WO2014145109A1 - Sugar-linker-drug conjugates - Google Patents

Sugar-linker-drug conjugates Download PDF

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Publication number
WO2014145109A1
WO2014145109A1 PCT/US2014/029793 US2014029793W WO2014145109A1 WO 2014145109 A1 WO2014145109 A1 WO 2014145109A1 US 2014029793 W US2014029793 W US 2014029793W WO 2014145109 A1 WO2014145109 A1 WO 2014145109A1
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Prior art keywords
conjugate according
group
ethyl acetate
nhr
previous
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PCT/US2014/029793
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French (fr)
Inventor
Sidney Hecht
Rakesh PAUL
Chenhong TANG
Manikandadas MADATHIL
Chandraball BHATTACHARYA
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Arizona Board Of Regents, For And On Behalf Of, Arizona State University
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Application filed by Arizona Board Of Regents, For And On Behalf Of, Arizona State University filed Critical Arizona Board Of Regents, For And On Behalf Of, Arizona State University
Priority to US14/776,396 priority Critical patent/US9919055B2/en
Priority to EP14764855.4A priority patent/EP2968381A4/en
Priority to AU2014233404A priority patent/AU2014233404A1/en
Publication of WO2014145109A1 publication Critical patent/WO2014145109A1/en
Priority to AU2018274904A priority patent/AU2018274904A1/en

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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
    • A61K47/54—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 organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure relates to sugar-linker-drug conjugates, compositions comprising them and methods of using ⁇ hem,
  • the bleomycins are a family of glyeopeptkle-derived antitumor antibiotics used clinically for the treatment, of squamous ceil carcinomas and malignant lymphomas. [Levi, J, A. et ai.., J. Clin. Oncol 1993, 1.1, 1300;
  • BLM has been, recognized for its ability to target tumors and shown to act as a tumor-imaging agent.
  • A. is:
  • j is selected from the group consisting of H, OH, SH, N3 ⁇ 4, OR4,
  • each i is selected from the group consisting of B, G-Cs alkyi, C ; - C( > alkenyl and Ca-Q a!kynyi;
  • each R s is selected from the group consisting of Q-Q, alkyi, C3 ⁇ 4-C $ alkenyl and QrG, alkynyl;
  • R;i is selected ftom the group consisting o H, OB, SH, Nf3 ⁇ 4 5 OR4,
  • R' is selected from the group consisting of H, OB and NBR 4 ;
  • B is a Spacer Unit
  • n is an integer selected from 1 to 3;
  • L is absent or a Linker
  • D is a Drug Unit having one or more chemically reactive functional groups selecieci from the group consisting of a primary or secondary amine, hydroxy!, suSfhydryL carboxyl, aldehyde and ketone.
  • A is:
  • A is selected from the group consisting of:
  • A is:
  • Rs is selected from the group consisting of R, OH, 0C(0)R 4 . OCONHR 4 , and OCONR 4 R 5 .
  • R 2 is selected from the group consisting of H, OH, OC(0)R4, OCONHR4, OCONR4R5, OCSNHR4, NHCONHR4, HCONRiRs, OCHjCONHRi, and OCH.2CONR4R5.
  • R* is selected from the group consisting of H, OH,
  • R' is IS or OH.
  • each R 4 is selected from the group consisting of H, niethvi. and ethvt. ⁇ 90191 in some embodiments, each R s is aeiecied from the group consisting of methyl, ethyl, and isobiUyl
  • A is selected from the group consisting of:
  • A is selected from the group consisting of:
  • A is selected from the group consisting of
  • B is a Spacer Unit selected from the group consisting of a bond, CrC» aikyl, C rC?o alkenyl, CrQ» alkynyl, atyl, heterearyl, heterocyclyl, C 3 -C5 cycloalkyl an oligoaikylene glycol an. oligopeptide and a dendriraer.
  • the Spacer Unit is X-CL'-Y ⁇ -tr-Z
  • X is C3 ⁇ 4 or O
  • L ! is C Cft alkyi
  • Y is (X S intercept or NR y , wherein R* is hydrogen or C Cs alkyi;
  • tti is an integer selected from 1 to 10; 1/ is €:-0>» alkyl, C Cas a!kenyf, Cj- ao alkviiyi, aryl, heteroaryl, heterocyclyl, C : Cj cyc!oalfcyJ; aad
  • Z is absent, O, R X , S, C(0), SCO), S(0 ⁇ 2 , OC(0), N ⁇ R*)C(0), N(R*)S(0), N(R x )S(0 ⁇ 2 , 0(0)0, C(0)N(R*) 5 S(0)N(R s ), S(0> 2 N(R s X 00(0)0, OC(0)N(R*X N(R*)C(0)0, N(R s )C(0) (R s ), or N(R s )S(0) 2 .NCR x ), wherein
  • each R* is independently hydrogen or Q-CV > alkyl.
  • X is O
  • L ! is C 2 -C4 alkyl
  • L ⁇ is CrQ, alkyl
  • Z is a bond O NR*, S, C(0), 5(0), S(0> 2 , or N(R K )C(0).
  • the Spacer Unit is 0-(CH CH -0) ffi - C3 ⁇ 4CH Z, wherein Z is O, (H), S or NCR x )C(0), R x is H and m is an integer selected from I to 20.
  • the Spacer Unit is 0 ⁇ CH>CH 2 ⁇ 3 ⁇ 4- O -CHjCH , wherein Z is Q, N(H), or S and ni is an integer seiected from 1 to 20.
  • the Spacer Unit is 0-(CHjCH.r0) ni - CJ3 ⁇ 4CH 2 -Z, wherein Z is C(0) or S(0)i and m is an integer selected from 1 to 20.
  • the linker L is absent.
  • the Spacer Unit is ⁇ -( ⁇ ⁇ ) ⁇ -1 - ⁇ , wherein X is O, L ! is C2-C4 alkyl; L ⁇ is C C ⁇ s alkyl; and Z is a bond, O, NR S , S, C(0), SCO), S(0) 2 , or NCR s )C(0).
  • X is O
  • L f is C 6 -03 ⁇ 4 alky ⁇
  • L " is CVQ, alkyl: and Z is
  • the linker L is a non-cleavahle linker.
  • L is CE ⁇ L ' *-F-L 4 ) P -C ⁇ 5 -G
  • each E is bond, O, NR ⁇ S, CCO), S(0), S ⁇ 0) 2 , OCC ), N(R x ⁇ C(0), N(R x )S(0), N(R x )S(0) 2> 0(0)0, C(0)N ⁇ R*X SiO)N(R x ), S(0) 2 N(R x ), OC(0)0, OCCO) (R x ), (R x ⁇ CCO)0, N(R x )C(0 ⁇ N(R * X or NCR x )S(0) 2 N(R x );
  • each L * is CV , alkyl
  • each F is bond, O, NR S , S, CiO), SCO), S(0) 2 , OC(0), NCR*)C(OX NCR x )S(0) 5 N ⁇ R s )S ⁇ 0) 2 , C(0)0, C(0)N(R x ), S(0)N(R*X S(0) 2 NiR x X OC(0)0, OC(0)N(R*X (R x )CCO)0 5 N(R*)C(0)N(R*X or CR x )S(0) 2 N(R 3 ⁇ 4 );
  • each if is Co-Ce alkyl; Is I, 2 or 3, when p is 2 or 3, then each (E-1 -F *) group m be the same or different;
  • q is 0 or 1 ;
  • G is a bond, (X NR*, S, Ct ' O), S(0), S( ⁇ 3 ⁇ 4 OQO N(R £ )C(Ox N(R s )S(0), NflR x )S(0) 2 , C(Op, C(0)N(R*X S(0)N ⁇ R x ), S ⁇ P) 2 N(R* ⁇ , 0C(0)0, OC(0) (R x X N(R x ⁇ C(0)0 5 N(R x )C(0 ⁇ N(R s X ⁇ *)8( ⁇ ) 2 ⁇ 0** NR3 ⁇ 4iO)L 5 NR ⁇ NR*CXO) ' L $ NR*C(OX NR*CX.O)L 5 0 or NR s C(0)L 5 C(0);
  • L 3 is Cj-Q, alky!
  • each R x is as defined above.
  • E is NR* or CfO
  • L is C.i ⁇ C(, alkyi
  • F i a bond, NR ⁇ N(R*)C ⁇ 0X GC(0), C(0)0 or C(0)N(R x ); and G is O, S, C(O) or NR* ' .
  • E is R ; or CfO
  • each is C 4 -C 6 alkyi
  • each L '1 is C0-C2 alkyi
  • p 2;
  • each F is a bond, NR ⁇ N(R x )C(0), OCfO), CfO)0 or C(0)N(R x );
  • G is O, S. C(O) or NR S .
  • E is NR* or CfO
  • each V is Q r C 2 alkyi
  • each L is C0-C5 alkyi
  • p 2;
  • each F is a bond, R ⁇ N(R x )C(0), OC(0), CfO)0 or C(0)N(R x );
  • G is O, S, C(OX NR*, NR3 ⁇ 4(0)L 5 NR s ! NR s C(0)L i R ,i C(0) ! NR3 ⁇ 4(0)L 5 O or NR3 ⁇ 4(0)L 5 C(0).
  • E is NR* or CfO
  • each L 3 is C Cs alkvl; each L 4 is Co-C> alkyl;
  • p 3;
  • each F is a bond, NR ⁇ MCR*)C ⁇ Q), OC(OX C(0)0 or C(0)N ⁇ R*); q is ⁇ ;
  • G is NR3 ⁇ 4(0)L 5 NR x or NR3 ⁇ 4(0)I/NR s C(0).
  • the linker L is a cleavable linker.
  • L is a cleavable linker
  • it is cleavable by a method selecied from the group consisting of acid-induced cleavage, light-induced cieavage, peptidase- induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.
  • the cleavable linker comprises a hydraxone, a cathepsin-B-cleavable peptide, a disulfide or an ester bond.
  • n is I .
  • n is 2.
  • n 3.
  • D is a drug sel ected from the group consisting of a cytotoxic drug, a cytostatic drug and antiproliferative drug.
  • D is an antitumor agent, cytotoxic or otherwise.
  • D is an inhibitor of a cellular metabolic event.
  • D can be an enzyme or protein inhibitor, such as an Hsp Inhibitor or a protei kinase inhibitor.
  • D is an amino containing drug selected from the group consisting of mitomycin-C. mitomycin- A, daimorubicin, doxorubicin, N- (5,5-diacetox.ypentyl)doxorubkm, aminopterin, actkon cin, bleomycin, 9-aniino eamptothecin, N* -acetyl spermidine, l-(2 clrioroethyl.)-l,2-dimethaeesu!fonyl liydrazide, tallysomycin, methotrexate, amsacrin, cis-platin, mercaptopuritte and derivatives thereof.
  • mitomycin-C mitomycin- A, daimorubicin, doxorubicin, N- (5,5-diacetox.ypentyl)doxorubkm, aminopterin, actkon cin, bleomycin, 9
  • D is a hydroxy! containing drug selected irom the group consisting of etoposide, camptothecin, taxol, esperamicin, L$-dihydroxy- hicyclof 7.3.1 jtrideca ⁇ 4, -diene ⁇ 2 >-diyne-i 3-or!e, angoidine, doxorubicin, morpliofino-doxonibicin, ⁇ (5,5-diacetoxypentyl)doxorabici.n, vincristine, vinblastine, bleomycin, tenyposide, podophyliotoxin and derivatives thereo 00 71 in some embodiments, D is a sulfhydryl containing drug selected from the group consisting of esperamicm, 6-mercaptoptsririe, and derivatives thereof.
  • D is a carboxyl containing drug selected from (he group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, nitrogen mustard drugs, chlorambucil, meiphaUm and derivatives thereof.
  • D is methotrexate or a derivative thereof.
  • D is camptothecin or a derivate thereof.
  • D is a nitrogen mustard drug or a derivative thereof.
  • D is chlorambucil or a derivative thereof, in other aspects, D is melphaian or a derivative thereof.
  • 0 is an aldehyde containing drug, such as, einnama!dehyde, inosine dialdehyde, and diglycoaidehyde.
  • D is a ketone containing drag, such as an anihracycline or t epothilone.
  • the conjugate is represented by formula:
  • the conjugate is represented by fonntila:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by fonntila:
  • the conjugate is represented by formula: ct
  • the conjugate is represetiied by formula:
  • the conjugate is represented by fonmtla:
  • the conjugate is represented by formula:
  • the present disclosure also provides a pharmaceutical composition comprising a conjugate of formula 0) and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a conjugate of formula 0) and a pharmaceutically acceptable carrier.
  • !0066J The present disclosure also provides a method of treating cancer in a patient comprising administering to a patient in need thereof a conjugate of formula (I) or a pharmaceutical composition thereof.
  • the sugar moiety binds to a cancer cell
  • the drug is reieasable from the sugar moiety at or nea the cancer cell by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell
  • the present disclosure also provides a method of reducing the toxic side effects of administering a drug to treat cancer, comprising administering to a patient an effective amount of a conjugate of formula t i) or a pharmaceutical composition thereo in some aspects of this embodiment, the sugar moiety binds to a cancer cell, the drag is reieasable from the sugar moiet at or near the cancer cell by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell.
  • a!kenyi refers to a straight or branched chain hydrocarbon containing from 2 to 10 carbons, unless otherwise specified, and containing at. least one carbon-carbon double bond.
  • Representative examples of aiketiyt include, but are not limited to, ethenyl, 2-propetiyl, 2-methy! ⁇ 2 ⁇ propenyl, 3-butenyI, 4-pentenyl, 5-hexenyf 2-faeptenyJ, 2-methyl-l-heptenyl, 3-decenyI, and 3 ,7-di methy loc ia-2,6-dieny I .
  • alkoxy refers to an aikyl group, as defined herein, appended to a parent moiety via an oxygen atom.
  • Representative examples of alfcoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, sec-buioxy, iso-kitoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentyioxy, n- hexyioxy, 3-meihylhexyIaxy, 2,2-dimethylpentoxy, 2,3 ⁇ dimetbylpen.toxy !
  • alkoxyalkyp as used herein, means an alkoxy group, as defined herein, appended to a parent moiety via an alky! group, as defined herein.
  • Representative examples of alkoxyalkyl include, but are not limited to,
  • alkyl as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, unless otherwise specified.
  • Representative examples of alky! include, but are not limited to, methyl, ethyl, n ⁇ propyl, iso -propyl, o-butyS, sec-butyl iso-buty tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methyihexyi, 2,2-diniethylpentyI, 2,3 ⁇ dimethylpentyl n- hepi l, n-octyl, n-nonyl, and n-decyl.
  • an "alkyi" group is a linking group between two other moieties, then it ma also be a straight or branched chain; examples include, but are not limited to -CHr, -
  • aU ynyl as used herein, means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond.
  • Representative examples of a!kynyl include, but are not limited, to acetylenyl, i-propynyl, 2-propynyl, 3-butynyl 2-penty.nyi and 1- butynyl.
  • aryl as used herein, means a phenyl (i.e., monocyclic aryl), a bicychc ring system containing at least one phenyl ring or an aromatic bicyciic ring containing only carbon atoms in the aromatic bicyciic ring system.
  • the bicyciic aryl can be axulenyl, naphthyl, or a phenyl (base ting) fused to a monocyclic cycioaikyl, a monocyclic cycloalkenyi, or a monocyclic heterocyclyl
  • the bicyciic aryl is attached to the parent molecular moiety through any carbon atom contained within the base ring, or any carbon, atom with the tiapihyt or azulenyl ring.
  • bicyciic ar ls include, but are not limited to, a/uleny naphthyl, dihydroinden-l-yl, dihydro den-2-yl, dihydfoi «den-3-yl 5 dihydroinden-4-yl, 2,3-dihydroindol ⁇ 4-yl, 2,3 ⁇ dihydroindol-5-yl, 2,3-dihydroindol- 6 ⁇ yl, 2,3-di.hydroindol-7 ⁇ yl, inden-l -y!, inden-2-yl, inden-3 ⁇ yl, inden-4-yl, dihydroiiaphthaien-2-yl, dihydronaphthaien-3-yi, dihydronaphthalen-4-yl, dih droiiaphi!ialen- i ⁇ yl, $,6,7,8-teirahydronaphihalett
  • cycloalkyl as used herein, means a monocyclic or bicyclic ring system.
  • Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated (i.e., cy oalkanyl) or unsaturated (i.e., cycloalkenyi), but not aromatic.
  • Examples of monocyclic cycloalky!s include cyclopropyl, cyclobaiyl, cyclopentyl, cyclopentenyl,
  • cyclohexyL cyclohexenyl
  • eyclohepiyi and cyclooctyl.
  • monocyclic cycloalkyl groups are folly saturated
  • Bicychc cycloalkyl groups are a monocyclic cycloalkyl ring (bas ring) fused to one ring selected from the group consisting of a phenyl .ring, a monocyclic cycloalkyl, a monocyclic cycloalkenyi, a monocyclic heterocyciyi, and a monocyclic heteroaryl.
  • the bicyclic cycloalkyl is attached to the parent molecular moiely through any carbon atom contained within the base ring.
  • bicyclic cycloalkyl groups are a monocyclic cycloalkyl ring (base ring) fused to one ring selected from the group consisting of a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyi, a 5 or 6 membered monocyclic heterocyciyi, and a 5 or 6 membered monocyclic heteroaryl.
  • heteroaryl as used herein, means a monocyclic or bicyclic heteroaryl ring system.
  • the monocyclic heteroaryl can be a 5 or 6 membered ring.
  • the 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom.
  • the 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms.
  • the 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or arty nitrogen atom contained within the heteroaryl.
  • monocyclic heteroaryl include, but are not limited to, fury!, im!dazo!yi, Isoxazolyl, isothiazo!yl, oxadiazolyl, oxaxol l, pyridinyl, pyridazinyl, pyrimidmyJ, pyraziny!, pyraxoly!, pyrrolyl, tetrazoiy ' t, thiadiaxolyl, thia olyl, thienyl triaxolyl, and tt inyl.
  • the bicyclic heteroaryl consists of a monocyclic heteroaryl ring (base ring) fused to a phenyl, a monocyclic cy loalkyi, a monocyclic cycloalkenyl,. a. monocyclic heterocyclyl, or a monocyclic heteroaryl
  • the bicyclic heteroaryl contains a fused cycloalkyl cycloalkenyl, or heterocyclyl ring, then the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.
  • bicyclic heteroaryl is a monocyclic heteroary] fused to a phenyl ring or a monocyclic heteroaryl
  • the bicyclic heteroaryl group is connected io the parent molecular moiety through an carbon atom or nitrogen atom within the bicyclic ring system.
  • Representative examples of bicyclic heteroaryl include, but are not limited to, benzimklazo!yi, benxofuranyl,
  • the bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to a phenyl ring, a 5 or 6 niembered monocyclic cycloalkyl, a 5 or 6 niembered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl.
  • heterocyclyl as used herein, means a monocyclic or bicyclic heterocycle.
  • the monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
  • the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S.
  • the 5 membered ring ca contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, and S.
  • the monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle.
  • Representative examples of monocyclic heterocycle include, but are not limited to, a .etsdiayL azepanyl, aziridinyl, diaxepanyl, 1 ,3-dioxanyl, 1,3- dioxolanyl, 1 ,3-dithioianyi, 1 ,3-dMiiaoyl, imidazo!inyl, imidazoiidinyl, isothiazolinyi, isothiazolidin l, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiaxolinyl, oxadiazolidinyL oxaxolinyl, oxazoHdinyl, piperazinyi, piperidinyl, pyran l, pyrazolmyl,
  • the bicyclic heterocycle is a monocyclic heterocycle ring (base ring) fused to a. phenyl, a monocyclic cycioalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle. or a monocyclic heteroaryl.
  • the bicyclic heterocycle is connected to the parent, molecular moiety through any carbon atom or any nitrogen atom contained withi the base ring.
  • bicyclic heierocydes are a monocyclic heterocycle ring (base ring) fused to a phenyl, a 5 or 6 merohered monocyclic cycioalkyl, a 5 or 6 membered monocyclic cycloalkenyl. a 5 or 6 membered monocyclic heterocycle, or a 5 or 6 membered monocycl ic heteroaryl.
  • bicyclic lieterocyc!yls include, but are not limited to, 2,3-dihydroben/.ofurmi-2-yL 2,3- dihydrobenzofuran-3-yl, indolin-i-yl, mdolin-2-yl, indolin-3-yl, 2,3- dihydroben.zotliiei5-2-yL decahydroqumolinyl, decahydroisoquinoHnyl, octahydro- !.H-mdolyL and octahydrobei ofiiranyl.
  • nitno as used herein, means a -NOj group.
  • ''saturated means the referenced chemical structure does not contain any multiple carbon-carbon bonds.
  • a saturated cycioalkyl group as defined herein includes cyclohexyl, cvciopropyl, and the like.
  • the terra "unsaturated” as used herein means the referenced chemical structure contains at least, one multiple carbon-carbon bond, but is not aromatic.
  • an unsaturated cycioalkyl group as defined herein includes cyclohexenyt cyclopentenyi, cyclohexadienyl, and the like.
  • oligoaiky!ene glycol refers to a linear oKgoalkylene glycol, a branched oligoalkylene glycol, and a comb-oJigoaiky!ene glycol, each comprising from about ⁇ to 1000 repeat units.
  • an oligoaJkyteae glycol is a linear oHgoaikylene glycol
  • oligopeptide refers to a peptide with fewer than about 20 amino acid residues.
  • dendrimer refers to a highly branched polymer o oligomer having a well-defined chemicai structure comprising a core and a given number of generations of branches, or spindles, and end groups.
  • the generations of spindles consist of structural units that are identical for the same generation of spindles and that may be identical or different, for different, generations of spindles.
  • the generations of spindles extend radially in a geometrical progression from the core.
  • the end groups of a dendrimer from the Nth generation are the end functional groups of the spindles of the Nth generation or end. generation.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomerie, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enanti meric, diastereomerie, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.
  • Ri is selected from ihe group consisting of R, OH, SH, NH 3 ⁇ 4 OR 4 , OC(0)R 4 , OC ⁇ 0)NHR , OC(0 ⁇ N3 ⁇ 4R 5t OC(S)NHR_,, 0C(S)NR 4 R5, SC(0)NHR 4> SC(0) R4R;, HC(0) HR4, NHCfO ⁇ NR 4 R s> NHC(S) HR4, NHC(S)NR4R 3 ⁇ 4 , NBC(N)NHR4, NHC( )NR,R 5 . OCH 2 C ⁇ 0)NHR 4 , OCH 2 C(0)NR 4 R 5 ,
  • each R4 is selected from the group consisting of H, Cj-Ce alky I, C? ⁇ C3 ⁇ 4 alkenyl and C G, alkynyl;
  • each R 5 is selected from the group consisting of CrQ aifcyt C * aikenyi and C?-C f , alkynyl;
  • R.2 is selected from the group consisting of H, OH, SH, NR 2t O *, 0C(0)R 4 , OC(0)NHR , OC(0 ⁇ NR 4 R 5 , 0C(S)NHR 4 , OCfS) R 4 R 5! SC(0)NHR 4 , SC(0)NR 4 R s , HC(0) HR 4> NHC(0)NR4R.5, NHC(S)NHR 4 , NHC(S)NR 4 Rs, NIIC( )NHRi, NHC(N)NR 4 R 55 0CB 2 C(0)NHR. j OCH 2 C(0)NR. 4 R5,
  • s is selected from the group consisting of H, OH, S , NH 2s OR4, 0C(0)R 4 , OC(0)NHR 4 , OC(0 ⁇ NR 4 R 5 , 0C(S)NHR 4 , OCfS) R 4 R 5! SC(0)NHR 4 , SC(0)NR 4 R s , BC(0) HR4, NHC(0) R(R5, NHC(S)NHR 4 , BC(S ⁇ NR 4 Rs, NHC( )NHRt, NHC(N)NR 4 R5 5 OCB 2 C(0)NHRi, OC 2 C(0)NR 4 R 5 ,
  • R ' is selected from the group consisting of H, OH and NBR.j;
  • B is a Spacer Unit
  • n is an integer selected from 1 to 3 ;
  • L is absent or a Linker
  • D is a Drug Unit having one or more chemically reactive functional groups selected from the group consisting of a primary or secondary amine, hydroxy !, su!fhydryl, carboxy!, aldehyde and ketone. f0090j In some embodiment
  • A is:
  • A is:
  • Rs is selected frora the group consisting of R, OH, 0C(0)R 4 . OCONHR 4 , and OCONR 4 R 5 .
  • R 2 is selected from the group consisting of H, OH, OC(0)R4, OCONHR4, OCONR4R5, OCSNHR4, NHCONHR4, HCONRiRs, OCHjCONHRi, and OCH.2CONR4R5.
  • R* is selected from the group consisting of H, OH,
  • R' is I! or OH.
  • each R 4 is selected from the group consisting of H, raethvi. and ethvt. 10103) in some embodiments, each R ? is selected from the group consisting of methyl ethyl, and isob tyi
  • a ss selected from the group consisting of:
  • A is selected from the group consisting of:
  • A is
  • B is a Spacer Unit selected from the group consisting of a bond, CrC» alkyi, C rC?o alkenyl, C5-C2 alkynyl, atyl, heterearyl, heterocyclyl, C3-C5 cycloalkyl an oligoaikylene glycol an. oligopeptide and a dendrirner.
  • the Spacer Unit is X-(.L'-Y)as-L"-Z,
  • X is C3 ⁇ 4 or O
  • L ! is Cs-Cft alkyl
  • Y is O, S intercept or NR y , wherein R* is hydrogen or Ci ⁇ C « alkyi;
  • m is an integer selected from 1 to 10; 1/ is €:-0>» alkyl, C Cas a!kenyf, Cj- ao alkviiyi, aryl, heteroatyi heterocyclyl, C : Cj cyc!oaU yl; aad
  • Z is absent, O, R X , S, C(0), SCO), S(0 ⁇ 2 , OC(0), N ⁇ R*)C(0), N(R*)S(0), N(R*)S(0 ⁇ 2 , 0(0)0, C(0)N(R*) 5 S(0)N(R s ), S(0) 2 N ⁇ R s X 00(0)0, OC(0)N(R*X N(R*)C(0)0, N(R s )C(0) (R s ), or N(R s )S(0) 2 .NCR x ), wherein
  • each R* is independently hydrogen or Q-CV > alkyl.
  • X is O
  • L ! is C 2 -C4 alkyl
  • L ⁇ is CrQ, alkyl
  • Z is a bond O, NR*, S, C(0), 5(0), S(0> 2 , or N(R K )C(0).
  • the Spacer Unit is 0-(CH CH> « 0)m ⁇ CHjCH Z, wherein Z is O, (H), S or NCR x )C(0), R x is H and m is an integer selected irom I to 20.
  • the Spacer Unit is 0 ⁇ CH>CH 2 ⁇ 3 ⁇ 4- O -CHjCH , wherein Z is Q, N(H), or S and ni is an integer seiected from 1 to 20.
  • the Spacer Unit is 0-(CHjCH.r0) ni - CJ3 ⁇ 4CH 2 -Z, wherein Z is C(0) or S(0)i and m is an integer selected from 1 to 20.
  • the linker L is absent.
  • the Spacer Unit is ⁇ -( ⁇ ⁇ ) ⁇ -1 - ⁇ , wherein X is O, L ! is C2-C4 alkyl; L ⁇ is Ci-Cs alkyl; and Z is a bond, O, NR S , S, C(0), SCO), S(0) 2 , or NCR s )C(0).
  • X is O, L f is C 6 -03 ⁇ 4 alky ⁇ ; L " is Ci-C 6 alkyl: and Z is
  • the Sinker L is a non-cleavahle linker.
  • L is (E ⁇ L :> -F ) P -C ⁇ -G,
  • each E is bond, O, NR ⁇ S, CCO), S(0), S ⁇ 0) 2 , GC(0), N(R* ⁇ C(0), N(R x )S(0), NCR x )S(0> 2> 0(0)0, C(0)N ⁇ R*X SiO)N(R x ), SCOfeNfR*), 0C(0)0, OCCO) (R x ), (R x ⁇ C(0)0, N(R x )C(0 ⁇ N(R * X or NCR x )S(0) 2 N(R x );
  • each L * is CVQ, alkyl
  • each F is bond, O, NR S , S, C(0), SCO), S(0) 2 , 0C(0), NCR*)C(OX NCR ⁇ SCO), N ⁇ R s )S ⁇ 0) 2 , C(0)0, C(0)N(R x ), S(0)N(R*X S(0) 2 NiR x X 0C(0)0, OC(0) (R x X (R x )CCO)0 5 N(R*)C(0)N(R*X or CR x )S(0) 2 N(R 3 ⁇ 4 );
  • each (E-I '-F-L*) group may be the same or different;
  • q is 0 or 1 ;
  • G is a bond, (X NR*, S, Ct ' O), S(0), S( ⁇ 3 ⁇ 4 OQO N(R £ )C(Ox N(R s )S(0), NflR x )S(0) 2 , C(Op, C(0)N(R*X S(0)N ⁇ R x ), S ⁇ P) 2 N(R* ⁇ , 0C(0)0, OC(0) (R x X N(R x ⁇ C(0)0 5 N(R x )C(0 ⁇ N(R s X ⁇ *)8( ⁇ ) 2 ⁇ 0** NR3 ⁇ 4iO)L 5 NR ⁇ NR*CXO)L $ NR*C(OX NR*CX.O)L 5 0 or NR s C(0)L 5 C(0);
  • L 3 is Cj-Q, alky!
  • each R x is as defined above.
  • E is NR* or CfO
  • L ' is C.i ⁇ C(, alkyi
  • F i a bond, NR ⁇ N(R*)C ⁇ 0X GC(0), C(0)0 or C(0)N(R x ); and G is O, S, C(O) or NR * .
  • E is NR ; or CfO
  • each is C 4 -C 6 alkyi
  • each L '1 is C0-C2 alkyi
  • p 2;
  • each F is a bond, ' NR 1 , N(R x )C(0), OCfO), CfO)0 or C(0)N(R x );
  • G is O, S. C(O) or NR S .
  • E is NR* or Cf O );
  • each V is Q r C 2 alkyi
  • each L is C0-C5 alkyi
  • p 2;
  • each F is a bond, NR ⁇ N(R x )C(0), OC(0), CfO)0 or C(0)N(R x );
  • G is O, S, C(OX NR*, NR3 ⁇ 4(0)L 5 NR s ! NR s C(0)L i R ,i C(0) ! NR3 ⁇ 4(0)L 5 O or NR3 ⁇ 4(0)L 5 C(0).
  • E is NR* or CfO
  • each L 3 is C Cs alkvl; each L 4 is Co-C> alkyl;
  • p 3;
  • each F is a bond, N ⁇ HCR ⁇ CCO), OC(OX C(0)0 or C(0)N ⁇ R*); q is ⁇ ;
  • G is NR3 ⁇ 4(0)L 5 NR x or NR3 ⁇ 4(0)L1 ⁇ 4 s C(0).
  • the linker L is a cleavable linker.
  • L is a cleavable linker
  • it is cleavable by a method selecied from the group consisting of acid-induced cieavage, light-induced cleavage, peptidase- induced cleavage, esterase-iiiduced cleavage, and disulfide bond cleavage,
  • the cleavable linker comprises a hydrazone, a cathepsin-B-cieavabie peptide, a disulfide or an ester bond.
  • n is 1.
  • n is 2.
  • n 3.
  • the sugar-linker drug conjugates of the present disclosure are effective for the usual purposes for which the corresponding drugs are effective, and have superior efficacy because of the ability, inherent in the sugar, to transport the drug to the desired cell where it is of particular benefit.
  • the conjugates of the disclosure can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may b a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a protein such as tumor necrosis factor.
  • the term "Drug" refers to any pharmacologically active agent capable of arresting cell growth, or killing the cell in which i is present.
  • the drug can be selected from the group consisting of a cytotoxic drug, a cytostatic drug, antiproliferative drug and antitumor agent.
  • the drug is a cytotoxic drug.
  • D is an antitumor agent, in some aspects of this embodiment, D is an inhibitor of a cellular metabolic event.
  • D can be an enzyme or protein inhibitor, such, as an Hsp 0 inhibitor or a protein kinase inhibitor.
  • D is an amino containing drug selected from the group consisting of mitomycin-C, mitomycin- A, daunombicm, doxorubicin, N- (3,5-diacetoxypentyl)doxorabici», anunopterin, actmoraycin, bleomycin, 9-amino eamptoihecin, N s -acetyl spermidine, i-(2 chlot»ethy).)- i ,2-dimethanesulfonyl hydrazide, iallysomycin, methotrexate, amsacrin, cis-platin, mercapiopurine and derivatives thereof.
  • D is a hydroxy! containing drug selected from the group consisting of etoposide, camptoihecin, iaxol, esperamicin, i,8 ⁇ dihydroxy- bicyclo[7.3.1 jirideea ⁇ 4,9-diene ⁇ 2,6-diyne- ⁇ 3-orte, angyidine, doxorubicin, mo ⁇ holin -d xorubic ⁇ ;l N-(5,5-diacetoxypent I)d xoru kiB, vincristine, vinblastine, bleomycin, teniposide, podophyilotoxin and derivatives thereof.
  • drug selected from the group consisting of etoposide, camptoihecin, iaxol, esperamicin, i,8 ⁇ dihydroxy- bicyclo[7.3.1 jirideea ⁇ 4,9-diene ⁇ 2,6-diyne- ⁇ 3-or
  • 0 is a suJfhydryl containing drug selected from the group consisting of esperamicin, 6-mercaptoputirte, and derivatives thereof.
  • D is a carbox l containing drug selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, nitrogen mustard drugs, chlorambucil, melphaian and derivatives thereof.
  • D is methotrexate or a derivative thereof.
  • D is campiothecm or a derivative thereof.
  • D is a nitrogen mustard drug or a derivaiive thereof.
  • D is chlorambucil or a derivaiive thereof, in other aspects, D is melphaian or a derivative thereof.
  • D is an aldehyde containing drug, such as, cinnamaldehyde, inosine dialdehyde, and. diglycoaidehyde.
  • D is a ketone containing drag, such as
  • the conjugate is represented by foroniia:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjagaie is represented by fonntila:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • the conjugate is represented by formula:
  • fee conjugate is represented by formula:
  • a "pharmaceutically acceptable salt'” means any non-toxic salt of a conjugate of this disclosure that, upon administration to a patient, is capable of providing, either directly or indirectly, a conjugate of this disclosure.
  • Phannaeeiitieali acceptable salts of the conjugates of this disclosure include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • suitable acid salts hiclude acetate, adipale, alginate, aspartate, benzoate, benzenesuifonate, bisulfaie, butyrate, citrate, camphorate, caniphorsu!fbnate, cyciopeiitanepropionate, digluconate, dodecylsulfaie , ethanesu!fonaie.
  • Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), asmmmium and N ' (CM. alkyl ' k salts.
  • alkali metal e.g., sodium and potassium
  • alkaline earth metal e.g., magnesium
  • asmmmium e.g., magnesium
  • N ' CM. alkyl ' k salts.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate of formula (I) as described above and a pharmaceutically acceptable carrier.
  • compositions include, but are not limited to, ion exchangers, alumina, aluminum, stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial gjyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisillcate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium, carboxymethyfceilulose, polyaerylate
  • “prophy tactically effective amount” refers to an amount effective in preventing o substantially lessening a disease or disorder in a patient. 01571
  • the term "patient”, as used herein, means an animal., preferably a mammal, and most preferably, a mouse, rat, other rodent, rabbit, dog, cat, swine, cattle, sheep, horse, or primate, and even more preferably, a human.
  • compositions of the present disclosure may be administered orally, parenteral ly, by inhalation spray, topically, rec tally, nasally, buccal l.y, vaginally or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra-articuiar, intra-synovial,
  • compositions of this invention may be administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 3 ,3-hotanediol.
  • the acceptable vehicles and solvents thai may be employed are water. Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including
  • Fatty acids such as oleic acid and its glyceri.de derivatives are useful in the preparatio of injectables, as are natural
  • oils such as olive oil or castor oil, especially in their polyoxyeihylated versions.
  • oils solutions or suspensions may also contain a .long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of
  • compositions including emulsions and suspensions.
  • Other commonly used surfactants such as T weens.
  • Spans and other emulsifying agents or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous sospessions or solutions.
  • carriers commonly used include lactose and com starch.
  • Lubricating agents, such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • lactose lactose and dried cornstarch.
  • the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • compositions of this disclosure may be administered in the form of suppositories for rectal
  • thai is solid at room iemperaiure but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non- irritating excipient thai is solid at room iemperaiure but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non- irritating excipient thai is solid at room iemperaiure but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • Such materials include cocoa, butter, beeswax and polyethylene glycols.
  • compositions of this disclosure may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation, (see above) or in a suitable enema formulation. Topically- transdermal patches may also be used.
  • compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil , liquid petrolatum, white petrolatum, propylene glycol, polyoxyethy!ene,
  • the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monosfearate, polysorbate 60, cetyl esters wax, vaearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutically acceptable compositions may be formulated as micromxed suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as betizylalkonium chloride.
  • the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
  • compositions of this disclosure may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubtiizi.ri.g or dispersing agents.
  • compositions of this disclosure are formulated for oral administration.
  • Dosage levels of between about 0.0 i and about 100 mg/kg tody weight per day, preferably between 0.5 and about 75 mg/kg body weight per day and most preferably between about i and 50 mg/kg body weight per day of the active ingredient compound are useful in a monotherapy for the prevention and treatment of cancer.
  • the pharmaceutical compositions of this disclosure will be administered from about 1 to 5 times per day or alternatively, as a continuous infusion. Or, alternatively, the compositions of the present disclosure may be administered in a pulsatile formulation. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated a nd the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • such preparations contain from about 20% to about 80% active compound,
  • compositions of this disclosure comprise a combination of a compound of the present disclosure and one or more additional therapeutic agents
  • both the compound and the additional agent should be present at dosage levels of between about 1 % to 80% of the dosage normally administered m a monotherapy regime
  • a maintenance dose of a compound, composition or combination of this disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • additional therapeutic agents which are normally administered to treat or prevent that condition, may also be present in the compositions of this invention.
  • additional therapeutic agents that, are normally administered to trea or prevent a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated”.
  • Additional therapeutic agents that ma be combined with the conjugates of the present disclosure include, but are not limited to, GleevecTM, adria rycra, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, iaxoi, interferons, platinum derivatives, alkylating drugs (mechlorethamrae,
  • compositions according to this invention comprise a combination of a compound of the present disclosure and. another therapeutic or prophylactic agent.
  • the additional therapeutic agents described above may be administered separately, as pan of a multiple dosage regimen, from the sogar-linker-drog conjugate containing composition. Al ternati vely, these agents may be part of a single dosage form, mixed together with sugar-linker-drag conjugate in a single composition.
  • ⁇ 01771 T e present disclosure provides a method of using the conjugates of the present disclosure or a pharmaceutically acceptable composition comprising a conjugate.
  • the present disclosure provides a method of treating or preventing cancer in a patient
  • the method comprises administering to a patient in .need thereof a conjugate as described, above or a pharmaceutical composition as described above.
  • cancer includes, but is not limited to the following cancers: breast; ovary; cervix; prostate; testis, genitourinary tract;
  • esophagus larynx, glioblastoma: neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, small ceil, carcinoma, lung adenocarcinoma; bone; colon, adenoma; pancreas, adenocarcinoma; thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma; seminoma,; melanoma; sarcoma: bladder carcinoma; liver carcinoma and biliary passages; kidney carcinoma; myeloid disorders; lymphoid disorders, Hodgkio's, hairy cells; buccal cavity and pharynx (oral), lip, tongue, month, pharynx; small intestine; colon-rectum, large intestine, rectum; brain and central nervous system; and leukemia.
  • the sugar moiety of the conjugate binds to a cancer cell.
  • the drug is released from the sugar moiety at or near the cancer cell by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell,
  • the present disclosure provides a method of reducing the toxic side effects of admini tering a drug to treat cancer.
  • the method comprises administering to a patient an effective amount of a conjugate as described above or a pharmaceutical composition as described above.
