CN108289964B - Novel linkers and their use for specific coupling of drugs and biomolecules - Google Patents

Novel linkers and their use for specific coupling of drugs and biomolecules Download PDF

Info

Publication number
CN108289964B
CN108289964B CN201580082582.5A CN201580082582A CN108289964B CN 108289964 B CN108289964 B CN 108289964B CN 201580082582 A CN201580082582 A CN 201580082582A CN 108289964 B CN108289964 B CN 108289964B
Authority
CN
China
Prior art keywords
cell
acid
drug
binding agent
receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201580082582.5A
Other languages
Chinese (zh)
Other versions
CN108289964A (en
Inventor
赵珞博永新
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Dac Biotech Co Ltd
Original Assignee
Hangzhou Dac Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Dac Biotech Co Ltd filed Critical Hangzhou Dac Biotech Co Ltd
Priority to CN202210788010.0A priority Critical patent/CN115300640A/en
Publication of CN108289964A publication Critical patent/CN108289964A/en
Application granted granted Critical
Publication of CN108289964B publication Critical patent/CN108289964B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/08—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D263/16—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D263/18—Oxygen atoms
    • C07D263/20—Oxygen atoms attached in position 2
    • C07D263/26—Oxygen atoms attached in position 2 with hetero atoms or acyl radicals directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/60—Two oxygen atoms, e.g. succinic anhydride
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Abstract

The present invention relates to a novel class of linkers containing 2, 3-disubstituted succinic acid or 2-monosubstituted or 2, 3-disubstituted fumaric or maleic acid (trans (E) -or cis (Z) -butenedioic acid) groups, which link two or more small molecule drugs/cytotoxic agents by specific coupling reactions with bis-thiol groups on cell binding molecules. The invention also relates to methods of making such linkers, methods of making homogeneous conjugates using such linkers, and the use of these conjugates for the treatment of cancer, infection, and autoimmune disease.

Description

Novel linkers and their use for specific coupling of drugs and biomolecules
Technical Field
The invention relates to a novel bridge connector, wherein each connector is connected with two micromolecule medicines, and the connector specifically conjugates cell-bound molecules and the micromolecule medicines, especially cytotoxic agents, under the action of sulfur atom pairs on the cell-bound molecules. The invention also relates to a method of preparing a cell-binding agent-drug (cytotoxic agent) conjugate: one is to modify the drug small molecule with such linker and then react with the cell-binding agent, and the other is to modify the cell-binding agent with such linker and then react with the drug small molecule.
Technical Field
A major problem with chemotherapeutic drugs is their narrow therapeutic window, which often does not distinguish between normal and malignant cells, and can cause toxic side effects after use, even resulting in tolerable doses below the clinically effective dose. In contrast, immunotherapy, represented by monoclonal antibodies (mabs), can specifically bind to certain proteins or malignant cells, leaving normal cells unharmed, with fewer side effects and a larger therapeutic window than chemotherapy. Monoclonal antibodies (mabs) can attack malignant tumor cells by various mechanisms, such as 1) to make Cancer cells more visible to the immune system (Villaruz l.c. et al, trans Lung Cancer Res, 2014, 3, 2-14; camacho l.h. cancer Med 2015, 4, 661-72); 2) block growth signals (Dillman r.o. cancer Biother Radiopharm, 2011, 26, 1-64; ferris, R.L et al, J Clin Oncol, 2010, 28, 4390-9); 3) prevent neovascularization (Arrilaga-Romany, I. et al, Expert Opin Investig Drugs, 2014, 23, 199-; 4) radiation cancer cells (Chapuy, B et al, Biotechnol J.2007, 2, 1435-43); 5) delivering chemotherapeutic drugs to cancer cells (Chari R J, Acc Chem res, 2008, 41, 98-107; mullard A., Nature Reviews Drug Discovery 2013, 12, 329-; zhao R J, j.med.chem 2012, 55, 766-; 6) the enzyme is delivered to cancer cells (Francis R.J., et al, Br.J. cancer 2002, 87, 600-7). Antibody-drug conjugate (ADC) technology is one of the above strategies, and the ability to target drugs to cancer cells through the precise targeting ability of antibodies, leaving normal cells substantially unaffected, has been well developed over the last two decades. In particular, since Adcetris (brentuximab vedotin) was approved by the U.S. FDA in 2011 and Kadcyla (ado-trastuzumab emtansine) was approved in 2013, explosive developments were obtained for the use of antibody-drug conjugates (ADC) as a means for targeted cancer therapy, with almost all major pharmaceutical and biotechnology companies involved therein (Chari, R. et al, Angew. chem., int. Ed.2014,53, 3796-3827; Sieser, E.L. et al, Annu Rev Med.2013,64, 15-29; Mehrling, T.Future Oncol,2015,11, 549). According to the statistics of the website www.clinictrails.gov, more than 50 ADC drugs have been currently in clinical trial.
First generation ADC drugs, including kadcyl and Adcetris, were prepared by non-selectively coupling cytotoxic drugs to the native lysine amino group on antibodies, or to the thiol group of cysteine residues between antibody chains. Due to the presence of more than 50 surface-exposed lysines and 8 hinge region cysteines in the IgG1 antibody, this non-selective coupling approach can result in random cross-coupling reactions of toxic drugs occurring in almost all regions of the antibody molecule, and in particular, in the generation of a wide variety of ADC populations with a broad distribution of drug-antibody ratios (DAR) (Wang L et al, Protein sci.2005,14,2436; Hamblett, k.j. et al, clin.cancer res.2004,10,7063). Some undesirable ADC subsets have disadvantages of short circulating half-lives, low efficiency, high potential off-target toxicity, and uncertain Pharmacokinetic (PK) profiles in vivo (Hamblett, k.j. et al, clin. cancer res.2004,10, 7063-. Furthermore, it is very challenging to maintain batch-to-batch consistency for ADCs produced by this traditional coupling method, requiring higher production quality control capabilities (Wakankar, a. mabs,2011,3,161-.
Therefore, many biotechnology companies and research institutes have been working on the development of novel site-directed conjugation methods for ADC drugs. Some site-directed ADC drug preparation methods have also emerged in recent years (Panowski, S,2014, mAbs 6,34), including: introduction of unpaired cysteines on antibodies, such as Genentech's THIOMAB antibody (Junutula, j.r. et al, clin.cancer res.2010, 16,4769; Junutula, j.r. et al, Nat biotechnol.200826,925-32; US patent 8,309,300; 7,855,275; 7,521,541; 7,723,485; WO 2008/141044); or the introduction of a glutamine tag, which can be recognized by transglutaminase (mTG) amplified with Streptomyces mobaraensis (Strop, P., Bioconjugate chem.,2014,25,855-862; Strop, P. et al, chem. biol.2013,20,161-167; U.S. Pat. No. 8,871,908) or by bacterially derived transglutaminase (MTGase) (Dennler, P. et al, Bioconjugate. chem.2014,25,569-578; U.S. Pat. No. 20130189287; 7,893,019); introduction of thiol-L-fucose (Dennler, P. et al, Bioconjugate Chemistry 2014,25, 569; Okeley, N.M. et al, Bioconjugate chem.2013,24,1650); introduction of unnatural amino acids by mutagenesis (Axup, J.Y. et al, Proc. Natl.Acad.Sci.2012,109,16101-16106; Zimmerman, E.S., et al, bioconjug.Chem.2014,25,351-361; Wu, P. et al, Proc. Natl.Acad.Sci.2009,106,3000-3005; Rabuka, D. et al, nat. Protoc.2012,7,1052-67; U.S. Pat. No. 8,778,631; 20100184135; WO 2010/081110; WO2006/069246,2007/059312; U.S. Pat. No. 7,332,571,7,696,312; 7,638,299; WO 2007/130453; U.S. Pat. No. 7,632,492; 7,829,659); introduction of selenocysteine (Hofer, T. et al Biochemistry 2009,48, 12047-12057; U.S. Pat. No. 8,916,159); converting cysteines in the CXPXR consensus sequence to formylglycine (FGly) with a Formylglycine Generating Enzyme (FGE) (Drake, P.M., et al, bioconjugate, chem.2014,25,1331-; or by glycoengineering with galactose or sialyltransferase (Zhou, Q. et al bioconjugate. chem.,2014,25,510-520, U.S. Pat. No. 20140294867). Homogeneous products can be produced by the methods described above, but they all require antibody engineering and re-optimization of cell culture conditions. Furthermore, the expression encoded by the unnatural amino acid gene is often not as high as desired (Tian, f. et al, 2014, proc. natl.acad.sci.u.s.a.111,1766-71), which has a significant impact on the cost of the product. Furthermore, ADC drugs obtained by cysteine conjugation are often of limited stability in the circulation, resulting in premature breakdown of the toxic small molecule load before reaching the tumor cells (Junutula, j.r. et al nat. biotechnol.2008,26,925-32).
The disulfide bond structure of the four subtypes of IgG antibodies was well known in the sixties of the last century (Milstein C.Biochem J1966,101: 338-351; Pink JR, Milstein C.Nature 1967,214: 92-94; Frangone B, Milstein C.Nature 1967,216: 939-941; Pink JR, Milstein C.Nature 1967,216: 941-942; Frangone B et al Biochem J1968, 106, 15-21; Frangone B, Milstein C.J Mol Biol 1968; 33: 893-906; Edelman GM et al Proc Natl Acad Sci USA 1969; 63: 78-85; Frangone B, et al Nature 196,221:145- & ltCHEM & gt 148, Spiegelberg, H.L. et al Biochemistry,1975,10, 2157-63). Disulfide bonds play an important role in the structure, stability and biological function of IgG molecules. Four subclasses of IgG antibodies 1 ,IgG 2 ,IgG 3 And IgG 4 Contains a total of 12 intrachain disulfide bonds per IgG molecule; each disulfide bond is associated with a separate IgG domain. The two heavy chains are linked by different numbers of disulfide bonds at the hinge region: IgG 1 And IgG 4 Is 2, IgG 2 4, IgG 3 11 pieces of the feed. In IgG 1 In addition, the last cysteine in the light chain is disulfide bonded to the 5 th cysteine in the heavy chain. In IgG 2 ,IgG 3 And IgG 4 The last cysteine on the light chain forms a disulfide bond with the 3 rd cysteine on the heavy chain (Liu, H.; May, K.; 2012, mAbs 4, 17-23). Human IgG was known from reduction experiments, alkylation and LC-MS analysis 1 The ease of disulfide bond cleavage on antibodies (Liu, H, et al anal. chem.,2010,82, 5219-5226). The interchain disulfide bonds are more susceptible to reductive cleavage than the intrachain disulfide bonds, and the disulfide bonds between the light and heavy chains are more susceptible to reductive cleavage than the disulfide bonds between the two heavy chains. The interchain disulfide bond in the upper part of the two heavy chains is more easily cleaved than in the lower one. Furthermore, the disulfide bond in the CH2 domain is the most easily reduced. The disulfide bonds of the VL, CL, VH, and CH1 domains have similar moderate cleavability, while the disulfide bond of the CH3 domain is the least susceptible to reduction (Liu, H et al, anal. chem.,2010,82, 5219-.
Based on human IgG 1 The more easily cleavable interchain disulfide bonds of antibodies, many institutes and companies have employed chemical site-directed ligation strategies to reduce the interchain disulfide bonds of natural antibodies and then bridge them to re-crosslink them together, for example, using maleimide compounds (NGMs) known as next generation, i.e., bromo-or dibromo-maleimides (Schumacher, F.F., et al, org.Biomol.Chem.2014,12,7261-7269), using dialkylating reagents to form a three-carbon bridge (Badescu, G., et al, bioconju.Chem.2014, 25,1124-1136, WO2013/190272, WO2014/064424), using disubstituted heteroaryl bridges (U.S. Pat. No. 2015/0105539), or via bismaleimides as bridges (WO 2014/114) 207). For a long time, we have also used bromomaleimide and dibromomaleimide as linkers to couple drugs and antibodies (WO2014/009774, PCT/IB 2012/053554). However, the bridge linker design described above is designed to couple one cytotoxic molecule to a pair of disulfide bonds, and since the number of disulfide bonds available for conjugate coupling on an antibody is limited (about 2 pairs), ADC drug DAR values below 2 are produced in most cases.
Given that the efficacy of ADCs drugs is limited by the number of toxic small molecules that ultimately reach tumor cells, it is desirable to have DAR values greater than 3 in order to increase the therapeutic index of ADCs (Epenetos, a.a. et al, Cancer res.,1986,46, 3183-. The novel disulfide bridge linker of the invention not only can link 2 or more small molecule drugs on each linker, realize higher DAR (not less than 4), but also can selectively re-bridge interchain disulfide bonds reduced by TCEP or DTT on the antibody surface. Excess reductively produced sulfhydryl groups, which cannot be bound by the bridging linker, can be oxidatively regenerated later in the coupling reaction by an oxide, such as dehydroascorbic acid (DHAA) or Cu (II), to regenerate disulfide bonds. Theoretically, the reduced disulfide bond is re-bonded, so that the obtained ADC is more stable and has a longer half-life than the traditional sulfhydryl-linked ADC.
Furthermore, the literature reports that thioether-bond containing "ring-opened" succinimide ring linkers have higher in vitro stability, superior PK properties and potency than monothiol maleimide conjugated ADCs (Tumey L.N et al, Bioconjug. chem.2014, 25, 1871-80; lyonr. p et al, Nat. biotechnol.2014, 32, 1059-62) because the latter can lose the conjugated drug by retroro-Michael reaction (Shen b.q et al, Nat biotechnol.2012, 30, 184-9; Tumey L.N et al, bioconju chem.2014, 25, 1871-80). Similarly, the bridge linkers of the present invention contain 2, 3-disubstituted succinic acid groups or 2-monosubstituted or 2, 3-disubstituted fumaric or maleic acid (trans (E) or cis (Z) -butenedioic acid) groups, with less potential for loss of the drug molecule load than unhydrolyzed bromo or dibromomaleimide linker homologues.
In other words, the method of the invention can be used to prepare immunoconjugates, carrying drug combinations, in particular different drugs, and specifically delivering them to specific target sites, the conjugate molecules being highly homogeneous with good batch-to-batch consistency. The main advantages of such immunoconjugates include: simultaneously, a plurality of medicines are delivered in a targeted way, and the malignant tumor cells are acted by synergistic targeting; combining drugs that act at different stages of the cell cycle to increase the number of tumor cells exposed to a particular drug or effect; minimizing exposure of the drug to non-target cells, tissues or organs; precise control of drug payload and drug ratio results in a homogenous and uniform final product. In short, the bridge linker of the present invention can produce specific homogeneous and homogeneous ADC drugs in a simple manner.
Summary of the invention
The invention relates to a linker containing a 2, 3-disubstituted succinic acid group or a 2-monosubstituted or 2, 3-disubstituted fumaric or maleic acid (trans (E) or cis (Z) -butenedioic acid) group, and 2 small molecule drugs are connected to a cell binding agent (such as an antibody). Preferred cell binding molecule-linker-drug conjugate structures can be represented as:
Figure BDA0001580863800000051
wherein Cb is a cell binding agent, L is a linker comprising a succinic, fumaric or maleic acid group, Drug1 and Drug2 are small Drug molecules, n is an integer from 1 to 30, 2 sulfur elements bridge Cb to L, and 2 or more Drug molecules are covalently linked per bridge linker L. The advantages of using such linkers in cell binding molecule-drug conjugates are: a) covalently cross-linking (re-bridging) sulfhydryl groups on cell binding agents (e.g., antibodies) that are reduced open disulfide bonds, which is beneficial for maintaining the stability of the conjugate; b) toxic small molecules/drugs can be attached at specific locations of the cell binding agent, such as interchain disulfide bond sites of IgG antibodies, resulting in a homogeneous ADC drug.
In one aspect, the linker structure of the invention can be represented by formula (I)
Figure BDA0001580863800000052
Wherein:
Figure BDA0001580863800000053
Is an optional single bond;
Figure BDA0001580863800000054
is a single bond or a double bond;
u and U' are the same or different leaving groups that may be substituted with a sulfhydryl group, including but not limited to: halogen (F, Cl, Br, I), methanesulfonyl (Ms), p-toluenesulfonyl (Ts), trifluoromethanesulfonyl (Tf), trifluoromethanesulfonate, nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, pentafluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl-5-phenylisoxazole-3' -sulfonate, or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate;
when in use
Figure BDA0001580863800000055
When it is a single bond, U and U' are not H, when
Figure BDA0001580863800000056
When the double bond is one, U or U' can be H, but is not H at the same time;
Z 1 and Z 2 Are the same or different functional groups capable of reacting with a toxic drug to form a disulfide bond, an ether bond, an ester bond, a thioether bond, a thioester bond, a peptide bond, a hydrazone bond, a carbamate bond, a carbonate bond, an amine bond (secondary, tertiary or quaternary), an imine bond, a heterocycloalkyl, a heteroaryl, an alkoxyoxime bond or an amide bond;
R 1 and R 2 Are the same, different or default straight chain alkyl groups of 1-6 carbon atoms, 3 to 6 carbon atoms Branched or cyclic alkyl, straight chain, branched or cyclic alkenyl or alkynyl of a seed, or ester, ether, amide or polyethoxy (OCH) group of 1-6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to about 1000, or a combination of these groups;
in addition, R 1 And R 2 Is a chain structure containing C, N, O, S, Si and P atoms, optimally contains 0-500 atoms, and is covalently connected with X 1 Or X 2 And Z 1 Or Z 2 ；R 1 And R 2 Are combined in all possible chemical ways, such as to form an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkyl, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, polypeptide, acyloxyamine, hydroxamic acid, or a combination of these groups;
X 1 and X 2 Independently selected from NH, N (R) 3 ) O, S or CH 2 ；R 3 Is H, a straight chain alkyl group of 1 to 6 carbon atoms, a branched or cyclic alkyl group of 3 to 6 carbon atoms, a straight chain, branched or cyclic alkenyl or alkynyl group, or an ester, ether, amide or polyethoxy unit (OCH) of 1 to 6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to 1000, or a combination of these groups.
