethyl magnesium bromide, NaHCC>3 is sodium bicarbonate, Na2C03 is sodium carbonate, Na2S04 is sodium sulfate, EDC'HCl is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, DCC is N,N- dicyclohexylcarbodiimide, HOBt is hydroxybenzotriazole, PyBOP is benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate, EEDQ is N-efhoxycarbonyl-2-efhoxy- 1,2-dihydroquinoline, DBAD is di-tert-butyl azodicarboxylate, Cbz is benzyloxy carbamate, Val is L-valine, OSu is succinate ester, Cit is L-citroline, Arg is arginine, HMC is 7- hydroxy-4-methylcoumarin, AMC is 7-amino-4-methylcoumarin, cone is concentrated, DTT is dithiothreitol, EDTA is ethylenediamine tetraacetic acid, ELISA is enzyme-linked immunosorbent assay, KOAc is potassium acetate, M+ is molecular ion, [M+H]+ is protonated molecular ion, PBS is phosphate buffered saline, PH is pleckstrin homology, PPh3 is triphenylphosphine, PPM is parts per million, SPR is surface plasmon resonance, TEA is triethylamine, and TMS is tetramethylsilane.
Biotage Isolera® One and CombiFlash® (Teledyne Isco) Automated Flash
Purification Systems were used for the purification of crude products using the eluent combination mentioned in the respective procedures. Flash Chromatography was performed using silica gel (60-120 and 230-400 mesh) from Swambe chemicals, with nitrogen and/or compressed air. Preparative thin-layer chromatography was carried out using silica gel (GF 1500 μΜ 20 x 20 cm and GF 2000 μΜ 20 x 20 cm Prep-scored plates from Analtech, Inc. Newark, DE, USA). Thin-layer chromatography was carried out using pre-coated silica gel sheets (Merck 60 F254). Visual detection was performed with ultraviolet light, p- anisaldehyde stain, ninhydrin stain, potassium permanganate stain, or iodine. Reactions at lower temperature were performed by using cold baths, e.g., H20/ice at 0°C, and acetone/dry ice at -78°C. XH NMR spectra were recorded at 300 MHz with a Bruker 300 DPX and 400 MHz with a Bruker 400 AVI, AVII & AVIII (unless otherwise noted) at ambient temperature, using tetramethylsilane as internal reference. The chemical shift values are quoted in δ (parts per million). Mass spectra of all the intermediates and final compounds were recorded using Acquity® UPLC-SQD (Waters) & Agilent 1100 & Agilent 1200 LCMS with 6130 SQD machines. HPLC spectra were recorded using Agilent 1100 and 1200 series with Quaternary pump systems with diode array detector (DAD) detection using Atlantis CI 8, Hypesil™ BDS column, Sunfire™ CI 8 column, Xbridge™ C 18 and Zorbax® C18 (50 mm x 4.6 mm x 5μ), (150 mm x 4.6 mm x 3μ) & (250 mm x 4.6 mm x 5μ) columns. LCMS spectra were recorded using Agilent 1100 and 1200 series with Quaternary pump systems with diode array detector (DAD) detection using Atlantis® CI 8, Xterra® C8 and Zorbax® C18 (50 mm x 4.6 mm x 5μ) and (150 mm x 4.6 mm x 3μ) columns. UPLC spectra were recorded using Waters Acquity® UPLC-SQD systems with diode array detector (DAD) detection using Acquity® BEH C18 and HSS T3 C18 (50 mm x 2.1 mm x 1.7μ) & (75 mm x 2.1 mm x 1.8μ) columns. A mobile phase of 0.01% of formic acid or TFA with acetonitrile and 0.01% of formic acid or TFA with water with a flow rate of 0.5 mL/min or 1.0 mL/min or 1.5 mL/min at temperature of RT or 40 °C was used.
The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively.
As used herein, the term "about" means a variability of 10 % from the reference given, unless otherwise specified.
Example 1
Figure imgf000088_0001
11 N-Boc-CBI (12) Boc-Pro-CBI (2a) A. tert-Butyl (4-hydroxynaphthalen-2-yl)carbamate 6
A toluene (250 mL) solution of 1 ,3-dihydroxy naphthalene 4 (50 g, 0.3123 mol) and diphenyl methylamine (69.92 mL, 0.406 mol) was heated at 100°C for 6 h in a sealed tube. After that reaction mass was transfer to a par shaker and were added di-tert-butyl dicarbonate (204 mL, 0.94 mol), palladium hydroxide (17 g; 20% Pd(OH)2/C) and dioxane: water (4: 1 , 250 mL). The mixture was shaken at 60 psi hydrogen pressure for 48 h. The mixture was filtered through Celite® bed and filtrate was concentrated under reduced pressure to yield the crude product. The crude residue was subjected to column chromatography on silica gel (60-120 mesh) using ethyl acetate-hexane (10%) as eluent to yield compound 6 (51 g, 87% HPLC purity) in 63% yield over two steps; XH NMR (400 MHz, CDC13): δ 8.10 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.43-7.47 (m, 1H), 7.34-7.38 (m, 1H), 7.27 (s, 1H), 7.19 (s, 1H), 6.66 (s, 1H), 6.22 (br s, 1H), 1.57 (s, 9H); MS calcd. for C15H17N03: 259.1, Found: 160.2 (M + 1-Boc). B. tert-Butyl (4-(benzyloxy)naphthalen-2-yl)carbamate 7
To a 150 mL anhydrous DMF solution of compound 6 (51 g, 0.196 mol) was successively added benzyl bromide (28.12 mL, 0.236 mol), potassium carbonate (40.5 g, 0.294 mol) and terra butyl ammonium iodide (0.72 g, 0.00196 mol). After 3 h stirring at RT, the mixture was poured over ice-cold water (500 mL) and extracted with ethyl acetate (3 x 300 mL). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The brown color solid (51 g, 74.2%, 77% HPLC purity) was used in the next step without further purification; H NMR (300 MHz, DMSO- d6): δ 9.50 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 7.0 Hz, 2H), 7.29-7.44 (m, 5H), 7.21 (s, 1H), 5.21 (s, 2H), 1.45 (s, 9H); MS calcd. for C22H23NO3: 349.1, Found: 350.2 (M + 1).
C. tert-Butyl (4-(benzyloxy)-l-iodonaphthalen-2-yl)carbamate 8
Compound 7 (51 g, 0.145 mol) was dissolved in THF (300 mL) in a 3-necked round bottom flask under nitrogen atmosphere and cooled to -20°C followed by addition of p- toluenesulfonic acid (0.35 g,0.0018 mol). After 10 min, N-iodosuccinimide (39.3 g, 0.175 mol) in THF (200 mL) was added drop wise over 30 min, ensuring that the temperature was maintained at -20°C. After additional stirring at -20°C for 2 h, the mixture was allowed to warm to RT and stirred for an additional 1.5 h. The mixture was poured over saturated sodium bicarbonate solution (500 mL) and extracted with ethyl acetate (3 x 300 mL). The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude material obtained was purified by silica gel (60-120 mesh) column chromatography (5% ethyl acetate in hexane) to obtained product 8 (47 g, 67.7%, 95.8% HPLC purity) as a light brown solid; XH NMR (300 MHz, DMSO-d6): δ 8.72 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.63 (t, J = 8.3 Hz, 1H), 7.34-7.56 (m, 5H), 7.27 (s, 1H), 5.28 (s, 2H), 1.43 (s, 9H); MS calcd. for C22H22INO3: 475.1, Found: 476.3 (M + 1).
D. (R)-tert-butyl (4-(benzyloxy)-l-iodonaphthalen-2-yl)(oxiran-2-ylmethyl)carbamate 9 To a DMF (350 mL) solution of compound 8 (35.1 g, 0.07389 mol) was added 60% sodium hydride (11.82 g, 0.2956 mol) at 0°C and the mixture was stirred at 0°C for 10 min. (s)-(+)-Glycidal nosylate (31.61 g, 0.1219 mol) was then added, ensuring that the temperature was maintained at 0°C and stirred for additional 30 min at 0°C before warming to RT. After 12 h, the mixture was poured over saturated aqueous ammonium chloride solution (200 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were washed with water (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude material thus obtained was subjected to purification by silica gel (60-120 mesh) column chromatography using 15% ethyl acetate in hexane as eluent to yield the product 9 as pale yellow solid (32.6 g, 83.3% yield with 78.5% HPLC purity); XH NMR (400 MHz, DMSO-d6, mixture of rotamers, signed with a and b): 8.22 (dd, J = 8.2, 1.7 Hz, 1H x 2), 8.14 (dd, 7 = 8.1 , 3.4 Hz, 1Η χ 2), 7.69 (t, / = 8.16 Hz, 1Η χ 2), 7.61 (t, 7 = 7.12 Hz, 1Η χ 2), 7.59-7.54 (m, 2H x 2), 7.41-7.31 (m, 3H x 2), 7.18 (s, IH of a), 7.09 (s, IH of b), 5.35 (s, 2H x 2), 3.94 (dd, J = 14.4, 3.8 Hz, 1H of a), 3.86 (dd, J = 14.6, 5.4 Hz, 1H of b), 3.48 (dd, J = 14.8, 5.2 Hz, 1H of a), 3.35-3.30 (m, 1H of b), 3.29-3.23 (m, 1H of a), 3.18-3.12 (m, 1H of b), 2.67-2.61 (m, 1H x 2), 2.38-2.32(m, 1H x 2), 1.26 (s, 9H x 2); MS calcd. for C25H26INO4: 531.1, Found: 554.3 (M + Na).
E. (S)-tert-butyl 6-(benzyloxy)-2-hydroxy-2,3-dihydrobenzo[f]quinoline-4(lH)- carboxylate 10
To an anhydrous THF (330 mL) solution of compound 9 (32.5 g, 0.061205 mol) was added ethyl magnesium bromide (3.0 M in ether) (81.6 mL, 0.2448 mol) at RT. After 30 minutes, the mixture was poured over saturated aqueous ammonium chloride solution (300 mL) at 0°C and extracted with ethyl acetate (3 x 150 mL). The combined organic layers were washed with water (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel (60-120 mesh) column chromatography using 20% ethyl acetate in hexane as eluent to yield compound 10 (17.5 g, 90.16% UPLC purity) in 70.6% yield; H NMR (300 MHz, CDC13): δ 8.32 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.83-7.35 (m, 7H), 7.26 (d, / = 2.0 Hz, 1H), 5.24 (s, 2H), 4.46-4.39 (m, 1H), 3.90-3.81 (m, 2H), 3.39 (dd, J = 17.1, 6.1 Hz 1H), 3.04 (dd, J = 17.1, 5.0 Hz, 1H), 1.56 (s, 9H), OH proton did not appear; MS calcd. for C25H27NO4: 405.2, Found: 306.6 (M + 1-Boc). F. (S)-tert-butyl 2,6-dihydroxy-2,3-dihydrobenzo[f]quinoline-4(lH)-carboxylate 11
To a stirred solution of compound 10 (12.2 g, 0.0301 mol) in THF:MeOH (9: 1, 610 mL) was added ammonium formate (94.7 g, 1.506 mol) and 10% palladium on carbon (6.1 g). The mixture was heated at 70°C for 12 h. The mixture was filtered through a Celite® bed and the filtrate was concentrated under reduced pressure to yield the crude product. The crude material was dissolved in ethyl acetate and washed with water and concentrated to afford product 11 as pale yellow gummy solid (9.0 g, 95%, UPLC purity, 87% yield); H NMR (400 MHz, acetone-d6): δ 8.88 (s, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.52 (t, / = 7.2 Hz, 1H), 7.42 (t, / = 7.00 Hz, 1H), 4.27 (s, 1H), 7.23 (s, 1H), 3.99 (d, J = 11.8 Hz, 1H), 3.52 (dd, J = 16.2, 6.8 Hz, 1H), 3.37 (dd, J = 16.8, 5.8 Hz, 1H), 2.92-2.82 (m, 2H), 1.56 (s, 9H); MS calcd. for Ci8H2iN04: 315.1 , Found: 316.4 (M + 1).
G. (8bR,9aS)-tert-butyl 4-oxo-9,9a-dihydro-lH-benzo[e]cyclopropa[c]indole-2(4H)- carboxylate 12
To a stirred solution of compound 11 (9.0 g, 0.0284 mol) in toluene was added 1, 1 - (azodicarbonyl) dipiperidine (36.04 g, 0.143 mol) at RT. Tributyl phosphine in 50% ethyl acetate solution (35.67 mL, 0.143 mol) was then added drop wise over a period of 10 min and resulting mixture was stirred for additional 30 min. The solvent was removed under reduced pressure and the residue was subjected to purification by silica gel (60-120 mesh) column chromatography. Compound 12 was obtained as pale brown gummy solid (5.2 g, 95% UPLC purity) in 61% yield; XH NMR (400 MHz, acetone-d6): δ 8.07 (dd, J =7.8, 1.01 Hz, 1H), 7.55 (td, / = 7.8, 1.4 Hz, 1H), 7.41 (td, J = 8.6, 1.1 Hz, 1H), 7.12(d, / = 7.8 Hz, 1H), 6.79 (s, 1H), 4.06-4.05 (t, / = 4.9 Hz, 2H), 3.05-3.09 (m, 1H), 1.68 (dd, / = 5.8, 3.1 Hz, 1H), 1.51 (dd, J = 4.5, 1.1 Hz, 1H), 1.55 (s, 9H); MS calcd. for Ci8H19N03: 297.1 , Found: 298.4 (M + 1). H. Boc-Pro-CBI 2a
Compound 12 (5.2 g, 0.0175 mol) was treated with HCl in ethyl acetate (200 mL, ~4 N) at -78°C. After 30 min at -78°C, TLC analysis indicated the consumption of compound 12 and formation of the non-polar compound. The mixture was then warmed to 23 °C and stirred for 1 h. The TLC analysis indicated the conversion of the non-polar spot into polar spot indicated the formation of amine product. The solvent and HCl gas were removed under a stream of nitrogen to yield compound 2 (CBI) as green colored solid (3.8 g, 84% UPLC purity, 93% yield). 1H NMR (300 MHz, DMSO-d6): δ 10.94 (s, 1H), 8.17 (d, J = 8.13 Hz, 1H), 7.91 (d, / = 8.4 Hz, 1H), 7.60 (t, / = 7.1 Hz, 1H), 7.47 (t, / = 7.0 Hz, 1H), 6.87 (s, 1H), 4.29-4.21 (m, 1H), 4.027 (dd, J = 10.9, 3.2 Hz, 1H), 3.76-3.92 (m, 3H), one proton did not appear; MS calcd. for C13H12C1N0: 233.1, Found: 234.2 (M + 1-Boc).
The amine salt 2 (CBI) was dissolved in THF:water (4: 1 ; 40 mL). Triethyl amine (1.0 eq.) and Boc20 (1.0 eq.) were added successively and mixture was stirred at RT. After 4 h, volatiles were removed under reduced pressure and the crude material was purified by silica gel (60-120 mesh) column chromatography to provide compound 2a. Yield 4.6 g, 97.5% LCMS purity; XH NMR (400 MHz, DMSO-d6): δ 10.36 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.72 (br s, 1H), 7.59-7.44 (m, 1H), 7.29-7.26 (m, 1H), 4.10- 3.94 (m, 4H), 3.74 (t, J = 7.6 Hz, 1H), 1.54 (s, 9H); MS calcd. for Ci8H2oClN03: 333.1, Found: 234.2 (M + 1-Boc).
E
Figure imgf000093_0001
11 N-Boc-CBI (12) Pro-CBI (2)
Step A:
A toluene solution of 1 ,3-dihydroxy naphthalene 4 and diphenyl methylamine was heated at 100°C for 4 to 6 h in a sealed tube. After that reaction mass was transfer to a par shaker and were added di-tert-butyl dicarbonate, palladium hydroxide (20% Pd(OH)2/C) and dioxane: water. The mixture was shaken at 60 psi hydrogen pressure for 4 to 6 h.
Step B:
To an anhydrous DMF solution of compound 6 were successively added benzyl bromide, potassium carbonate and tetrabutyl ammonium iodide. After 2 to 3 h stirring at RT, the mixture was poured over ice-cold water and extracted with ethyl acetate.
Step C:
Compound 7 was dissolved in 300 mL of tetrahydrofuran (THF) in 3-necked round bottom flask under nitrogen atmosphere and cooled to -20 °C followed by addition of p- toluenesulfonic acid. After 10 min N-iodosuccinimide in THF was added drop wise over a period of 30 min such that the temperature was maintained at -20°C. After additional stirring at -20 °C for 2 h, the mixture was allowed to warm to RT and stirred for an additional 1.5 h to obtain desired iodo product 8. Step D:
To a DMF solution of compound 8 was added 60% sodium hydride at 0°C and the mixture was stirred at 0°C for 10 min. Then (s)-(+)-Glycidal nosylate was added such that the temperature was maintained at 0°C and stirred for additional 30 min at 0°C before warming up to RT. After 12 h, the mixture was poured over saturated aqueous ammonium chloride solution and extracted with ethyl acetate to yield the desired product 9 as pale yellow solid.
Step E:
To a THF (anhydrous) solution of epoxide compound 9 was added ethyl magnesium bromide (3.0 M in ether) at RT. After 30 minutes to 1 h, the mixture was poured over saturated aqueous ammonium chloride solution at 0°C and extracted with ethyl acetate to yield compound 10.
Step F:
To a stirred solution of compound 10 in THF:MeOH (9:1) was added ammonium formate and 10% palladium on carbon. The mixture was heated at 70°C to 80°C for 20 h to afford product 11.
Step G:
To a stirred solution of compound 11 in toluene was added 1,1 -(azodicarbonyl) dipiperidine (ADDP) at RT. Then tributyl phosphine in 50% ethyl acetate solution was added drop wise over a period of 10 min and resulting mixture was stirred for additional 30 min to provide compound 12 (N-Boc-CBI) (J.P. Lajiness and D.L. Boger, J. Org. Chem. 2011, 76, 583-587).
Step H:
Compound 12 was treated with HCl in ethyl acetate (~4 N) at -78 °C. After 30 min at -78°C, thin layer chromatography (TLC) analysis indicated the consumption of compound 12 and formation of non-polar compound. Then the mixture was warmed to 23 °C and stirred for 30 minutes to 1 h. The TLC analysis indicated the conversion of the non-polar spot into polar spot indicated the formation of amine product as the HCl salt. The solvent and HCl gas was removed under a stream of nitrogen to yield compound 2 (Pro-CBI).
Figure imgf000095_0001
Figure imgf000095_0002
37 DMMI (3)
Example 3 describes the synthesis of compound 3 ("DMMI"). To a stirred solution of 3-hydroxy-4-methoxybenzaldehyde 5 in DMF was added potassium carbonate and 1, 2- dichloroethane. The mixture was stirred at 70°C for 16 h. The mixture was cooled down to RT and 1,2-dichloroethane was removed under reduced pressure to yield compound 35.
To a DMSO solution of methyl chloroacetate was added sodium azide portion wise. After 24 h stirring at RT, water (300 mL) was added and the mixture was extracted with diethyl ether. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuum. Then a solution of aldehyde 35 in MeOH was added to a methanolic solution of methyl azidoacetate. The mixture was cooled to -30°C and sodium methoxide in MeOH was added over 30 min. The mixture was warmed to 0°C and diluted with MeOH and was stirred at 0°C for 16 h to give the desired product 36.
A solution of azido derivative 36 in toluene was heated at reflux for 6 h to 8 h to yield compound 37.
To a solution of compound 37 in water was added 40% aqueous dimethyl amine solution and sodium carbonate. The mixture was stirred at 100 °C for 20 h. The mixture was cooled to RT and solvent was removed under reduced pressure. The residue obtained was dissolved in water (300 mL) and the resulting solution was acidified with HCl. The solution was then evaporated and the residue was purified by preparative HPLC to provide 3 (DMMI). xample 4: Synthesis of Pro CBI-DMMI (1)
Figure imgf000096_0001
Pro-CBI (2) DMMI (3) Pro CBI-DMMI (1)
Coupling of 2 (Pro-CBI) and 3 (DMMI) was accomplished by an EDC coupling reaction to provide 1 (Pro CBI-DMMI), as outlined in Scheme 3.
Figure imgf000096_0002
A. 2-hydroxy-2-(4-nitrophenyl)acetonitrile 14
To a stirred solution of 4-nitro benzaldehyde 13 (13 g, 0.086 mol) in DCM (130 mL), zinc iodide (2.7 g, 0.0086 mol) and trimethylsilyl cyanide (16.15 mL, 0.12 mol) were added at RT. The mixture was refluxed for 3 h. TLC analysis showed the completion of starting material and formation of new non-polar compound. 1 N hydrochloric acid (70 mL) was then added and mixture was heated at 55°C. After 4 h, the mixture was cooled to RT, diluted with water (100 mL) and extracted with DCM (300 mL x 3). The combined organic layers was washed with brine solution and dried over anhydrous Na2S04. The solvent was removed under reduced pressure and resulting crude material (21 g, 43% UPLC purity) was used in the next step without further purification; MS calcd. for C8H6N203: 178.0, Found: 177.1 (M- 1). B. 2-Hydroxy-2-(4-nitrophenyl)acetic acid 15
To a stirred acetic acid (100 mL) solution of compound 14 (21 g) was added 10 N hydrochloric acid (100 mL) and the mixture was heated at 100°C. After 6 h of heating at 100°C, the mixture was cooled to RT and was concentrated at reduced pressure to obtain crude solid. The crude material (27 g) was used in the next step without further purification.
C. Methyl -2-hydroxy-2-(4-nitrophenyl)acetate) 16
To a stirred MeOH (100 mL) solution of crude compound 15 (22 g) was added concentrated H2SO4 (8 mL) and the mixture was heated at 85°C. After 6 h, the mixture was cooled to RT and MeOH was removed under reduced pressure. The resulting crude ester material was dissolved in ethyl acetate, washed with water, brine solution and dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure and crude ester product was purified using column chromatography (silica gel, EtOAc/hexane). Yield 11.3 g (62% over four steps, 0.054 mol), 96% UPLC purity; XH NMR (300 MHz, DMSO-d6): δ 8.21 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 6.45 (d, J = 5.2 Hz, 1H), 5.36 (d, J = 5.0 Hz, 1H), 3.61 (s, 3H); MS calcd. for C9H9N05: 211.0, Found: 212.2 (M + 1).
D. PAMA methyl ester (para-aminomandelic acid, methyl ester; methyl 2-(4- aminophenyl)-2-hydroxyacetate) 17
A MeOH (60 mL) solution of compound 16 (6.0 g) was charged 1.20 g Pd/C (10%
Pd/C) under nitrogen atmosphere. The mixture was then flushed with H2 gas and the mixture was stirred under H2 balloon pressure for 16 h. The palladium catalyst was removed using Celite® bed, and the Celite® bed was washed with MeOH (20 mL x 3). MeOH was removed under reduced pressure to obtain 5.1 g (0.028 mol) of the compound 17 in 99% yield; H NMR (400 MHz, DMSO-d6): δ 7.01 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz, 2H), 5.70 (d, J = 5.2 Hz, 1H), 5.10 (s, 2H), 4.90 (d, 7 = 5.2 Hz, 1H), 3.57 (s, 3H); MS calcd. for &>ΗηΝ03: 181.1, Found: 182.3 (M + 1).
E. Compound 18
To a DCM:THF (1 :1, v/v) solution of compound 17 (1.2 g, 6.62 mmol) were added
Fmoc-Cit-OH (3.1 g, 7.95 mmol) and EEDQ (4.9 g, 19.88 mmol) successively at RT. After 16 h stirring at RT, volatiles were removed under reduced pressure and crude material was purified by column chromatography (silica gel) using DCM/MeOH. Yield 2.6 g (4.64 mmol, 70%, 98% UPLC purity); XH NMR (300 MHz, DMSO-d6): δ 10.05 (s, 1H), 7.87 (d, / = 7.5 Hz, 2H), 7.73-7.65 (m, 3H), 7.57 (d, 7 = 8.2 Hz, 2H), 7.42-7.29 (m, 6H), 6.0 (d, J = 3.8 Hz, 2H) 5.42 (s, 2H), 5.05 (d, J = 5.2 Hz, 1H), 4.24 (t, J = 5.2 Hz, 2H), 3.57 (s, 3H), 3.15 (d, J = 5.3 Hz, 2H), 3.01-2.94 (m, 2H), 1.62-1.21 (m, 4H); MS calcd. for C30H32N4O7: 560.2, Found: 561.4 (M + 1).
F. Compound 19
To a stirred solution of triphenyl phosphine (0.70 g, 2.67 mmol) in dry tetrahydrofuran (25 mL) was added DBAD (di-tert-butyl azodicarboxylate; 0.614 g, 2.67 mmol) at 0°C followed by HMC (0.235 g, 1.33 mmol). The mixture was stirred at 0°C for 15 min before addition of compound 18 (0.5 g, 0.892 mmol). Triethylamine (0.4 mL, 02.67 mmol) was then added to the mixture and was stirred for 16 h at RT. Volatiles were removed under reduced pressure and crude product was purified by column chromatography (silica gel) in ethyl acetate/Hexane. Two fractions containing compound 19 were collected one with 87% UPLC purity (0.15 g) and other with 54% UPLC purity (0.27 g); MS calcd. for C40H38N4O9: 718.3, Found: 719.4 (M + 1).
G. Compound 20a, Cbz-Val-Cit-PAMA-(methyl carboxylate)-HMC
To a stirred DMF (10 mL) solution of compound 19 (0.20 g, 0.297 mmol) was added piperidine (0.2 mL) at 0°C and solution was stirred for 1.5 h at RT. The DMF was removed under reduced pressure at 50°C. The crude material was triturated with diethyl ether to yield 0.12 g of crude material with 52% UPLC purity. The crude material was dissolved in DMF (2.5 mL) and Cbz-Val-OSu (0.168 g, 0.36 mmol) and diisopropyl ethylamine (0.13 mL, 0.725 mmol) were then added successively at 0°C. The mixture was stirred at RT for 16 h under N2 before addition of cold water (10 mL). The solid was filtered, washed with cold water and dried. The crude solid was purified by preparative HPLC method to obtain 75 mg of compound 20a; HPLC purity 99% (Rt = 10.28 min); System: Agilent 1200; detector: DAD max chromatogram (210-400 nm); column: Atlantis dC18 (250 x 4.6 mm, 5μ): A: water + 0.1% TFA; B: acetonitrile; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 10.17 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 7.8 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.36-7.28 (m, 6H), 7.05.70 (m, 2H), 6.23 (d, J = 0.8 Hz, 1H), 6.22 (s, 1H), 5.98 (s, 1H), 5.42 (s, 2H), 5.03 (s, 2H), 4.40 (q, J = 6.0 Hz, 1H), 3.91 (t, J = 8.4 Hz, 1H), 3.77 (s, 3H), 3.02- 2.89 (m, 2H), 2.39 (s, 3H), 2.00-1.91 (m, 1H), 1.69-1.57(m, 2H), 1.42-1.37 (m, 2H), 0.87 (d, 7 = 6.8 Hz, 3H), 0.83 (d, / = 6.8 Hz, 3H); MS calcd. for C38H43N5O10: 729.3, Found: 730.4 (M + 1).