  • the sugar moiety of the conjugate binds to a cancer cell.
  • the drug is released from the sugar moiety at or near the cancer cell, by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell
  • the methods of this disclosure that utilize compositions that do not contain an additional therapeutic agent comprise the additional step of separately administering to said patient an additional therapeutic agent.
  • additional therapeutic agents are administered separately, they may be administered to the patient, prior to, sequentially with or following administration of the compositions of this disclosure.
  • TLC chromatograms were developed by immersing the plates in 2.5% potassium permanganate in ethanol or 2% anisaldehyde + 5% sulfuric acid + 1.5% glacial acetic acid, in ethanol, followed by heating, or else visualised by UV irradiation (254 am). Melting points were recorded on a MelTemp apparatus and are uncorrected. Tetrahydrofuran was distilled from sodiiun henzophenone ketyl and dichloromethaiie from calcium hydride. ⁇ and L 'C NMR spectra were recorded on a Gemini 300 or Varian Inova 400, or on a Varian Inova 500 spectrometer, using CTK3 ⁇ 4 as solvent and internal standard, unless otherwise indicated.
  • TX-di-ester-BLM-disaccharide 15
  • a solution containing 1 mg ( 1 .2 pmol) of compound 13 in 1 : 1 TFA-CHCI.2 was shaken at room temperature for 1 h and concentrated under diminished pressure. The residue was co- evaporated with five 2-raL portions of toluene and dissolved in 0.1 mL of anhydrous D SO. To this solution was added 2,5 mg (5.2 umoi) of 34, 20 pL (15 rag, 0.1 ⁇ mniol) of DIPEA and 15 mg (39 praol) of HATU and the reaction mixture was stirred at room temperature for 24 h.
  • reaction was purified on an Econosil Os reversed phase semi-preparative (250 10 mm, if ) pm) HPLC column using 0.1% aq. TEA and CH 3 C mobile phases. A linear gradient was employed (99: 1 0.1% aq. TFA ⁇ CH 3 CN-»45:55 0.1% aq. TFA --CH.5CN) over a period of 30 min with a flow rate of 3 mL'tnia. Fractions containing the desired product eluted at 22.
  • the residue was suspended in three 3 -niL portions of dicMoromethane and filtered through a pad ofCelite 545* : .
  • the organic layer was washed with 40 mL of I aq Ki, dried (MgSC and then concentrated under diminished pressure to afford the crude aldehyde 17.
  • the aldehyde was used for the nest reaction immediately.
  • the reaction mixture was stirred at 20 : 'C for 15 min, filtered through a pad of Celite 545* and then concentrated under diminished pressure to remove acetonitrile and most of the water (bath temperature not exceeding 40 ⁇ 'C) to afford a crude residue.
  • the brown residue was triturated with four 50-mL portions of ether and the liquid phase was pipetted and filtered through a pad of Florist!* ' (60-100 mesh) to afford a colorless solution. After a further washing of Fiorisii* with 50 mL of ethyl acetate, the combined organic layer was concentrated under diminished pressure to yield the crude aldehyde 21 as a brown syrup, which was used immediatel for the next reaction.
  • reaction mixture was allowed to warm to room temperature slowly. After 2 h, silica gel TLC analysis (1 :3 ethyl acetate- hexanes) indicaied complete consumption of starting material (3 ⁇ 4- 0.80) and formation of two products 3 ⁇ 4 0.40 and 3 ⁇ 4- 0.30).
  • the reaction ' mixture was quenched by the dropwise addition of 50 ml., of methanol and the mixture was diluted with 250 mL of dichloromethane.
  • the organic layer was washed with 200 mL of 10% solution of Rochelle's salt and brine and then dried ( gSCV ⁇ . The organic layer was filtered and the filtrate was concentrated trader diminished pressure.
  • the resul ing residue was purified by flash column chrom tography (1 :3 ethyl acetate-hexanes) to afford the xmdesired compound methyl 4,6-0- benzy ⁇ idene-3- ⁇ beRzyl-fi-.D-mannopyranoside (R( 0.30) and the desired methyl 4/!- -benzylid.ene-2-0-benzyl-a-D-mannopyranoside (24) as a colorless crystalline solid: yield 3.0 g (41 % . ); silica gel TLC #f 0,40 (1 :3 ethyl acetate- hexanes); ' H NMR.
  • the reaction mixture was stirred at 60 ' ' "C under diminished pressure for 1 Si, allowed to cool to room temperature and then poured into a stirriag mixture of 120 mL of ethyl acetate and 1 0 mL satd a NaHCOs.
  • the organic layer was washed with three 50-mL portions of brine and dried (Mg$i3 ⁇ 4 ⁇ .
  • the solvent was concentrated under diminished pressure to afford a crude residue.
  • the residue was applied to a silica gel column (30 x 5 cm). Ehition with 4: 1 ethyl acetaie-hexai .es afforded aceta!
  • reaction mixture was stirred at 0 "C for 2 fa and then poured into a mixture of 300 mL of ethyl acetate and 150 mL of said aq NaHCOj.
  • the aqueous and organic layers were separated and the organic layer was washed with three 50-roL portions of disti lled water and brine and then dried (MgSO-j). he solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column.
  • Example 7 Synthesis of niannose donor 40.
  • reaction mixture vvas diluted with 800 mL of ethyl acetate.
  • the organic layer was washed with three 250-mL portions of water and dried (MgSQ ).
  • the organic laye was concentrated under diminished pressure to afford a crude residue.
  • the residue was crystallized from 1 :7 dichloroniethane-hexanes to afford acetai 41 as a colorless solid: yield.
  • reaction mixture was then quenched by the addi tion of 500 mg of ' Dowex 50x resin, shaken, for 15 min and filtered.
  • Pd/C To the sotoiion of the crude product in methanol was added Pd/C and 3 ⁇ 4 gas was bubbled through for 1 h. The complete consumption of starting material was confirmed by MALDl-TOF mass spectral analysis.
  • the reaction mixture was filtered through Celite 545 s " and then concentrated trader diminished pressure to afford 57, which was used for the next reaction; HRMS (APCI), m/z 473, 1 86 (M + H) '!' (C
  • H N (CDGb) ⁇ 1.92 is, 311), 2.00-2.01 (in, 6H, J - 2.8 Hz,), 2.04 (s, 3H, J 5.3 Hz), 2.08 (d, 6H, J - 1.9 Hz), 2.12 (s, 3B), 3.61 (ddd, I H, ,/ - 12.7, 9.6 and 3.3 Hz), 3,84-3.95 (ra, 2H), 3,96-4.20 (m, 4H), 4.26-4.37 (m, 2H), 4.59 (t, 1 H, J - 10.4 Hz,), 4.90-5. 8 (m.
  • the reaction mixture was stirred for 20 mm at which time it was poured, into a two phase solution of 70 mL of ethyl acetate and 43 mL of said aq. NaHCOj, The organic layer was washed with two 50-ra ' L portions of brine, dried (MgSO-j) and concentrated under diminished pressure to afford a crude residue.
  • the residue was purified by flash chromatography on a silica gel column (25 * 3 cm). Elution with 3:1 ethyl acetate-hexanes afforded 65 as a colorless oil: yield 275 mg (51 %); silica gel TLC / .26 (3: 1 ethyl acetete-hexanes).
  • niaiumpyranosyl)-p-l..-gulopyranosyl Diphenyi Phosphate 66.
  • disaccharide 65 a solution containing 62.0 mg (0.09 mmol) of disaccharide 65 in 1,0 mL of anh DMF was added 12.0 nig (0.13 mmoi) of hydrazine acetate.
  • the reaction mixture was stirred at room temperature for 2.5 h and quenched by the addition of 15 mL of ethyl aceiate.
  • the organic solution was washed with 10 mL of water, said aq NaHC ⁇ 3 ⁇ 4, brine and dried iMgSO. ⁇ ).
  • the reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetate and 20 mL of satd aq NaHC ⁇ 3 ⁇ 4, The aqueous and organic layers were separated and the organic layer was washed with three 10-mL portions of water and brine and then dried (MgSC ). The solvent, was concentrated, under diminished pressure to afford a crude residue.
  • altropyranosyl « ⁇ l «L>gtt1opyranos»de (77).
  • ester 76 To a solution containing 73.0 mg (0.09 mmol) of ester 76 in 2 mL of nh THF was added a solution of 0,7 mL of anh THF saturated with N3 ⁇ 4 at 0 °C. The reaction mixture was allowed to warm to room temperature and then stirred for 2.5 h at which time silica gel TLC analysis indicated that the reaction was complete. The solvent was concentrated under diminished pressure to afford a crude residise. The residue was applied to a silica gel column (20 x 3 cm). Eluiion with 3:1.
  • reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetate and 20 mL of said aq aMCC
  • the aqueous and organic layers were separated and fee organic layer was washed with three 1 -raL portions of distilled water and brine and then dried (MgS(3 ⁇ 4).
  • the solvent was concentrated under diminished pressure to afford a crude residue.
  • the residue was applied to a silica gel column (25 x 2 cm).
  • mannopvranose (86) To a solution containing 0.55 g (1 .60 mmoi) of 85 in 5.6 ml, of pyridine were added 0.77 g (6.30 rnmol) of DMAP and 1.30 g
  • reaction mixture was stirred at 0 C C for 17 rain at which time it was poured into a two-phase solution of EtOAc (60 mL) and saturated aq NaHCCh (25 mL). The organic layer was washed with two 20-raL portions of brine, dried ( ajSO, ⁇ ), filtered and concentrated under diminished pressure.
  • the reaction mixture was stirred at 0 °C for 1.7 rain, at which time it was poured into a two-phase solution of EtOAc (50 ml,) and saturated aq NaHCOj (20 mL). The organic layer was washed, with two 20- L portions of brine, dried ( a ⁇ SC ⁇ ), filtered and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (25 * 2 em).
  • the ethyl acetate layer was washed with two 40-trsL portions of 2 M aq HCI, two 30- m.L portions of IhO, and 20 mL of brine, then dried over anhydrous ajSC , filtered and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (1:2 * 3 cm).
  • trimer BLM monosaccharide 106 as a colorless oil: yield 15 mg (43%); silica gel TLC R f 0.56 (4:4:1 chioroform-acetone-methanol); mass spectrum (MALDi), m/z 1730.76 (M + Na) * ; mass spectrum (TOF), m/z 854.3351 (M + 2H) *'* requires m/z 854.3357).
  • Triiner BLM menosacehar e-Mraker (107). To a solution of 4.2 mg (2.46 ⁇ ) of 106 in 2 ml, of auh methanol was added 0.2 raL of 25% w/w freshly prepared solution of sodiitm methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 2.5 h, and the complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for 15 min and filtered; mass spectrum (MALDl), m/z 1351.40 (M + Na ⁇ ' (theoretical m/z 1328.56).
  • reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for 15 mir> and filtered; mass spectrum (MALDI), m/z 424.24 (M ⁇ * ⁇ Naf; Mass spectrum (APCi), m/z 402.1759 (M + HY
  • the reaction mixture was purified on an Eeonosil C;s reversed phase semi-preparative RPLC column. (250 ⁇ 1 mm, 10 ⁇ ) using aq 0.1% TFA and CH?CN mobile phases.
  • a linear gradient was employed (99:1 0.1% aq TFA-CH3CN -> 60:40 0.1% aq TFA-CHjCN) over a period of 18 rain at a flow rate of 4.5 mL-Zmin.
  • the tractions containing the desired product elated at 17.9 min (monitoring at 364 nm) and were collected, frozen and iyophiiized to give 123 as a light yellow solid: yield 0.5 mg (12% over two steps).
  • a methotrexate resistant cell line, DU i45(MT ' X) was developed from parental DU 145 cells by exposure to increasing concentrations (starting at 0, 1 ⁇ ) of methotrexate over a period of 6 mouths.
  • DU J45( TX) was at least 250- fold less sensitive to methotrexate than parental cells. After five passages in drug-free medium, the resistant " ceils retained their drag resistance, suggesting the stability of the cell line.
  • the human prostate cancer cell lines DU-1 5 was cultured in MEM media with glutamine and supplemented with 10% fetal bovine serum, penicillin (KKHJ/mL) and streptomycin (lOOU/mL) in air enriched with 5% €(3 ⁇ 4 at 37° C.

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Abstract

The present disclosure relates to sugar-linker-drug conjugates, of the formula [A-B-]n-L-D, wherein A is a saccharide; B is a spacer, n is an integer selected from 1 to 3; L is a linker group and D is a drug having a chemically reactive functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde and ketone. Pharmaceutical compositions comprising the conjugates and methods of using thern are also provided.

Description

SUGAR-LINKER-DRUG CONJUGATES
CROSS REFERENCE TO RELATED APPLICATION
[OOOlj This application claims the benefit U.S. .Provisional Application.
No. 61/801 ,202, filed March 15, 2013. which is incorporated by reference herein in its entirety.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
{0002] This invention was made with government support trader ROI CA J 40471 awarded by the National Insiitutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
Ι.ΘΘ03] The present disclosure relates to sugar-linker-drug conjugates, compositions comprising them and methods of using {hem,
BACKGROUND Of THE INVENTIO
(O0O4| The bleomycins (BLMs) are a family of glyeopeptkle-derived antitumor antibiotics used clinically for the treatment, of squamous ceil carcinomas and malignant lymphomas. [Levi, J, A. et ai.., J. Clin. Oncol 1993, 1.1, 1300;
Bleomycia Chemotherapy; Slide, B. I., RoxeneweigjM., Carter, S, K., Eds.;
Academic Press; Orlando, FL. 1 85.] Their antitumor activity is thought to result from selective oxidative cleavage of 5'~GC~3' and 5'~GT-3' sequences i DNA and possibly also from oxidative degradation of R.NA. {Holmes, C. E. et aL
I Biochemistr 1993, 32, 4293; Kane, S. A.; Heeht, S.M. Prog. Nocieic Acid Res. Mo1. Biol 1994, 49, 313; Claussea, C. A.; Long, E. C. Chem. Rev. 1 99, 99, 2797: Hecht, S. M. J. Nat. Prod. 2000, 63, 158; Abraham, A. T. et al, Chem. Biol. 2003, 10, 5; Chen, J.; Slubbe, J. Nat. Rev. Cancer 2005, 5, 102; Tao, Z. P.; Konislii, , et al., J. Am. Chem. Soc. 2006, 128, 1.4806]. In addition to its antitumor activity, BLM has been, recognized for its ability to target tumors and shown to act as a tumor-imaging agent. Jones, S. E,; Lilian, Ό. L.; O'Mara, R. E,; Durie, B. G,; Salmon, S. E. Med. Pediatr. Oncol. 1975, 1, 1 1 ; Silverstein, M. J.; Venna, R. C; Greenfield, L.; Morton, D. L. Cancer 1976, 37, 36; Bekerman, C; Moran, E. M.; Mofifer, P. B.; Hendrix, R. W,; Gottschalk, A, Radiology 1977, 123, 687; Burton, I. E.; Todd, J. H.; Tomer, R. L. Br. J. Radiol. 1977, 50, SOS; Goodwin, D. A.;
Meares, C. F.; DeRiemer, L. H.; Diamanti, C. 1.; Goode, R. L.; Baumert, J. .£., Jr.; Sartoris, D. J.; Lanfieri, R. L.; Fawcett, H. D. J. Nad. Med. 1981 , 22, 787; Stem, P. IT; Hdpera, S. £.; Hagan, P. L.; Howell, S. B.; Dabbs, J. E.; Gordon, R.M. J, Nail, Cancer Inst. 1981 , 66, 807 j.
SUMMARY OF THE INVENTION
|0005] The present disclosure provides a sugar-linker-drug conjugate of formula
A-8|-L~D (!) or a pharmaceutically acceptable sail thereof,
wherein A. is:
Figure imgf000003_0001
j is selected from the group consisting of H, OH, SH, N¾, OR4,
OC(0)R4, OC(0)NHR , OC(0)NRiR¾ OC(S)NHR , OClS NRj s- SC(0) HR SC(0}NR4R5! NHC(0) HR45 NHOO NRiRs, NHC(S)NH1 NHC(S)NR4R?5 NHC(N)NHR4, NHCCN)NR4R5, OCH2C(0)NHR , OCH2C(0)NR R55
"7 OC¾C(S)NHR.1f OCHjC(S) R4R5? SCH2C(0)NHR4i SC¾e(0) R4R¾
NHCH2C(0)NH 4,NHC¾C{0)NR R5, NHCH2C(S}NHR4 and
Figure imgf000004_0001
each i is selected from the group consisting of B, G-Cs alkyi, C;- C(> alkenyl and Ca-Q a!kynyi;
each Rs is selected from the group consisting of Q-Q, alkyi, C¾-C$ alkenyl and QrG, alkynyl;
is selected from the group consisting of H, OH, SH, Ni¾ OR4, OC(0)R4, OC(0} HR4, 0C(0) R4R.s, OCtfQNHR*, OC(S)NR4R5, SC(0)NHR> SC(0) R4R5, HC(0)NHR4, NHC(G)NR4Rs, HC(S)NHR , NMC(S)NR R5, NHC(N)NHR4, NHC(N)NRR5, OCH2C(0)NHR4, OCH2C(0)NR R5i
OCH2C(S)NH ! OCH2Cf S)NR4 5, SCH2C(0)NH.R4, SC^CCOJN ^, NHCH2C{0) HR,NHCH2C(0)NRR5> NHCJ¾C(S)NHR and
Figure imgf000004_0002
R;i is selected ftom the group consisting o H, OB, SH, Nf¾5 OR4,
0C(0)R4, OC(0)NHR4- OC(0) R4 ;, OC(S)NHR4, 0C(S) R4R;, SC(0)NHR4, SC(0)NR 5, HC{0)N.HR4, NHC(0)NR4R5, NHC(S)NHR ! NHC(S} R4R5, NBC(N)NHR4, NBC(N)NR4R5:, OCH2C(0)NBR4, OC¾C(0)NR4R5!
OC¾C(S)NHR45 OCH2C(S)NR4R5; SCH2C(0)NHR4, SCH2C{0) R4Rj,
HCH2C{0) HR,NHCH2C(0)N 4R5, NHCJ¾C(S)NHR and
Figure imgf000004_0003
R' is selected from the group consisting of H, OB and NBR4;
B is a Spacer Unit;
n is an integer selected from 1 to 3;
L is absent or a Linker; and
D is a Drug Unit having one or more chemically reactive functional groups selecieci from the group consisting of a primary or secondary amine, hydroxy!, suSfhydryL carboxyl, aldehyde and ketone.
{00O6j in some embodiments, A is:
Figure imgf000005_0001
10007 J In alternative embodimen
Figure imgf000005_0002
fM)0S| in some embodiments, A is selected from the group consisting of:
Figure imgf000005_0003
{9909j in some embodiments, A
Figure imgf000005_0004
|00l Of In alternative embodiments, A is:
Figure imgf000006_0001
|ΘΘΙ ί f In alternative embodimen
Figure imgf000006_0002
|00i 2| In alternative embodimen
Figure imgf000006_0003
|0O13j In alternative embodimen
Figure imgf000006_0004
{9014} In some erabodiinents, Rs is selected from the group consisting of R, OH, 0C(0)R4. OCONHR4, and OCONR4R5.
[°00:15| I11 some embodiments, R2 is selected from the group consisting of H, OH, OC(0)R4, OCONHR4, OCONR4R5, OCSNHR4, NHCONHR4, HCONRiRs, OCHjCONHRi, and OCH.2CONR4R5.
{0016| in some embodiments, R* is selected from the group consisting of H, OH,
0C(0)R4, aBd 0tONffit .
{0017} In some erabodiinents, R' is IS or OH.
{0018} In some embodiments, each R4 is selected from the group consisting of H, niethvi. and ethvt. {90191 in some embodiments, each Rs is aeiecied from the group consisting of methyl, ethyl, and isobiUyl
[0020| In some embodittients, A is selected from the group consisting of:
Figure imgf000007_0001
4S R4 = H
53 s H^.
Figure imgf000007_0002
64 R4 = H 76 R4 = H
65 4 = CH3 77 R4 « CH3
(00 1] In some embodiments, A is selected from the group consisting of:
Figure imgf000008_0001
10? >V* N QS ^ H 111 Rs = M
Figure imgf000008_0002
{00221 ¾ some embodiments, A is selected from the group consisting of
Figure imgf000008_0003
[00231 In some embodiments, B is a Spacer Unit selected from the group consisting of a bond, CrC» aikyl, C rC?o alkenyl, CrQ» alkynyl, atyl, heterearyl, heterocyclyl, C3-C5 cycloalkyl an oligoaikylene glycol an. oligopeptide and a dendriraer.
{002 j in some embodiments, the Spacer Unit is X-CL'-Y^-tr-Z,
wherein X is C¾ or O;
L! is C Cft alkyi;
Y is (X S„ or NRy, wherein R* is hydrogen or C Cs alkyi;
tti is an integer selected from 1 to 10; 1/ is€:-0>» alkyl, C Cas a!kenyf, Cj- ao alkviiyi, aryl, heteroaryl, heterocyclyl, C: Cj cyc!oalfcyJ; aad
Z is absent, O, RX, S, C(0), SCO), S(0}2, OC(0), N<R*)C(0), N(R*)S(0), N(Rx)S(0}2, 0(0)0, C(0)N(R*)5 S(0)N(Rs), S(0>2N(RsX 00(0)0, OC(0)N(R*X N(R*)C(0)0, N(Rs)C(0) (Rs), or N(Rs)S(0)2.NCRx), wherein
each R* is independently hydrogen or Q-CV> alkyl.
{0025| In some aspects of this embodiment, X is O, L! is C2-C4 alkyl; L~ is CrQ, alkyl; and Z is a bond O NR*, S, C(0), 5(0), S(0>2, or N(RK)C(0).
[0026} In some aspects of this embodiment, the Spacer Unit is 0-(CH CH -0)ffi- C¾CH Z, wherein Z is O, (H), S or NCRx)C(0), Rx is H and m is an integer selected from I to 20.
100271 In other aspects of this embodiment, the Spacer Unit is 0^CH>CH2<¾- O -CHjCH , wherein Z is Q, N(H), or S and ni is an integer seiected from 1 to 20.
{0028} In some aspects of this embodiments, the Spacer Unit is 0-(CHjCH.r0)ni- CJ¾CH2-Z, wherein Z is C(0) or S(0)i and m is an integer selected from 1 to 20.
[0029} In some embodiments, the linker L is absent.
[0030} In some aspects of the embodiments in w hich the linker L is absent, the Spacer Unit is Χ-(ΐ Υ)∞-1 -Ζ, wherein X is O, L! is C2-C4 alkyl; L~ is C C<s alkyl; and Z is a bond, O, NRS, S, C(0), SCO), S(0)2, or NCRs)C(0). In some of these embodiments, X is O, Lf is C6-0¾ alky}; L" is CVQ, alkyl: and Z is
N(Rx)C(0).
[0031} In other embodiments, the linker L is a non-cleavahle linker.
[0032} In some aspects of this embodiment, L is CE~L'*-F-L4)P-C<5-G,
wherein each E is bond, O, NR\ S, CCO), S(0), S{0)2, OCC ), N(Rx}C(0), N(Rx)S(0), N(Rx)S(0)2> 0(0)0, C(0)N<R*X SiO)N(Rx), S(0)2N(Rx), OC(0)0, OCCO) (Rx), (Rx}CCO)0, N(Rx)C(0}N(R*X or NCRx)S(0)2N(Rx);
each L* is CV , alkyl;
each F is bond, O, NRS, S, CiO), SCO), S(0)2, OC(0), NCR*)C(OX NCRx)S(0)5 N{Rs)S{0)2, C(0)0, C(0)N(Rx), S(0)N(R*X S(0)2NiRxX OC(0)0, OC(0)N(R*X (Rx)CCO)05 N(R*)C(0)N(R*X or CRx)S(0)2N(R¾);
each if is Co-Ce alkyl; Is I, 2 or 3, when p is 2 or 3, then each (E-1 -F *) group m be the same or different;
q is 0 or 1 ; and
G is a bond, (X NR*, S, Ct'O), S(0), S(<¾ OQO N(R£)C(Ox N(Rs)S(0), NflRx)S(0)2, C(Op, C(0)N(R*X S(0)N{Rx), S<P)2N(R*}, 0C(0)0, OC(0) (RxX N(Rx}C(0)05 N(Rx)C(0}N(RsX ^*)8(Ο)2Ν0** NR¾iO)L5NR\ NR*CXO)'L$NR*C(OX NR*CX.O)L50 or NRsC(0)L5C(0);
herein L3 is Cj-Q, alky! and
each Rx is as defined above.
[00331 In some aspects of this embodiment, E is NR* or CfO);
L is C.i~C(, alkyi;
is C0-C2 alkyi;
p is ;
F i a bond, NR\ N(R*)C{0X GC(0), C(0)0 or C(0)N(Rx); and G is O, S, C(O) or NR*'.
[0034] in some aspecis of this eitibodiment, E is R ; or CfO);
each is C4-C6 alkyi;
each L'1 is C0-C2 alkyi;
p is 2;
each F is a bond, NR\ N(Rx)C(0), OCfO), CfO)0 or C(0)N(Rx); and
G is O, S. C(O) or NRS.
[00351 i some aspects of this embodiment, E is NR* or CfO);
each V is QrC2 alkyi;
each L is C0-C5 alkyi;
p is 2;
each F is a bond, R\ N(Rx)C(0), OC(0), CfO)0 or C(0)N(Rx); and
G is O, S, C(OX NR*, NR¾(0)L5NRs ! NRsC(0)Li R,iC(0)! NR¾(0)L5O or NR¾(0)L5C(0).
[0036] In some aspects of this embodiment, E is NR* or CfO);
each L3 is C Cs alkvl; each L4 is Co-C> alkyl;
p is 3;
each F is a bond, NR\ MCR*)C{Q), OC(OX C(0)0 or C(0)N<R*); q is Ϊ ; and
G is NR¾(0)L5NRx or NR¾(0)I/NRsC(0).
[003?| in some embodiments, the linker L is a cleavable linker.
[0038] in embodiments wherein L is a cleavable linker, it is cleavable by a method selecied from the group consisting of acid-induced cleavage, light-induced cieavage, peptidase- induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.
|ΘΘ39| In some embodiments, the cleavable linker comprises a hydraxone, a cathepsin-B-cleavable peptide, a disulfide or an ester bond.
[0040] in some embodiments, n is I .
{00 1! in some embodiments, n is 2.
[0042] in some embodiments, n is 3.
[0043] in some embodiments, D is a drug sel ected from the group consisting of a cytotoxic drug, a cytostatic drug and antiproliferative drug.
[0044] In some embodiments, D is an antitumor agent, cytotoxic or otherwise. In some aspects of this embodiment, D is an inhibitor of a cellular metabolic event. D can be an enzyme or protein inhibitor, such as an Hsp Inhibitor or a protei kinase inhibitor.
[0045] in some embodiments, D is an amino containing drug selected from the group consisting of mitomycin-C. mitomycin- A, daimorubicin, doxorubicin, N- (5,5-diacetox.ypentyl)doxorubkm, aminopterin, actkon cin, bleomycin, 9-aniino eamptothecin, N* -acetyl spermidine, l-(2 clrioroethyl.)-l,2-dimethaeesu!fonyl liydrazide, tallysomycin, methotrexate, amsacrin, cis-platin, mercaptopuritte and derivatives thereof.
[0046] in some embodiments, D is a hydroxy! containing drug selected irom the group consisting of etoposide, camptothecin, taxol, esperamicin, L$-dihydroxy- hicyclof 7.3.1 jtrideca~4, -diene~2 >-diyne-i 3-or!e, angoidine, doxorubicin, morpliofino-doxonibicin, ~(5,5-diacetoxypentyl)doxorabici.n, vincristine, vinblastine, bleomycin, tenyposide, podophyliotoxin and derivatives thereo 00 71 in some embodiments, D is a sulfhydryl containing drug selected from the group consisting of esperamicm, 6-mercaptoptsririe, and derivatives thereof.
[0048| In some embodiments, D is a carboxyl containing drug selected from (he group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, nitrogen mustard drugs, chlorambucil, meiphaUm and derivatives thereof.
{0049} In some embodiments, D is methotrexate or a derivative thereof. In other embodiments, D is camptothecin or a derivate thereof. In yet other embodiments, D is a nitrogen mustard drug or a derivative thereof. In some aspects of this embodiment, D is chlorambucil or a derivative thereof, in other aspects, D is melphaian or a derivative thereof.
(00501 -In some embodiments, 0 is an aldehyde containing drug, such as, einnama!dehyde, inosine dialdehyde, and diglycoaidehyde.
{00511 to some embodiments, D is a ketone containing drag, such as an anihracycline or t epothilone.
[00521 i s
Figure imgf000012_0001
[00531 In some embodiments, the conjugate is represented by formula:
Figure imgf000013_0001
{ S4j in some embodiments, the conjugate is represented by fonntila:
Figure imgf000013_0002
{ 0055) to some embodiments, the conjugate is represented by formula:
1.2.
Figure imgf000014_0001
jf)056| In some embodiments, the conjugate is represented by formula:
Figure imgf000014_0002
{0057| In some embodiments, the conjugate is represented by formula:
Figure imgf000014_0003
(00581 In some embodiments, the conjugate is represented by formula:
Figure imgf000015_0001
{00591 to some embodiments, the conjugate is represented by formula:
Figure imgf000015_0002
{006Oj in some embodiments, the conjugate is represented by fonntila:
Figure imgf000015_0003
{0061] in some embodiments, the conjugate is represented by formula: ct
H
NO'
HO.
OH
{0062] In some embodiments, the conjugate is represetiied by formula:
Figure imgf000016_0001
ΘΘ63] in some embodiments, the conjugate is represented by fonmtla:
Figure imgf000016_0002
{ΘΘ64] in some embodiments, the conjugate is represented by formula:
Figure imgf000017_0001
(ΘΘ65| The present disclosure also provides a pharmaceutical composition comprising a conjugate of formula 0) and a pharmaceutically acceptable carrier. !0066J The present disclosure also provides a method of treating cancer in a patient comprising administering to a patient in need thereof a conjugate of formula (I) or a pharmaceutical composition thereof. In some aspects of this embodiment, the sugar moiety binds to a cancer cell, the drug is reieasable from the sugar moiety at or nea the cancer cell by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell
Ι.0Θ67] The present disclosure also provides a method of reducing the toxic side effects of administering a drug to treat cancer, comprising administering to a patient an effective amount of a conjugate of formula t i) or a pharmaceutical composition thereo in some aspects of this embodiment, the sugar moiety binds to a cancer cell, the drag is reieasable from the sugar moiet at or near the cancer cell by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell. DETAILED DESCRIPTION OF THE INVENTION
|0068| In order that the invention herein described may be folly understood, the following detailed description is set forth.
{0069} Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples are illustrative only, and are not intended to be limiting. All publications, patents and other documents mentioned herein are incorporated by reference in their entirety.
[00701 Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers,
100711 The term "a" or "an" may mean more than one of an item.
{0072] The 'terms "and" and "or" may refer to either the conjunctive or disjunctive and .mean "and/or".
{0073 j The term "about" means within plus or minus 1 % of a stated value. For example, "about 1.00" would refer to any number between 90 and .1 1 .
[0074| The term "a!kenyi" as used herein, refers to a straight or branched chain hydrocarbon containing from 2 to 10 carbons, unless otherwise specified, and containing at. least one carbon-carbon double bond. Representative examples of aiketiyt include, but are not limited to, ethenyl, 2-propetiyl, 2-methy!~2~propenyl, 3-butenyI, 4-pentenyl, 5-hexenyf 2-faeptenyJ, 2-methyl-l-heptenyl, 3-decenyI, and 3 ,7-di methy loc ia-2,6-dieny I .
[0075j The term "alkoxy" refers to an aikyl group, as defined herein, appended to a parent moiety via an oxygen atom. Representative examples of alfcoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, sec-buioxy, iso-kitoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentyioxy, n- hexyioxy, 3-meihylhexyIaxy, 2,2-dimethylpentoxy, 2,3~dimetbylpen.toxy! n- hepty'ioxy, n-octyloxy, n-nonyloxy, and n-decoxy. f 00761 The term "alkoxyalkyp as used herein, means an alkoxy group, as defined herein, appended to a parent moiety via an alky! group, as defined herein. Representative examples of alkoxyalkyl include, but are not limited to,
methoxyraethy methoxyeth l, ethoxymethyl, ethoxyethyl, n-propoxymethyi, iso- propoxymethyl, ii-butox meihyl, sec-butoxymeihyl, iso~butoxymethyl and tert- butoxymethyl.
{00771 The term "alkyl" as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, unless otherwise specified. Representative examples of alky! include, but are not limited to, methyl, ethyl, n~ propyl, iso -propyl, o-butyS, sec-butyl iso-buty tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methyihexyi, 2,2-diniethylpentyI, 2,3~dimethylpentyl n- hepi l, n-octyl, n-nonyl, and n-decyl. When an "alkyi" group is a linking group between two other moieties, then it ma also be a straight or branched chain; examples include, but are not limited to -CHr, -CHjCHjCHC CHj)-,
Figure imgf000019_0001
100781 The term "aU ynyl" as used herein, means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of a!kynyl include, but are not limited, to acetylenyl, i-propynyl, 2-propynyl, 3-butynyl 2-penty.nyi and 1- butynyl.
{00791 The term "aryl " as used herein, means a phenyl (i.e., monocyclic aryl), a bicychc ring system containing at least one phenyl ring or an aromatic bicyciic ring containing only carbon atoms in the aromatic bicyciic ring system. The bicyciic aryl can be axulenyl, naphthyl, or a phenyl (base ting) fused to a monocyclic cycioaikyl, a monocyclic cycloalkenyi, or a monocyclic heterocyclyl The bicyciic aryl is attached to the parent molecular moiety through any carbon atom contained within the base ring, or any carbon, atom with the tiapihyt or azulenyl ring.
Representative examples of the bicyciic ar ls include, but are not limited to, a/uleny naphthyl, dihydroinden-l-yl, dihydro den-2-yl, dihydfoi«den-3-yl5 dihydroinden-4-yl, 2,3-dihydroindol~4-yl, 2,3~dihydroindol-5-yl, 2,3-dihydroindol- 6~yl, 2,3-di.hydroindol-7~yl, inden-l -y!, inden-2-yl, inden-3~yl, inden-4-yl, dihydroiiaphthaien-2-yl, dihydronaphthaien-3-yi, dihydronaphthalen-4-yl, dih droiiaphi!ialen- i~yl, $,6,7,8-teirahydronaphihalett-.l -yf 5,6,7,8- tetrahydro.naphthalen~2-yl, 2,3-dihydroben¾oJ¾ran-4-yl, 2!3-dihydrobenzofuran-5- yl, 2,3-dihydro en2ofuran-6-yI, 2,3-dihydro enzofuran-7-yl, ben oj d|[ 1 ,3]dioxol- 4-yl, benzo[d] 1 ,3 jdioxol-5-yI, 2,3-dihydro enzo(bj[ 1 ,4]dioxan-5-yl, and 2,3- dihydroben£o[b][ 1 ,4]dioxan~6-yl. In certain embodiments, the bicyclic ary! is (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyi, or a 5 or 6 tnembered moiiocyciic heterocyciyi.
[ΘΘ80| The term "cycloalkyl" as used herein, means a monocyclic or bicyclic ring system. Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated (i.e., cy oalkanyl) or unsaturated (i.e., cycloalkenyi), but not aromatic. Examples of monocyclic cycloalky!s include cyclopropyl, cyclobaiyl, cyclopentyl, cyclopentenyl,
cyclohexyL cyclohexenyl, eyclohepiyi, and cyclooctyl. In certain embodiments, monocyclic cycloalkyl groups are folly saturated, Bicychc cycloalkyl groups are a monocyclic cycloalkyl ring (bas ring) fused to one ring selected from the group consisting of a phenyl .ring, a monocyclic cycloalkyl, a monocyclic cycloalkenyi, a monocyclic heterocyciyi, and a monocyclic heteroaryl. The bicyclic cycloalkyl is attached to the parent molecular moiely through any carbon atom contained within the base ring. In certain embodiments, bicyclic cycloalkyl groups are a monocyclic cycloalkyl ring (base ring) fused to one ring selected from the group consisting of a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyi, a 5 or 6 membered monocyclic heterocyciyi, and a 5 or 6 membered monocyclic heteroaryl.
[0081 J The term "heteroaryl" as used herein, means a monocyclic or bicyclic heteroaryl ring system. The monocyclic heteroaryl can be a 5 or 6 membered ring. The 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom. The 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms. The 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or arty nitrogen atom contained within the heteroaryl. Representative examples of monocyclic heteroaryl include, but are not limited to, fury!, im!dazo!yi, Isoxazolyl, isothiazo!yl, oxadiazolyl, oxaxol l, pyridinyl, pyridazinyl, pyrimidmyJ, pyraziny!, pyraxoly!, pyrrolyl, tetrazoiy't, thiadiaxolyl, thia olyl, thienyl triaxolyl, and tt inyl. The bicyclic heteroaryl consists of a monocyclic heteroaryl ring (base ring) fused to a phenyl, a monocyclic cy loalkyi, a monocyclic cycloalkenyl,. a. monocyclic heterocyclyl, or a monocyclic heteroaryl When the bicyclic heteroaryl contains a fused cycloalkyl cycloalkenyl, or heterocyclyl ring, then the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring. When the bicyclic heteroaryl is a monocyclic heteroary] fused to a phenyl ring or a monocyclic heteroaryl, then the bicyclic heteroaryl group is connected io the parent molecular moiety through an carbon atom or nitrogen atom within the bicyclic ring system. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimklazo!yi, benxofuranyl,
benzothienyl, benzoxadiazolyL benzoxatliiadiazolyl ben othiazolyl cinnolinyl, 5,6-dihy roquinolin-2-yl, 5}6-dihydroisoquinolhi-.l - l, i½ropyridinyL indazolyh indolyl, isoquinolinyl, naphthyridinyl, quinolinyl purirryl, 5,6,7,8- teirahydroqu.molin-2-yi, 5,6,7,8-tetrahydroquinoiin-3-yt 5,6,7,8- teirahydroqirinohn-4-yl 5 >,7,8-ietrahydroisoquinoliri-]-yl, and ihienopyridinyl. In certain embodiments, the bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to a phenyl ring, a 5 or 6 niembered monocyclic cycloalkyl, a 5 or 6 niembered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl.
|0082] The term "heterocyclyl" as used herein, means a monocyclic or bicyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S. The 5 membered ring ca contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, and S. The monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle. Representative examples of monocyclic heterocycle include, but are not limited to, a .etsdiayL azepanyl, aziridinyl, diaxepanyl, 1 ,3-dioxanyl, 1,3- dioxolanyl, 1 ,3-dithioianyi, 1 ,3-dMiiaoyl, imidazo!inyl, imidazoiidinyl, isothiazolinyi, isothiazolidin l, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiaxolinyl, oxadiazolidinyL oxaxolinyl, oxazoHdinyl, piperazinyi, piperidinyl, pyran l, pyrazolmyl, pvrazolidinyl, pyrroltnyl, pyrrolidinyi, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, ihiadiazolidinyl, thiazolinyl, th.iazoIid.inyl, thioraorpholinyl, ihiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle ring (base ring) fused to a. phenyl, a monocyclic cycioalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle. or a monocyclic heteroaryl. The bicyclic heterocycle is connected to the parent, molecular moiety through any carbon atom or any nitrogen atom contained withi the base ring. In certain embodiments, bicyclic heierocydes are a monocyclic heterocycle ring (base ring) fused to a phenyl, a 5 or 6 merohered monocyclic cycioalkyl, a 5 or 6 membered monocyclic cycloalkenyl. a 5 or 6 membered monocyclic heterocycle, or a 5 or 6 membered monocycl ic heteroaryl. Representative ex amples of bicyclic lieterocyc!yls include, but are not limited to, 2,3-dihydroben/.ofurmi-2-yL 2,3- dihydrobenzofuran-3-yl, indolin-i-yl, mdolin-2-yl, indolin-3-yl, 2,3- dihydroben.zotliiei5-2-yL decahydroqumolinyl, decahydroisoquinoHnyl, octahydro- !.H-mdolyL and octahydrobei ofiiranyl.