In another aspect, the cell-binding agent-Drug conjugate of the present invention can be represented by structural formula (II), wherein the cell-binding agent Cb, the Drug 1 And Drug 2 Have been reacted separately with the bridge linker ends:
Figure BDA0001580863800000061
cb is a cell-binding agent, most preferably an antibody;
within the brackets are linker-drug components coupled to the cell binding molecule through a pair of sulfur atoms. Preferably the sulfur atom pair is a sulfhydryl pair formed by opening of an inter-chain disulfide bond in a cell-binding agent by a reducing agent such as DTT and/or TCEP;
Drug 1 and Drug 2 Are identical orDifferent cytotoxic agents linked to the cell binding agent by a bridge linker with a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, cycloheteroalkyl, heteroaryl, alkoxyoxime, or amide bond;
n is 1 to 30;
Figure BDA0001580863800000062
R 1 ,R 2 ,X 1 and X 2 The definition of (A) is the same as that of the structural formula (I).
In another aspect, the invention includes a modified cell-binding agent, represented by formula (III), wherein the cell-binding agent Cb, a pair of sulfhydryl groups produced by reduction of disulfide bonds therein, has reacted with a bridge linker comprising a functional group Z capable of further reaction with a small drug molecule 1 And Z 2 ：
Figure BDA0001580863800000063
Wherein
Figure BDA0001580863800000064
Cb,Z 1 ,Z 2 ,n,R 1 ,R 2 ,X 1 And X 2 Are as defined for formulae (I) and (II).
Still further, the present invention includes a modified Drug molecule, which can be represented by formula (IV), wherein the Drug is a Drug 1 And Drug 2 Having reacted with the linker of formula (I), still retains a 2, 3-disubstituted succinic acid group or a 2-monosubstituted or 2, 3-disubstituted fumaric or maleic acid (trans (E) or cis (Z) -butenedioic acid) group capable of reacting with the sulfur atom pair on the cell-binding agent:
Figure BDA0001580863800000065
Wherein
Figure BDA0001580863800000071
Drug 1 ,Drug 2 ,U,U’,R 1 ,R 2 ,X 1 And X 2 Are as defined for formulae (I) and (II).
The invention also includes a method of preparing a cell binding molecule-Drug conjugate of formula (II), wherein the Drug is 1 And Drug 2 Linked by a bridge linker and a cell binding agent.
The invention also includes a method of making a modified cell binding molecule of formula (III) wherein the cell binding molecule has been reacted with a bridge linker of formula (I).
The invention also includes a method of making a modified drug small molecule as shown in formula (IV) wherein the drug molecule has been reacted with a bridge linker as in formula (I).
Description of the figures
FIG. 1 Synthesis of a polyethylene glycol-containing bridge linker and conjugation of antibodies and drugs via amide bonds therein
FIG. 2 Synthesis of a bridge linker containing polyethylene glycol and conjugation of antibodies and drugs via amide bonds therein
FIG. 3 Synthesis of a bridging linker containing polyethylene glycol and conjugation of antibodies and drugs via oxime linkage therein
FIG. 4 Synthesis of a bridge linker containing polyethylene glycol and conjugation of antibodies and drugs through hydrazone bonds therein
FIG. 5 Synthesis of a polyethylene glycol-containing bridge linker and conjugation of antibody and drug through amide bond therein (two different drugs attached to each linker molecule)
FIG. 6 Synthesis of bridge linkers and conjugation of antibodies and drugs via hindered amide bonds therein (two different drugs attached to each linker molecule)
FIG. 7 Synthesis of a bridge linker containing a polypeptide or polyethylene glycol and conjugation of two identical/different drugs to each antibody through a hydrazone bond therein
FIG. 8 Synthesis of conjugated conjugates of MMAE, Tubulysin and PBD cytotoxic agents
FIG. 9 Synthesis of Conjugatable PBD, MMAE, Tubulysin D cytotoxic Agents
FIG. 10 Synthesis of conjugates of cell binding molecule-tubulysin homologs connected by bridging linkers
FIG. 11 conjugate coupling of linker containing both PBD dimer homolog and Tubulysin B homolog to antibody and conjugate coupling of linker containing both MMAE homolog and Tubulysin D homolog to antibody
FIG. 12 conjugate coupling of a linker comprising both PBD dimer homolog and MMAF homolog to an antibody and conjugate coupling of a linker comprising both PBD dimer homolog and Tubulysin B homolog to an antibody
FIG. 13 conjugate coupling of a linker containing both maytansine homolog and Tubulysin B homolog to an antibody
FIG. 14 conjugation of a linker containing both maytansine homologs and PBD dimer homologs to antibodies and conjugation of a linker containing two Tubulysin B homologs to antibodies
FIG. 15 conjugation of a linker comprising two MMAF homologs and polyethylene glycol to an antibody and conjugation of a linker comprising two Tubulysin B homologs and polyethylene glycol to an antibody
FIG. 16 comparison of the antitumor effects of conjugates 127,129 and 142 with T-DM1 on a human gastric tumor N87 cell model, 3mg/kg i.v. for one injection. None of the four conjugates caused weight loss in the animals (on the graph). The tumor volume is more than 1500mm 3 And the disease was too severe, animals in the control group were sacrificed on day 37. All three compounds 127,129 and 142 outperformed T-DM 1: all 6/6 animals in the group with compounds 127 and 129 had no measurable tumor at all from day 13 to day 60 (end of experiment). All 6/6 animals in the group with compound 142 had no measurable tumor at day 21, 2/6 animals had visible (measurable) tumor growth at day 48, and inhibited tumor growth for more than 55 days. In contrast, T-DM1 at a dose of 3mg/kg failed to completely eradicate the tumor, inhibiting tumor growth for only about 28 days.
Detailed description of the invention
Definition of
"alkyl" means containing from 1 to 8 carbon atomsLinear or branched aliphatic hydrocarbons. "branched" means that one or more lower carbon number alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3, 3-dimethylpentyl, 2,3, 4-trimethylpentyl, 3-methylhexyl, 2, 2-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 3, 5-dimethylhexyl, 2, 4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, isooctyl. A C 1 -C 8 Alkyl groups may be unsubstituted or substituted with one or more groups including, but not limited to, -C 1 -C 8 Alkyl, -O- (C) 1 -C 8 Alkyl), aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH 2 ,-C(O)NHR',-C(O)N(R') 2 ,-NHC(O)R',-SR',-S(O) 2 R ', -S (O) R', -OH, halogen, -N 3 ,-NH 2 ,-NH(R'),-N(R') 2 and-CN; wherein each R' is independently selected from-C 1 -C 8 Alkyl groups and aryl groups. "halogen" means a fluorine, chlorine, bromine, iodine atom, preferably a fluorine and chlorine atom.
"heteroalkyl" means C 2 -C 8 Alkyl, wherein one to four carbon atoms are independently replaced by O, S and N atoms.
"carbocycle" refers to a saturated or unsaturated ring, monocyclic having 3 to 8 carbon atoms or bicyclic having 7 to 13 carbon atoms. Monocyclic carbocycles contain 3 to 6 atoms, typically 5 to 6 atoms. Bicyclic carbocycles containing 7 to 12 atoms to form [4,5 ]],[5,5],[5,6]Or [6,6 ]]Bicyclic systems, or 9 to 10 atoms, forming [5,6 ]]Or [6,6 ]]A bicyclic ring system. Typical C 3 -C 8 Including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1, 3-cycloheptadienyl, 1,3, 5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.
“C 3 -C 8 Carbocycle "means a 3, 4,5, 6, 7 or 8 membered non-aromatic carbocyclic ring containing either saturation or unsaturation. A C 3 -C 8 The carbocycle may be unsubstituted or substituted with one or more groups including, but not limited to, -C 1 -C 8 Alkyl, -O- (C) 1 -C 8 Alkyl), aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH 2 ,-C(O)NHR',-C(O)N(R') 2 ,-NHC(O)R',-SR',-S(O)R',-S(O) 2 R', -OH, halogen, -N 3 ,-NH 2 ,-NH(R'),-N(R') 2 and-CN, wherein each R' is independently selected from-C 1 -C 8 Alkyl groups and aryl groups.
"alkenyl" refers to straight or branched chain aliphatic hydrocarbons containing 2 to 8 carbon atoms with a carbon-carbon double bond. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methyl-2-butenyl, n-pentenyl, hexenyl, heptenyl, octenyl.
"alkynyl" refers to a straight or branched chain aliphatic hydrocarbon containing from 2 to 8 carbon atoms with a carbon-carbon triple bond. Examples of alkenyl include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentenyl, hexynyl, heptynyl, octynyl.
"alkylene" refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1 to 18 carbon atoms containing two monovalent radical centers derived from the removal of two hydrogens on the same or different carbons of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH) 2 -, 1, 2-Ethyl (-CH) 2 CH 2 -, 1, 3-propyl (-CH) 2 CH 2 CH 2 -, 1, 4-butyl (-CH) 2 CH 2 CH 2 CH 2 -) and the like.
"alkenylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms containing two monovalent radical centers derived from a parent olefin by the removal of two hydrogens on the same or different carbons. Typical alkenylene groups include, but are not limited to, 1, 2-vinyl (-CH ═ CH-).
"alkynylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2 to 18 carbon atoms containing two monovalent radical centers derived from the removal of two hydrogens on the same or different carbons of a parent alkyne. Typical alkynylene groups include, but are not limited to, ethynyl, propynyl, and 4-pentynyl.
"aryl" or "arylalkyl" refers to an aromatic or heteroaromatic group, consisting of one or several rings, containing from 3 to 14 carbon atoms, preferably from 6 to 10 carbon atoms. Heteroaryl refers to the substitution of one or more carbon atoms (preferably 1,2, 3 or 4 carbon atoms) on the aromatic ring with O, N, Si, Se, P or S (preferably O, S and N). "aryl" OR Ar also means that one OR more hydrogen atoms on the aromatic ring are each independently replaced by-R ', halogen, -OR ', OR-SR ', -NR ' R ", -N ═ NR ', -N ═ R ', -NR ' R", -NO 2 ,-S(O)R’,-S(O) 2 R’,-S(O) 2 OR’,-OS(O) 2 OR ', -PR ' R ', -P (O) R ', -P (OR ') (OR '), -P (O) (OR ') OR-OP (O ') (OR ') "), wherein R ', R ' are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl OR pharmaceutically acceptable salts.
"heterocycle" is a ring system in which 1 to 4 atoms are each independently replaced by heteroatoms such as O, N, S, Se, and P, preferably O, N, and S. "heterocyclic" is also defined in The Handbook of Chemistry and Physics,78th Edition, CRC Press, Inc.,1997-1998, p.225to 226. Preferred non-aromatic heterocycles include, but are not limited to, epoxy, vinylimino, pyrrolidine, pyrazolidinyl, alkylimidazole, oxiranyl, tetrahydrofuran, dioxolane, doxorubicin, dioxanyl, dioxolane, gua, piperazine, morpholine, pyran, imidazoline, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyran, dithiane, thiomorpholine, dihydropyran, pyraclostrobin, tetrahydropyridine, dihydropyridine, tetrahydropyrimidine, dihydrothiopyran, hexamethyleneimine and the structures resulting from their condensation with phenyl.
"heteroaryl" or heteroaromatic refers to 5to 14, preferably 5to 10, aromatic hetero-, mono-, bi-or polycyclic rings, including pyrrole, pyridine, pyrazole, pyrimidine, pyrazine, tetrazolyl thienyl, indole, quinoline, purine, imidazolyl, thiophene, thiazole, benzothiazole, furan, benzofuran, 1,2, 4-triazole, isothiazole, triazole, tetrazole, isoquinoline, benzothiophene, isobenzofuran, pyrazole, carbazole, benzimidazole, isoxazole, pyridine nitroxide, and ring structures resulting from their condensation with phenyl.
"alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocycle" and the like also refer to the corresponding "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene", and "heterocyclylene" and the like, free of two hydrogen atoms.
"aralkyl" refers to an acyclic alkane radical wherein one is joined to a carbon atom, usually a terminal or sp 3 The carbon atom to which the hydrogen atom is attached is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, phenyl, 2-phenyl-1-ethyl, 2-phenyl-1-vinyl, naphthylmethyl, 2-naphthyl-1-ethyl, 2-naphthyl-1-vinyl, naphthylphenyl, 2-naphthylbenzene-1-ethyl, and the like.
"Heteroaralkyl" means an acyclic alkyl radical in which one is terminal or sp with a carbon atom 3 The carbon atom to which the hydrogen atom is attached is replaced by a heteroaryl group. Typical heteroaralkyl groups include, but are not limited to, 2-benzimidazolyl, 2-furoethyl and the like.
Examples of "hydroxy protecting groups" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilylether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetyl ester, substituted acetyl ester, 2, 2-dimethylpropionate, benzoate ester, methylsulfonate ester, and p-toluenesulfonate ester.
"leaving group" means a functional group that can be substituted with another group. Leaving groups well known in the art include, but are not limited to, halo (chloro, bromo, iodo), methanesulfonyl, p-toluenesulfonyl, trifluoromethanesulfonyl, trifluoromethanesulfonate.
The following abbreviations are used in the present invention and are defined as: boc, tert-butoxycarbonyl; BroP, tris (dimethylamino) phosphine bromide hexafluorophosphate; CDI, carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethylpyrocarbonate; DMA, N-dimethylacetamide; DMAP, p-dimethylaminopyridine; DMF, N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, 2- (7-benzotriazol oxide) -N, N' -tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminophenyl; PBS, phosphate buffer (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, trichloroethyl phosphate; TFA, trifluoroacetic acid; THF, tetrahydrofuran; val, valine.
"pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce deleterious, allergic, or other untoward effects when administered to an animal or human, as appropriate.
"pharmaceutically acceptable solvate" or "solvate" refers to the disclosed compound and one or more solvent molecules associated therewith. Solvents in pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
"pharmaceutically acceptable excipients" include any carrier, diluent, adjuvant or excipient, such as protective or antioxidant agents, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents in pharmaceutically active compositions is well known in the art. Any conventional vehicle or agent, except those incompatible with the active ingredient, is contemplated for use in the therapeutic compositions. Auxiliary active ingredients may also be added to make suitable therapeutic compositions.
"pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds that form salts by reacting the parent compound with an acid or a base. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts formed with the parent compound, e.g., non-toxic inorganic or organic acids. For example, conventional non-toxic salts include derivatives of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared with organic acids such as acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, sulfonic acid, benzenesulfonic acid, glucose, glutamic acid, benzoic acid, salicylic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, maleic organic acids, lactic acid, and the like. Other salts include ammonium salts such as trimethylamine, meglumine, pyrrolethoxide and the like, and metal salts such as sodium, potassium, calcium, zinc and magnesium salts.
The pharmaceutically acceptable salts of this patent can be synthesized from the parent, which contains an acid or base, by conventional chemical methods. In general, such salts can be formed by adding an equivalent amount of the appropriate base or acid to the free acid or base of the parent compound in water or an organic solvent, or a mixture of two solvents. In general, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences,17 th ed., Mack Publishing Company, Easton, PA,1985, p.1418, incorporated herein by reference.
"administration" refers to any mode of transfer, delivery, introduction or delivery of a drug or other agent to a subject, including oral, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration, and also encompasses administration using devices or apparatus which may employ active or passive transport and which may be slow release or rapid release delivery devices.
The novel conjugate disclosed in the present invention uses a bridge linker. Partial linkers and their synthesis are shown in FIGS. 1 to 15.
Bridge connector
The synthesis of the bridge linker, and the preparation of the drug-cell binding molecule conjugate in this patent are shown in FIGS. 1-15. The bridge connection contains two elements: a) functional groups capable of reacting with thiol pairs on the cell binding agent to form covalent thioether linkages comprising a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, a 2-monosubstituted or 2, 3-disubstituted maleic acid group; b) a functional group capable of reacting with a drug molecule, including but not limited to disulfide, maleimide, haloacetyl, aldehyde, ketone, azide, amine, alkoxyamine, hydrazine, vinylsulfonyl, acid halide, acrylic group, and/or anhydride. A bridge linker comprising a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, a 2-monosubstituted or 2, 3-disubstituted maleic acid group may be formed by direct condensation of a 2, 3-disubstituted succinic acid, a 2-monosubstituted or 2, 3-disubstituted fumaric acid, a 2-monosubstituted or 2, 3-disubstituted maleic acid and an amine, alcohol or thiol group to form an amide, ester or thioether bond. Examples of the synthesis of these bridge linkers can be seen in figures 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 and 15.
Preferably, the bridge linker has the structure shown in (I):
Figure BDA0001580863800000121
Wherein:
Figure BDA0001580863800000122
is an optional single bond;
Figure BDA0001580863800000123
is a single bond or a double bond;
when in use
Figure BDA0001580863800000124
When it is a single bond, U and U' are not H, when
Figure BDA0001580863800000125
When the double bond is a bond, U or U' can be H, but is not H at the same time;
Figure BDA0001580863800000126
is a component capable of reacting with the thiol atom pair on the cell binding agent and may be a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, a 2-monosubstituted or 2, 3-disubstituted maleic acid group; the sulfur atom pair is preferably a thiol group formed by reduction of a cell-binding agent interchain disulfide bond with a reducing agent such as Dithiothreitol (DTT), Dithioerythritol (DTE), L-Glutathione (GSH), and tris (2-carboxyethyl) phosphine (TCEP), or/and β -mercaptoethanol (β -Me, 2-ME).