Figure imgf000099_0001
A. Compound 21
To a stirred DMF (10 mL) solution of compound 19 (0.4 g, 0.56 mmol, 54% UPLC purity) was added concentrated ammonium hydroxide solution (10 mL) and the mixture was stirred for 1 h at RT. Volatiles were removed under reduced pressure and crude gummy material was triturated with diethyl ether to obtain crude material (0.20 g, 40% UPLC purity) which was used in next step without further purification; MS calcd. for C39H37N5O8: 703.3, Found: 704.4 (M + 1). B. Compound 22, Cbz-Val-Cit-PAMA-(amide)-HMC
To a stirred DMF (10 mL) solution of compound 21 (0.2 g with 40% UPLC purity, 0.42 mmol) were added Cbz-Val-OSu (0.22 g, 0.625 mmol), di-isopropyl ethylamine (0.21 mL, 1.24 mmol) successively at 0°C. After 16 h of stirring at RT, the mixture was diluted with cold water (20 mL) and sonicated for 2 min. The solid was filtered, washed with cold water and dried. The crude solid was purified by preparative HPLC to afford 15 mg of compound 22; HPLC purity 96% (Rt = 8.53 min); System: Agilent 1200; detector: DAD max chromatogram (210-400 nm); column: Atlantis dC18 (250 x 4.6 mm), 5μ: A: water + 0.1% TFA; B: acetonitrile; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.59 (d, J = 6.8 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.36-7.28 (m, 6H), 7.05.70 (m, 2H), 6.95 (s, 1H), 6.62 (s, 1H), 6.22 (s, 1H), 5.98 (s, 1H), 5.76 (s, 1H), 5.42 (s, 2H), 5.04 (s, 2H), 4.40 (q, J = 6.0 Hz, 1H), 3.91 (t, J = 8.4 Hz, 1H), 3.77 (s, 3H), 3.01-2.89 (m, 2H), 2.38 (s, 3H), 1.99-1.91 (m, 1H), 1.69-1.57(m, 2H), 1.42-1.37 (m, 2H), 0.88-0.84 (m, 6H); MS calcd. for C37H42N6O9: 714.3, Found: 715.3 (M + 1). Example 7: Preparation of Cbz-Val-Cit-PAMA-(PEG-amide)-HMC (23)
Figure imgf000100_0001
20a 23
To a stirred MeOH: water (v/v, 1 :1, 10 mL) solution of compound 20a (0.4 g, 50% HPLC purity, 0.54 mmol) was added LiOH.H20 (0.067 g, 1.64 mmol). After 2 h, MeOH was removed and the solution was diluted with water (10 mL) and washed with ethyl acetate (20 mL). The water layer was acidified with 1.5 N HC1, extracted with ethyl acetate (20 mL x 3), the combined organic layers were dried over anhydrous sodium sulfate and concentrated to obtained 0.1 g of crude acid (37% HPLC purity). The crude acid was dissolved in DMF (5 mL) and CH3OCH2CH2OCH2CH2NH2 (0.097 g, 0.40 mmol), PyBop (0.138 g, 0.265 mmol) - DIPEA (0.06 mL, 0.40 mmol) were added successively at 0°C. After 16 h, the mixture was diluted with cold water (20 mL), extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated. Crude product was purified by preparative HPLC to obtain 4 mg of compound 23. HPLC purity 90% (Rt = 9.22 min); System: Agilent 1200; detector: DAD max chromatogram (210-400 nm); column: Atlantis dC18 (250 x 4.6 mm), 5μ: A: water + 0.1% TFA; B: acetonitrile; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 8.49 (t, / = 5.8 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.36-7.28 (m, 7H), 7.03 (dd, / = 8.8, 2.5 Hz, 1H), 6.95 (d, / = 2.4 Hz, 1H), 6.22 (d, J = 1.2 Hz, 1H), 5.98 (t, / = 5.7 Hz, 1H), 5.84 (s, 1H), 5.42 (s, 2H), 5.03 (s, 2H), 4.41 (q, / = 6.0 Hz, 1H), 3.92 (t, J = 6.7 Hz, 1H), 3.34-3.21 (m, 6H), 3.20 (s, 3H), 3.09-2.89 (m, 4H), 2.38 (s, 3H), 1.99-1.94 (m, 1H), 1.69-1.57 (m, 2H), 1.45-1.36 (m, 2H), 0.90-0.82 (m, 6H); MS calcd. for C42H52N6O11: 816.4, Found: 817.5 (M + 1).
Figure imgf000101_0001
A. Compound 20
To a DCM:THF:MeOH (1 : 1 : 1 , v/v) solution of compound 17 (para-amino mandelic acid methyl ester; 6.2 g, 0.034 mol) were added Cbz-Val-Cit-OH (20.0 g, 0.050 mol) and N- ethoxycarbonyl-2-ethoxy- l,2-dihydroquinoline (EEDQ) (16.0 g, 0.06 mol) successively at RT. After 16 h stirring at RT, volatiles were removed under reduced pressure and crude material was purified by column chromatography (silica gel) using DCM/MeOH. Yield 4.2 g (7.3 mmol, 21.5%, 94% UPLC purity; XH NMR (400 MHz, DMSO-d6): δ 10.04 (s, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.56 (d, J = 7.3 Hz, 2H), 7.36-7.30 (m, 8H), 5.97 (s, 2H), 5.41 (s, 2H), 5.07 (s, 1H), 5.03 (s, 2H), 4.40 (q, J = 7.8 Hz, 1H), 3.91 (t, J = 7.7 Hz, 1H), 3.58 (s, 3H), 3.03-2.92 (m, 2H), 1.97 (q, J = 6.6 Hz, 1H), 1.68-1.56 (m, 2H), 1.50-1.32 (m, 2H), 0.88 (d, J = 7.6 Hz, 3H), 0.85 (d, J = 7.4 Hz, 3H); MS calcd. for CzsHsvNjOg: 571.3, Found: 572.4 (M + 1).
B. Compound 24, Cbz-Val-Cit-PAMA-(PEG-Amide)-OH
To a 10 mL MeOH:water (10: 1 , v/v) solution of compound 20 (1.0 g, 1.75 mmol) was added LiOH.H20 (0.147 g, 3.50 mmol) and stirred for 2 h at RT. After the disappearance of the starting material 20 (TLC analysis), the mixture was neutralized with acidic resin. Resin was filtered off and removal of MeOH yielded crude acid. The crude acid (0.900 g) was dissolved in DMF (10 mL) and NH2CH2CH2OCH2CH2OCH3 (0.571 g, 4.85 mmol), benzotriazol-l -yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (1.68 g, 3.23 mmol), DIPEA (0.86 mL, 4.85 mmol) were added successively at 0°C. The mixture was stirred for 16 h at RT under nitrogen atmosphere. The mixture was diluted with cold water (50 mL), extracted with 10% butanol in ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography (silica gel) using DCM/MeOH. Compound 24 was obtained in 46% yield (0.49 g) with 90% UPLC purity; XH NMR (300 MHz, DMSO-d6): δ 10.00 (s, 1H), 8.05 (d, J = 7.40 Hz, 1H), 7.95-7.85 (m, 1H), 7.51-7.59 (m, 1H), 7.38-7.25 (m, 7H), 6.11 (d, J = 1.80 Hz, 1H), 6.09-6.10 (m, 1H), 5.38 (d, J = 12.60 Hz, 2H), 5.02 (s, 2H), 4.83 (d, 7 = 4.50 Hz, 1H), 4.34-4.45 (m, 1H), 4.16-4.27 (m, 1H), 3.81-3.90 (m, 1H), 3.55 (t, J = 1.80 Hz, 2H), 3.46 (t, J = 3.30 Hz, 2H), 3.41-3.24 (m, 4H), 3.23 (s, 3H), 3.01-2.91 (m, 2H), 2.01-1.90 (m, 2H), 1.69-1.31 (m, 4H), 0.82-0.87 (m, 6H); MS calcd. for C32H46N609: 658.3, Found: 659.7 (M + 1).
C. Compound 25, Cbz-Val-Cit-PAMA-(PEG-Amide)-OC(0)-AMC
To a DMF solution of compound 24 (0.1 g, 0.152 mmol) and DIPEA (0.039 mL, 0.228 mmol) was added freshly prepared isocyanate of AMC (0.036 g, 0.182 mmol) at RT under nitrogen atmosphere. Volatiles were removed under reduced pressure and crude material was purified by prep HPLC to afford 8 mg of compound 25 (91% HPLC purity); 400 MHz, DMSO-d6: δ 10.60 (s, 1H), 10.28 (s, 1H), 8.47 (t, J = 8.6 Hz, 1H), 8.32-8.28 (m, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.70-7.60 (m, 3H), 7.56 (d, J = 2.0 Hz, 1H), 7.49-7.42 (m, 3H), 7.35-7.29 (m, 5H), 6.25 (s, 1H), 6.19 (s, 1H), 5.88 (s, 1H), 5.49 (s, 2H), 5.04 (s, 2H), 4.45-4.35 (m, 1H), 3.98-3.90 (m, 1H), 3.44-3.49 (m, 4H), 3.29-3.19 (m, 2H), 3.20 (s, 3H), 3.05-2.90 (m, 3H), 2.39 (s, 3H), 2.03-1.92 (m, 2H), 1.69-1.31 (m, 4H), 0.90-0.80 (m, 6H); MS calcd. for C43H53N7O12: 859.4, Found: 860.2 (M + Na). Examp
Figure imgf000102_0001
To a MeOH:water (10: 1, v/v) solution (30 mL) of compound 20 (3.0 g, 0.0052 mol) was added LiOH.H20 (0.44 g, 0.0105 mol) at RT. After completion of the starting material (TLC analysis), the mixture was neutralized with acidic resin. The resin was removed by filtration and removal of MeOH yielded crude acid. The crude acid was dissolved in DMF (30 mL) and NH2CH2CH2OCH2CH2OCH2CH2NHB oc (1.8 g, 0.0064 mol), PyBOP (4.2 g, 0.0080 mol), DIPEA (1.3 mL, 0.0107 mol) were added successively at 0°C. The mixture was stirred for 18 h at RT under nitrogen atmosphere. The mixture was diluted with minimum amount of ice water, and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated. Crude product was purified by column chromatography (silica gel) using DCM/MeOH. Compound 26 was obtained in 61% yield (2.7 g) with 90% UPLC purity; XH NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 8.51 (s, 1H), 8.12 (d, 7 = 7.4 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.39-7.31 (m, 6H), 7.04 (dd, 7 = 8.8, 2.40, Hz, 1H), 6.95 (d, 7 = 2.40 Hz, 1H), 6.82-6.78 (m, 1H), 6.22 (s, 1H), 6.00- 5.93 (m, 1H), 5.84 (s, 1H), 5.42 (s, 2H), 5.03 (s, 2H), 4.40 (t, 7 = 7.6 Hz, 1H), 3.95-3.89 (m, 1H), 3.49-3.44 (m, 8H), 3.29-3.19 (m, 2H), 3.09-2.88 (m, 4H), 2.38 (s, 3H), 2.03-1.92 (m, 2H), 1.69-1.31 (m, 3H), 1.36 (s, 9H), 0.80-0.90 (m, 6H); MS calcd. for C48H63N7Oi3: 945.4, Found: 968.4 (M + Na).
Figure imgf000103_0001
A. Compound 28, Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-HMC
To a THF:water (10: 1, v/v; 3 mL) solution of compound 20 (0.1 g, 0.13 mmol) was added LiOH.H20 (0.035 g, 0.82 mmol) at 0°C at RT for 2 h. After consumption of starting material 20 (TLC analysis), the mixture was neutralized with citric acid. The solid was removed using filtration and removal of MeOH yielded crude acid. The crude acid was dissolved in DMF (3.0 mL) and NH2CH2CH2OCH2CH2OCH2CH2NHB0C (0.04 g, 0.16 mol), PyBOP (0.10 g, 0.20 mmol), DIPEA (0.03 mL, 0.27 mmol) were added successively at 0°C. The mixture was stirred for 18 h at RT under nitrogen atmosphere. The mixture was diluted with minimum amount of ice water, and extracted with ethyl acetate (3 x 25 mL). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated. Crude product was purified by column chromatography (silica gel) using DCM/MeOH. Compound 28 was obtained in 76% yield (0.10 g) with 77% UPLC purity; XH NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 8.51 (s, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.39-7.31 (m, 6H), 7.04 (dd, J = 8.8, 2.40, Hz, 1H), 6.95 (d, J = 2.40 Hz, 1H), 6.82-6.78 (m, 1H), 6.22 (s, 1H), 6.00-5.93 (m, 1H), 5.84 (s, 1H), 5.42 (s, 2H), 5.03 (s, 2H), 4.40 (t, J = 7.6 Hz, 1H), 3.95-3.89 (m, 1H), 3.49-3.44 (m, 8H), 3.29-3.19 (m, 2H), 3.09-2.88 (m, 4H), 2.38 (s, 3H), 2.03-1.92 (m, 2H), 1.69-1.31 (m, 3H), 1.36 (s, 9H), 0.80-0.90 (m, 6H); MS calcd. for C48H63N7Oi3: 945.4, Found: 968.4 (M + Na).
B. Compound 29, Cbz-Val-Cit-PAMA-(MB-PEG3-amide)-HMC
To a DCM solution of compound 28 (0.080 g, 0.029 mmol) was added TFA (0.10 mL) at -10°C. After completion of the reaction, DCM was removed under a nitrogen stream. The crude material was dissolved in DMF (2 mL). To the resulting solution was added succinate ester 27 (52 mg, 0.189 mmol) and DIPEA (0.04 mL, 0.37 mmol) at RT. After completion of the reaction (monitored using LCMS), DMF was removed under reduced pressure and crude material obtained was purified using prep HPLC to afford 20 mg of compound 29 (0.006 mmol, 91% LCMS purity, 85% HPLC purity) in 21% Yield; HPLC purity 85% (Rt = 8.84 min); System: Agilent 1200; detector: DAD max chromatogram (210- 400 nm); column: Atlantis dC18(250 x 4.6 mm), 5μ: A: water + 0.1% TFA; B: acetonitrile; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; 400 MHz, DMSO-d6: δ 10.09 (s, 1H), 8.49 (t, / = 5.6 Hz, 1H), 8.10 (d, 7 = 7.4 Hz, 1H), 7.82 (t, J = 5.5 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.35- 7.29 (m, 7H), 7.02 (dd, 7 = 8.8, 2.4 Hz, 1H), 6.99 (s, 2H), 6.94 (d, 7 = 2.4 Hz, 1H), 6.21 (d, 7 = 1.1 Hz, 1H), 5.96 (t, 7 = 4.8 Hz, 1H), 5.83 (s, 1H), 5.41 (s, 2H), 5.03 (s, 2H), 4.40 (q, 7 = 5.8 Hz, 1H), 3.91 (t, 7 = 5.6 Hz, 1H), 3.49-3.21 (m, 11H), 3.24 (t, 7 = 4.96 Hz, 2H), 3.08- 2.90 (m, 2H), 2.37 (s, 3H), 2.03 (t, 7 = 7.2 Hz, 2H), 2.00-1.90 (m, 1H), 1.67 (p, 7 = 7.4 Hz, 2H), 1.29-1.13 (4 H), 0.92-0.80 (m, 6H); MS calcd. for C51H62N8014: 1010.2, Found: 1012.2 (M + 2).
Example 11: Cbz-Val-Cit-PAMA-(MB-PEG4-amide)-HMC (31)
Figure imgf000105_0001
31
A. Compound 30, Cbz-Val-Cit-PAMA-(Boc-PEG4-amide)-HMC
To a THF: water (10: 1, v/v, 6 mL) solution of compound 20 (0.3 g, 0.41 mmol) was added LiOH.H20 (0.051 g, 1.2 mmol) at 0°C and stirred at RT for 3 h. After the disappearance of compound 20 from the mixture (TLC analysis), it was neutralized with citric acid. The solid material was removed by filtration and removal of MeOH yielded (0.25 g) crude acid. The crude acid was dissolved in DMF (5.0 mL) and NH2CH2CH2OCH2CH2O- CH2CH2OCH2CH2NHB0C (0.12 g, 0.41 mol), PyBOP (0.27 g, 0.52 mmol), DIPEA (0.12 mL, 0.69 mmol) were added successively at 0°C. The mixture was stirred for 18 h at RT under nitrogen atmosphere. The mixture was diluted with minimum amount of ice water, and extracted with ethyl acetate (3 x 25 mL). Combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated. Crude product was purified by column chromatography (silica gel) using DCM/MeOH. Compound 30 was obtained in 67% yield (0.27 g); XH NMR (400 MHz, DMSO-d6): δ 10.1 (s, 1H), 8.5 (s, 1H), 8.12 (d, J = 7.3 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 8.70 Hz, 2H), 7.48 (d, J = 7.96 Hz, 2H), 7.38-7.31 (m, 8H), 7.03 (d, 7 = 8.10 Hz, 1H), 6.95 (s, 1H), 6.78-6.72 (m, 1H), 6.22 (s, 1H), 6.00-5.93 (m, 1H), 5.84 (s, 1H), 5.42 (s, 2H), 5.03 (s, 2H), 4.45-4.38 (m, 1H), 3.49-3.44 (m, 8H), 3.40-3.33 (m, 4 H), 3.10-2.90 (m, 6H), 2.38 (s, 3H), 2.00-1.90 (m, 3H), 1.75-1.71 (m, 1H), 1.36 (s, 9H), 0.90-0.80 (m, 6H); MS calcd. for C5oH67N7014: 989.5, Found: 240.3 (M + 1).
B . Compound 31 , Cbz- Val-Cit-PAMA-(MB -PEG4-amide)-HMC
To a DCM solution of compound 30 (0.16 g, 0.16 mmol) was added TFA (0.5 mL) at
-10°C for 2 h. After completion of the reaction, DCM was removed under a nitrogen stream. The crude material was obtained as a TFA salt (0.15 g) with 79% LCMS purity, and was then dissolved in DMF (2 mL). To the resulting solution was added succinate ester 27 (90 mg, 0.33 mmol) and DIPEA (0.057 mL, 0.33 mmol) at RT. After completion of the reaction (monitored using LCMS), DMF was removed under reduced pressure and the crude material was purified using prep HPLC to afford 25 mg of compound 31 (94% LCMS purity, 91% HPLC purity) in 14% yield; HPLC purity 91% (Rt = 8.95 min); System: Agilent 1200; detector: DAD max chromatogram (210-400 nm); column: Atlantis dC18(250 x 4.6)mm, 5μ: A: water + 0.1% TFA; B: acetonitrile; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.49 (t, J = 5.6 Hz, 1H), 8.11 (d, 7 = 7.2 Hz, 1H), 7.83 (t, J = 5.4 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.35-7.29 (m, 7H), 7.02 (dd, J = 8.8, 2.4 Hz, 1H), 6.99 (s, 2H), 6.94 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 1.1 Hz, 1H), 5.97 (t, J = 5.2 Hz, 1H), 5.83 (s, 1H), 5.41 (s, 2H), 5.01 (s, 2H), 4.40 (q, J = 5.8 Hz, 1H), 3.9 (t, J = 5.6 Hz, 1H), 3.56 (s, 1H), 3.49-3.32 (m, 12H), 3.29-3.22 (m, 2H), 3.19 (q, J = 5.7 Hz, 2H), 3.06-2.90 (m, 2H), 2.37 (s, 3H), 2.03 (t, / = 7.2 Hz, 2H), 2.00-1.90 (m, 1H), 1.67 (p, / = 7.4 Hz, 2H), 1.29-1.13 (4 H), 0.93-0.81 (m, 6H); MS calcd. for C53H66N8Oi5: 1054.5, Found: 1056.4 (M + 2).
Table 1
Illustrative PAMA Linker Com ounds with Surrogate Drug
Figure imgf000107_0001
The compounds listed in Table 1 incorporate 7-hydroxy-4-methylcoumarin (HMC) as a surrogate for a hydroxy-linked drug, and 7-amino-4-methylcoumarin (AMC) as a surrogate for an amine-linked drug, and variations in the PAMA linker structure. These compounds were assayed using a fluorescence assay method as described below (Example 155) to determine the amount of surrogate prodrug/drug released under different conditions and for variations in the PAMA linker structure of formula (IX). These compounds are also used as synthetic intermediates to prepare a conjugate of formula (I), in which HMC or AMC is incorporated as a surrogate drug/prodrug.
The compounds listed in Table 2 and Table 3 are prepared in a similar manner to the Examples described above. These compounds also incorporate 7-hydroxy-4-methylcoumarin (HMC) as a surrogate for a hydroxy-linked drug, and 7-amino-4-methylcoumarin (AMC) as a surrogate for an amine-linked drug, and they further incorporate variations in the PAMA linker structure of formula (IX). These compounds are assayed using the fluorescence assay method as described below (Example 155) to determine the amount of surrogate prodrug/drug released under different conditions and for different variations in the PAMA linker structure of formula (IX). The compounds listed in Table 2 and Table 3 are also used as synthetic intermediates to prepare a conjugate of formula (I), in which HMC or AMC is incorporated as a surrogate drug/prodrug.
Figure imgf000108_0001
Figure imgf000109_0001
107
Figure imgf000110_0001
108 Table 3
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000113_0002
A. Trichloroacetimidate (32)
To a DMF (10 mL) solution of compound 26 (2.0 g, 2.54 mmol) was added CS2CO3 (1.6 g, 5.08 mmol) at RT. The mixture was cooled to 0°C and CC13CN (2.5 mL, 25.40 mmol) was added. The mixture was allowed to warm to RT over 2 h and stirred for additional 4 h. The mixture was poured over water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL). The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel (60-120 mesh) column chromatography using 10% MeOH in DCM as eluent to yield compound 32 (1.8 g) in 76% yield. Compound 32 was used in next step without further purifications. lH NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1H), 9.50 (s, 1H), 8.19 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.4 Hz, 1H), 7.30-7.40 (m, 5H), 6.75 (s, 1H), 5.94-5.98 (m, 2H), 5.41 (s, 2H), 5.03 (s, 2H), 4.40-4.43 (m, 1H), 3.92 (t, J = 7.9 Hz, 1H), 3.51-3.44 (m, 4H), 3.36-3.33 (m, 4H), 3.29-3.18 (m, 2H), 3.08-2.94 (m, 6H), 2.00-1.91 (m, 1H), 1.73-1.35 (m, 4H), 1.36 (s, 9H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H); MS calcd. for G10H57CI3N8O11 : 930.3, Found: 953.2 (M + Na).
B. Compound 33
To a suspension of 4A molecular sieves (0.2 g) in dry CH3CN were added compound 32 (1.04 g, 1.1 mmol) and compound 2a (Boc-Pro-CBI) (0.25 g, 0.75 mmol) at RT. After stirring at RT for 2 h, the mixture was cooled to -10°C and BF3-efher (0.136 mL, 1.1 mmol) was added. The mixture was allowed to warm to RT over 2 h and stirred for additional 16 h. An additional 0.40 mL of BF3- ether was added and solution was stirred at RT overnight. The mixture was neutralized using NEt3 (2 mL) and the molecule sieves were removed by filtration. Volatiles were removed under reduced pressure and resulting crude material was purified by silica gel (60-120 mesh) column chromatography using DCM:MeOH: NH3 as eluent to afford compound 33 (0.2 g, 61% LCMS purity) in 27% yield; MS calcd. for C46H59CIN8O9: 902.4, Found: 903.2 (M + H). C. Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-Pro-CBI (34)
To a MeOH solution of compound 33 (0.40 g, 0.44 mmol) was added Boc20 (0.091 mL, 0.39 mmol) and NEt3 (0.10 mL, 0.88 mmol) at RT. After 4 h, volatiles were removed under reduced pressure and resulting crude material was purified by silica gel (60-120 mesh) column chromatography using DCM:MeOH as eluent to yield compound 34 (0.20 g, 45% yield, 97% LCMS purity); MS calcd. for CSIHOTCINSOU: 1002.5, Found: 1003.4 (M + H).
Examp
Figure imgf000114_0001
Figure imgf000114_0002
A. 3-(2-chloroethoxy)-4-methoxybenzaldeliyde (35)
To a stirred solution of 3-hydroxy-4-methoxybenzaldeliyde 5 (25 g, 0.1664 mol) in DMF was added potassium carbonate (113.5 g, 0.8223 mol) and 1 ,2-dichloroethane (98.96 mL, 3.2894 mol). The mixture was stirred at 70°C for 16 h. The mixture was cooled to RT and 1 ,2-dichloroethane was removed under reduced pressure. The slurry was poured onto ice and extracted with diethyl ether (3 x 500 mL) and ethyl acetate (3 x 400 mL). The combined organic layer was successively washed with water (3 x 500 mL) and brine (2 x 300 mL), dried over anhydrous sodium sulfate and solvent was removed under vacuum. The crude compound was purified by crystallization using 3 : 1 ratio of ethyl acetate and hexanes to yield compound 35 as white solid (27.5 g, 78.17% yield with 98% HPLC purity); XH NMR (300 MHz, DMSO-d6): δ 9.82 (s, 1H), 7.58 (dd, J = 8.3, 1.6 Hz, 1H), 7.41 (d, J = 1.6 Hz, 1H), 7.19 (d, / = 8.3 Hz, 1H), 4.30 (t, / = 5.3 Hz, 2H), 3.95 (t, / = 4.9 Hz, 2H), 3.82 (s, 3H), 3.85 (s, 3H); MS calcd. for CioHnClCb: 214.0, Found: 215.2 (M + 1). B. Methyl 2-Azido-3-[3-(2-chloroethoxy)-4-methoxyphenyl] acrylate (36)
To a DMSO (270 mL) solution of methyl chloroacetate (99.23 mL, 1.1265 mol) was added sodium azide (110.07 g, 1.6934 mol) portion wise. After 24 h of stirring at RT, water (300 mL) was added and the mixture was extracted with diethyl ether (3 x 500 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to 250 mL. A solution of aldehyde 35 (26.55 g, 0.1241 mol) in MeOH (250 mL) was then added. The mixture was cooled to -30°C and sodium methoxide (30% in MeOH, 0.9305 mol, 167.5 mL) solution was added over 30 min. The mixture was warmed to 0°C, diluted with MeOH (250 mL) and stirred at 0°C for 16 h. Water (200 mL) was added to the mixture and extracted with DCM (3 x 500 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to give compound 36 as pale yellow solid (27.4 g, 71 % yield with 94% HPLC purity); XH NMR (300 MHz, DMSO-d6): δ 7.53 (s, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.89 (s, 1H), 4.24 (t, / = 5.3 Hz, 2H), 3.93 (t, J = 4.9 Hz, 2H), 3.82 (s, 3H), 3.80 (s, 3H); MS calcd. for
Figure imgf000115_0001
311.1, Found: 284.2 (cyclized form).