{O083| The term nitno" as used herein, means a -NOj group.
{008 1 The term ''saturated" as used herein means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycioalkyl group as defined herein includes cyclohexyl, cvciopropyl, and the like. The terra "unsaturated" as used herein means the referenced chemical structure contains at least, one multiple carbon-carbon bond, but is not aromatic. For example, an unsaturated cycioalkyl group as defined herein includes cyclohexenyt cyclopentenyi, cyclohexadienyl, and the like.
{0085] The term "oligoaiky!ene glycol" refers to a linear oKgoalkylene glycol, a branched oligoalkylene glycol, and a comb-oJigoaiky!ene glycol, each comprising from about ί to 1000 repeat units. In certain embodiments, an oligoaJkyteae glycol is a linear oHgoaikylene glycol
[0086| The term "oligopeptide" refers to a peptide with fewer than about 20 amino acid residues.
[§087| The term "dendrimer' refers to a highly branched polymer o oligomer having a well-defined chemicai structure comprising a core and a given number of generations of branches, or spindles, and end groups. The generations of spindles consist of structural units that are identical for the same generation of spindles and that may be identical or different, for different, generations of spindles. The generations of spindles extend radially in a geometrical progression from the core. The end groups of a dendrimer from the Nth generation are the end functional groups of the spindles of the Nth generation or end. generation.
{0088] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomerie, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enanti meric, diastereomerie, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.
Sugar-Llnker- mg Conjugates
0089| The present disclosure provides a sugar-linker-drag conjugate of formula
0):
Figure imgf000023_0001
or a pharmaceutically acceptable salt thereof.
wherein A is;
Figure imgf000023_0002
Ri is selected from ihe group consisting of R, OH, SH, NH¾ OR4, OC(0)R4, OC<0)NHR , OC(0}N¾R 5t OC(S)NHR_,, 0C(S)NR4R5, SC(0)NHR4> SC(0) R4R;, HC(0) HR4, NHCfO}NR4Rs> NHC(S) HR4, NHC(S)NR4R¾, NBC(N)NHR4, NHC( )NR,R5. OCH2C<0)NHR4, OCH2C(0)NR4R5,
OCH2C(S)NHR45 OCH2C(S) R4 5; SCH2C(0)NHR4, SCH2C(0)NR4R5, • HCH2C(0) HR4, NHCH2C(O)NR4R5! NHCH2C(S)NHR4 and
Figure imgf000024_0001
each R4 is selected from the group consisting of H, Cj-Ce alky I, C?~ C¾ alkenyl and C G, alkynyl;
each R5 is selected from the group consisting of CrQ aifcyt C * aikenyi and C?-Cf, alkynyl;
R.2 is selected from the group consisting of H, OH, SH, NR2t O *, 0C(0)R4, OC(0)NHR , OC(0}NR4R5, 0C(S)NHR4, OCfS) R4R5! SC(0)NHR4, SC(0)NR4Rs, HC(0) HR4> NHC(0)NR4R.5, NHC(S)NHR4, NHC(S)NR4Rs, NIIC( )NHRi, NHC(N)NR4R55 0CB2C(0)NHR. j OCH2C(0)NR.4R5,
OCH2C(S)NHR4, . OCH2C{S)NR4Rs, SC¾C(0) HR45 SCH2C(0)NR4Rs,
.HCH2C(0)NHR4,NHC¾C(0)NR R5, NHCH2C(S]NHR4 and
Figure imgf000024_0002
s is selected from the group consisting of H, OH, S , NH2s OR4, 0C(0)R4, OC(0)NHR4, OC(0}NR4R5, 0C(S)NHR4, OCfS) R4R5! SC(0)NHR4, SC(0)NR4Rs, BC(0) HR4, NHC(0) R(R5, NHC(S)NHR4, BC(S}NR4Rs, NHC( )NHRt, NHC(N)NR4R55 OCB2C(0)NHRi, OC 2C(0)NR4R5,
OCR2C(S)NHR4, OCH>C(S)NR4R5> SCH2C(0)NHR4, SCH2C(0)NR4R¾
NHCH2C(0)NHR4, HCH,CfO)NR4R;, NHCH2C{S}NHR4 and
Figure imgf000024_0003
R ' is selected from the group consisting of H, OH and NBR.j;
B is a Spacer Unit;
n is an integer selected from 1 to 3 ;
L is absent or a Linker: and
D is a Drug Unit having one or more chemically reactive functional groups selected from the group consisting of a primary or secondary amine, hydroxy !, su!fhydryl, carboxy!, aldehyde and ketone. f0090j In some embodiment
(ΘΘ9
{009 f:
Figure imgf000025_0001
£0093 j in some embodiments.
Figure imgf000025_0002
[099 1 in alternative embodiments, A is:
Figure imgf000026_0001
|ΘΘ95| In alternative embodimen
Figure imgf000026_0002
|0096| In alternative embodiments, A
Figure imgf000026_0003
|Θ097{ In alternative embodiments, A is:
Figure imgf000026_0004
in some embodiinents, Rs is selected frora the group consisting of R, OH, 0C(0)R4. OCONHR4, and OCONR4R5.
00 j In some embodiments, R2 is selected from the group consisting of H, OH, OC(0)R4, OCONHR4, OCONR4R5, OCSNHR4, NHCONHR4, HCONRiRs, OCHjCONHRi, and OCH.2CONR4R5.
{OlOOj in some embodiments, R* is selected from the group consisting of H, OH,
OC(0)R4, and OCONHR4.
{OlOIj in some embodiments, R' is I! or OH.
|01O2| in some embodiments, each R4 is selected from the group consisting of H, raethvi. and ethvt. 10103) in some embodiments, each R? is selected from the group consisting of methyl ethyl, and isob tyi
[θ ί 04f In some embodiments, A ss selected from the group consisting of:
Figure imgf000027_0001
64 = H 76 R4 = H
77 R4 = CHj
|01 OSj In some embodiments, A is selected from the group consisting of:
Figure imgf000028_0001
10? R, » H 109 ^ H 111 Rs = M
0S ¾ ~ CH¾ 110 CHj
Figure imgf000028_0002
{01O61 In some embodiments, A is
Figure imgf000028_0003
|0J 07 f In some embodiments, B is a Spacer Unit selected from the group consisting of a bond, CrC» alkyi, C rC?o alkenyl, C5-C2 alkynyl, atyl, heterearyl, heterocyclyl, C3-C5 cycloalkyl an oligoaikylene glycol an. oligopeptide and a dendrirner.
[0l08j in some embodiments, the Spacer Unit is X-(.L'-Y)as-L"-Z,
wherein X is C¾ or O;
L! is Cs-Cft alkyl;
Y is O, S„ or NRy, wherein R* is hydrogen or Ci~C« alkyi;
m is an integer selected from 1 to 10; 1/ is€:-0>» alkyl, C Cas a!kenyf, Cj- ao alkviiyi, aryl, heteroatyi heterocyclyl, C: Cj cyc!oaU yl; aad
Z is absent, O, RX, S, C(0), SCO), S(0}2, OC(0), N<R*)C(0), N(R*)S(0), N(R*)S(0}2, 0(0)0, C(0)N(R*)5 S(0)N(Rs), S(0)2N{RsX 00(0)0, OC(0)N(R*X N(R*)C(0)0, N(Rs)C(0) (Rs), or N(Rs)S(0)2.NCRx), wherein
each R* is independently hydrogen or Q-CV> alkyl.
{0109} In some aspects of this embodiment, X is O, L! is C2-C4 alkyl; L~ is CrQ, alkyl; and Z is a bond O, NR*, S, C(0), 5(0), S(0>2, or N(RK)C(0).
I'O lOj In some aspects of this embodiment, the Spacer Unit is 0-(CH CH>«0)m~ CHjCH Z, wherein Z is O, (H), S or NCRx)C(0), Rx is H and m is an integer selected irom I to 20.
10111| in other aspects of this embodiment, the Spacer Unit is 0^CH>CH2<¾- O -CHjCH , wherein Z is Q, N(H), or S and ni is an integer seiected from 1 to 20.
{0112} In some aspects of this embodiments, the Spacer Unit is 0-(CHjCH.r0)ni- CJ¾CH2-Z, wherein Z is C(0) or S(0)i and m is an integer selected from 1 to 20.
[01 U| in some embodiments, the linker L is absent.
| 01 14| In some aspects of the embodiments in w hich the linker L is absent, the Spacer Unit is Χ-(ΐ Υ)∞-1 -Ζ, wherein X is O, L! is C2-C4 alkyl; L~ is Ci-Cs alkyl; and Z is a bond, O, NRS, S, C(0), SCO), S(0)2, or NCRs)C(0). In some of these embodiments, X is O, Lf is C6-0¾ alky}; L" is Ci-C6 alkyl: and Z is
N(R*)Ci0).
{0115} In other embodiments, the Sinker L is a non-cleavahle linker.
{0116} In some aspects of this embodiment, L is (E~L:>-F )P-C<{-G,
wherein each E is bond, O, NR\ S, CCO), S(0), S{0)2, GC(0), N(R*}C(0), N(Rx)S(0), NCRx)S(0>2> 0(0)0, C(0)N<R*X SiO)N(Rx), SCOfeNfR*), 0C(0)0, OCCO) (Rx), (Rx}C(0)0, N(Rx)C(0}N(R*X or NCRx)S(0)2N(Rx);
each L* is CVQ, alkyl;
each F is bond, O, NRS, S, C(0), SCO), S(0)2, 0C(0), NCR*)C(OX NCR^SCO), N{Rs)S{0)2, C(0)0, C(0)N(Rx), S(0)N(R*X S(0)2NiRxX 0C(0)0, OC(0) (RxX (Rx)CCO)05 N(R*)C(0)N(R*X or CRx)S(0)2N(R¾);
each if is Co-Ce alkyl; is 1, 2 or 3 when p is 2 or 3. then each (E-I '-F-L*) group may be the same or different;
q is 0 or 1 ; and
G is a bond, (X NR*, S, Ct'O), S(0), S(<¾ OQO N(R£)C(Ox N(Rs)S(0), NflRx)S(0)2, C(Op, C(0)N(R*X S(0)N{Rx), S<P)2N(R*}, 0C(0)0, OC(0) (RxX N(Rx}C(0)05 N(Rx)C(0}N(RsX ^*)8(Ο)2Ν0** NR¾iO)L5NR\ NR*CXO)L$NR*C(OX NR*CX.O)L50 or NRsC(0)L5C(0);
herein L3 is Cj-Q, alky! and
each Rx is as defined above.
[0J 1 ?f In some aspects of this embodiment, E is NR* or CfO);
L' is C.i~C(, alkyi;
is C0-C2 alkyi;
p is ;
F i a bond, NR\ N(R*)C{0X GC(0), C(0)0 or C(0)N(Rx); and G is O, S, C(O) or NR*.
[0118] in some aspecis of this eitibodiment, E is NR; or CfO);
each is C4-C6 alkyi;
each L'1 is C0-C2 alkyi;
p is 2;
each F is a bond, 'NR1, N(Rx)C(0), OCfO), CfO)0 or C(0)N(Rx); and
G is O, S. C(O) or NRS.
[0119] In some as pec ts of this embodimen t, E is NR* or Cf O );
each V is QrC2 alkyi;
each L is C0-C5 alkyi;
p is 2;
each F is a bond, NR\ N(Rx)C(0), OC(0), CfO)0 or C(0)N(Rx); and
G is O, S, C(OX NR*, NR¾(0)L5NRs ! NRsC(0)Li R,iC(0)! NR¾(0)L5O or NR¾(0)L5C(0).
[0120] In some aspects of this embodiment, E is NR* or CfO);
each L3 is C Cs alkvl; each L4 is Co-C> alkyl;
p is 3;
each F is a bond, N \ HCR^CCO), OC(OX C(0)0 or C(0)N<R*); q is Ϊ ; and
G is NR¾(0)L5NRx or NR¾(0)L¼ sC(0).
{0121] In some embodiments, the linker L is a cleavable linker.
{0122] in embodiments wherein L is a cleavable linker, it is cleavable by a method selecied from the group consisting of acid-induced cieavage, light-induced cleavage, peptidase- induced cleavage, esterase-iiiduced cleavage, and disulfide bond cleavage,
(0123 j In some embodiments, the cleavable linker comprises a hydrazone, a cathepsin-B-cieavabie peptide, a disulfide or an ester bond.
{0124] in some embodiments, n is 1.
{0125] in some embodiments, n is 2.
{0126] in some embodiments, n is 3.
[0127] The sugar-linker drug conjugates of the present disclosure are effective for the usual purposes for which the corresponding drugs are effective, and have superior efficacy because of the ability, inherent in the sugar, to transport the drug to the desired cell where it is of particular benefit. Further, because the conjugates of the disclosure can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may b a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a protein such as tumor necrosis factor. In some embodiments, the term "Drug" refers to any pharmacologically active agent capable of arresting cell growth, or killing the cell in which i is present. The drug can be selected from the group consisting of a cytotoxic drug, a cytostatic drug, antiproliferative drug and antitumor agent. In some embodiments, the drug is a cytotoxic drug.
{0128] in alternative embodiments, D is an antitumor agent, in some aspects of this embodiment, D is an inhibitor of a cellular metabolic event. D can be an enzyme or protein inhibitor, such, as an Hsp 0 inhibitor or a protein kinase inhibitor. [0129] in some embodiments, D is an amino containing drug selected from the group consisting of mitomycin-C, mitomycin- A, daunombicm, doxorubicin, N- (3,5-diacetoxypentyl)doxorabici», anunopterin, actmoraycin, bleomycin, 9-amino eamptoihecin, Ns -acetyl spermidine, i-(2 chlot»ethy).)- i ,2-dimethanesulfonyl hydrazide, iallysomycin, methotrexate, amsacrin, cis-platin, mercapiopurine and derivatives thereof.
[0130] in some embodiments, D is a hydroxy! containing drug selected from the group consisting of etoposide, camptoihecin, iaxol, esperamicin, i,8~dihydroxy- bicyclo[7.3.1 jirideea~4,9-diene~2,6-diyne- ί 3-orte, angyidine, doxorubicin, moφholin -d xorubicί ;l N-(5,5-diacetoxypent I)d xoru kiB, vincristine, vinblastine, bleomycin, teniposide, podophyilotoxin and derivatives thereof.
101311 I some embodiments, 0 is a suJfhydryl containing drug selected from the group consisting of esperamicin, 6-mercaptoputirte, and derivatives thereof.
{01321 to some embodiments, D is a carbox l containing drug selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, nitrogen mustard drugs, chlorambucil, melphaian and derivatives thereof.
(01331 In some embodiments, D is methotrexate or a derivative thereof. In alternate embodiments, D is campiothecm or a derivative thereof. In yet other embodiments, D is a nitrogen mustard drug or a derivaiive thereof. In some aspects of this embodiment, D is chlorambucil or a derivaiive thereof, in other aspects, D is melphaian or a derivative thereof.
[01341 to some embodiments, D is an aldehyde containing drug, such as, cinnamaldehyde, inosine dialdehyde, and. diglycoaidehyde.
[0135| In some embodiments, D is a ketone containing drag, such as
antferacycfine or an epothilone.
[01365 to some embodiments, the conjugate represented by formula'.
Figure imgf000032_0001
[01371 to some embodiments, the conjugate represented by formula:
Figure imgf000033_0001
|0 I38| ID some embodiments, the conjugate is represented by foroniia:
Figure imgf000033_0002
[0139] la some embodiments, the conjugate is represented by formula:
Figure imgf000034_0001
{0140] In some embodiments, the conjugate is represented by formula:
Figure imgf000034_0002
f 03411 In some embodiments, the conjugate is represented by formula:
Figure imgf000034_0003
0'J42'| In some embodiments, the conjugate is represented by formula:
Figure imgf000035_0001
{0143] in some embodiments, the conjugate is represented by formula:
Figure imgf000035_0002
{0144] in some embodiments, the conjagaie is represented by fonntila:
Figure imgf000035_0003
{0145] in some embodiments, the conjugate is represented by formula:
Figure imgf000036_0001
[01461 In some embodiments, the conjugate is represented by formula:
Figure imgf000036_0002
|0I47| in some embodiments, the conjugate is represented by formula:
Figure imgf000036_0003
(01 81 In some embodiments, fee conjugate is represented by formula:
Figure imgf000037_0001
(0J 9f The conjugates of the present disclosure can exist in their free form or, where appropriate, as pharmaceutically acceptable salts thereof.
10150] A "pharmaceutically acceptable salt'" means any non-toxic salt of a conjugate of this disclosure that, upon administration to a patient, is capable of providing, either directly or indirectly, a conjugate of this disclosure.
|0 51| Phannaeeiitieali acceptable salts of the conjugates of this disclosure include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts hiclude acetate, adipale, alginate, aspartate, benzoate, benzenesuifonate, bisulfaie, butyrate, citrate, camphorate, caniphorsu!fbnate, cyciopeiitanepropionate, digluconate, dodecylsulfaie , ethanesu!fonaie. formate, fumarate, glucoheptauoate, glycerophosphate, glyeolate, hemisulfate, heplanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2~ hydroxyethanesulfonate, lactate, maleate, malooate, methanesulfonate, 2- naphthalenesuifoBate, ntcotioate, nitrate, oxalate, pairaoaie, pectinate, persulfaie, 3-phenylpropionate, phosphate, picrale, pivaiate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, iosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in tire prepara tion of salts useful as mter ediaies in obtaining the compounds of the disclosure and their pharmaceutically acceptable acid addition salts.
(01521 Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), asmmmium and N ' (CM. alkyl'k salts. This disclosure also envisions the quatetnization of any basic nitrogen-containing groups of the conjugates disclosed herein. Water or oil- soluble or dispersible products ma be obtained by such quaternization.
{0153) One of skill in the ar would recognize that a variety of conjugates of the present disclosure may be prepared according to methods known in the art, and the synthetic Examples set forth below.
Compositions
(01541 The present disclosure provides a pharmaceutical composition comprising a conjugate of formula (I) as described above and a pharmaceutically acceptable carrier.
{0155} The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the conipoiind with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles thai may be used in the present compositions include, but are not limited to, ion exchangers, alumina, aluminum, stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial gjyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisillcate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium, carboxymethyfceilulose, polyaerylates, waxes, polyethylene-poiyoxypropylene-block polymers,
polyethylene glycol and wool fat,
{0156} The term "pharmaceutically effective amount" relets to an amount required to confer a therapeutic effect on the treated patient. The terra
"prophy tactically effective amount" refers to an amount effective in preventing o substantially lessening a disease or disorder in a patient. 01571 The term "patient", as used herein, means an animal., preferably a mammal, and most preferably, a mouse, rat, other rodent, rabbit, dog, cat, swine, cattle, sheep, horse, or primate, and even more preferably, a human.
|0J S8j The compositions of the present disclosure may be administered orally, parenteral ly, by inhalation spray, topically, rec tally, nasally, buccal l.y, vaginally or via an implanted reservoir. The terra "parenteral", as used herein, includes subcutaneous, intravenous, intramuscular, intra-articuiar, intra-synovial,
mtrastemal, intrathecal, intrahepatic, intralesionai and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 3 ,3-hotanediol. Among the acceptable vehicles and solvents thai may be employed are water. Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
(01591 For this purpose, any bland fixed oil may be employed including
synthetic mono- or di~giycerid.es. Fatty acids, such as oleic acid and its glyceri.de derivatives are useful in the preparatio of injectables, as are natural
pharmaceuiically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyeihylated versions. These oil solutions or suspensions may also contain a .long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as T weens. Spans and other emulsifying agents or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
0160J The pharmaceutically acceptable compositions of this disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous sospessions or solutions. la the case of tabiets tor oral use, carriers commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required .for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
{0161] Alternatively, the pharmaceutically acceptable compositions of this disclosure may be administered in the form of suppositories for rectal
administration. These can be prepared by mixing the agent with a suitable non- irritating excipient thai is solid at room iemperaiure but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa, butter, beeswax and polyethylene glycols.
{01621 The pharmaceutically acceptable compositions of this disclosure may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
{0163] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation, (see above) or in a suitable enema formulation. Topically- transdermal patches may also be used.
{0164] For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil , liquid petrolatum, white petrolatum, propylene glycol, polyoxyethy!ene,
polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monosfearate, polysorbate 60, cetyl esters wax, ceiearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. |0165j For ophthalmk use, the pharmaceutically acceptable compositions may be formulated as micromxed suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as betizylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
{0166| The pharmaceutically acceptable compositions of this disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubtiizi.ri.g or dispersing agents.
{0167] Most preferably, the pharmaceutically acceptable compositions of this disclosure are formulated for oral administration.
{0168] Dosage levels of between about 0.0 i and about 100 mg/kg tody weight per day, preferably between 0.5 and about 75 mg/kg body weight per day and most preferably between about i and 50 mg/kg body weight per day of the active ingredient compound are useful in a monotherapy for the prevention and treatment of cancer.
{0169] Typically, the pharmaceutical compositions of this disclosure will be administered from about 1 to 5 times per day or alternatively, as a continuous infusion. Or, alternatively, the compositions of the present disclosure may be administered in a pulsatile formulation. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated a nd the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound,
{0170J When the compositions of this disclosure comprise a combination of a compound of the present disclosure and one or more additional therapeutic agents, both the compound and the additional agent should be present at dosage levels of between about 1 % to 80% of the dosage normally administered m a monotherapy regime,
[0171 j Upon, improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
[Oi ?2| As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens Cor any particular patient will depend upon a variety of factors, including the activity of the specific compound, employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the se verit and course of the disease, and the patient's disposition, to the disease and the judgment of the treating physician,
[0173] Depending upon the particular condition, or disease, to be treated or prevented, additional therapeutic agents, which are normally administered to treat or prevent that condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that, are normally administered to trea or prevent a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated".
[01741 Additional therapeutic agents that ma be combined with the conjugates of the present disclosure include, but are not limited to, Gleevec™, adria rycra, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, iaxoi, interferons, platinum derivatives, alkylating drugs (mechlorethamrae,
chlorambucil, Cyclophosphamide, Melphalan, ifosiamide), antimetabolites {Methotrexate), purine antagonists and pyrimidme antagonists (6-Mercaptoparine, 5-Fluorouracil, Cyiarabile, Gemcitabme), spindle poisons (Vinblastine,
Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (Etoposide, Irmotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin), nitrosoureas
(Carmustine, Lomusiine), inorganic ions (Cisplatin, Carboplatin), enzymes (Asparaginase), and hormones (Tamoxifen. Leuproiide, Flutamide, and
Megestro!), Gleevec.TML, adriaroycut dexaraethasone, and cyclophosphamide. For a more comprehensive discussion of updated cancer therapies see, http://www.nci.nih.gov/., a list of the FDA approved oncology drugs at
htt : v id .go cder/c ncer/dru h tft¾me. ιr^. and The Merck Manual, Seventeenth Ed, 1 99, the en tire contents of which, are hereby incorporated by reference. .
{0175) The compounds of the present disclosure may also be co-administered with other cytotoxic agents to increase the effect of therapy or prophylaxis. When the compounds of this disclosure are administered in combination therapies with other agents, they may be adminisiered sequentially or concurrently to the patient Alternatively, pharmaceutical or prophylactic compositions according to this invention comprise a combination of a compound of the present disclosure and. another therapeutic or prophylactic agent.
{0176} The additional therapeutic agents described above may be administered separately, as pan of a multiple dosage regimen, from the sogar-linker-drog conjugate containing composition. Al ternati vely, these agents may be part of a single dosage form, mixed together with sugar-linker-drag conjugate in a single composition.
Methods Of Use
{01771 T e present disclosure provides a method of using the conjugates of the present disclosure or a pharmaceutically acceptable composition comprising a conjugate.
{0178} In some embodiments, the present disclosure provides a method of treating or preventing cancer in a patient The method comprises administering to a patient in .need thereof a conjugate as described, above or a pharmaceutical composition as described above.
{0179} The term "cancer," as used herein, includes, but is not limited to the following cancers: breast; ovary; cervix; prostate; testis, genitourinary tract;
esophagus; larynx, glioblastoma: neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, small ceil, carcinoma, lung adenocarcinoma; bone; colon, adenoma; pancreas, adenocarcinoma; thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma; seminoma,; melanoma; sarcoma: bladder carcinoma; liver carcinoma and biliary passages; kidney carcinoma; myeloid disorders; lymphoid disorders, Hodgkio's, hairy cells; buccal cavity and pharynx (oral), lip, tongue, month, pharynx; small intestine; colon-rectum, large intestine, rectum; brain and central nervous system; and leukemia.
{0180| in some aspects of this embodiment, the sugar moiety of the conjugate binds to a cancer cell. The drug is released from the sugar moiety at or near the cancer cell by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell,
(01811 In alternate embodiments, the present disclosure provides a method of reducing the toxic side effects of admini tering a drug to treat cancer. The method comprises administering to a patient an effective amount of a conjugate as described above or a pharmaceutical composition as described above.
{0182} in some aspects of this embodiment, the sugar moiety of the conjugate binds to a cancer cell. The drug is released from the sugar moiety at or near the cancer cell, by cleavage of the linker, and the drug, when released, is cytotoxic or cytostatic to the cancer cell
(0183] in an alternate embodiment, the methods of this disclosure that utilize compositions that do not contain an additional therapeutic agent, comprise the additional step of separately administering to said patient an additional therapeutic agent. When these additional therapeutic agents are administered separately, they may be administered to the patient, prior to, sequentially with or following administration of the compositions of this disclosure.
[0184| In order that this invention be more fully understood, the following examples are set forth. These example are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
Examples
{0185} The compounds and methods of the disclosure are illustrated further by the following examples, which are provided for illustrative purposes and not intended to be construed as limiting the disclosure i scope or spirit to the specific compounds and methods described in them. |018 j The chemicals were ail ACS reagent grade and were used without further purification. The reactions were carried out under an argon atmosphere unless specified. Flash column chromatography was carried out using silica gel (Silieye'Se J0030B, 60 particle size, 230-400 mesh), applying a low pressure stream of nitrogen. Analytical thin layer chromatographic separations were carried out on glass plates coated with silica ge! (60 particle size F254, Si!iCycle TLG-R1001 ΓΒ- 323). The TLC chromatograms were developed by immersing the plates in 2.5% potassium permanganate in ethanol or 2% anisaldehyde + 5% sulfuric acid + 1.5% glacial acetic acid, in ethanol, followed by heating, or else visualised by UV irradiation (254 am). Melting points were recorded on a MelTemp apparatus and are uncorrected. Tetrahydrofuran was distilled from sodiiun henzophenone ketyl and dichloromethaiie from calcium hydride. Ή and L'C NMR spectra were recorded on a Gemini 300 or Varian Inova 400, or on a Varian Inova 500 spectrometer, using CTK¾ as solvent and internal standard, unless otherwise indicated. Ή NMR chemical shifts were reported relative to residual CHt¾ at 7,26 ppm, or to residual DMSO-t/j at 2.50 ppm; f~'C NMR shifts were reported relative to the central line ofCJX¾ at 77.16 ppm, or to "C DMSO-i¾ at
39.51 ppra. Splitting patterns are designated as s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; di. doublet of triplets; m, rouitiplet; q, quartet", quin, quintet. Cyanine dyes were obtained from our collaborators at Genera! Electric, High resolution mass spectrometric data was obtained at the Michigan State Mass Spectrometry Facility or at the Arizona State University CLAS High Resolution Mass Spectrometry Facility.
0187j Example 1. In order to permit, a study of the efficacy of the BLM- disaccha.ri.de as a tumor targeting vehicle for cytotoxic agents, APA (4-ararno-4- deoxy- 1.0-i¥-methylpter ic acid) was conjugated to the BLM.-disaccharide
(Scheme 1). 2,4-Diamino-6-(hydroxymethyl)pieridine hydrochloride (1) was treated with triphenylphosphme dibromide to generate the aikyi bromide in situ: this was coupled with 4-i¥-rnethylarriir!obeii/.oic acid to obtain APA (2) in 56% yield. Compound 3 was condensed with APA t provide the APA-BLM- disaccharide conjugate 4 in 37% yield. Conjugate 5 was designed to be a tumor selective drug delivery vehicle. Once inside the ceil, the ceihtlar esterases should release the cytotoxic drug, in. this case APA, from the conjugate. The synthesis started with the esterificaiioii of APA 2 with /e -btuyl 6-hydroxyhexanoate (7) to obtain compound 8 in 60% yield. The /en-bui i group was removed by the use of CF.*€OGH in dichlororoetharie and the resulting acid was condensed with compound s to afford the conjugate 5 in 53% yield.
{0188] Scheme L Synthesis of AP A-B'LM-disaccharide conjugates 4 and 5.
Figure imgf000046_0001
4
Figure imgf000046_0002
APA ester-BLM-disaccharitte
5
Figure imgf000047_0001
|0J 89j 4»Araino»4-deoxy-l O-A^nethyipterok Acid (APA, 2) ( ra!ovec. I; Spencer, G.; Blair, A. H.; Mammen, M; Singh, M,; Ghose, T. J. Med Chem., 1989, 32, 2426). A mixture of 249 rag (0.59 mraol) dibromotripheaylphosphorane and 45,0 mg (0.20 tnmol) 2,4-diaraino-6-(hydr )xymethyl)pteridin.e hydrochloride (1) in 1 .5 mL of anhydrous dmieihylacetamide was stirred at room iemperatare for 24 h under an argon atmosphere. To the reaction mixture were added 41 mg (0.27 mmol) of 4-(methylamino)benzoic acid and 0,16 mL { 116 mg, 0,90 mmol) of DIPEA and the reaction mixture was stirred at room temperature for 48 h, and then at 60 '"'C for 24 fi. The cooled reaction mixture was poured into 25 mL of 0.33 M aq NaOH and the precipitate was filtered. The filtrate was adjusted to H 5.5 with 10% acetic acid and the resulting precipitate was collected through filtration, washed wi h water and dried under diminished pressure at 80 C overnight to obtain 2 as an orange solid: yield 42 mg (56%); silica gel TLC ¾- 0.47 (5:4: 1 chlorofornv-methanol-water); lH NM (DMSO-<&) o 3.19 (s, 3H), 4.76 (s, 2H), 6.61 (s, 2H>, 6.78 (d, 21i J- 8.9 Hz), 7.72 (d, 2H, J- 8.7 Hz) and 8.56 (s, 1H).
Figure imgf000047_0002
{01901 APA-BLM-disacehande (4). To a solution containing 3.5 mg
(7.4 pmol) of BLM-di saccharide linker 3, 2.5 mg (7,6 umol) of 6 and 3.0 μΐ (2.2 mg; 17 μχηοϊ) of DIPEA in 0.12 mL of anhydrous DMSO was added 4.3 rag (I S μχηοϊ) ofRATU. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified on an Econosil Cm reversed phase semi- preparative (250 x 10 mm, 10 μηι) HPLC column using 0.1% aq TFA and Q¾C mobile phases. A inear gradient was employed (99; 1 0. i% aq TPA-C¾CN -> 45:55 0.1 % aq TFA~CH;;C ) over a period of 30 min. at a flow rate of 3 mL/min. Fractions containing the desired product eiuted at 26.5 min (monitoring at 292 nm) and were collected, frozen, and lyophilized to give APA-BLM-disaccharide conjugate 4 as a yellow solid: yield 2.2 mg (37%); mass spectrum (ESI), m/z 780.3168 (M +
Figure imgf000048_0001
[0191 J 4-Amine-4-tieoxy-10-A'-nietlij lpteroic Acid 6-{fef-/-B«toxy)-6- oxohexjl Ester (8). To a solution of 36 mg (0.2 mraol) of 7, 12 mg (37 pmol) of 6 and 45 mg (0.4 mrno'l) of DMAP in I mh of anhydrous DMSO was added 39 rag (0.2 mmol) of DCC and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was filtered and the filtrate was purified on a C? s reversed phase semi -preparative (250 χ 10 mm, 10 um) HPLC column using 0.1% aq TFA and CH3CN mobile phases. A linear gradient was employed (99: 1 0.1% aq TFA~CH;¾CN■» 1:99 0.1 % aq TFA-€H3CN) over a period of 30 min at a flow rate of 3 mL/min. Fractions containing the desired product eiuted at 25.8 min (monitoring at 292 ran) and were collected, frozen, and iyophilked to give 8 as a yellow solid: yield 1 1 mg (60%); Ή MR (CD¾CN) 5 1.40 (m, 9H)5 1.58 (m, 2H), 1.70 (m, 2.H.), 2, 19 (t, 2H, J— 7.3 Hz), 3.24 (s, 3HI 4J 9 (i, 2H, = 6.4 11/), 4.84 (s, 2H), 5.45 (s, 2H), 6.79 (m, 2H), 7.26 (s, IH), 7.72 (s, IB), 7.8 Cm, 2H) and 8.73 (s, I H); mass spectrum (ES!), m/z 496.2676 (M■<■ Η'
Figure imgf000048_0002
equires m/z 496.2667).
Figure imgf000049_0001
|0I92| APA ester-BL -tlisaeehaiide (5). A solution containing 2.8 mg (5.6 μηιοί) of compound 8 in 1 : 1 TFA-C¾C¾ was shaken at room temperature for I h and concentrated under diminished pressure. The residiie was co-evaporated with five 2-mL portions of toluene and dissolved .in 0.1 ml, of anhydrous DMSO. To tiiis solution were added 2.9 mg (6,2 μηιοΙ) of 3 (Boger, D, 1..; Honda, T. J. Am. Chem. Soc, 1994, i itf, 5647; and Doridoni, A,; Marra, A.; assi, A. J. Org. C m. 1997, 62, 6261) 2.0 μΐ, (1.5 rag; 12 μηιοΐ} ofDlPEA and 3.5 mg (9.3 umol) of HATU. The reaction mixture was stirred at room temperature for 1 h. The reaction was purified on an Econosil reversed phase semi-preparative (250 χ 10 mm, 10 μητ) SIPLC column using 0.1% aq TFA and CH3CN mobile phases, A linear gradient was employed (99: 1. 0.1 % aq TFA-C¾CN -> 45:55 0.1% aq TFA- C¾CN) o ver a period of 30 min at a flow rate of 3 mL/min, Fractions containing the desired product eiuted at 20.7 min (monitoring ai 292 am) and were collected, frozen, and lyophiii ed to give APA-BLM-disaccharide conjugate 5 as a yellow solid: yield 2,2 mg (53%); mass spectrum (ESI), m/z 894.3853 (M ÷ Ef
(C,¾H*5 90i4 requires m/z 894.3840).
|0J 93j Example 2: Synthesis of MTX-di-ester-BLM-disaecbaride (15).
(01941 Scheme 2,
Figure imgf000050_0001
MTX-d^este(-8L -d!S;ac;ohar(de (15) j.0195] Synthesis of the MTX-sugar conjugate 15 started with the attachment of linker 7 with glutamic acid. Frooc-G1u(/m-butyl)OH (9) was treated with trifiuoroacetic acid to deproteci the /ert-buty! ester and the resulting diacid was condensed with compound 7 to afford compound 11. The Fmoc group was removed by the use of piperidine in DMF. Compound 12 was then condensed with AFA (2) to provide the MTX derivative 13 equipped, with two linkers. Finally the iert-buty\ esters were removed by the use of trifiuoroacetic acid and the resulting intermediate was coupled with the bleomycin disaccharide linker 3 to afford the MTX conjugate I S in 30% yield.
BuOOC
FmocHN . QO
Figure imgf000050_0002
{0196| Fmoc- !ti-tli -terth ittyl ester (11). A solution containing 43 mg
{0.10 ntmol) of compound 9 in 1 m.L of 1 : 1 TPA-CI LO? was shaken at room temperature for 1 and concentrated under diminished pressure. The residue was co-evaporated with five 2-mL portions of toluene and dissolved m 1.5 mL of anhydrous€!-¾€¾. To this solution was added 75 mg (0.40 mmol) of alcohol 10, 6.1 mg (0.50 mmol) ofDMAP and 82 rag (0.40 mmol) of DCC and the reaction mixture was stirred at room temperature for 48 h„ The reaction mixture was concentrated under diminished. ressure and the residue was suspended in 5 ml . of acetonitri!e. The suspension was filtered and the filtrate was concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (18 2 cm). Elution with 4:1 faexanes-ethyi acetate gave compound 1.1. as a colorless oil: yield 25 mg (35%); silica gel TLC Rf 0.76 (1: 1 hexanes-ethyl acetate); ¾ NMR (CDCb) δ 1.41 (m, 24H), 1.98 (m, 2H), 2.20 (m, 6H), 2.40 (m, 3H), 3.64 (t, 1H, J - 6.4 Hz), 4.14 (m, 6H), 4.39 (m, 3H), 5.51 (d, IH, </ = 8.1 Hz), 7.31 (t, 2H, J - 7.4 Hz), 7.39 (t, 2H, J~ 7.4 Hz), 7.59 (d, 2H, J= 3.7 Hz) and 7.76 id, 2H, J ~ 7.5 Hz); mass spectrum (MALDt), m/z 732.47 (M + Na
(CsoHsiNOioNa requires m/z 732.3 ).
Figure imgf000051_0001
(01971 MTX-di-fcr/hut l ester (13). To a solution of 25 mg (35 ηιοΐ) of compound 11 in 0,5 mL of anhydrous DMF was added 0.1 mL of piperidine and the solution was stirred at room temperature for 1 h. The reaction mixture was concentrated under diminished pressure. The residue was purified by flash
chromatography on a silica gel column (5 χ 3 cm). Elation with 4: 1 hexanes-ethyl acetate- 100% ethyl acetate gave compound 12 as a colorless oil: yield 12 mg (70%); mass spectrum (MALD1), m/z 510.36 CM + Na)';' (CjsHtsNOs arequires m/z 510.30). To a solution containing 10 mg (20 pmol) of the amine 12, 5 mg (15 μηιοΐ) of A? A (2) and 25 pL (18 mg, 0.14 mmol) o f Df P EA in 0.25 mL of anhydrous DMSO was added 30 mg (79 uraol) of HATU and the reaction mixture was stirred at room temperature for 48 h. The reaciion mixture was filtered and the filtrate was purified on a C m reversed phase serai-preparative (250 * 10 mm, 10 ura) HPLC column using 0.1% aq. TFA and C¾CN mobile phases. A linear gradient was employed (99: 1 0.1 % aq TFA- ¼CN-» 1 :99 0.1 % aq TFA~CH.CN) over a period of 30 min with a flow rate of 3 mL/rom. Fractions containing the desired product eluted at 20.1 min (monitoring at 292 nm) and were collected, frozen, and lyophilized to give 13 as an orange solid: yield 2.5 mg (20%); mass spectrum (MALDf), m 795.29 (M + H) G«H»NaO» requires m/z 795.44), m/z 817.28 (M + Naf (C^Hss sO^ a requires m z 817.42).
Figure imgf000052_0001
{0198} TX-di-ester-BLM-disaccharide (15). A solution containing 1 mg ( 1 .2 pmol) of compound 13 in 1 : 1 TFA-CHCI.2 was shaken at room temperature for 1 h and concentrated under diminished pressure. The residue was co- evaporated with five 2-raL portions of toluene and dissolved in 0.1 mL of anhydrous D SO. To this solution was added 2,5 mg (5.2 umoi) of 34, 20 pL (15 rag, 0.1 Ϊ mniol) of DIPEA and 15 mg (39 praol) of HATU and the reaction mixture was stirred at room temperature for 24 h. The reaction was purified on an Econosil Os reversed phase semi-preparative (250 10 mm, if) pm) HPLC column using 0.1% aq. TEA and CH3C mobile phases. A linear gradient was employed (99: 1 0.1% aq. TFA~CH3CN-»45:55 0.1% aq. TFA --CH.5CN) over a period of 30 min with a flow rate of 3 mL'tnia. Fractions containing the desired product eluted at 22. 1 min (monitoring at 292 nm) and were collected, frozen, and lyophilized to give MTX-di-ester-BLM-disaccharide conjugate 15 as a yellow solid: yield 0.6 mg (30%); mass spectrum ( ALDI), m/z 1 1 3.29 (M + H)+
Figure imgf000052_0002
|01 j Example 3: Synthesis of gulose acceptor 23.