U and U' are the same or different leaving groups that may be substituted with a sulfhydryl group, including but not limited to: halogen (F, Cl, Br, I), methanesulfonyl (Ms), p-toluenesulfonyl (Ts), trifluoromethanesulfonyl (Tf), trifluoromethanesulfonate, nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, pentafluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl-5-phenylisoxazole-3' -sulfonate, or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate;
Z 1 And Z 2 Are the same or different functional groups capable of reacting with a toxic drug to form a disulfide bond, an ether bond, an ester bond, a thioether bond, a thioester bond, a peptide bond, a hydrazone bond, a carbamate bond, a carbonate bond, an amine bond (secondary, tertiary or quaternary), an imine bond, a heterocycloalkyl, a heteroaryl, an alkoxyoxime bond or an amide bond;
R 1 and R 2 Is the same, different or default straight-chain alkyl group with 1 to 6 carbon atoms, branched chain or cycloalkyl group with 3 to 6 carbon atoms, straight chain, branched chain or cycloalkenyl or alkynyl, or ester group, ether group, amide group or polyethoxy (OCH) group with 1 to 6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to about 1000, or a combination of these groups;
in addition, R 1 And R 2 Is a chain structure containing C, N, O, S, Si and P atoms, optimally contains 0-500 atoms, and is covalently connected with X 1 Or X 2 And Z 1 Or Z 2 ；R 1 And R 2 Are combined in all possible chemical ways, such as to form an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkyl, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, polypeptide, acyloxyamine, hydroxamic acid, or a combination of these groups;
X 1 and X 2 Independently selected from NH, N (R) 3 ) O, S or CH 2 ；R 3 Is H, a straight chain alkyl group of 1 to 6 carbon atoms, a branched or cyclic alkyl group of 3 to 6 carbon atoms, a straight chain, branched or cyclic alkenyl or alkynyl group, or an ester, ether, amide or polyethoxy unit (OCH) of 1 to 6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to 1000, or a combination of these groups.
In one embodiment, R 1 ，R 2 And R 3 Are chain structures comprising C, N, O, S, Si, and P atoms covalently linked to cell binding molecules and/or conjugated drugs. The individual atoms forming the bridge linker are joined in all possible chemical ways, such as forming alkylene, alkenylene, alkynylene, ether, polyoxyalkyl, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, polyurethane, amino acid, acyloxyamine, hydroxamic acid, and others. In addition, it is to be understood that each atom forming the linker may be saturated or unsaturated, may be a radical, may be annulated to each other to form a divalent ring structure, including cycloalkyl, cyclic ether, cyclic amine, arylene, heteroarylene, and the like.
Functional group Z 1 And Z 2 Examples of (a) include, a group that can be linked to a cytotoxic drug to form a disulfide bond, a thioether, a mercaptoester, a peptide, a hydrazine, an ether, an ester, a carbamate, a carbonate, an oxime, or an amide bond. Such functional groups include, but are not limited to, sulfhydryl, disulfide, amino, carboxyl, aldehyde, carbonyl, maleimide, haloacetyl, hydrazine, alkoxyamino, and/or hydroxyl groups.
Functional groups that can react with the terminal amino group of the drug/cytotoxic agent include, but are not limited to: n-hydroxysuccinimide ester, p-nitrophenyl ester, dinitrophenyl ester, pentafluorophenyl ester, an acid chloride or an acid anhydride; functional groups that react with a terminal thiol group include, but are not limited to: pyridyl disulfide, nitropyridine disulfide, maleimide, haloacetate, oxadiazole phenyl mesylate (ODA), acid chlorides and anhydrides; functional groups that react with terminal ketone or aldehyde groups include, but are not limited to: amines, alkoxyamines, hydrazines, acyloxyamines or hydrazides; functional groups that react with the terminal azide include, but are not limited to, alkynyl groups. Examples of such functional groups are:
Figure BDA0001580863800000141
n-hydroxysuccinimide ester;
Figure BDA0001580863800000142
a maleimide group;
Figure BDA0001580863800000143
a disulfide;
Figure BDA0001580863800000144
a haloacetyl group;
Figure BDA0001580863800000145
an acid halide;
Figure BDA0001580863800000146
a vinyl sulfonyl group;
Figure BDA0001580863800000147
an acryloyl group;
Figure BDA0001580863800000148
2- (p-toluenesulfonyloxy) acetyl;
Figure BDA0001580863800000149
2- (methylsulfonyloxy) acetyl;
Figure BDA00015808638000001410
2- (nitrophenoxy) acetyl;
Figure BDA00015808638000001411
2- (dinitrophenoxy) acetyl;
Figure BDA00015808638000001412
2- (fluorophenoxy) acetyl;
Figure BDA00015808638000001413
2- (difluorophenoxy) acetyl;
Figure BDA00015808638000001414
2- (trifluoromethanesulfonyloxy) acetyl;
Figure BDA00015808638000001415
a ketone or an aldehyde;
Figure BDA00015808638000001416
2- (pentafluorophenoxy) acetyl;
Figure BDA00015808638000001417
oxadiazole phenyl methane sulfonate (ODA);
Figure BDA00015808638000001418
An acid anhydride;
Figure BDA00015808638000001419
an alkoxyamine;
Figure BDA00015808638000001420
nitrine;
Figure BDA00015808638000001421
alkynyl or
Figure BDA00015808638000001422
A hydrazine. Wherein, X 1 Is F, Cl, Br, I or a leaving group; x 2 Is O, NH, N (R) 1 ) Or CH 2 ；R 3 And R 5 Is H, R 1 Aryl, heteroaryl or one or more H independently by-R 1 Halogen, -OR 1 、-SR 1 、-NR 1 R 2 、-NO 2 、-S(O)R 1 、-S(O) 2 R 1 or-COOR 1 Substituted aryl; leaving groups include nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethylsulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, self-anhydrides, or anhydrides with other anhydrides such as acetic anhydride, formic anhydride, or polypeptide condensation or Mitsunobu reaction intermediates.
In a preferred embodiment, R 1 ，R 2 And R 3 Is a linear alkyl or polyethoxy (OCH) radical having 1-6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to about 100.
The key step in the synthesis of a bridge linker comprising a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, a 2-monosubstituted or 2, 3-disubstituted maleic acid group is the direct condensation of a 2, 3-disubstituted succinic acid, a 2-monosubstituted or 2, 3-disubstituted fumaric acid, a 2-monosubstituted or 2, 3-disubstituted maleic acid or a derivative thereof and an amine (primary or secondary amine), alcohol or thiol group at the end of the other component as shown in (Ia):
Figure BDA0001580863800000151
Wherein X in formula (Ia) is X in formula (I) 1 Or X 2 Is NH, N (R) 3 ) O, or S; r is R in formula (I) 1 And/or R 2 ，R 3 The definition of (A) is also the same as that of formula (I).
Lv1 and Lv2 are OH, F, Cl, Br, I, nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, optionally substituted with one another,Trifluoromethylsulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, self-anhydrides or anhydrides with other anhydrides such as acetic anhydride, formic anhydride; or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate; the condensing agent comprises: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), Dicyclohexylcarbodiimide (DCC), N, N '-Diisopropylcarbodiimide (DIC), 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC or CME-CDI), Carbonyldiimidazole (CDI), TBTU (O-benzotriazol-N, N, N', N '-tetramethyluronium tetrafluoroborate), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinylphosphates (PyBOP), Diethylpyrocarbonate (DEPC), N, N, N', n ' -Tetramethylchloroformamidine hexafluorophosphate, 2- (7-benzotriazol oxide) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU), 1- [ (dimethylamine) (morpholinyl) methylene ]-1[1,2,3]Triazolo [4,5-b]1-pyridin-3-yloxyhexafluorophosphate (HDMA), 2-chloro-1, 3-dimethylimidazolium hexafluorophosphate (CIP), chlorotriazolylphosphonium hexafluorophosphate (PyCloP), bis (tetramethylene) fluorocarboxamide (BTFFH), N, N, N ', N' -tetramethyl-thio- (1-oxo-2-pyridinyl) thiouronium hexafluorophosphate, 2- (2-pyridinone-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TPTU), thio- (1-oxo-2-pyridinyl) -N, N, N ', N' -tetramethylthiouronium hexafluorophosphate, oxy- [ (ethoxycarbonyl) cyanomethylamine]-N, N, N ', N ' -tetramethylthiourea Hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate (COMU), (benzotriazol-1-yloxy) dipyrrolidine carbohexafluorophosphate (HBPyU), N-benzyl-N ' -cyclohexylcarbodiimide (or supported on a polymer), dipyrrolidinyl (N-succinimidyloxy) carbonium hexafluorophosphate (HSPyU), 1- (chloro-1-pyrrolidinylmethylene) pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1, 3-dimethylimidazole tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipiperidine carbohexafluorophosphate (HBPipU), 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium tetrafluoroborate (TCTU), tris (dimethyl) bromide Amino) phosphine hexafluorophosphate (BroP), 1-n-propyl phosphoric anhydride (PPACA,
Figure BDA0001580863800000164
) 2-isocyanoethylmorpholine (MEI), N' -tetramethylurea-oxy- (N-succinimidyl) Hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy- [ (ethoxycarbonyl) cyanomethylamine]-N, N '-tetramethylthionurebetrafluoroborate (TOTU), 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate (TSTU), N' -tetramethyl-oxy- (3, 4-dihydro-4-oxo-1, 2, 3-benzotriazin-3-yl) urea tetrafluoroborate (TDBTU), azodicarbonyl dipiperidine (ADD), bis (4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD).
Specific examples of synthetic bridge connectors are shown in FIGS. 1-10. In general, a linker component containing a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, a 2-monosubstituted or 2, 3-disubstituted maleic acid group is condensed with another linker component containing a functional group capable of coupling to a drug molecule.
Cell-binding agent-drug conjugates
The conjugates of the invention may be represented by the following structural formula:
Figure BDA0001580863800000161
wherein Cb is a cell binding agent, L is a linker comprising a succinic, fumaric or maleic acid group, Drug 1 And Drug 2 Is a small drug molecule, n is an integer from 1 to 30, 2 sulfur bridges Cb to L, and 2 or more drug molecules are covalently linked per bridge linker L.
The bridge linker L may be composed of one or more linker components. Typical linker components include: 6-Maleimidohexanoic acid (MC), 3-Maleimidopropionic acid (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af), p-aminoBenzyloxycarbonyl (PAB), 4-thiopentanoic acid (SPP), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid (MCC), (4-acetyl) aminobenzoic acid (SIAB), 4-thiobutanoic acid (SPDB), 4-thio-2-hydroxysulfonylbutanoic acid (2-Sulfo-SPDB), one or more ethoxy-CH 2 CH 2 O-units (EO or PEO), and also other linkers well known in the art, some of which are listed herein.
Examples of linkers comprising these components are:
Figure BDA0001580863800000162
(containing 6-Maleimidohexanoic acid MC)
Figure BDA0001580863800000163
(including 3-Maleimidopropionic acid MP)
Figure BDA0001580863800000171
(including p-aminobenzyloxycarbonyl group PAB)
Figure BDA0001580863800000172
Figure BDA0001580863800000173
Figure BDA0001580863800000174
(containing Ethylmaleimide ME)
Figure BDA0001580863800000175
(including valine-citrulline)
Figure BDA0001580863800000176
(comprising 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid MCC)
Figure BDA0001580863800000177
Figure BDA0001580863800000178
(comprising (4-acetyl) aminobenzoic acid)
Figure BDA0001580863800000179
(containing 4-thio-2-hydroxysulfonylbutanoic acid, 2-thio-SPDB)
Preferred conjugates are of formula (II):
Figure BDA00015808638000001710
wherein:
cb is a cell binding agent, most preferably an antibody, attached to Drug via a pair of sulfur atoms (sulfhydryl groups) 1 And Drug 2 . The thiol groups which can undergo conjugation are typically generated by reduction of the interchain disulfide bond of the cell-binding agent by a reducing agent, such as Dithiothreitol (DTT), Dithioerythritol (DTE), L-Glutathione (GSH), and tris (2-carboxyethyl) phosphine (TCEP), or/and β -mercaptoethanol (β -Me, 2-ME).
Drug 1 And Drug 2 The same or different cytotoxic agents are linked to the cell binding agent by a bridging linker through an alkyl, alkylene, alkenylene, alkynylene, ether, polyalkoxy, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, carbamate, amino acid, peptide, acyloxyamine, hydroxamic acid, disulfide bond, thioether, thioester, carbamate, carbonate, heterocycle, heteroalkyl, heteroaryl or alkoxyoxime bond, and combinations thereof.
n is 1 to 30;
Figure BDA0001580863800000181
R 1 ，R 2 ，X 1 and X 2 The definition of (A) is the same as that of the structural formula (I).
See the following more detailed description, Drug 1 And Drug 2 Can be any small molecule drug including, but not limited to, tubulysin, calicheamicin, auristatin, maytansine, CC-1065 homolog, morpholino, doxorubicin, taxanes, cryptophycins, epothilones, benzodiazepine series dimers (e.g., Pyrrole Benzodiazepine (PBD) or tomaymycin, indole benzodiazepine, imidazole benzodiazepine, or oxazole benzodiazepine dimer).
For the synthesis of conjugate conjugates, the cell binding agent may be first modified with the bridge linker of this patent, e.g., wherein the disulfide bond is first reduced to produce a pair of free thiols, and then reacted with the bridge linker of formula (I) in an aqueous phase at pH 5-9, with or without the addition of 0-30% water-miscible organic solvents, such as: n, N-dimethylacetamide, N-dimethylformamide, ethanol, methanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, dioxane, propylene glycol, or ethylene glycol to introduce Z 1 And Z 2 The reactive group can be a disulfide bond, a maleimide group, a haloacetyl group, an azide, a 1-alkyne, a ketone, an aldehyde, an alkoxyamino group, or a hydrazide. The reactive group of the cytotoxic molecule then reacts with the modified cell-binding agent. For example, the synthesis of disulfide-linked cell-binding agent-drug conjugates is achieved by disulfide bond exchange between disulfide bonds in the modified cell-binding agent and a drug containing a free thiol group; the synthesis of the thioether bond-bound cell-binder-drug conjugate is realized by the reaction of a cell-binding agent modified by maleimide group or haloacetyl group or ethylsulfone and a free sulfhydryl-containing drug; synthesis of conjugates containing acid-labile hydrazones in the molecule can be achieved by reaction of a hydrazide group and a carbonyl group in a linker, as is well known in the art (see P.Hamann et al, Cancer Res.1993, 53, 3336-za 334; B.Laguzza et al, J.Med.Chem., 1959, 32, 548-555; P.trail et al, Cancer Res., 1997, 57, 100-105); synthesis of triazole group-containing conjugates can be achieved by click chemistry (dipolar cycloaddition) of 1-alkynes on a linker with azide group-containing drugs (Lutz, J-F. et al, adv. drug Del. Rev.2008, 60, 958- 970; sletten, e.m., et al, acc, chem, research 2011, 44, 666-.
Alternatively, the drug small molecule can be reacted with a modified cell binding molecule-linker structure as shown in structural formula (III) that has been coupled to a cell binding agent and contains a reactive functional group. For example, a thiol-containing drug can be reacted with maleimide, haloacetyl, or ethylsulfonyl groups on the linker of formula (III) in a buffer at pH 5.0-9.0 to form thioether-linked conjugate conjugates. The sulfhydryl-containing drug can undergo disulfide bond exchange with a pyridine disulfide fragment on the linker of formula (III) to form a disulfide bond-linked conjugate. The drug containing hydroxyl or sulfhydryl can react with halogen on the connecting body in the structural formula (III), especially halogen on alpha halogenated carboxylic acid, under the condition of weak base, such as pH value 7.5-9.5, to obtain ether or thioether bond linked conjugate. The hydroxyl-containing drug can also be condensed into ester with carboxyl on the linker in the structural formula (I) under the action of a condensing agent of EDC or DCC. The drug-modified bridge linker is then coupled to a cell binding molecule. The amino-containing drug can react with carboxylic acid esters of NHS, imidazole, nitrophenol, N-hydroxysuccinimide, phenol, dinitrophenol, pentafluorophenol, 2,3,5, 6-tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid on the linker in the structural formula (III) to obtain the amide bond linked conjugate.
The conjugate may be purified by standard biochemical methods, such as gel filtration, adsorption chromatography, ion exchange or dialysis with Sephadex G25 or Sephacryl S300. In some cases, for example, small molecule cell binding agents (e.g., folic acid, melanocyte stimulating hormone, EGF, etc.) can be purified by HPLC, medium pressure column chromatography, ion exchange chromatography, or other chromatographic methods after binding to the small molecule drug.
Modified cell binding agents/molecules
Preferably, the structure of the cell binding agent modified with the linker of the invention is of formula (III):
Figure BDA0001580863800000191
wherein
Figure BDA0001580863800000192
Cb,Z 1 ,Z 2 ,n,R 1 ,R 2 ,X 1 And X 2 The same as defined in structural formulae (I) and (II).