C. Methyl 5-(2-chloroethoxy)-6-methoxy-lH-indole-2-carboxylate (37)
A solution of azido derivative 36 (27.3 g, 0.0877 mol) in toluene (300 mL) was heated at reflux for 6 h. The mixture was then cooled to RT and toluene was removed under reduced pressure. The gummy solid was purified by crystallization using 2: 1 ratio of hexanes and ethyl acetate. The solid was filtered and again washed with hexane to yield compound 37 as pale-yellow solid (15.4 g, 62% yield with 96% HPLC purity); XH NMR (300 MHz, DMSO-d6): δ 11.66 (s, 1H), 7.14 (s, 1H), 7.01 (m, 1H), 6.89 (s, 1H), 4.22-4.18 (m, 2H), 3.95-3.91 (m, 2H), 3.82 (s, 3H), 3.81 (s, 3H); MS calcd. for Ci3H14ClN04: 283.1, Found: 284.2 (M + 1).
D. 5-[2-(Dimethylamino)ethoxy]-6-methoxy-lH-indole-2-carboxylic (DMMI 3)
To a solution of compound 37 (5 g, 0.01766 mol) in water (450 mL) was added 40% aqueous dimethyl amine solution (45 mL, 0.3533 mol) and sodium carbonate (4.679 g, 0.04415 mol). The mixture was stirred at 100°C for 20 h. The mixture was cooled to RT and solvent was removed under reduced pressure. The residue was dissolved in water (300 mL) and the resulting solution was acidified with 1.5 N HC1. The solution was then evaporated to dryness and the resulting crude product purified by preparative HPLC to yield a black solid (1.5 g, 30% yield with 93% HPLC purity); XH NMR (300 MHz, DMSO-d6): δ 11.55 (s, 1H), 9.68 (br s, 1H), 7.24 (s, 1H), 9.96 (d, J = 1.6 Hz, 1H), 6.91 (s, 1H), 4.25 (t, J = 4.9 Hz, 2H), 3.55-3.49 (m, 2H), 3.80 (s, 3H), 2.90 (s, 3H), 2.89 (s, 3H); MS calcd. for ^Η18Ν204: 278.1, Found: 279.0 (M + 1). e)-Pro-CBI-DMMI (40)
Figure imgf000116_0001
3 38
Figure imgf000116_0002
A. Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-Pro-CBI-DMMI (39)
To a DMF (2 mL) solution of DMMI 3 (0.2 g, 0.72 mmol) was added 1,1'- carbonyldiimidazole (0.165 g, 0.93 mmol) portion wise at 0°C. After 2 h of stirring at RT, the mixture was transferred to stirred DMF solution of amine 34 (0.14 g, 0.140 mmol) and sodium carbonate (0.148 g, 1.40 mmol). The reaction was continued for another 16 h. Progress of the reaction was monitored using LCMS. After completion of the reaction, solid was removed by filtration and solution was neutralized with formic acid. DMF was removed using reduced pressure and crude material was purified using prep HPLC to obtain 39 as pale yellow solid (0.075 g, 98% LCMS purity) in 42.6% yield; XH NMR (400 MHz, DMSO-d6): mixture of diastereomers; δ 11.69 (s, 1H), 11.60 (s, 1H), 10.02 (s, 1H), 9.85 (s, 1H), 8.46 (s, 1H), 8.20 (s 1H), 8.20 (s, 1H x 2), 8.13 (d, J = 7.4 Hz, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.92 (t, 7 = 7.8 Hz, 1H), 7.84 (t, / = 5.5 Hz, 1H), 7.63-7.59 (m, 1H x 2), 7.43-7.25 (m, 8H x 2), 7.20- 7.10 (m, 3H x 2), 7.06 (d, 7 = 7.2 Hz, 1H x 2), 6.96 (s, 1H), 6.95 (s, 1H), 6.78-6.72 (m, 1H x 2), 6.55 (d, J = 7.6 Hz, 1H x 2), 5.97 (d, J = 4.00 Hz, 1H x 2), 5.43 (s, 2H), 5.41 (s, 2H), 5.03 (s, 2H), 5.02 (s, 2H), 4.81-4.72 (m, 1H x 2), 4.50-4.30 (m, 2H x 2), 4.13-4.01 (m, 3H x 2), 3.90 (q, J = 8.0 Hz, 2H x 2), 3.82 (s, 3H), 3.81 (s, 3H), 3.75-3.23 (m, 11H x 2), 3.19-2.85 (m, 6H x 2), 2.73 (t, J = 5.08 Hz, 2H x2), 2.29 (s, 6H), 2.28 (s, 6H), 2.01-1.90 (m, 1H x 2), 1.70- 1.30 (m, 4H x 2), 1.34 (s, 9H), 1.33 (s, 9H), 0.91-0.77 (m, 6H x 2); MS calcd. for CesHfflClNioOw: 1262.6, Found: 1264.6 (M + 2).
B. Cbz-Val-Cit-PAMA-(MB-PEG3-amide)-Pro-CBI-DMMI (40)
To an ethyl acetate suspension of 39 (0.030 g, 0.024 mmol) was added HC1 (0.4 mL) in EtOAc (~4 N) at -10°C for 2 h. After completion of the reaction, EtOAc was removed under a nitrogen stream. The crude material was dissolved in DMF (2 mL). To the resulting solution was added succinate ester 27 (16.6 mg, 0.059 mmol) and sodium carbonate (6.3 mg, 0.059 mmol) at RT. After completion of the reaction (about 10 h) the mixture was monitored using LCMS. DMF was removed under reduced pressure and crude material was purified using prep HPLC to afford 8 mg of compound 40 (0.006 mmol, 91% HPLC purity) in 25.3% Yield; XH NMR (400 MHz, DMSO-d6): mixture of diastereomers; δ 11.70 (s, 1H), 11.61 (s, 1H), 10.02 (s, 1H), 9.85 (s, 1H), 8.46 (s, 1H), 8.22 (s 1H), 8.10 (d, J = 7.4 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.93-7.82 (m, 2H x 2), 7.61 (d, J = 8.1 Hz, 1H x 2), 7.48-7.32 (m, 9H x 2), 7.21-7.06 (m, 4H x 2), 6.98 (d, J = 1.4 Hz, 1H x 2), 6.96 (d, J = 1.6 Hz, 1H x 2), 6.74 (s, 1H), 6.57 (d, J = 7.6 Hz, 1H x 2), 6.59 (s, 1H), 5.98 (s, 1H x 2), 5.42 (s, 2H), 5.41 (s, 2H), 5.04 (s, 2H), 5.03 (s, 2H), 4.81-4.74 (m, 1H x 2), 4.53-4.32 (m, 2H x 2), 4.14-4.03 (m, 3H x 2), 3.90 (q, J = 8.0 Hz, 1H x 2), 3.82 (s, 3H), 3.81 (s, 3H), 3.62-3.23 (m, 12H x 2), 3.19-2.85 (m, 8H x 2), 2.66 (t, J = 5.1 Hz, 2H x2), 2.27 (s, 6H), 2.26 (s, 6H), 2.10-1.90 (m, 3H x 2), 1.71-1.28 (m, 6H x 2), 0.89-0.81 (m, 6H x 2); MS calcd. for CBUBCINHOIS: 1327.6, Found: 1328.6 (M + 1).
Figure imgf000118_0001
20 41
To a MeOH:water (5: 1, v/v) solution (60 mL) of compound 20 (3.0 g, 0.0052 mol) was added LiOH.H20 (0.44 g, 0.0105 mol) at 0°C, and the mixture was stirred at RT for 3 h. After consumption of starting material 20 (TLC analysis), the mixture was cooled to 0°C and neutralized with acidic resin (Purolite Ultraclean™ UCW9126). The resin was removed by filtration and removal of MeOH yielded crude acid. The crude acid was dissolved in DMF (30 mL) and NH2CH2CH2OCH2CH2OCH2CH2NHB oc (1.8 g, 0.0064 mol), PyBOP (4.2 g, 0.0080 mol), DIPEA (1.85 mL, 0.0107 mol) were added successively at 0°C. The mixture was stirred for 16 h at RT under nitrogen atmosphere. Volatiles were removed under reduced pressure. Crude product was purified by column chromatography (silica gel) using DCM/MeOH. Compound 41 was obtained in 61 % yield (1.8 g); XH NMR (400 MHz, MeOD): δ 7.97 (s, 1H), 7.55 (d, J = 4.00 Hz, 2H), 7.34-7.36 (m, 7H), 5.09 (s, 2H), 4.91 (s, 1H), 3.92-3.97 (m, 1H), 3.60-3.61 (m, 7H), 3.54 (d, J = 5.20 Hz, 4H), 3.49 (t, J = 1.60 Hz, 1H), 3.12-3.13 (m, 12H), 2.98 (s, 3H), 2.86 (s, 3H), 2.84-2.85 (m, 1H), 1.82-1.83 (m, 1H), 1.43 (s, 9H), 0.93-0.95 (m, 6H); MS calcd. for C4oH6iN7Oi2: 831.96, Found: 832.4.
Figure imgf000119_0001
A. Trichloroacetimidate (42)
To a DMF (10 mL) solution of alcohol 41 (4.8 g, 0.50 mmol) was added Cs2C03 (3.7 g, 0.01 lmol) at RT. The mixture was cooled to 0 °C and CC13CN (8.3 g, 0.057 mol) was added. The mixture was allowed to warm to RT and stirred for 4 h. The mixture was filtered and DMF were removed under reduced pressure to provide 42. The crude product was purified by silica gel (60-120 mesh) column chromatography using 10% MeOH in DCM as eluent to yield compound 42 (3.1 g) in 55% yield; XH NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1H), 9.50 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 8.80 Hz, 2H), 7.45 (d, J = 8.40 Hz, 1H), 7.30-7.40 (m, 5H), 6.75 (s, 1H), 5.94-5.98 (m, 1H), 5.42 (s, 2H), 5.03 (s, 2H), 4.41- 4.42 (m, 1H), 3.93 (t, J = 7.20 Hz, 1H), 3.45-3.49 (m, 6H), 3.36-3.33 (m, 6H), 3.25-3.15 (m, 2H), 3.10-2.91 (m, 8H), 2.01-1.90 (m, 1H), 1.72-1.36 (m, 4H), 1.36 (s, 9H), 0.88-0.82 (m, 6H); MS calcd. for C42H61Cl4N8Oi2: 974.3, Found: 1011.2 (M + CI, negative mode).
B. Diamine (43)
To a suspension of 4A molecular sieves (0.2 g) in dry CH3CN was added Boc-Pro- CBI (0.3 g, 0.90 mmol) and trichloroacetimidate 42 (1.3 g, 1.35 mmol) at RT. After stirring at RT for 2 h, the mixture was cooled to -10°C and BF3.ether (0.255 g, 1.8 mmol) was added. The mixture was allowed to warm to RT and stirred overnight. The mixture was neutralized using NEt3 (2 mL) and the molecular sieves removed by filtration. Volatiles were removed under reduced pressure and resulting crude material was purified by silica gel (60-120 mesh) column chromatography using DCM:MeOH:NH3 solution as eluent to afford compound 43 (0.25 g) in 61% purity by LCMS; MS calcd. for C48H63ClN8O10: 946.4, Found: 947.4 (M + H).
C. Cbz-Val-Cit-PAMA-(Boc-PEG4-amide)-Pro-CBI (44)
To a MeOH solution of compound 43 (0.20 g, 0.211 mmol) was added NEt3 (0.06 mL, 0.422 mmol) cooled to 0°C. Boc20 (0.037 mL, 0.168 mmol) was added to this mixture. The mixture was stirred at RT for 12 h. Volatiles were removed under reduced pressure and resulting crude material was purified by silica gel (60-120 mesh) column chromatography using DCM:MeOH as eluent to yield compound 44 (0.16g, 85% yield, 72% LCMS purity); MS calcd. for CssHviClNgOiz: 1046.5, Found: 1047.4 (M + H).
Figure imgf000120_0001
Figure imgf000120_0002
A. Cbz-Val-Cit-PAMA-(Boc-PEG4-amide)-Pro-CBI-DMMI (45)
To a DMF (2 mL) solution of DMMI compound 3 (0.2 g, 0.72 mmol) was added Ι,Γ-carbonyldiimidazole (0.151 g, 0.93 mmol) portion wise at 0°C. After 2 h of stirring at RT, the mixture was transferred to stirred DMF solution of amine 44 (0.16 g, 0.15 mmol) and sodium carbonate (0.162 g, 1.52 mmol). The mixture was stirred for 12 h at 45°C. Progress of the reaction was monitored using LCMS. After completion of the reaction, the solid was removed by filtration and the solution was neutralized with formic acid. DMF was removed using reduced pressure and crude material was purified using prep HPLC to obtain compound 45 as a pale yellow solid (0.030 g, 96% LCMS purity) in 15% yield; XH NMR (300 MHz, DMSO-d6): mixtures of diastereomers assigned as a and b; δ 11.68 (s, 1H, a), 11.59 (s, 1H, b), 11.00 (s, 1H, a), 8.83 (s, 1H, b) 8.45 (s, 1H), 8.21 (s, 2H), 8.09-8.02 (m, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.45-7.20 (m, 10H), 7.20-7.10 (m, 3H), 7.05 (d, J = 8.25 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.75 (s, 2H), 6.54 (d, J = 8.0 Hz, 1H), 5.96 (s, 1H), 5.41 (d, 7 = 2.4 Hz, 2H), 5.02 (d, J = 3.6 Hz, 2H), 4.82-4.69 (m, 1H), 4.52- 4.28 (m, 2H), 4.08-4.01 (m, 2H), 3.93-3.85 (m, 2H), 3.80 (d, J = 2.4 Hz, 3H), 3.72-3.65 (m, 1H), 3.52-3.18 (m, 14H), 3.08-2.92 (m, 3H), 2.70 (t, J = 6.90 Hz, 2H), 2.24 (s, 6H), 2.00- 1.91 (m, 1H), 1.68-1.33 (m, 4H), 1.33 (s, 9H), 0.85-0.77 (m, 6H); MS calcd. for CevHgvClNioOis: 1306.6, Found: 1309.6 (M + 3).
B. Cbz-Val-Cit-PAMA-(MB-PEG4-amide)-Pro-CBI-DMMI (46)
To an EtOAc suspension of compound 45 (0.01 g, 0.007 mmol) was added HC1 (0.1 mL) in EtOAc (~4 N) at -10°C and stirred for 4 h. After completion of the reaction EtOAc was removed under a nitrogen stream. The crude material was dissolved in DMF (2 mL). To the resulting solution was added succinate ester 27 (0.0046 g, 0.016 mmol) and sodium carbonate (0.0018 g, 0.016 mmol) at RT for 12 h. After completion of the reaction (monitored using LCMS), DMF was removed under reduced pressure and crude material was purified using prep HPLC to afford 3 mg of compound 46 (89% LCMS purity) in 27% yield; XH NMR (400 MHz, DMSO-d6): mixture of diastereomers; δ 11.72 (s, 1H), 11.62 (s, 1H), 11.49 (s, 1H), 10.44 (s, 1H), 10.02 (s, 1H), 9.86 (s, 1H), 8.46 (s, 1H), 8.23 (s 1H), 8.12- 8.05 (m, 1H x 2), 7.93-7.82 (m, 2H x 2), 7.61 (d, J = 8.1 Hz, 1H x 2), 7.39-7.29 (m, 8H x 2), 7.21-6.95 (m, 8H x 2), 6.73 (s, 1H), 6.56 (d, J = 7.6 Hz, 1H x 2), 6.57 (s, 1H), 5.98 (s, 1H x 2), 5.43 (s, 2H), 5.42 (s, 2H), 5.04 (s, 2H), 5.03 (s, 2H), 4.82-4.73 (m, 1H x 2), 4.53-4.32 (m, 3H x 2), 4.10-4.03 (m, 3H x 2), 3.90 (q, J = 8.0 Hz, 1H x 2), 3.82 (s, 3H), 3.81 (s, 3H), 3.60- 3.19 (m, 14H x 2), 3.19-2.86 (m, 8H x 2), 2.78-2.70 (m, 2H x 2), 2.31 (s, 6H), 2.30 (s, 6H), 2.10-1.90 (m, 3H x 2), 1.79-1.29 (m, 6H x 2), 0.89-0.81 (m, 6H x 2); MS calcd. for CvoHgeClNnOie: 1371.6, Found: 1372.6 (M + 1).
Example 18: Synthesis of spirocyclic amine used for Examples 18-23 (Preparation A)
Figure imgf000122_0001
47 48 49
Figure imgf000122_0002
A. Compound 49
To a DCM (40 mL) solution of compound 47 (5.00 g, 26.45 mmol) and ethyl cyanoacetate (5.64 mL, 52.91 mmol) was added NEt3 (11.13 mL, 79.30 mmol) and 4A molecular sieves (3 g) at RT. After 12 h of stirring at RT, TLC analysis showed completed consumption of starting material. The mixture was filtered through a Celite® bed and volatiles were removed under reduced pressure to provide compound 48 in 8.4 g. H NMR (400 MHz, CDC13): δ 7.27-7.37 (m, 5H), 4.26 (q, J = 7.7 Hz, 2H), 3.56 (s, 2H), 3.16 (t, J = 5.7 Hz, 2H), 2.81 (t, J = 5.8 Hz, 2H), 2.59 (t, J = 5.8 Hz, 2H), 2.59 (t, J = 5.8 Hz, 2H), 1.37 (t, / = 7.7 Hz, 3H). MS calcd. for C17H20N2O2: 284.2, Found: 284.7.
Compound 48 was dissolved in ethanol (38 mL) and water (7.5 mL) and KCN (4.85 g, 73.94 mmol) was added before heating the mixture to 80°C. After 24 h of heating at 80°C, ethanol was removed under reduced pressure and the resulting solution was extracted with ethyl acetate (300 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated to provide crude material. Yield was 4.60 g (72%); XH NMR (400 MHz, CDCI3): δ 7.28-7.37 (m, 5H), 3.57 (s, 2H), 2.95 (d, J = 12.6 Hz, 2H), 2.72 (s, 2H), 2.36 (t, J = 12.5 Hz, 2H), 2.07 (dd, J = 13.2, 2.3 Hz, 2H), 1.77 (td, / = 13.2, 3.8 Hz, 2H). MS calcd. for Ci5H17N3: 239.1, Found: 240.3 (M + 1). B. Compound 50
A sealed tube was charged with compound 49 (11.30 g, 47.28 mmol) and concentrated HCl (175 mL). The tube was closed with cap and was heated in oil bath at 100°C for 72 h. Water was then removed under reduced pressure and residual water was removed as via azeotroping using toluene. Diacid 50 was obtained in 76% yield (10.00 g); MS calcd. for C15H19N04: 277.1, Found: 277.2.
C. Compound 51
To DMF (60 mL) solution of compound 50 (6.00 g, 21.66 mmol) was added DCC (4.46 g, 21.66 mmol) under N2. After stirring at RT for 2.5 h, 4-methoxy benzyl amine (2.67 g, 19.49 mmol) and NEt3 (6.07 mL, 43.20 mmol) were added. After 3 h, solid was filtered off using a Celite® pad and DMF was removed under reduced pressure. The crude material was purified using silica column chromatography to obtain 3.7 g of white solid (9.33 mmol). The product was dissolved in Ac20 (70 mL) and sodium acetate (3.83 g, 46.71 mmol) was added. The mixture was heated at 100°C for 3 h. Acetic anhydride was removed under reduced pressure, diluted with water (200 mL) and extracted with ethyl acetate (300 mL x 3). Combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated to provide crude material. The crude product was purified by column chromatography (silica gel) in DCM/MeOH. The yield of compound 51 was 71% (2.5 g); XH NMR (400 MHz, DMSO-d6): δ 7.23-7.34 (m, 5H), 7.16 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 4.47 (s, 2H), 3.73 (s, 3H), 3.46 (s, 2H), 2.74 (d, J = 11.6 Hz, 2H), 2.6 (s,2H), 1.99 (t, 7 = 11.4 Hz, 2H), 1.79 (t, J = 12.3 Hz, 2H), 1.51 (d, J = 12.5 Hz, 2H). MS calcd. for C23H26N2O3: 378.2, Found: 379.0 (M + 1). D. Spirocyclic amine 52
To a stirred solution of compound 51 (0.50 g, 1.33 mmol) in acetic acid: 1,4-dioxane: water (1.5: 1.5:1, v/v) was charged with palladium hydroxide (20%; 0.15 g) under N2 atmosphere. Nitrogen atmosphere was replaced with hydrogen and the mixture was stirred for 6 h under hydrogen atmosphere (balloon pressure). The catalyst was removed by filtration through a Celite® pad and volatiles were removed under reduced pressure to provide crude spirocyclic amine 52. Column chromatography on silica afforded pure compound 52 (0.36 g) in 94% yield; XH NMR (300 MHz, DMSO-d6): δ 7.14 (d, J = 8.6 Hz, 2H), 6.87 (d, / = 8.6 Hz, 2H), 4.46 (s, 2H), 3.70 (s, 3H), 3.02 (t, / = 9.3 Hz, 2H), 2.68 (t, J = 2.0 Hz, 2H), 1.88 (s, 2H), 1.76 (t, 7 = 12.7 Hz, 2H), 1.52 (d, J = 13.2 Hz, 2H). MS calcd. for C16H20N2O3: 288.1, Found: 290.3 (M + 2).
E. Compound 53
To acetonitrile: water (5: 1) solution (6.0 mL) of compound 51 (0.50 g, 1.33 mmol) was added eerie ammonium nitrate (1.67 g, 3.05 mmol) at RT. After 16 h, acetonitrile was removed under reduced pressure and the residue was diluted with water washed with ethyl acetate (50 mL x 3). The aqueous layer was basified with NaHCC>3, extracted with ethyl acetate (100 mL x 3) and the combined organic layer was washed with brine solution, dried over anhydrous sodium sulfate concentrated to obtain 0.22 g of compound 53 (64% yield); XH NMR (400 MHz, DMSO-d6): δ 11.11 (s, 1H), 7.22-7.33 (m, 5H), 3.44 (s, 2H), 2.73 (d, J = 11.8 Hz, 2H), 2.50 (t, / = 1.8 Hz, 2H), 1.95 (t, 7 =10.8 Hz, 2H), 1.74 (t, / = 12.6 Hz, 2H), 1.52 d, J = 12.2 Hz, 2H). MS calcd. for ¾Η18Ν202: 258.1, Found: 260.5 (M + 2).
Figure imgf000124_0001
A. Compound 54
To a DMF (5.0 mL) solution of spirocyclic amine 52 (0.330 g, 1.14 mmol) were added compound 32 (0.390 g, 1.37 mmol) and K2C03 (0.474 g, 3.43 mmol) under N2 atmosphere. The mixture was heated at 100°C for 12 h with stirring. The mixture was then cooled RT and diluted with water. The mixture was extracted with ethyl acetate (100 mL x 3), and combined organic layers were washed with brine solution, dried (over anhydrous sodium sulfate) and concentrated. The crude product was purified by column chromatography (silica gel) using DCM/MeOH as eluent. The yield was 52% (0.32 g); H NMR (DMSO-d6, 400 MHz) 11.63 (s, 1H), 7.16-7.13 (m, 3H), 6.99 (d, J = 1.6 Hz, 1H), 6.88-6.86 (m, 3H), 4.47 (s, 2H), 4.03 (t, J = 7.0 Hz, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 3.71 (s, 3H), 2.92-2.89 (m, 2H), 2.72-2.69 (m, 2H), 2.66 (s, 2H), 2.11-2.08 (m, 2H), 1.82-1.79 (m, 2H), 1.50-1.45 (m, 2H); MS calcd. for C29H33N3O7: 535.2, Found: 536.5 (M+l).
B. Compound 55
To pyridine solution (4 mL) of spirocyclic compound 54 (0.18 g, 0.33 mmol) was added Lil (0.27 g, 2.02 mmol) under N2 atmosphere. The mixture was heated at 200°C in a microwave reactor for 30 min. The mixture was then cooled to RT and pyridine was removed under reduced pressure. The crude product was purified by column chromatography (silica gel) using DCM/MeOH. The yield was 0.116 g (63% Purity by UPLC); XNMR (DMSO-d6, 300 MHz) 11.39 (s, 1H), 7.16-7.12 (m, 3H), 6.87-6.83 (m, 4H), 4.46 (s, 2H), 4.02 (t, J = 7.0 Hz, 2H), 3.76 (s, 3H), 3.70 (s, 3H), 2.92-2.89 (m, 2H), 2.73-2.70 (m, 2H), 2.70 (s, 2H), 2.15-2.11 (m, 2H), 1.89-1.80 (m, 2H), 1.57-1.53 (m, 2H); MS calcd. for C28H31N3O7: 521.2, Found: 521.5.