Figure imgf000053_0001
{0201] 2^,4,5-l)i- - opropylidene-L-xylose Diethyl Dithioacetal (16). To a suspension of 8,00 g (53,3 mmoi) of L-xylose in 3.2 mL of cone HCi was added, with vigorous magnetic stirring, 1 1.8 mL (10.1 g, 160 mmol) of etlianetfaiol. Stirring was continued at room temperature until the two layer mixture gave a homogenous solution (usually afier 15-20 mm) which was then dilirted with 1 0 mL of acetone. After stirring for 5 h, the solution was neutralized with satd aq NH OH .solution and co-evaporated with six 20-mL portions of toloene to afford a crude residue. The residue was applied to a silica gel column (28 x 5 cm). EHuion with 1 : 1 ethyl aceiaie-hexanes gave 16 as a colorless syrup: yield 13,4 g (75%);
[«3D * 57.2 (<■' L8 6¾), lit. cf]D + 51 .3 c 1.8, C<¾>; silica gei TLC ¾■ 0.59 (3: 1 ethyl acetate-iiexaries); Ή NMR (CDC 3) 0 1.23-1.28 (m, 611), 1 .36 (s, 3H), 1 .41 (s, 6H), 1 .45 (s, 3H), 2.68-2.77 (m, 4H), 3.91 (dd, 2H, J - 9.8 and 4.5 Hz), 4.02- 4.06 (m, 1H), 4.13 (dd, 1 E, J ::: 5.3 and 2.1 Hz) and 4.31-4.34 (m, 2H>; C NMR (CDC ) 5 14.26, 14.34, 24.9, 25.3, 25.6, 26.1 , 27.1. 27.3, 53.0, 65.9, 75.2, 78.7, 80.1. 109.5 and Π 0.0.
Figure imgf000054_0001
(02O2| 2,3,4,5-l>i-0-isopr py deti -ii i?/i}Y o-L-xyiose (17). To a stirred solution containing 2.60 g (7.70 mmol) of thioacetal 16 in 26 mL of acetone diluted with 2.6 roL of water was added 3,80 g (17,7 mmol) of yellow mercur (H> oxide and 3.80 g ( 1 3.9 mmol) of mercuric(O) chloride. The reaction mixture was stirred at 55 *C for 2 h and then allowed to cool to room temperature. The solvent, was filtered through a pad ofCelite 545* and concentrated under diminished pressure to afford a crude residue. The residue was suspended in three 3 -niL portions of dicMoromethane and filtered through a pad ofCelite 545*:. The organic layer was washed with 40 mL of I aq Ki, dried (MgSC and then concentrated under diminished pressure to afford the crude aldehyde 17. The aldehyde was used for the nest reaction immediately.
Figure imgf000054_0002
{0203] 2-(TriraethyisiJyl)t a¾t)le (18). A 500-mL, four-necked, round- bottomed flask, containing a magnetic stirring bar, was equipped with two 1 0- raL, pressure-equalizing dropping funnels and a low-temperature thermometer. The anh apparatus was filled with argon and kept under a slightly positive pressure during the entire reaction. The flask was charged with 80 mL of freshly distilled Et jO and 42 mL (67 mmol) of a 1.6 M solution of »-BuLi in hexane. One of the two dropping funnels was charged with 5.5 mL (10 g, 6 mmol) of 2- bromoihiazo!e in 20 ml, of EfcO and the other with 7.7 ml (6.6 g, 61 mmol) of ch!orotrimethylsilane in 20 mL of EtjO. The reaction flask was cooled to -78 in an acetone bath. While ihe solution in the flask was stirred, 2~bromot azole was added dropwise over a period of I h. After 20 mm of additional stirring, ehiorotrimethylsilane was added dropwise over 30 min and the stirring was continued for a period of i h at -78 "C. The resulting mixture was then allowed to wa m up to room temperature. A satd aq aHCO? was added and the mixture was transferred into a i L separatory funnel. The organic layer was recovered and the aqueous layer was extracted with two 200-raL portions of E¾0. The combined organic layer was dried (NajSO ), filtered, and concentrated under diminished pressure with the external bath iemperature not exceeding 40 °C. The residue was distilled from a 1 -mL flask at diminished pressure in a Classen apparatus. The distillation was carried out under diminished pressure at 45 'r'C after a forerun at 25 °C consisting mainly of bromobutane was collected. The pure product. 18 was isolated as a colorless oil: yield 7,3 g (76%); SH NMR (CDC ) § 0.39 ($, I2H), 7.50 (111 d? J■■■■■■ 3.0 Hz) and 8.09 (111 d, J === 2.9 Hz); !¾ NMR. (C£X¾) O 1.03, 127.3, 145.6 and 174,2.
Figure imgf000055_0001
To a stirred solution containing 2,22 g (9,65 mmol) of crude aldehyde 17 in 38 mL ofanh dichloromethane cooled to -20 °C was added 2,00 mL (i .97 gs 12,5 mmol.) of 2-(trimethylsilyl)thiazole (18) dropwise over a period of 15 min. The solution was stirred at 0 °C for I h and then concentrated under diminished pressure to afford a crude residue. The residue was dissolved in 38 ml, of nh THF and treated with 3,0 g (9.65 mmol) of ??-Bu NF«3H20 at 20 °C for 30 min and then concentrated under diminished pressure. The residue was diluted by the addition of 250 mL of dichloromelharie. The organic layer was washed with three 50-mL portions of water, dried (NajS04) and then concentrated under diminished pressure to yield compound 19 as a crude residue. Recrystailizaiion of the residue from cyclohexane afforded alcohol 1 as a colorless crystalline solid: yield 1.94 g (64% over two steps); [a}0+ 18.2 (c I A, CHC ), lit. [afo÷ 18.5 (<? 1.1, CHC¾); silic gel TLC ¾0.49 (1:1 ethyl acetate-hexanes); ¾ M'R (CDC¾) 31.28 is, 3H), 1.36 (s, 6H), 1.40 (s, 3H), 3.67 (i, IH, J = 6.6 Hz), 3.79-3.84 (m, 2H), 4.12 (dd, 1H,J = 7.2 and 3.6 Hz), 4.31-4.34 (m, IH), 4.56 (brs, 1H), 5.10(4 IH,J=5.5Hz), 7.30 (d, 1 H, J■■■■■■ 3.2 Hz) and 7.71 (d. III ./=== 3.2 Hz); nC NMR (CDC¾) δ 25.6, 26.1, 27.07, 27.1.3, 65.7, 71..7, 75.5, 77.4, 79.8, 1.09.5, 1.1.0.2, 119.7, 142.1 and 170.9.
Figure imgf000056_0001
|0205] l,2,3,4- s-0-(l-Methy^
t iV3Eolyl-{S$)-B-xylitol (20), To a solution containing 1.94 g (6,15 mmol) of alcohol 1 in anh MF cooled to 0 °C was added 0.49 g (60% dispersion in oil, 1.2,3 mmol) ofNaR poriionwise and the reaction mixture was stirred at 0 °C for 0.5 h. To this solution was then added 1.10 mL ( 1 ,58 g, 9.20 mmol) of benzyl bromide and the reaction mixture was stirred at room temperature for 0.5 fa. The reaction mixture was quenched by the addition of 1.2 mL of methanol, stirred for 10 min and then diluted with 40 mL of distilled water. The aqueous layer was extracted with three 100-roL portions of ether. The combined organic layer was dried (MgSO,}) and concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 * 4 cm). Elation with 6:1 ethyl acetaie-hexanes gave ether 20 as a colorless solid: yield 2.26 g (91%); [a]D .--32.2 (c ΪΛ, CHC¾), hi jojn -32.3 (e 1.1, CHC¾); silica gel TLC /?f0.36 (9:1 toluene-met anol); lH NMR (CDCI3) 31.20 (s, 3H), 1.25 (s, 3H), 1.29 (s, 3H), I.33(s, 3H), 3,62-3.68 i'ro, IH), 3.75-3.8 (m.111).3.89-3.93 (m, IH), 3.96- 3.99 (m, IH), 4.35 (dd, 1H,J= 7.3 and 2,5 Hz), 4.44 id, 1H,J= 12.1 Hz), 4.63 (d, IH, ,/~ 12.1 Hz), 4.80 (d, IH, .7-4.8 Hz), 7.21-7,28 (m, 5H), 7.32 (d, 1H,J= 3.2 Hz) and 7.78 (d, 1H,J - 3.2 Hz); !3C NMR. (CDC¾> δ 14.0, 25.5, 26.03, 26.05, 26.7, 27,0, 65.5, 72.2, 75.5, 77,7, 78.5, 79.4, 109.4, 110,3, 120,1, 127,9, 128.1, 128.3, 136.8, 142.4 and 168.9.
Figure imgf000057_0001
{0206J Z'^Bemy ^^^^^O'koprop Udm^ld kyd& .^iost (21). A solution containing 0.61 g ( 1.50 mrnol) of O-benzyl ether 2Θ nd 2.80 g of activated 4A molecular sieves dissolved in 15 mL of anh acetonitrile was stirred at 20 °C for 10 mint and then 0,22 mL (329 mg, 1.95 mmol) of methyl inflate was added drop wise. The suspension was stirred at room temperature for 15 mm and then concentrated under diminished pressure to afford the crude Λ- methyUhia¾oHum salt. To a stirred solution of the erode N-roethylthiazoliura salt in 15 mL of methanol cooled to 0 °C was added 0. 12 g (3,30 mmol) of sodium borohydride. The reaction mixture was stirred at room temperature for 5 min and diluted with 5 mL of acetone. Th solvent was filtered through a pad of Celite 34S¾' and concentrated under diminished pressure to afford a crude mixture of thiazolidines. This was dissolved in 14 mL of acetonitriie and 1.4 mL of water and treated under vigorous stirring with 0.96 g (12.0 mmol) of CuO and 0.26 g (1.50 mmol) of CuC *2HL>Q. The reaction mixture was stirred at 20 :'C for 15 min, filtered through a pad of Celite 545* and then concentrated under diminished pressure to remove acetonitrile and most of the water (bath temperature not exceeding 40 ~'C) to afford a crude residue. The brown residue was triturated with four 50-mL portions of ether and the liquid phase was pipetted and filtered through a pad of Florist!*' (60-100 mesh) to afford a colorless solution. After a further washing of Fiorisii* with 50 mL of ethyl acetate, the combined organic layer was concentrated under diminished pressure to yield the crude aldehyde 21 as a brown syrup, which was used immediatel for the next reaction.
Figure imgf000057_0002
02071 l^ ^Yetra-O-a ^t l-l^^enzj'l-i^ ttlo yranose (22). A solution containing 470 mg (.! .34 mmoi) of the crude aldehyde 21 was dissolved in 7.4 mL of glacial acetic acid and 1. mL of distilled water and stirred at 100 for 40 rain. The reaction mixture was then concentrated by co-evaporation three times with toluene to afford the crude 2-O-benzyl-L-gulose as a mixture of β-pyranose, a~ pyranose and furanose forms. A solution of the erode residue and 0. 16 g (1.34 mmoi) of DMAP in 3.4 mL of pyridine and 3.4 mL of acetic anhydride was stirred at 20 °C .for 12 h and concentrated under diminished pressure to yield a brown syrup. The crude residue was applied to a silica gel column (38 x 3 cm). E! tion with 3: 1 ethyl acetate-hexanes gave 22 as a yellow oil: yield 1.56 g (64% over two steps); silica gel TLC /?f 0.44 (1 : 1 ethyl acetate-hexanes); SH NMR. (CDCb) 52.01 (s, 3H), 2.05 (s, 3H), 2.08 (s, 311), 2.1 1 Css 3H)S 3.64 (dd, I IL J - 8,3 and 4.9 Hz), 3.98-4.13 (m, 2H), 4.24-4.32 (m, IH), 4.49 (d, 1 H, J ~ 1 1 .9 Hz), 4.63 (d, l.H, ,/ = 1 1,9 Hz), 4.95 (dd, IH, ,/ = 3.9 and 2.5 Hz), 5.43-5.45 (m, 1 H), 5.89 (d, IH, J -8.3 Hz) and 7.23-7.34 Cm, 5H),
Figure imgf000058_0001
|0208j L3?4-6-Tt*trii- ~iicetyl-L- alopyra}u>se (23). To a solution containing 1,47 g (3.35 mmoi) of 22 hi 23 mL of ethyl acetate was added 0.73 g of 10% Pd/C and the reaction mixture was stirred overnight under 1 atm of ¾. The solvent was filtered through a pad of Celite 545*' and concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (15 χ 4 cm). Eiution with 1 : 1 ethyl acetate-hexanes afforded 23 as a 77:20:3 mixture of ct- pyrattose, β-pyranose and furanose forras as determined by 5H NMR: yield 1.02 g (87%); silica gel TLC % 0.52 (ethyl acetate); !H M (CtX¾) 6 1.91 (s, 3H), 2.00 (s, 3H), 2.03 (s, 6H), 3.22-3.52 (br s, 1 H), 3.80 (dd, IH, J = 8.4 and 3.5 Hz), 3.91-3.97 Cm, 1 H), 3.99-4.04 (m, H), 4.14-4.19 (m, I H), 4.82-4.88 (in, IH), 5.19 (U I H, J™ 3.6 Hz) and 5.70 (d, IH, J ~ 8.4 Hz); !3C NMR (CDCh) 5 20.4, 20.5, 20.6, 20.8, 61 .6, 66.2, 67.5, 69.5, 70.9, 92.1, 169.4, 1.69.6, 169.7 and 170.5. 1 091 Exam le 4: Synthesis of mannose donor 12.
Figure imgf000059_0001
27
Figure imgf000059_0002
0211 j Methyl ,6^B ti2^i deae»2* «i> nz ]« X»i »inattitopyraito$ide (24), To a solution containing 5.00 g (26.0 mmol) of methyl a-D-mannopyranoside and 60.0 mg (0.26 mmol) of camphorsulfonic acid in 75 mL of DMF was added dropwise 9.7 mL (9.8 g, 65 mmol) of benzaldebyde dimethyl acetal. The resulting solution was heated to 60 CC on a rotary evaporator under a pressure of 250 mbar. After
3 h, analysis by silica gel TLC ( 1 ;3 ethyl acetate-hexanes) indicated complete conversion of starting material (Rt~ 0,0) to two products ih 0.50 and 0,80). To the reaction mixture was then added 4.90 mL (4.90 g, 32,4 mmol) of henzaklehyde dimethyl acetal and 30.0 mg (0.13 mmol) of camphorsulfonic acid. The reaction mixture was stirred under diminished pressure. After 2 h, silica gel TLC ( 1 ;3 ethyl acetate -hexanes) indicated the formation of a single product (A 0.80). The solvent was concentrated under diminished pressure, the residue was co-evaporated with 50 mL of toluene and then dissolved in 100 mL of dichioromethane. The organic layer was washed with 50 mL of sat aq NaHCO? and brine. The organic phase was then dried { gS Xi), filtered and concentrated under diminished pressure. The resulting crude mixture of endo and exo dibenzylidene derivatives was dissolved in 1 0 mL of freshly distilled toluene and cooled to -40 °C under an argon atmosphere. Then 65 ml. of DISAL (1 M. solution ίη toluene, 64.9 mmol) was added slowly to the reaction mixture. The reaction mixture was allowed to warm to room temperature slowly. After 2 h, silica gel TLC analysis (1 :3 ethyl acetate- hexanes) indicaied complete consumption of starting material (¾- 0.80) and formation of two products ¾ 0.40 and ¾- 0.30). The reaction 'mixture was quenched by the dropwise addition of 50 ml., of methanol and the mixture was diluted with 250 mL of dichloromethane. The organic layer was washed with 200 mL of 10% solution of Rochelle's salt and brine and then dried ( gSCV}. The organic layer was filtered and the filtrate was concentrated trader diminished pressure. The resul ing residue was purified by flash column chrom tography (1 :3 ethyl acetate-hexanes) to afford the xmdesired compound methyl 4,6-0- benzy}idene-3- ~beRzyl-fi-.D-mannopyranoside (R( 0.30) and the desired methyl 4/!- -benzylid.ene-2-0-benzyl-a-D-mannopyranoside (24) as a colorless crystalline solid: yield 3.0 g (41 %.); silica gel TLC #f 0,40 (1 :3 ethyl acetate- hexanes); ' H NMR. (CDCis δ 3,34 (s, 3H), 3.79-3.82 (ra, 3H), 3.96 (t, 1 H, J = 8.0 Hz), 4.10-4.12 (m, 1H), 4.26-4.27 (ra, ! H), 4.72-4.75 (ra, 3H), 5.53 (s, ΪΗ), 7.33- 7.41 (m, mi) and 7,42-7.55 (ra, 2H).
Figure imgf000060_0001
[02121 i,3, ,6-Tetr-t~ ~-u:et l-2-0^enz ^^ (25). To a solution containing 3.57 g (9.5 mmol) of acetal 24 in 70 mL of Ac-jO was added, a catalytic amount of H2SG4 and the reaction mixture was stirred at 25 °C for 40 tnin. The reaction mixture was poured into a stirring mixture of 100 mL of ethyl acetate and 80 mL of said aq NaHC<¾. The organic and aqueous layers were separated and the organic layer was washed with 60 mL of brine and dried (M'gS04). The organic layer was filtered and concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column. ( 7 x 5 cm). Ehition with 2: 1 ethyl acetate-hexanes afforded 25 as a yellow oil: yield 3.35 g (80%); silica gel TLC i¾- 0.66 (1 : 1 ethyl acetate-hexanes); ! B NMR (CDClj) δ 1 .98 (s, 3HX 2.04 (s, 3H)S 2.0S (s, 3H), 2, 12 ($, 3H), 3,82 (dd, 1H, J= 3.2 and 2.2 Hz), 4.01 (ddd, 1 H, ::: 10.0, 4.8 and 2,3 Hz), 4.08-4. 15 (rn, I B), 4,23- 4.28 (m, 1H.K 4.56-4.76 (m, 2H), 5.19 (dd, 1 H, J- 1 .0 and 3.3 Hz), 5.43-5.52 (m, J.H), 6J 8 (d, IH, ,/ - 1.9 Hz) and 7.27-7.38 (m, 5H) i?C NMR (CDCb) 5 20.8, 20.90, 20.93, 21.1 , 62.4, 66.0, 70.7, 71.1 , 73.0, 74.0, 13, 128.1 , 128.2, 128.6, 337.3, 168.8, 169.6, 170.4 and 170.9,
Figure imgf000061_0001
[02131 3,4,6-Tri-O-acety i-2-0-beaz.y!-a-D-i»annopyranoside (26). To a solution containin 1.13 g (2.58 mmol) of compound 25 in 21 nit of anh DMF was added 286 rng (3 JO mmol) of hydrazine acetate. The reaction mixture was stirred at room temperature for ί .5 h and quenched, by the addition of 1 0 m'L of ethyl acetate. The organic layer was washed with three 50-mL portions of brine and dried (MgS(¾). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 χ 3 cm). Eltttion with 1 :2 ethyl acetate-hexanes afforded pyranoside 26 as a colorless oil: yield 793 mg (73%); silica gel TLC R{ (),23 (1 : 1 ethyl acetate -hexanes); 5H NMR (CDCh) δ 1.97 is, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 3.81 -3.87 (m, 1 H), 4.05-4.17 (m, 2H), 4.20 (dt, 1H, 9.3 and 4.7 Hz), 4.56-4.63 (m, 3H), 5.21-5.33 fm, 2¾ 5.40 (l, 1H, J = 9.9 Hz) and. 7.21-7.36 (m, 5H); ¾ NMR iCDCh) δ 20.57, 20,58, 20.7, 62.7, 66.6, 68.2, 70.9, 72,8, 75,6, 92.2, 127.70, 127.72, 128.2, 137.6, 169.8, 170.2 and .1 1 .1 ; mass spectrum (APCI), m/z 397.1498 (M - M (Ci.jHjjOy requires 397.1498).
Figure imgf000061_0002
[0214] S^^TH^acety l^ eoz l^D^miittjt-ep ranes l Diphenyl
Phosphate (27). To a stirred solution containing 793 mg (2.00 mmol) of 26 in
1.20 mL of anh dic loromethane was added 305 mg (2.50 mmol) of 4- dimethylaminopyridine (DMAP), 3.00 ml (2. i 7 g, 21.6 mmol) of EtjN and 4.00 mL (5,20 g, 1.9.2 mmol) of diphenyl chlorophosphate. The reaction mixiure was stirred at 0 X for 2 and poured into a stirring mixture of 300 mL of ethyl acetate and 150 mL of satd aq NaHCC . The aqueous and organic layers were separated and the organic layer was washed with three 50»mL portions of water and brine and then dried (MgSO ). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 χ 3 cm). Elutioa with 1 :2 ethyl acetate-fiexanes afforded 27 as a colorless oil: yield 508 rag (40%); silica gel TLC ¾· 0.44 (1 : 1 ethyl acetate- hexanes);'H NMR CDC ) 5 2. 17 (s, 3H), 2.20 is, 3H), 2.23 is, 3H), 4.10-4.25 (m, 3H), 4.42 (dd, Ϊ Η, J = 12.2 and 3.9 Hz), 4.76-4.88 (ni, 2H), 5.49 (d, IB, J = 8.0 Hz), 5.73 it, i ll J- 10.1 Hz), 6.21 (d, I H, J - 5.7 Hz) and 7.33-7.62 (m, 15H); C NMR (CDCb) δ 20.39, 20.46, 20.53, 61.7, 65.3, 69,8, 70.8, 73.1 , 74.4, 96.6, 1 19.9, 120.05, 120.09, 1.20.14, 124.59, 125.63, 127.8, 127.9, 128.3, 129.3, 129.8, 136.8, 149.9, 150.1, 150.8, 169,3, 169,8 and 170,53: mass spectrum (APCi), m/z 629, 1788 (M -f Hf
Figure imgf000062_0001
requires 629, .1 88).
[0215] Example 5: Synthesis of raannose donor 32.
|02I 6| Scheme 5
Figure imgf000062_0002
m-■ D-methyiffla nnopyranosKfe 28 29
Figure imgf000062_0003
30 31 32
Figure imgf000062_0004
[0217] Meth M,6- '-beiia^iiden«>a-D--maiinop>rranoside (28), To a solution, containing 7.00 g (36.0 mrnol) of a-D-mannopyranoside in 85 mL of DMF was added 5,60 mL (5.68 g, 37.3 mmoi) of ben/aldehyde dimethyl acetal and a catalytic amount of p-TsOH. The reaction mixture was stirred at 60 ''"C under diminished pressure for 1 Si, allowed to cool to room temperature and then poured into a stirriag mixture of 120 mL of ethyl acetate and 1 0 mL satd a NaHCOs. The organic layer was washed with three 50-mL portions of brine and dried (Mg$i¾}. The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (30 x 5 cm). Ehition with 4: 1 ethyl acetaie-hexai .es afforded aceta! 28 as a colorless solid: yield 7.1 g (70%); silica gel TLC ¾ 0,31 (.1 : 1 ethyl acetate-hexanes); SH NMR (CDCk) δ 3.38 (s, 3H), 3.78 (m, 2HX 3.87 (m, I H), 3.98 (m, 2H), 4.25 (m, 1HX 4.72 (d, Hi), 5.55 (s, IH), 7.36 (m, 3H) and 7,47 (m, 2H); UC NMR (CDCIj) 5 55.2, 63.3, 68.8, 69.0, 71.1 , 79.0, 101.6, 102.4 129.5 and 137,4.
Figure imgf000063_0001
(02181 Methyl 4,6- -Ben/:yijdeiie-3- -i>t i¾yl-«-l>-manRopyranostde (29). To a solution containing 2.00 g (7..1 mmol) of aceiai 28 in 60 mL of methanol was added 1.94 g (7.79 mmol) of B¾SnO, The solution was heated to reflux for 1.5 h affording a clear solution. The solvent was concentrated under diminished pressure and the resulting solid was dried under vacuum overnight:. The white residue was dissolved m 6 mL of DMF and treated with i .69 mL (2.43 g. 14.2 mmol) of benzyl bromide and then warmed to 100 *C for 30 rain. The cooled reaction mixture was poured into a stirred mixture of 90 mL of ethyl acetate and 60 mL of said aq NaHCCh. The organic layer was separated and washed with 60 mL of brine and dried (MgSO*). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (30 χ 5 cm). Elutioii with 3:7 ethyl acetate- hexartes afforded acetai 29 as a colorless oil: yield 1.93 g (73%); silica gel TLC ¾ 0,30 (3:7 ethyl acetate-hexanes); Ή NMR.
(CDCh) o 3,38 (s, 3H), 3.77 (m, 3H), 4.05 (m, 2H), 4.27 (m, IH), 4,70 (m, 2H), 4.84 (HI IH), 5.62 (s, I H) and 7.28-7.52 (ra, 10H); ¾ NMR (CDC ) δ 55.2, 60.7, 63.5, 65.4, 69.1 , 70.1 , 73.2, 75.8, 79.0, 101.3, i0L8, 126.3, 127.2, 527.8, 128.1 1 , .128.1 , 1 28.5, 128.7, 129.2, 137.8 and 138.2.
Figure imgf000063_0002
1.0219] l,2,4,,6-Teira-0-aeei>i^ (30). To a solution containing 1 ,93 g (4.40 mmol) of acetal 29 in 30 mL of Ac¾0 was added a catalytic amount of H2SO4 and the solution was stirred at room temperature for 40 rain. The reaction mixture was quenched by the addition of 120 mL of ethyl acetate and 80 mL of said aq NaHCOj. The organic and aqueous layers were separated and the organic layer was washed with brine and dried (MgStXs). The solvent was concentrated under diminished pressure to afford a erode residue.. The residue was applied to a silica gel column (30 χ 5 cm). Elation with 2: 1 ethyl acetate-hexanes afforded pyranoside 30 as a yellow oil: yield 1.94 g (85%); silica gel TLC ¾ 0.34 (3:7 ethyl acetate-hexanes); ] H NM (CDCU) δ 2.02 (s, 3H), 2.07 (s, 3H), 2.11 (s, 3H), 2.15 (s, 3H), 3.83 (dd, IH, J - 9.7 and 3.4 Hz), 3.90 <m5 1 H), 4.04 (m, IH), 4.1.9 i m, 1 Π). 4.41 (ro, 1H), 4.64 (m, 11 I K 5.24 On, I H), 5.34 (dd, H i. ./ 3.4 and 2.1 Hz), 6.09 (d, IH, J = 2.0 Hz) and 7.24-7,37 (m, 5H); ¾ NMR (C'DC ) δ 14.4, 20.98, 21 ,08, 21.13, 62.6, 67.0, 67.2, 71 ,0, 71.7, 74.3, 91.2, 128.0, 128.2, 128.6,
137.6, 168.3, 169.8, 170.2 and .171.0.
Figure imgf000064_0001
[02201 2,4,6-Tri-i -a€eiyl-3- -bt^i¾yl-« >-inaniu)pyranosyi Diphenvl Phosphate (32). To a solution containing 1 ,40 g (3, 1 mmo!) of acetate 30 in 25 mL of DMF was added 353 mg (3.83 mmol) of hydrazine acetate. The solution was stirred at room temperature for 1,5 h and quenched by the addition of .1 0 mL of ethyl acetate. The organic phase was wa ed xvith three 50-m.L portions of brine and dried (MgSQ . The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 * 4 cm). Elation with 1 :2 ethyl acetate- hexanes afforded monosaccharide 31 as a colorless oil This material was used tor the next reaction immediately: yield 968 mg (76%); fH NMR (CDC¾) 0 1 ,95 (s, 3H), 2.02 (s, 3H), 2.10 (s, 3H), 3,90 (dd, I H, J:::: 9.7 and 3,3 Hz), 4.00-4.1 1 (m. 2H), 4.16 (ddd, IH, J~ 12.3, 7.7 and 4.6 Hz), 4.33 (s, IH), 4.38 (dd, 1 H, J= 12.3 and 4.3 Hz), 4.60 (d, I H, J= 12.2 Hz), 5.1 -5.23 (ro, 2H), 5,28-5.33 (m, IH) and 7.18-7.31 (m, 5H); C NMR (CDC ) 0 14.2, 20.78, 20.85, 21 .0, 60.6, 62,9, 67.5, 68.5, 68.8, 71.4, 74.0, 92.3, 1 27.78, 127.83, 128.4,
137.7, 169.9, 170.6 and .171.1. |0221) To a stirred solution containing 968 mg (2.44 mmol) of pymnoside 3J in 144 mL of anh dichtoromethane was added 372 mg (3.05 mmol) of D.MAP, 3.67 mL (2.66 g, 26.3 mmol) of EfcN and 4.83 mL (6.26 g, 23.4 ramol) of diphenyl chlorophosphate. The reaction mixture was stirred at 0 aC for 2 h and poured into a mixture of 300 mL of ethyl acetate and 150 mL of sa d aq NaHCO;;. The organic layer was washed with three 50-mL portions of water and brine and then dried (MgS04). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica ge! column (20 * 4 cm). Elution with .1:2 ethyl acet.aie~.hexan.es afforded 32 as a colorless oil: yield 737 mg (48%); silica gel TLC ¾ 0.38 (1 : ϊ ethyl acetate-hexanes); ¾ NMR (CDC¾) ø 1.93 (s, 3H), 1.97 (s, 3H), 2.10 (s, 3H), 3.84 (dd, 1H, J = 9.7 and 3.3 Hz,), 3,89-4.03 (m, 2H), 4.10-4.20 (m, 1 HL 4.33 (d, iH, ,/- 12.1 Hz), 4.57 (ds 1H, J:::: 12.1 Hz), 5.27 (I, Hi, J'~ 10.0 Hz), 5.38 (dd, IH, J=~ 8. and 6.2 Hz), 5.91 (dd. Hi J ~ 6.4 and 1.6 Hz) and 7.16-7.38 (m, I 5H); ] SC NMR (CDC¾) δ 20.5, 20.62, 20.67, 61.8, 66.2, 67.2, 67.3, 70.9, 71 ,5, 73.4, 77,4, 96.5, 1 9.90, 1 19.95, 125,67, 125.71, 127,9, 128.3, 129.85, 137.2. 150.08, 150.15, 169.3, 169.6 and 170.4; mass spectrum (APCI), m/z 629.1770 (M + H (CsjiLwOnP requires 629.1788).
{02221 Example 6: Synthesis of mannose donor 35.
{02231 Scheme 6
Figure imgf000065_0001
a-O-methyimannopyfaiiceiije 28 33
Figure imgf000065_0002
34 35
Figure imgf000065_0003
|0224j i,2,3»6-Tetra- ~i^^^^^ (33). To a stirred solution containing 5.43 g (19.2 mmol) of acetal 28 in 50 mi, ofanh THF was added 58.0 niL (57.6 raraol) of a 1 M solution of'BHs in THF and 7.48 g (57.6 .mmol) of anh CoC at roo.ro temperature. The reaction mixture was stirred for 1 5 rain at room temperature and quenched by the addition of 100 mL of ethyl acetate. The organic phase was filtered and the filtrate was treated with 20 ml, of 20% aq solution ofNaBRj. The solution was again filtered and washed successively with sat aq NaHCOj and water, and then dried (MgSO.f). The solution was concentrated under diminished pressure to afford a erode residue.. To a soiution containing 3.44 g (12.1 mmol) of the crude residue in 85 mL of Ac20 was added a catalytic amount of H2SQ . The soiution was stirred at room temperature for 1.2 h. The reaction mixture was quenched by the addition of 120 mL of ethyl acetate and 80 ml, of satd. aq NaHCCK. The organic and aqueous layers were separated and the organic layer was washed with brine and dried (MgSGi). The soiution was conceiitrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column. (30 χ 5 cm). Elation with 2:1 ethyl aceiate~.hexan.es afforded pyranoside 33 as a yellow oil: yield 1.17 g (22% over two steps); silica gel TLC & 0.26 (2: 1 ethyl acetate -- hexaaes); Ή NMR iCDC ) δ 2.00 (s, 3H), 2.08 (s, 3H), 2.13 (s, 3H), 2, 15 is, 3H) 3,87 (t 1 H, ,/ = 9:7), 3.99 (di, HI J- 9.9 and 3.4 I¾ 4.32 (d, 2H, J ==== 3.5 Hz), 4,59 (d, Hi J- 1 1.2 Hz), 4,70 (d, I B, J- 10.8 Hz), 5.26 (dd, 1H, J- 3.3 and 2.1 Hz), 5,37 (dd, Hi J- 9.5 and 3.4 Hz), 6.04 (i, 1 H, J - 6.1 Hz), and 7,24-7.38 (m, 5H); ¾ NMR (CDC ) δ 20.92, 20,97, 20.99, 21.04, 62.9, 8.9, 71.6, 71 ,8, 72.6, 75.2, 90.8, 1.27,9, 128.3, 128.7, 137.5, 168.4, 169,8, 169.9 and 170.8.
Figure imgf000066_0001
|u225| 2,3 -Tri- -a er> -4-0-benzyl-a,p-D-niannopyran{»se (34), To a stirred soiution containing 1.09 g (2.49 mmol) of acetate 33 in 20 mL of anfa DMF was added 274 mg (2.98 mmol) of hydrazine acetate. The reaction mixture was stirred at room temperature for 1.5 h and quenched by the addition of 100 mL of ethyl acetate. The organic layer was washed with three 50-mL portions of brine and dried (MgS<¾). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 x 3 cm).
Elation with 1 :2 ethyl acetate-hexanes afforded pyranoside 34 as a colorless oil: yield 884 mg (90%); silica gel TLC ¾ 0.36 (1 : 1 ethyl acmte~h.exan.es); SH NMR (CD<¾) 6 1.92 (s, 3H), 2.0 ! (s, 3H), 2.08 (s, 3H), 3.77 (t, I H, J = 1 0.0 Hz), 4. 1 1 (ddd, ill, J :::: 7, 4.1 and 2.1 Hz), 4 J 7-4,34 (m, 2H), 4.69-4.48 (m, 3H), 5.09 (ss Hi), 5. 17-5.23 (m, IH). 5.33-5.38 (m, i l) and 7. 1 8-7.32 (m, SH); ! 3C NMR (CDCU) δ 20.69, 2073, 63. J , 69.2, 70.5, 71.5, 72.8, 74.6, 77.4, 91 .8, 127.6, 127.8, 128.3, 137.5, 170.0, 1 70.2 and 171.0; HRMS (AFtl), m z 397. 1483 (M ÷ Hf (CJ HSSOP requires nth 397. 1498),
Figure imgf000067_0001
{02261 2,3,6-Tri-O-acetyM- -benzyl-a-0-inan»opyra»osyl Oiphenyl Phosphate (35). To a stirred solution eomakring 812 trig (2.05 mraol of 34 in 80 mL of anh dichloromethane was added 3.13 mg (2.56 mmol) of DMAP and 3.10 mL (2.25 g, 22.1 mmol) of Et;;N, 4.10 mL (5.33 g, 1 .7 mmol) of diphenyl ehlorophosphate. The reaction mixture was stirred at 0 "C for 2 fa and then poured into a mixture of 300 mL of ethyl acetate and 150 mL of said aq NaHCOj. The aqueous and organic layers were separated and the organic layer was washed with three 50-roL portions of disti lled water and brine and then dried (MgSO-j). he solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column. (20 x 4 cm), .Elution with 1 :2 ethyl acetate-hexanes afforded 35 as a colorless oil: yield 857 mg (66%); silica gel TLC ftf 0.29 (1 :1 ethyl acetaie-hexanes); *H NMR (CDC%) 3 1.93 (s, 3H); 1.96 (s, 3H), 2.09 (s, 3H), 3.80 (i, I H, 9.6 }¾ 3,91 -4.12 (ra, 2H), 4.18 (dd, IH, 12.2 and 4.2 Hz), 4.50-4.68 (m, 2H)« 5.27-5.38 (m, 2H), 5.80 (d, 1H, J = 6.1 Hz) and 7.1 1- 7.38 (m, 1 5H); l3C NMR (CDC ) δ 2074, 20.9, 62.4, 69.1 , 70.9, 71.8, 72. L 75.0, 77.4, 96.3, 120.1 , 120.4, 1257, 125.9, 127.9, 128.2, 128,6, 129.9, 130.0, 137.3, 150. 1 , 150.3, 169.5, 169.6 and 170.5; HRMS (APCI), m/z 629.1794 <M +
(C3 {HM032.P requires m/z 629.1788).
[0227] Example 7: Synthesis of niannose donor 40.
[02281 Scheme 7
Figure imgf000068_0001
38 3B
BySra ine acetate
DMP. r.l ftnff
(quant ) n^Noc Y 3AC {¾¾J H ?*iOeO'sv' y"' OAt
OAc
3S
Figure imgf000068_0002
[0229] ,2 i6-Tetra~ ~-u>etyl-o-I)-maniM>pyraiu>skie (36). To a solution of 3 .63 g (3.72 ramol) of 33 in 33 mL of ethyl acetate was added a 308 rag of
Pd(OH)j/C and {lie reaction was placed under 1 aim of lh overnight The catalyst was removed by filtration through a pad of Ce!iie 545* and concenirated under diminished pressure. The residue was purified by flas chromatography on a silica gel column (5 x 20 cm). Elution with 2: 1 hexanes-ethyl acetate afforded 36 as a colorless oil: yield 1 .09 g (84%); silica gel TLC 0.25 (1 : 1 ethyl acetate- hexanes). JH NMR (CDCb) δ 2.06 (s, 3H), 2.12 (s, 6H), 2.14 (s, 3H), 2.94 (br s, IH), 3,83-3.92 Cm, 2H), 4.24-4.27 (m, IH), 4.50-4.54 (m, 1H), 5. 18-5.23 (m, 2H), 6.04 (d, IH, J= 1.6 Hz).
Figure imgf000068_0003
mannopyranoside (37). To a solution of 1 ,74 g (5,00 mmol) of 36 in 17.8 mL of pyridine was added 2,44 g (20.0 mmol) of DMAP and 4.03 g (20.0 mmol) of p- rritropheiiyl chloroformate. The reaction was stirred at 40 <!C for 2.5 b at which time it was poured into a two phase mixture of 50 rat ethyl acetate and 1 mL of water. The organic layer was washed with three 25-m'L portions of I HO, 5 mL of satd aq. NaHC<½ and 25 mL of brine. The solution was dried (MgSO*) and concentrated under diminished pressure to afford a crude residue. The residue was purified by flash chromatography on a silica gel column (5 x 28 cm). Ehition with T.2 ethyl acetate -hexanes afforded 37 as a white foam: yield L 1 g (74%); siika gel TLC / 0.21 (2: 1 ethyl aeetate-hexanes). Ή NMR (CDC¾) δ 2.05 (s, 3H), 2 J 1 (s, 3H), 2.18 (s, 3H), 2.19 (s, 3H), 4.15-4.20 (m, 2H), 4.53-4.58 (m, 1H}( 5.23 (t, lH, J= 9.9 Hz), 5.32-5.33 (m, IH), 5.45 (d.d, iH, J = 10.1 , 3.5 H¾), 6.12 (d, IH, ./ - 1.9 Hz), 7.38 id. 211, J - 9.2 Hz), 8.29 (d, 2H, J - 8.8 Hz); "C NMR (CDCT) δ 20.66, 20.69, 20.8, 61.6, 68.3, 68.6, 70.1 70.9, 0.4, 121 ,6, 125.4, 145.7, 151.7, 155.1 , 1 7.9. 169.5, 169.8, 170.6..