In a preferred embodiment, Z 1 And Z 2 Is dithio, maleimido, haloacetyl, alkoxyamine, azide, ketone, aldehyde, hydrazine, N-hydroxysuccinimide ester or a carboxylic ester of phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid. Z 1 And Z 2 Can react with cytotoxic drugs to form thioethers, hydrazones, amides, alkyloximes, carbamates, esters, ethers, or heteroaromatic rings. As described in structural formula (II), the modified cell-binding agent can be prepared by reacting the cell-binding agent with the bridge linker described in structural formula (I).
In order to achieve efficient linkage of the alkenyl functional group on the bridging linker in formula (I) to a cell binding agent, especially a pair of free thiol groups in an antibody, a small amount of organic cosolvent is added to dissolve the structure in formula (III) in water well in the reaction system or after the reaction is completed. In modifying the cell binding agent, the cross-linker (bridge linker) of formula (I) is first dissolved in a polar organic solvent that is miscible with water, for example: various alcohols such as methanol, ethanol and propanol, acetone, acetonitrile, Tetrahydrofuran (THF), 1, 4-dioxane, Dimethylformamide (DMF), Dimethylacetamide (DMA) or Dimethylsulfoxide (DMSO); the solution concentration is slightly higher, e.g., 1-500 mM. Meanwhile, a cell binding agent, such as an antibody, is dissolved in a buffer solution with pH of 5-9.5, preferably 6-8.5 at a concentration of 1-35 mg/ml, and reacted with 1-20 equivalents of TCEP or DTT for 20 minutes to 12 hours. After reduction, the DTT is removed by purification on a SEC column, and the TCEP can be purified on a SEC column or can be carried on to the next step without purification. Furthermore, antibodies or other cell-binding agents reduced with TCEP can be carried out in the presence of a bridge linker of formula (I), in which case conjugation to the cell-binding agent is carried out simultaneously with the reduction of TCEP.
The reaction to modify the cell binding agent is generally carried out in a buffer at a pH of 6 to 9, preferably 6.5 to 7.5, and may be any buffer salt system without nucleophilicity in this pH range. Typical buffers include phosphate, triethanolamine hydrochloride, HEPES, and MOPS buffers, and may contain other ingredients such as cyclodextrins, sucrose, and salts such as sodium chloride and potassium chloride. The solution of the bridge linker of formula (I) is added to the reduced cell-binding agent solution and incubated at 4 to 45 deg.C, preferably room temperature, and the progress of the reaction is monitored by measuring the decrease in absorbance at 254nm, or the increase in absorbance at 280nm, or any other suitable change in absorbance at a wavelength. After completion of the reaction, the modified cell-binding agent may be purified according to conventional methods, for example using gel filtration chromatography or adsorption chromatography.
The extent to which the cell-binding agent is modified can be determined by measuring the uv absorbance of the nitro-pyrithione, dinitropyridine disulfide, pyrithione, formamide pyridine disulfide, and dimethylamide pyridine disulfide groups produced by the reaction. If the conjugate has no chromophore, it can be determined analytically by LC-MS or more preferably UPLC-QTOF mass spectrometry, or Capillary Electrophoresis Mass Spectrometry (CEMS). The bridge linkers of the present invention may contain various functional groups for reacting with various drugs, particularly cytotoxic drugs having suitable substituted functional groups. For example, a modified cell-binding agent containing an amino or hydroxyl substituent can be reacted with a drug containing an N-hydroxysuccinimide (NHS) ester, and a modified thiol-containing cell-binding agent can be reacted with a drug containing a maleimide or haloacetyl group. In addition, cell-binding agents modified to contain a carbonyl group (aldehyde or ketone group) can react with drugs containing hydrazide or alkoxyamine. Those skilled in the art can readily determine what linker to use based on the reactivity of their functional groups.
Modified cytotoxic drugs
The cytotoxic drug structure modified with the bridge linker of the invention is preferably (IV):
Figure BDA0001580863800000211
wherein
Figure BDA0001580863800000212
U,U’,Drug 1 ,Drug 2 ,R 1 ,R 2 ,X 1 And X 2 Are as defined for formulae (I) and (II).
The modified drug with the structure (IV) is obtained by the reaction of drug molecules and a connecting body with the structure of the formula (I), wherein the modified drug contains a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, and a 2-monosubstituted or 2, 3-disubstituted maleic acid group functional group. But for drugs containing sulfhydryl groups, or drugs that are linked to cell binding molecules by thioether-, thioester-or disulfide-containing linkages, Drug 1 Or Drug 2 Preferably first through a thioether, thioester or disulfide bond with R 1 Or R 2 Ligation followed by synthesis of R 1 -Drug 1 Or R 2 -Drug 2 The component (IV) is assembled with 2, 3-disubstituted succinic acid, 2-monosubstituted or 2, 3-disubstituted fumaric acid, 2-monosubstituted or 2, 3-disubstituted maleic acid to form the bridge linker modified drug (IV).
For example, a thiol-containing drug can be reacted with R in a buffer at neutral pH 1 Or R 2 Maleimide on the component reacts to form a thioether-bonded R 1 -Drug 1 Or R 2 -Drug 2 And then condensed with 2, 3-disubstituted succinic acid, 2-monosubstituted or 2, 3-disubstituted fumaric acid, 2-monosubstituted or 2, 3-disubstituted maleic acid to form the modified drug comprising thioether bond as shown in formula (IV). The hydroxyl-containing drug can be reacted with R containing halogen, p-toluenesulfonic acid or mesylate under the weak base condition 1 Or R 2 The components react to obtain ether bondsAnd R is 1 -Drug 1 Or R 2 -Drug 2 And then condensed with 2, 3-disubstituted succinic acid, 2-monosubstituted or 2, 3-disubstituted fumaric acid, 2-monosubstituted or 2, 3-disubstituted maleic acid to form a modified drug comprising thioether linkages as in formula (IV). The drug containing hydroxyl can be condensed with the linker with carboxyl in the structure (I) under the action of dehydrating agent such as EDC or DCC to obtain the modified drug containing ester bond in the structural formula (IV). The sulfhydryl-containing drug may be reacted with R 1 Or R 2 Reacting maleimide, vinylsulfone or haloacetyl on the component to generate the R combined by thioether bond 1 -Drug 1 Or R 2 -Drug 2 And then condensed with 2, 3-disubstituted succinic acid, 2-monosubstituted or 2, 3-disubstituted fumaric acid, 2-monosubstituted or 2, 3-disubstituted maleic acid to form the modified drug comprising thioether bond as shown in formula (IV). Similarly, amino group-containing drugs can also be condensed with the carboxyl groups on the bridge linker (I) to give modified drugs containing amide bonds as in formula (IV). The modified drug may be purified by standard methods, such as silica gel or alumina column chromatography, recrystallization, preparative thin layer chromatography, ion exchange chromatography or high performance liquid chromatography.
Cell binding agents
The cell-binding agents of the invention, including conjugates and modified cell-binding agents, may be any of a variety of molecules now known or later discovered that are capable of binding, complexing or otherwise reacting with a cell fragment of therapeutic interest or that are biologically modified.
Cell-binding agents include, but are not limited to, large molecular weight proteins, such as whole antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies, e.g., bispecific antibodies); a single chain antibody; antibody fragments such as Fab, Fab ', F (ab') 2 ,F v (Parham, J.Immunol.1983,131, 2895-2902); fragments produced by the Fab expression library, anti-idiotype (anti-Id) antibodies; a CDR; a bivalent antibody; trivalent antibodies and epitope-binding fragments of any of the above antibodies that immunospecifically bind to a cancer cell antigen; a viral antigen; microbial antigens or proteins produced by the immune system, capable of recognizing and bindingSpecific antigens or having the desired biological activity (Miller et al J. of Immunology 2003,170, 4854-4861); interferons (e.g., type I, II, III); a polypeptide; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon- γ (IFN- γ); hormones such as insulin, TRH (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgen and estrogen; growth factors and colony stimulating factors, such as Epidermal Growth Factor (EGF), granulocyte macrophage colony stimulating factor (GM-CSF), Transforming Growth Factors (TGF) such as TGF α, TGF β, insulin and insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 1984,5, 155-); vaccinia Growth Factor (VGF); fibroblast Growth Factor (FGF); a small molecular weight protein; a polypeptide; peptides and peptide hormones, such as bombesin, gastrin-releasing peptide; platelet-derived growth factors; interleukins and cytokines, for example, interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte macrophage colony stimulating factor (GM-CSF); vitamins, such as folic acid; apoproteins and glycoproteins, such as transferrin (O' Keefe et al, J.biol.chem.1985260932-937); carbohydrate binding proteins or lipoproteins, such as lectins; a cellular nutrient-delivery molecule; small molecule inhibitors such as Prostate Specific Membrane Antigen (PSMA) inhibitors and small molecule Tyrosine Kinase Inhibitors (TKIs), non-peptides or any other cell binding molecule or substance such as bioactive polymers (Dhar, et al, proc.natl.acad.sci.2008,105,17356-61), bioactive dendrimers (Lee, et al, nat.biotechnol.2005,23,1517-26; Almutairi, et al; proc.natl.acad.sci.2009,106,685-90), nanoparticles (Liong, et al, ACS Nano,2008,19, 1309-12; Medarova, et al, nat.med.2007,13,372-7; Javier, et al, Bioconjugate chem.2008,19,1309-12), liposomes (nai, et al, curr.phars. 10,2981-9) and viral coat (fluhne.327, shin-71, U.71).
In general, monoclonal antibodies are preferred as cell surface binding agents if appropriate monoclonal antibodies are available. The antibody may be murine, human, humanized, chimeric or derived from other species.
The production of antibodies for use in the present invention includes in vivo or in vitro methods or combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well known in the art, for example, as described in U.S. patent 4,493,795. Monoclonal antibodies are typically prepared by fusing myeloma cells with spleen cells of mice that have been immunized with the desired antigen(s) (ii)
Figure BDA0001580863800000221
G; milstein, C.Nature 1975,256: 495-497). The detailed procedure is described in "Antibodies- -A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988)", which is incorporated herein by reference. Specifically, a mouse, rat, hamster, or any other mammal may be immunized with an antigen of interest, such as an intact target cell, an antigen isolated from a target cell, an intact virus, an inactivated whole virus, and viral proteins. Spleen cells are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused cells were screened for sensitivity to HAT (hypoxanthine-aminopterin-thymidine). Hybridomas that embody the monoclonal antibodies of the invention can be determined by their ability to immunoreact with specific receptors or to inhibit the activity of receptors on target cells.
The production of monoclonal antibodies for use in the present invention is carried out in monoclonal hybridoma cultures comprising a nutrient medium and hybridomas secreting antibody molecules with the appropriate antigen specificity. The culture is maintained under suitable conditions for a period of time sufficient for the hybridomas to secrete the antibody molecules into the culture medium. The antibody-containing medium is then collected. Antibody molecules are further separated using techniques well known in the art, such as protein A affinity chromatography, anionic, cationic, hydrophobic or size exclusion chromatography (particularly by protein A affinity chromatography and size exclusion chromatography), centrifugation, differential solubility or any other standard technique for purifying proteins.
Media useful for preparing these compositions are well known in the art and are commercially available, including synthetic media. An example of a synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al, Virol.1959,8,396) supplemented with 4.5g/ml glucose, 0-20mM glutamine, 0-20% fetal bovine serum, several ppm of heavy metals or/and heavy metal salts such as Cu, Mn, Fe or Zn, and antifoams such as polyoxyethylene-polyoxypropylene block copolymers.
Alternatively, antibody-producing cell lines can be obtained by techniques other than fusion, such as transformation of neoplastic DNA into B lymphocytes, or transfection of a neoplastic virus, such as epstein-barr virus (EBV, also known as human herpesvirus 4(HHV-4)) or kaposi's sarcoma-associated herpesvirus (KSHV), see U.S. patent 4,341,761; 4,399,121, respectively; 4,427,783, respectively; 4,444,887; 4,451,570, respectively; 4,466,917, respectively; 4,472,500, respectively; 4,491,632, respectively; 4,493,890. Monoclonal antibodies can also be prepared by anti-receptor peptides or peptides containing terminal carboxyl groups, as is well known in the art, for example, see the Niman et al, Proc. Natl. Acad. Sci. USA,1983,80: 4949-4953; geysen et al, Proc. Natl. Acad. Sci. USA,1985,82: 178-; lei et al biochemistry 1995,34(20): 6675-6688. In general, anti-receptor peptides or peptide homologs as immunogens for generating monoclonal antibodies against receptor peptides can be used alone or linked to an immunogenic carrier.
Monoclonal antibodies useful as binding molecules in the present invention may also be obtained by other techniques known in the art. Particularly useful are methods of making fully human antibodies. One method is phage display technology, which uses an affinity enrichment format and can be used to select for human antibodies that specifically bind to an antigen. Phage display techniques are also described in detail in the literature, and the construction and screening of phage display libraries is also well known in the art, as described in Dente et al, Gene.1994,148(1): 7-13; littlet et al, Biotechnol adv.1994,12(3): 539-55; clackson et al, Nature 1991,352: 264-; huse et al, Science 1989,246: 1275-.
Monoclonal antibodies produced by hybridomas fused to non-human, e.g., mouse, cells can be humanized to avoid production of human anti-mouse antibodies. Common antibody humanization methods are complementarity determining region grafting techniques, which have also been described in detail, e.g., U.S. Pat. nos. 5,859,205 and 6,797,492; liu et al, Immunol Rev.2008,222: 9-27; almagro et al, Front biosci.2008,13: 1619-33; lazar et al, MolImmunol.2007,44(8): 1986-98; li et al, Proc. Natl. Acad. Sci. U S A.2006,103(10):3557-62, incorporated herein by reference. Whole human antibodies can also be prepared by immunizing a transgenic mouse, rabbit, monkey or other mammal carrying a large portion of the heavy light chain of human globin with an immunogen. Examples of such mice are: xenomouse (Abgenix/Amgen), HuMAb-Mouse (Metarex/BMS) and VelociMouse (Regeneron), see U.S. Pat. Nos. 6,596,541,6,207,418,6,150,584,6,111,166,6,075,181,5,922,545,5,661,016,5,545,806,5,436,149 and 5,569,825. For human therapy, the mouse variable region and the human constant region can also be fused to form a "chimeric antibody" that is significantly less immunogenic in humans than the mouse monoclonal antibody (Kipriyanov et al, MolBiotechnol.2004,26: 39-60; Houdbine, CurropinBiotechnol.2002,13: 625-9). In addition, site-directed mutagenesis of the variable region of an antibody can result in antibodies with higher affinity and specificity (Brannigan et al, Nat Rev Mol Cell biol.2002,3: 964-70; Adams et al, J Immunol methods.1999,231:249-60), and alterations in the constant region of an antibody can enhance its effector functions mediating binding and cytotoxicity.
Immunospecific antibodies for malignant cell antigens may also be obtained commercially or produced by any known method, such as chemical synthesis or recombinant expression techniques. Nucleotide sequence encoding antibodies immunospecific for malignant tumor cell antigens are commercially available, for example from GenBank databases or similar databases, literature publications, or by routine cloning and sequencing.
In addition to antibodies, a peptide or protein that interacts (binds, blocks, targets, or otherwise) with an epitope or corresponding receptor on the target cell may also serve as a binding molecule. These peptides or proteins may be any random peptides or proteins that have an affinity for an epitope or a corresponding receptor and are not necessarily immunoglobulin family members. These peptides can be isolated by techniques similar to phage display antibodies (Szardnings, J Recept Signal Transmission Res.2003; 23(4): 307-49). Peptides obtained from random peptide libraries can be used similarly to antibodies and antibody fragments. The peptide or protein binding molecule may be coupled or linked to a macromolecule or other substance, including but not limited to albumin, polymers, liposomes, nanoparticles, dendrimers, so long as such linkage retains the antigen binding specificity of the peptide or protein.