C. Compound 56
To DMF (3 mL) solution of Pro-CBI 2 (0.04g, 0.171 mmol) were added compound 55 (0.17 g, 0.206 mmol) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC HC1) (0.16 g, 0.858 mmol) at 0°C. After 16 h of stirring RT, the mixture was diluted with water. The resulting mixture was concentrated using a freeze drier. After complete removal of DMF, crude product was purified by preparative HPLC (Column: Kromasil® C18; mobile phase: water (0.06% HC1) and acetonitrile) method to obtain 0.007 g of compound 56 (5% yield); HPLC purity 97% (Rt = 15.3 min); System: Agilent 1100 HPLC; detector: ELS, Waters 2420; column: Hypersil® BDS CI 8 (250 x 4.6) mm 5μ; eluents: A: water + 0.06% HC1; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.55 (s, 1H), 10.43 (s, 1H), 10.34 (s, 1H), 8.11 (d, J = 8.32 Hz, 1H), 7.97 (s, 1H), 7.84 (d, J = 8.28 Hz, 1H), 7.50 (t, J = 7.36 Hz, 1H), 7.35 (t, J = 7.72 Hz, 1H), 7.30 (s, 1H), 7.17 (d, J = 8.56 Hz, 2H), 7.08 (d, J = 1.48 Hz, 1H), 7.01 (s, 1H), 6.87 (d, J = 8.64 Hz, 2H), 4.77 (t, 7 = 7.20 Hz, 1H), 4.49-4.51 (m, 3H), 4.40-4.33 (m, 1H), 4.27-4.20 (m, 1H), 4.02 d, J = 8.24 Hz, 1H), 3.81-3.83 (m, 1H), 3.83 (s, 3H), 3.63-3.69 (m, 2H), 3.71 (s, 3H), 3.59-3.54 (m, 2H), 3.35-3.29 (m, 1H), 3.18-3.14 (m, 2H), 2.84 (s, 2H), 2.19-2.16 (m, 2H), 2.00-1.87 (m, 2H). MS calcd. for C41H41CIN4O7: 736.3, Found: 737.1 (M + 1)
Figure imgf000126_0001
A. Compound 57
To DMF (4.0 mL) solution of spirocyclic amine 53 (0.168 g, 0.658 mmol) were added compound 32 (0.203 g, 0.716 mmol) and K2C03 (0.224 g 1.62 mmol) under N2 atmosphere. The mixture was heated at 100°C for 12 h while stirring. The mixture was then cooled to RT and diluted with ice cold water. The resultant solid was filtered. The yield was 80% (0.264 g); XH NMR (300 MHz, DMSO-d6): δ 11.63 (s, 1H), 7.22-7.24 (m, 5H), 7.09 (s, 1H), 6.97 (s, 1H), 6.83 (s, 1H), 4.04 (t, J = 5.9 Hz, 2H), 3.81 (s, 3H), 3.74 (s, 3H), 3.44 (s, 2H), 2.73 (d, J = 10.6 Hz, 2H), 2.59 (s, 2H), 2.57-2.49 (m 2H), 1.97 (t, 7 = 11.4 Hz, 2H), 1.79 (t, J =11.2 Hz, 2H), 1.49 (d, J = 12.7 Hz, 2H); MS calcd. for C28H31N306: 505.2, Found: 506.1 (M + 1)
B. Compound 58
To pyridine (1.5 mL) solution of spirocyclic derivative 57 (0.08 g, 0.158 mmol) was added Lil (0.127 g, 0.950 mmol) under N2 atmosphere. The mixture was heated at 200°C in a microwave reactor for 30 min. The mixture was then cooled RT and pyridine was removed under reduced pressure. The crude product was poured into water and the aqueous layer was acidified using 1.5N HC1. The black solid formed was filtered and washed with water to yield 0.05 g of compound 58; MS calcd. for C27H29N3O6: 491.2, Found: 491.6 C. Compound 59
To a DMF (3 mL) solution of Pro-CBI 2 (0.02g, 0.085 mmol) were added compound 58 (0.055 g, 0.103 mmol) and EDC.HCl (0.049 g, 0.257 mmol) at 0°C. After 16 h of stirring RT, the mixture was cooled RT and diluted with water. The mixture was extracted with ethyl acetate (100 mL x 3), the combined organic layers were washed with brine solution, dried (over anhydrous sodium sulfate) and concentrated. The crude product was purified by preparative HPLC (Column: Kromasil® C18; mobile phase: water (0.06% HC1) and acetonitrile) method to obtain 0.004 g of compound 59 (6% yield); HPLC purity 94.3% (Rt = 15.4 min); System: Shimadzu LC20AD; detector: PDA max chromatogram (210-400 nm); column: Hypersil® BDS CI 8 (250 x 4.6) mm 5μ; eluents: A: water + 0.06% HC1; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.50 (s, 1H), 10.44 (s, 1H), 10.24 (br s, 1H), 8.12 (d, / = 8.2 Hz, 1H), 7.98 (s, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.63-7.47 (m, 5H), 7.37 (t, / = 8.0 Hz, 2H), 7.18 (s, 1H), 7.05 (d, J = 1.80 Hz, 1H), 6.96 (s, 1H), 4.77 (t, / = 10.1 Hz, 1H), 4.53 (d, / = 9.6 Hz, 1H), 4.31 (s, 1H), 4.22 (t, J = 4.8 Hz, 1H), 4.10 (t, J = 5.8 Hz, 2H), 4.03 (dd, J = 11.2, 3.1 Hz, 1H), 3.86-3.84 (m, 2H), 3.79 (s, 3H), 3.44 (s, 2H), 3.02-3.11 (m, 2H), 2.80 (s, 2H), 2.10- 2.21 (m, 2H), 2.00-1.96 (m, 2H), 1.84 (d, J = 12.00 Hz, 2H), one extra proton from HC1 salt; MS calcd. for C40H39CIN4O6: 706.3, Found: 707.6 (M + 1)
Figure imgf000127_0001
A. Acid 60
To an ethanol: water (1 :1, v/v; 40 mL) solution of ester 32 (2.0 g, 7.06 mmol) was added CS2CO3 (3.44 g, 10.60 mmol) and resulting solution was stirred at RT for 12 h. The ethanol was removed under reduced pressure. The aqueous layer was acidified with 1.5N HC1. The solid removed by filtration and dried to obtain 1.88 g of compound 60 (99% yield); XH NMR (300 MHz, DMSO-d6): δ 12.64 (s, 1H), 11.48 (s, 1H), 7.13 (s, 1H), 6.93 (s, 1H), 6.87 (s, 1H), 4.19 (t, / = 7.8 Hz, 2H), 3.90 (t, J = 9.5 Hz, 2H), 3.79 (s, 3H); MS calcd. for Ci2H12ClN04: 269.0, Found: 270.2 (M + 1) B. Benzyl ester 61
To a DCM (15.0 mL) solution of compound 60 (0.50 g, 1.85 mmol) were added benzyl alcohol (0.24 g, 2.23 mmol) and DCC (0.46g, 2.23 mmol), DMAP (0.054 g, 0.464 mmol) at 0°C. After 16 h of stirring RT, the solid was removed by filtration and solvent was removed under reduced pressure. Crude material was purified by silica column chromatography to obtain 0.53 g of compound 61 (79% yield); XH NMR (400 MHz, DMSO- d6): δ 11.67 (s, 1H), 7.47 (d, J = 7.3 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.14 (s, 1H), 7.06 (s, 1H), 6.91 (s, 1H), 5.34 (s, 2H), 4.20 (t, / = 5.40 Hz, 2H), 3.91 (t, J = 14.88 Hz, 2H), 3.78 (s, 3H); MS calcd. for Ci9H18ClN04: 359.1, Found: 360.3 (M + 1) C. Compound 62
To a DMF (5.0 mL) solution of spirocyclic amine 53 (0.144 g, 0.558 mmol) were added compound 61 (0.2 g, 0.558 mmol) and K2C03 (0.192 g, 1.39 mmol) under N2 atmosphere. The mixture was heated at 100°C for 6 h under stirring. The mixture was cooled to RT and was diluted with ice water. The solid was removed by filtration and dried to obtain 0.30 g of compound 62 (92% yield); XH NMR (400 MHz, DMSO-d6): δ 11.67 (s, 1H), 7.46 (d, / = 7.0 Hz, 2H), 7.40 (t, / = 7.6 Hz, 2H), 7.36 (t, / = 7.6 Hz, 1H), 7.17-7.22 (m, 5H), 7.15 (s, 1H), 7.03 (s, 1H), 6.85 (s, 1H), 5.33 (s, 2H), 4.04 (t, J = 6.0 Hz, 2H), 3.69 (s, 3H), 2.72 (d, / = 5.60 Hz, 2H), 2.59 (s, 2H), 1.98 (t, / = 8.00 Hz, 2H), 1.82-1.76 (m, 2H), 1.71-1.68 (m, 2H), 1.60-1.57 (m, 2H), 1.52-1.47 (m, 2H); MS calcd. for Cs^NsOe,: 581.3, Found: 582.2 (M + 1)
D. Compound 63
To an acetic acid: 1,4-dioxane: water (2:2: 1, v/v) solution of benzyl ester 62 (0.29 g, 0.499 mmol) was charged palladium hydroxide (20%, 0.15 g) under N2 atmosphere. Nitrogen atmosphere was replaced with hydrogen and the mixture was stirred for 3 h under hydrogen atmosphere (balloon pressure). The catalyst was removed using a Celite® pad and volatiles were removed under reduced pressure to provide spirocyclic amine 63 (0.25 g) which was used in next step without further purification; H NMR (300 MHz, DMSO-d6): δ 11.10 (s, 1H), 7.04 (s, 1H), 6.84 (s, 1H), 6.70 (s, 1H), 4.02 (t, 7 = 6.00 Hz, 2H), 5.57 (d, 7 = 10.2 Hz, 1H), 3.81-3.75 (m, 2H), 3.75 (s, 3H), 3.00-2.87 (m, 2H), 2.75-2.66 (m, 2H), 2.67 (s, 2H), 1.79-1.46 (m, 6H); MS calcd. for C20H23N3O6: 401.2, Found: 402.4 (M + 1).
E. Compound 64
To the stirred solution of amine 63 (0.116 g, 0.289 mmol) in MeOH (5 mL) were added acetic acid (0.05 mL), formaldehyde (0.017 g, 0.578 mmol) and the mixture was stirred at RT for 1 h. The mixture was then charged with sodium triacetoxy borohydride (0.245 g, 1.15 mmol) at 0°C. After 20 min, the cooling bath was removed and mixture was stirred for 12 h at RT. Volatiles were removed under reduced pressure and crude product was purified by column chromatography (silica gel). Yield 0.04 g of compound 64 (62% purity by LCMS); XH NMR (300 MHz, DMSO-d6): δ 11.47 (s, 1H), 7.09 (s, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 5.57 (d, 7 = 7.92 Hz, 1H), 4.04 (t, 7 = 5.6 Hz, 2H), 3.79-3.73 (m, 2H), 3.73 (s, 3H), 3.08 (s, 3H), 2.75 (s, 2H), 2.76-2.67 (m, 2H), 2.47-2.20 (m, 4H), 1.83-1.71 (m, 2H); MS calcd. for C21H25N3O6: 415.2, Found: 416.2 (M + 1)
F. Compound 65
To DMF (3 mL) solution of compound 64 (0.04g, 0.096 mmol) was added Pro-CBI 2 (0.018g, 0.077 mmol) and EDC.HC1 (0.074g, 0.385 mmol) at 0°C. After 16 h of stirring at RT, the mixture was diluted with water. The resulting mixture was concentrated using a freeze drier. After complete removal of DMF, crude product was purified by preparative HPLC (Column: Kromasil® CI 8; mobile phase: water (0.06% HC1) and acetonitrile) to obtain 0.010 g of compound 65 (16% yield); HPLC purity 85% (Rt = 14.55 min); System: Agilent 1200 HPLC; detector: DAD max chromatogram (210-400 nm); column: Kromasil® C18 (4.6 x 250 mm, 5μ); eluents: A: water + 0.06% HC1; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.48 (s, 1H), 10.62 (br s, 1H), 10.43 (s, 1H), 8.11 (d, 7 = 8.3 Hz, 1H), 7.97 (s, 1H), 7.83 (d, 7 = 8.3 Hz, 1H), 7.51 (t, 7 = 8.0 Hz, 1H), δ 7.35 (t, 7 = 7.6 Hz, 1H), 7.19 (s, 1H), 7.05 (s, 1H), 6.97 (s, 1H), 4.77 (t, 7 = 9.3 Hz, 1H), 4.52 (d, 7 = 11.0 Hz, 1H), 4.21 (br s, 1H), 4.10 (t, 7 = 5.8 Hz, 2H), 4.02 (dd, 7 = 11.0, 2.8 Hz, 1H), 3.77-3.79 (m, 1H), 3.77 (m, 3H), 3.06-3.01 (m, 2H), 2.78 (s, 2H), 2.66-2.79 (m, 5H), 2.15 (t, J = 11.4 Hz, 2H), 1.98-2.00 (m, 2H), 1.80 (d, J = 12.3 Hz, 2H), one extra proton from HC1 salt; MS calcd. for C34H35CIN4O6: 630.2, Found: 631.2 (M + 1).
Figure imgf000130_0001
A. Spirocyclic amine 66
To a THF (10 mL) solution of spirocyclic compound 53 (0.80 g, 3.22 mmol) was added L1AIH4 (12.9 mL, IN solution in THF, 12.90 mmol) at 0°C under nitrogen atmosphere. The mixture was then heated at 65°C for 3.5 h. The mixture was quenched with 10% aqueous NaOH solution, diluted with water, and extracted with ethyl acetate, (200 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and volatiles were removed under reduced pressure. Compound 66 was obtained in 74% yield (0.65g 2.83 mmol); XH NMR (300 MHz, DMSO-d6): δ 7.21-7.27 (m, 6H), 3.40 (s, 2H), 2.73 (t, J = 7.02 Hz, 2H), 2.48 (s, 2H), 2.27-2.20 (m, 4H), 2.27-0.00 (m, 6H); MS calcd. for ¾Η22Ν2: 230.2, Found: 231.3 (M + 1
B. Compound 67
To a DMF (5 mL) solution of the spiroamine 66 (0.35 g, 1.52 mmol) were added compound 32 (0.473 g, 1.67 mmol) and K2CO3 (0.419 g, 3.04 mmol) at RT. The mixture was then heated at 100°C for 4 h. The mixture was cooled to RT, diluted with water and extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Crude material was purified by column chromatography (silica gel, Ethyl acetate/pet. ether as eluent). The yield of compound 67 was 0.42 g (0.88 mmol, 58% Yield); XH NMR (400 MHz, DMSO-d6): δ 11.65 (s, 1H), 7.30-0.00 (m, 5H), 7.14 (s, 1H), 7.00 (s, 1H), 6.88 (s, 1H), 4.06-4.10 (m, 4H), 3.87 (s, 3H), 3.80 (s, 3H), 3.55-3.32 (m, 4H), 3.42 (s, 2H), 2.29-2.32 (m, 4H), 1.90-1.90 (m, 6H); MS calcd. for C28H35N3O4: 477.3, Found: 478.6 (M + 1).
C. Compound 68
To an acetic acid: 1,4-dioxane: water (2:2: 1, v/v; 10 mL) solution of compound 67
(0.42 g, 0.88 mmol) was added 0.15 g of palladium hydroxide (20%) under N2 atmosphere. Nitrogen atmosphere was replaced with hydrogen and the mixture stirred for 16 h under hydrogen atmosphere (balloon pressure). The catalyst was removed using a Celite® pad and volatiles were removed under reduced pressure to provide amine 68 (0.500 g, 1.29 mmol) in 97% yield; XH NMR (300 MHz, CDCI3): δ 9.09 (s, 1H), 7.27 (s, 1H), 7.10 (d, J = 3.7 Hz, 1H), 6.87 (s, 1H), 4.28-4.22 (m, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.24-3.02 (m, 6H), 3.09 (s, 2H), 3.05-3.00 (m, 3H), 1.94-1.81 (m, 6H); MS calcd. for C21H29N3O4: 387.2, Found: 388.5 (M + 1). D. Compound 69
To the stirred solution of amine 68 (0.5 g, 1.29 mmol) in MeOH (10 mL) were added acetic acid (0.2 mL), formalin solution (40% in water, 2.58 mmol, 0.2 mL) and sodium cyanoborahydride (0.162 g, 1.26 mmol) at 0°C. The mixture was stirred for 1 h at 0°C. Volatiles were removed under reduced pressure and crude product was purified by column chromatography (silica gel). 0.26 g of compound 69 was obtained (0.65 mmol, 50% yield); XH NMR (300 MHz, DMSO-d6): δ 11.63 (s, 1H), 7.11 (s, 1H), 6.99 (d, J = 1.6 Hz, 1H), 6.86 (s, 1H), 4.02 (t, / = 5.9 Hz, 2H), 3.78 (s, 3H), 3.73 (s, 3H), 2.83-2.79 (m, 2H), 2.69-2.51 (m, 6H), 2.46 (s, 2H), 2.88 (s, 3H), 1.46-1.68 (m, 6H); MS calcd. for C22H31N3O4: 401.2, Found: 402.2 (M + 1)
E. Compound 70
To a MeOH: water (1: 1, v/v; 10 mL) solution of compound 69 (0.26 g, 0.65 mmol) was added LiOH.H20 (0.159 g, 3.89 mmol) and resulting mixture was stirred at RT for 12 h. The mixture was acidified with HC1, and then volatiles were removed under reduced pressure to yield 0.20 g (0.52 mmol, 74%) of crude compound 70; XH NMR (400 MHz, DMSO-de): δ 11.75-11.77 (m, 1H), 11.55 (s, 1H), 10.96-10.98 (m, 1H), 7.21 (s, 1H), 6.97 (s, 1H), 6.93 (s, 1H), 4.32-4.26 (m, 2H), 3.83 (s, 3H), 3.83-3.11 (m, 9H), 2.69 (s, 5H), 2.09-1.92 (m, 6H); MS calcd. for C21H29N3O4: 387.2, Found: 388.5 (M + 1)
F. Compound 71
To a DMF (5 mL) solution of compound 70 (0.20 g, 0.52 mmol) were added Pro- CBI 2 (0.096 g, 0.413 mmol) and EDC.HCl (0.493 g, 2.58 mmol) at 0°C, and the solution stirred for 16 h at RT. The mixture was diluted with water, and concentrated using a freeze drier. After complete removal of DMF, crude product was purified by preparative HPLC (Column: Kromasil® C18; mobile phase: water (0.06% HC1) and acetonitrile). Yield 0.014 g (0.023 mmol, 4.5% yield); HPLC purity 92% (Rt = 13.50 min); System: Agilent 1200 HPLC; detector: DAD max chromatogram (210-400 nm); column: Kromasil® C18 (250 x 4.6 mm, 5μ); eluents: A: water + 0.06% HC1; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.59 (s, 1H), 11.42 (s, 1H), 10.57 (s, 1H), 10.45 (s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.97 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.29 (s, 1H), 7.08 (s, 1H), 7.01 (s, 1H), 4.77 (t, / = 10.0 Hz, 1H), 4.54 (d, J = 10.8 Hz, 1H), 4.36 (d, J = 4.3 Hz, 2H), 4.25 (s, 1H), 4.00 (dd, J = 10.2, 2.7 Hz, 1H), 3.84 (s, 3H), 3.84-3.82 (m, 1H), 3.82-3.75 (m, 2H), 3.59-3.51 (m, 4H), 3.45-3.31 (m, 2H), 3.14-2.92 (m, 2H), 2.70 (d, / = 4.6 Hz, 3H), 2.09-1.84 (m, 6H), two extra protons from HC1 salt; MS calcd. for C34H39CIN4O4: 602.3, Found: 603.0 (M + 1)
Table 4: Synthesis of Cytotoxic Compounds (Examples 19-26)
Figure imgf000132_0001
Figure imgf000133_0001
131
Figure imgf000134_0001
Example 27: Synthesis of spirocyclic amine compound 75 (Preparation B)
Figure imgf000134_0002
72 73 74 75 A. Diol 73
To a THF (30 mL) solution of diacid 72 (3.00 g, 10.83 mmol) was added LiAlH4 (27 mL (1.6 M solution in THF, 43.20 mmol) at 0°C under N2 atmosphere. The mixture was heated at 80°C for 12 h, cooled to 0°C and poured over 10% aqueous NaOH solution. The solution was extracted with ethyl acetate (200 mL x 3) and the combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate. Volatiles were removed under reduced pressure to provide 2.10 g of compound 73 (8.42 mmol, 56% yield); H NMR (400 MHz, DMSO-d6): δ 7.23-7.33 (m, 5H), 4.53 (t, J = 5.3 Hz, 1H), 4.41 (t, J = 4.9 Hz, 1H), 3.41-3.43 (m, 4H), 3.21 (d, 7 = 5.4 Hz, 2H), 2.25-2.35 (m, 4H), 1.48 (t, / = 7.3 Hz, 2H), 1.36-1.43 (m, 2H), 1.28-1.34 (m, 2H); MS calcd. for C15H23NO2: 249.2, Found: 250.6 (M + 1)
B. Compound 74
To a THF (20 mL) solution containing triphenylphosphine (0.789 g, 3.01 mmol) and diol 73 (0.50 g, 2.01 mmol) was added slowly THF (2 mL) solution of DBAD (Di-tert- butylazedodicarboxylate) (0.692 g, 3.01 mmol) at 0°C under N2 atmosphere. The mixture was stirred at 0°C for 1.5 h before warming to RT. After 16 h, volatiles were removed under reduced pressure and residue was dissolved in DCM (300 mL). The DCM solution was washed with 1.5 N aqueous HCl (50 mL). The aqueous layer was basified to pH 10 using IN NaOH solution and then extracted with ethyl acetate (100 mL x 3). Combined ethyl acetate layer was washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The spirocyclic 74 was obtained in 86% yield (0.40 g, 1.73 mmol); XH NMR (400 MHz, DMSO-d6): δ 7.21-7.33 (m, 5H), 3.70 (t, J = 7.2 Hz, 2H), 3.43 (s, 2H), 3.41 (s, 2H), 2.25-2.35 (m, 4H), 1.65 (t, / = 7.2 Hz, 2H), 1.49-1.41 (m, 4H); MS calcd. for C15H21NO: 231.2, Found: 232.4 (M + 1)
C. Compound 75
To an acetic acid: 1,4-dioxane: water (4:4: 1, v/v, 18 mL) solution of compound 74 (0.4 g, 1.73 mmol) was added 0.15 g of palladium hydroxide (20%) under N2 atmosphere. Nitrogen atmosphere was replaced with hydrogen and the mixture was stirred for 16 h under hydrogen atmosphere (balloon pressure). The catalyst was removed using a Celite® pad and volatiles were removed under reduced pressure to provide amine 75 (0.20 g, 1.42 mmol) in 82% yield; XH NMR (400 MHz, DMSO-d6): δ 5.81 (br s, 1H), 3.70 (t, J = 9.4 Hz, 2H), 3.41 (s, 2H), 2.77-2.79 (m, 4H), 1.68 (t, J = 9.4 Hz, 2H), 1.48-1.50 (m, 4H); MS calcd. for C8H15NO: 141.1, Found: 142.2 (M + 1).
Example 28: Synthesis of spirocyclic amine compound 82
Bn
Figure imgf000135_0001
66 82a 82
A. Compound 82a
To a stirred solution of amine 66 (0.30 g, 1.30 mmol) in MeOH (10 mL) were added acetic acid (0.1 mL), formaldehyde (0.078 g, 2.60 mmol) and sodium cyanoborahydride (0.242 g, 3.91 mmol) at 0°C. After 20 min, the cooling bath was removed and the mixture was stirred for 4 h at RT. Volatiles were removed under reduced pressure and crude product 82a was purified by column chromatography (silica gel). Yield 0.2 g (62%); H NMR (300 MHz, CDCI3): δ 7.26-7.35 (m, 5H), 3.54 (s, 2H), 3.02 (t, / = 7.2 Hz, 2H), 2.80 (s, 2H), 2.64 (s, 3H), 2.2-2.55 (m, 4H), 1.87 (t, J = 7.2 Hz, 2H), 1.80-1.69 (m, 4H); MS calcd. for C16H24N2: 244.2, Found: 245.2 (M + 1).
B. Compound 82
To an acetic acid: 1,4-dioxane: water (2:2: 1, v/v; 5 mL) solution of compound 82a
(0.2 g, 0.82 mmol) was added palladium hydroxide (20%; of) under N2 atmosphere. Nitrogen atmosphere was replaced with hydrogen and mixture was stirred for 16 h under hydrogen atmosphere (balloon pressure). The catalyst was removed using a Celite® pad and volatiles were removed under reduced pressure to provide amine 82 0.125 g (yield 99%); XH NMR (300 MHz, CDCI3): δ 7.38-7.34 (m, 1H), 3.22-3.03 (m, 6H), 2.70 (s, 3H), 2.66 (s, 2H), 2.06-1.81 (m, 6H); MS calcd. for C9H18N2: 154.1, Found: 155.2 (M + 1).
Example 29: Synthesis of spirocyclic amines
OH OH OMs
/ \ / 1) Pd(OH)2, H2 /— \ , / MsCI, DIPEA Na2S, DMF
BnN X »► BocN X »- BocN
S / V-OH 2 ?i) R Bnofc.2 n0, n DiIPPFEAA \— / \ V--OOHH V-OMs
76 77
Figure imgf000136_0001
80 81
A. Compound 76
To an acetic acid: 1,4-dioxane: water (2:2: 1, v/v; 20 mL) solution of compound 73 (0.90 g, 3.61 mmol) was added 0.30 g of palladium hydroxide (20%) under N2 atmosphere. Nitrogen atmosphere was replaced with hydrogen and the mixture stirred for 16 h under hydrogen atmosphere (balloon pressure). The catalyst was removed using a Celite® pad and volatiles were removed under reduced pressure to provide 0.574 g of crude amine. Crude material was dissolved in THF:water (1: 1, v/v; 10 mL), Boc anhydride (0.9 mL, 3.97 mmol) and triethyl amine (0.9 mL, 7.22 mmol) were added at 0°C for 1 h. The mixture was warmed to RT and was stirred for 16 h. Volatiles were removed under reduced pressure and crude material was purified by column chromatography (silica gel). Compound 76 was obtained in 54% yield (0.51 g); XH NMR (300 MHz, CDC13): δ 3.77 (t, J = 7.1 Hz, 2H), 3.53 (s, 2H), 3.41-3.36 (m, 4H), 1.67 (t, / = 7.1 Hz, 2H), 1.51-1.45 (m, 2H), 1.45 (s, 9H), 1.33-1.35 (m, 2H), two OH protons did not appear; MS calcd. for C13H25NO4: 259.2, Found: 160.2 (M + 1- Boc).
B. Compound 77
To a DCM (10 mL) solution of compound 76 (0.51 g, 1.97 mmol) was added TEA (1.8 mL, 17.80 mmol), followed by methane sulfonylchloride (0.53 mL, 6.89 mmol) at 0°C under N2 atmosphere. The mixture was warmed to RT and stirred for 2 h. Ice cold water was then added to the mixture, and extracted with DCM (200 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous Na2S04 and concentrated. Compound 77 was obtained in 1.00 g yield, and was used in next step without further purification; XH NMR (300 MHz, CDCI3): δ 4.35 (t, / = 6.6 Hz, 2H), 3.69 (s, 2H), 3.47-3.41 (m, 4H), 3.07 (s, 3H), 3.02 (s, 3H), 1.95 (t, J = 8.8 Hz, 2H), 1.57-1.52 (m, 4H), 1.46 (s, 9H); MS calcd. for CijHzgNOgSz: 415.1, Found: 316.0 (M + 1-Boc).