Figure imgf000069_0001
(02311 l,2^,&~Tetra~0-acet !~ -O-(c^^
(38). To a solution of 2.02 g (3.93 mmol) of 37 hi 107 mL of dichloromeihane was added a solution of 37 mL of a h THF satd with. Hj at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred at room temperature overnight. The solution was concentrated under diminished pressure and the residue was purified by flash chromatography on a siiica gel column (3 x 15 cm). Eiution with 1 : 1 ethyl acetate- hexanes afforded 38 as a white foam; yield 1.22 g (87%); silica gei TLC R, 0. I 2 (1 : 1 hexanes-ethyl acetate). *H NMR (CDCL) 2.03 (s, 3H), 2.09 (s, 3H), 2.16 (s, 3H), 2.17 (s, 3H), 4.00-4.05 (m, Hi), 4.15*4.19 (m, I H), 4.26-4.31 (m, IH), 4.73 (br s, 2H), 5.19 (t, 1 Η, ,/ = 10. i Hz), 5.24-5.25 (n% 1 H), 5.34-5.37 (m, IH), 6.07 (d, 1 H, ~ 1.9 Hz); ¾ NMR (CDCU) B 20.68, 20.72, 20.76, 20.85, 62.3, 66.7, 68.4, 68.6, 70.7, 90.6, 154.9, 168.0, 169.8, 170.1 , 170.7; mass spectrum (APCi), miz 392.1203 (M + H)* (C15.H22NO1 t requires mk
392.1 193).
Figure imgf000070_0001
[0232 j 2,3.6- ri-<.>-actnyl-4- -(carbafn{)yloxy>-«~l)~inatu}opyranosidc 09), To a solution containing 553 rag (1.41 mmoi) of 38 in.9.20 mL of anh DMF was added 182 nag (1.98 mmo!) of hydrazine acetate. The solution was stirred at 25 for 2 h and then treated with 120 mL of ethyl acetate. The organic solution was washed with \ 20 nxL of water, 120 ml of said aq. NaHCO,?, 120 mL of brine, and then dried (MgSO^), The solvent was concentrated under diminished pressure to afford 39 as a white foam: yield 501 mg (quant.,); silica gel TLC R, 0.28 ( 1 :3 hexanes-ethyl acetate). *H NMR (CDCI3) 5 2.00 (s, 3Hk 2.08 is. Mil 2.14 (8, 3H), 4.17-4.24 (m, 3H), 4.58-4.64 (br s, 1H), 5.10 (t, 1.H, 9.6 Hz:), 5.07-5.1 5 (br s, 2H), 5.20-5.22 (m, 2H), 5.37-5.41 (m, IE),
Figure imgf000070_0002
{0233] 2 ,6-Tri- -acetyM- -(carbaitt«y!o^
Diphenyl Phosphate (40). To a solution of 496 mg (1.42 mraol.) of 39 in 29 mL of dichloromethane at 0 °C was added 217 rag ( i .78 mmol) of ΌΜΑΡ, 2.1 mL 05.0 mraol) of Et*N, and 2.8 mL f 13.6 ramol) of diphenyl phosphoryl chloride under an argon atmosphere. The reaction mixture was stirred for 1 .5 h and the solution was poured into a two phase mixture of 43 mL of ethyl acetate and 20 mL of said aq NaHCCh. The organic layer was washed with two 20-raL portions of brine, dried (MgSCXi) and concentrated under diminished pressure to afford a crude residue. The residue was purified by flash chromatography on a silica gei column (3 x 20 cm). Elution with 2:3 hexanes-eth l acetate afforded 40 as a colorless oil: yield 460 mg (56%); silica gel TLC R 0.33 (1 :3 hexanes-ethyl. acetate). 3H NMR
(CDClj) δ 2.06 (s, 3HX 2.12 (s, 3H), 2.24 (s, 3H), 4, 15-4.19 (ra, 1H), 4.28-4.32 (m. !H), 4,37-4.41 Cm, 1H), 4.80 (s, ! H), 4.82-4.90 (br s, 2H), 5.21-5.30 (m, 1 H), 5.41- 5.50 (m, IB), 5,95-5.97 (m, H), 7.24-736 (m, 6H), 7.44-7.48 (m, 4H): L,C NMR (400 MHz, CIX¾) δ 20,56, 20.62, 20.7, 61.9, 66.3, 68,0, 68.7, 68.8, 70,8, 96,0, 120.01. 120.05, 120.16, 120.21 , 125.7, 125.85, 125.86, 129.93, 129.99, 169.6, 169.9, 170,6; mass spectrum (APCI), m/z 582.1387 (M + H)* (C25¾ Qt;;P requires m/z 582, 1377),
1.0234] Example 8: Synthesis .f altrose donor 32*
(02351 Scheme 8
Figure imgf000071_0001
u-D-mettsylgkicopyrarsoside 41 42
B
Figure imgf000071_0002
|0236| Mi hyl-4,6- -beBaEySi(Je«e-«-I -g!«copyraiiosiile (41). To a solution containing 1 .0 g (51 ,5 mmoi) of a-D-raethyl glueopyranoside in 200 mL of acetonitriie was added 14.0 mL (14.2 g, 92.7 tnmo!) of benxaldehyde dimethyl acetai and 600 rag (2,57 mmoi) of camphor si Ionic acid. The reaction mixture was heated to reflux for 20 mm and then allowed to cool to room temperature and neutralized by the addition of 400 uJL of triethySamine. The reaction mixture vvas diluted with 800 mL of ethyl acetate. The organic layer was washed with three 250-mL portions of water and dried (MgSQ ). The organic laye was concentrated under diminished pressure to afford a crude residue. The residue was crystallized from 1 :7 dichloroniethane-hexanes to afford acetai 41 as a colorless solid: yield. 9.48 g (65%); silica gel TLC Rf 0.1 (2: i ethyl acetate-hexanes); fH NMR (CDC ) 5 3.45-3.47 (m, 4H), 3.63 (dd, IH, J" 9.1 and 3.9 Hz), 3.71-3.85 (m5 2H), 3.93 (t, 1H, J '= 9.2 .z), 4.29 (dd, 1H, J= 9.7 and 4.3 Hz), 4.80 (d, JH, = 3.9 Hz), 3.53 (s, I H) aad 7.33-7.53 (m, 5H); :¾ NMR (CDCfe) δ 55.7, 62.5, 69. L 72.0, 73.0, 81.0, 99.9, 102.1 , 126.4, 137.2.
Figure imgf000072_0001
{ 0237) Methyl 2,3-Aii iytlro-4,6- -beiizyi-a~D-Hiaiiiiopyrau side (42). To a solution containing 2,44 a (60% m oil dispersion, 60.9 mmol) of Nail in 290 mL of anh DMF at 0 °C was added 8.20 (29.0 mmol) of aeetaS 41 under an argon, atmosphere. The reaction mixiure as stirred at room iemperature for 0.5 h. To the above stirred solution at 0 UC was then added 7.10 g (31 .9 mmol) of<¥- tosy [imidazole. The suspension was stirred at room temperature for 1 a. The reaction mixture was poured with stirring into 2.5 L of ice - cold water and the resulting solid was filtered and washed with water to afford a crude residue. The residue so obtained was triturated with methanol to obtain the epoxide 42 as a colorless solid: yield 1.83 g (24%); silica gel TLC ¾ 0.68 (1 : 1 ethyl
acetate-hexanes); Ή NMR (CDC ) d 3.17 (d, 1H, J- 3.6 Hz), 3.45-3.49 (m, 4H), 3.64-3.79 (m, 3H), 4.21 -4.32 (m, ΓΗ), 4.91 is, IH), 5.57 (s, 1H), 7.35-7.53 (m, 5H); BC NMR (CD(¾) δ 50.7, 54.0, 55.9, 61.8, 69.6, 75.0, 97.0, 102.6, 126.3, 128.5, 129.4 and 137.2.
Figure imgf000072_0002
{0238} Methyl 4?6" ~Benzylide] e^ ^>b BzyJ*a<<I>-anropyrano$ de (43), A solution containing 214 rag (9.32 mmol) of sodium metal i 2.9 mL of anh benzyl alcohol was heated (·-- ! 00 "C) until all of the sodium metal bad dissolved. The cooled solution was treated with, i .07 g (4.05 mmol) of anhydromannopyranoside 42. The reaction mixiure was then heated, to reflux for 1 mm, cooled and diluted by the addition of 20 mL of ether. The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 x 5 cm). Elution with 1 ;4 ethyl aceiaie-hexanes afforded aceta! 43 as a colorless solid: yield 723 mg (48%); silica gel TLC R/H550: i ethyl
acetate-hexanes); }H NMR (CDC15) δ 2.30 ( M), 3.42 < , 3H), 3.77 (t, 1 H, J = 10.3 Hz), 3.84 (t, 1H, J= 2.8 Hz), 3.93 (& 1H, J= 2.8 Hz), 3.98 (dtJH, J- 9.3 and 4.6 Hz), 4.28-4.45 (m, 2H). 4.55 (d, I H, J = 6.0 Hz), 4.70-4.90 (m, 2H), 5.56 (s, Hi) and 7.23-7.53 (m, ί OH); C NMR. (CDCI3) 5 55.8, 58.7, 69.4, 70.2, 72.9, 74.9, 77.2, 102.0, 102.4, 126.3, 127.5, 1.27.7, 128.30, 128.36, 129.1 , 137.7 and
338.7.
Figure imgf000073_0001
|0239] Methyl~3~ ~beiixyl-H~I)-alirepyraRostdi' (44). To a solution containing
1.67 g (4.48 mmoi) of aeetal 43 in 4.2 mL of methanol was added 43.0 mg (0.22 mmoi) of Holuenesulfonic acid monohydrate at 0 °C, The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was quenched by the additiosi of ί .90 mL ( .38 g, 1.3.4 mmoi) of triethylamine and concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (10 x 3 cm), Elution with 5: 1 ethyl acetate -hexaiies afforded methyl pynmoside 44 as a colorless oil: yield 1.22 g (96%); silica gel
TLC ¾ 0.17 {ethyl acetate); Ή NMR (CD<¾) 0 3.01 (d, I H, J - 9.3 Hz), 3.33 is. 3H), 3.53 (d, 1H, J:::: 15.3 Hz), 3.70-3.77 (m, 2H), 3.80 (dt, 2H, J -»- 8.8 and 4.3 Hz), 3.96 (s, 2H), 4.40-4.78 (m, 4H) and 7.21 -7.35 (m, 5H); i NMR fCDCl. 5 55.5, 61.9, 63.4, 67.3, 69.2, 127.9, 128.0, 128.5 and 138.0.
Figure imgf000073_0002
|024Of l,2 ,6-Tetra- -ace¾ -3-0-benzyl-a*p-D-altroj3yranosi(le (45), To a solution containing 532 mg (.1.87 mmoi) of meth l pyranoside 44 in 13 ml, of A jO was added a catalytic amount of H SO4. The solution was stirred overnight at room temperature. The reaction mixture was then poured into a stirred mixture of 120 mL of ethyl acetate and 80 mL of satd aq NaHCOs. The organic and aqueous layers were separated and the organic layer was washed with brine and dried (MgS<¾). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (30 χ 3 era).
Elution with 1 :2 ethyl acetate-hexanes afforded the product 45 as 3:2 mixture of a and p anomers as determined by ¾ NMR; yield 705 mg (86%); silica gel TLC 1¾/0.5S (1 :1 ethyl acetate-hexanes): a anonier f H NMR (CDC S δ 2.01 (s, 3H), 2.06-2.09 (m, 6H), 2.1.4 (s, 3H), 3.96 (t, I H, J = 3.2 Hz), 4.1 1-4.16 (m, I H), 4.24- 4.37 (HI 2H), 4.55-4.75 (m, 2H), 5.03-5.09 (ra, IH), 5.29 (s, IH), 5.99 (d, IH, ,/ === 1 1.3 Hz) and 7.27-7.38 (m, 5H); UC NMR (CDCJ*) δ 20.9 L 20.92, 21.04, 21.05, 62.6, 66.3, 66.6, 68.0, 72.46, 72.49, 91.4, 1.27.8, 128.1, 128.5, 137.5, .169.0, 169.7, 1 9.8 and. Ϊ 70.9; HRMS (APCi), m/z 379, 1387 (M - CH OOV (CAOs requires m/z 379.1393).
Figure imgf000074_0001
{0241J 2 ,6-Tri-0-acetyM-0-benzyl-a,p-B-altropyraaositIe (46). To a solution containing 1.93 g (4,40 mraol) of monosaccharide 45 in 35 mL of anh DMF was added 486 mg (5.28 mmol) of hydrazine acetate. The reaction mixture was stirred at room temperature for 1.5 h and quenched by the addition of 100 mL of ethyl acetate. The organic layer was then washed with three 50-mL portions of brine and dried (MgSQt). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 x 4 cm). Elution with 1 :2 ethyl acetate-hexanes afforded 46 as a colorless oil. The product was isolated as a mixture of anomers as analyzed by ¾ NMR; yield 837 rag (48%); silica gel TLC i?f 0. (1 :l ethyl acetate-hexanes); !H NMR CDClj) § 1.95 (s, 3H), L96 (s, 3H), 2.05 (s, 3H), 2.07 is, 3H), 2.1 1 (s, 3H), 2.15 (s, 3H), 3.73-3.95 (br s, I H), 3.98-4.05 (m, IH), 4.09 (d, Ul, J === 8.6 Hz), 4.12-4.27 (m, 4H), 4.32 (dt5 IH, / - 14.2 and 7 J Hz), 4.36-4,46 (m, IH). 4.54-4.75 (m, 4H), 4.89-4.94 (m, 2H), 4.96-5.08 (m, 4H), 5.24 (t, 1H, J= 12.1 Hz) and 7.41 -7.27 (m, l OH); s¾ NMR (CDC¾) δ 20.80, 20.82, 20.86, 20.98, 21.02, 62.9, 63.2, 64.1, 66.2, 66.9, 68.3, 70.0, 70.3, 72.9, 73.3, 73.8, 74,2, 91.6, 92.8, 1.28..1, 128.2, .128.4, 128.5. 128.7, 128.8, 136.2, 137.3, 169.73, 169.78, 169.83, 170.4, 170.95 and .170.96; HRMS (A.PCI), m/z 379.1394 (M - OH) " (Cis>H 5Og requires m z
379.1393).
Figure imgf000075_0001
{0242 j Z^^Tri^ -ae i^f^iM!nz l^f attropyraiiosyi Diphenyl
Phosphate (47). To a stirred solution containing 637 nig (1.61 mraol) of pyranoside 36 m 2.7 mL ofanh dichloromeihane was added 1.21 mL (1.6 M, 1.93 mol) of rt-BuLi solution at -78 °C. The reaction mixture was stirred at this temperature for 1 mm and 40 μΐ. (520 mg, 1 .93 mmol) of diphenyl
chlorophosphate was added dropwise. The reaction mixture was stirred at -78 °C for an additional 10 min aod then poured into a mixture of 20 mL of ethyl acetate and 10 mL of said aq NaHCO;?. The organic and aqueous layers were separated and the organic layer was washed with three 10-mL portions of water and brine and then dried (MgS<¼). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 x 3 cm). Elation wit 1 :2 ethyl aeetaie-hexanes afforded phosphate ester 47 as a colorless oil: yield 324 mg (32%); 121 mg ofunreacted starting material was also recovered; silica gel TLC _¾ 0.40 (1 : 1 ethyl acetate-hexanes); ¾ NMR <CDC¾) δ 1.97 fs, 3H), 1 .98 (s, 3H), 2.00 (d, 3H, J --- 2.1 Hz), 3.99 (dd, 1H, J- 6.3 and J Hz), 4.05- 4.28 (m, 3H), 4.50-4.62 (ni, 2H), 5.13 (dd, 1 H, J - 7.0 and 3.2 Hz), 5.19 (dd, IE, J = 6.4 and 2.2 Hz), 5.96 (dd, 1 R /» 7.1 and 2.2 Hz) and 7.12-7.36 (m, 15H); BC NMR (CDC¾) δ 20.74, 20.76, 20.9, 62.8, 66.9, 68.20, 6S.28, 71.6, 72.94, 72.97, 95.5, 120.30, 120.35, .125.7, 128.0, 128.2, 128.5, 129.8, 129.9, 137.1 , 150.2, 150.4, 169.9 and 170.6; HRMS (APCl), m/z 569.1598 (M - CH3COO)* (ΟΛΑο requires m/z 569, 1576). |0243) Example 9: Synthesis of C2 modified maimose dlsaceharkk-iinkers 57 and 58c
[0244 S Scheme
Figure imgf000076_0001
Figure imgf000076_0002
¾ i- )-:
se
Figure imgf000076_0003
10245) 1 A6- etra-0^cetyl~2^ 3A^iri^acet l«2-C^I e»2yl^^
HUinnopyraiii Sx J-fi-L-guiopyranose (48), To a stirred solution containing 234 mg (0.67 mmo.l) of glycosyl acceptor 23 and.508 mg ( 1. i 7 mmol) of g!ycosyl donor 27 in 4.8 niL of anh dichloromethane at 0 °C, was added 244 (300 mg, 1.35 mmol) of TMSOTf, The reaction mixture was stirred at 0 for 10 min at which time it was poured into a two phase mixture of 30 mL of ethyl acetate and 30 mL of said aq affCO?. The organic and aqueous layers were separated and the organic layer was washed with two 20-mL portions of brine and dried (MgSO ). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (30 y- 3 cm). Elation with 2: 1 ethyl acetate hexanes afforded compound 48 as a colorless oil: yield 302 nig (62%); silica gel TLC Hf 0.2 (1 ; 1 ethyl acetate-hexanes); Ή NM (CDC%) δ 1.84 {s, 3H>, 1.94 (s, 3H), 1.99 (s, 3H), 2.04 (s, 3H), 2.08 (s, 3H), 2.09 (in, 6H), 3.51-3.61 (m, 1M), 3.87-4.23 im, 5H), 4.31 (t, 1H, J === 6.3 Hz,), 4.44-4.47 (m, I H), 4.56-4.69 (m, IH), 4.80-4.97 Cm, 2H), 5.02-5.07 (m, 2H), 5.27-5.47 (m, 2H), 5.78 (d, IH, 8.5 Hz,) and 7. 16-7.36 (ro, 5H); lsC NMR (CDCH) S 20, 1 , 20.63, 20,66, 20.67, 20.69, 20.72, 61.3, 2.2, 65.3, 65.7, 66,0, 67,7, 68.8, 69.2, 70.4, 1 ,3, 72.2, 73.9, 90.6, 94.2, 127.7, 128.1, 128.2, 137.6, 168.7, 169.36, 169.37, 169.4, 170.0, 170.3 and 170.6; mass spectrum (APC1), m/z 727,2453 (M ÷ H) ' (C.;.;¾Ots requires 727.2450).
Figure imgf000077_0001
(02461 A^1.etra-f cetyl-2- 3A^H^-acetyi-2-O »- iti^o h ny i^r ameylha^D-maiiao yraaosyl^p-L-^ilopyranose (49). To a solution containing 200 mg (0.27 mrool) of disaccharide 48 in 38 mL of ethyl acetate was added a catal tic amount of PdCOH /C and the reaction mixture was stirred overnight under 1 aim of JT. The solvent was filtered through a pad of Ce!ite 545* and the filtrate was concentrated, under diminished pressure to afford a crude residue. The residue was used for the next reaction; silica gel TLC R? 0.08 ( J. ; S ethyl acetate-- hexanes).
[0247| To a solution, containing 1 8 mg (0.31 raraol) of the crude residue in 1.2 mL of anh pyridine was added .151 mg ( 1 .24 mmo!) of DMAP and 276 mg (.1.24 mmol) of ?-mtrophe.nyl chloroforraate. The reaction mixture was stirred at 40 °C overnight at which time it was poured into a mix toe of 30 mL ethyl acetate and 10 mL of M30, The organic and aqueous layers were separated and the organic layer was washed with three iO-mL portions of 1 N ECl and 10 mL of said aq NaHCOs and then brine. The solution was dried (MgSO*) and filtered and the filtrate was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 3 cm). Eluiion with 1 : 1 ethyl aeetate-bexanes afforded 4 as a colorless foam: yield 211 mg (96% over two sieps); silica gel TLC R{ 0,30 (I : I. ethyl aceiate-hexanes); !H NMR <CDC%) 51.98 (m, m\ 2,03 (s, 6H), 2.10 (s, 3H), 2.12 (s, 3H), 2.14 (s, 3H), 2.17 (s, 3H), 3.96- 4.1 (m, 2H), 4.19-4.29 (ffi, 2K), 4.35 (t, IH, J = 6.5 Hz), 4.96-5.03 (m, 2H), 5.06- 5.23 (m, 311), 5.27-5.40 (m, 215), 5.44 (t, 111 3.0 Hz), 5.88 (d, IH, J:::: 8.4 Hz), 7.39 (d, 2H, J ^ 8.0 Hz) and 8.26 (d, 211 J~ 9.1 .Hz); {3C NMR (CDC ) δ 20.70, 20.72, 20.75, 20.76, 20.9, 61.3, 62.0, 65.5, 65.7, 67.8, 68.8, 69.4, 70.1 , 7 .4, 73.5, 90.6, 94.5, 121.7, 25.4, 145.6, 149.8, 151.6, 155.3, 168.7, 169.3, 169.5, 169.7, 1 9.7, 170.5 and 170.6; HRMS (APCI), m/z 802.2053 (M + B)' (C33H40NO22 requires m/z 802.2042).
Figure imgf000078_0001
|024S| 13,4i6-Teira- -ac tyi-2-<: -f3,4,6-tri~^acety!-2-0-(c
manaopyranosyl^p-L-giiiopyranose (SO).T'o a solution containing 94 mg (0.12 mmol) of 49 irs .0 niL of dicbloromethane was added 2.2 nil, of THF saturated with ¾. The reaction .mixture was stirred at room temperature for 3 h. The solvent was concentrated under diminished pressure to afford a crude residue. The residue was purified by flash chromatography on a siiica gei column (2.5 x .15 era). Elution with. 3:1 ethyl acetale-hexan.es afforded 50 as a white foam: yield 73 mg (92%); silica gei TLC K{ 0.13 (3:1 ethyl acetate-hexanes). Ή NMR
(CDCls) δ 1.98 (s, 3H), 2.02 (s, 3H), 2.04 (s, 3H); 2. 1 (s, 3H), 2.13 (s, 3H1 2.14 (s, 3H), 2.18 (s, 3H), 3.98 (dd, 1H, J- 8.4, 3.3 Hz), 4.06-4.1 1 (in, 2H), 4.14-445 (m, 1H), 4. 1.7-4.19 (m, IH), 4.22-4.27 (m, 1H), 4.33-4.37 (m, IH), 4.85 (br s, 2B), 4.95-4.97 (m, I H), 5.00-5.02 (m, 2H), 5.08-5.14 (m, IH), 5.22-5.27 (m, IH), 5.44 (t, IH, J~ 3.6 Hz), 5.89 (d,lH, J - 8.4 Hz); "C NMR. (CEX¾) 0 20.62, 20.65, 20.68, 20,71 , 20.82, 61.35, 62.1.1, 65.59, , 65,74, 67.59, 68.88, 69, 10, 69.43, 69.79, 71.30, 90.67, 95.29, , 154.88, 168,68, 169.21 , 169.64,1 9.87, 170,43, 170.53; mass spectrum (APCI), ml∑ 680.2026 (M ÷ VL
Figure imgf000079_0001
requires mlz 680.2038).
Figure imgf000079_0002
{02491 !^A^Te i¾-0-acet>!l-2-<M3A6-trR>-a€etyI-2-i -
(meih lcarbam yl)-o-D-mani )pvran s> l)-p-l. -gulopyi ano.se (51 ). To a solution containing 201 rag (0.25 mmol) of nitrophenyl. ester 49 in. 6 mh of aah THF was added dropwise at 0 °C I 25 pL (2 M solutio in THF5 0.25 mraol) of CH3 H2. The reaction mixture was stirred at room temperature for 15 h at which time silica gel TLC analysis indicated thai the reaction was complete. The solvent was concentrated under dinvinished pressure to afford a crude residue. The residue was applied to a silica gel column (25 ' 3 cm). Eiution with 1:1 ethyl
acetate-hexanes afforded disaccharide 51 as a colorless oil: yield 134 mg (77%); silica gel TLC 0.14 (1 : 1 ethyl acetate-hexanes); !H MR. (CDCb) δ 1.94 (s, 3H), 1.98-2.15 (m, 18H), 2.75 (d, 3H, J ~ 3.7 Hz), 3,93-4.13 (m, 4H), 4.18-4.22 (m, 2H), 4.30-4.33 im, IE), 4.87-5.10 (m, 4H), 5.17-5.21 (m, 2H) and 5.33 <m, 2H); nC NMR (CDC ) 5 20.62, 20.63, 20.68, 20.72, 20.75, 20,77, 20.85, 27.6, 61 .4, 62.0, 65.9, 67.6, 68.0, 70.5, 71.4, 90.7, 93.2, 155.38, 155.40, 1 55.49, 169.24, 169.27, 169.30, 170.50, 170.51 , 170.6 and 170.9; HRMS (APCI), m/z 694.2169 (M + H}' (C2sH4o O{9 requires m/z 694.21 5).
Figure imgf000079_0003
|025O| 3, ,6-Tfi-^aeet l-2- ^3^^
manuopyrano.?yt)~p ^gulopyra«osyl Dfphenyi Phosphate (52). To a solution containing 66 nig (0.10 mmoi) of disaccharide SO in 1.0 ml. of anh DMF was added .13.0 mg (0.14 ramol) ofhydraxine acetate. The reaction mixture was stirred at room temperature for 3 Si and quenched by the addition of 14 mL of ethyl acetate. The organic solution was washed with 12 raL of water, 12 mL of said aq. NaHCO¾, I 2 raL of brine and then dried (MgSO^. The solvent was concentrated under diminished pressure to afford the crude product as a light yellow oil: yield 56 mg (90%); silica gei TLC R? 0,23 (1 :4 hexanes-ethyl acetaie). MALD1, m/z 660.18 for (M Na)'". The residue was used for next reaction.
[0251. J To a stirred solution containing 56.0 mg (0.09 mmoi) of the crude residue in 3.30 mL of anh dichioromethane was added 13,0 mg (0.1 1 mmoi) of DMAP, 133 μΐ, (96 mg, 0.95 mmoi) of Et3 and 176 L (229 mg, 0.85 mmoi) of diphenyi chlorophosphate. The reaction mixture was stirred at 0 °C for 2 h and poured into a mixture of 5 mL of ethyl acetate and 5 mL of satd aq NaHCO;?. The organic layer was washed with three 10-mL portions of water and brine and then dried ( gSO.j). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 x 2 cm). Elation with 2: 1 ethyl acetate-hexanes afforded phosphate ester 52 as a colorless oil: yield 36 mg (47% over two steps); silica gel TLC R/0.18 (2; 1 ethyl acetate-hexanes); lH NMR (CDOb) δ 1.77 (s, 3H), 2.03 ($, 3H); 2.05 (s, 3H), 2.13 (s, 3H), 2.21 (s, 3H), 2.27 (s, 3H), 4.03-4.10 (m, 2B1 4.14-4.21 (m, 2H), 4.24-4.28 (m, I ff), 4,36-4.42 (m, 2H), 4.87 (hr s, 2H), 5.05-5.10 (m, 3H), 5.24-5.27 (m, I H), 5.29-5.35 (m, 1H), 5.51 -5,53 (m, IH), 5.75-5.79 (ms I H), 7.22-7,28 (m, 2H), 7.32-7.43 (m, 8H). iSC NMR (CDCIj) δ 20.3, 20,63. 20.64, 20.68, 20.71 , 61.2, 61 .8, 65,3, 65.6, 67.4, 69.0, 69.1 , 69.5, 71.1. 71.2, 71 .6, 95.6, 96.17, 96.22, 120.19, 120.24, 125.57, 125.71 , 125.70, 129.6, 129.9, 150.05, 150.10, 154,9, 169.2, 169.6, 169.7, 170.4, 170,6; mass spectrum (APCI), mlz 870.2224 (M + Η (C37H45 NQ21P requires mlz 870,2222),
Figure imgf000081_0001
|0252| 3,4,6~Tri- -aceiyl-2- -(3,4,6^^^
l>-iiia«iiopyraiiosyl)-p-L~giiiopyra«osy! Diphenyl Phosphate (53). To a solution containing 108 mg (0.16 nimol} of disaccharide 51 in 1.2 ml, of anh D F was added 1.7.0 nig (0.1.9 mroo'f) of hydrazine acetate. The reaction mixture was stirred at room temperature for 1.5 h and quenched by the addition of 20 mh of ethyl acetate. The organic solution was washed with three I 0-mL portions of brine and dried (MgSOj). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was used for the next reaction.
[0253 j To a stirred solution containing 90,0 mg (0.14 mmol) of the crude residue in 8.2 roL of anh dichloromethane was added 2.1.0 mg (0.17 mmol) of D AP, 210 pL (1.52 mg, 1.49 mmol) of E¾N and 270 uL (351 mg, 1.32 mmol) of diphen l chlorophosphate. The reaction mixture was stirred at 0 °C for 2 h and poured into a mixture of 40 ml, of ethyl acetate and 20 niL of said aq NaHCOj. The organic layer was washed with three 10~mL portions of water and brine and then dried (MgSQ*). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 χ 3 era). Eiution with 2:1 ethyl acelate-hexanes afforded phosphate ester 53 as a colorless oil; yield 82 mg (56% over two steps); silica gel TLC R 0.1.8 (2:1. ethyl acetate-hexanes); !H NMR (CDC ) δ L67 (s, 3H), 1.94 (d, 6H, J- 7. Hz), 2.01 (s, 3H), 2.1 1 is, 3H), 2. J 6 (s, 3H), 2.76 (s, 3H), 3.89-4.39 (m, 7H), 4.75~5.05(m, 4H), 5.10-5.30 (m, 2H), 5.44 (s, 1 H), 5.68 (s, lH) and 7.1 1-7.39 (m, 0H); nC NMR (CDC¾) δ 20.4, 20.70, 20.76, 20.8, 20.9, 27.7, 61.2, 62.0, 65.5, 65.8, 67.5, 69.1, 69.3, 69.4, 71.4, 71.5, 71.7, 95.9, 96.34, 120.31 , 120.33, 125.6, 125.72, 125.78, 125.83, 129.7, 1.30.0, 155.4, 169.3, 169.7, 169.8, 170.4, 170.67 and 170.68; HRMS (APCI), m/z 884,2371 (M -f Hf (C^H^NO^ requires m/z 884.2378).
Figure imgf000082_0001
[02541 Benzyl 2 ~(2~Hy drox v eth ox v )et h vlca rbam ate (54). To a solution containing 1.01 g (9.61 mmol) of 2-(2-aniinoethoxy)ethanol in 1 0 mL of THF at room temperature was added 1.34 mL (9.61 mmol) of EtjN and 1.49 mL (1.78 g, 10.6 nunol) of CB Ci. The reaction mixture was stirred for 1 h and was then diluted with 250 mL of ethyl acetate. The organic layer was washed with two 250- mL portions of¾0, two 250-mL portions of brine, and was then dried (MgSi¾} and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (30 *· 4 cm). Elution with 9:1 ethyl acetate hexaues afforded alcohol 54 as a colorless oil: yield 2.21 g (96%); silica gel TLC / 0.30 (9: 1 ethyl acetate--hexaaes); SH NMR (CDCK) 3.30 (m, 2H), 3.45 On, 4H), 3.52 (s, 1H), 3.62 (m, 2H), 5.03 (s, 2H), 5.86 (m, 1H) and 7.27 (m, 5H); t3C NMR (CDCfe) 40.5, 61 .1 , 66.3, 69.7, 72.0, 127.72; 127.75, 128.1 , 1 36.3 and 156.5.
Figure imgf000082_0002
(02551 3 ,6-Tri- -acetyI-2- -(3^6-tri- -acei> -2- -(carbantoyI}-«-D- mannopyranosyl)-tt,p~L-gulopyranosyl Benzyl 2~(2-Eihoxy)ethyleaf hamate (55). To a stiffed solution containing 31.0 mg (0.04 mmol) of phosphate ester 52 in 3.9 mL of anh dichlorometh.ane was added a solution of 9.40 mg (0.04 mmol) of CBz linker 54 in 4.5 mL of anh dichlororaethane at 0 °C. To the cooled reaction mixture was then added 41 ,0 uL (51 ,0 mg, 0,23 mmol) of TMSOTf and the reaction mixture was stirred at 0 °C fo 15 min at which time it was poured into a mixture of 20 mL of ethyl acetate and 20 mL of said aq aHCO;;. The aqueous and organic layers were separated and the organic layer was washed with three 10-mL portions of water and brine, and then dried. (MgSO*). The solvent was concentrated under diminished pressure to afford a erode residue. The residue was applied to a silica gel column (12 x 2 cm). Elution with 3: 1 ethyl acetate~-hexan.es afforded disaechaiide-Uiiker conjugate 55 as a colorless oil: yield 12 nig (39 %): silica gel TLC % 0.12 (3: 1 ethyi acetate-hexanes); fH NMR. (CDCi3) 5 1.99 (s, 3H), 2.02 (s, 2H), 2.04 (s, 3H), 2.07 (s, 3H), 2.09 <s, 3H), 2. 1.2 (s, 3H), 3.37-3.42 (ms 2H), 3.55- 3.65 (.m, 3H), 3.67-3.69 (m, 2H), 3.83-3.88 fm, 1H), 3.97 (t 1H, J = 3.9 Hz), 4.03- 4.09 (m, 2H), 4.10-4.15 (m, 1H), 4.28 (dd, 1H, J- 1 1.9, 5.2 Hz), 4.46 (t, 1H, J = 6.6 Hz), 4.77-4.90 (or s. 2H), 4.93 (d, J - 3.9 Hz, 1H), 5.03-5.06 (m, 3E\ 5.09 (s, 2H), 5.23-5.29 (m, 3H), 5.46-5.48 (m, 1H), 7.28-7.37 fm, 5H); '¾ NMR (CDCb) δ 20.62, 20.65, 20.72, 20.76, 40,9, 62.1 , 62.5, 63.8, 65,8, 66.1, 66.6, 67.6, 68.6, 68.7, 69.0, 70.07, 70.16, 79.3, 7] .0, 77.2, 97.0, 97.6, 128.10, 128.18, 128.5, 136.5, 155.0. 169.3, 169.72, 169.73, 170.0, 1 70.56, 170.59; mass spectrum (APCI), mfz 859.2987 (M + Hf (
Figure imgf000083_0001
[02561 3,4,6-Tri-i -a€eiyl-2- -(3^6~tri-i -acety!~2~ -(methykar
D-i«aiii}Opyrai}Os 1)-a,p-L-gu!opyranos l Benzyl 2-(2-Etfooxy)ethyiearbamate (56). To a stirred solution containing 90,0 mg (0.10 mmol) of phosphate ester 53 in 1 .1 rnL of anh dichloromelhane was added a solution of 22.0 mg (0,09 mmol) of CBz linker 54 in 1 .1 ml, of anh dichbromethane at 0 CC. To the cooled reaction mixture was then added 33,0 μL· (41 ,0 mg, 0, 18 mmol) of MSOTf and the reaction mixture was stirred at 0 V'C for 15 min at which time it was poured into a mixture of 20 nil, of ethyl acetate and 20 rnL of said aq NaHCOj. The aqueous and organic layers were separated and the organic layer was washed with three lO-jiiL portions of water and brine, then dried (MgSO*). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied ιο a silica gel column (25 x 3 cm). Elation with 12: 12 1 ethyi
acetate - hexanes -methanol afforded disaccharide -linker conjugate 56 as a colorless oil: yield 56 mg (63%); silica gel TLC R{ 0.20 (12: 12: i ethyl
acetate~½xaties~raethano1); Ή NMR iCDC ) § 1.96 (s, 3H), 2.00 (s, 3H), 2.01 (a, 3M), 2.05-2.08 <m, 6H), 2.10 (s, 3H)5 2.78 (d, 31 J- 4.6 Hz), 3.38 (d, 2H, J==== 4.4 Hz), 3.51-3.70 (m, 4H), 3.78-3.87 (ra, ! H), 3.95 (d, .1H, 3.5 Hz), 4.00-4.15 (m, 4H), 4,20-4.30 (m, 2H), 4.45 it, 1H, J = 6, 1 Hz), 4.89-5.12 (m, 6H), 5.20-5.30 (ra, 3H), 5.42-5.49 (m, 1H), 5.46 (s, 1H) and 7.27-7.38 (m, 5H); i 5C NMR (CDC ) 520.7.1 , 20.73, 20.77, 20.80, 20.84, 20.88, 27.7, 62.3, 62.7, 63.9, 66.0, 66.3, 66.7, 68.7, 68.9, 69.2, 70.1 , 70.2, 70.4, 97.2, 97,9, 128.21 , 128.23, 128.28, 128.59, 128.61, 136.7, 155.5, 169.4, 169.80, 169.84, 170.0, 170.66 and 170.69; HRM'S (APCI), m z 873.3166 (M + if { AN.Gjs requires m/z 873.3141 ).
Figure imgf000084_0001
|0257| {2- -carbamoyi-a-D-maH!H>pyra«osy ~«4^-L-gulopyranosy^ 2-(2- amiitoethoxy)eti*aiioI (57). To a solution of 2.20 rag (2.60 umol) of compound SS in 1 niL of anli methanol was added a freshly prepared soluiion of 0.4 M sodium methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 3 !i, and the complete consumption of sorting material was confirmed by MALDl-TOF mass spectra! analysis. The reaction mixture was then quenched by the addi tion of 500 mg of'Dowex 50x resin, shaken, for 15 min and filtered. To the sotoiion of the crude product in methanol was added Pd/C and ¾ gas was bubbled through for 1 h. The complete consumption of starting material was confirmed by MALDl-TOF mass spectral analysis. The reaction mixture was filtered through Celite 545 s" and then concentrated trader diminished pressure to afford 57, which was used for the next reaction; HRMS (APCI), m/z 473, 1 86 (M + H)'!' (C|7¾N20{ 3 requires /z 473.1 83).
Figure imgf000085_0001
2-(2-amiiH>et xy)ethaiio{ (58). To a solution of 4,40 mg (5.00 μηιοΐ) of compound 56 in 2 ml, of anh methanol was added a freshly prepared solution of 0.4 M sodium methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 3 h, and the complete consumption of starting material was confirmed by MALDl-TOF mass spectral analysis. Hie reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for 15 min and filtered. To the solution of the crude product in methanol was then added Pd C and ¾ gas was bubbled through for 1 h. The complete consumption of starting material was confirmed by MALDl-TOF mass spectral analysis. The reaction mixture was filtered through Ce!Ue 545'*" and concentrated under diminished pressure to afford 58, which was used for the next reaction; HRMS (APCi), /z 487.2140 (M + Mf
Figure imgf000085_0002
requires m/z 487.2139).
0259| Example 10; Synthesis of modified mannose dlsaccharide-linker
[0260| Scheme 10
Figure imgf000086_0001
64
Figure imgf000086_0002
{02611 1^4»6-Teti¾-0^cetyl-2^2y^
iiUHino !ranos )~ji-L~guIopyranose (59). To a stirred solution containing
340 mg (0.98 mmoi) of gulose acceptor 23 and 737 .rag ( 1.1 ? ramoi) of mannose donor 32 in 7.0 mL of anh dichloromet ane cooled to 0 CC was added 352 iL (526 mg, 1.95 mmol) of TMSQTf at 0 °C The reaction mixture was stirred for 10 mill at which time it was poured into a mix Cure of 30 mL of ethyl acetate and 30 mL of said aq aHC(¾, The organic and aqueous layers were separated and the organic layer was washed with two 20-mL portions of brine and dried {Mg$Q4). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (30 χ 3 cm). Elation with 2: 1 ethyl aceiate-hexanes afforded disaccharkie 59 as a colorless oil: yield 407 mg (57%); silica gel TLC /?f .31 ( 1 ; 1 ethyl acetale-hexanes); !H N (CDGb) δ 1.92 is, 311), 2.00-2.01 (in, 6H, J - 2.8 Hz,), 2.04 (s, 3H, J 5.3 Hz), 2.08 (d, 6H, J - 1.9 Hz), 2.12 (s, 3B), 3.61 (ddd, I H, ,/ - 12.7, 9.6 and 3.3 Hz), 3,84-3.95 (ra, 2H), 3,96-4.20 (m, 4H), 4.26-4.37 (m, 2H), 4.59 (t, 1 H, J - 10.4 Hz,), 4.90-5. 8 (m. 4H), 5.39 (dd, Ϊ , J - U .I and 3.3 Hz), 5.86 (d, 1H, J" 8.3 Hz) and 7.24 (m, SH); , 3C NMR (CDC ) 6 20.56, 20.59, 20.61, 20.64, 20.65, 20.75, 20.78, 61.4, 2.3, 65.5, 66.9, 67.2, 67.5, 69.4, 71.3, 73.8, 90.5, 95.1, 127.6, 127.7, 127.9, 128.3, 137.4, 168.7, 1 68.8, 168.9, 169.1 , 169.4, 169.6, 1 70.3 and 170.4; mass spectrum (APCI), m/z 727.2444 (M + H)÷ (Cu^ s requires 727.2450).