Examples of antibodies on conjugates for the treatment of cancer, autoimmune and/or infectious diseases, and drug molecules linked by the bridging linker of this patent include, but are not limited to, 3F8 (anti-GD 2), abazumab (anti-CA-125), abciximab (anti-CD 41 (integrin α -IIb), adalimumab (anti-TNF- α), Adecatumumab (anti-EpCAM, CD326), aphidimab (anti-TNF- α), afutumumab (anti-CD 20), alilizumab (anti-VEGFR 2), ALD518 (anti-IL-6), Alemtuzumab (Campath, mabcappath, anti-CD 52), altumumab (anti-CEA), antatumomab (anti-TAG-72), ankizumab (anti-imab-638, anti-IL-13), Apolizumab (anti-HLA-CEA), azithromycin (anti-axuzumab), azithromycin-DR-CD 3862), azithromycin-adalimumab (anti-CD 62L), RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rheumas factor), Bapineuzumab (anti- β amyloid), Basiliximab (Simulect, anti-CD 25 (alpha chain of IL-2 receptor)), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD 22), Bellinumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD 125), Bertilimumab (anti-CCL 11(eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromimab (Fibri Scirunt, anti-fibrin II beta chain), Bivazuumab (anti-CD-3544), Blinizumab (Avastin-C), anti-Catualab (anti-CD 3558), anti-Catulimab (anti-CD 3655-CA-C), anti-Catulimab (anti-C3658, anti-Catulimab (anti-CD 3655, anti-CA-C3645, anti-Catulimab (anti-CA 3655), cetuximab (erbitux, IMC-C225, anti-EGFR), Cituzumab bogatox (anti-EpCAM), Cixuumumab (anti-IGF-1), Clenolixumab (anti-CD 4), Clivatuzumab-mab (anti-MUC 1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti-CD 40), D aclizumab (Zenapax, anti-CD 25(IL-2 receptor alpha chain)), Daratumumab (anti-CD 38 (cyclic ADP ribohydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphoma cells), Dorlimomab, Dorliximab, Ecromeximab (anti-GD 3 ganglioside), Eculizumab (Soliris, anti-C5), Edobaoma (anti-endotoxin), Edrocolomab (Panorex, MAb17-1A, anti-EpCAM), Efalizumab (Raptiva, anti-CD 11A), Efungumab (Mycogranb, anti-Hsp 90), Elotuzumab (anti-SLAMF 7), siumumab (anti-LFIL-6), Enlimob (anti-CD-2), anti-Epsilozumab (anti-Epsilozumab), anti-interference-Epsilozumab (anti-HER-Epsilozumab) 3), anti-Epsilozumab (anti-Epsilozumab), anti-HER-Epsilozumab (anti-CD-TNF-2) antigen (anti-Epsilozumab-CD-EPO-CD-6853), anti-Epsilozumab (anti-Epsilozumab), anti-Epsilozumab), anti-Epsilozumab-EPO-IFN-EPO-IFN-EPO-beta-3), anti-Epsilomab (anti-Epsilomab-E-IFN-beta-E-beta-2), anti-E-beta-E-beta-2), anti-E-beta-E-2), anti-E-2-E-2), anti-E-FAbix-E-2-E (anti-E-FAbix-E-2), anti-E-FAB-E-2), anti-E-2-E-2), anti-E-FAB-E (anti-E (anti-E-2), anti-E (anti-E-2), anti-E (E-, felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-. gamma.), Foravizumab (anti-rabies glycoprotein), Fresolimumab (anti-TGF-. beta.), Galiximab (anti-CD 80), Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD 147 (baisin)), Gemtuzumab (anti-CD 33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-NMB), Golomab (Simponin, anti-alpha), miliximab (anti-CD 23(IgE receptor)), Izulizumab (anti-CD 4), Ibriumumab (anti-CD 58), anti-myocardial syncytial 37152, anti-TNF-CD 35125 (anti-CD 3552), anti-TNF-IgG 3645 (anti-TNF-. alpha), anti-TNF-IFN 30 (anti-TNF-. gamma), anti-TNF-. gamma., anti-TNF 30, anti-TNF-. gamma. (anti-TNF-. beta. (anti-TNF 30), Izodiac 30, anti-TNF-. gamma., TNF-. gamma. (anti-TNF-. beta. (anti-TNF-. 30), anti-TNF-. beta. (anti-TNF-. beta.) (anti-TNF-IFN, anti-TNF-IFN-. beta. (anti-TNF-IFN 369), and its-IFN-. beta. (anti-IFN-. beta.) (anti-TNF-IFN, anti-TNF-IFN-. beta. (anti-gamma., TNF-20), keliximab (anti-CD 4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalezumab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2), Lexatuzumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD 33), lumimumab (anti-CD 40), lumimumab (anti-CD 23(IgE receptor), Mapatumab (anti-TRAIL-R1), Maximazumab (anti-T-cell receptor), matuzumab (anti-EGFR), Mepolizumab (Bosatura, anti-IL-5), Metalimumab (anti-TGF beta 1), Milatuzumab (anti-CD 74), Mintumomab (anti-TAG-72), Mitumomab (anti-BE-5) C-2, anti-GD 3 ganglioside), Morolimumab (anti-rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3(Orthoclone OKT3, anti-CD 3), Nacolomab (anti-C242), Naptumomab (anti-5T 4), natalizumab (Tysabri, anti-integrin alpha 4), nebuzumab (anti-endotoxin), Netumumab (anti-EGFR), Nerelimomab (anti-TNF-alpha), Nimotuzumab (Theramim, Theraloc, anti-EGFR), Notumomab, Ocreluzumab (anti-CD 20), Olilimumab (Afolimomab, anti-Afolimomab A-1(CD11a)), Arzerrab, anti-CD 20), Olaramuzumab (anti-PDGF-Ab), anti-adenovirus Ab, anti-Epiolimumab (Ab-X-Ab), anti-Epiolimumab (Ab-X-Ab-X-Ab), anti-Ab-X-Ab, Ab-Ab, Ab-Ab, Ab-Ab, Ab-e, Ab-e, Ab-e, Ab-, (ii) paclobutrazumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC 1), Pertuzumab (Omnitarg, 2C4, anti-HER 2/neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-D4), Pritumumab (anti-vimentin), PRO140 (anti-CCR 5) Racotumomab (1E10, anti-N-glycolylneuraminic acid (NeuGc, NGNA) -ganglioside GM3), Rafivirumab (anti-rabies glycoprotein), Ramucirumab (anti-VEGFR 2), Ravinibizumab (Lucentis, anti-VEGF-A), Ratubacumab (anti-antitoxin, protective antigen), Regaviumamab (anti-rabies glycoprotein B), Reslilizumab (anti-Vegfirhizumab) (anti-IFN-5), anti-Rituzumab (anti-IFN-R-23), anti-VEGF-A), Ratuzumab (anti-CD 46154, anti-IFN-CD L), anti-Rituzumab (anti-TNF-LR), anti-TNF-VEGF-2, anti-TNF-MAb (anti-VEGF-CD 46154), anti-TNF-G-2), anti-TNF-2), anti-rat-2, anti-rat (anti-rat-2), anti-rat (anti-rat-C, anti-rat (anti-rat-C, anti-rat-C, anti-rat-C, anti-rat-C, anti-rat-, satumomab (anti-TAG-72), Seviruzumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), sifaxmab (anti-IFN-. alpha.), Siltuximab (anti-IL-6), Siplizumab (anti-CD 2), Smart MI95 (anti-CD 33), Solanezumab (anti-beta amyloid), Sonepcizumab (anti-sphingosine-1-phosphate), Sontuzumab (anti-epidialin), Stamulumab (anti-myostatin), Sulesomab (Leucoscan, anti-NCA-90 (granulocyte antigen)), Tacatazumab (anti-alpha fetoprotein), Tadocizumab (anti-integrin. alpha. b. beta.3), Talizumab (anti-IgE), Tanemab (anti-NGF), Taplimumab (anti-NGF), Taplitmab (anti-CD 19), Tabrotuzumab (anti-CD 19-NGF) ) Tefibuzumab (Aurexis, (anti-aggregatory factor A)), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD 40), Teplizumab (anti-CD 3), TGN1412 (anti-CD 28), Ticilimumab (Tremelimumab, anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, IL-6 receptor), Totalizumab (anti-CD 154(CD40L)), Tositumumab (anti-CD 20), trastuzumab (herceptin, anti-HER 2/neu), Tremelimab (anti-4), Tucotuzumab (anti-CAM), Tucoruzumab (anti-AArivirus-CAM), Tuvotuzumab (anti-AAC-11), anti-AAzezumab (anti-AAvkipedia-CTLA-11), anti-hepatitis-VAkinumab (anti-CTLA-4), anti-CTLA-11, anti-CTLA-11 (anti-CTLA-2), and TAX-13), vitaxin (anti-angiointegrin avb3), Volociximab (anti-integrin. alpha.5. beta.1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zaluteumab (HuMax-EGFR, Zanolimumab (HuMax-CD4, anti-CD 4), Ziralimumab (anti-CD 147 (baisin)), Zolinmomab (anti-CD 5), Enasipu
Figure BDA0001580863800000271
Alefacept
Figure BDA0001580863800000272
Abatacept
Figure BDA0001580863800000273
Rilonacept (Arcalalyst), 14F7 (anti-IRP-2 (IRP-2), 14G2a (anti-GD 2 ganglioside, Nat. Cancer Inst., treatment of melanoma and solid tumors), J591 (anti-PSMA, west Cornell institute of medicine, treatment of prostate Cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma associated antigen), Sorin radiofacci SRL (Nat. Italy, treatment of melanoma), COL-1 (anti-CEACAM 3, CGM1, Nat Cancer Inst., treatment of colorectal Cancer and gastric Cancer), CYT-356 (anti-IRP-2 (IRP-2), 14G2a (anti-GD 2 ganglioside, Nat. Cancer Inst., treatment of melanoma and solid tumors), J591 (anti-PSMA, anti-HMW-MAA (high molecular weight melanoma associated antigen), Sorin radiofacci SRL (anti-melanoma), COL-1 (anti-CEACAM 3, CGM1, Nat Cancer Inst., treatment of colorectal Cancer, gastric Cancer), CYT-356 (anti-356), (CYT-S, and/S)
Figure BDA0001580863800000274
For the treatment of prostate cancer), HNK20(OraVax inc. for the treatment of respiratory syncytial virus infection), immrarit (from immunol)dics, therapy NHL), Lym-1 (anti-HLA-DR 10, Peregrine Pharm), MAK-195F (anti-TNF (tumor necrosis factor, TNFA, TNF- α, TNFSF2, from Abbott/Knoll, therapy of septic shock), MEDI-500(T10B9, anti-CD 3, TR α β (T cell receptor α/β), from medimmunene Inc, for graft versus host disease), RING SCAN (anti-TAG 72 (tumor-associated glycoprotein 72), from Neoprobe corp., for breast, colon and rectal cancer), Avicidin (anti-EPCAM (epithelial cell adhesion molecule)), anti-TAG cstd1 (tumor-associated calcium signaling 1), anti-GA 733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial 2), anti-KSA, KS1/4 antigen, M4S, tumor antigen 17-1A, CD326 (NeoRx), treatment of colon cancer, ovarian cancer, prostate cancer and NHL), LymphoCide (from immunology), Smart ID10 (from Protein Design Labs), Oncolym (from Techniclone Inc), Allomune (from BioTransplant), anti-VEGF (from Genentech); CEAcide (from Immunodics), IMC-1C11 (from ImClone Systems) and Cetuximab (from ImClone).
Other antibodies that may act as cell binding molecules/ligands include, but are not limited to, antibodies to the following antigens: aminopeptidase N (CD13), annexin A1, B7-H3(CD276, various cancers), CA125 (ovarian cancer), CA15-3 (various cancers), CA19-9 (various cancers), L6 (various cancers), Lewis Y (various cancers), Lewis X (various cancers), alpha-fetoprotein (various cancers), CA242 (colorectal cancer), placental alkaline phosphatase (various cancers), prostate specific antigen (prostate cancer), prostatic acid phosphatase (prostate cancer), epidermal growth factor (various cancers), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma, lung cancer, breast cancer, stomach cancer, ovarian cancer, autoimmune disease, malignant ascites), CD19 (B-cell malignancy), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin lymphoma), CD33 (leukemia, autoimmune disease), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Meike cell carcinoma, as well as liquid tumors, multiple myeloma), CD66e (various cancers), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (various cancers), mucin (various cancers), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell and other lung cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEM 5 (carcinoembryonic antigen, CEA, CD66e) (breast, colorectal cancer and lung cancer), DLL4, EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4(CD184, hematological tumors, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder cancer, head and neck cancer, colon cancer, NHL prostate cancer, ovarian cancer), ERBB2 (epidermal growth factor receptor 2, lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (various cancers), G-28 (cell surface antigen lipids, melanoma), GD3 idiotype (respective cancers), heat shock proteins (various cancers), HER1 (lung cancer, stomach cancer), HER2 (breast cancer, lung cancer and ovarian cancer), HLA-DR10(NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (various cancers), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptors (interleukin 2 receptors, T cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castleman's disease, interleukin 6 dependent tumors), integrins (α v β 3, α 5 β 1, α 6 β 4, α ll β 3, α 5 β 5, α v β 5, various cancers), MAGE-1 (various cancers), MAGE-2 (various cancers), MAGE-3 (various cancers), MAGE 4 (various cancers), anti-transferrin receptor (various cancers), p97 (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, non-Hodgkin B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and alpha gastrointestinal tract cancer), MUC16 CA (125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, small cell lung cancer, NHL), Nucleolin, Neu oncogene products (cancers respectively), P21 (various cancers), anti- (N-glycolylneuraminic acid) antibody binding sites (breast cancer, melanoma), PLAP testis alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate cancer), PSA (prostate cancer), ROBO4, ROBO 72 (tumor-associated glycoprotein 72, AML, stomach cancer, colorectal cancer, ovarian cancer), T cell transmembrane proteins (various cancers), Tie (CD202B), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, various cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (TAG cell glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1(CD106, melanoma), VEGF, VEGF-a, VEGF-2(CD309) (various cancers). Other tumor-associated, antigens recognized by antibodies have been summarized and reviewed (Gerber, et al, mAbs 2009, 1:3, 247-.
The cell binding agent, preferably an antibody, can be any agent that is capable of antagonizing tumor cells, virally infected cells, microbially infected cells, parasitically infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes. More specifically, the cell binding agent may be any drug/molecule capable of resisting one of the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD14, CD14, CD14, CD14, CD 6856854, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD 6856854, CD14, CD14, CD14, CD14, CD14, CD14, CD14 6856856854, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD, CD274, CD276(B7-H3), CD303, CD304, CD309, CD326, 4-1BB, 5AC, 5T4(Trophoblast glycoprotein, TPBG, WNT-activated inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, α integrin, α v β 6, aminopeptidase N, amyloid β, androgen receptor, angiogenesis promoting protein factor 2, angiogenesis promoting protein factor 3, annexin A1, anthrax toxin protective antigen, anti-metastasis protein receptor, AOC3(VAP-1), B7-H3, Bacillus anthracis, BAFF (B cell activator), B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen), MUC16, MUC-IX (CAIX-38IX), CALA 31 AG, CALA 31, carbonic anhydrase IX, cardiac myoglobin, CCL11(C-C fragment chemokine 11), CCR4(C-C chemokine receptor 4, CD194), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2(CCK2R), CLDN18(Claudin-18), clusterin A, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4(CD184), C-X-C chemokine receptor 4, cyclic ribonuclease, cyclin B2, 5961B glycoprotein 8, cytomegalovirus B, Dabitran, ADP 4 (ADP), DPP-delta-peptide-like peptidase 4 (DPP-D634), DR5 (death receptor 5), Escherichia coli shiga toxin type-1, Escherichia coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain protein 7), EGFR, EGFRII, EGFRvIII, endoglin (CD105), endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, F protein of respiratory syncytial virus, coiled receptor, GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype GD, GlobH, Glica 3, n-glycolylneuraminic acid, GM3, the GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase C receptor, thermostable enterotoxin receptor (hSTAR), heat shock proteins, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3(ERBB-3), IgG4, HGF/SF (hepatocyte growth factor/scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-. gamma., influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2R (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2), integrin (. alpha.4,. IIb. beta.3,. alpha.v. beta.3,. alpha.4. beta.7,. alpha.5. beta.1,. alpha.6. beta.4,. alpha.7. beta.3,. alpha.5. beta.5, alpha. v. beta.5), interferon-. gamma. inducible protein, ITGA2, ITGB2, KIR2D, LCK, Leman, Leguy-antigen, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or Glycosyl Inhibitory Factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSMSMesothelin, MUC1 (mucin 1, cell surface-associated (MUC1) or Polymorphic Epithelial Mucin (PEM)), MUC1-KLH, MUC16(CA125), 1 (monocyte protein 1), MelanA/MART1, IAP, MPG, MS4A1, CN, myelin associated glycoprotein, Netasol glycoprotein, Netas-22, phospholipid 17, chemotactic antigen (ASN-Na-22), NGF, neuronal apoptosis-regulating protease 1, NOGO-A, Notch receptors, nucleolin, Neu oncogene products, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low density lipoprotein), OY-TES1, P21, A P53 non-mutant, P97, PAP, anti- (N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1(PD-1, programmed cell death protein 1, CD279), PDGF-R alphA (alphA platelet-derived growth factor receptor), PDGFR-betA, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor betA, sodium phosphate co-transporter, PMEL17, poly, protease 3(PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosA, PSMA, PSA, PSCA, rabies glycoprotein, RHD (Rh polypeptide 1(RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7(SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial prostate antigen 1), STEAP2, TGF, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor vascular endothelial marker 1), TENB2, Tenascon C (TN-C), TGF-alpha, TGF-beta (transforming growth factor beta 1, TGF-beta 2 (TEM-2), Tie 202b), tie2, TIM-1(CDX-014), Tn, TNF, TNF- α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5(DR5)), tumor-associated calcium signaling sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1(CD106), VEGF, VEGF-A, VEGF-2(CD309), VEGFR-1, VEGFR2, vimentin, WT1, XAGE 1, cells expressing any insulin growth factor receptor, or any epidermal growth factor receptor.
In another specific example, cell-binding agent-drug conjugates linked by the bridge linker of this patent are useful for cancer targeting therapy. Target cancers include, but are not limited to, adrenocortical carcinoma, anal carcinoma, bladder carcinoma, brain tumors (brain stem glioma, cerebellar astrocytoma, brain astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and hypothalamic glioma), breast carcinoma, carcinoid tumors, gastrointestinal cancer, unknown small cell carcinoma, cervical carcinoma, colon carcinoma, endometrial carcinoma, esophageal carcinoma, extrahepatic bile duct carcinoma, ewing family tumor (PNET), intracranial germ cell tumors, eye carcinoma, intraocular melanoma, gallbladder carcinoma, gastric carcinoma (stomach carcinoma), extragonadal germ cell tumors, peritrophoblastoma, head and neck carcinoma, hypopharynx carcinoma, islet cell carcinoma, renal carcinoma (renal cell carcinoma), leukemia (acute lymphocyte, acute myeloid, chronic lymphocyte, chronic granulocyte, hair cell), colon carcinoma, bladder carcinoma, and other cell, Lip and oral cancers, liver cancer, lung cancer (non-small cell, small cell), lymphoma (aids-related, central nervous system, cutaneous T-cell, hodgkin's disease, non-hodgkin's disease), malignant mesothelioma, melanoma, merkel cell carcinoma, metastatic squamous neck cancer and occult primary cancer, multiple myeloma and other plasma cell tumors, mycosis fungoides, myelodysplastic syndrome, myelodysplastic disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low malignancy), pancreatic cancer (exocrine, islet cell carcinoma), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, prostate rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), colon cancer, Salivary gland cancer, seiili syndrome, skin cancer (cutaneous T cell lymphoma, kaposi's sarcoma, melanoma), small intestine tumor, soft tissue sarcoma, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, urinary tract cancer, uterine cancer, unusual juvenile cancer, vaginal tumor, vulval tumor, and wilms tumor.