C. Compound 78
To a DMF (10 mL) solution of compound 77 (1.00 g, 2.41 mmol) was added Na2S.H20 (0.231 g, 2.41 mmol) at RT and the mixture was heated to 105°C for 6 h. The mixture was cooled to RT and cold water (200 mL) was added. The resulting mixture was extracted with ethyl acetate (300 mL x 3) and the combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate, and concentrated. The yield of compound 78 was 82% (0.51 g); XH NMR (300 MHz, CDCI3): δ 3.60-3.28 (m, 6H), 3.05 (s, 2H), 2.02-1.76 (m, 6H), 1.45 (s, 9H); MS calcd. for C13H23NO2S: 257.1, Found: 258.0 (M + 1).
D. Spirocyclic amine 79
To a ethyl acetate (5 mL) solution of compound 78 (0.4 g, 1.55 mmol) was added HC1 solution (~4 M in ethyl acetate; 5 mL) at 0°C, then stirred for 3 h at RT. Volatiles were removed under reduced pressure and crude material was purified by triturating with diethyl ether and dried. Spirocyclic compound 79 was obtained as a crude product (0.275 g), and used in the next step without further purification; XH NMR (300 MHz, DMSO-d6): δ 8.80 (br s, 1H), 3.74-3.63 (m, 2H), 3.22 (s, 2H), 3.04 (br s, 4H), 1.90 (s, 2H), 1.65 (br s, 4H). E. Compound 80
To a THF (16 mL) solution of compound 78 (0.40 g, 1.55 mmol) in a sealed tube were added ¾(¾ solution (9.3 mL 33% in water, 9.34 mmol), water (0.5 mL) and Na2W04.2H20 (1.53 g, 4.60 mmol). The sealed tube was closed with a cap and heated at 60°C for 16 h. The mixture was cooled to RT and diluted with water (200 mL) and was extracted with ethyl acetate (300 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate, and concentrated. Compound 80 was obtained in 98% yield (0.45 g); XH NMR (400 MHz, CDC13): δ 3.79-3.75 (m, 2H), 3.19 (t, J = 7.6 Hz, 2H), 3.14-3.07 (m, 2H), 3.03 (s, 2H), 2.13 (t, / = 7.6 Hz, 2H), 1.66-1.60 (m, 4H), 1.49 (s, 9H); MS calcd. for C13H23NO4S: 289.1, Found: 190.2 (M + 1-Boc).
F. Spirocyclic amine 81
To a ethyl acetate (5 mL) solution of compound 80 (0.45 g, 1.55 mmol) was added HCl solution (~4 M in ethyl acetate; 5 mL) at 0°C, which was stirred for 3 h at RT. Volatiles were removed under reduced pressure and crude product was purified by triturating with diethyl ether and drying. Spirocyclic amine 81 was obtained as the HCl salt (0.30 g); XH NMR (400 MHz, DMSO-d6): δ 8.82 (s, 1H), 3.22 (t, J = 7.6 Hz, 2H), 3.17 (s, 2H), 3.08-3.15 (m, 2H), 2.9-3.02 (m, 2H), 2.04 (t, / = 7.6 Hz, 2H), 1.80-1.88 (m, 2H), 1.70-1.80 (m, 2H). Exa
Figure imgf000138_0001
32 75; X = 0 83; X = 0
82; X = W-Me 84; X = W-Me
79; X = S 85; X = S
81 ; X = S02 86; X = S02
Figure imgf000138_0002
89; X = S 92: X = /V-Me
90; X = S02 93: X = S
94: X = S02
Example 30: Synthesis of compound 91
A. Compound 83 To a DMF (5 mL) solution of amine 75 (0.245 g, 1.77 mmol) were added compound 32 (0.501 g, 1.77 mmol) and K2C03 (0.489 g, 3.54 mmol) at RT. The mixture was heated at 100°C for 4 h. The mixture was cooled to RT, diluted with water and extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Crude material was purified by column chromatography (silica gel, ethyl acetate/pet. ether as eluent). The Yield of compound 83 was 0.12 g (17%); XH NMR (300 MHz, DMSO-d6): δ 11.60 (s, 1H), 7.12 (s, 1H), 6.99 (d, J = 1.6 Hz, 1H), 6.86 (s, 1H), 4.02 (t, J = 2.9 Hz, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 3.70 (t, / = 7.2 Hz, 2H), 3.40 (s, 2H), 2.68 (t, / = 6.1 Hz, 2H), 2.44-2.48 (m, 4H), 1.65 (t, J = 7.2 Hz, 2H), 1.51-0.00 (m, 4H); MS calcd. for C21H28N2O5: 388.2, Found: 389.0 (M + 1-Boc).
B. Compound 87
To a MeOH:water (1: 1, v/v; 5 mL) solution of compound 83 (0.12 g, 0.31 mmol) was added LiOH.H20 (0.038 g, 0.93 mmol) and resulting mixture was stirred at RT for 2 h. The mixture was acidified with HC1, and then volatiles were removed under reduced pressure to yield 0.20 g of crude compound 87 as the HC1 salt; XH NMR (400 MHz, DMSO- d6): δ 11.30 (s, 1H), 7.09 (s, 1H), 6.86 (s, 1H), 6.84 (s, 1H), 4.02 (t, J = 6.00 Hz, 2H), 3.76 (s, 3H), 3.71 (t, / = 6.8 Hz, 2H), 3.41 (s, 2H), 2.69 (t, / = 6.00 Hz, 2H), 2.46-2.49 (m, 4H), 1.64 (t, J = 6.8 Hz, 2H), 1.50-1.51 (m, 4H); MS calcd. for C21H28N2O5: 388.2, Found: 389.0 (M + 1-Boc).
C. Compound 91
To a DMF (5 mL) solution of compound 87 (0.115 g, 0.31 mmol) were added Pro- CBI 2 (0.06 g, 0.25 mmol) and EDC.HCl (0.245 g, 1.28 mmol) at 0°C, and the mixture then stirred for 16 h at RT. The mixture was diluted with water, and concentrated using a freeze drier. After complete removal of DMF, crude product 91 was purified by preparative HPLC (Column: Kromasil® CI 8; mobile phase: water (0.06% HC1) and acetonitrile). Yield 0.008 g (4.4%); HPLC purity 95.3% (Rt = 9.85 min); System: Agilent 1200 HPLC; detector: DAD max chromatogram (210-400 nm); column: Atlantis® dC18 (250 x 4.6 mm, 5μ); eluents: A: water + 0.1% TFA; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.57 (s, 1H), 10.58 (s, 1H), 10.47 (s, 1H), 8.12 (d, J = 8.3 Hz, 1H), 8.00 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.52 (t, / = 7.1 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.31 (s, 1H), 7.09 (s, 1H), 7.02 (s, 1H), 4.78 (t, / = 9.8 Hz, 1H), 4.53 (d, J = 9.8 Hz, 1H), 4.40 (s, 2H), 4.22 (t, J = 2.5 Hz, 1H), 4.03 (dd, J = 11.0, 3.0 Hz, 1H), 3.84 (s, 3H), 3.84-3.74 (m, 3H), 3.61-3.45 (m, 4H), 3.37-3.12 (m, 4H), 1.98-1.70 (m, 6H), one extra proton from HC1 salt; MS calcd. for C33H36CIN3O5: 589.2, Found: 590.2 (M + 1). Example 31: Synthesis of Compound 92
A. Compound 84
This compound was prepared from spirocyclic amine 82 using experimental procedure similar to the preparation of compound 83; H NMR (300 MHz, DMSO-d6): δ 11.63 (s, 1H), 7.11 (s, 1H), 6.99 (d, J = 1.5 Hz, 1H), 6.86 (s, 1H), 4.02 (t, J = 5.9 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 2.82-2.68 (m, 2H), 2.63-2.52 (m, 6H), 2.48-2.39 (m, 2H), 1.99 (s, 3H), 1.74-1.57 (m, 6H); MS calcd. for C22H31N3O4: 401.2, Found: 402.6 (M + 1).
B. Compound 88
This compound was prepared from ester 84 using experimental procedure similar to the preparation of compound 87. The acid was used in next step without characterization.
C. Compound 92
This compound was prepared from compounds 88 and Pro-CBI 2 using experimental procedure similar to the preparation of compound 91 in Example 27. HPLC purity 87% (Rt = 7.52 min); System: Agilent 1200 HPLC; detector: DAD max chromatogram (210-400 nm); column: Atlantis dC18 (250 x 4.6) mm, 5μ; eluents: A: water + 0.1% TFA; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.56 (s, 1H), 11.1-11.3 (m, 1H), 10.5-107 (m, 1H), 10.44 (s, 1H), 8.11 (d, / = 8.3 Hz, 1H), 7.98 (s, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.62 Hz, 1H), 7.30 (s, 1H), 7.08 (d, / = 1.8 Hz, 1H), 7.02 (s, 1H), 4.77 (t, / = 10 Hz, 1H), 4.58 (d, J = 10.8 Hz, 1H), 4.40 (s, 2H), 4.21 (s, 1H), 4.01-4.04 (dd, J = 11.2, 2.8, 1H), 3.84 (s, 3H), 3.84-3.80 (m, 1H), 3.80-3.01 (m, 9H), 2.92-2.89 (m, 1H), 2.80-2.77 (m, 3H), 1.94-2.00 (m, 2H), 1.84-1.87 (m, 4H), two extra protons from HC1 salt; MS calcd. for Cs^ClN^: 602.3, Found: 603.6 (M + 1)
Example 32: Synthesis of Compound 93
A. Compound 85
This compound was prepared from spirocyclic amine 79 using the experimental procedure similar to the preparation of compound 83; H NMR (300 MHz, CDCI3): δ 8.80 (s, IH), 7.16 (s, IH), 7.11 (s, IH), 6.87 (s, IH), 4.51 (t, J = 4.5 Hz, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.69-3.62 (m, 2H), 3.49-3.41 (m, 2H), 3.22-3.11 (m, 2H), 2.90-2.83 (m, 2H), 2.70-2.66 (m, 2H), 2.39-2.21 (m, 2H), 1.91-1.76 (m, 4H); MS calcd. for C21H28N2O4S: 404.2, Found: 405.1 (M + 1).
B. Compound 89
This compound was prepared from ester 85 using the experimental procedure similar to the preparation of compound 87; XH NMR (400 MHz, DMSO-d6): δ 11.93 (s, IH), 11.55 (s, IH), 7.23 (s, IH), 6.97 (s, IH), 6.91 (s, IH), 4.34-4.29 (m, 2H), 3.81 (s, 3H), 2.83-2.70 (m, 4H), 2.69-2.63 (m, 4H), 2.39-02.34 (m, 2H), 1.30-1.20 (m, 6H); MS calcd. for C20H26N2O4S: 390.2, Found: 391.5 (M + 1).
C. Compound 93
This compound was prepared from compounds 89 and Pro-CBI 2 using the experimental procedure similar to the preparation of compound 91; HPLC purity 70% (Rt = 10.62 min); System: Agilent 1200 HPLC; detector: DAD max chromatogram (210-400 nm); column: Hypersil® BDS C18 (250 x 4.6 mm, 5μ); eluents: A: water + 0.06% HC1; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.55 (s, IH), 10.44 (s, IH), 10.43 (br s, IH), 8.11 (d, / = 8.3 Hz, IH), 7.98 (s, IH), 7.84 (d, J = 8.3 Hz, IH), 7.52 (t, J = 7.2 Hz, IH), 7.35 (t, J = 7.5 Hz, IH), 7.30 (s, IH), 7.09 (d, J = 1.8 Hz, IH), 7.02 (s, IH), 4.77 (t, / = 9.4 Hz, IH), 4.53 (d, J = 9.4 Hz, IH), 4.39 (s, 2H), 4.22 (br s, IH), 4.03 (dd, J = 9.5, 2.8 Hz, IH), 3.86-3.83 (m, IH), 3.84 (s, 3H), 3.60-3.50 (m, 4H), 3.24-3.16 (m, 2H), 2.96-2.78 (m, 4H), 1.75-2.02 (m, 6H), one extra proton from HC1 salt; MS calcd. for C33H36CIN3O4S: 605.2, Found: 606.2 (M + 1).
Example 33: Synthesis of Compound 94
A. Compound 86
This compound was prepared from spirocyclic amine 81 using the experimental procedure similar to the preparation of compound 83; H NMR (400 MHz, CDC13): δ 8.79 (s, IH), 7.14 (s, IH), 7.12 (s, IH), 6.90 (s, IH), 4.49-4.42 (m, 2H), 3.99 (s, 3H), 3.94 (s, 3H), 3.68-3.62 (m, 2H), 3.59-3.49 (m, 4H), 3.28-3.16 (m, 2H), 3.09 (s, 2H), 2.62-2.51 (m, 2H), 2.29-2.21 (m, 2H), 2.08-1.98 (m, 2H); MS calcd. for CziHzgNzOeS: 436.2, Found: 437.6 (M + 1). B. Compound 90
This compound was prepared from ester 86 using the experimental procedure similar to the preparation of compound 87; XH NMR (400 MHz, CD3OD): δ 7.29 (s, 1H), 7.09 (s, 1H), 7.06 (s, 1H), 4.40 (t, J = 4.9 Hz, 2H), 3.95 (s, 3H), 3.62-3.65 (m, 4H), 3.59-3.43 (m, 2H), 3.32-3.32 (m, 2H), 3.20-3.17 (m, 2H), 2.36-2.12 (m, 6H), NH proton and COOH proton did not appear; MS calcd. for C2oH26N206S: 422.2, Found: 423.6 (M + 1).
C. Compound 94
This compound was prepared from compounds 90 and Pro-CBI 2 using the experimental procedure similar to the preparation of Compound 91; HPLC purity 99% (Rt = 9.32 min); System: Agilent 1200 HPLC; detector: DAD max chromatogram (210-400 nm); column: Hypersil® BDS C18 (250 x 4.6 mm, 5μ); eluents: A: water + 0.06% HC1; B: MeOH; linear gradient: 0-15 min 30 to 100% B; 15 -20 min 100% B; flow: 1.0 mL/min; XH NMR (400 MHz, DMSO-d6): δ 11.58 (s, 1H), 10.53 (s, 1H), 10.46 (s, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.99 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.1 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.31 (s, 1H), 7.10 (s, 1H), 7.03 (s, 1H), 4.78 (t, / = 10.2 Hz, 1H), 4.54 (d, J = 10.2 Hz, 1H), 4.39 (q, / = 4.9 Hz, 2H), 4.23 (t, / = 2.5 Hz, 1H), 4.04 (dd, J = 11.0, 3.0 Hz, 1H), 3.86- 3.83 (m, 1H), 3.84 (s, 3H), 3.65-3.51 (m, 4H), 3.34-3.26 (m, 4H), 3.19-3.11 (m, 2H), 2.09- 2.02 (m, 4H), 1.99-1.90 (m, 2H), one extra proton from HC1 salt; MS calcd. for C33H36CIN3O6S: 637.2, Found: 638.7 (M + 1)
In a similar manner, the following compounds are prepared using commercially availa
where
Figure imgf000142_0001
Figure imgf000143_0001
A. Cbz-Val-Cit-PAMA-(Alloc-PEG3-amide)-OH (95)
To a 30 mL methanol: water (10: 1, v/v) solution of compound 20 (10.00 g, 17.51 mol) was added LiOH.H20 (2.21 g, 52.53 mmol) at 0°C. After the disappearance of the starting material 20 (TLC analysis), the mixture was neutralized with acidic resin (20 g). The resin was filtered off and removal of methanol yielded crude acid. The crude acid was dissolved in DMF (30 mL) and NH2CH2CH2OCH2CH2OCH2CH2NHAII0C (5.60 g, 20.46 mmol), PyBOP (13.3 g, 25.58 mole), DIPEA (8.75 mL, 51.16 mmol) were added successively at 0°C. The mixture was stirred for 16 h at RT under nitrogen atmosphere. The mixture was diluted with ice water, stirred for 1 h, the solid was filtered off and washed with diethyl ether to obtained compound 95 as a light brown colored solid. Yield was 58% (7.60 g) with 86% LCMS purity; XH NMR (400 MHz, DMSO-d6): δ 9.99 (s, 1H), 8.08 (d, / = 7.6 Hz, 1H), 7.89 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.35-7.29 (m, 7H), 7.20 (s, 1H), 6.11 (s, 1H), 5.97-5.85 (m, 2H), 5.40 (s, 2H), 5.26 (d, J = 18.20 Hz, 1H), 5.15 (d, J = 10.40 Hz, 1H), 5.03 (s, 2H), 4.84 (s, 1H), 4.44-4.38 (m, 3H), 3.95-3.89 (m, 1H), 3.49-3.44 (m, 8H), 3.29-3.19 (m, 2H), 3.09-2.88 (m, 4H), 2.03-1.92 (m, 2H), 1.69-1.31 (m, 4H), 0.80-0.90 (m, 6H); MS calcd. for CsvHssNvOn: 771.4, Found: 772.4 (M + 1).
B. Trichloroacetimidate (96)
To a DMF (25 mL) solution of alcohol 95 (5.30 g, 6.87 mmol) was added Cs2C03
(4.50 g, 13.74 mmol) at RT. The mixture was cooled to 0°C and CC13CN (9.89 g, 68.74mmol) was added. The mixture was allowed to warm to RT over 2 h and stirred for additional 4 h. The mixture was poured over ice cooled water (100 mL), stirred for 1 h, the solid was filtered off and washed with diethyl ether to obtained compound 96 as a brown solid. The yield was 77% (4.80 g); MS calcd. for C39H52CI3N8O11: 914.3, Found: 754.4 (M- CF3CONH).
C. Cbz-Val-Cit-PAMA-(Alloc-PEG3-amide)-Pro-CBI (97)
To a suspension of 1.0 g 4 A molecular sieves (MS) in dry CH3CN was added trichloroacetimidate 96 (8.36 g, 9.12 mmol) and Boc-Pro-CBI 2a (1.52 g, 4.56 mmol) at RT. After stirring at RT for 2 h, the mixture was cooled to -10 °C and BF3.ether (5.9 mL, 47.92 mmol) was added. The mixture was allowed to warm to RT over 2 h and stirred for additional 4 h. Additional BF3 ether (0.40 mL) was added and solution was stirred at RT overnight. The mixture was neutralized using NEt3 and the separated mass was filtered off. Volatiles were removed under reduced pressure and resulting crude material was purified by silica gel (60-120 mesh) column chromatography using dichloromethane (DCM):MeOH solution as eluent to afford the compound 97 (2.20 g, 59% LCMS purity) in 48.8% yield; XH NMR (400 MHz, DMSO-d6): mixture of diastereomer, δ 10.1, 10.08 (s, 1H), 9.79 (s, 1H), 8.34-8.31 (m, 1H), 8.19-8.03 (m, 5H), 7.90-7.86 (m, 1H), 7.80 (d, 7 = 7.7 Hz, 1H), 7.52-7.35 (m, 6H), 7.29-7.21 (m, 20H), 7.09-7.04 (m, 2H), 6.52 (d, 7 = 8.2 Hz, 2H), 6.00-5.82 (m, 4H), 5.46-5.41 (m, 4H), 5.27-5.22 (m, 2H), 5.16-5.12 (m, 3H), 5.02 (s, 2H), 5.01 (s, 2H), 4.56- 4.31 (m, 6H), 3.99-3.83 (m, 4H), 3.70-3.61 (m, 2H), 3.50-2.82 (m, 35H), 2.00-1.90 (m, 2H), 1.71-1.22 (m, 8H), 0.87-0.79 (m, 12H); MS calcd. for CjoHesClNgOn: 986.4, Found: 987.0 (M + H).
d-91) (100)
Figure imgf000145_0001
A, B. Cbz-Val-Cit-PAMA-(Alloc-PEG3-amide)-(compound 91) (99)
To a DMF (3 mL) solution of compound 87 (0.40 g, 1.069 mmol) was added 1,1 '- carbonyldiimidazole (0.26 g, 1.604 mmol) portion-wise at 0°C. After 2 h of stirring at RT, the mixture (containing 98) was transferred to a stirred DMF solution of amine 97 (0.70 g, 0.709 mmol) and sodium carbonate (0.751 g, 7.092 mmol). Progress of the reaction was monitored using LCMS. After completion of the reaction, the mixture was poured into ice cold water stirred for 30 minutes, and the separated solid was filtered off and purified by silica gel (60-120 mesh) column chromatography using dichloromethane (DCM):MeOH solution as eluent to afford compound 99 (0.388 g, 65% LCMS purity) in 40.7% yield; MS calcd. for CVOHSVCINK J: 1342.6, Found: 1343.6 (M + 1).
C. Cbz-Val-Cit-PAMA-(MB-PEG3-amide)-(compound 91) (100): To a DCM solution of 99 (0.388 g, 0.288 mmol) was added Pd(PPh3)4 (0.02 g) and 1,3-dimethylbarbituric acid (DMBA) (0.019 g, 0.125 mmol), the mixture was stirred at RT for 2 h. After completion of reaction, DCM was removed under vacuum. The crude material obtained was purified by silica gel (60-120 mesh) column chromatography using dichloromethane (DCM):MeOH: NH3 solution as eluent to afford (0.197 g, 74% LCMS purity) amine. To a DMF (3 mL) solution of amine (0.197 g, 0.156 mmol) was added succinate ester 27 (0.109 g, 0.39 mmol) and sodium carbonate (0.033 g, 0.312 mmol) at RT. After completion of the reaction (monitored using LCMS), the mixture was poured over ice cold water and extracted with dichloromethane (3x 50 mL). The combined organic layers were dried over anhydrous Na2S04 and concentrated under reduced pressure to obtain crude material. The crude material was purified using prep HPLC to obtain 100 as a pale yellow solid (0.075 g, 95% LCMS purity) 18.2% Yield; MS calcd. for C74H90CIN11O16: 1423.6, Found: 1424.4 (M + 1).
Example 36: Synthesis of Cbz-Val-Cit-PAMA-(Iodoacetylaminobutanoyl-PEG3- amide)-(compound 91) (102)
Figure imgf000146_0001
To a 0.1 M borate buffer (pH 8.0):dioxane (1: 1, v/v, 2.0 mL) solution of amine 101 (0.020 g, 0.016 mmol) was added succinate ester 103 (0.012 g, 0.032 mmol) and sodium carbonate (0.0033 g, 0.032 mmol) at RT. After completion of the reaction (monitored using LCMS), DMF was removed under reduced pressure to obtain crude material. The crude material was purified using prep HPLC to obtain compound 102 as a pale yellow solid (0.005 g, 90% LCMS purity) 20.1% Yield; MS calcd. for C72H91ClIN110i5: 1511.5, Found: 1512.4 (M + 1). Example 37: Conjugation of anti-5T4 scFv-Fc antibody to a PAMA-Linker-Drug to form an anti-5T4 antibody drug conjugate ("anti-5T4 scADC"):
Anti-5T4 scFv-Fc engineered to present two Cysteine residues for site-specific conjugation was covalently linked to Cbz-Val-Cit-PAMA-(MB-PEG3-amide)-Pro-CBI- DMMI (compound 40), to form the corresponding anti-5T4 single-chain antibody drug conjugate ("anti-5T4 scADC").
A. Anti-5T4 scFv-Fc antibody
Anti-5T4 single chain antibody-Fc fusion protein ("anti-5T4 scFv-Fc") was prepared in CHO DG44 cells, as disclosed in US Provisional Application Number 61/835,858, filed June 17, 2013, herein incorporated by reference. SEQ ID NO: A
5T4-specific murine scFv:
EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSPGKGLEWIGRINPNNG VTLYNQKFKDKATLTVDKSSTTAYMELRSLTSEDSAVYYCARSTMITNYVMDYWG QGTSVTVSSGGGGSGGGGSGGGGSSIVMTQTPTSLLVSAGDRVTITCKASQSVSNDV AWYQQKPGQSPKLLISYTSSRYAGVPDRFTGSGSGTDFTLTISSVQAEDAAVYFCQQ DYNSPPTFGGGTKLEIK
SEQ ID NO: B
5T4-specific humanized scFv:
EVQLVESGGGLVQPGGSLRLSCKASGYSFTGYYMHWVRQAPGKGLEWVSRINPNN GVTLYNQKFKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTMITNYVMDYW GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG DRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYTSSRYAGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQDYNSPPTFGGGTKLEIK
SEQ ID NO: C
Human Hinge-CH2-CH3 (Fcgammal):
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: Y
Site-specific conjugation- 1 :
ASTC
SEQ ID NO: Z
No site-specific conjugation- 1 ASTX
SEQ ID NO: D
Chimeric anti-5T4 scFv-Fc:
EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSPGKGLEWIGRINPNNG VTLYNQKFKDKATLTVDKSSTTAYMELRSLTSEDSAVYYCARSTMITNYVMDYWG QGTSVTVSSGGGGSGGGGSGGGGSSIVMTQTPTSLLVSAGDRVTITCKASQSVSNDV AWYQQKPGQSPKLLISYTSSRYAGVPDRFTGSGSGTDFTLTISSVQAEDAAVYFCQQ DYNSPPTFGGGTKLEIKASTCEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: E
Humanized anti-5T4 scFv-Fc (ASTC):
EVQLVESGGGLVQPGGSLRLSCKASGYSFTGYYMHWVRQAPGKGLEWVSRINPNN GVTLYNQKFKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTMITNYVMDYW GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG DRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYTSSRYAGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQDYNSPPTFGGGTKLEIKASTCEPKSSDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: F
Humanized anti-5T4 scFv-Fc (ASTX):
EVQLVESGGGLVQPGGSLRLSCKASGYSFTGYYMHWVRQAPGKGLEWVSRINPNN GVTLYNQKFKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTMITNYVMDYW GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG DRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYTSSRYAGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQDYNSPPTFGGGTKLEIKASTXEPKSSDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B. Conjugation of Anti-5T4 scFv-Fc to a PAMA-Linker-Drug
Prior to the conjugation reaction, reduction of the scFv-Fc was performed with 0.8mM DTT in 20mM sodium phosphate, pH 7.2, 150mM NaCl at 3 mg/mL protein concentration for 45 min to break any possible adducts of the engineered Cys residue with glutathione or cysteines. The reduced scFv-Fc sample was then subjected to desalting in 50mM Tris-Cl, 150 mM NaCl, 250 mM Arginine (pH 8.2) to remove DTT using Hi-Prep 26/10 Desalting (GE Healthcare) column. Buffer exchanged scFv-Fc was then concentrated to 3 mg/mL and stirred slowly for 30 min to allow oxidative regeneration of the native disulfides.
scFv-Fc prepared in the above step was incubated in the presence of Cbz- Val-Cit-PAMA-(MB-PEG3-amide)-Pro-CBI-DMMI (compound 40) at 15-fold molar excess for 90 min with slow stirring for conjugation to occur. The conjugated scFv-Fc ("anti-5T4 scADC") was desalted to 20 mM PBS (phosphate buffer saline) containing 10% glycerol. Anti-5T4 scADC was analyzed by SDS-PAGE (reducing and non-reducing), MALDI-MS (Bruker Autoflex™ III), LC-ESI-MS (Bruker HCT Ultra) and SE-HPLC. Sinapinic acid in the presence of 50% acetonitrile and 0.1% TFA was used to formulate samples for MALDI. For LC-ESI-MS, a Zorbax® 300sb-C3 column was used for reverse phase chromatography.