Figure imgf000087_0001
102621 l^^Tetra-0-acet>'l-2^M2A^t«-i>-acet t^ -<(p- ill trophenyi)cai'bamo l)~a-B~mamiopyra nos O-p-L-giilopyranose (60), To a solution containing 470 nig (0.56 mmol) of disaceharide 59 in 40 mL of ethyl acetate was added a catalytic amount of Pd(OH);/C and the reaction mixture was stirred overnight under i aim of ¾. The solvent was Ulcered through a pad of C lite 545*' and the filtrate was concentrated under diminished pressure to afford crude residue. The residue was used for the next reaction; silica gel TLC .¾ 0.16 (.1 :2 ethyl acetate-hexanes); mass spectrum (APCI), m/z 637.1 93 (M ÷ H) ' (C¾!½Ojs requires 637. 1 80).
{0263] To a solution containing 338 mg (0.53 mmol) of the crude residue i 2 mL of pyridine was added 259 mg (2.12 mmol) of DMAP and 471 mg
(2.12 mmol) of/i-nitrophenyi chloroformate. The reaction mixture was stirred at 40 °C overnight at which time it was poured into a mixture of 30 mL of ethyl acetate and 10 mL of distilled water. The organic and aqueous layers were separated and the organic layer was washed with three 10-mL portions of 1 if CI and 10 mL of satd aq aHCC , The organic layer was then washed with brine and dried {MgSCy. The solvent was concentrated under diminished pressure to afford a crude residue. The residue applied to a silica gel column (25 * 3 cm). Elation with 1 :1 ethyl acetate-hexanes afforded the ester 60 as a colorless foam: yield 320 mg (71 % over two steps); silica gel TLC i¾- 0.24 ( i ; l ethyl acetate-hexanes); Ή NMR (CDC ) S 1.99 (s, 3H), 2.05 (s, 3H), 2.06-2.14 (m, 15H), 3.95 (dd, IH, J - 8.4 and 3.0 Hz), 3.99-4.16 (m, 4H), 4.16-4.2? (m, 2.H), 4.30 (dd, 1H, J - 1 5.0 and 8.7 Hz,), 5.21 -5.35 (ni, 2H), 5.39 (dd, 1H, «/ = 14.8 and H .5 Hz), 4.91-5.08 (m, 2H), 5.84 (cl i l l. ./ 8.4 Hz), 7.33 (d, 2H, J~ 9.0 Hz) and 8.21 (d, 2H, J =:: 9.0 Hz); , C NMR (CDClx) 6 20.57, 20.63, 20.64, 20.70, 20.71 , 20.8, 61.3, 61.9, 65.3, 65.5, 67.6, 67.7, 69.2, 69.8, 71 .3, 74.3, 90.5, 94.9, .122.0, 125.3, 145.6, 151.4, 155.2, 168.6, 1 9.2, 169.37, 169.41 , 169.7, .170.36 and 170.43; mass spectrum CAPCI), m z 742.1841 (M-AcOH)" (CsiHMNOa) requires 742. 831).
Figure imgf000088_0001
1026 S 1 ,3,4,6-Tet ra- -acety !~2~ -(2(4,6~tt i~6 -aeety!-3- ~
Figure imgf000088_0002
solution containing 320 rog (0.40 mmol) of di saccharide 60 in 12 inL of THF was added 200 uL (0.4 mmo!) of 2 M methylaraine in THF at 0 °C. The reaction mixture was stirred at room temperature for 15 h at which time silica gel TLC analysis indicated that the reaction was complete. The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 * 3 cm). Elution with 1 : 1 ethyl acetate-hexanes afforded disaeeharide 61 as a colorless oil: yield 239 rag (86%); silica gei TLC ¾· 0.17 (1 ; i ethyl acetate-hexanes); lE NMR (CDCfe) δ 1 .98 (d, 6H, J ~ 7.5 Hz), 2,03-2.1 1 (m, 1.2H), 2.13 (d, 3H, J - 8.8 Hz), 2.69 (d, 3H, J = 4.2 Hz), 3.88-4.22 (m, 6H), 4.31 (t 1 H, ,/ = 6.0 Hz), 4.67 (d, H i. J = 4, 1 Hz), 4.89-5.01 (m, 2H), 5.00-5.10 (m, 2H), 5.12-5.20 (m, HI), 5.38 (s, Hi) and 5.82 (d, i.H, 8.3 Hz,); C NMR (CDCfe.) δ 20.66, 20.69, 20,7 , 20,79, 27. 6, 61.4, 62, 1 , 65.4, 66.0, 67.7, 69.17, 69.27, 69.33, 69.38, 71.31 , 77.36, 90.6, 94,8, 155.4, 168.6, 169.2, 169,4, 169.8, 170,42 and 170.49; mass spectrum (APCl), m/z 694.2206 ( ÷ Hf (C^H^NQ^ requires 694,2195),
Figure imgf000089_0001
[Θ265| 3,4 >-Tn- -acetyi-2-0^^
»-i«a«nopyraiiosyi)-P-L-««lopyranosyi Biphenyl Phosphate (62), To a solution containing 65.0 mg (0.09 mmol) of disaccharide 6:1 in 0,8 raL of anh D F was added 1.1.0 mg (0. 1 mmol) of hydrazine acetaie. The reaction mixture was stirred at room temperature for 1.5 h and quenched by the addition of 20 mL of ethyl acetate. The organic layer was washed with three 10-niL portions of brine and dried (MgSG*). The solvent was concentrated under diminished pressure to afford a erode residue which was used for next reaction; mass spectrum (APC ), m/z 652.2086 (M + H) ! (C¾f¾NOi ¾ requires 652.2089),
|0266f To a stirred solution containing 43.0 mg (0.0? mrnol) of the crude residue in 4.0 mL of anh dichloromethane was added 1 .0 mg (0.08 mmol) of DMAF and 100 uL (72.0 mg, 0.7.1 mmol) of E N and 13 i uL (170 mg, 0.06 mmol) of diphenyl chlorophosphate. The reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetaie and 20 mL of said aq
NaHCOj. The organic and aqueous layers were separated and the organic layer was washed with three 10-mL portions of water and brine and then dried (M'gSC>4), The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column. (25 χ 3 cm). Ektion with 2: 1 ethyl aceiate-hexanes afforded phosphate ester 62 as a colorless oil: yield 44 mg (76% over two steps); silica gel TLC /if 0.25 (3: 1 ethyl aeetafe-hexarses): Ή NM
(CDC ) 3 1.70 (s, 3f¾ 1 .98 (s, 3H), 2.06 (s, 3H), 2.12 (d, 6H, J~ 1 1.4 Hz), 2.21 (s, 3H), 2.75 (d, 311, J - 4.5 Hz), 3.93-4.22 (m, 5H), 4.25-4.40 (m, 2H), 4.56 (d, H, 4.6 Hz), 4.93-5.05 (m, 2H), 5.120.24 (m, 2H.)t 5.29 (s, 1H)5 5.44 (s( 111), 5.65-5.73 (m, IH) and 7.13-7.40 (m, 10H); °C NMR (CDCU) δ 20.5, 20.9, 27.7, 36.7, 61.3, 62.0, 65,7, 67.5, 69.2, 69.4, 69.7, 7i ,2, 71 .3, 71.7, 95.6, 96,29, 96.34, 120,36, 120.41 , 125.7, 125.8, 129.7, 130.0, 150.2, 150.3, 150.4, 150.5, 155.3, 169.36. 169.42, 169.49, 169.9, 170.5 and 170.7; mass spectrum (APO),
884.2369 (M + Hf (
Figure imgf000090_0001
{0267j 3 ,6-Tri- -acetyI-2- -(2^6- i- -acei> -3-f -(methykarbai»0yi)-«- D-rfia«ii pyraii sy!)~a.,p~L-g»!opyraiH)sy! Benzyl 2-(2-Eihoxy)ethykarbaniate (63). To a stirred solution containing 44 nig (50 praol) of the phosphate ester 62 in 0.6 mL of anh dichloromethane was added a solution of 1 1 rag (40 μ«κ>1) of the
CBz-protecied Sinker 54 in 0,6 mL of anh dichloromethane at 0 °C. To the cooled reaction mixture was added 16 jiL (20 mg, 90 μτηοΐ) of TMSOTf and the reaction mixture was stirred at 0 *C for 15 rain. The reaction mixture was poured into a mixture of 10 mL ethyl acetate and 10 mL said aq NaHC<¾. The organic and aqueous layers were separated and the organic layer was washed with three 10-mL portions of water and brine and then dried (MgS<¾). The organic layer was concentrated under diminished pressure to afford a cntde residue. 'T he residue was applied to a silica gel column (25 χ 3 cm). Elation with 12: 12: 1 ethyl
acetate—hexanes-raeihanol afforded linker conjugate 63 as a colorless oil. The product was isolated as a (5:3) mixture of anomers: yield 32 mg (73%); silica gel TLC i/ .1 1 ( 12: 12: 1 ethyl acetate-hexarjes-methanol); 'H NM'R (CDCl. (major anoraer) 3 2.03 (s, 3E), 2.05 (s, 3H), 2.06-2.15 (in, J2H), 2.71 (d, 3H, J~ 4.8 Ez), 3.40 (s, .Hi), 3.51-3.74 (m, 6H), 3.79-3.89 (ra, 1 H), 3.92-4.01 (m, 1H), 3.99-4.21 (m, 4H), 4.21 -4.41 (m, 2H), 4.55-4.63 (m, 2H), 4.89-5.04 (m, 211), 5.09 (d, 2H, J:~ 5.6 Hz), 5.12-5.30 (m, 3H), 5.32-5.41 (m, 1H), 5.65-5.73 (m, 1 H) and 7.27-7.39 (m, 5H); UC MR (CDCL) δ 20.78, 20.83, 20.87, 20.91 , 20.93, 20.98, 21.0, 27.67, 27.69, 40.9, 41.1. 53.6, 61.8, 61.9, 62.3, 62.7, 63.9, 65.6, 65.7, 66.1, 66.4, 66.7, 67.9, 68.0, 68.6, 68.8, 69.0, 69.3, 69.5, 69.72, 69.76. 70.0, 70.1 , 70.3, 70.4, 70.52, 70.55, 70.7, 72.3, 97.1, 97.2, 120.38, 120.43, 128.2, 128.3, 128.60, 128.65, 129.8, 130.0, 136.8, 155.7. 156.7, 169.33, 169.37, 169.39, 169.47, 169.54, 169.6, 170.0, 170.5, 170.6, .170.7, 170.8 and 170.9; mass spectrum (APCI), m/z 873.3150 (M + H)* (C3S.H53 2O21 requires 873.3141).
Figure imgf000091_0001
{0268] ( ?^methyicarbamoy!)HK-D-m
2-(2-amiiioeth{iiLy) tiiaaol (64), To a solution of 5,80 m (6.60 pmol) of compound 63 in. 2 ml of aah methanol was added a freshly prepared solution of 0.4 M sodium methoxide in. methanol. The reaction mixture was allowed to stir at room temperature for 3 h, and the complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for .15 rain and filtered. To the solution of the crude product in methanol was added Pd C and H? gas was bubbled through, for 1 h. The complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was filtered through Ceiite 545* and then concentrated under diminished pressure to afford 64, which was used for the next reaction. H MS (APCl), m/z 487.2133 (M + Hf (Cj«¾j ;;0!.s requires m/z 487.2139).
[0269 | Example 1 1 : Synthesis of€4 modified mannose disaccharide-linker
73 and 74
|0270J Schem
Figure imgf000091_0002
Figure imgf000092_0001
Figure imgf000092_0002
[02711 ,3,4,6-Tetra~ ~-u>etyI-2- -(2,3,6 ri- -aeety{-4-f -(carbaiR tiiantiopyraiiosyl)-p-L-gulopyranose (65). To activated molecular sieves, a solution of 460 mg (0,79 mmoi) of 23 in 5.10 mL of dichloromethane and 1 1 mg (0.33 mmoi) of 40 in 4. SO mL of dichloromethane were added. The solution was cooled to 0 °C and was (hen treated with 220 μΐ, ( i .22 mmoi) ofTMSOTf. The reaction mixture was stirred for 20 mm at which time it was poured, into a two phase solution of 70 mL of ethyl acetate and 43 mL of said aq. NaHCOj, The organic layer was washed with two 50-ra'L portions of brine, dried (MgSO-j) and concentrated under diminished pressure to afford a crude residue. The residue was purified by flash chromatography on a silica gel column (25 * 3 cm). Elution with 3:1 ethyl acetate-hexanes afforded 65 as a colorless oil: yield 275 mg (51 %); silica gel TLC / .26 (3: 1 ethyl acetete-hexanes). lH NMR (CDCk) δ 1.99 (s, 3H), 2.05 ($, 3H), 2. 12 ($, 3H), 2.13 (s, 6H), 2.14 (s, 3H), 2.18 (s, 3H), 3.97-4.00 (m, lH), 4.03-4.16 (m, 2H), 4.26-4.28 (m, IB), 433-4.3? (m, IB), 4.73 (br s, 2H), 4.94- 4.97 (m, IB), 4.99-5.01 (m, I H), 5.06-5.09 (m, 2H), 5.13-5.15 (n% 2H\ 5.14-5.1 5 (m, IH), 5.43 ft, 111 J - 3.6 ! !/}, 5.88 (d. I H, J - 8.3 Hz), BC NMR (CDC!,)
20.56, 20.63, 20,68, 20.69, 20.7, 20.8, 20.9, 1.3, 62,2, 65.5, 65.89, 66.9, 67,0,
67.57, 67.60, 68.66, 68.71 , 69.4, 69.8, 7.1 .3, 90.6, 95, 1 , .155.0, 155.2, 168.7, 169,24, 169.26, 169.5, 1.70.0, 170.4, 170.6; mass spectrum (FAB), m/z 680.2045 (M + H) ' (C2?Hss 0i¾> requires miz 680,2038),
Figure imgf000093_0001
niaiumpyranosyl)-p-l..-gulopyranosyl Diphenyi Phosphate (66). To a solution containing 62.0 mg (0.09 mmol) of disaccharide 65 in 1,0 mL of anh DMF was added 12.0 nig (0.13 mmoi) of hydrazine acetate. The reaction mixture was stirred at room temperature for 2.5 h and quenched by the addition of 15 mL of ethyl aceiate. The organic solution was washed with 10 mL of water, said aq NaHC<¾, brine and dried iMgSO.}). The solvent was concentrated trader diminished pressure to afford the product as a yellow oil: yield 51 mg (88%); silica gel TLC Rf(k 1 (1 :3 hexanes-ethyl acetate). lH NMR (400 MHz, CDCfe) δ 2.00 (s, 3H), 2.07 (s, 3H), 2.1.2 (s, 3H), 2.13 (s, 3H), 2.14 (s, 3H), 2.1.7 (s, 3H), 3.74-3,7? (in, IH), 4.1 1 -4.19 (m, 2H), 4.23-4.26 (m, 2H), 4.33-4.38 (m, IH), 4,53-4.56 (br s, 2H), 4.94-4.95 (m, IH), 4.97-5.01 (m, 2H}« 5,09-5.15 (m, 3H), 5.26-5.30 (m, I H), 5.39 (t, IH, J= 3.6 Hz), MALDi, m/z 660.1 for (M + Na)*. The crude residue was used for the next reaction,
10273 { To a stirred solution containing ! .0 mg (0.1 mraol) of the crude residue in 3.00 mL of anh dichloromethane was added 1 ,0 mg (0, 12 mmol) of DMAP, .147 μ.Ι... (106 .mg, 1.04 mmol) of Et3 and 1 4 μΐ- (252 mg, 0.94 mmol) of diphenyi chlorophosphate. The reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetate and 20 mL of satd aq NaHC<¾, The aqueous and organic layers were separated and the organic layer was washed with three 10-mL portions of water and brine and then dried (MgSC ). The solvent, was concentrated, under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (20 x 2 cm), Elution with 2: 1 ethyl acetate-hexanes afforded the phosphate ester 66 as a colorless oil: yield.41 mg (52% over two steps); silica gel TLC 0;23 (3: 1 ethyl acetate-hexanes); !H NMR (CDC1. δ 1.95 (s. 3H), 1.97 (s, 3.H), 2.07 (s, 3H>, 2.1 1 (s, 3H), 2.13 (s, 3H), 2.20 (s, 3H), 3.95- 4.06 (m, 2.H.), 4,08-4.15 (m, !H), 4.17-4:2 ! (ni, 2H), 4.25-4.35 (in, 2H), 4.94-5.00 (m, 2H), 5.05-5.13 (m, 3H), 5.20-5.23 (m, 1H), 5.40-5.45 (br s, 2H), 5.700, 1H, J ==== 8.0 Hz), 7.15-7.21 (ra, 4H), 7.28-7.38 (m? 6H); °C NMR (CDCb) 20,62.20.68, 20.76, 61..1.3, 1.98, 65.42, 66.41 , 67.39, 68.60, 68.92, 69.21 , 71.58, 95.06, 96.19, 120.18, 120.23, 120.44, 120.49, 125.59, 125,66, 129,66, 129,93, 154.86, 169.28, 169.48, 169.80, 170.40, 170.63; mass spectrum (APC!), mis 870.2230 (M + E (CJ7H45NO21P requires m/z 870.2222).
Figure imgf000094_0001
|0274| 3 ,6-Tri- -acetyI-2- -(2,3,6-tri- -acet 4-4- -(carbatnoyI}-«-D- iiiatmopyranosyl)~«,{i~L-gukipyraiiosyf Benzyl 2~(2-Ethoxy)ethyicarbainate
(67), To a stirred solution containing 27,0 rag (0.03 mmol) of phosphate ester 66 in 3.9 mL of anii dichloromethane was added a solution of 8.20 mg (0.03 mmol) of CBz-proieeied Sinker 54 in 3.9 mL of aab dichloromethane at 0 °C. To the cooled reaction mixture was then added 8.20 μΐ,. (10,1 mg, 0.04 mmol) of T SOTf, The reaction mixture was stirred at.0 °C for 15 mrn and then poured into a mixture of 20 mL of ethyl acetate and 4 mL of said aq NaHCOj. The aqueous and. organic layers were separated and the organic layer was washed with three 10-mL portions of water and brine and then dried (MgSO. - The solvent was concentrated under diminished pressure to afford a crude residue. 'Hie residue was applied to a silica gel column (12 x 2 cm). Ehuion with 3:1 ethyl acetate-hexanes afforded 67 as a colorless oil. The product, isolated as a mixture of anomers: yield 7 mg (26%); silica gel TJX Rf OA 1 (4:1 ethyl acetate-hexanes ; H NMR (CDCl?) 6 1 ,99 (s, 3H), 2.04 is, mi 2.09 (s, 3H), 2.10 (s, 3H), 2.12 (s, 3H), 2.13 (s, 3H), 3.33-3.45 (br s, 2H), 3.56-3.65 (m, 211), 3.67-3.73 (ra, 2H), 3.82-3.88 (m, 1H), 3.96 (t, 1 H, J = 4.0 Hz), 4.03-4.11 (m, 3H), 4.12-4.19 (m, 2M), 4.30 (dd, IE., J - 12.0, 5.7 Hz), 4.42 (t, 1H, J= 6.5 Hz), 4.93-4,98 (m, 3H), 5.00-5.03 (m( 1 H), 5.07 (s, 2H\ 5.12- 5.17 (m, 2H), 5.24-5.30 (m, 3H), 7.30-7.36 (m, 5H); l3C NMR (CDQj) 20.81 , 20.89, 20.90, 20.93, 21.0, 29.8, 41 .2, 62.3, 62.9, 63.8, 65.7, 67.0, 67. 1, 68.1 , 68.69, 68.72, 69.6, 70. i , 71.1 , 77.5, 97.2, 97.6, 128,32, 128,38, 128.7, 136.5, 155.5, 156.9, 169.5, 169.8, 1 9.9, 170,2, 170.7, 170,8; mass spectrum (APCI), m!z
859.2975 (M -f Hf (Cntibi jCta requires mlz 859.2984).
Figure imgf000095_0001
mannopyranosyI)-p-L-gutopyranose (68). To a stirred solution containing
217 nig (0.62 nunoJ) of gulose acceptor 23 and 471 rag (0.75 mrooi) of raannose donor 35 in 4.50 niL of mh dichloromethane cooled to 0 *C was added 230 uL (283 rag, 1.25 rmrsol) of TMSOTf. The reaction mixture was stirred at 0 for 10 min and ihen poured into a mixture of 30 ml, of ethyl acetate and 30 raL of said aq NaHCOj. The aqueous and organic layers were separated and the organic layer was washed with two 20-mL poriioiis of brine and dried (MgSO*). The solvent was concentrated under dirmoished pressure to afford a erode residue. The residue was applied to a silica gel column (30 x 3 cm). Elation with 2: 1 ethyl acetate-hexanes afforded 68 as a colorless oil: yield 330 mg (73%); silica gel TLC li 0.25 (1 : 1 ethyl acetate-hexanes); ]B NMR (CDC ) δ .1 .92 (s, 3H), 2.02 (s, 3H), 2.07 (t, 6H, J - 3.2 Hz), 2.08-2.1 1 (m, 6H), 2.15 (d, 3H, J = 3.7 Hz), 3.70-3,83 (m, 1H), 3.92- 4.18 fin, 4H), 4,23-4.40 (m, 2H), 4.50-4.71 (m, 2E), 4.S9 (ddJJi, ./ === 7.2 and 1 .7 Hz), 4.96-4.99 (m, 1H), 5.01-5.10 (ni, 2H), 5.1 -5.16 (ra, 1 H), 5.35-5.45 (m, 1H), 5.85 (d, 1H, = 8,4 Hz) and 7, 1 -7.34 (m, 5H); S 3C NMR (CDC!.?) § 20.68, 20.71 , 20.73. 20.79, 20.84, 20.88, 20.9, 61.4, 65.6, 67.7, 69.1, 69.5, 703, 71.3, 71.7, 72.4, 74.8, 90.7, 95.0, 127.6, 127.89, 127.99, 128.46, 128.49, 137.6, 168.8, 169.32, 3 9.36, 1 9.4, 169.7, 1 70,5 and 170.6; HRMS CAPCT), m/z 727.2439 ( + Hf requires m/z 727.2450).
Figure imgf000096_0001
[0276] l,3A -Tetra-0-aeer i^^
iHiropb<inyl)carbamoyi)-a-{^HU»Hiopyranosyi)-p-L-g»lopyran se (69). To a solution containing 140 mg (0.1 mnio!) of disaceharide 68 in 13 raL of ethyl acetate was added a catalytic amount of Pd(OH);/€ and the reaction mixture was stirred overnight under i aim of ¾. The solvent was filtered through a pad of Celiie 545* and the filtrate was concentrated under diminished pressure to afford a crude residue. The residue was used for the next reaction; silica gei TLC ¾ 0.08 (1 .1 ethyl acetate-hexanes).
{0277J To a solution containing 120 mg (0.1 mmoi) of the crude residue in 2.0 niL of anh pyridine was added 92.0 mg (0.76 mmol) of DMAP and 168 mg (0.76 mmol) of /-nUrophenyl chloroformate. The reaction mixture was stirred at 40 °C overnight and then, poured into a mixture of 30 ml, of ethyl acetate and 10 mL of H;>0. The aqueous and organic layers were separated and the organic layer was washed with three 10-m.L portions of 1 N HC! and 10 ml of said aq NaHCO.5 and brine. The organic solution was dried (MgSO*) and concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 3 cm). E!ulion with 1 : 1 ethyl acetate-.hexao.es afforded eater 69 as a colorless foam: yield 121 mg (78% over two steps): silica gel TLC R? 0.30 (1 : 1 ethyl acetate-hexanes); Ή NMR (CDC¾) o 1.98 (s, 3H), 2.03 (s, 3H), 2. 1 id, 6.H., J = 5.0 Hz), 2.14 (s, 3Hj, 2.19 (d, 3H, ,/ = 5.4 Hz), 3.99 (dd, 1 H, J= 8.4 and 3.3 Hz), 4.02-4.25 (m, 4H), 4.27 (d, 1 H, J~ 2.4 Hz), 4.35 (t, M, J~ 6.0 Hz), 4.46-4.55 (m, 2.H), 4.93-5.01 (m, 2H), 5. i 1-5.18 (m, 2H), 5.24 (dd, IB, J - 10.1 and 3.3 Hz), 5.32 (dd. Hi, J - 7.7 and 4.3 H¾), 5.43 (t, 1H, J= 3.5 Hz), 5.89 (d, I H, J - 8.5 Hz), 7.29-7.39 (m, 2H) and 8.25 (t, 2H, J - 6.0 Hz); ,3C N R (CDCfe) 5 20.69, 20.71 , 21.0, 61.3, 6.1.7, 65.6, 67.7, 68.6, 68.8, 70.0, 71,3, 71.4, 90.6, 5.1, 121.7, 125.4, 145.7, 151.8, 155.2, 168.7, 169.29, 169.33, 169.38, 169.58, 169.65, 169.7, 169.8, 1.70.44, 170.46 and 170.58; HRMS (APC1), m z 802.2035 (M + H
Figure imgf000097_0001
(02781 ^^etra-f cetyl-2- -<2^^H- -acetyi- -0- (meih lcarbam yl)-o-D-mani )p ran syl)-P-l.-gul pyi 5)n .se (70). To a solution containing 121 rag (0.15 mmol) of 69 in 3.2 raL of anb THF was added 76,0 μΐ (0, 15 mraol) of a 2 solution. ofC¾NH2 in Τϊ-IF at 0 X. The re-action mixture was stirred at room temperature for 15 h at which time silica gel TLC analysis indicated that the reaciioii was complete. The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 3 cm). Elution with 1:1 ethyl acetate - hexanes afforded disaccharide 70 as a colorless oi!: yield 90 mg (86%); silica gel TLC ¾ 0.14 (1 : 1 ethyl acetate-hexanes); !H NMR (CDCfe) δ 1 .96 (t, 3H, J= 3.4 Hz), 2.04 (d, 3H, J - 6.4 Hz), 2.1 1 (dd, 12H, J~ 5.4 and 2.8 Hz), 2. 7 (d, 3H, J - 2.5 Hz), 2.76 (d, 3H, J■■■■■ 4.8 Hz), 3.97 (dd, 1 H, J = 8.4 and 3.2 Hz), 4.00-4.39 (m, 3H), 4.48-4.80 (m, IH), 4.93 (d, Ϊ H, ./ = 7.2 Hz), 4.99 (dd, 1 H, J = 7.0 and 4.4 Hz), 5.04-5.10 (m, 2H), 5.08-5.17 (m, 2H}« 5,29 (dd, IH, J= 13.2 and 9.8 Hz), 5.42 (t, iH, ,/ = 3,5 Hz), 5,87 (d, 1 H, J ~ 8.4 Hz) and 6.28 (d, IH, ,/= 4.2 Hz); UC NMR (CDCh) δ 20.68, 20.75, 20.76, 20.80, 20.82, 20.84, 27.8, 61.5, 1.8, 62.5, 62.7, 65.6, 66.0, 66.3, 66.8, 67.8, 68.9, 69.75, 69.79, 71.4, 0.7, 169.3, 169.59, 169.61, 169.65, 170.53, 170.55 and 170.7; HRMS (APC1), m/z 694.2199 (M + BY (C^H^ O^ requires m/z 694.2195).
Figure imgf000098_0001
|Θ2?9| 3,4,6-Tri-0-acetyi-2- 2,3,6-tri^
»-i«a«nopyraiiosyi)-P-L-««lopyranosyi Bipheoyl Phosphate (71). To a solution containing 44.0 mg (0.06 mmoi) of disaccharide 70 in 0,50 mL of anh DMF was added 7.00 nig (0.08 mmoi) of hydrazine acetate. The reaction mixture was stirred at room temperature for 1.5 h and quenched by the addition of 20 mL of ethyl acetate. The organic solution was washed with three 10-mL - portions of brine and dried (MgSO j), The solvent was concentrated under diminished pressure to afford a crude residue. "The crude residue was used for the next reaction.
102801 To a stirred solution containing 43,0 mg (0.07 mmoi) of the crude residue in 4.00 mL of anh dich!oromethane was added 10,0 mg (0.08 mmoi) of DMAP, 100 μΤ (72.0 mg, 0,71 mmoi) of Ei¾N and 130 pL ( 160 mg, 0.63 mmoi) of diphenyl chlorophosphale. The reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetate and 20 mL of said aq
NaHCCb. The aqueous and organic layers were separated, and the organic layer was washed, with three 1 -niL portions of water and brine and then dried (MgSO<f), The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 3 cm). Elution with 2:1 ethyl acetate-hexanes afforded the phosphate ester 71. as a colorless oil: yield 38 mg (69% over two steps); silica gei TLC 0.48 (2: 1 ethyl acetate-hexanes); Ή MR (CDC ) δ 1 .95 (s, 3B), 2.00 (s, 3H), 2.09 (s, 3H)S 2, 12 (s, 3H), 2, 15 (s, 3H), 2,21 (s, 3H), 2.57 (d, 3H, J- 4.0 Hz), 3.70 (s, 1 H), 4.03 (s, 2H% 4.15 (d, 2H, J~ 9.6 Hz), 4.24 (d, 2H, ./ = 12,2 Hz), 4.32-4.38 (m, 1H), 4.99 (d, 2H, ./ = 12.6 Hz), 5.05- 5.25 ( , 2H), 5.30 (s, 1HX 5.45 ($, ΪΗ), 5.71 (d, IH, J= 7,4 Hz) and 7.19-7.41 (m, 10H); ¾ NMR (CDCb) δ 20.77, 20.83, 20.89, 20.93, 27,6, 61 .3, 62.3, 65.6, 66.3, 67.5, 68,8, 69.2, 69.5, 70.7, 70.8, 71.7, 5.1 , 96.4, 1.20,4, 125,7, .129.8, 130.0, 1.50.4. 155.4, 16937, 169.39, 169.6, 169.9, 170.5 and 170.73, 170.76: HRMS (APCl), m/z 884.2381 (M + H} : ΟΑτΝΟ,^Ρ requires /z 884.2378).
Figure imgf000099_0001
{02811 3A6-Tri-0-acetyl-2-i -^^^
D-ma«ii pyraii sy!)~a.,p~L-g»!opyraiH)sy! Benzyl 2^2-Ethoxy)er.hyJcarbamate
(72). To a stirred solution containing 38. mg (0.04 mmol) of phosphate ester 71 in 0.5 raL of anii dichioromethane was added a solution of 10.0 mg {0.04 mmol) of CBz-proieeied Sinker 54 in 0,5 mL of anh dichioromethane at 0 °C. To the cooled reaction mixture was then added 14.0 μί, (17.0 mg, 0.08 mmol) of TMSOTf. The reaction mixture was stirred at 0 °C for 15 min and then poured into a mixture of 20 mL of ethy l acetate and 20 mL of satd aq NaHCOj. The aqueous and organic layers were separated and the organic layer was washed with three 10-m.L portions of water and brine and then dried (MgSOj). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 3 cm). Etation with 12: 12:1 ethyl aceta-e-hexanes-methanol afforded 72 as a coiorless oil. The product isolated as a mixture of anomers: yield 19 mg (51%); silica gel TLC Rf0.l4 (12: 12: 1 ethyl acetate~hexaoe«~melhanol); lH NMR (CDCb) δ ! .92-2. I 4 (m, 1 8H), 2.71 (t, 3H, - 4A Hz), 3.40 id, 3H, J = 4.9 Hz), 3.52-3.77 (ra, 8H), 3.85 (ddJ B, </= 8.4 and 3.2 Hz), 3.95 it 1 H, J = 3.9 Hz), 4.27 (dd, 21·!, J~~ 13.4 and 7.3 Hz), 4.40 (t, 1 H, J ::: 6.4 Hz), 4.88-5.04 (m, 3H), 5.05-5.22 (m, 6H), 5.25 (dd, 1 H, J = 7.3 and 3.6 Hz) and 7.28-7.40 (m, 5H); ¾ NMR (CDC1. δ 20.78, 20.83 20.85, 20.87, 20.92, 20,95, 27.7, 61.9, 62.3, 63.1 , 63.8, 65.7, 66.8, 66,9, 68.1 , 68.7, 68.8, 69.6, 69,8, 70.2, 71.0, 72.3, 7,2, 97.5, 128.27, 128.33, 128.65, 128.67, 169.5, 169.7, 169.8, 169.9, 170.57, 170.63 and 170.7; HRMS (APCI), m/z 873.3142 (M + H)" (Cj«H53 202j requires m z
873.3141 ).
Figure imgf000100_0001
(0282 j {4- -earbai»oy{-a-D-maiittopyranosyl)-tt,P-L-gulopyra«osyl 2-(2- am.n.oethoxy)ethanol (73). To a solution containing 2.20 rag (2.56 pmol) of compound 67 in i ml, of anh. methanol was added a freshly prepared solution of 0.4 M sodium methoxide in methanol. The reaction mixture was aliowed to stir at room temperature for 3 h, and the complete consumption of starting material was confirmed by MALD1.-TOF mass spectra! analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex SOx resin, shaken for 15 min and filtered. To the solution of the crude product in methanol was then added Pd C and ¾ gas was bubbled through for 1 h. The complete consumption of starting material was confirmed by MALDI-IOF mass spectral analysis. The reaction mixture was filtered through Celite 545'*" and then concentrated under diminished pressure to afford 73, which was used for the next reaction; HRMS (APCi), /z 473.1972 (M + Mf (Ci?H3?N20i3 requires m/z 473.1983).
Figure imgf000100_0002
£0283 | (4> >(methykarbamo> )>a-D>mannopyranosyl)-a,^>L'-gul»pyran»sy!
2-t2-amiiu>ethoxy)ethan«l (74). To a solution containing 2.70 mg (3.1 0 μτηοΐ) of 72 in 2 mL of anh methanol was added a freshly prepared solution of 0.4 M sodium methoxide in methanol. The reaction mixture was aliowed to stir at room temperature for 3 Si. and the complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex SOx resin, shaken for 15 min and filtered. To the solution of the crude product in methanol was added Pd/C and H; gas was bubbled througii for I h. Hie complete consumption of starting material was confirmed by 'ALDi-TOF mass spectral analysis. The reaction mixture was filtered through Celite 545* and concentrated under diminished pressure to afford 74, which was used for the next reaction; HRMS (APCi), m/z 487.2153 (M + E (Ci»l¼ ¼Ou requires m/∑ 487,2139).
(02841 Example 12: Synthesis of C3 modified altrose disaccha ride-linker $3 and 84
0285| Scheme 12
Figure imgf000101_0001
78 R * Ms
Figure imgf000101_0002
78 » H 81 f » H
= Me S2 R = Me
Figure imgf000101_0003
S3f¾ ~ H
84 R - Me
Figure imgf000102_0001
|Θ286| l,3 ,6-Tetra-0-acet I-2-O 2,4,6 ri-0-acet !»3-0^enzyi-a-D- altropyranosyl)«}l«L>gtt1opyrau0se (75). To a stirred solution containing 180 rag (0.52 rmno!) ofgulose acceptor 23 and 324 rag (0.52 raol) of aHrose donor 47 in 3.70 mL of anh dich!oromelhane at 0 *C was added 190 ,uL (234 rag, 1 .03 mraol) ofTMSOTf. The reaction mixture was stirred at 0 °C for 10 mm at which time it was poured into a mixture of 30 mL of ethyl acetate and 30 ml of said aq
NaHt'Cb. The aqueous and organic layers were separated, and the organic layer was washed with two 20-mL portions of brine and dried (MgSO«f), The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (30 x 3 cm). Elation with 1 :2 ethyl aeeiaie-hexanes afforded disaccharide 75 as a colorless oil: yield 1 9 mg (40%); silica gel TLC f 0.24 ( 1 ; 1 ethyl acetat -- hexanes); J H NM (CDOU) δ 1.93 is, 3H>, 1.97 (s, 3H>, 2.00 (s, 3H), 2.02 <$, 3H), 2.04-2.06 (m, 6H), 2.08 (s, 3H), 3.72-3,83 (m, 1H), 3.94-4.16 (m, 2H), 4. ί 6-4.35 (ms 3H), 4.35-4.62 (m, 3H), 4.79-5.01 (m, 4H), 5.24 (d, 1H, J- 0.4 Hz), 5.35-5.42 (m, 1H), 5.90 (d, IH, J - 8,4 Hz) and 7.15-7.30 (HI, 5H); 13C NMR (CDC¾) 3 20.7, 20.80, 20.81 , 20.86, 20.89, 21.0, 61.6, 62.6, 6:5.3, 65.5, 66.4, 67.8, 68.4, 68.8, 72.0, 72.7, 90.6, 95.4, 127.4, 127.6, 127.9, 128.5, 137.7, 169.0, 169.2, 169.4, 169.5, 169.9, 170.5, 170.7; HRMS (APCi)t m z
667.2230 (M - CHsCOO)'' (CsiH-wOt* requires m/z 667,2238).
Figure imgf000102_0002
{02871 l.A^Tctra-0-a «t 1-2^M2^tH-i>-acetyl-3- -( p- nift'o hen i)c»rfoamo l) X-D" itro riiaosyl ^-L'' eiop raiio$e (76). To a solution containing 190 mg (0.26 mmol) of disaceharide 75 in 18 mL of ethyl acetate was added a catalytic amount of Pd(OH);/C and the reaction mixture was stirred overnight under 1 aim of ¾. The solvent was filtered through a pad of Celite 545* and the filtrate was concentrated under diminished pressure to afford crude residue. The crude product was used for the next reaction; siiica gel TLC Rf 0.12 (1 :1 ethyl acetate -hexanes).
{0288| To a solution containing 1 8 mg (0.31 nitnol) of the crude residue in L I mL of anh pyridine was added 151 mg ( i ..24 mmol) of DMAP and 280 mg ("1.24 mmol) of i-nitropheny! chloroformate. The reaction mixture was stirred at 40 °C overnight and then, poured into a mixture of 30 mL ethyl acetate and 10 ml, of FLO. The aqueous and organic layers were separated and the organic layer was washed wit three 10-mL portions of I N HC1 and 10 mL of satd a NaHCO;; and brine. The solvent was dried (MgSQt) and then concentrated under diminished pressure to afford a crude residue. The residue was applied to a siiica gel column (25 3 cm). Elution with 1 : i ethyl acetate-hexanes afforded ester 76 as a colorless foam: yield 177 m (71 % over two steps); silica gel TLC ¾ 0.28 (1: 1 ethyl acetate-hexanes); Ή N R (CDCfe.) δ 2.02 (s, 3H), 2.04 (s> 3H), 2.09 (s, 3H), 2.10 is, 3H), 2.1.2 s, 3H), 2.13 (s, 3H), 2.14 (s, 3H), 3.99-4.17 (m, 3H), 4.23-4.38 On, 2H), 4.41 -4.50 (m, 1H), 4.89-5.02 (m, 2H), 5.02-5.13 (ra, 2H), 5.20 idf M, J ■■■■■■ 10.4 and 5.2 Hz), 5.25-5.34 (m, H¾ 5.43 (L IH, J === 3,5 Hz), 5.94 (d, 1H» J= 8.4 Hz), 7.42 (t, 2H, J = 7.1 Hz) and 8.22-8.30 (m, 2H); C NMR (CDCL) δ 20.66, 20.71 , 20.72, 20.76, 20.9, 61.5, 62.2, 64.7, 65.1 , 65.4, 67.6, 68.1 , 68.6, 7 .3, 72.1 , 90.5, 94.5, 121.4, 125.4, 136.0, 145.6, 149.8, 151.6, 155.2, 168.8, 168.9, 169.1 , 1.69.3, 169.5, 170.4 and 1.70.6; HRMS (APCI), mk 742.1 85 i (M - CH3COO)* (C¾tH¼NO?.o requires m z 742.1 31 ).