In another specific embodiment, cell-binding agent-drug conjugates linked by the bridge linkers of this patent are useful as compositions and methods for treating or preventing autoimmune diseases. Autoimmune diseases include, but are not limited to, Achlorhydradra autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukocytitis, Addison's disease, azoospermia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome, anti-dysenzymic syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune multiple endocrine syndrome types I, II and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo disease/Balo homosclerosis, bechets syndrome, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, Creutzfeldt-Jakob disease, ST syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytosis vasculitis, Degoid's disease, Dercuum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, discoid lupus erythematosus, eczema, endometriosis, anchorage-dependent arthritis, Eosinopophilus fasciitis, epidermolysis pilosus, nodular erythema, idiopathic mixed cryoglobulinemia, Erwinian syndrome, fibrodysplastic ossification, fibromyalgia, fibrotic myositis, fibrotic alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch's purpura, hepatitis of pregnancy, hidradenitis suppurativa, Huss syndrome (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Bergey's disease), inclusion body myositis, inflammatory demyelinating polyneuritis, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambertian-Eton myasthenia gravis syndrome, leukocyte clastic vasculitis, lichen planus, sclerosclerosis, Linear IgA disease (LAD), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis), lupus hepatitis, lupus erythematosus, Majeed's syndrome, Meniere's disease, microscopic polyarteritis, Miller-Fisher syndrome, mixed connective tissue disease, morphosis, Mohammerd-Huberman disease, Mkocurie syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, lethargy, neuromyelitis optica (Devic's disease), neuromuscular sclerosis, eyelid cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Hui rheumatism, PANDAS (a pediatric autoimmune neuropsychiatric disease associated with streptococci), paraneal cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, parsonage planitis, pemphigus vulgaris, anemia, peripheral encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, gangrenous dermatitis, pure red cell aplasia, Rasmussen encephalitis, Raynaud's phenomenon, recurrent polychondritis, Reiter's syndrome, restless leg syndrome, posterior neurofibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler's syndrome, Schnierler syndrome, Schniemann-strander syndrome, scleritis, scleroderma, Sjogren's syndrome, spondyloarthropathy, mucoviscidosis, Still disease, stiff person syndrome, subacute endocarditis disease, suzak syndrome, Sweet syndrome, chorea minor, sympathetic anemia, Takayasu arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, wegener's granulomatosis, wilson's syndrome, wiskott-aldrich syndrome.
In another specific embodiment, the binding molecules linked to the drug molecules through the bridging linker of the present invention, on the conjugates for the treatment or prevention of autoimmune diseases, include, but are not limited to, anti-elastin antibodies, Abys anti-epithelial cell antibodies, anti-basement membrane type IV collagen antibodies, antinuclear antibodies, anti-ds DNA, anti-ss DNA, anti-cardiolipin antibody IgM, IgG, anti-celiac disease antibodies, anti-phospholipid antibody IgK, IgG, anti-SM antibodies, anti-mitochondrial antibodies, thyroid antibodies, microsomal antibodies, T cell antibodies, thyroglobulin antibodies, anti-SCL-70, anti-Jo, anti-u.sub.1 RNP, anti-La/SSB, anti-SSA, anti-SSB, anti-parietal cell antibodies, anti-histone, anti-RNP, C-ANCA, P-ANCA, anti-centromere, anti-fibrinogen, anti-GBM antibodies, anti-ganglioside antibodies, anti-desmein 3 antibodies, anti-p 62 antibodies, anti-sp 100 antibodies, anti-mitochondrial (M2) antibodies, rheumatoid factor antibodies, anti-MCV antibodies, anti-topoisomerase antibodies, anti-neutrophil cytoplasmic (cANCA) antibodies.
In certain preferred embodiments, the binding molecules on the conjugates of the present invention bind to a receptor or receptor complex expressed on activated lymphocytes associated with autoimmune diseases. The receptor or receptor complex comprises, a member of the immunoglobulin superfamily (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD147, CD152/CTLA-4, PD-1 or ICOS), a member of the TNF receptor superfamily (e.g., CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3), an integrin, a cytokine receptor, a major histocompatibility factor receptor, a lectin (C, S-I) or a complement control type I protein.
In another embodiment, useful cell binding ligands immunospecific for viral or microbial antigens are humanized or human monoclonal antibodies. "viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV Tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen) capable of eliciting an immune response. "microbial antigens" include, but are not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide or lipid molecule capable of eliciting an immune response (e.g., bacterial, fungal, pathogenic protozoan or yeast polypeptides, including, e.g., LPS and capsular polysaccharides). Examples of antibodies that may be used to treat viral or microbial infections include, but are not limited to: palivizumab, which is a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542, a CD4 fusion antibody, used to treat HIV infection; ostevir, a human antibody used in the treatment of hepatitis B virus; PROTVIR, a humanized IgG1 antibody for the treatment of cytomegalovirus, and anti-LPS antibodies.
The cell binding molecule-drug conjugates made by the bridge linkers of this patent are useful for treating infectious diseases. These infectious diseases include, but are not limited to, acinetobacter infection, actinomycosis, african narcolepsy (african trypanosomiasis), aids (acquired immunodeficiency syndrome), amebiasis, anaplasmosis, anthrax, yersinia haemolytica infection, argentina hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, bacillus cereus infection, bacterial pneumonia, bacterial vaginitis, bacteroidal infection, venosasis, ascaris infection, BK viral infection, black birth control disease, human blastocyst protozoa infection, blastomycosis, viia hemorrhagic fever, borrelia infection, botulism (and infantile botulism), brazilian hemorrhagic fever, brucellosis, burkholderia infection, bruxiella ulcer, calicivirus infection (norovirus and saporovirus), campylobacteriosis, candidiasis (candidiasis, thrush), cat scratch disease, cellulitis, Chagas disease (trypanosomiasis americana), ascomycetes, chicken pox, chlamydia pneumoniae infection, cholera, glioblastoma, clonorchiasis sinensis, clostridium difficile infection, coccidioidomycosis, colorado tick fever, common cold (acute viral nasopharyngitis, acute rhinitis), creutzfeldt-jakob disease, crimean-congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larva migratory, cyclosporinosis, enterobacter infection, enterovirus infection, epidemic typhus, erythema infectioum (fifth disease), acute eruption, fascioliasis, fatal familial insomnia, filariasis, clostridium capsulatum food poisoning, free living amoeba infection, clostridium infection, aeronevus necrotica (fusobacterial myonecrosis), filariasis, germann-straussler-scherrella-scherreri syndrome, giardiasis, melioidosis, gonorrhea, granulomatous diarrhea (fifth disease), group a streptococcal infection, group B streptococcal infection, haemophilus influenzae infection, hand-foot-and-mouth disease (HFMD), hantavirus pulmonary syndrome, helicobacter pylori infection, hemolytic uremic syndrome, renal syndrome hemorrhagic fever, hepatitis a, hepatitis B, hepatitis c, hepatitis d, hepatitis e, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papilloma virus infection, human parainfluenza virus infection, membranous taenia disease, epstein-barr virus infectious mononucleosis (mononucleosis), influenza, isospora, kawasaki disease, keratitis, gigerbilosis, kuru, lassa fever, Legionella disease (Backward legionnaires 'disease), Legionella disease (Pontiake fever), Leishmaniasis, Lyme disease, lymphofilariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, melioidomycosis (Whitman's disease), meningitis, meningococcosis, posterior genital trematosis, microsporosis, molluscum contagiosum, parotitis, mouse typhus (endemic typhus), mycoplasmal pneumonia, foot edema, myiasis, neonatal conjunctivitis (neonatal eye disease), variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardia disease, onchocerciasis (Heanopheles), paracoccidioidomycosis (southern Eimeria), paragonimiasis, paragonitis, Pasteuresis, head lice, body lice, pubic louse, pelvic inflammatory disease, pertussis, pneumonic infection, pneumococcal disease, poliomyelitis, previtamin infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite heat, respiratory syncytial virus infection, nosemosis, rhinovirus infection, rickettsia pox, rift valley heat, rocky mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, septicemia, shigellasis (Bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporothrix, staphylococcal food poisoning, staphylococcus aureus infection, strongylosis, syphilis, taeniasis, tetanus, tinea barbarum (Barber itch), scalp tinea, tinea corporis, tinea cruris, tinea manuum, harbourne, tinea pedis (tinea pedis), onychomycosis (onycis), tinea versicolor, ascariasis (eye larva migration), toxocariasis (visceral larval transmigration), toxoplasmosis, trichinosis, trichomoniasis, trichiasis (whipworm infection), tuberculosis, tularemia, ureaplasma urealyticum infection, venezuelan equine encephalitis, venezuelan hemorrhagic fever, viral pneumonia, west nile fever, leukosarcoidosis (tinea alba), yersinia pseudotuberculosis, yersinia pestis enteropathy, yellow fever, zygomycosis.
The cell binding agents of the invention, more preferably antibodies, are directed against pathogenic strains including, but not limited to, Acinetobacter baumannii, Actinomyces israelii, Actinomyces and Propionibacterium, Trypanosoma brucei, HIV (human immunodeficiency virus), entamoeba histolytica, Anaplasmacytes, Bacillus anthracis, Vibrio haemolyticus, Hunnins, ascaris, Aspergillus, Astroviridae, Babesia, Bacillus cereus, various bacteria, Bacteroides, Escherichia coli, ascaris, BK virus, Oesophaga, Protozoa hominis, Blastomyces dermatitidis, Marulovirus, Borrelia, Clostridium botulinum, Sinomenii, Brucella, typically Burkholderia cepacis and other Burkholderia species, Mycobacteria ulceraceae, Calicidae, Campylobacter, typically Candida albicans and other Candida species, Burmannia, group A streptococci and staphylococci, Trypanosoma cruzi, Haemophilus ducreyi, VZV, Chlamydia trachomatis, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Climiya-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, hookeria brasiliensis, various parasites, Cyclosporidium, Taenia ribbon, Cytomegalovirus, dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) -flavivirus, Bifidobacterium fragilis, Corynebacterium diphtheriae, cestode, Melilonella, Ebola, Echinococcus, Enterococcus, Enterovirus, Rickettsia prosii, Brucella parvovirus B19, human herpesvirus 6 and human herpesvirus 7, fasciola gingivalis, Pediobolus hepatica and Pectinopsis megafasciola, FFI virus, Hyperperfringens, Clostridium prions, clostridium, other clostridia, geotrichum candidum, GSS prion, giardia enterica, burkholderia, bacillus spinosus and candida, gonococcus, klebsiella granulomatosa, streptococcus pyogenes, streptococcus agalactiae, haemophilus influenzae, enteroviruses, mainly coxsackie a and enterovirus 71, innominate virus, helicobacter pylori, escherichia coli O157: h7, Bunyaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus 1, herpes simplex virus 2, histoplasma capsulatum, duodenal adenoma and Chlamydomonas ampullatus, human bocavirus, ehrlichia, phagocytophile haemophilus, human metapneumovirus, Ehrlichia chalcone, human papilloma virus, human parainfluenza virus, Taenia miniata and Thymenia amesii, Epidera virus, Orthomyxoviridae family, Isospora beijerinckii, Chryseobacterium, Klebsiella pneumoniae, Legionella pneumophila, Leishmania, Mycobacterium leprae and Mycobacterium tuberculosis, Leptospira, monocytogenes, Listeria borrelia, Borrelia borrelia and other species of the genera, spanish and Malathia, lymphocytic choriomeningitis virus (LCMV), Plasmodium, Marburg, measles, Burkholderia pseudomallei, Neisseria meningitidis, retrograduate schistosomiasis, Microsporozoales, Molluscum Contagiosum (MCV), mumps, Rickettsia typhi, Mycoplasma pneumoniae, multiple bacterial and fungal parasitic dipteran larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prions, Nocardia and other Nocardia genera, Spanish, Paeoniaceae, Simania para and other subgenericosida genera, Pasteurella, head lice, human pediculus humanus, Bordetella pertussis Yersinia pestis, Streptococcus pneumoniae, pneumococci, poliovirus, Prevotella, Neisseria, JC virus, Chlamydia psittaci, Coxiella pneumoniae, rabies virus, S.unicus and Spirosoma, respiratory syncytial virus, nosema, rhinovirus, Rickettsia, Leptosphaeria, Rickettsia, rotavirus, rubella, Salmonella, SARS coronavirus, human scabies, Haemophilus, somatic cell, Shigella, varicella zoster virus, Notopterygium smallpox or smallpox, Trichosporon aureus, Staphylococcus aureus, Streptococcus pyogenes, Torulopsis, Treponema pallidum, tapeworm, tetanus, ringworm, uranium, tinea genus, Epidermophyton floccosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton rubrum, Venezon venenum , Trichophyton genus, Toxophyte or Toxolodon, Toxoplasma gondii, Trichophyton vaginalis, Mycobacterium tuberculosis, France Langerella, urea and equine encephalitis viruses, venezuelan equine encephalitis viruses, vibrio cholerae, melon naipoto viruses, west nile virus, beigelii filariosis, yersinia pseudotuberculosis, yersinia enterocolitica, yellow fever viruses, mucorales order (mucormycosis) and entomomycetales order (entomomycetous mycosis), mucorales order pseudomonas aeruginosa, campylobacter (vibrio), aeromonas, ehrlichia, yersinia, shigella, salmonella typhi, sargassum, treponema pernici, borrelia burgdorferi, spirochete, pneumocystis carinii, brucella abortus, brucella, mycoplasma, rickettsia pusturti, rickettsia, chlamydia, pathogenic fungi (aspergillus fumigatus, candida albicans, histoplasma capsulatus), protozoa (amoeba immaturus, tenas Trichomonas, Hominis Trichomonas, Trypanosoma gambiense, Trypanosoma rhodesiense, Leishmania rosenbergii, Leishmania tropicalis, Leishmania brasiliensis, Pneumocystis pneumoniae, Plasmodium vivax, Plasmodium falciparum, or Helminiththths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma Egypti and hookworm).
Other antibodies useful as cell-binding agents in the present patent for the treatment of viral diseases include, but are not limited to, antibodies to the following pathogenic viral antigens: poxviruses; herpes virus; an adenovirus; a small yellow virus; enteroviruses; picornavirus; parvovirus; reovirus; a retrovirus; an influenza virus; a parainfluenza virus; parotitis; measles; respiratory syncytial virus; rubella; arbovirus virus; a rhabdovirus; salmonella; non-a/non-b hepatitis virus; a rhinovirus; a coronavirus; a rotordo virus; oncogenic viruses, such as HBV (hepatocellular carcinoma), human papilloma virus (cervical cancer, anal carcinoma), kaposi's sarcoma-associated herpes virus (kaposi's sarcoma), human herpes virus type four (nasopharyngeal carcinoma, burkitt's lymphoma, primary central nervous system lymphoma), virus (merkel cell carcinoma), SV40 (simian virus 40), HCV (hepatocellular carcinoma), HTLV-1 (adult T-cell leukemia/lymphoma); immune disorders result in viruses such as human immunodeficiency virus (aids); central nervous system viruses, such as JCV (progressive multifocal leukoencephalopathy), hepatitis c virus (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), subacroviral encephalitis, orthomyxovirus (encephalitis), RV (rabies), probovirus, herpesvirus meningitis, ramusch hunter syndrome type II, poliovirus (poliovirus, post-polio syndrome), HTLV-1 (tropical palsy)); cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis), cardiovascular viruses, such as CBV (pericarditis, myocarditis), respiratory/acute viral intranasal inflammation/viral pneumonia, such as Epstein-Barr virus (EBV infection/infectious mononucleosis), cytomegalovirus, Severe coronavirus (severe acute respiratory syndrome) or orthomyxovirus, influenza a/b/c (influenza/avian influenza), paramyxovirus, human parainfluenza virus, RSV (human respiratory syncytial virus), hMPV, digestive system viruses (mumps virus, cytomegalovirus (cytomegalovirus esophagitis), adenovirus (adenovirus infection), rotavirus, norwalk virus, astrovirus, coronavirus, hepatitis B virus, CBV, hepatitis A virus, hepatitis C virus, hepatitis d virus, hepatitis e virus, HGV); urogenital viruses, such as BK virus, MuV (mumps).
Still further, the invention also includes a conjugate coupled to a bridge and an acceptable carrier, diluent or excipient for treating cancer, infection or autoimmune disease. Methods of treating cancer, infections and autoimmune diseases can be performed in vitro, in vivo or ex vivo. Examples of in vitro uses include treating a cell culture with it to kill all cells except for variants that do not express the target antigen; or to kill variants that express the undesired antigen. Examples of ex vivo use include treatment of Hematopoietic Stem Cells (HSCs) to kill diseased or malignant tumor cells prior to transplantation (HSCT). For example, tumor cells or lymphocytes are removed from bone marrow prior to autologous transplantation in the treatment of cancer or in the treatment of autoimmune diseases, or T cells and other lymphocytes are removed from allogeneic bone marrow or tissue prior to transplantation in order to prevent graft versus host disease. Such clinical ex vivo treatment may be carried out as follows: bone marrow is harvested from a patient or other individual and then incubated in a serum-containing medium to which the conjugate of the invention is added at a concentration ranging from about 1pM to 0.1mM at about 37 ℃ for about 30 minutes to about 48 hours. The specific drug concentration and incubation time should be determined by a skilled clinician. After incubation, the bone marrow cells are washed with serum-containing medium and administered to the patient intravenously according to known methods. In the case of patients who have received additional treatment (e.g., ablative chemotherapy or whole body radiation therapy) between bone marrow harvest and reinfusion of the treated cells, the treated bone marrow cells should be cryopreserved in liquid nitrogen using standard medical equipment.