C. Characterization of the anti-5T4 scADC
LC-ESI-MS analysis of the scADC demonstrated clear deconvolution of the detected charge states into two peaks. A peak corresponding to MW (major fraction) of
54669.6 Da corresponded to the monomer of non-conjugated anti-5T4 scFv-Fc. The second peak corresponding to MW 56000.7 Da was obtained, in which an increment of 1331 Da matched the molecular weight of the fragment added by conjugation with Cbz-Val-Cit- PAMA-(MB-PEG3-amide)-Pro-CBI-DMMI (Figure 7a). This observation confirmed that a single conjugation event occurred in each monomer of the anti-5T4 scFv-Fc. In addition, Figure 7b shows relative peak intensity of >80% for the conjugated scFv-Fc indicating the conjugation efficiency to be greater than 80%. Non-reducing SDS-PAGE of the scADC yielded a band at position corresponding to the dimeric scFv-Fc (anti-5T4 scADC) at -110 kDa. Negligible intensity of the monomeric band at -55 kDa indicated near complete formation of the covalently dimerized protein after air oxidation and conjugation. Overall, these analyses indicate that this sample of anti-5T4 scADC incorporated a distribution of 0, 1 and 2 linker-drug groups per scFvFc antibody, with an average of > 1.6 linker-drug groups per scFvFc antibody. That is, the drug-to-antibody ratio (DAR) for this sample was determined to be ca. 1.6. The structure of this anti-5T4 scADC is comprised as follows:
Figure imgf000150_0001
where Ab-S- is derived from anti-5T4 scFvFc (i.e., Ab-SH is anti-5T4 scFvFc), p" ranges from 0 to 2 in this sample, and the average p" = ca. 1.6.
Affinity of anti-5T4 scADC to 5T4 was determined by SPR analysis using a BIAcore™ T200 instrument (GE Healthcare). Briefly, protein A affinity purified 5T4- extracellular domain-human Fc fusion protein extracellular was covalently immobilized on a BIAcore™ CM5 sensor chip by amine coupling method using reagents and instructions provided by the manufacturer. In the binding study, anti-5T4 scADC was serially diluted to a concentration series and flowed over the immobilized antigen for a fixed period of time, followed by flow of buffer to dissociate the antigen. At the end of the dissociation cycle, regeneration of chip the surface was carried out at low pH. The resulting sensorgrams were fit to a 1: 1 Langmuir binding model using the BIAevaluation™ software (GE Healthcare) and the kinetics parameters such as association rate, dissociation rate and affinity were estimated. From this analysis, binding affinity of anti-5T4 scADC to 5T4 antigen was estimated in the range of 30-85 pM under the experimental conditions used. This affinity is in close agreement with non-conjugated anti-5T4 scFv-Fc antibody affinity for 5T4.
Fluorescence-activated cell sorting (FACS) analysis was carried out to determine cell binding of the anti-5T4 scADC, by using a MDA-MB-231 breast carcinoma cell line that over-expressed 5T4 (generated by stable transfection). Selective binding of the anti-5T4 scADC to the 5T4-high-expressing (transfectant) MDA-MB-231 cells versus the native 5T4-low-expressing MDA-MB-231 cells was observed, in close agreement to observations for the non-conjugated scFv-Fc antibody.
D. Cell growth inhibition by the anti-5T4 scADC
An evaluation of the cytotoxic potency of the anti-5T4 scADC in vitro, against 5T4-expressing cancer cells, was conducted by using a MDA-MB-231 breast carcinoma cell line that over-expressed 5T4 (generated by stable transfection). Recombinant MDA-MB-231 cells over-expressing 5T4 (disclosed in US Provisional Application Number 61/835,858, filed June 17, 2013) were used to study the cell growth inhibition potential of anti-5T4 scADC. Native MDA-MB-231 cells (ATCC) were used as negative control due to their extremely low basal expression levels of 5T4. Cell were routinely cultured as monolayers in nutrient medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin G and 100 μg/mL streptomycin sulfate at 37 °C under 5% C02 atmosphere. Cells of an exponentially-growing monolayer culture were harvested and plated at a concentration of 4000 cells/well in 96 well cell culture plate and allowed to grow overnight under standard cell growth conditions. Cells were treated with the scADC and other drug molecules for 96 h after which the culture medium was replaced with 1 X MTS reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (Promega). After 3 h incubation at 37 °C absorbance of individual wells was read at 490 nm in a spectrophotometer. Control cultures were incubated in medium containing 0.5% (v/v) DMSO. IC50 values were determined after analysis of the drug response curve with four parameter non-linear regression equation using GraphPad Prism® software.
As summarized in Table 5, an IC50 value of 0.8 nM was obtained for the anti- 5T4 scADC. Non-conjugated scFv-Fc antibody did not result in any cell growth inhibition of the recombinant MDA-MB-231 cells. Native MDA-MB-231 cells, previously confirmed to have extremely low basal expression of 5T4 were also used in the cell growth inhibition assays. The 5T4-low-expressing cell line was approximately 100-fold less sensitive to growth inhibition by the anti-5T4 scADC.
Table 5: Cell growth inhibition IC50 of anti 5T4 scADC against MDA-MB-231 and
MDA-MB-231 (5T4-overexpressing transfectant) cell lines
Figure imgf000151_0001
Example 38: Conjugation of anti-HER2 scFv-Fc to a PAMA-Linker-Drug to form an anti-HER2 antibody drug conjugate (anti-HER2 scADC-vl) In a similar manner to Example 37, anti-HER2 scFv-Fc engineered to present two cysteine residues for site-specific conjugation was covalently linked to Cbz-Val-Cit-PAMA- (MB-PEG3-amide)-Pro-CBI-DMMI (compound 40), to form the corresponding anti-HER2 single-chain antibody drug conjugate ("anti-HER2 scADC-vl "). In this reaction, purified anti-HER2 scFv-Fc, freshly reduced in the presence of 600 μΜ DTT at 3 mg/mL protein concentration in 20 mM sodium phosphate, 150 mM NaCl, pH 7.0 for 45 minutes, was desalted using a Hiprep® 26/10 column (GE Healthcare) into 50 mM Tris, 50 mM NaCl, 125 mM arginine (pH 7) and was then air-oxidized for 20 min with slow stirring. The anti- HER2 scFv-Fc reduced and re-oxidized in the above reaction was treated with 10-fold molar excess of the linker-drug molecule 40 for 90 min to generate the conjugate anti-HER2 scADC-vl , which was then buffer-exchanged into 20 mM PBS, 10% glycerol at pH 7.2 for storage.
In a variation to the above procedure, the anti-HER2 scADC-vl generated was further purified using a Hi-trap Butyl sepharose FF (GE Healthcare) column. The scADC was mixed with equal volume of 50 mM Tris-Cl pH-7.0, 1.5M ammonium sulfate and clarified by filtration prior to loading on to the column. Proteins bound to the column were eluted with a linear gradient of 50 mM Tris-Cl, pH 7. Each collected fraction was analyzed by MALDI mass spectrometry to identify the fractions containing scADC, which were pooled. The pooled scADC was buffer exchanged into 20 mM PBS, 10% glycerol, pH 7.
A. Characterization of the anti-HER2 scADC-vl
LC-ESI mass spectrometric analysis as described in Example 37 detected greater than 65% scADC in the final preparation with 2 linker-drug molecules per the scFv- Fc dimer.
Surface Plasmon Resonance (BIAcore) analysis of the anti-HER2 scADC binding kinetics to purified HER-2 extracellular domain-Fc fusion protein indicated similar affinity of the scADC to that of the unconjugated anti-HER2 scFv-Fc.
Overall, the analyses performed indicate that this sample of anti-HER2 scADC-vl incorporated a distribution of 0, 1 and 2 linker-drug groups per anti-HER2 scFv- Fc antibody, with an average of > 1.3 linker-drug groups per scFv-Fc antibody. The structure of this anti-HER2 scADC-vl is comprised as follows:
Figure imgf000153_0001
where Ab-S- is derived from anti-HER2 scFv-Fc (i.e., Ab-SH is anti-HER2 scFv-Fc), and p" ranges from 0 (unconjugated antibody) to 2 in this sample, with the average p" = ca. 1.3.
B. In vivo Tumor growth inhibition by the anti-HER2 scADC-vl
Athymic male & female nude mice (Hsd: Athymic Nude-Foxnl™) 5-6 weeks old, weighing 20-22 g were obtained from Harlan, Netherlands. Animals were taken care as per the Regulations of Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), Government of India and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) compliance. The 'Form B' for carrying out animal experimentation was reviewed and approved by the Institutional Animal Ethics Committee (IAEC Protocol Approval No: SYNGENE/IAEC/349/10-2012). Animals were maintained in a controlled environment with 22 ± 3°C temperature, 50 ± 20 % humidity, a light/dark cycle of 12 hours each and 15-20 fresh air changes per hour. Animals were housed group wise and autoclaved corncob was used as a bedding material. The animals were fed, ad libitum, with certified Irradiated Laboratory Rodent Diet during the study period. The animals were kept under acclimatization in the experimental room for a period of at least 5 days. Animals were individually numbered and the cage cards indicating the experiment, study number, date of tumor implantation, date of randomization, tumor type, mouse strain, gender, and individual mouse number were displayed to corresponding cages. After randomization, group identity, test compound, dosage, schedule and route of administration were added.
Preparation of tumor cells: All procedures were performed in laminar flow hood following sterile techniques. Cancer cells SKOV-3 (Ovarian) with 70-80% confluent and viability of >90 % were chosen for the study. Cancer cells SKOV-3 (5 X 106 cells) were resuspended in 200 of PBS or serum free media containing 50% of matrigel kept in ice. Subcutaneous injection of cells: Nude mice (Hsd: Athymic Nude-Foxnl™) housed in Individual Ventilated Cages (IVCs) were used. Cancer cell line (SKOV-3) was propagated in the animals by injecting the cancer cells subcutaneously in the flanks or back of the animals. The implanted area was monitored for growth of tumor. Once the tumor attained palpable and required volume (TV~ 200mm3), animals were randomized based on tumor volume, and dosing of the test samples was initiated (Day 0). The tumor volume was determined by two-dimensional measurement with a caliper on the day of randomization (Day 0) and then once every three days (i.e. on the same days on which mice were weighed). Using a vernier caliper the length (1) and width (w or b) of the tumor was measured. Tumor volume (TV) was calculated using the following formula: Tumor Volume (mm3) = L X W2 / 2, where L = Length (mm) and W = Width (mm). The scADC test sample was dissolved in sterile lx PBS, which resulted in a clear solution and was administered intravenously via tail vein. The test solution was freshly prepared on the days of administration and the dose volume was kept at 5 mL/kg body weight. For each animal group, separate new syringe and needles were used.
Clinical signs and body weight: Animals were observed individually for visible general clinical signs once every three days during the study period. All the animals were checked for morbidity and mortality. Body weights were measured once every three days during the study period. The % change in body weights of individual mice was calculated.
Antitumor Activity: Antitumor activity was evaluated as maximum tumor volume inhibition versus the vehicle (formulation buffer) control group. Data evaluation was performed using statistical software Graph Pad version 5.
Results: In a xenograft experiment with the SKOV-3 cell line, anti-HER2 scFv-Fc (unconjugated antibody) and anti-HER2 scADC-vl were dosed to the tumor-bearing mice at 2 mg/kg IV, Q7D x 3, i.e., dosing once every seven days for a total of three doses. Anti-HER2 scADC-vl therapy demonstrated significant antitumor activity (inhibition of tumor growth), measured up to Day 33, in comparison to vehicle-treated control animals. Anti-HER2 scFv-Fc antibody provided no significant antitumor activity, in comparison to vehicle-treated control animals. There was no significant body weight loss measured in vehicle-treated control group, anti-HER2 scFv-Fc treated group, and the anti-HER2 scADC- vl treated group during the experiment period (up to Day 33). All animals were observed to be active and healthy. Anti-HER2 scADC-vl therapy was relatively well tolerated at the tested dose level with no mortality. Moreover, there were no visible signs of abnormal behavior or any adverse clinical symptoms during treatment.
Example 39: Conjugation of anti-HER2 scFv-Fc to a PAMA-Linker-Drug to form an anti-HER2 antibody drug conjugate (anti-HER2 scADC-v2)
In a similar manner to Example 37, anti-HER2 scFv-Fc engineered to present two
Figure imgf000155_0001
which incorporates the PAMA linker and the duocarmycin analog prodrug compound 91. This conjugation process formed the corresponding anti-HER2 single-chain antibody drug conjugate ("anti-HER2 scADC-v2"). This conjugation process included the generation of reduced anti-HER2 scFv-Fc in the presence of 800 μΜ DTT for 45 min at room temperature in 20 mM sodium phosphate, 150 mM NaCl, pH 7.0 followed by desalting into 50 mM Tris, 50 mM NaCl, 125 mM Arginine, pH 7.0 and air oxidation with slow stirring. The reduced, air-oxidized anti-HER2 scFv-Fc was diluted in conjugation buffer (50 mM Tris, 50 mM NaCl, 125 mM Arginine, pH 7.0) to a final concentration of 0.03 mg/mL. To the preparation of 500 mL of the above dilute protein, 2.5 mL of the linker-drug molecule at 1.5 mg/mL in DMF was added. The conjugation reaction was allowed to proceed for 1.5 h at room temperature with slow rocking. The conjugated protein was purified by butyl sepharose chromatography in a manner similar to Example 38.
A. Characterization of the anti-HER2 scADC-v2 LC-ESI-mass spectrometric analysis of anti-HER2 scADC-v2 following purification by butyl sepharose chromatography indicated the preparation to be almost entirely composed of scFv-Fc conjugated to 2 linker-drug molecules per the scFv-Fc dimer. Measurement of affinity of this anti-HER2 scADC-v2 against HER2 extracellular domain-Fc fusion protein in a SPR analysis similar to those described above confirmed similar affinity of the scADC as that of the unconjugated anti-HER2 scFv-Fc.
Overall, the analyses performed indicate that this sample of anti-HER2 scADC-v2 incorporated ~2 linker-drug groups per anti-HER2 scFv-Fc antibody. The structure of this anti-HER2 scADC-v2 is comprised as follows:
Figure imgf000156_0001
where Ab-S- is derived from anti-HER2 scFv-Fc (i.e., Ab-SH is anti-HER2 scFv-Fc), and p" = 2.
D. Cell growth inhibition by the anti-HER2 scADC-v2
An evaluation of the cytotoxic potency of the anti-HER2 scADC-v2 in vitro, was carried out against HER2-expressing cancer cells. The cytotoxicity assay was carried out in 96-well plates; approximately five thousand cells were seeded in 100 μΕ of medium in each well of the 96-well plates. After 24 h, cells were treated with different concentrations of the anti-HER2 scADC-v2. 96 h after the treatment, media was removed from the wells, and 10 μΕ of cell titre aqueous reagent was added to 100 μΕ of media in each well. Plates were incubated for 3 hours and absorbance in each well at 490 nm was read using an ELISA plate reader. IC50 values were determined after analysis of the drug response curve with four parameter non-linear regression equation using GraphPad Prism® software. Average IC50 values of 4.0 nM and 40 nM were determined for the anti-HER2 scADC-v2 in NCI-N87 and SKBR3 tumor cell lines, respectively. In comparison, the non-conjugated anti-HER2 scFv- Fc antibody did not result in any cell growth inhibition in these two cell lines.
B. In vivo Tumor growth inhibition by the anti-HER2 scADC-v2
In a similar manner to the procedure described in Example 38, anti-HER2 scADC-v2 was evaluated in HER2-expressing tumor xenograft models, for example SKOV3 tumor xenograft model. Following a procedure similar to that described in Example 38, the anti-HER2 scADC-v2 was administered at a dose of 3 mg/kg mouse weight, IV, followed by 1 mg/kg IV Q4D x 4. Antitumor activity was assessed based on the observed inhibition of tumor growth or tumor suppression, in comparison to mice treated with vehicle (formulation buffer). Approximately 100% tumor growth inhibition by the anti-HER2 scADC-v2 was observed on Day 18. In this SKOV3 model, the non-conjugated anti-HER2 scFv-Fc antibody was also active, but the percent tumor growth inhibition observed on Day 18 was significantly less than that observed for that observed for the anti-HER2 scADC-v2.
Example 40: Conjugation of anti-HER2 IgG (trastuzumab) to a PAMA-Linker-Drug, where the drug is the amine-linked cytotoxic drug MMAE, to form an anti-HER2 antibody drug conjugate (anti-HER2 ADC-v3)
In a similar manner to Example 37, the anti-HER2 IgG antibody trastuzumab was
Figure imgf000157_0001
103 (R6 = H); 104 (R6 = CH3)
This compound 103 incorporates the PAMA linker and the amine-linked cytotoxic drug N- methyl auristatin E (MMAE). Compound 104 is a related MMAE-drug/PAMA-linker compound that is also used to prepare antibody-drug conjugates.
B. Preparation of the Drug-Linker Compound 103
The drug-linker compound 103 was prepared by the following steps.
Figure imgf000158_0001
Figure imgf000158_0002
103-C
Preparation of compound 103-b: To a 10 mL DMF solution of compound 103-a (2.2 g, 3.49 mmol) was added bis(4-nitro phenyl)carbonate (2.12 g, 6.99 mmol) and DIPEA (0.9 mL, 5.24 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 3 h. After consumption of starting material 1 (TLC analysis), the reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (50 mL x 3). Combined organic phases were dried over anhydrous Na2S04 and concentrated under reduced pressure. The crude solid obtained was stirred with dichloromethane and filtered to obtained 2.2 g (85%) of 103-b; 72% purity (LCMS); MS calcd. for C35H4oN6Oi2: 736.3, Found: 737.7 (M + 1).
Preparation of compound 103-c: To a solution of N-methyl auristatin E (MMAE) (0.10 g, 0.139 mmol; Concords Biosystems, San Diego, CA) in 1.5 mL DMF was added HOBt (0.01 g, 0.069 mmol) and DIPEA (0.035 mL, 0.209 mmol) at RT. The reaction mixture was cooled to 0°C and compound 2 (0.113 g, 0.155 mmol) was added. The reaction mixture was stirred at RT for 48 h, and then additional HOBt (0.01 g, 0.069 mmol) and compound 103-b (0.051 g, 0.069 mmol) were added. The reaction mixture was stirred for an additional 12 h. The reaction mixture was poured over ice-cold water (30 mL). The solid that formed was filtered off and washed with water and hexane, dried under reduced pressure, and then purified by column chromatography (silica gel) using dichloromethane/methanol as eluent. The compound 103-c was obtained (0.16 g, 87%) with 62% purity (LCMS); MS calcd. for C68Hio2N1oOi6: 1314.7, Found: 1315.6 (M + 1).
Figure imgf000159_0001
103-f
Preparation of compound 103-e: To a 10 mL DMF solution of tert-butyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate (1.0 g, 4.03 mmol) and hydroxysuccinyl ester 103-d (1.35 g, 4.83 mmol) at 0 °C was added Na2C03 (0.641 g, 6.04 mmol). The reaction mixture was allowed to warm to RT and stirred for 12 h. The solid was filtered off and the filtrate was diluted with water (100 mL) and extracted with ethyl acetate (70 mL x 3). Combined organic phases were dried over anhydrous Na2S04, concentrated under reduced pressure and then purified by silica gel (60-120 mesh) column chromatography using 5% methanol in dichloromethane as eluent to yield compound 103-e (0.88 g, 53%) with 99% purity (LCMS); XH NMR (400 MHz, DMSO-d6): δ 7.86 (s, 1H), 7.01 (s, 2H), 6.78 (s, 1H), 3.49 (br s, 5H), 3.40-3.34 (m, 5H), 3.17 (t, J = 5.80 Hz, 2H), 3.05 (t, J = 5.88 Hz, 2H), 2.05 (t, J = 7.68 Hz, 2H), 1.70 (p, J = 7.32 Hz, 2H), 1.37 (s, 9H); MS calcd. for C19H31N3O7: 413.2, Found: 414.2 (M + 1).
Preparation of compound 103-f: To a DCM solution of 103-e (0.08 g, 0.193 mmol) at 0 °C was added 0.4 mL TFA. After completion of the reaction (TLC analysis), the solvent was removed under reduced pressure. The residual solid was washed with ether to provide compound 103-f (0.06 g, 99%); MS calcd. for C14H23N3O5: 313.2, Found: 314.0 (M + 1).
Figure imgf000160_0001
Preparation of drug-linker compound 103: To a solution of compound 103-c (0.158 g, 0.120 mmol) in 6.6 mL THF: water (10: 1, v/v) at 0 °C was added LiOH.H20 (0.015 g, 0.360 mmol). After consumption of starting material 103-c (TLC analysis), the reaction mixture was neutralized by addition of acidic H+ resin. The resin material was filtered off and methanol was removed under reduced pressure to provide the crude carboxylic acid derivative of compound 103-c. The crude carboxylic acid was dissolved in DMF (5 mL) and amine 103-f (0.055 g, 0.161 mmol), PyBOP (0.084 g, 0.161mmol) and DIPEA (0.037 mL, 0.215 mmol) were added successively at 0 °C. The reaction mixture was stirred for 3 h at RT. The reaction mixture was diluted with ice water and extracted with ethyl acetate (3 x 25 mL). Combined organic phases were washed with brine solution, dried over anhydrous sodium sulphate, and concentrated under reduced pressure. The residual solid obtained was purified by preparative HPLC, to provide the drug-linker compound 103 (0.056 g, 29%); HPLC purity 99% (Rt = 15.3 min); System: Agilent 1200 HPLC; detector: PDA max chromatogram (210-400 nm); column: Column-XTERRA RP18 (150 x 4.6 mm) mm 5 μιη; eluents: A: water + 0.1% HCOOH; B: acetonitrile; linear gradient: 0-15 min 5 to 80% B; 15-20 min 80 to 100% B; 20-25 min 100% B; flow: 0.8 mL/min; XH NMR (400 MHz, DMSO-d6): δ 10.10-10.07 (m, 1H), 8.49-8.42 (m, 1H), 8.20-8.12 (m, 2H), 7.68-7.54 (m, 3H), 7.44-7.13 (m, 13H), 7.00 (s, 2H), 5.98 (s, 1H), 5.43 (s, 3H), 4.80-4.32 (m, 4H), 4.22-3.89 (m, 4H), 3.53-2.80 (m, 31H), 2.31-1.32 (m, 21H), 1.10-0.60 (m, 35H); MS calcd. for C81H121N1302o: 1595.9, Found (negative-mode MS): 1595.6 [M - H] , 1632.6 [M + CI]".
B. Preparation of the Drug-Linker Compound 104
O
,NHBoc
HpN' ,NHBoc
F,C N
104-a 104-b
Figure imgf000161_0001
Synthesis of trifluoroacetamide 104-b: To a 90 mL THF solution of tert-butyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate 104-a (9.0 g, 0.036 mmol) was added Et3N (7.3 g, 0.072 mmol) and trifluoroacetic anhydride (7.5 mL, 0.054 mmol) drop-wise at 0 °C. The reaction mixture was allowed to warm to RT and stirred for 12 h. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried over anhydrous Na2S04, concentrated under reduced pressure, and the residue was purified by silica gel (60-120 mesh) column chromatography to provide compound 104-b (11.0 g, 88%); XH NMR (300 MHz, CDC13): δ 7.26 (br s, 1H), 4.92 (br s, 1H), 3.63-3.49 (m, 10H), 3.32 (br s, 2H), 1.44 (s, 9H); MS calcd. for C13H23F3N2O5: 344.2, Found: 245.0 (M + 1-Boc).
Preparation of compound 104-c: To a 15 mL acetone solution of trifluoroacetamide 104-b (1.2 g, 0.0033 mmol), K2C03 (0.52 g, 0.0038 mmol) was added and then dimethyl sulfate (0.36 mL, 0.0038 mmol) at RT. The reaction mixture was heated at reflux for 12 h. The reaction mixture was then cooled to RT, diluted with water (20 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were dried over anhydrous Na2S04, concentrated under reduced pressure, and the residue was purified by silica gel (60-120 mesh) column chromatograp to provide compond 104-c (0.8 g, 64%); H NMR (300 MHz, CDCI3): δ 6.73 (br s, 1H), 3.60-3.47 (m, 8H), 3.37-3.34 (m, 2H), 3.13 (s, 3H), 3.12-2.99 (m, 2H), 1.44(s, 9H); MS calcd. for C14H25F3N2O5: 358.2, Found: 259.0 (M + 1-Boc). Preparation of compound 104-d: To a 10 mL DMF solution of N-methyl trifluoroacetamide 104-c (1.3 g, 0.0036 mmol) at 0 °C was added aqueous ammonia (10 mL). The reaction mixture was allowed to warm to RT and then stirred for 12 h. Volatiles were removed under reduced pressure and the residue was purified by silica gel (60-120 mesh) column chromatography to provide compound 104-d (0.61 g, 64%); XH NMR (300 MHz, CDC13): δ 8.32 (br s, 1H), 6.79 (br s, 1H), 3.63-3.52 (m, 6H), 3.37-3.35 (m, 2H), 3.08-3.02 (m, 4H), 2.71 (s, 3H), 1.33 (s, 9H); MS calcd. for C12H26N2O4: 262.2, Found: 263.0 (M + 1).