Figure imgf000104_0001
0289| 1^4,6-Tetra-0-ace†yi-2-0-<2,4^
altropyranosyl)«}l«L>gtt1opyranos»de (77). To a solution containing 73.0 mg (0.09 mmol) of ester 76 in 2 mL of nh THF was added a solution of 0,7 mL of anh THF saturated with N¾ at 0 °C. The reaction mixture was allowed to warm to room temperature and then stirred for 2.5 h at which time silica gel TLC analysis indicated that the reaction was complete. The solvent was concentrated under diminished pressure to afford a crude residise. The residue was applied to a silica gel column (20 x 3 cm). Eluiion with 3:1. ethyl acetate-hexanes afforded disaccharide 77 as a colorless oil: yield 44 mg (71 %); silica gel TLC /ir0.38 (ethyl acetate); !H NM.R (CDC13) δ 2.00 (s, 3H), 2.05 (s, 3H), 2. i 1 (s, 6 ), 2. 13 (s, 3H), 2, 16 (s, 3H), 2.17 (s, 3H), 3.9 (dd, 1H, J = 8.1 and 3.3 Hz), 4.02-4.3 (m, 7H), 4.75 (d, IH, 3.3 Hz), 4,82-4.9 (m, 2H), 4,99-5.12 (m, 2H), 5.13 (dd, ΪΗ, J~ 7.8 and 4.4 Hz), 5.44 (i IH> J===: 3.7 Hz) and 6.1 1 id, 1 H, J = 8.1 Hz); "C NMR (OX¾) δ 20.72, 20.75, 20.79, 20.82, 20.83, 20.87, 21.2, 61..8, 62.4, 4.6, 64.9, 65.5, 66.8, 67.6, 69,0, 69.5, 71.7, 91.0, 94.4, 155.6, 168.9, 169.3, 169.4, 169.6, 170.2, 170.5 and 170.7; H MS (APCl), /z 680.2039 ( + Hf (C>-1½NC½ requires m/z 680.2038).
Figure imgf000104_0002
|029O| 3,4 »-Trf~ ~acetyl-2~6^(2,4,6-Tri- ~acetyi-3-O-c8rl ara0
aiiropyranosyl)-p-L-gtilopyraBosyI Diphen i Phosphate (79). To a solution containing 44.0 mg (60.0 pmol) of disaccharide 77 in.0.50 mL of anh DMF was added 7.00 rag (80,0 prnol) of hydrazine acetate. The reaction mixture was stirred at room, temperature for .1.5 h and. then quenched by the addition of 20 mL of ethyl acetate. The organic layer was washed with three 10-mL portions of brine and dried (MgSClj). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was used for the next reaction.
(02911 To a stirred solution containing 1 .0 mg (60,0 μηιοΙ) of the crude residue in 4.00 mL of anh dichloromethane was added 10.0 rag (80.0 grnol) of D.MAP, 100 (72.0 mg, 0,68 rnmol) ofEtsN and 125 L (162 tag, 0.61 mmol) of diplieoyl chiorophosphate at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetate and 20 mL of said aq aMCC The aqueous and organic layers were separated and fee organic layer was washed with three 1 -raL portions of distilled water and brine and then dried (MgS(¾). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 2 cm). Elution with 2: 1 ethyl aceta.e--hexan.es afforded phosphate ester 79 as a colorless oil: yield 3 1 mg (55% over two steps); silica gel TLC /ΐ/0,30 (2: 1 ethyl acetate-hexanes); JH NMR (CDCIs) 5 1.83 (s, 3H), 1.98 (s, 3H), 2.04 (s, 3H), 2. 1.2 (d, 3H, J~ 2.8 Hz), 2.15 (d, 6E, J * 3.9 Hz), 3.98-4.0 (in, 2H), 4.09-4.25 (m, 4H), 4.26-4.36 (m, 2E 4.66 (d, 1 E, J■■■■■■ 9.8 Hz), 4.83 (d, 1 H, J === 2.1 Hz), 4.91 id, 1 H, J === 6,4 Hz), 5.03 (t, i.H, ,/ - 5,7 Hz), 5.09-5.19 (m, 2H), 5.45 (d, ΓΗ, J™ 3.2 Hz), 5.74 (t, ΓΗ, ~ 8.0 Hz) and 7.09-7.41 (m, iOH); l3C NMR. (CDC13) S 20.62, 20.66, 20.77, 20.83, 20.88, 6 i ,6, 62.2, 64.5, 64.7, 65. L 67.1 , 67.3, 68.9, 71.7, 94.1, 120.28, 120.32, 120.37, 1 25.98, 125.99, 126.23, 126.24, 129,93, 129.94, 130.1 , 155.9, 168.8, 169.0, 169.3, 169.5, 170.4, and 170.8; HRMS (APCI), m/z 870.2230 (M. ÷ Hf C3?H 5 02iP requir
Figure imgf000105_0001
}0292| ,4 )-i'n-f -acetyJ-2- -(2,4,6-'rri-6^acetyI-3- arbainovl-«-0- aitropyranosyl)-^-L-gutopyranosyl Benzyl 2-(2-Ettioxy)ethykarba«iate (81). To a stirred solution containing 31 mg (40 μηιοΙ) of phosphate ester 79 in 0.45 ml of anh dichlororaethane was added a solution of 8,0 mg (30 μπιοί) of CBz- protected linker 54 in 0.45 raL of anh dichloromethane at 0 °C. To the reaction mixture was added 12 uJL (15 rag, 80 umol) of TMSOTf and the reaction mixture was stirred at 0 °C for 15 min. The reaction mixture was poured into a mixture of 1 niL of ethyl acetate and 10 raL said aq NaHCQ,¾. The a ueous and organic layers were separated and the organic layer was washed with three 10-niL portions of water and brine and then dried (MgSO.j)- The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica, gel column (25 x 2 cm). E!ution with 12:12:1 ethyl acetate-hexanes-methanol afforded 81 as a colorless oil: yield 15 mg (48%): silica gel TLC .¾ 0.17 (11:1 1:1 ethyl acetate-hexanes-methanol); lH NMR (OX¾) 3 iH NMR (CDC¾) δ 1 .95- 2.07 (m, 6.R), 2.07-2.15 (m, 12H), 3.4! (t, 2.H,J - 9,5 Hz), 3.59 (d, 2R ./- 5,0 Hz), 3.61-3.71 (m, 3H), 3.87 (di, iH, J= 12.8 and 6.5 Hz), 3.94-4.04 (m, IH), 4.04-4.20 (m, 3H), 4.2 ί -4,26 (ms I H), 4.36-4.48 (m, IH), 4.49-4.60 (m, 1H), 4.75 (d 1 H, J■■■■■■ 7.5 Hz), 4.84-5.05 (m, 411), 5.05-5.20 (m, 4H), 5.21-5.29 (ffl, I H), 5.32- 5.49 (m, 2tt) and 7.27-7.38 (m, 5H) ; °C NMR (CDC ) δ 20.75, 20.77, 20.82, 20.85, 20.88, 20.92, 0.9, 62.1, 62.3, 62.6, 65.1, 65.2, 66.9, 67.8, 68.1, 68.5, 68.6, 69.2, 70.37, 70.45, 99.5, 128.3, 128.4, 128.5, 128.7, 136.6, 155.7, 169.0, 169.4, 169.61. 169.65, 170.6, 170.82 and 170.89: HRMS (APC!), m/z 859.2973 (M + Hf
Figure imgf000106_0001
102931 1 ,3 ,6-Tetra-0-acetyl-2-0-(2,4,6^ri- -3cetyi-3-0- (inethylcarbaraoyl)-«-D-a1tro|>yranosyl)-p-L~gu!opyra«ose (78). To a solution containing 86.0 nig (0.11 mmol) of ester 76 in 2.4 m.L of anh THF was added 54.0 pL (0.11. mmol) of a 2 M solution of CH¾NH ? in THF at 0 -:C. The reaction mixture was stirred at room temperature for 1 h at which time analysis by silica gel TLC indicated that, the reaction was complete. The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column. (35 x 2 cm). Elution with 2: 1 ethyl acetate-hexaaes afforded disaccharide 78 as a colorless oil: yield 3.1 mg (42%); silica gel TLC ¾- 0, 13 (3: 1 ethyl acetate-hexanes); H NMR (CDC¾) δ 2.01 is, 3H), 2.05 (s, 3H), 2. i 1 (s, 6H), 2.13 (s, 3H), 2.15 (s, 3H), 2.16 (s, 3H), 2.79 (d, 3H, J- 4.7 Hz), 3.98 (dd, 1H, J 8.0 and 3.3 Hz), 4.04-4.30 (m, 4H), 4.33 (dt 1H, «/ = 12 J and 6.1 Hz), 4.71 - 4.77(ra, 1HX 4.84-4.95(1», 1 H), 5.06 (dd, 2H, J*> 10.1 and 6.6 Hi), 5.1 1-5.19 (ra, I H), 5.21-5.41 (m, 2H), 5.43 (dd, Hi </ ::: 10.0 and 6.3 Hz) and 6.10 (d, 1H, J~- 8.0 Hz); lsC M.R (CDC53) 6 20.77, 20.81 , 20.82, 20.85, 20.88, 20.9, 21.3, 27.8, 61.8, 62.5, 64.8, 65.0, 65.5, 66.4, 66.7, 67,6, 69,2, 71.6, 91 .1 , 94.7, 155.9, 169.0, 169.3, 169.4, 169.6, 170.1 , 1 70.5 and 170.8; HRMS (APCI), »3ώ 9 .2204 (M ÷ Hf
(C2SH4(*NOi9 requires m z 694.21 5).
Figure imgf000107_0001
[02941 3,4,6~Tri- -aceiy5-2- -(2,4,6 ri^^
l)-aitropyran .svl)-P-l.-g»l pyran svl Diphenyl Phosphate (80), To a solution containing 31 .0 mg (40.0 μτηοΐ) of disaccharide 78 in 0.5 mL of anh DMF was added 5.00 mg (50.0 μιηο!) of hydrazine acetate. The reaction mixture was stirred at room temperature for 1 ,5 h and then quenched by the addition of 20 mL of ethyl acetate. The organic solution was washed with three i0-mL portions of brine and dried (MgS<¾). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was used for the next reaction.
[02951 To a stirred solution containing 22.0 mg (30.0 umo!) of the residue in 2 mL of anh dichloromethane was added 6.00 mg (40.0 μηιοί) of DMAP, 52.0 pL (38.0 mg, 370 pmoi) ofEtsN and 70.0 uL (91.0 mg, 330 umoi) ofdiphenyl chlorophosphate at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then poured into a mixture of 40 mL of ethyl acetate and 20 ml of satd aq NaHCOs. The aqueous and organic layers were separated and the organic layer was washed with three 10-mL portions of distilled water and brine and then dried (MgSCXt). The solvent was concentrated under diminished pressure to afford a crude residiie. The residue was applied to a silica gel column (25 x 2 cm). Elution with 2: 1 ethyl acetate-hesanes afforded phosphate ester 80 as a colorless oil: yield 7.0 mg (1 7% over two steps); silica gel TLC Rf 0.28 (3: 1 ethyl acetate-hexanes); !H NMR (CDC ) 6 1.85 (s, 3H), 1.98 (s, 3H), 2.04 (s, 3H), 2.12 (s, 3H), 2.15 (d, 6H, J = 2.5 Hz), 2.63 (d, m, J■■■■■■ 4.7 Hz), 3.98-4.08 (in, 2H)} 4,09-4.26 (m, 3H), 4.30 (t, UL J ■■■■■ 6.1 Hz), 4.63 (d, 1 It, J - 10.5 Hz), 4.80 (d, 1 H, === 3.0 Hz), 4.89 (s, 1H\ 5.00- 5.06 (m, !H), 5.13 (dd, 1H, J= 10.5 and 3.1 Hz), 5. 1 8 (d, fH, J = 3.0 Hz), 5.45 id, 1 H, J ~ 2,9 Hz), 5.73 (t, Ϊ Η, J - 8.0 Hz), 6.46 (d, 1 H, J - 4.8 Hz.) and 7.12-7.40 (ra, 10H); C NMR (CDCi3) δ 20.67, 20.72, 20.77, 20.8, 20.9, 27.4, 61.6, 62.3, 64.67, 64.72, 65.1 , 66.7, 67.2, 69.1 , 71.7, 4.2, 96.52, 96.56, 120.1 , 120.2, 120.32, 120.37, 126.0, 126.1, .129.9, 130.1 , 156.1, 168.8, 169.0, 169.4, 169.5, 170.5 and 370.8; FIRMS (APCI requires m/z 884.2378).
Figure imgf000108_0001
[0296] 3, »6-Tri-O-a∞tyi-2-0^2 ,6-tr^^
D-alfrop ranos l}-o4 -I -g«l<>pv t anf.»svl Benzyl 2-<2~Ethoxy)ethykarbainate (82), To a stirred solution containing 17 mg (19 prnol) of phosphate ester SO in 0.25 rnL of anh dichloromeihane was added a solution of 5.0 mg (17 μτηοΐ) of €Bz~protected linker 54 in 0.25 ml, of anh dichloromeihane at 0 'C. To the reaction mixture was added 7.0 pL (8.6 mg, 34 pmol) of TMSOTf The reaction mixture was stirred at 0 °C for 15 rain and then poured into a .mixture of 10 rnL ethyl acetate and 10 mL satd aq NaHC<¾. The aqueous and organic layers were separated and the organic layer was washed with three 10-raL portions of distilled water and brine aid then dried (MgSOf). The solvent was concentrated under diminished pressure to afford a crude residue. The residue was applied to a silica gel column (25 x 2 cm). Elation with 12:1.2:1 ethyl ac«tate--hexaaes--met¾aiTol afforded 82 as a colorless oil: yield 10 mg (59%); silica, gel TLC ¾ 0, 14 (I 1 : 11 : 1 ethyl acetate-hexanes-methaiiol); Ή N R (CDCK) δ 1.97 id, 3H, J■■■■■■ 8,6 Hz), 2.04 (d, ,J » 4.2 Hz), 2.07-2.15 (m, 12H), 2,75 (d, 3H, J - 4.7 Hz), 3.34-3.44 (ra, 2H), 3.51-3.70 (ni 8H), 3.72 (dd5 HI J ::: 10.3 and 5.6 Hz), 3.82-3.93 (m, I H),
3.95- 4.25 (m, 3M), 4.26-4.56 (m, IH), 4.63 (d, IH, 7.2 Hz), 4.86-5.02 (rn, 1 H),
4.96- 5.28 (ra, 6H), 5.33-5.5! (m, IH), 5.83 (d, IH, J - 4.7 Hz) and 7.27-7.39 (ra, 5H); °C NMR (CDCb) δ 20.79, 20.84, 20.86, 20.89. 20.93, 21.0, 29.8, 41.0, 61.9, 62.2, 62.3, 62,7, 62.9, 65.26, 65.33, 66.9, 67, 1, 70.2, 70.4, 70.5. 72,3, 128.3, 128.4, 128.66, 128.67, 136.6, 169.61 , 169.65, 169.68, 170.6, 170.7, .170.8 and 170.9; HRMS (APC m/z 8 ffi& 873.3141).
Figure imgf000109_0001
|029?f {3- -carbainoyl-0.-D-aitr pyraaosyl)- ,p-L-giito|>yraiu syl 2-{2- aminoerhoxy)ethanoI (83), To a solution containing 2.40 mg (2.80 μηιοΐ) of compound 8 J. in 2 ml, of anh methanol was added a freshly prepared solution of 0. M sodium methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 3 h, and the complete consumption of starting material was confirmed by MAL.D1.-TO.F mass spectral analysis. The reaction mixture was then, quenched by the addition of 500 mg of'Dowex S0x resin, shaken for 15 rain and filtered. To the solution of the crude product in methanol was then added Pd/C and ¾ gas was bubbled through for 1 h. The complete consumption of starting material was confirmed by MALDl-TOF mass spectral analysis. The reaction was filtered through Celite 545*' and then concentrated under diminished pressure to afford 83, which was used for the next reaction. HRMS (APCI), m/z 473.1978 (M + H)! (C{?¾,?N20i3 requires m/z 473.1 83).
1.08
Figure imgf000110_0001
j0298] (4~ ~(nH1tfiykarbainoyi)~a-D~altrop rsiiifts l)~««p-L-g»l py 2- (2-aminoettaoxy)ethanol (84). To a solution containing i . mg (1, 10 μηιοΐ) of compound 82 in 2 mL of anh methanol was added a freshly prepared solution of 0.4 M sodium methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 3 k arid (he complete con sumption of starting materia! was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 300 mg of Dowex 50x resin, shaken for 15 min and filtered. To the solution of the crude product in methanol was added Pd C and H? gas was bubbled through, for 1 h. The complete consumption of starting material was confirmed by M ALDI-TOF mass spectral analysis. The reaction mixture was filtered through Ceiite 545* and then concentrated under diminished pressure to afford 84, which was used for the next reaction. H MS (APC , m/z 487.2143 (M + Η.Γ (CjjtHisNjOu requires m z 487,2139).
J0299J Example 13: Synthesis of bleomycin disaccharide linker 3
(03001 Scheme 13
Figure imgf000110_0002
(03011 l^^^-Teira- -ac t !^-D-maniiopyranose (85). To a solution containing 0.88 g (2.00 nimoi) of compound 30 in 24 mL of ethy l acetate was added a catalytic amount of Pd(OHh/C and the reaction was maintained under 1 atm of ¾(g) overnight. The catalyst was removed by filtration through a pad of Ceiite and the filtrate was concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (20■< 3 cm). Elutso with 75% ethyl acetate in hexanes afforded compound 85 as a colorless oil: yield 550 mg (79%); silica gel TLC A\ 0.11 (i : l ethyl acetate-hexanes); Ή NMR (CIX¾) o 2.04 (s, 3H), 2.09 fs, 3H), 2.13 (s, 3H), 2.15 (s, 3H), 2.97 (s, JH), 3.95 (m, 1H), 4.04 (m, 1 H>, 4.09 (is, 1H), 4.19 (dd, 1H, >/ - 12.3 and 4.8 Hz), 5.07 (m, 1 H), 5.13 (rn, 1 H) and 5.99 ( , I H); nC NMR iCDCfe) δ 20.7, 20.8, 62.3, 68.0, 68.6, 70.3, 70.9, 90.4, 168, .170.9.
Figure imgf000111_0001
{©3021 i 2,4,6-Tetra-0-aeet l~3-0^(^
mannopvranose (86), To a solution containing 0.55 g (1 .60 mmoi) of 85 in 5.6 ml, of pyridine were added 0.77 g (6.30 rnmol) of DMAP and 1.30 g
(6.30 mmoi) of p-nitropheiryS diloroiorroate. The reaction mixtur was stirred at 40 °C for 2 h at which time it was poured into a two-phase solution of 40 mL of ethyl acetate and 10 mL of ¾0. The organic layer was washed successively wit three 10-mL portions of i N HCi, it) mL of satd aq NaHCO.; and 1.0 mL of brine. The solution was dried ( ajSQ*) and concentrated under diminished pressure. The residue was purified by ilash chromatography on a silica gei column (20 x 3 cm). Elation with 50% ethyl acetate in hexanes afforded compound 86 as a yellow oil: yield 0.66 g (81%); silica gei TLC R{ 0.58 (1 :1 ethyl acetate-hexanes); fH NMR (CDCfe) 0 2.0? (s, 3H), 2.09 (s, 3H)5 2.14 (s, 3H), 2.1 ? (s, 3H), 4.08 (rn, 2H), 4.25 (in, 1H), 5.15 (dd, Ϊ H), 5.41. (m:, 2H), 6.1 1 (s, IH), 7.34 (d, 2H) and 8.23 id, 2H); l NMR (CDC 3) 6 20.6, 20.9, 61 .8, 64.9, 67.4, 70.5, 74.1 , 90.5, 121.8, 125.2, 145.5, 151.6, 155.1 . 167.8, 169.3, 169.9 and 170,5.
Figure imgf000111_0002
(0303 S 1^4,6>Tetra> >acety^3^^ari>amo l^~l>>inatt}io y a}io$e (87). To a solution of 0.51 g ( 1.31 mniol) of carbonate 86 in 27 mL ofanh CHbCL was added 15 mL of THF that had been saturated with H3 (g). The solution was stirred at
1.10 room temperature for 1.5 h (at which time silica gel TLC analysis indicated that the reaction was complete). The solution was concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (1 χ 3 cm). Ei li n with 3: 1 ~> 1 :2 hexanes -ethyl acetate afforded corn-pound 87 as a colorless oil: yield 355 mg ( 1 %); silica gel TLC ¾ 0.10 (1 : i hexanes-ethyl acetate). Ή NMR (CDCU) δ 2.02 (s, 3H), 2.04 is, 3H), 2.1 1 (s, 3H), 2.12 (s, 3H), 4.04 (ni, 2H), 4.22 (dd, 1H, J= 12. and 5.0 Hz), 5.03 (br s, 2H), 5.24 ( , 3H) and 6.03 (d, 1 H, J■■■■■■ 1 .? Hz); UC NMR (CDCIj) 6 20.6, 20.6, 20.7, 6 .9, 65.4, 68.6, 69.4, 70.4, 90.3, 155.2, 168.0 170.5.
Figure imgf000112_0001
{0304J 2 ,6-Tri- -carham;oyI-«, >-D~inaiiitopyrano$e (88), To a solution of 365 mg (0.93 mmo!) of compound 87 in 10.5 mL of dry DMF was added 120 mg ( 1 , 1 mmol) of acetate salt of hydrazine. The reaction mixture was stirred at room temperature for 1 h (at which time silica gel TLC analysis indicated that 87 had been consumed) arid diluted with 80 mL of ethyl acetate. The solution was washed with three 25-mL portions of brine and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (15 3 cm). Elution with 1 : 1 hexanes-ethyl acetate afforded compound 88 as a colorless oil: yield 285 mg (87%); silica gel TLC ¾ 0,24 (1 :1 hexanes-ethyl acetate). Ή NMR (CDO ) δ 2.06 (s, 3.H), 2.09 (s, 3H), 2.15 <st 3H), 4.1 (m, 1H), 4.23 (m, 2H), 4.83 (s, 2H) and 5.25
Figure imgf000112_0002
j 305 j 2,4 )- fn-< -acetyL3- -carbamoy}-«-D-oranttopyra«osy! Diphenyi Phosphate (89). To a solution of 1 0 mg (0.46 mmol) of intermediate 88, 64.0 mg
(0.57 mmol) of DMAP and 640 pL (468 mg; 4.63 mmol) of E¾N in 12.0 mL of CH2CI2 at 0 *C was added dropwise 0.95 mL (1.23 g; 4.6 mmol of diphenyl chlorophosphate. The solution was stirred at 0 °C for 1.5 h and was poured into a two-phase solution of EtOAc ( 100 mL) and sattsi'aied aq NaHCC {40 mL). The organic layer was washed with two 30-mL portions of brine, dried over NaaSCj, filtered and concentraied under diminished pressure. The residue was purified by flash chromatography on a silica gei column (15 χ 3 cm). Eluiion with 2: 1 1:2 hexanes- -ethyl acetate afforded the phosphate ester 89 as a colorless oti: yield 201 mg (75%); silica gel TLC ¾ 0. I (2:3 hexanes-etbyl acetate). ¾ NMR (CDCI3) δ 1.95 (s, 3H), 2.03 (s, 3H), 2.12 (s, 3H), 3.91 (d, 1H, J = 12.4 and 2.2 Hz), 4.08 (m, 1 H), 4.17 (dd, 1H, 12.4 and 4.7 Hz). 4.66 (br s, 2H), 5,30 (m, 3H), 5.87 (dd, 1 H, J- 6.5 and 1.6 Hz) and 7.28 (m, I OH); nC NMR (CDCI3) δ 20.6, 20.7, 20.7, 6.1.7, 65.3, 69.0, 69, 1 , 69.2, 70.7, 96.0, 96.1 , 120.1 , 120.1 , 120.2, 120.3, 1.25.8, 125.9, 130.0, 129.0, 1.50 169.5, 169.8 and 170.6.
Figure imgf000113_0001
{03061 , ,6-Tetra-0-acet I~2^M »^tn^ac yl^ H:ar amoyl-^-D- iiiaonopyranos !l)~«-L-g»lopyraiiostde (90), To a round bottom flask containing 200 mg (0.34 mmoi) of 89 was added a solution of 95.0 mg (0.27 mrao1) of 23 in 3.80 mL of anhydrous C¾Ch. The solution was cooled to 0 aC and to it was added 98.0 μΐ, ( 120 rag; 0.55 mmol) of TMSOTf dropwise. The reaction mixture was stirred at 0 CC for 17 rain at which time it was poured into a two-phase solution of EtOAc (60 mL) and saturated aq NaHCCh (25 mL). The organic layer was washed with two 20-raL portions of brine, dried ( ajSO,}), filtered and concentrated under diminished pressure. The residue was purified by Hash chromatography on a silica gei column (25 χ 2 cm), Elution with 3:2 - 1 :3 hexanes -ethyl acetate afforded the disaccharide 90 as colorless oil: yield 1 15 mg (62%); silica gei TLC Aj f 0.38 (1 :4 hexanes-ethyl acetate); fH NMR (CDCI3) 0 2.05 (s, 3H), 2.06 (s, 31:1), 2.07 (s, 3H), 2.14 (s, 6H), 2.16 (s, 3!:I), 2.20 (s, 3H), 3.98 (άά, 1H, J~ 8.4 and 3.3 Hz), 4.19 (m, 2H), 4.38 (m, 1HL 4.85 is, 2H), 5.13 (m, 7H), 5.45 (m, I H) and 5.88 (d, 1H, ./ = 8.4 Hz); 1 SC NM (CDC¾) 3 14.4, 20.9, 21.0, 21.0, 21.2, 21.3, 60.6, 61.6, 62.3, 65.7, 66.1 , 67.9, 69.3, 69.4, 69.9, 71.5, 90.8, 95,2, 155.4, 170.7 and 170.8.
Figure imgf000114_0001
mannopyranosyI)-«-L- i-opyrano$y]. Diphenyl Phosphate (91). To a solution containing 1 12 mg (0. 165 mmol) of 90 in 0.80 mL of anhydrous DMF was added 21 mg (0.23 mmol) of the acetate salt of hydrazine. The reaction mixture was siiiTed at room temperature for 1 h and quenched by the addition of 60 mL of ethyl acetate. The organic layer was washed with three ! O-raL portions of brine and dried (NajSO*). The solvent was filtered and then concentrated under diminished pressure to afford the deacetylated intermediate as a crude ressdoe which was used for next reaction without further purification.
{0308j To a solution of 1 15 mg of the crude residue, 26.0 mg f 0.21 mmol) of DM.AP and 242 pL (177 mg, 1 .75 mmol) ofEtjN in 16.5 mL of anhydrous Ci¾C¾ at 0 °C was added 0.33 mL (428 mg, 1.59 mmol) of diphenyl chlorophosphate dropwise. The solution was stirred at 0 ftC for 1 ,5 h and was then poured into a two-phase solution of EtOAc (80 mL) and saturated aq NaHCX soln (30 mL). The organic layer was washed with three 25-roL portions of H>0, two 25-mL portions of brine, then dried over a^SO^, filtered, and concentrated under diminished pressure. The residue was purifsed by Hash chromatography on a silica gel column (22 x 2 cm). Eiuiion with 1 : 1 -> 1 :3 hexanes-ethyi acetate afforded compound 91 a colorless oil; yield 121 rog (84%); 5H NMR (CDCIj) 6 L70 (s, 3H), 1.97 (s, 3H), 2,05 (s, 3H), 2.1 1 (s, 3H), 2.13 (s, 3H), 2.19 (s, 3H), 4.13 (ra, 5H), 4.31 (m, 2H), 4.76 (s, 2M), 4.96 (m, 1 E), 4.98 (m, 1H), 5.18 (m, 3tT), 5.43 (m, 1 H), 5.69 (m, IH) and 7.25 (ra, 10H); ,5C NMR (CDC ) δ 20.2, 20.6, 20.7, 61.1 , 61.7, 65.3, 65.4, 67.3, 9.0, 69.8, 71.5, 95.3, 96..1 , 120.1 , 120.2, 125.5, 129.6, 129.8, 129.9, 155.0, 169.2, 169.3, 169.7, 1 70.3 and 170.5.
Figure imgf000115_0001
(03091 3 ,6-Tri- -acetyI-2- K2A6- i- -acet> -3- -cai t)antoyl-«- - iTiaiinopYriinosyI)~«-lj-giiJop raiH)sy! benzyl 2~(2~ethoxy)eiiiyicar aoiaie (92). To a solution of 78 mg ( 1 μηιοί) of J and 19 mg (79 μηιοΐ) of 54 in 2.4 n L of anhydrous CH2C12 was added 28 uL (34 mg, 0.16 mmol) of TMSOTf at 0 aC. The reaction mixture was stirred at 0 °C for 1.7 rain, at which time it was poured into a two-phase solution of EtOAc (50 ml,) and saturated aq NaHCOj (20 mL). The organic layer was washed, with two 20- L portions of brine, dried ( a^SC^), filtered and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (25 * 2 em). Elation with 15;32; 1 - 1.1 :36:1 hexanes-ethyl acetate— methanol afforded compound 92 as a colorless oil: yield 62 mg (80%); silica gei TLC /if 0.30 (1 :4 hexanes-etliyl acetate); Ή NMR (CDCfe) δ 2.03 (s, 6E), 2.07 (s, 3H), 2.09 (s, 3H), 2.1 1 (s, 3H), 2.12 (s, 3H), 3.54 (m, 8H), 3,83 (m, I H), 3.96 (m, ΓΗ), 4.05 (in, 4H), 4,25 (m, iH), 4.46 (ra, I B), 4.69 (s, I H), 4.91. (m, I H), 5.12 (m, 8H), 5.61 (ra, I H) and 7.34 (m, 5H); ¾ NMR (CDC1. S 20.6, 20.7, 20,8, 29.6, 40.9, 62.1, 62.5, 63,7, 65.5, 66 J , 66.6, 67.6, 68.5, 69.1 , 69,6, 69.7, 70.0, 70,3, 70.6, 97.0, 97.1, 128. 1, 128,2, 128.4, 136.5, 156.5, 169.3, 1 9,5, 1 9.8 and 170.5.
Figure imgf000115_0002
|03l 0j (3^^»r »m lHZ-l>-manttopyratto^)>at[^L-- ulopyrano$yl 2~(2-
Figure imgf000116_0001
(3), To a solution containing 15.00 mg (8.06 μ¾ηο1) of cornpoimd 92 in 5 mL ofanh methanol was added a freshly prepared solution of 0.4 M sodium methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 3 h, and the complete consumption of starting material was confirmed by MALDi-TGF mass spectral analysis. The reaction mixture was then quenched by the addition of 300 mg of Dowex 5 x resin, shaken for 15 min and filtered. To the solution of the crude product in methanol was added Pd/C and lh gas was bubbled through for 1 h. The complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was filtered through Celite 545''" and then concentrated under diminished pressure to afford 3, which was used for the next reaction. Mass spectrum (MALD1), m/z 473.35 (M + ΉΫ, 495.32 (M + a (CisHjs jOjs requires m/z 472.19).
{031 Ij Example 14: Synthesis of bleomycin disaccharide-trimer linker 102
[03121 Scheme 14
Figure imgf000116_0002
Figure imgf000117_0001
{0313} 4-(2-^/-B«toxycarboijyl-ethyl)-4-!»ilro-heptanedH>k Acid Wi~terf- butyl Ester (94). To a solution of 2.1.4 ml (2.43 g; 39.8 mmol) of nitromethane in 10 iiiL of dimeihoxyeihane at 65 °C was added 0.4 of 40% aq
teirabutylmnmonium hydroxide soln and the reaction mixture was heated to 75 °C. To the reaction, mixture was added dropwise 18.2 mL (125 mmol) of /erf-buiyi acryJaie (93). To this mixture was added 0.8 mL of 40% aq tetrabutylarnraoni una hydroxide soin in portions over a period of i fa. The reaction mixture was stirred at 75 °C for 2 h. The reaction mixture was concentrated under diminished pressure and the residue was diluted in 100 mL of diethyl ether. The ether layer was washed with two 30~mL portions of 10% aq citric acid soln, two 30-mL portions of sat aq NaHCG.? soln, 20 mL of brine, then dried over anhydrous NajSO*, filtered and concentrated under diminished pressure. The residue was ^crystallized .from absolute ethaitol to afford compound 94 as colorless needles: yield 16.1 g (91%); mp 92-94 ¾C, !H NMR (CIX¾) δ 1.43 (s, 2?H) and 2.19 (m, 12H); C NMR. (CDC1. δ 28.2, 29.9, 30,5, 81.3, 92.3 and 171.2,
{0314} 4-Ann«o-4-(2-fe/,r-butoxycarbonySetlwl)heptanedioic Acid D ert- butyl Ester (95). A mixture of 1.02 g (2.29.mmol) of compound 94. -6 mL of Tl~ Raney Ni (suspension in ethano!) and 1 S mi,, of absolute ethanoi was shaken in Parr shaker at room temperature and 52 psi H: for 72 h. The reaction mixture was liilered through a pad of Celiie and the filtrate was concentrated under diminished pressure to afford the amine 95 as a waxy solid which was used directly in the next step: yield 0.88 g (92%); silica gel TLC ¾ 0.14 (1:3 hexanes-ethyl acetate); Ή NMR (CDC ) 5 1.42 (s, 27H), 1.58 (t, 6H, J= 8.4 H¾) and 2.22 ft, 6H, J = 8.4 Hz); "C NMR (CDCb) and 173.0.
Figure imgf000118_0001
{0315 j -(3~Be zyJoxycarb0iiyiainM^
b«toxycarb iiyi-ethyl)-iiepia«edioic Acid Bi-fert butyi Ester (97), To a solution of 0.84 g (2.02 rnmol) of compound 95 and 0.43 g (.1.91 rnmol) of€ ζ-β~ alanine (96) in 15 mL of dry DMF were added 0.74 g (1.95 mmol) of ΗΑΤϋ and 0.82 g (3.82 nimol) of proton sponge. The resulting yellow mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under diminished pressure and the residue was dissolved is 80 ml, of ethyl acetate. The ethyl acetate layer was washed with two 40-trsL portions of 2 M aq HCI, two 30- m.L portions of IhO, and 20 mL of brine, then dried over anhydrous ajSC , filtered and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (1:2 * 3 cm). Elation with 1 : 1 hexanes-ethyl acetate gave compound 97 as a colorless solid: yield 1 , 17 g (98%); silica gel TLC _¾ 0.400:5 hexanes-ethyl acetate); ¾ NMR CDCh) δ 1.42 (s, 27H), 1.94 (t, 6H, J '= 8. Bz), 2.19 (t 6H, J = 8.4 Hz), 2.34 (m, 2B1 3.44 (m, 2H), 5.09 (s, 2H), 5.57 (brs, 1H), 5.99 (brs, 1H) and 7.32 (m, 5H); 13C NMR (CDC ) 28.1, 29.8, 30.0, 36.8, 37.3, 57.8, 66.6, 80.8, 128.0, 128.5, 136.7, 156.6, 70.9 and ] 72.9; mass spectrum (ESI), m/z 621.3753 (M + H)* (C55H53 2O9 requires m/z 621.3746).
Figure imgf000118_0002
hep anediotc Acid (98). A solution of 1.21 g ( 1.93 mmol) of 97 in 25 mL of formic acid was stirred at room temperature for 12 h. The reaction mixture was concentrated under diminished pressure. The residue was co-evaporated with six 10-mL portions of toluene to afford the tri-acid 98 as colorless oil: yield 0.91 g (.1 0%); iH NMR (C¾OD) δ 2.01 (m, 6H), 2.26 (m, 6H), 2.40 fro, 2H), 336 (m, 2H), 5.07 is, 2H) and 7,31 (m, 5H); l3C NMR (DM$Q-d6) δ 28.1, 29.0, 36.2, 37.3, 56.4, 65.2, 127.71 , 127.75, 137.2, 156.0, 170.0 and 374.5; mass spectrum (ESI), m z 453.1886 (M. + Hf (C2iH29N><¾ requires m/z 453.1868).
4 3~Beaz Iox carbo«yta^
yloxj carbonyI)ethyi|iieptaiiedioic Acid Bi$-(A-hydroxysuccinlmide) Ester (99). To a solution, of 0.48 g (1.06 mmol) of compound 98 and 0.44 g (3.82 mmol) ofA-bydroxysuccmimide in 9.00 mL of dry THF at 0 C was added drop wise a solution of 0.83 g (4.03 mmol) of CC in 2,00 mL of dry THF. The reaction mixture was stirred at 5 ¾C for 1 h. The reaction mix Care was concentrated under diminished pressure and the residue was suspended in 10 mL of aceioaitrile. The suspension was filtered and the filtrate was concentrated under diminished pressure. The residue was the purified by crystallization from absolute ethanol to afford 99 as colorless crystals: yield 366 mg (46%); } H NMR (CD3CN) 5 2.08 (m, 6H), 2.3.1 (m, 2H), 2.58 (m, 6H), 2.74 (s, I 2H), 3.28 (m, 2H), 5.02 (s, 2H), 5.73 (brs, I H), 6.10 (brs, 1 H) and 7.32 (m, 5H); C NMR (CD3CN) 3 25.9, 26.3, 29.5, 37.0, 37.9, 58.0, 66.7, .128.6, 128.7, 129.3, 138.3, 157.2, 169.8, 171.0 aud 172.1 ; mass spectrum (ESI), m/z 744.2342 (M + H)'
Figure imgf000119_0002
requires m/z
744.2359).
U 8
Figure imgf000120_0001
|0317| TrimerBLM-disaccharide (101). H gas was bubbled through a mixture containing 18 mg (21 pmol) of 92 and a catalytic amount ofPd C in 5.0 mL of dry THF for 45 min. The reaction mixture was filtered through a pad of Celite arid the filtrate was concentrated under diminished pressure to obtain crude 100 as a colorless oil, which was used immediately in the next step: crude yield 14 mg; mass spectrum (MALDl) m/z 725.28 (M + H) ' (theoretical m/z 725.26).
{0318j To a solution containing 14 mg ( 19 pmol) of 100 and 20 tiL ( 15 mg, 0.14 nii ol) of t iethylamme in J .5 mL of dry DMF was added 1.6 mg (2.2 μπ οϊ) of 99 and the mixture was stirred at room temperature for 20 h. The reaction mixture was concentrated under dimioished pressure. The residue was purified by flash chromatography OH a silica gel column (14 χ 1 cm), Eluiio with 32:15:1 ~3* 1 1: 10: i cMorofomi-acetone-meihanol afforded trimerBLM-disaccharide 101 as a. colorless oil: yield 4,5 rag (81 %); silica gel TLC flf 0.60 (4:4:1 chloroform---- acetone-methanol); mass spectrum (MALDl), m/z 2595.11 (M + NaV" (theoretical ma 2594.90); mass spectrum (ESI), m/z 1.297.4575 (M + H ÷ Na)"
(CtosHissNsO^ requires mh { 297.4529}.
Figure imgf000121_0001
|0319J TrimerBLM-disaeeharide tinker (102). To a solution of 5.0 mg (1,94 μπιοΐ) of 101 in 2 niL of dry MeQH was added 0.3 mL of a 25% w/w solu tion of NaGMe in MeOH, The reaction mixture was shaken at room temperature for 2 h. One hundred rag of Dowex SOW resin was added and die .mixture was shaken at room temperature for 30 mm. The mixture was filtered., diluted to 5 mL with methanol and a catalytic amount of Pd C was added, ¾ gas was bubbled through the mixture for 3 mm and the mixture was filtered. The filtrate was concentrated to obtai compound 102 as colorless solid: crude yield 2.6 mg (80%).