In clinical in vivo use, the conjugates linked to the connectors of the present patent can be supplied as solutions or lyophilized solids that can be reconstituted in sterile water for injection. Examples of conjugate administration regimens are as follows: the conjugate was administered intravenously once a week for 8 to 20 weeks. A bolus dose is given in 50-500mL of physiological saline to which human serum albumin (e.g., 0.5 to 5mL of a concentrated solution of human serum albumin, 100mg/mL) can be added. The intravenous dose will be about 50 μ g to 20mg/kg (body weight) per week (10 μ g to 200mg/kg per injection). The patient can receive the second course of treatment 4-20 weeks after treatment. The specific clinical regimen for the route of administration, excipients, diluents, dosage, number of times, etc., can be determined by a skilled clinician.
Examples of medical conditions that can be treated with in vivo or ex vivo methods include malignancies of any type of cancer, autoimmune diseases, graft rejection and infections (viral, bacterial or parasitic).
The amount of conjugate required to achieve the desired biological effect will vary depending upon a variety of factors including the chemical identity, potency and bioavailability of the conjugate, the type of disease, the patient's ethnicity, the patient's disease progression, the route of administration, all factors determining the required dose, mode of administration and dosage regimen.
In general, the conjugates of the invention can be formulated for injection in an aqueous physiological buffer solution containing 0.1 to 10% w/v conjugate. Typical doses range from 1. mu.g/kg to 1g/kg (body weight) 1 time per day. Preferred dosage ranges are from 0.01mg/kg to 20mg/kg body weight per day or weekly, or infant equivalent. The preferred dosage of the drug to be administered may depend on such factors as the type and extent of progression of the disease or condition, the overall health status of the particular patient, the relative biological efficacy of the selected compound, the formulation of the drug, the route of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the drug-designated delivery route, as well as the rate of administration (bolus or continuous infusion) and the dosage regimen (number of repetitions in a given time period).
The conjugates of the invention can also be administered in unit dosage form, where the term "unit dose" refers to a single dose that can be administered to a patient and can be readily handled and packaged while the active conjugate itself, or a pharmaceutically acceptable composition as described below, remains a physically and chemically stable unit dose. Typical total daily doses range from 0.01 to 100mg/kg body weight. As a general guideline, the unit dosage for humans ranges from 1mg to 3000mg daily or weekly, or 2 weeks or monthly. The unit dosage range is preferably 1 to 500mg, once to four times a week, more preferably 1mg to 100mg, once a week. The conjugates provided herein can be formulated into pharmaceutical compositions by mixing with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be administered orally, e.g., as a medicament in the form of a tablet, simple capsule or soft gel capsule; or intranasally, such as powders, nasal drops or aerosols; or dermally, such as with a topical ointment, cream, lotion, gel or spray or via a transdermal patch.
Drug/cytotoxic agent
Drugs that can be conjugated to the cell binding molecules of the present invention are small molecule drugs that include cytotoxic agents, and can be attached, or modified, to the cell binding agent. The "small molecule drug" in the present invention broadly refers to an organic, inorganic or organometallic compound having a molecular weight of 100 to 1800, more preferably 120 to 1400. Such small molecule drugs are well described in the literature, such as WO05058367A2 and U.S. Pat. No. 4,956,303, etc., which are incorporated herein by reference. Small molecule drugs include known drugs and drugs to be disclosed.
Known drugs include, but are not limited to:
1) chemotherapeutic agents: a) alkylating agents, such as nitrogen mustards: chlorpheniramine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, dimethoxyamine hydrochloride, mechlorethamine oxide, amlodipine hydrochloride, mycophenolic acid, dulcitol, pipobroman, neomechlorethamine, benzene mustard cholesterol, prednimustine, thiotepa, triamcinolone pair, uracil; CC-1065 (including its aldorexin, kazelaixin and bizelaixin synthetic homologs); duocarmycin (including the synthetic homologs KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., dimers of Pyrrolobenzodiazepine (PBD) or tolmetin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidobenzodiazepine); nitrosoureas (carmustine, lomustine, fusin chloride, fotemustine, nimustine) Statin, lamotrigine); alkyl sulfonates (busulfan, endosulfan and sulfur); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines, such as chromanone, carotenone, metoclopramide and lindopa; ethyleneimine and methyl melamine, including hexamethylmelamine, triethylenetriamine, triethylphosphoramide, triethylenethiophosphoramide and trimethylolmethylamine; b) plant alkaloid: such as vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, catharanthine); taxoids (paclitaxel, docetaxel) and homologs thereof; maytansine (DM1, DM2, DM3, DM4, maytansine and ansamycin) and homologs thereof; cryptophycin (especially cryptophycin 1 and cryptophycin 8); epothilone, juncecrogol, discodermolide, bryozoalactone, dolastatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictyin, spongistatin; c) DNA topoisomerase inhibitors, such as etoposide (9-aminocamptothecin, camptothecin, clinostat, daunomycin, etoposide phosphate, irinotecan, mitoxantrone, noroxadiargyl, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycin (mitomycin C); d) antimetabolites, such as antifolates, DHFR inhibitors (methotrexate, trexate, denoxate, pteropterin, aminopterin (4-aminobenzoic acid), or other folate homologs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazolofuran, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine homologues, uracil homologues (ancitabine, azacitidine, 6-azauracil, capecitabine (hiloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (Tomudex), cytosine homologues (cytarabine, cytosine arabinoside, fludarabine), purine homologues (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine), folic acid supplements, such as, for example, loxelin, e) hormone therapy agents, such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (Gostaurin) Leuprolide acetate); anti-androgens (bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 homologues (CB1093, EB1089KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin a); cytokines (interferon- α, interferon- γ, Tumor Necrosis Factor (TNF), TNF-containing human proteins); f) kinase inhibitors, such as BIBW 2992 (anti-EGFR/Erb 2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR 2), mubritinib, ponatinib (AP 34), bafetinib (INNO-406), bosutinib (sk24ni-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, felinib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, istussin; g) antibiotics, such as enediynes antibiotics (calicheamicin, particularly calicheamicin γ 1, δ 1, α 1 and β 1 (see J.Med. Chem.1996,39 (11)), 2103-, triiron doxorubicin, adriamycin, streptozotocin, tubercidin, ubenimex, setastatin, zorubicin; h) others, such as polyketides (annonaceous acetogenins), in particular bullatacin and bullatacinone; gemcitabine, epoxygenase (e.g. kaferousome) Cloth), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovivin-7, Xegeva, Provenge, Yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, pelomycin), anthracyclines (e.g., daunorubicin), doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g., verapamil), Ca 2+ Inhibitors of ATPase (e.g., thapsigargin), inhibitors of histone deacetylase (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, entinostat, SB939, remininostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide a; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane, acetoglucuronolactone, aldphosphoramide, aminolevulinic acid, amsacrine, arabinoside, bestraucil, bison, edatraxate, defofamine, meclocine, disazoquinone, eflornithine (DFMO), efamitine, etiloamine, ethylgluconic acid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, diaminenitracridine, pentostatin, mechlorethamine, pirarubicin, podophyllic acid, 2-ethylhydrazine, procarbazine;
Figure BDA0001580863800000411
Piperazinedione propane; rhizomycin; (iv) Wenzuo; spiro germanium; geobacillus azavor; a tri-imine quinone; trichlorotriethylamine; trichothecenes (in particular T-2 toxin, verrucomicin A, bacillocin A and anguidine), polyurethanes, siRNAs, antisense drugs and nucleolytic enzymes.
2) Autoimmune disease agents, including but not limited to, cyclosporine, cyclosporin a, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucoloridazole, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus.
3) Anti-infectious disease agents, including but not limited to a) aminoglycosides: amikacin, astemicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminoxykanamycin, dibekacin, tobramycin), neomycin (framycin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methyltautomycin; b) amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycin: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephem: cephem (loracarbef), cephalosporacetonitrile, ampicillin, cephradine, cefadroxil, cephalonine, ceftazidime, cephalothin or cephalotaxin, cephalexin, cephramycin, cefamandole, cefapirin, azapiromidin, fluxazole cephalosporin, sporocetone, azolin cephalosporin, cefbuperazone, cefcapene, cefixime, cefpodoxime, cefixime, cefoxitin, cefprozil, cefetaxeme, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefpira, cefetamet, cefepime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, thienam, cefotaxime, cefozopran, cefazolin, cephalexin, cefepime, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, cefotiarin, ceftizoxime, cefprozil, ceftriaxone, cefuroxime, ceftizoxime, cephamycins (cefoxitin, cefotetan, cefcyanazole), oxacephems (flomoxef, cephalosporins); f) glycopeptide: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin, g) glycylcyclin: such as tigecycline, h) beta-lactamase inhibitors: penicillane (sulbactam, tazobactam), oxapenem (clavulanic acid); i) lincosamide: clindamycin, lincomycin; j) lipopeptides: daptomycin, a54145, Calcium Dependent Antibiotic (CDA); k) macrolides: azithromycin, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolide (telithromycin, sequoyimycin), midecamycin, mickamycin, oleandomycin, rifamycin (isoniazid, rifampin, rifabutin, rifapentine), ropiniromycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), oleandomycin acetate, telithromycin; l) monocyclic amines: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, silocillin, bacampicillin, ampicillin, doxorubicin), azlocillin, benzylpenicillin, benzathine phenoxymethylpenicillin, cloxacillin, procaine penicillin (metilin), mezlocillin, methicillin, nafcillin, oxacillin, acemethicillin, penicillin, nafcillin, piperacillin, ampicillin, sulfonicillin, temocillin, ticarcillin; o) a polypeptide: bacitracin, colistin, polymyxin B, p) quinolones: alatrefloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, gatifloxacin, gemifloxacin, grepafloxacin, carnotrexacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) streptogramins: pristinamycin, quinupristin/dalfopristin, r) sulfonamides: sulfonamides: sulfonamides, sulfadiazine, sulfasalazine, sulfisoxazole, tamoxifen, trimethoprim-sulfamethoxazole (sulfamethoxazole); s) steroid antibacterial drugs: such as fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, cimeticycline, demeclocycline, ramoxiline, mecycline, methacycline, minocycline, oxytetracycline, penicillin V kalipecycline, pyrrolidinemethyltetracycline, tetracycline, glycylcycline (such as tigecycline): u) other types of antibiotics: annonaceous acetogenins, arsine, bacteroidal terpineol inhibitors (bacilli), DANAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, saxifragol, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalitide, metronidazole, mupirocin, NAM synthesis inhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, pratensomycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, echinacon.
4) Antiviral drugs: a) entry/fusion inhibitors: apaviralo, maraviroc, vicrivroc, gp41 (enfuvirtide), PRO 140, CD4 (abalizumab); b) integrase inhibitors: raltegravir, elvite-gravir, globoid dna a; c) maturation inhibitors: bevirimat, vivocon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; E) nucleosides and nucleotides: abacavir, adefovir, armocivir, abciximab, brivudine, cidofovir, cladribine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluxacillin (5-FU), 3 '-fluoro-substituted 2', 3 '-deoxynucleoside homologs (such as 3, 3' -fluoro-2 ', 3' -dideoxythymidine (FLT) and 3 '-fluoro-2', 3 '-dideoxyguanosine (FLG), fomivirsen, 9-guanine, idoxuridine, lamivudine (3TC), 1-nucleosides (such as β -1-thymidine and β -1-2' -deoxycytidine), penciclovir, racivir, ribavirin, dilastatin, vudine (d4T), talivirin (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleoside: amantadine, atitidine, carboprvirine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, pegylated interferon, lovirine, lodenosine, methidathiozone, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod (R-848), acetimidamantadine; g) protease inhibitors: amprenavir atazanavir, boceprevir, daronavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) other types of antiviral drugs: antioxidase, arbidol, kalarotide, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, anabolic inhibitor, ribavirin, seliciclib.
5) Drugs linked through the bridges of the present invention also include radioisotopes. Examples of radioactive isotopes (radionuclides) are 3 H， 11 C， 14 C， 18 F， 32 P， 35 S， 64 Cu， 68 Ga， 86 Y， 99 Tc， 111 In， 123 I， 124 I，125I， 131 I， 133 Xe， 177 Lu， 211 At or 213 And (4) Bi. The radioisotope labeled antibodies may be used in receptor targeted imaging experiments or in targeted therapy as antibody-drug conjugates of the invention (Wu et al Nature Biotechnology 2005,23(9): 1137-1146). Cell binding molecules, such as antibodies, may be labeled by linking the ligand reagent to a linker of the present patent. Ligands can be bound, chelated, or complexed to radioactive metals using methods described in the literature (Current Protocols in Immunology, Volumes 1and 2, origin et al, ed.wiley-Interscience, New York, n.y., Pubs. (1991)). Chelating ligands that can complex metal ions include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX), among others.
6) A pharmaceutically acceptable salt, acid or derivative thereof of any of the above.
In another example, the drugs of structural formulae (II) and (IV) may be chromogenic molecules, and the conjugates may be used to detect, monitor or study the interaction of cell-binding molecules with target cells. The chromonic molecule can absorb a light, such as ultraviolet, fluorescent, infrared, near infrared, or visible light; the chromophoric molecules include yellow pigment, red blood cell, iridescent pigment, white blood cell, melanin and blue-green pigment, fluorescent molecule (fluorescent chemical substance capable of absorbing light and emitting light), visual light transduction molecule, photon molecule, luminescent molecule and fluorescein compound.
The chromonic molecule can be selected from, but is not limited to, non-protein organic fluorophores such as xanthene derivatives (fluorescein, rhodamine, Oregon Green, eosin, and Texas Red); cyanine derivatives (cyanines, indocarbocyanines, oxacyanines, thiacyanines, and merocyanines); squaric acid derivatives and ring-substituted squaric acids, including Seta, SeTau and Square dyes; naphthalene derivatives (dansyl and sodium fluorosilicate derivatives); coumarin derivatives; oxadiazole derivatives (pyridyloxazole, nitrobenzoxazole and benzooxadiazole); anthracene derivatives (anthraquinones, including DRAQ5, DRAQ7 and CyTRAK orange); pyrene derivatives (cascade blue, etc.); oxazine derivatives (nile red, nile blue, cresyl violet, oxazine 170, etc.); acridine derivatives (flavonol flavin, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (auramine, crystal violet, malachite green) and tetrapyrrole derivatives (porphin, phthalocyanine, bilirubin).
The chromogenic molecule is selected from any of the homologues and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluProbes (Interchim), Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandy), HiLyte Fluor (Anaspec), Seta, Setau and Square dyes (Biosearch Technologies), SureLight dyes (APC, RPEPercP, Phyobilisomes) (Columbia Biosciences), APCXL, RPE, BPE (Phoco-Biotech).
Examples of widely used fluorescent compounds that can be reacted or coupled to the linkers of the present invention are: allophycocyanin (APC), annatto, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, hydroxycoumarin, lissamine rhodamine B, lucifer yellow, Me-methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugates, PE-R-Phycoerythrin (PE), Red 613, Seta-555-Azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta 680-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, Seu-380-NHS, Seta-405-maleimide, Seta-NHS 405-NHS 425-Seu-NHS, Seta-647-S, texas Red, TRITC, TruRed, X-Rhodamine.
Fluorescent compounds which can be linked to the linker of the invention for the study of nucleic acids or proteins are selected from the following compounds or derivatives thereof: 7-AAD (7-Aminoactinomycin D, CG-selective), acridine orange, chromomycin A3, CyTRAK orange (Biostatus), DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, Hoechst33342, LDS 751, mithramycin, Propidium Iodide (PI), SYTOX blue, SYTOX green, SYTOX orange, thiazole orange, TO-PRO, cyanine dye monomers, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Fluorescent compounds which can be linked to the linker of the invention for the study of cells are selected from the following compounds or derivatives thereof: DCFH (2', 7' -dichlorodihydrofluorescein, oxidized form), DHR (dihydrorhodamine 123, oxidized form, photocatalytic oxidation), Fluo-3(AM ester, pH >6), Fluo-4(AM ester, pH7.2), Indo-1(AM ester, low/high calcium (Ca 2+)), SNARF (pH 6/9). Preferred fluorescent compounds are selected from: allophycocyanin (APC), AmCyan1 (tetramer, Clontech), AsRed2 (tetramer, Clontech), Cirsium green (monomer, MBL), Azurite, B-phycoerythrin (BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP", Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation) GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRed1, J-Red, Katusha, Kusara, Ssanra Orange (monomer, McOcofP, mCopofp, mClont, mClostrich, monomer, MBherry 635, Khryse 635, Tahryse, Tahrysen-5, Tamcorphan, Tamcitabine, Tamconse, Tamcrofen, Tamcfargey 2, Tamcrofen, Tamcfargex, Tachy, Tsange (S65, Tachy, Tachytrix, Tachy, Tachytrix, Tachy, Tachytrix, Tachy, Tachytrix, T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFPP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP (dimer, Evrogen), Venus, wild-type GFP type, YPet, ZsGreen1 (tetramer, Clontech), zsyollow 1 (tetramer, Clontech).