Figure imgf000162_0001
Preparation of compound 104-e: To a solution of compound 103-c (0.15 g, 0.114 mmol) in 4.4 mL MeOH:water (10:1, v/v) at 0 °C was added LiOH.H20 (0.014 g, 0.34mmol). After consumption of starting material 103-c (TLC analysis), the reaction mixture was neutralized by the addition of acidic H+ resin. The resin material was filtered off and methanol was removed under reduced pressure to provide the crude carboxylic acid derivative of compound 103-c. The crude carboxylic acid was dissolved in DMF (3 mL) and amine 104-d (0.0407 g, 0.155 mmol), PyBOP (0.081 g, 0.155 mmol) and DIPEA (0.035 mL, 0.207 mmol) were added successively at 0 °C. The reaction mixture was stirred for 16 h at RT. Reaction mixture was diluted with ice water and the precipitated solid material was filtered and washed with water (20 mL x 2) to provide compound 104-e (0.115 g, 64%), which was used in the next step without further purification; purity 63% (LCMS); MS calcd. for C79H124N12O19: 1544.91, Found: 1546.8 (M + 1). Preparation of drug-linker compound 104: To a EtOAc suspension of compound 104-e (0.095 g, 0.0615 mmol) was added 1.5 mL HC1 in EtOAc (~4 N) at -10 °C. After completion of the reaction, EtOAc was removed under nitrogen stream. The residual material was dissolved in 2 mL DMF, and then the hydroxysuccinyl ester 103-d (0.034 g, 0.121 mmol) and sodium carbonate (0.012 g, 0.110 mmol) were added at room temperature. The reaction mixture was stirred for 12 h. After completion of the reaction (monitored using LCMS), the reaction mixture was diluted with ice water and extracted with ethyl acetate (3 x 25 mL). The combined organic phases were washed with brine solution, dried over anhydrous sodium sulphate, and concentrated. The crude material obtained was purified by preparative HPLC to afford drug-linker compound 104 (0.034 g, 32%); purity 98% (LCMS); MS calcd. for C82H123N13O20: 1609.90, Found: 1611.6 (M + 1). Compound 104 is a drug-linker compound, related to 103, that can also be used for conjugation to a targeting ligand such as an scFvFc or IgG antibody.
C. Conjugation of anti-HER2 IgG antibody to a PAMA-Linker-MMAE
The conjugation process formed an anti-HER2 IgG antibody drug conjugate ("anti-HER2 ADC-v3") incorporating the PAMA linker, and MMAE as the cytotoxic drug. Trastuzumab (T-mAb) was obtained from a commercial source and was purified by gel filtration chromatography before conjugation. T-mAb (0.2 mg/mL) was treated with 3 molar equivalents of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) in 0.025 M sodium borate pH 8, 0.025 M NaCl, 1 mM diethylenetriamine penta-acetic acid (DTP A) for 2 h at 37 °C. The mixture was then cooled to 4 °C. The partially reduced T-mAb was then treated with 6 molar equivalents of drug-linker compound 103 for 1 h at 4°C. The conjugated and unconjugated fractions were purified by Butyl Sepharose chromatography on a Hi-Trap Butyl HP (5 mL) column (GE Healthcare) equilibrated with 50 mM Tris-Cl pH 7.5, 750 mM Ammonium Sulfate. Bound proteins were eluted using a linear gradient of water in 20 column volumes. Each peak eluted from the Butyl Sepharose column was analyzed by SDS- PAGE and LC-ESI-MS to identify those corresponding to conjugated antibody. Purified ADC fractions were pooled and buffer exchanged into sodium phosphate buffer, pH 7.4 150 mM sodium chloride and 5% glycerol.
D. Characterization of the anti-HER2 ADC-v3 For LC-ESI-MS analysis, about 20 μg of purified anti-HER2 scADC-v3 was reduced with 10 mM di-thiothreitol (DTT) at 37°C for 30 min. A portion (5μg) of the reduced sample was loaded on to an Zorbax 300SB-C3 HPLC column (Agilent) equilibrated with 0.1% formic acid using an Agilent 1200 Series HPLC interfaced with Bruker HCT Ultra ESI-MS ion trap system. A linear gradient of acetonitrile was used for elution of protein. The mass spectrometric data was acquired using Esquire Control software and analyzed by DataAnalysis software. Analysis of conjugated T-mAb LC-ESI-MS data in a manner similar to that described in Example 37 indicated that the sample of conjugated T- mAb consisted of major fractions containing either 2 or 4 conjugation events per antibody molecule giving rise to an approximate average drug to antibody ratio (DAR) of 3.
Analysis of binding kinetics of T-mAb drug conjugate to HER2 extra cellular domain was conducted using surface plasmon resonance based methods as described in Examples 37 and 38. This analysis indicated that the affinities of T-mAb and T-mAb drug conjugate anti-HER2 ADC-v3 were similar (KD <30 pM) for HER-2 extracellular domain.
Overall, the anti-HER2 ADC-v3 was determined to incorporate a distribution of about 2 to about 4 linker-drug groups per anti-HER2 IgG antibody. The structure of this anti-HER2 ADC-v3 is comprised as follows:
Figure imgf000164_0001
where R6 is H, Ab-S- is derived from trastuzumab (i.e., Ab-SH is trastuzumab), and p ranges from about 2 to about 4 in this sample, and the average p is about 3.
C. Cell growth inhibition by the anti-HER2 ADC-v3
Characterization of the antibody drug conjugate to demonstrate in vitro cytotoxic activity growth inhibition was carried out by methods known to those skilled in the art, for example, using methods similar to those described for Examples 37 to 39. Average IC50 values of 0.09 nM and 0.3 nM were obtained for the T-mAb drug conjugate against HER2 -expressing NCI-N87 gastric carcinoma and SKBR-3 breast carcinoma cell lines, respectively. Tested under identical conditions, the unconjugated T-mAb did not have any effect on the cell growth kinetics of these cell lines for the range of concentrations used. In comparison, IC50 values of 57 nM (NCI-N87) and 57 nM (SKBR3) were determined for Doxorubicin. In vitro growth of another breast carcinoma cell line, MDA-MB-231, which was characterized to have low expression of the target HER2 antigen, was not affected by either T-mAb and T-mAb MMAE conjugate.
D. In vivo Tumor growth inhibition by the anti-HER2 ADC-v3 In a similar manner to the procedure described in Example 38, anti-HER2
ADC-v3 was evaluated in HER2-expressing tumor xenograft models, for example SKOV3 tumor xenograft model. Following a procedure similar to that described in Example 38, the anti-HER2 ADC-v3 was administered at a dose of 3 mg/kg mouse weight, IV, on Day 0, followed by 1 mg/kg IV Q4D x 4 (on Days 4, 8, & 12). Antitumor activity was assessed based on the observed inhibition of tumor growth or tumor suppression, in comparison to mice treated with vehicle (formulation buffer). Approximately 100% tumor growth inhibition by the anti-HER2 ADC-v3 was observed on Day 18. In this SKOV3 model, the non-conjugated Trastuzumab antibody was also highly active. Example 41: Conjugation of anti-HER2 IgG (trastuzumab) to a PAMA-Linker-Drug to form an anti-HER2 antibody drug conjugate (anti-HER2 ADC-v4)
In a similar manner to Example 40, anti-HER2 IgG (trastuzumab) was covalently linked to compound 100:
Figure imgf000166_0001
which incorporates the PAMA linker and the duocarmycin analog prodrug compound 91. This conjugation process formed the corresponding anti-HER2 antibody drug conjugate ("anti-HER2 ADC-v4").
A. Conjugation of anti-HER2 IgG antibody to a PAMA-Linker-Drug
Trastuzumab (0.2 mg/mL) was treated with 3 fold molar equivalents of TCEP (tris-(2-carboxyethyl)phosphine hydrochloride) in 0.025 M sodium borate pH 8, 0.025M NaCl, 1 mM DTPA (diethylenetriamine penta-acetic acid) for 2 h at 37 °C. The mixture was then cooled to 4 °C. Partially reduced Trastuzumab from the above reaction was treated with 6 fold molar equivalents of linker-drug 91 for 1 h at 4 °C. The conjugated protein was purified by butyl sepharose chromatography in a manner similar to Example 38. B. Characterization of the anti-HER2 ADC-v4
LC-ESI-mass spectrometric analysis of anti-HER2 scADC-v4 following purification by butyl sepharose chromatography indicated the preparation to be a heterogenous mixture of ADCs with DAR values from about 2 to about 6. An average DAR of 3.7, calculated from the weighted average of LC-ESI-MS peaks, was obtained for the anti- HER2 ADC-v4.
Measurement of affinity of this anti-HER2 ADC-v4 against HER2 extracellular domain-Fc fusion protein in a SPR analysis similar to those described above confirmed similar affinity of the ADC (KD <30 pM) as that of the unconjugated T-mAb (KD <30 pM).
Overall, the analyses performed indicate that this sample of anti-HER2 ADC- v4 incorporated ~2 to ~6 linker-drug groups per anti-HER2 Trastuzumab IgG antibody, with an average of 3.7 linker-drug groups per Trastuzumab antibody. The structure of this anti- HER2 ADC-v4 is comprised as follows:
P where Ab-S- is derived from Trastuzumab, and p" = 2 to 6, with average p" = 3.7.
C. Cell growth inhibition by the anti-HER2 ADC-v4
Characterization of the antibody drug conjugate to demonstrate in vitro cytotoxic activity growth inhibition was carried out by methods known to those skilled in the art, for example, using methods similar to those described for Examples 37 to 39. Average IC50 values of 0.07 nM and 0.3 nM were determined for the anti-HER2 ADC-v4 in NCI-N87 and SKBR3 HER2-expressing tumor cell lines, respectively. In comparison, the non- conjugated Trastuzumab IgG antibody did not result in any cell growth inhibition in these two cell lines.
D. In vivo Tumor growth inhibition by the anti-HER2 ADC-v4
In a similar manner to the procedure described in Example 38, anti-HER2 ADC-v4 was evaluated in HER2-expressing tumor xenograft models, for example SKOV3 tumor xenograft model. Following a procedure similar to that described in Example 38, the anti-HER2 ADC-v4 was administered at a dose of 3 mg/kg mouse weight, IV, on Day 0, followed by 1 mg/kg IV Q4D x 4 (on Days 4, 8, 12). Antitumor activity was assessed based on the observed inhibition of tumor growth or tumor suppression, in comparison to mice treated with vehicle (formulation buffer). Approximately 100% tumor growth inhibition by the anti-HER2 ADC-v4 was observed on Day 18. In this SKOV3 model, the non-conjugated Trastuzumab antibody was also highly active. In an NCI-N87 tumor xenograft model, treatment with the anti-HER2 ADC-v4 at 5 mg/kg IV Q4D x 4 caused strong tumor growth inhibition (ca. 80% on Day 21) that was significantly greater than that caused by the non- conjugated Trastuzumab antibody also dosed at 5 mg/kg IV Q4D x 4. Using the conjugation procedures described in the preceding examples, the illustrative conjugates of the following Table 6 are prepared in a similar manner.
Table 6
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
= N(CH3), = 1 to 6 = 1 to 6
Figure imgf000182_0001
= 1 to 6
6
6 6
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
= 1, p" = 1 = 2, p" = 1 = 3, p" = 1
Figure imgf000186_0001
6
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
= 3, p" = 1 = 1 to 6 6
Figure imgf000191_0001
6
p" = 1 to 6
p" = 1 to 6
p" = 1 to 6
= 1, p" = 1
Figure imgf000192_0001
= 2, p" = 1
= 3, p" = 1
= 1, p" = 1
= 2, p" = 1
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Example 155: Cathepsin B Assay for Demonstration of Drug Release from the PAMA Linker
A. Drug release assay with fluorescence detection
The fluorescence drug release assay was carried out in 96-well black opaque plates (Greiner) in a final volume of 100 μί, in duplicate. Each well contained 25 mM Sodium acetate/1 mM EDTA buffer (pH 5.0), 40 nM activated human liver Cathepsin B (Enzo Life Sciences) and 40 μΜ test substrate (Trigger-linker molecules). (Cathepsin B was activated at 37°C for 15 minutes in activation buffer: 25 mM Sodium acetate, pH 5.0 containing 30 mM DTT and 15 mM EDTA.) Final DMSO concentration in the assay was 4%, and at this concentration DMSO had no significant effect on enzyme activity (assessed using a standard peptide substrate Cbz-Phe-Arg-AMC). After adding all components, the assay plate was incubated at 37°C and relative fluorescence units (RFU) were measured in the plate reader (Flexstation®, Molecular Devices or Envision Multilabel, Perkin Elmer; set in a kinetic mode) up to 6 hours (Ex/Em 380/460 nm). Control incubations included assay buffer without enzyme (buffer blank, pH 5.0) and substrate incubated in assay buffer in the absence of enzyme (substrate blank at pH 5.0). For each time point, substrate blank value was subtracted from the test values to get the final value. RFU for each time point was plotted against time to obtain a time versus RFU curve (progress curve).
As a positive control or reference compound in these assays, Cbz-Val-Cit-
PABE-HMC or Cbz-Val-Cit-PABC-AMC was included (see table below). Using the above described fluorescence assay protocol, Cbz-Val-Cit-PAMA-(methyl-carboxylate)-HMC (Fig. l), Cbz-Val-Cit-PAMA-(amide)-HMC (Fig. 2), Cbz-Val-Cit-PAMA-(PEG-amide)- HMC (Fig. 3), Cbz-Val-Cit-PAMA-(PEG-amide)-OC(0)-AMC (Fig. 4), Cbz-Val-Cit- PAMA-(MB-PEG3-amide)-HMC (Fig. 5), and Cbz-Val-Cit-PAMA-(MB-PEG4-amide)- HMC (Fig. 5) were evaluated and release of the surrogate hydroxy-linked drug HMC and the surrogate amine-linked drug AMC were demonstrated. The results are shown in Figs. 1 to 5.
Figure imgf000200_0001
B. Drug release assay with LC -MS/MS detection
Drug release from PAMA derived linkers carrying the duocarmycin analog Pro CBI-DMMI as the prodrug was determined in the Cathepsin B assay using LC-MS/MS methodology, specifically developed for quantitative estimation of the CBI-DMMI drug. The assay was carried out in duplicates (n=2) at 37 °C in a final volume of 1 mL and contained 25 mM Sodium acetate/1 mM EDTA buffer (pH 5.0), 40 nM activated human liver Cathepsin B (Enzo Life Sciences) and 40 μΜ test substrate. At different time -points, 100 aliquots were withdrawn and the reaction was stopped by adding 100 μΕ of acetonitrile containing tolbutamide as internal standard. The sample was centrifuged for 5 minutes at 4000 rpm and the supernatant was analyzed by LC-MS/MS. Amount of drug payload released was estimated using a calibration curve generated using CBI-DMMI.
Following this protocol, Cbz-Val-Cit-PAMA-(PEG3-amide)-Pro-CBI- DMMI, Cbz-Val-Cit-PAMA-(Boc-PEG3-amide)-Pro-CBI-DMMI, and Cbz-Val-Cit-PAMA- (Boc-PEG4-amide)-Pro-CBI-DMMI (Fig. 6) were evaluated and release of the hydroxy- linked prodrug Pro CBI-DMMI and conversion to the duocarmycin analog drug CBI-DMMI was demonstrated. It was also demonstrated (Fig. 6) that these three compounds were stable (no significant release/formation of CBI-DMMI over time) under the assay conditions in the absence of cathepsin B.
Figure imgf000201_0001
Figure imgf000202_0001
C. Drug release assay with mass spectrometry detection
A sample of anti-5T4 scADC (Example 37) was incubated in the presence of 40 nM pre-activated (50 mM sodium acetate, 1 mM DTT at pH 5) Cathepsin B in a reaction mixture containing 50 mM sodium acetate at pH 5 for 2 h at 37°C. This resulted in the formation of a new signal in the MALDI mass spectrum recorded using sinapinic acid matrix, with m/z corresponding to [M+H]+ for Pro CBI-DMMI. Under the same conditions in the absence of Cathepsin B, there was no signal observed for the mass of Pro CBI-DMMI.
D. Drug release assay from ADC with LC-MS/MS detection
Drug release was determined for antibody drug conjugate (ADC) incorporating PAMA linker carrying a duocarmycin analog related to Pro-CBI-DMMI as prodrug. The assay was carried out in duplicate. ADC protein (10 μg) was incubated with activated human liver Cathepsin B enzyme (20 nM) in assay buffer (pH 5.0) at 37°C.
Incubation that contained ADC protein but no Cathepsin B enzyme was included as control. At different time -points, 50 μΐ^ aliquots were taken and mixed with 150 μΐ^ of acetonitrile containing 500 ng/mL of Tolbutamide as internal standard. Samples were centrifuged at 13,000 rpm for 5 minutes and the supernatants were analyzed by LC-MS/MS.
Following this protocol, anti-HER2 scADC-v2 was assayed and formation of the cyclopropa[c]benzo[e]indol-4-one (CBI) analog of compound 91 (compound A in the following figure) was measured using specific multiple reaction monitoring (MRM) parameters developed in LC-MS/MS. It was also demonstrated that this ADC was stable (no significant release/formation of the CBI analog compound A over time) under the assay conditions in the absence of cathepsin B. The results are summarized in Figure 7.
Figure imgf000203_0001
Example 156: Evaluation of cytotoxicity of Pro-Duocarmycin analogs in in vitro cell- based assays
In vitro cytotoxic properties of various pro-duocarmycin analogs were determined in cell-based cytotoxicity assays. The assay was carried out in 96-well plates in a final volume of 200 μΐ^ using 5000 cells/well. Cells were treated with varying concentrations of drugs for 72 hours in triplicates (n=3). At the end of treatment, cell viability was determined using CCK-8 kit (Dojindo Laboratories). Dose for 50% inhibition (IC50) was calculated using GraphPad Prism® 5 software using 4-P fit. The list of human tumor cell lines and assay conditions used in the study is given in Table 7 and 8. The cytotoxicity of compounds 1 and 91 were also evaluated in additional cell lines, in a similar manner but with a 96-hour incubation; the results are listed in Table 8; both compounds were found to have sub- nanomolar IC50 values in all cell lines tested.
Table 7: Cytotoxicity of various compounds in multiple tumor cell lines
Figure imgf000203_0002
Table 8: Cytotoxicity of compounds 1 and 91 in multiple tumor cell lines (96 h incubation)
Figure imgf000204_0001
NT: not tested.
Figure imgf000204_0002
Example 157: Generation of anti HER2 and HERl scFv-Fc Immunoglobulins
A. These immunoglobulins were engineered on the basis following amino acid and nucleic acid sequences: 1. Anti-HER2 (4D5) scFv-Fc (15mer linker); SEQ ID NO: AA2:
MGWSCIILFLVATATGAHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR QAPGKGLEWVARIYPTNGYTRYADSVKGRFTLSADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLS ASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGT DFTLTIS SLQPEDFATYYCQQHYTTPPTFGQGTKVEIKASTCEPKS SDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
This sequence is encoded by SEQ ID NO AA1, which follows:
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACAGGCGCGCACT CTGAGGTGCAGCTGGTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCC TGAGACTGTCCTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCACTG GGTCCGACAGGCTCCAGGCAAGGGCCTGGAATGGGTGGCCCGGATCTACCCCAC CAACGGCTACACCAGATACGCCGACAGCGTGAAGGGCCGGTTCACCCTGTCCGC CGACACCTCCAAGAACACCGCCTACCTGCAGATGAACTCCCTGCGGGCCGAGGA CACCGCCGTGTACTACTGCAGCCGGTGGGGCGGAGACGGCTTCTACGCCATGGA CGTGTGGGGCCAGGGCACCCTGGTCACAGTGTCTAGCGGTGGAGGCGGAAGTGG AGGGGGAGGATCTGGCGGTGGAGGATCCGACATTCAGATGACCCAGTCCCCCTC CAGCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACATGCAGAGCCAGCCA GGACGTGAACACCGCCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCA AGCTGCTGATCTACAGCGCCTCCTTCCTGGAGAGCGGCGTGCCCTCCAGATTCTC CGGCTCTAGGTCCGGCACCGACTTCACACTGACCATCTCCAGCCTGCAGCCCGA GGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCCCACCTTCGGC CAGGGCACCAAGGTGGAGATCAAGGCTAGCACATGCGAGCCCAAGTCCTCCGA CAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCCTCC GTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCG AAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCA ATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAG GAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG GACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCT GCCCCC ATC GA AA AGACC ATC AGC A AGGCC A AGGGCC AGCCCC GC GAGCCTC A GGTGTACACCCTGCCTCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCT GACCTGCCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTC CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGA CGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCA GGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA
2. Anti-Herl (Vec) scFv-Fc (15mer linker): SEQ ID NO: AB2
MGWSCIILFLVATATGAHSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWT WIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYY C VRDRVTGAFDIWGQGTM VTVS S GGGGS GGGGS GGGGSDIQMTQSPS S LS AS VGD RVTITCQAS QDIS NYLNWYQQKPGKAPKLLI YD ASNLETGVPSRFS GS GS GTDFTFTI SSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKASTCEPKSSDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
This amino acid sequence is encoded by SEQ ID NO: AB1, which follows:
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACAGGCGCGCACT CTCAGGTGCAGCTGCAGGAATCCGGCCCCGGACTGGTGAAGCCTAGCGAGACCC TGAGCCTGACCTGCACCGTGTCCGGCGGCTCCGTGAGCTCCGGCGACTACTACT GGACCTGGATTCGACAGAGCCCAGGCAAGGGCCTGGAATGGATCGGCCACATCT ACTACAGCGGCAACACCAACTACAACCCCAGCCTGAAGAGCCGCCTGACCATCT CCATCGACACCTCCAAGACCCAGTTCAGCCTGAAGCTGAGCTCCGTGACCGCCG CCGACACCGCCATCTACTACTGCGTGCGGGACAGAGTGACCGGCGCTTTCGACA TCTGGGGCCAGGGCACCATGGTCACAGTGTCTAGCGGTGGAGGCGGAAGTGGA GGGGGAGGATCTGGCGGTGGAGGATCCGACATTCAGATGACCCAGTCCCCCTCC AGCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACATGCCAGGCCAGCCAG GACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAG CTGCTGATCTACGACGCCTCCAACCTGGAGACCGGCGTGCCCTCCAGATTCTCCG GCTCTGGCTCCGGCACCGACTTCACATTCACCATCTCCAGCCTGCAGCCCGAGGA CATCGCCACCTACTTCTGCCAGCACTTCGACCACCTGCCCCTGGCCTTCGGCGGA GGCACCAAGGTGGAGATCAAGGCTAGCACATGCGAGCCCAAGTCCTCCGACAA GACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCCTCCGTG TTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAA GTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAAT TGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGA ACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGC CCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCTCAGG TGTACACCCTGCCTCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGA CCTGCCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCA ACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACG GCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGG GCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA
3. Anti-Herl (Vec) scFv-Fc with complete IgG 1 framework (15mer linker): SEQ ID NO: AC2:
MGWSCIILFLVATATGAHSEVQLVESGGGLVQPGGSLRLSCTVSGGSVSSGDYYWT WIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISADTSKNTAYLQMNSLRAEDTAV YYCVRDRVTGAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG DRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQHFDHLPLAFGQGTKVEIKASTCEPKSSDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
This sequence is encoded by SEQ ID NO: AC1, which follows: ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACAGCCACCGGCGCTCACT CCGAAGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTGCAGCCTGGCGGCTCTC TGAGACTGTCCTGCACCGTGTCCGGCGGCTCCGTGTCCTCCGGCGACTACTACTG GACCTGGATCAGACAGTCCCCCGGCAAGGGCCTGGAATGGATCGGCCACATCTA CTACTCCGGCAACACCAACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCTC CGCCGACACCTCCAAGAACACCGCCTACCTCCAGATGAACTCCCTGCGGGCCGA GGAC ACCGCC GTGT ACTACTGC GTGC GGGAC AG AGTG ACC GGC GCCTTC GATAT CTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGAGGGGGAGGATCTGGCG GCGGAGGAAGTGGCGGAGGCGGCTCCGATATCCAGATGACCCAGTCCCCCTCCA GCCTGTCCGCCTCCGTGGGCGATAGAGTGACCATCACCTGTCAGGCCTCCCAGG ACATCTCCAACTACCTGAATTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGC TGCTGATCTACGACGCCTCCAACCTGGAAACCGGCGTGCCCTCCCGGTTCTCCGG CTCCAGATCTGGCACCGACTTCACCCTGACCATCAGCTCCCTCCAGCCTGAGGAC TTCGCCACCTACTACTGCCAGCACTTCGACCATCTGCCCCTGGCCTTCGGACAGG GCACC AAGGTGGAAATC AAGGCTAGC AC ATGCGAGCCC AAGTCCTCCGAC AAG ACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCCTCCGTGT TCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAG TGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAA CAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC TGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCC CCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCTCAGGT GTACACCCTGCCTCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGAC CTGCCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAA CGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGG CTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGG CAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCA GAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA
B. Brief description on generation of the constructs
These gene sequences were manipulated to maximize expression of their gene products by codon optimization for expression in CHO cells. The synthetic coding sequences were assembled by methods known in the art.
pTT5 expression vector was obtained from National Research Council,
Canada was used to clone genes for transient expression studies. Restriction enzymes and Phusion polymerase enzyme used in the study were procured from New England Biolabs, USA. T4 DNA Ligase and TaqDNA polymerase were procured from Bangalore Genei, India. 1Kb DNA ladder, Shrimp Alkaline Phosphatse and INSTAClone T/A cloning kit containing vector pTZ57R/T were purchased from Fermentas. Primers for gene synthesis, PCR and sequencing were obtained either from IDT or Eurofins, India. Sequencing of constructs was carried out using Bigdye® terminator V 3.1 cycle sequencing kit, Applied Biosystems, USA. Plasmid preparation at various scales was carried out using appropriate kits (GenElute™ plasmid mini prep kit - Sigma, USA; Plasmid maxi, midi and mega kit - Qiagen, USA). For gel extraction, gen elute gel Extraction kit (Sigma, USA) was used. E. coli Omnimax cells and Zero-Blunt TOPO cloning kit were procured from Invitrogen, USA. Quick change site directed mutagenesis kit was procured from Stratagene. The codon optimized framework region along with Fc molecules used are disclosed in US Provisional Application Number 61/835,858, filed June 17, 2013, herein incorporated by reference.
Codon optimized immuno fusion molecules were cloned in pTT5 and UCOE (CET1019HS-Puro) vectors for transient and stable expression respectively.