{&320j Example 15: Synthesis of Monosaccharide-iinker 104
{03211 Scheme 15 1} MisOWte.
Figure imgf000122_0001
0322) 2,4,6-tri- -aeeryi-3~0-(carbam Benzyl 2-
(Erhoxy)ethylcarbamate (103). To a aoSoiion of 121 mg (0.21 ramoi) of 89 and
45 mg (0.19 niraol) of 54 in 3.5 r»L of anhydrous CHjCh was added 68 pL (83 mg, 0.38 rarool) of TMSOTf at 0 °C, The reaction mixture was stirred at 0 °C for 20.rain, at which time it was poured into a two-phase solution, of EtOAc (70 mL) and saturated aq NaHCO* (28 mL), The organic layer was washed with two 28-mL portions of brine, dried (NasSO*), filtered and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (25 * 2,5 era). Elution with 1: 1 1:2 -> 1 :3 hexanes-ethyl acetate afforded compound 103 as a colorless oil; yield 95 mg (80%); silica gei TLC ¾- 0.26 (1 :3 hexanes-ethyl acetate); Ή NMR (CDCU) δ 2.02 (s, 3H), 2.08 (s, 3H), 2.1.2 is, 3H), 3.39 (m, 2H), 3.54 (TO, 2H), 3.64 (m, 3H), 3.79 (m, 1 HQ, 4.08 (m, 2H)t 4.26 Cm, !H), 4.71. (br s, 2H), 4.91 (s, IB), 5.10 (s, 2H), 5.25 (m, 3H), 5.37 (br s, 1H) and 7.35 (m, 5H); {3C NMR (CDC ) 8 20.8, 20.9, 21.0, 41.1 , 62.7, 66.5, 66.8, 67,3, 68,5, 70.0, 70.1, 70.3, 70.4, 77,4, 97.6, 128.1, 128,2, 128,6, .136.8, .155.3, 1.70.1, 170,2, 170.8.
Figure imgf000122_0002
{0323) 3- -(carbamoyi)-«-0-snaneopyraiJOsyi 2-(2-amittoet oxy)etIiaitol 104. To a solution of 4.60 rag (8.06 praol) of compound 103 in 2 mL of anfa methanol was added a freshly prepared solution of 0.4 M sodium merhoxide in methanol. The reaction mixture was allowed to stir at room temperature for 3 h, and the complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for IS mm and filtered: mass spectrum (MALDI), mfz 467.27 (M ·*· Na} ' {theoretical m 444..17). To the solution of the crude product in methanol was added Pd C and ¾ gas was bubbled through for 1 fa. The complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was filtered through Celile 545* and then concentrated under diminished pressure to afford 104, which was used for the next reaction; mass spectrum (MALDI), m/z 31 1.12 (M +· H)* (theoretical m/z 310.14). (03241 .Example 16: Synthesis of monosaecharide-trimer linker 107
Figure imgf000123_0001
RsHNOC"'" -''t -'"'" 'CONH "
Figure imgf000123_0002
CONHR"
107
12:
Figure imgf000124_0001
[0326j Trimer BL -mon ©saccharide (1.Θ6). H gas was babbled through a mixture containing 36 mg (21 μηιοΐ) of J 03 and a catalytic amount of Pd C in 6 mL of dry THF for 45 rain. The reaction mixture was filtered through a pad of Celtic and the filtrate was concentrated under diminished pressure to obtain crude 105 as a colorless oil, which was used immediately in the next step: crude yield 27 mg (99%); silica gel TLC ii 0.29 (1 :3 hexanes-ethyl acetate); mass spectrum (MALDi), m/∑ 459.26 (M + Ma)"'; mass spectrum (APCI), 437.1768 (M - Hf (CnH29N2Ou requires m/2437.1772).
|02 6| To a solution containing 27 mg (61.8 μνηοί) of 105 in 0,53 mL of dry DMF, 13 pL (0,09 mmoi) of triethylamine was added 15.2 mg (204 praol) of 99 were added and stirred al room temperature for 24 h. The reaction mixture was concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (1.5x1 cm). Elution with
1 : 12: 1 -> 1 1 : 12: 1 chioroform-acetone-methanol afforded trimer BLM monosaccharide 106 as a colorless oil: yield 15 mg (43%); silica gel TLC Rf 0.56 (4:4:1 chioroform-acetone-methanol); mass spectrum (MALDi), m/z 1730.76 (M + Na)*; mass spectrum (TOF), m/z 854.3351 (M + 2H)*'*
Figure imgf000124_0002
requires m/z 854.3357).
Figure imgf000125_0001
[0327j Triiner BLM menosacehar e-Mraker (107). To a solution of 4.2 mg (2.46 μηιοΐ) of 106 in 2 ml, of auh methanol was added 0.2 raL of 25% w/w freshly prepared solution of sodiitm methoxide in methanol. The reaction mixture was allowed to stir at room temperature for 2.5 h, and the complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for 15 min and filtered; mass spectrum (MALDl), m/z 1351.40 (M + Na}' (theoretical m/z 1328.56). To the solution of the crude product in methanol was added Pd C and U-> gas was bubbled through for 45 min. The complete consumption of starting materia! was confirmed by M ALDI-TOF mass speciral analysis. The reaction mixture was filtered through Cefite 545* and then
concentrated under dknioished pressure to afford 2.9 mg of 107 (quant.), which was used for the next reaction; mass spectrum (MALDl), m/z 1217.62 (M + Na)*; mass spectrum (TOP), m/z 1229,4961 (M + CI)" (C H^NsO^Cl requires m/z 1229.4927).
[03281 Exam le 17: Synthesis of decarbamoyl monosaccharide linker 112 |0329| Scheme 17 M
Figure imgf000126_0001
109
I . NaOMe. MsOH NHCfc* 2. Ewwex SO
3. paic. m
Figure imgf000126_0002
118 111
Figure imgf000126_0003
112
Figure imgf000126_0004
{0330] Fcnta- -acctyf-iZ-D-niami p ranose (108). To a solution containing 1.00 g (5.15 rnmol) of O-mefhyl-ct-D-mannopyranose in 18.9 mL of Ao;G, was added a catalytic amount of H SO,s, and {he solution was stirred at room
temperature for 12 . The reaction mixture was poured into a stirred mixture of 150 niL of ethyl acetate and 80 mL of said aq NaHCOj. The organic phase was separated and washed with 40 mL of satd aq NaIiC<¾, 30 mL of brine, then dried (NajSO.j) and concentrated under diminished pressure. The residue was purified fay flash chromatography on a silica gel column (5x18 cm). Elntion with 5: 1 -^3: 1 hexanes-ethyl acetate afforded 108 as a colorless oil: yield 1.97 g (98%); silica gel TLC Rt 0.60 ( 1 ;2 hexaoes-ethyi acetate); Ή NMR. (CDCS3) 61.86 (s, 3H), I .91 (s, 3H), 1.95 (m5 3H), 2.04 (m, 6H)5 3.94 (m, 2H), 4..I3 (mt iH), 5.1 2 (s, IH), 5.20 ( , 2H) and 5.94 (s, H); 13C NMR (CDCfe) 3 20.40, 20.43, 20.47, 20.53, 20.6, 1.9, 65.3, 68.1, 68.6, 70.4, 76.8, 169.5, 1 9.7 and 170.3.
Figure imgf000126_0005
|0331j l^^^Tetra-O- cet l-^D-tnattnopy aiios i Bipheuji Phosphate (110), To a solution of 525 rag (1.34 mmol) 308 in 8. j niL of dry DMF, was added 370 mg (1.88 mmol) of hydrazine acetate. The reaction was stirred at room temperature for 2 h until analysis by silica gel TLC indicated it was complete. The reaction mixture was diluted with 50 mL of ethyl acetate and washed with three 20-mL portions of brine. The aq layer was re-extracted with three 30-m'L portions of ethyl acetaie. The combined organic layer was dried
Figure imgf000127_0001
and concentrated under diminished pressure and dried to afford compound 109 as a colorless oil; yield 397 mg (85%); silica gel. TLC Rf 0.39 (3:1 hexanes-ethy! acetate).
|Θ332| To a solution of 397 mg (1.14 mmol) of 109 in 16,5 mL of dry CM ;>0;?, 180 mg (1.47 mmol ) of DMAP and 1.6 ml (1 1.4 mmol) of B . The react ion mixture was stirred for 1 mm, followed by the addition of 2.3 ml. (10.9 mmol} of diphenyl chJorophosphate drop wise at °C. The solution was siirred at 0 X for 1.5 h and was poured into a two-phase solution of EtOAc (200 mL) and saturated aq NaHCOs soln (80 mL). The organic layer was washed with two 50-mL portions of brine, dried over N aSC^. filtered, and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (18x5 cm). Ekiiion with 3:1 ~>2:1 hexanes -ethyl acetaie afforded compound 110 as a colorless oil: yield 424 mg (54% over two steps); silica gel TLC Rt- 0.54 (3: 1 hexanes-ethyl acetate): Ή M.R (CDCU) 5 2.04 (s, 3H), 2.06 (s, 3H), 2.10 (s, 3H), 2.22 (s, 3H), 3.98 (dd IH, J ~ 12.4 and 2.0 Hz), 4.14 (ra, IH), 4.25 (dd, 1H, J ~ 12.4 and 4.8 Hz), 5.40 (ra, 3H)5 5.92 (dd, l.H, ,/ ::: 6.8 and 1.6 Hz), 7.28-7.33 (m, 6H) and 7.40-7.45 (m, 4H); ¾ NMR (CDCI3) δ 20.70, 20.72, 20.78, 20.84, 61.8, 65.2, 68.3, 68.7, 68.8, 70.9, 96.17, 96.22, 120.18, 120.22, 120.3, 120.4, 125.90, 125.91, 126.0, 130.1, 130.2, 169.6, 169.9 and 170.7.
Figure imgf000127_0002
{OSSSJ 2 i4,6-Tetra- -acet> -«-0-mannt)pyrant)syl benxyl 2-(2- ctho:sy)eihjkarbainaie (til). To a solution of 300 mg (0.52 mmol) of phosphate ester 110 and 1 1 1 mg (0.46 mmol) of the alcohol 54 in 5.5 mL of anhydrous CHiC , was added 1 8 uL (207 mg, 0.93 mmol) of TMSOTf at 0 X,. The reaction was stirred at 0 aC for 18 mm and was then poured into a two-phase solution of EtOAc (1 0 ml) and saturated aq NaHCO } (40 mL). The organic layer was washed with two 40-raL portions of brine, dried { ajSQt), filtered and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica ge! column (2.5x25 cm). ESotion with 2: l-> 1 :2 hexanes-ethyl acetate afforded compound ill as a colorless oil: yield 1 10 rag {37%); silica gel TLC l¾/ 0.35 (1 :3 hexanes-ethyl acetate); !H N R <CDC¾) δ l ,6I (s, 1 H), 1.96 (s, 311), 1.98 ($, 3H), 2.06 (s, 3H), 2.11 (s, 311), 3.38 (m, 2H), 3.53 (m, 2.B.), 3.63 (m, 3H), 3.77 (m, TH), 4.05 (m, IB), 4.09 (m, 1H), 4.24 (dd, 1 H, J - 12.4 and 5.2 Hz), 4,87 (d, 1 H, J - 1.2 Hz), 5.08 (s, 2H), 5.22 (m, 1H), 5.26 (m, 1H), 5.31 (br s, 1 H), S.34 (m, 5H), 7.26-7.34 (m, 5H); °C NMR (CDC¾) δ 20.80, 20.82, 20.9, 21.0, 39.4, 41.1, 62.7, 66.4, 66,8, 67.2, 68.6, 69. 1 , 69.8, 70.1 , 70.4, 97.7, 128.2, 128.6, 136.8, 169.9, 170.0, 170.3, 170.8 and 170.9; mass spectrum (MALDI), m/z 592.34 (M + af ; mass spectrum (APCI), m/z 570.21 2 (M + E)"" (Cjfii-lsft Oi , requires m/z 570.2187).
Figure imgf000128_0001
[0334| Decarbamoyl BLM monosaccharide-d e Conjugate 112. To a solution of 8,9 mg (1 5.6 pmol) of compound ill in 2 mL of as methanol was added, 0.2 ml. of 25% / freshly prepared solutio of sodium meihoxide in methanol. The reaction mixture was allowed to stir at room temperature for 2.5 b, and the complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. The reaction mixture was then quenched by the addition of 500 mg of Dowex 50x resin, shaken for 15 mir> and filtered; mass spectrum (MALDI), m/z 424.24 (M ·*· Naf; Mass spectrum (APCi), m/z 402.1759 (M + HY
(CisH;¾NO<} requires m z 402 J 764). To the solution of the crude product in 5 mL methanol was added Pd/C and IT gas was bubbled through for 45 min. The complete consumption of starting material was confirmed by MALDI-TOF mass spectral analysis. Th reaction mixture was filtered through Ce!ite 545*" and then concentrated under diminished pressure to afford 112, which was used for the next
1.27 reaction; mass spectrum (MALDl), m/z 268.25 (M + HX 290.25 (M + Na)v; mas spectrum (AFCI), m z 268.1391 (M + f (Cj0H22NO? requires /z 268.1396).
[0335| Example 18: Synthesis of Cauiptotherin (CPT)-saceharide conjugates 1 15, 116, 117 and 118
[03361 Scheme 18
Figure imgf000129_0001
Figure imgf000130_0001
[03371 CPT-carbamate (1 4). To a solution of 40 mg (0. 1 1 mmol) of 113 m 0.42 raL of pyridine was added 56 mg (0.46 mmol) of DMAP and 92 mg
(0.46 mmol) of/niitrophenyl chloroformaie and was stirred at 40<J C overnight. The solution was cooled and poured into a two-phase solution of 210 mL EtOAc and 3 mL of H 0. The organic layer was washed with three 5 mL portions of IN HCL 5 mL of saturated aq. NaHCQj nd 3 nil- of brine. The solution was dried over MgSiXs, filtered, and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica ge! column (2.5x20 cm). Eiution with 15: ! ~>S: 1 chioroform-meihanoi afforded. 1.1.4 as a light yellow solid: yield 43 mg (73%); silica gel TLC ¾ 0.30 (?: i hexanes-ethyl acetate). Ή NMR (500 MHz, CDCI3) δ 1 .07 (ffi, 3H), 2.25 (m, IH), 2.37 <m, i H), 5.31 (m, 2E 5.42 (d, l H_ ,/ = 16.5 Hz), 5.72 (d, 1 H, J- 17.0 Hz), 7.40 (m, 2H), 7.70 (ra, .IH), 7.86 (m, I H), 7.96 (d, I H, J = 7.5 Hz), 8.23 (ra, 3H), 8.42 is, I H).
Figure imgf000130_0002
[0338 j CPT-carbaraate-monosaccharide (J 15) To 6 mg (1 .47 uraol) 104 in 1.75 ml, of THF-MeOH-DMSO (2:4: 1 }, was added 5 mg (9.74 μιηοΙ.) of 114 and stirred overnight. The reaction mixture was purified on an Econosil Ci« reversed phase serai-preparative (250 x 10 mm, 10 p.m) HPLC column using aq 0.1% TFA and€H iC mobile phases. A linear gradient was employed (99: 1 0.1 % aq TFA~€H3C -»60:40 0.1% aq TFA-CHjC ) over a period of 18 min at a flow rate of 4.5 niL/mtn. The fractions containing the desired product eluted at 1 .9 min (monitoring at 364 nm) and were collected, frozen and lyophilized to give 115 as a light yellow solid: yield .1.5 mg (22%); mass spectrum (MALDl), m/x 685.72 (M + Na .
Figure imgf000131_0001
[0339] CPT-carbarnate~dtsaccharide (116)» To 6.6 mg ( 13.97 pmol) 3 in 1.8 niL of THF-MeOH (2:1), was added 3.6 mg (7.01 μπ οϊ) of 114 and stirred overnight. The reaction mixture was purified on an Eeonosil C¾s reversed phase semi-preparative (250 x .10 mm, 10 μηι) MPLC column using aq 0.1% TFA and CHjCN mobile phases. A linear gradient was employed (99: 1 0.1% aq
TFA™C¾C - 60:40 0.1% aq TFA-CFfeCN) over a period of 18 mm at a flow rate of 4.5 mL/mtn. The fractions containing the desired product eluted at 13.8 rain (monitoring at 364 nm) and were collected, frozen and lyophiltzed to give 116 as a white solid; yield 1.8 mg (30%); mass spectrum (MALDlj, m z 869.43 (M + Na)f ; mass spectrum (ES!), m z 869.2706 CM + Na)"
Figure imgf000131_0002
mix 869.2705),
Figure imgf000131_0003
|034O| CPT arbaraate-iris«er-iMOoosaccharide (117). To 93 mg (7.78 pmol) 107 in 1.5 mL of THF-MeOH-D SO (2:4:1), was added 2 mg (3.90 μιηοϊ) of 114 and stirred overaight. The reaction mixture was purified on an EconosH Cn reversed phase semi-preparative (250 χ 10 mm, 10 μηι) HPLC column using aq 0.1% TFA and C¾CN mobile phases. A linear gradient was employed (99:1 0, 1% aq TFA~C%CN-»60:4O 0J% aq TFA--CH3CN) over a period of ] 8 min at a flow rate of 4.5 mL/min. The .tractions containing the desired product elated at 16,5 min (monitoring at 364 ran) and were collected, frozen and lyophilszed to give 117 as light yellow solid: yield .1.2 mg (20%); mass spectrum (MALDi), m z 1591.89 ( + , 1 07.88 ( + *.
Figure imgf000132_0001
[0341 S CPT-carbaraate-decarbainoySmoitosacciiaiide (t 1 §). To 5.2 mg ( I ,47 μτηοΐ) 112 in 1.5 mL of THF-MeOH (2;1 was added 5 mg (9,74 μηιοί) of 114 and stirred overaight. The reaction inrxtiire was purified on an Econosil C« reversed phase semi-preparative (250 χ 1 mm, 10 μηι) HPLC column using aq 0. % TFA and C¾CN mobile phases, A linear gradient was employed (99: 1 0.1 % aq l A-CH;sC - 60:40 0.1% aq TFA-CHjC ) over a period of 18 min at a flow rate of 4.5 mL/min. The fractions containing the desired product eiiited at 15.2 min (monitoring at 364 nm) and were collected, frozen and lyophili ed to give 118 as a white solid: yield 1.8 mg (29%); mass spectrum (APCI), m/z 642.2280 ( + Hf (C.nHjsNsOis requires m/z 642.2299).
|0342| Example 1 : Synthesis of Camptothecin (CPT)-$accharfde ester conjugates 122 and 123 [0343 j Scheme 1
Figure imgf000133_0001
¾ ¾3 120 121
Figure imgf000133_0002
X = 1G4 12a R = R. <17¾ over two steps) X = 3 123 - Rj ( 12% over two steps ί
Figure imgf000133_0003
Figure imgf000133_0004
[0344J tert-btityl CPT-succinate diester (120). To a solirtion of 198 nig (3.13 ramot) of 117 in 12 mL of dry CS¾(¾ was added 93 rag (0.76 mmo!) of DMAP, 0.18 mL (145 rag, 1.15 nimoi) o dusopropylcarbodiimide and 200 nig (0.57 ramoi) of 113. The solution was stirred at room temperature overflight, diluted with iO mL C¾C¾ and washed with 3 mL of 0.1 N HCI solution, then dried over MgSO, , .filtered, and concentrated under diminished pressure. The residue was crystallized from, methanol, filtered, washed with cold MeOH and. dried to afford 120 as a yellow solid: yield 170 rag (56%); silica gel TLC .¾■ 0.30 (12: 1. chloroform-MeOH); ¾ NMR (400 MHz, CDC¾) δ 0.99 (1, 3H, J- 7.2 Hz), 1 .36 is, 9H), 2. 15 (m, 1 H), 2.26 (m, IH), 2.56 (m, 2H), 2.78 (m, 2 ), 5.26 (d, 2H, ,./ = 3.2 .¾), 5.38 (d, 1 H, = 1 7.2 Hi 5.68 (d, IH, J = 1 7.2 Hz), 7.31 (s, IE), 7.66 Cm, 1 H), 7.82 (m. 1 E), 7.92 (d, 3 H, J ~ 8.4 Hz), 8.22 (d, 1 H, J = 8.4 Hz) and 8.37 (s. 1H); J?C NMR (CDCk) 6 7.75. 28.08, 29.27, 30.18, 31.90, 50.02, 67.15, 76.29, 81.06, 96.60, 120.19, .128.09, 128.26, 128.30, 128.57, 129.86, 130.68, 131.18, 146.19, 146.29, 149.03, 152,56, 157.52, 167.57, 171 .16 and 1 71 .64.
Figure imgf000134_0001
[03451 CPT-succinate acid (121) To 134 mg (0.27 mraol) 120 in 1.5 mL of CH2CI2, was added 0.6 μΐ. of TFA and the reaction mix toe was stirred for 5h. The reaction mixture was conceniraied under diminished pressure and ctysialiized from methanol, then filtered, washed with MeOH and. ether, and dried to afford Ϊ 21 as a pale yellow solid: yield 98 mg (82%); silica gel TLC ¾ 0.29 (12: 1 chloroform- MeOH). 5H NMR (400 MHz, DMSO~t¾) 8 1.05 (m, 3H), 2.29 (ni, 2H), 2.61 (m, 2H), 2.90 (m, 2H), 5.41 <d, 2H, 1.2 Hz), 5.62 ($, 2H), 7.26 (s, 1H), 7.84 (t, IH, J - 7.2 Hz), 8.00 (m, IH), 8.25 (d, 1H, J = 8.0 Hz), 8.30 (d, 1H, J = 8.4 Hz) and 8.80 (s, I H); C NMR (DMSO-t ) S 7,52, 28.37, 28.56, 30.39, 50.17, 66.29, 75.87, 95.1 1, 1 1 8.90, 127.68, 127.95, 128.50, 128.99, 129.75, 130.37, 131.52, 145.25, 145.90, 145.88, 152.37, 1.56.52, 167.16, 1 71 .26 and 172.97. {0346]
Figure imgf000135_0001
1 iimol) 121 in 0.15 mL of DMSO was added 1 .5 mg (13 μιηοΐ) of iV-hydroxysuccinimide and 1.7 ml. (1.4 mg, 1 1 pmol) of diisopropyicarbodiimide and die reaction mixture was stirred at room temperature. After 24 h, 3.5 pg (1 1 pmo!) of 104 in 0.5 ml of 1 : 1 0.2 M sodium phosphate buffer and DMSO was added, and the reaction mixture was stirred overnight. The reaction mixture was purified on an Econosi! Cm reversed phase semi-preparative HPLC column (250 ■* 10 ram. 10 μιη) using aq 0.3% TFA and CFbCN mobile phases. A linear gradient was employed (99:1 0.1% aq TFA-C¾CN -> 60:40 0.1% aq TFA-C¾CN) over a. period of 18 min at a flow rate of 4.5 ralJm n. The f actions containing the desired product elated at 18.8 mirt (monitoring at 364 nm) and were coliecled, frozen and lyophilized to give 122 as a light yello solid: yield 1.4 mg (17% over two steps); mass spectrum (MALDI), m/z 778.68 (M + K) \
Figure imgf000135_0002
|Θ347| CPT-carbamate^isaccharide (123). To 2.0 mg (4.5 μηΐοϊ) 121 in 0, ! 5 m'L of DMSO was added 1.0 mg (S.9 μιηοί) iV~hydroxysuccinirnide and 1.4 μΐ. ( 1, 1 mg, 8.9 μ∞ο1) of diisoprop lcaibodiimide and the reaction mixture was stirred at room temperature. After 24 h, 3.2 mg (6.77 μιηοί} of 3 in 0.4 mL of I : 1 0,2 M sodium phosphate buffer, pH 8,0, and DMSO was added, and then the reaction mixture was stirred overnight. The reaction mixture was purified on an Eeonosil C;s reversed phase semi-preparative RPLC column. (250 χ 1 mm, 10 μτη) using aq 0.1% TFA and CH?CN mobile phases. A linear gradient was employed (99:1 0.1% aq TFA-CH3CN -> 60:40 0.1% aq TFA-CHjCN) over a period of 18 rain at a flow rate of 4.5 mL-Zmin. The tractions containing the desired product elated at 17.9 min (monitoring at 364 nm) and were collected, frozen and iyophiiized to give 123 as a light yellow solid: yield 0.5 mg (12% over two steps).
{0348J Example 20: in Vitro Cell Growth Inhibition Tests
[0349J A methotrexate resistant cell line, DU i45(MT'X) was developed from parental DU 145 cells by exposure to increasing concentrations (starting at 0, 1 μΜ) of methotrexate over a period of 6 mouths. DU J45( TX) was at least 250- fold less sensitive to methotrexate than parental cells. After five passages in drug-free medium, the resistant" ceils retained their drag resistance, suggesting the stability of the cell line.
{0350} Cell culture. The human prostate cancer cell lines DU-1 5 (ATCC) was cultured in MEM media with glutamine and supplemented with 10% fetal bovine serum, penicillin (KKHJ/mL) and streptomycin (lOOU/mL) in air enriched with 5% €(¾ at 37° C.
(03511 In Vitro Cell Growth Inhibition Tests. Cell viability wa determined using the MTT assay. Briefly, cells were seeded in %-weIl plates at a density of 3000 cells per well in 0.1 mL MEM media supplemented with 10% fetal bovine serum, and test compounds were concomitantly added in a concentration range of 0,001-1000 μΜ. Dilutions were made using the culture medium from .1 mM stock solutions of the drugs in anhydrous DMSO. Controls were treated wit an equivalent amount of solvent, diluted as above, to assess the absence of toxicity due to the solvent. After 72 h of incubation, 15 μΐ. of 5 mg mL'1 MTT dye (3- 4,5- dimethylthiazol-2-y!]-2s5- diphenyltetrazoliurn bromide, sigma) was added for 4 h at 37'3C. Media was removed and the monolayer suspended in 0.15 mL of DMSO, after which the absorbance at 570 nm was measured using a microplate reader. The control value corresponding to untreated ceils was defined as 1 0% and the viability of treated samples was expressed as a percentage of the control .
Table 1. Cytotoxicity of MTX-Disaccharide Conjugates Toward MTX Resistant DU-145 Cells
Compound DU-145-MTX
(% cell survival)
Methotrexate (MTX)
100 H 82
1 μ.Μ 84
10 μ.Μ 83
Compound 5
100 uM 84
1 μΜ 69
10 μΜ 4 !
Compound 15
1 (H) tM 83
1 μΜ 60 Ι Ο μΜ 56
(0352| While particular materials, formulations, operational sequences, process parameters, and end products have been set .forth to describe and exemplify this invention, they are not intended to be limiting. Rather, it should be noted by those ordinarily skilled in the art that the written disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited, to the specific embodiments illustrated herein, but is limited only by the following claims.

Claims

What is Claimed is:
A Siigar-Iinker-drug conjugate of formula. (I)
A-BHL-D (I)
n
or a pharmaceutically acceptable salt thereof
wherein A is:
Figure imgf000138_0001
R} is selected from the group consisting of H, OH, SH, Ν¾, OR4, OC(0)R4, 0C(0)NHR4, OC(0)NR4R4. OC(S}NHR , OCiSJN +Rj, SC(0)NHR4, SC(0)NR4Rs> NHC(0) HRi, NHC(0}NRR5, NHC(S)Ni:iR4i MHCiS)NR4RS; NHC( } HR4, NHC(N)NR4R5, OC¾C(0) Rf OC¾C<0) R R5,
OCH2C(S)NHR4, OCH2C(S)NR4Rs, 80Η20(0}ΝΗ¾! SCH2C(0)NRR5, NBCH3C(0)NHR4,NHC¾C(0)NR4R5, HC¾C(S)NHR and
Figure imgf000138_0002
each R is selected from the group consisting of H, Ct-Q, alky!, (¾-
C7> alkenyl and€;■■ .'■, alkyn !;
each .5 is selected from the group consisting ofCj-O alkyl C2-G5 alkenyl and C>-( \ al.kyn.yl;
;> is selected from the group consisting of H, OH, SH, NH.?, OR4, OC(0)R! 0C(0)NHR4, OC(0)NR4R5, OC(S) HR , 0C(S)NRR5, SC{0)NHR4, SC(0)NRjR5, NHC(0)NHR4, H0O)NR4 .S, NI:iC(S)Niffi4, NHC(S) RR5, NHC(N)NHR4, NHC(N)NR4R:5, OCH2CfO) liR4, OC¾C(0)NR4R5i
OCH2C{S)NHR4, OCH2C(S)NRR5, SCH2C(0)NHR4, SCH2CfO) R4R5, NHCH2C(0)NHR4, NHC OJ fURs, NHC%C(S)NHR4 and
NHC¾C(S)NR4R5;
13? R;> is selected from ihe group consisting of H, OH, SH, NH¾ OR.*, OC(0)¾, OC(0)NHR , OC(0}N¾R 5t OCiS) HR j OC(S)NR4R5, SC(0)NHR4> SC(0) :R4R;, HC(0) HR4, NHC(0}NR4RS> NHC(S>NHR, NHC(S)NR4R¾, NBC(N)NHR4, HC(N)NRt¾ OCH2C(0)NHR4, OCH2C(0)NR4R55
OCH2C(S)NHR45 OCH2C(S) R4 5; SCH2C(0)NHR4, SCH2C(0)NR4R5, • HCH2C(0) HR4, NHCH2C(O)NR4R5! HCe2C(S)NHR4 and
Figure imgf000139_0001
R' is selected from the group consisting of H, OH and NHR*;
8 is a. Spacer Unit;
n is an integer selected from 1 to 3:
L is absent or a Lioker; and
D is a Drug Unit having a chemically reactive functional group selected from the group consisting of a primary or secondary amine, hydroxy]-, suMhydryl, carboxvi, aldehyde and ketone.
The conjugate according to claim L wherein A is
Figure imgf000139_0002
The conjugate according to claim L wherein A is selected from the group consisting of;
Figure imgf000139_0003
4. The conjugate according to any of the previous claims. wherein A is:
Figure imgf000140_0001
5, The conjugate according to any of claims 1 to 3, wherein A is:
Figure imgf000140_0002
6, The conjugate according to any of claims i to 3, wherein A is:
Figure imgf000140_0003
The conjugate according to any of claims 1 to 3, wherein A
IS:
Figure imgf000140_0004
8. The conjugate according to any of the previous claims, wherein Rs is selected from the group consisting of H, OH, OR*, 0C(0)K4, NHC( )NHR4, HC(N)NR*R5.. OC(0)R<, OCONHR4, and (XONR4RS;
9. The conjugate according to any of the previous claims, wherein Rj is selected from the group consisting of H, Oil, O . 0€(0)R , NHC(N)NMR4, NHC(N)NR R OC(0)Rj, OCO HR4, OCO t j, OCS HR , NHCONHR4, OCHjCONHRt, and OCH2CO R R5,
10. The conjugate according to any of the previous claims, wherein R3 is selected from the group consisting of H, OH, OR4, OCfO)R:¾.
HC( )NHR$, NHC(N}NR4R5l OC(0)R,, and OCONHR4.
11. The conjugate according to any of the previous claims, wherein R* is R or OH.
12. The conjugate according to any of the previous claims, wherein each. R4 is selected from the group consisting of H, .methyl and ethyl.
13. The conjugate according to any of the previous claims, wherein each R> is selected from the group consisting of methyl, ethyl, and isobutyl.
14, The conjugate according to any of the previous claims, wherein A is selected from the group consisting of:
Figure imgf000142_0001
SR = H 98fi==H
46K = CH3 S3 R ~ GH^
Figure imgf000142_0002
64 = H 76R»H
6 R 55 CH^ 77 R ~ CHj
15. The conjugate according to any of ihe previous claims, w'hereio A is selected from the group consisting of:
Figure imgf000142_0003
Figure imgf000142_0004
Figure imgf000142_0005
16. The conjugate according to any of the previous claims, wherein A is:
Figure imgf000143_0001
117
17. The conjugate according to any of the previous claims, wherein the Spacer Unit is selected from the group consisting of a bond. Cj-Cao alkyl, C C¾t alkenyl, C C>o alkynyl, aryl, heleroar l. heterocyc!yl. C3-C5 cycioatkyi an oligoalky!ene glycol, an oligopeptide and a dendrimer.
1 8. The conjugate according to any of claims i to 1 6, wherein the Spacer Unit is X-(L'-Y)fri-L":-Z,
wherein X is CHj or O;
L! is CrQy alkyl;
Y is O, S, or NR% wiierein R- is hydrogen or Cj-€¾ alkyl;
m is an integer selected frora 1 to it);
L~ is Cs-Cjo alkyl, CJ-CJO alkenyl, C Cjo alkynyl, aryl, heieroaryl. heierocyc!yl, C3-C5 cycloal'kyl; and
Z is absent O, NR\ S, C(0), S(0), S{0)¾ OCiO), N(Rx)C(0>, N(Rx)S(0)5 N(Rx}S(Ok 0(0)0, C(0)N(Rx), S(0)N(R*), S(0)2N(R*), 0C(0)0, OC(0)N(RK), N(Rx)C{0}05 N(Rx)C(0)N(Rx), or .N(Rx)S(0)2N(Rs)> wherein each R¾ is independently hydrogen or Cj-Ce alkyl.
1 , The conjugate according to claim 18, wherein X is O, V is C2-C4 alkyl; L? is C C<s alkyl; and Z is a bond, O, NRX, S, C(O), SCO), or S(0},.
20, The compound of claim 18, wherein the Spacer Unit is O- (CH;?CHj-0)m-CH2CH3-2, wherein Z is O, N(H), or S and m is an integer selected from 1 io 20.
21. The conjugate according to claim 18, wherein the Spacer Unit is 0-(CH2CH2-0)w-CH2CH ¾ wherein Z is C(0) or S(<%> and m is an integer selected from I to 20.
22. The conjugate according to claim any of the previous claims, wherein L is absent,
23. The conjugate according to any of claims 1 to 21, wherein L is a non-c!eavable linker.
24. The conjugate according to claim 23, wherein L is (E-L*-F-
L - .
wherein each E is bond, O, R\ S, C(O), SCO), S(0)2, OC(O), N(RX)CC ), N(Rx)S(0), N(R*)SCO)2, 0(0)0, O)N(Rx), S(0)NCR*}, SCO^MCR*), OC(0)Of OC(0}NiRs), N(R*)C(0)0, N(Rx)C(0)N(Rx), or N(Rx)S(0)2 f R );
each V is Ci~C* alkyi;
each F is bond, O, NR*. S, C(O), S(0), SCO)j, OCCO), N(R*)C(0\ N(R*)S(0), N(Rx)S(0}2, 0(0)0, C(0)N(R*)5 S(0)N(Rx), S(0)2N{Rs}, 0C(0)0S 0€(0)N(R N(R*)C{0)0; H(Rs)C 0) (Rx), or N(R*)S{0)2N(Rx);
each I.4 is QrC , alkyi;
p is 1 or 2; and
G is a bond. O, NRS, S, C(0), 5(0), S(0)2> OC(0), N(Rs)C(0), N(R.*)S(0), N(R*)S{0)2, C(0)0, C(0)N(Rx), S(0)N(R*), S(0)2N(Rx), 00(0)0, 0C(0) (R N(Rx)C(0)0, N(R*)C(0)N(Rx), or N(R*)S(0)2NCRx),
wherein each R is independently hydrogen or Ct-Q. alkyi.
25. The conjugate according to claim 24, wherein E is NR* or
C(0);
if is C -Cs alkyi;
L4 is C«rC¾ alkyi;
p is i;
F is a bond, R , N(Rx)CCO)t 0C(0), C(0}0 or C(0)N(R*); and G is 0, S. C{0) orNR*.
26. The conjugate according to claim 24, wherein L is E is NR* or C(0);
1 is t C( alky I;
Figure imgf000145_0001
p is 2;
F is a bond, NR", N(R*)C(0)- OC(0), C(O}0 or C(0)N(R*); and G is O, S, C(O) or NR*.
27. A conjugate represented by formula:
Figure imgf000145_0002
28. A conjugate represented by formula:
Figure imgf000146_0001
29. The conjugate according to any of claims 1 to 21 , wherein L is a cieavabie linker.
30. The conjugate according to claim 29, wherein the cieavabie linker is cieavabie by acid-induced cleavage, light-induced cleavage, peptidase- induced cleavage, esterase-indoced cleavage, and disulfide bond cleavage.
31. The conjugate according to claim 29, wherein the cieavabie linker comprises a hydraxone, a cathepsin-B-cleavable peptide, a disulfide or an ester bond.
32. The conjugate according to any of claims 29 to 31 , wherein n is L
33. The conjugate according lo any of claims- 29 to 31. wherein n is 2.
34. The conjugate according to any of claims 29 to 31, wherein n is 3.
35. The conjugate according to any of the previous claims, wherein D is a drag selected from the group consisting of a cytotoxic drug, a cytostatic drug and antiproliferative drug.
36. The conjugate according to any of the previous claims, wherein D is an antitumor agent.
37. The conjugate according to claim 36, wherein D is an inhibitor of a cellular metabolic event,
38. The conjugate according to claim, 37, wherein D is an enzyme or protein inhi itor, such as an Hsp9 inhibitor or a protein kinase inhibitor.
39. The conjugate according to any of the previous claims, wherein D is an amino containing drug selected from the group consisting of mitomycin-C, mitomycin- A, daunombicin, doxorubicin. N-(5,5- diacetoxypentyl)doxorttbicin, aminopterin, actioomycin, bleomycin, 9-amino eamptothecm, H* -acetyl spermidine, l-(2 chloroethy 1)- 1 ,2-dimethanesuJfonyl hydra ide, iallysomycin, methotrexate, amsaenn, cis-platm, mercaptopurine and derivatives thereof.
40. The conjugate according to any of the previous claims, wherein D is a hydroxyi. containing drug selected from the group consisting of etoposide, cam.ptoth.ecin, taxoL esperamicin, 1 ,8-dihydroxy-bicyc!o('7.3.1 jtrideea- 4,9-diene-2,6-diyne- 13-one, anguidine, doxorubicin, inorphoiino-doxorubicin, N- (5,5«diaceloxypenty1)doxorabkm, vincristine, vinblastine, bleomycin, teniposide, podophy'ilotoxin and derivati ves thereof.
41. The conjugate according to any of the previous claims, wherein D is a suKhydryl containing drug selected from the group consisting of esperamtcin, 6-mercaptopurine, or derivatives thereof.
42. The conjugate according to any of the previous claims, wherein D is carhoxyl containing drag selected .from the group consisting of methotrexate, campiothecin (ring-opened form of the lactone), butyric acid, re iiioie acid, and derivatives thereof.
43. The conjugate according to claim 41 , wherein D is methotrexate.
44. The conjugate according to any of the previous claims, wherein D is an aldehyde containing drag, such as, cinnanaaldehyde, inoske diaidehyde, and digiycoaldehyde.
45. The conjugate according to any of the previous claims, wherein D is a ketone containing drag, such as anthracycUne or an epotlul.one.
46. A pharmaceutical composition comprising a conjugate of formula (I) according to an of the previous claims and a pharma.ceutica.liy acceptable carrier.
47. A method of treating cancer in a patient comprising administering to a patient in need thereof a conjugate according io any of claims I - 45 or a pharmaceutical composition according to claim 46.
48. The method according to claim 47, wherei the sugar moiety binds to a cancer ceil, the drug is releasable from the sugar moiety at or near the cancer b cleavage of the linker and the drug, when released, is cytotoxic or cytostatic to the cancer cell .
49. A method of reducing the toxic side effects of administering a drug to treat cancer, comprising administering to a patient an effective amoom o a conjugate according to any of claims i-43 or a pharmaceutical composition according to claim 46.
50, The method according to claim 49, wherein, the sugar moiety binds to a cancer cell, the drug is releasable from the sugar moiety at or near the cancer by cleavage of the linker and the drug, when released, is cytotoxic or cytostatic to the cancer cell.
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