C. Gene synthesis, cloning and sequencing of constructs.
Gene synthesis was carried out by assembling the 49mer primers designed based on the codon optimized sequences by a PCR based assembly method. Gel eluted PCR products were A-tailed using Taq DNA polymerase and ligated to pTZ57R T vector by T/A cloning. The ligation mix was transformed into E. coli Omnimax cells followed by selection on LB Ampicillin agar plates 37°C for 16h. Recombinant clones were identified by colony PCR using vector specific Ml 3 Forward and reverse primers. Five to six randomly selected PCR positive colonies were inoculated in LB Ampicillin broth for plasmid isolation.
Plasmids isolated from overnight grown cultures were digested by Eco RI and Nhe I to confirm the release of inserts at expected size. Selected clones were sequenced using automated DNA sequencing (ABI 3130XL)
Based on the sequences clones with no or apparently no mutations were selected for further processing. Mutations, if any, in the genes were corrected by site directed mutagenesis using Quick change SDM kit. Corrections of the mutation were verified by sequencing
Sequence-corrected clones were further taken for sub-cloning into pTT5 vector. The scFv inserts were released from pTZ57R/T vector by restriction digestion using Eco RI and Nhe I enzymes. The released inserts were purified by gel elution and ligated to pTT5 vector backbone with Fc, generated as disclosed in US Provisional Application Number 61/835,858, filed June 17, 2013, herein incorporated by reference, digested with Eco RI and Nhe I enzymes. Recombinant E. coli Omnimax clones were screened using vector-specific primers by colony PCR. Positive colonies were used for plasmid isolation and confirmation of insert release. Selected clones were sequence confirmed and extra DNA was prepared by Maxi/Mega prep using the Qiagen columns and sequence confirmed which were further used for transient transfection. The scFv-Fc constructs were PCR amplified from the PTT5 clones generated for transient transfection using appropriate primers for the purpose of introducing BstBI (5') and Sal I (3') restriction sites. PCR products were analyzed by 1% agarose gel
electrophoresis and the specific products was eluted using Gen elute gel extraction columns. These products were further double digested with Bst BI and Sal I to generate sticky ends. Ligation was effected with CET1019HS-Puro vector backbones which were prepared by digestion with compatible enzymes and followed by treatment with Shrimp alkaline phosphatase.
The ligation mix was used to transform Stbl2™ Competent Cells followed by selection on LB Ampicillin agar plates at 27°C for 24hrs. Recombinant clones were identified by colony PCR using vector specific CET1019 HS Puro Vec FP1 and CET1019 HS Puro Vec RP2 primers. Randomly selected PCR positive colonies (3-4 Nos.) were inoculated in LB Ampicillin broth for plasmid isolation. Plasmids isolated from overnight cultures were digested by Bst BI and Sal I to confirm the release of inserts for expected size. Selected clones were sequenced by automated DNA sequencing (ABI 3130XL). DNA to be used for transfection was prepared by Maxi/Mega prep using the Qiagen columns and sequence confirmed. This DNA was further digested with Pvul enzyme to linearize the plasmid, checked on 1 % gel and was used for transfection.
All publications cited in this specification are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS:
1. A therapeutic drug and targeting conjugate of formula (I) comprising:
Figure imgf000210_0001
CO-(NH)p-(CH2)m-B{LG wherein:
L is a di-, tri- or tetra- amino acid chain;
X is: (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- which is the point of attachment to -OC(O)-,
(ii) -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -O- or -NR4- as the point of attachment to -N(H)C(0)-;
(iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or
(iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR4- which is part of the drug fragment or prodrug fragment;
p is 0 or 1 ;
m is 1 to 6 with the a proviso that, when m =1, p =0;
Figure imgf000210_0002
erein said LG has at least one thiol moiety or at least one amine moiety which forms the point of attachment to B;
p" is 1 to 6;
Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2;
R1 is -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, C C6 alkyl-,
or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8;
R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano R3 is H, Ci-C6 alkyl, C2-C6 hydroxyalkyl or C2-C6 perfluoroalkyl;
R4 is H, C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 perfluoroalkyl;
R6 is H, Ci-C6 alkyl, or C2-C3 hydroxyalkyl;
R7 is Ci-C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2N(Ci-C3 alkyl)2;
R8 is H, C1-C3 alkyl or C C3 hydroxyalkyl;
R9 and R10 are independently H or Ci-C6 alkyl;
R12, R13 and R14 are independently selected from H, C1-C6 alkyl or C2-C3 hydroxy alkyl; and
Z is an optional amine blocking group.
2. The therapeutic drug and targeting conjugate according to claim 1 , wherein the fragment of formula I char ture:
Figure imgf000211_0001
serves as a substrate for one or more proteases, and wherein following protease cleavage of formula I at the amide between the terminal carbonyl moiety of L and the -NH- of the 4- amino-phenyl moiety or the related amino-heterocyclic moiety, an electronic rearrangement or reaction proceeds and the drug fragment or prodrug fragment Y is released as the drug or prodrug Y-H.
3. The therapeutic drug and targeting conjugate according to claim 1, wherein the substrate is a substrate for a cathepsin selected from cathepsin B and cathepsin L.
4. The therapeutic drug and targeting conjugate according to claim 1 , wherein each of W, Wi, W2 and W3 is CR2, R1 is -CONR6CHR7CH2(OCH2CH2)nOCH2CHR8-, R2 is H, having formula IA:
Figure imgf000212_0001
5. The therapeutic drug and targeting conjugate according to claim 1, wherein when W is CR2, R2 is H, each of Wx, W2, and W3 are independently N or CR2, and R1 is - CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, having formula IB:
Figure imgf000212_0002
CO-(NH)p-(CH2)m-B LG
(IB)
P"
6. The therapeutic drug and targeting conjugate according to claim 1 , wherein W is absent, W1 is CR2 in which R2 is H and R1 is
-CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, having the following formula ICi or ICii:
Figure imgf000212_0003
7. The therapeutic drug and targeting conjugate according to claim 1, wherein LG comprises an immunoglobulin or a cytokine.
8. The therapeutic drug and targeting conjugate according to claim 7, wherein the cytokine is an interleukin.
9. The therapeutic drug and targeting conjugate according to claim 7, wherein the ligand targets CD19, CD20, CD30, CD33 CD38, CD52, or CD133, carcinogenic embryonic antigen (CEA), epidermal growth factor receptor-1 (EGFR or HER1 or erbBl), epidermal growth factor receptor (EGFRviii), human epidermal growth factor receptor-2 (HER2 or erbB2), epidermal growth factor receptor-3 (HER3 or erbB3), MET, insulin-like growth factor receptor 1 (IGF1R), platelet-derived growth factor receptor alpha and beta (PDGFRalpha and PDGFRbeta), EphrinA receptors 1-8 (EphAl-8), EphrinB receptors 1-6 (EphBl-6), folate receptor (FolRalpha), prostate specific membrane antigen (PSMA), MUC- 1, MUC- 16, high molecular weight melanoma-associated antigen (HMW-MAA) or chondroitin sulfate proteoglycan (CSPG), epithelial cell adhesion molecule (EPCAM), 5T4 oncofetal trophoblast glycoprotein, Tie-2, and vascular endothelial growth factor receptor-2 (VEGFR2).
10. The therapeutic drug and targeting conjugate according to claim 9, wherein the ligand targets oncofetal antigen or HER2.
11. The therapeutic drug and targeting conjugate according to claim 7, wherein LG is a monoclonal antibody, an immunoadhesin, a single chain Fv (scFv), single chain Fv- Fc antibody (scFv-Fc), or a functional antibody fragment (Fab' or F(ab')2).
12. The therapeutic drug and targeting conjugate according to claim 7, wherein LG is a therapeutically active immunoglobulin G.
13. The therapeutic drug and targeting conjugate according to claim 1, wherein X is (i) or (iii).
14. The therapeutic drug and targeting conjugate according to claim 1, wherein said drug or prodrug is selected from the group consisting of DNA damaging agents, microtubule disrupting agent, or cytotoxic proteins or polypeptides.
15. The therapeutic drug and targeting conjugate according to claim 14, wherein said drug is selected from the group consisting of mitomycin C, mitomycin A, daunorubicin, doxorubicin, aminopterin, bleomycin, 9-amino camptothecin, maytansine or an analog thereof, vincristine, vinblastine and other vinca derivatives, dolastatin or its derivatives, 1- (chloromethyl)-2,3-dihydro-lH-benzo[e]indol-5-ol, duocarmycin or analogs thereof, pyrrolobenzodiazepine (PBD) polyamide and dimer thereof; calicheamicin; auristatin and its derivatives, and tubulysins, paclitaxel, docitaxel, cabazetaxel, and tesetaxel.
16. The method according to claim 15 wherein the drug is selected from the group consisting of maytansine, monomethylauristatin F and monomethylauristatin E.
17. The therapeutic drug and targeting conjugate according to claim 1, wherein X is (i) or (iii) and the drug fragment or prodrug fragment Y is a DNA damaging agent having the structure of formula (III) or (IV), respectively, below:
Figure imgf000214_0001
where "Minor Groove Binder" is a DNA minor groove binding group which is a substituted 1H- indole-2-carbonyl group.
18. The therapeutic drug and targeting conjugate according to claim 17, wherein the Minor Groove Binder is selected from the group consisting of:
Figure imgf000215_0001
19. The therapeutic drug and targeting conjugate according to claim 18, wherein the Minor Groove Bind
Figure imgf000215_0002
20. The therapeutic drug and targeting conjugate according to claim 1 , wherein X is (i) or (iii) and the drug fragment or prodrug fragment Y is a DNA damaging agent having the structure of formula (III) or (IV:
Binder
wherein
Figure imgf000216_0001
(M) , and wherein Q is N(CH3)2 or is selected from the follow
Figure imgf000216_0002
Figure imgf000217_0001
Figure imgf000217_0002
215
Figure imgf000218_0001
(VII)
22. The therapeutic drug and targeting conjugate according claim 1 , wherein LG targets a cell surface molecule on a neoplastic cell.
23. The therapeutic drug and targeting conjugate according to claim 1 , wherein the drug is a cytotoxic protein or polypeptide.
24. The therapeutic drug and targeting conjugate according to claim 23, wherein the cytotoxic protein or polypeptide is selected from the group consisting of a ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, tumor necrosis factor alpha, and Pseudomonas exotoxin.
25. The therapeutic drug and targeting conjugate according to claim 1 , wherein Z is selected from the group consisting of C1-C6 acyl, optionally substituted aroyl, optionally substituted heteroaroyl, (aryl)alkyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C3-C6
heterocycloalkyl-carbonyl, (alkoxy)carbonyl, (aryloxy)carbonyl, (heteroaryloxy)carbonyl, (aryl alkoxy)carbonyl, (heteroaryl alkoxy)carbonyl, C3-C6 (cycloalkoxy)carbonyl, C3-C6 (heterocycloalkoxy)carbonyl, and RnNH-CO-, which is optionally linked to L via a peptide or an amino acid, wherein R11 is H, C1-C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, (aryl)alkyl, or
(heteroaryl)alkyl.
26. The therapeutic drug and targeting conjugate according to claim 25, wherein Z is carbobenzyloxy.
27. The therapeutic drug and targeting conjugate according to claim 25, wherein Z is acetyl, pyrroloyl, or t-butylcarbonyl.
28. The therapeutic drug and targeting conjugate according to claim 1 , wherein L is a dipeptide or tetrapeptide group.
29. The therapeutic drug and targeting conjugate according to claim 28, wherein L is selected from the group consisting of Val-Cit, Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly- Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe- Ala, Aln-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.
30. The therapeutic drug and targeting conjugate according to claim 29, wherein L is selected from -Val-Cit- or Gly-Gly-Phe-Gly.
31. The therapeutic drug and targeting conjugate according to any of claims 1 to 30, wherein p" is 1, 2, 3 or 4.
32. The therapeutic drug and targeting conjugate according to claim 31, wherein p" is 2.
33. A therapeutic drug and targeting conjugate comprising:
Figure imgf000219_0001
wherein:
L is selected from the group consisting of (a) Val-Cit or (b) Gly-Gly-Phe-Gly; X is -OC(0)Y, wherein Y is a drug or prodrug fragment having a -NR4- as the point of attachment; or a drug or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug or prodrug;
p is 0 or 1 ;
m is 1 to 6 with the a proviso that, when m =1, p =0;
LG is a ligand which targets a cell surface molecule;
Figure imgf000220_0001
B-LG is ; wherein said LG has at least one thiol moiety or at least one amine moiety which forms the point of attachment to B;
p" is 1 to 6;
R1 is -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8, wherein n is 1 to 8;
R2 is H, Ci-C6 alkyl, C C6 alkoxy, halo gen, or C1-C6 fluoroalkyl, cyano;
R3 is H, C1-C6 alkyl, C2-C6 hydroxyalkyl or C2-C6 -perfluoroalkyl;
R4 is H, C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 perfluoroalkyl;
R6 is H, Ci-C6 alkyl, or C2-C3 hydroxyalkyl;
R7 is Ci-C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(Ci-C3 alkyl)2;
R8 is H, C1-C3 alkyl or C C3 hydroxyalkyl;
R9 and R10 are independently H or C1-C6 alkyl;
R12, R13 and R14 are independently selected from H, Ci-C6 alkyl, C2-C3 hydroxyalkyl; and
Z is an optional amine blocking group selected from the group consisting of C1-C6 acyl, optionally substituted aroyl, optionally substituted heteroaroyl, (aryl)alkyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C3-C6 heterocycloalkyl-carbonyl, (alkoxy)carbonyl,
(aryloxy)carbonyl, (heteroaryloxy)carbonyl, (aryl alkoxy)carbonyl, (heteroaryl
alkoxy)carbonyl, C3-C6 (cycloalkoxy)carbonyl, C3-C6 (heterocycloalkoxy)carbonyl, and RnNH-CO-; and
R11 is H, C1-C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, (aryl)alkyl, or (heteroaryl)alkyl.
34. The therapeutic drug and targeting conjugate according to claim 33. X is (i) or (iii) and Y has the structure of formula (III) or (IV), respectively, below:
Figure imgf000221_0001
35. The therapeutic drug and targeting conjugate according to claim 34, X is (i) and Y has the structure of formula (VII) below:
Figure imgf000222_0001
36. A therapeutic drug and targeting conjugate selected from the group consisting of:
(a)
Figure imgf000222_0002
Figure imgf000222_0003
Figure imgf000223_0001
221
Figure imgf000224_0001
Figure imgf000224_0002
Figure imgf000224_0003
222
Figure imgf000225_0001
and
Figure imgf000225_0002
wherein LG is an immunoglobulin protein or polypeptide which targets a cell surface receptor, wherein LG contains at least one thiol group or at least one amine group that forms a point of attachment to the succinimide or the acetamide moiety; n is 1, 2 or 3 ; and p" is 1 , 2, 3 or 4.
37. The therapeutic drug and targeting conjugate according to claim 36, having the structure:
Figure imgf000225_0003
(b)
Figure imgf000226_0001
Figure imgf000226_0002
Figure imgf000226_0003
Figure imgf000226_0004
224
Figure imgf000227_0001
Figure imgf000227_0002
225
Figure imgf000228_0001
wherein LG is an immunoglobulin protein or polypeptide which targets a cell surface receptor, wherein LG contains at least two thiols group each of said two thiol groups forming a point of attachment to one of the two succinimide moieties of the structures; and n is 1, 2 or 3.
38. The therapeutic drug and targeting conjugate according to claim 36 or 37, wherein the immunoglobulin is selected from a monoclonal antibody, an immunoadhesin, a single chain (sc) FvFc antibody; or a functional Fab fragment.
39. The therapeutic drug and targeting conjugate according to claim 38, wherein the immunoglobulin is selected from the group consisting of antibodies directed against 5T4, AGS-5, CAIX, CD19, CD20, CD22, CD30, CD33, CD56, CD66e, CD70, CD74, CD79b, CD138, GPNMB, HER1, HER2, Mesothelin, MUC-1, PSCA, PSMA or SLC44A4 antigen..
40. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a conjugate according to any of claims 1 to 39.
41. A method of delivering a drug comprising the method of administering a pharmaceutically acceptable composition comprising a carrier and a conjugate according to any one of claims 1 to 39 to a subject.
42. The method according to claim 41, wherein said drug is formulated for parenteral delivery.
43. The method according to claim 41, wherein the delivery is intravenous or intraperitoneal.
44. An anti-neoplastic regimen comprising delivering a pharmaceutical composition according to claim 40.
45. A therapeutic drug and targeting conjugate according to any one of claims 1 to 40 for use in an anti-neoplastic regimen.
46. The therapeutic drug and targeting conjugate according to claim 45, wherein LG is selected from an anti-5T4 scFvFc, anti-HERl scFvFc or an anti-HER2 scFvFc.
47. The therapeutic drug and targeting conjugate according to claim 46, wherein the anti-5T4 scFvFc has the amino acid sequence of SEQ ID NO: A.
48. , comprising:
Figure imgf000229_0001
wherein
L is a di-, tri- or tetra- amino acid chain which forms a substrate specifically cleavable by a protease;
Z is an optional blocking group;
X and R1 are conjugatable groups;
Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2; and
R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano.
49. The compound of claim 48 wherein X is OH or NH2.
50. The compound of claim 49 wherein R1 is Ci-C6 alkyl-, - (CH2CH2OCH2CH20)n-, or -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or - (CH2)2NH(Ci-C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl.
51. A method of delivering an therapeutically active drug, said method comprising administering a therapeutic drug and targeting conjugate according to any one of claims 1 to 39, wherein said drug fragment or prodrug fragment is converted to a therapeutically active drug or prodrug following cleavage of the substrate fragment by a protease.
52. The method according to claim 51, wherein the protease is a lysosomal protease.
53. The method according to claim 52, wherein the lysosomal protease is Cathepsin B.
54. The method according to claim 51, wherein said active drug is selected from the group consisting of a DNA damaging agent, a microtubule disrupting agent, and a cytotoxic protein or polypeptide.
55. The method according to claim 54, wherein the drug is selected from the group consisting of duocarmycin or an analog thereof, pyrrolobenzodiazepine (PBD)- thereof, calicheamicin, an auristatin, a vinca alkaloid, a tubulysin, paclitaxel, docitaxel, cabazetaxel, a taxane, mitomycin C, mitomycin A, daunorubicin, doxorubicin, aminopterin, bleomycin, 9-amino camptothecin, maytansine, a maytansinoid, vincristine, a Dolastatin, and l-(chloromethyl)-2,3-dihydro-lH-benzo[e]indol-5-ol bound to a DNA minor groove binder.
56. The method according to claim 54, wherein the drug is selected from the group consisting of a maytansinoid, monomethylauristatin F and monomethylauristatin E.
57. The method according to claim 54, wherein said conjugate comprises a DNA alkylating agent drug or prodrug having the structure selected from the group consisting of: WO 2015/038426
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000232_0002
Figure imgf000232_0003
230
Figure imgf000233_0001
58. A cleavable para-amino mandelic acid (PAMA) derived linker useful for forming a conjugate between a drug fragment or prodrug fragment and a ligand, said linker having the structure IX,
Figure imgf000233_0002
(IX)
wherein
L is a di-, tri- or tetra- amino acid chain which forms a substrate specifically cleavable by a protease;
Z is an optional blocking group;
X is bound directly or indirectly to a drug fragment, prodrug fragment, a drug or prodrug;
Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2;
R1 is bound to the targeting moiety via a group having a formula selected from Ci-C6 alkyl-, -(CH2CH2OCH2CH20)n-, or -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(Ci-C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl; and R2 is H, Ci-C6 alkyl, Ci-C6 alkoxy, halogen, Ci-C6 fluoroalkyl, or cyano.
59. The linker according to claim 58, wherein the protease is a lysosomal protease.
60. The method according to claim 59, wherein the lysosomal protease is Cathepsin B.
61. A drug and targeting conjugate comprising the cleavable para-amino mandelic acid (PAMA) derived linker according to claim 58, wherein each of W, Wi, W2 and W3 is CR2, R1 is -CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, and R2 is H.
62. The drug and targeting conjugate according to claim 61, wherein the drug and targeting conjugate has the structure of formula IA:
Figure imgf000234_0001
wherein LG is an immunoglobulin selected from (a) an anti-5T4 scFvFc; (b) an anti-HERl scFvFc, and (c) an anti-HER2 scFvFc, which has at least one thiol moiety which forms the point of attachment to B; and B is an optionally substituted succinimide or acetamide moiety.
63. An anti-HERl scFv having the amino acid sequence of SEQ ID NO: AB1.
64. A polynucleotide encoding the anti-HERl scFv of claim 63.
65. An anti-HER2 scFv having the amino acid sequence of SEQ ID NO: AA2.
66. A polynucleotide encoding the anti-HER2 scFv of claim 65.
67. A synthetic process for preparing a tert-butyl (4-hydroxynaphfhalen-2- yl)carbamate, said process comprising:
(i) admixing 1,3-dihydroxynaphthalene and diphenyl methylamine in a toluene solution;
(ii) heating the toluene solution at 80°C to 125°C for about 4 to about 8 hours;
(iii) subsequently combining the reaction mixture with palladium hydroxide, di-tert butyl carbonate, and dioxane:water;
(iv) subsequently shaking the reaction mixture at 60-80 psi hydrogen gas pressure for about 24 to 48 hours;
(v) filtering the reaction mixture and concentrating the filtrate to yield crude tert-butyl (4-hydroxynaphthalen-2-yl)carbamate.
68. A synthetic process for preparing compound of formula 29a comprising:
(a) combining an alcohol having the structure 26a and cesium chloride in ound 26a has the structure:
Figure imgf000235_0001
26a
wherein Z is an amine blocking group and L is an amino acid chain selected from Val-Cit or Gly-Gly-Phe-Gly;
cooling the reaction mixture to about 0°C and adding trichloroacetonitrile; allowing the reaction mixture to warm to room temperature with stirring; pouring the reaction mixture over water and extracting with ethylacetate; washing the combined organic layers with water and brine, followed by separating the organic layer, drying, concentrating, and
purifying crude product by silica gel column chromatography using 10% methanol in dichloromethane as eluent to yield the trichloracetimidate product which has the structure 27a:
Figure imgf000236_0001
27a
combining the trichloracetimidate product 27a defined in (a) with
(Boc-Pro-CBI 2a)
Figure imgf000236_0002
and dry acetonitrile in a suspension of molecular sieves; cooling the reaction mixture to -10 °C to -20 C in the presence of a Lewis acid; neutralizing the reaction mixture; removing volatiles; and purifying by silica gel column chromatography using dichloromethane (DCM):MeOH: NH3 solution as eluent obtaining the compound of structure 28a:
Figure imgf000236_0003
28a
(c) combining the methanol solution of compound 28a, Boc20, and NEt3 at 0 °C and allowing the reaction to proceed; removing the volatiles and purifying the crude reaction product by silica gel column chromatography using DCM:MeOH as eluent to yield compound 29a
BocHN
Figure imgf000236_0004
29a
The method according to claim 68, wherein the Lewis acid is BF3.etherate.
70. A synthetic process for preparing compound of structural formula 34a useful in synthesis of a drug and targeting conjugate comprising a therapeutic drug associated with a cell specific targeting moiety via a linker which is specifically cleavable by a lysosomal protease, said process comprising:
a) combining carbonyldiimidazole and an optionally substituted Indole- 2-carboylic acid derivative in a DMF solution and stirring for about 2 hours at ambient temperature;
(b) combining the reaction mixture with a stirred DMF solution of amine 29a and sodium carbonate, wherein amine 29a has the structure:
BocHN
Figure imgf000237_0001
29a
wherein Z is an N-blocking group and L is a di-peptide, tri-peptide, or tetra- peptide fragment;
(c) filtering and removal of solid following the reaction and
Figure imgf000237_0002
71. A cytotoxic compound comprising a DNA alkylating agent drug or prodrug selected from the group consisting of:
Figure imgf000238_0001
236
Figure imgf000239_0001
Figure imgf000239_0002
237
Figure imgf000240_0001
Figure imgf000240_0002
238
Figure imgf000241_0001
74. A method of treating a disease or disorder associated with the presence of a specific cell surface molecule on cells of a subject, comprising administering to the subject a therapeutically effective amount of a therapeutic drug and targeting conjugate comprising at least one ligand that targets the cell surface molecule.
75. The method of claim 74, wherein the disease or disorder is a neoplastic disease.
76. The method of claim 75, wherein the neoplastic disease is cancer of the prostate, head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone, lung (small cell or non-small cell), colon, rectum, stomach, bladder, uterus, cervix, breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes, kidney (renal cancer), liver, intestines, pancreas, brain (e.g., glioblastoma), central nervous system, adrenal gland, or skin or a leukemia.
77. A method for producing a therapeutic drug and targeting conjugate, comprising the steps of providing a cleavable para-amino mandelic acid (PAMA) derived linker having the structure IX:
Figure imgf000242_0001
(IX)
wherein
L is a di-, tri- or tetra- amino acid chain;
Z is an optional amine blocking group;
Wi and W2 are independently N or CR2, and W is absent or present, provided that when W is absent, W3 is independently NR3, O or S, and when W is present, W and W3 are independently N or CR2, provided that at least one of Wi, W2, and W3 is CR2;
R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 fluoroalkyl, or cyano, and conjugating the cleavable para-amino mandelic acid (PAMA) derived linker to a targeting molecule through R1 and to a drug or prodrug fragment through X, wherein:
R1 is a group having a formula selected from
-CONR6CHR7CH2(OCH2CH2)„OCH2CHR8-, C C6 alkyl-, or -(CH2CH2OCH2CH20)n-, wherein n is 1 to 8, R6 is H, C C6 alkyl, or C2-C3 hydroxyalkyl; R7 is C C6 alkyl, C C3 hydroxyalkyl, or -(CH2)2NH(C C3 alkyl)2; R8 is H, C C3 alkyl or C C3 hydroxyalkyl; and
X is (i) -OC(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- which is the point of attachment to -OC(O)-, (ii) X is -N(H)C(0)Y, wherein Y is a drug fragment or prodrug fragment having a -NR4- or -O- as the point of attachment
to -N(H)C(0)-; (iii) a drug fragment or prodrug fragment Y bound to the a carbon via an oxygen which is part of the drug fragment or prodrug fragment; or (iv) a drug fragment or prodrug fragment Y bound to the a carbon via a -NR4- which is part of the drug fragment or prodrug fragment.
78. A kit comprising a conjugate according to any of claims 1 to 39, optionally containing instructions for administering the conjugate to a subject.
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