In another embodiment, the drug of formula (II) and (IV) may be a polyalkylene glycol, which when administered to a mammal, may extend the half-life of the cell-binding agent. Polyalkylene glycols include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol, and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are hydroxy PEGs with one end functionally activated (e.g., hydroxy PEGs with one end activated, including hydroxy PEG-carboxylic acid activated esters, hydroxy PEG-aldehydes, hydroxy PEG-amines, hydroxy PEG-hydrazides, hydroxy PEG-hydrazinoformates, hydroxy PEG-iodoacetamides, hydroxy PEG-maleimides, hydroxy PEG-ortho-methylpyridinyl disulfide, hydroxy PEG-oximes, hydroxy PEG-phenyl carbonates, hydroxy PEG-phenyl glyoxal, hydroxy PEG-thiazolidine-2-thiones, hydroxy PEG-thioesters, hydroxy PEG-thiols, hydroxy PEG-triazines, and hydroxy PEG-vinyl sulfones).
In certain embodiments, the polyalkylene glycol has a molecular weight of about 10Da to about 200kDa, preferably a molecular weight of about 88Da to about 40 kDa; having two branches, each branch having a molecular weight of about 88Da to about 40 kDa; more preferably, there are two branches, each of about 88Da to about 20 kDa. In one embodiment, the polyalkylene glycol is polyethylene glycol having a molecular weight of about 10kDa, 20kDa, or 40 kDa. In particular embodiments, the PEG is PEG 10kDa (linear or branched), PEG 20kDa (linear or branched) or PEG 40kDa (linear or branched). Many U.S. patents disclose the preparation of linear or branched "non-antigenic" PEG polymers and derivatives or conjugates thereof, see U.S. patent nos. 5,428,128; 5621039, respectively; 5622986; 5643575, respectively; 5728560, respectively; 5730990, respectively; 5738846, respectively; 5811076; 5824701, respectively; 5840900, respectively; 5880131, respectively; 5900402, respectively; 5902588, respectively; 5919455, respectively; 5951974, respectively; 5965119, respectively; 5965566, respectively; 5969040, respectively; 5981709, respectively; 6011042, respectively; 6042822, respectively; 6113906, respectively; 6127355, respectively; 6132713, respectively; 6,177,087, and 6,180,095. Antibody-polyalkylene glycol conjugates linked by a bridging linker have the structure as Pg 01:
Figure BDA0001580863800000461
Wherein the mAb is an antibody; n is 1 to 30; r 'and R' are independently H or CH 3 ；m 3 And m 4 Independently 0 to 5000;
Figure BDA0001580863800000462
X 1 ，X 2 ，R 1 ，R 2 and R 3 As defined in formulae (I) and (II); r is 4 Is OH, H, or R 1 Or R as defined in formula (I) 3 。
In another embodiment, preferred cytotoxic agents that are linked to the cell binding molecule via the linkers of this patent are tubulysin, maytansine, taxanes, CC-1065 homologs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., Pyrrolobenzodiazepine (PBD), tolmetin, indolopendrazine, imidazobenzothiadiazine or dimers of oxazolidobenzodiazepine), calicheamicin and enediyne antibiotics, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, auristatin (e.g., MMAE, MMAF, auristatin E, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB) and EFP (AEFP)), duocarmycin, thiotepa, vincristine, hemimitadine, navajamide, micin, thiostatin, dextran vitriol, microsclerominin, theonellamide, esperamicin, PNU-159682 and homologs and derivatives thereof.
Tubulysin is a preferred cytotoxic agent for conjugate coupling and may be prepared by methods known in the art, isolated or synthesized from natural sources, such as Balasubramanian, R.; et al.j.med.chem.,2009,52, 238-240. Wipf, p.; et al, org, lett, 2004,6, 4057-4060, Pando, o.; et al.j.am.chem.soc.,2011,133,7692-7695. Reddy, j.a.; pharmaceutical, 2009,6, 1518-1525. Raghavan, b.; et al.j.med.chem.,2008,51, 1530-1533, Patterson, a.w.; et al.j.org.chem.,2008,73, 4362-4369. Pando, o.; et al, org, lett, 2009,11(24), pp 5567-5569. Wipf, p.; et al, org, lett, 2007,9(8), 1605-1607. Friestad, g.k.; org.lett.,2004,6, pp 3249-3252. Hillary m.peltier, h.m.; et al.j.am.chem.soc.,2006,128,16018-16019. Chandrasekhar, s.; et al.j.org.chem.,2009,74, 9531-9534. Liu, y.; pharmaceutical, 2012,9, 168-175 Friestad, g.k., al.mol.pharmaceuticals, g.k.; et al org.lett.,2009,11, 1095-1098. Kubicek, k.; et al, Angew Chem Int Ed Engl,2010.49,4809-12.Chai, Y.; et al, Chem Biol,2010,17:296-309 Ullrich, A.; et al, Angew Chem Int Ed Engl,2009,48,4422-5.Sani, M.; et al, Angew Chem Int Ed Engl,2007,46,3526-9.Domling, A.; et al, Angew Chem Int Ed Engl,2006.45,7235-9. patent: zanda, m.; et al, can.pat.appl.ca 2710693(2011) Chai, y; eur.pat.appl.2174947(2010), PCT WO 2010034724, Leamon, c.; et al, PCT WO 2010033733, WO 2009002993, Ellman, J.; et al, PCT WO 2009134279; PCT WO 2009012958, US appl.20110263650,20110021568, Matschiner, g.; et al, PCT WO 2009095447.Vlahov, I.; et al, PCT WO 2009055562, WO 2008112873.Low, P.; et al, PCT WO 2009026177.Richter, W., PCT WO 2008138561.Kjems, J.; et al, PCT WO 2008125116.Davis, M.; et al, PCT WO 2008076333.diene, j.; et al, U.S. Pat. appl.20070041901, WO 2006096754, Matschiner, g.; et al, PCT WO 2006056464.Vaghefi, F.; et al,5PCT WO 2006033913.Doemling, a., ger. offen.de 102004030227; PCT WO 2004005327; WO 2004005326; wo2004005269.stanton, m.; et al, U.S. Pat. appl.pub.20040249130. hoefle, g.; et al, Ger.Offen.DE 10254439; DE 10241152; DE 10008089.Leung, d.; et al, WO 2002077036.Reichenbach, H.; et al, Ger.Offen.DE 19638870; wolfgang, r.; US 20120129779, Chen, h, US appl.20110027274. A preferred structure of tubulysin linked to a cell binding molecule is described in patent PCT/IB 2012/053554.
Examples of antibody-tubulysin conjugate structures linked by a bridging linker are T01, T02, T03, T04, T05, T06 and T07:
Figure BDA0001580863800000481
Figure BDA0001580863800000491
wherein the mAb is an antibody; z 3 And Z 3 ' independently is H, R 1 ，OP(O)(OM 1 )(OM 2 )，OCH 2 OP(O)(OM 1 )(OM 2 )，OSO 3 M 1 Or O-glycosides (glycosides, galactosides, mannosides, glucosides, fructosides, etc.), NH-glycosides, S-glycosides or CH 2-glycosides; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 (ii) a n is 1 to 30;
Figure BDA0001580863800000492
X 1 ，X 2 ，R 1 ，R 2 and R 3 The same as defined in formulae (I) and (II).
Calicheamicin and related enediyne antibiotics are preferred cytotoxic agents and are described in the following references: nicolaou, K.C. et al, Science 1992,256, 1172-; proc.Natl.Acad.Sci USA.1993,90,5881-; 5,053,394; 5,108,912, respectively; 5,264,586, respectively; 5,384,412, respectively; 5,606,040, respectively; 5,712,374; 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562, respectively; 6,124,310, respectively; 8,153,768. Examples of structures of antibody-calicheamicin homologues linked by a bridging linker are C01:
Figure BDA0001580863800000493
wherein the mAb is an antibody; n is 1 to 30;
Figure BDA0001580863800000501
X 1 ，X 2 ，R 1 ，R 2 and R 3 The same as defined in formulae (I) and (II).
Maytansine is the preferred cytotoxic agent in this patent, and maytansine and its homologs are described in the following U.S. patents: 4,256,746, respectively; 4,361,650, respectively; 4,307,016, respectively; 4,294,757, respectively; 4,294,757, respectively; 4,371,533, respectively; 4,424,219, respectively; 4,331,598, respectively; 4,450,254; 4,364,866, respectively; 4,313,946, respectively; 4,315,929, respectively; 4,362,663, respectively; 4,322,348, respectively; 4,371,533; 4,424,219; 5,208,020; 5,416,064; 5,208,020; 5,416,064; 6,333.410, respectively; 6,441,163; 6,716,821,7,276,497; 7,301,019, respectively; 7,303,749, respectively; 7,368,565; 7,411,063, respectively; 7,851,432, and 8,163,888. An example of an antibody-maytansine conjugate linked by a bridging linker is M01:
Figure BDA0001580863800000502
Wherein the mAb is an antibody; n is 1 to 30;
Figure BDA0001580863800000503
X 1 ，X 2 ，R 1 ，R 2 and R 3 The same as defined in formulae (I) and (II).
Taxanes, including paclitaxel (a cytotoxic natural product) and docetaxel (a semi-synthetic derivative) and homologs thereof, are preferred cytotoxic molecules of this patent and are described in the following references: kc. nicolaou et al, j.am. chem. soc.1995,117, 2409-2420; ojima et al, J.Med.chem.1996,39: 3889-; 1997,40, 267-278; 2002,45, 5620-5623; ojima et al, Proc.Natl.Acad.Sci.,1999,96: 4256-; kim et al, bull chem.soc, 1999,20, 1389-; miller, et al.J.Med.chem.,2004,47, 4802-; U.S. Pat. nos. 5,475,0115,728,849, 5,811,452; 6,340,701; 6,372,738; 6,391,913,6.436,931; 6,589,979, respectively; 6,596,757, respectively; 6,706,708; 7,008,942; 7,186,851, respectively; 7,217,819, respectively; 7,276,499, respectively; 7,598,290, and 7,667,054.
Examples of conjugate structures where the antibody-taxane is linked via a bridging linker are as Tx01, Tx02 and Tx 03:
Figure BDA0001580863800000511
wherein the mAb is an antibody; n is 1 to 30;
Figure BDA0001580863800000512
X 1 ，X 2 ，R 1 ，R 2 and R 3 The same as defined in formulae (I) and (II).
CC-1065 homologs and duocarmycin homologs are also preferred cytotoxic agents for attachment to the bridge linkers of this patent. Examples of CC-1065 homologs and duocarmycin homologs and their synthesis can be found in: warpehoski, et al, J.Med.chem.31:590-603(1988), D.Boger et al, J.org.chem; 66; 6654 and 6661, 2001; U.S. Pat. Nos. 4169888,4391904,4671958,4816567,4912227,4923990,4952394,4975278,4978757,4994578,5037993,5070092,5084468,5101038,5117006,5137877,5138059,5147786,5187186,5223409,5225539,5288514, 5324483483483, 53740, 5332837,5334528, 54035427908, 5475092,5495009 009, 5501, 5545806,5547667, 55825, 55798, 5573922, 55717, 5585089,5585499 499, 558587161, 55956017, 56229292929, 56375639,567763,567763,567763,567763,56607763,567763,56607763,7763,7763,7763,7763,77607763,7763,7763,7763,5935,337763,337763,977, 607763,979263,9748,9763,779, 607763,7763,7763,7763,7763,7763,7763,7763,7763,7763,978,976,976,779, 607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,978,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,3363,607763,3363,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,6064048,6064048,607763,607763,6064048,607763,6064048,607763,607763,607763,607763,6064048,607763,607763,607763,6064048,607763,6064048,6064048,607763,607763,6064048,607763,607763,607763,607763,607763,6064048,3363,6064048,6064048,6064048,6064048,606404607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,607763,6064046064048,607763,606404607763,606404607763,607763,607763,607763,607763,607763,6064048,6064048,6064048,6064048,6064048,6064048,6064048,607763,6064048,607763,6064048,607763,607763,607763,607763,607763,606404606404606404607763,607763,606404607763,3363,6064048,6064048,6064048,6064048,607763,6064048,6064048,6064048,6064048,6064048,607763,6064048,6064048,6064048,6064048,6064048,60. Examples of antibody-CC 1065 homolog structures linked via bridging linkers are as in CC01, CC02, and CC 03:
Figure BDA0001580863800000521
Wherein the mAb is an antibody; n is 1 to 30; z is a linear or branched member 4 And Z 4 ' is H, PO (OM) 1 )(OM 2 )，SO 3 M 1 ，CH 2 PO(OM 1 )(OM 2 )，CH 3 N(CH 2 CH 2 ) 2 NC(O)-，O(CH 2 CH 2 ) 2 NC(O)-，R 1 Or a glycoside; x 3 And X 3 ' is O, NH, NHC (O), OC (O), -C (O) O, R 1 Or by default;
Figure BDA0001580863800000531
X 1 ，X 2 ，R 1 ，R 2 ，M 1 and M 2 The same as defined in formulae (I) and (II).
Daunorubicin/doxorubicin homologs are also preferred cytotoxic agents linked to the bridge linkers of this patent. Preferred structures and their synthesis are referred to in the literature: hurwitz, E., et al, Cancer Res.1975, 35,1175-1181, Yang, H.M., and Reisfeld, R.A., Proc.Natl.Acad.Sci.1988,85, 1189-1193; pietersz, c.a., e., et al., Cancer res.1988,48, 926-one 9311; trouet, et al, 1982,79, 626-629; Z.Brich et al, J.controlled Release,1992,19, 245-; chen et al, syn, comm, 2003,33, 2377-; king et al, bioconj. chem.,1999,10, 279-one 288; king et al, j.med.chem.,2002,45, 4336-4343; kratz et al, J Med chem.2002,45,5523-33; kratz et al, Biol Pharm Bull. Jan.1998,21, 56-61; lau et al, bioorg.med.chem.1995,3, 1305-; scott et al, bioorg.med.l chem.lett.19966, 1491-1496; watanabe et al, Tokai J.Experimental Clin.Med.1990,15, 327-334; zhou et al, j.am.chem.soc.2004,126, 15656-7; WO 01/38318; us patent 5,106,951; 5,122,368, respectively; 5,146,064, respectively; 5,177,016, respectively; 5,208,323, respectively; 5,824,805; 6,146,658; 6,214,345; 7569358, respectively; 7,803,903, respectively; 8,084,586, respectively; 8,053,205. Examples of antibody-doxorubicin homolog structures linked by bridge linkers are Da01, Da02, Da03 and Da 04:
Figure BDA0001580863800000532
Figure BDA0001580863800000541
Wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independently H, O, NH, NHC (O) NH, C (O), R 1 Or OC (O);
Figure BDA0001580863800000542
X 1 ，X 2 ，R 1 and R 2 The same as defined in formulae (I) and (II).
Auristatins and dolastatins are preferred cytotoxic agents linked to a bridge linker. Auristatins (e.g., dolauristatin e (ae), auristatin eb (aeb), auristatin efp (aefp), monomethyl auristatin e (MMAE), monomethyl auristatin F (mmaf), Auristatin F Phenylenediamine (AFP) and phenylalanine variants of MMAE) are homologs of dolastatin and are described in: int.j.oncol.1999,15,367-72; molecular Cancer Therapeutics,2004,3(8), 921-932; us patent application 11134826,20060074008,2006022925, us patent 4414205,4753894,4764368,4816444,4879278,4943628,4978744,5122368,5165923,5169774,5286637,5410024,5521284,5530097,5554725,5585089,5599902,5629197,5635483,5654399,5663149,5665860,5708146,5714586,5741892,5767236,5767237,5780588,5821337,5840699,5965537,6004934,6033876,6034065,6048720,6054297,6054561,6124431,6143721,6162930,6214345,6239104,6323315,6342219,6342221,6407213,6569834,6620911,6639055,6884869,6913748,7090843,7091186,7097840,7098305,7098308,7498298,7375078,7462352,7553816,7659241,7662387,7745394,7754681,7829531,7837980,7837995,7902338,7964566,7964567,7851437,7994135. Examples of conjugate structures in which antibody-auristatin is linked by a bridging linker are as shown in Au01, Au02, Au03, Au04 and Au 05:
Figure BDA0001580863800000551
Wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)R 1 Or by default; x 4 And X 4 ' is independent CH 2 ，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; z 3 And Z 3 ' is independently H, R 1 ，OP(O)(OM 1 )(OM 2 )，OCH 2 OP(O)(OM 1 )(OM 2 )，NHR 1 ，OSO 3 M 1 Or O-glycoside (glycoside, galactoside, mannoside, glucoside, fructoside, etc.), NH-glycoside, S-glycoside or CH 2 -a glycoside; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；
Figure BDA0001580863800000561
X 1 ，X 2 ，R 1 ，R 2 And R 3 The same as defined in formulae (I) and (II).
Benzodiazepine dimers (e.g., dimers of Pyrrolobenzodiazepine (PBD), tolmetin, indolophenyldiazepine, imidazobenzothiadiazole, or oxazolidinobenzodiazepine) are preferred cytotoxic molecules of the present invention and are also described in the literature in the art: us patent 8,163,736; 8,153,627, respectively; 8,034,808, respectively; 7,834,005, respectively; 7,741,319, respectively;
7,704,924；7,691,848；7,678,787；7,612,062；7,608,615；7,557,099；7,528,128；7,528,126；
7,511,032；7,429,658；7,407,951；7,326,700；7,312,210；7,265,105；7,202,239；7,189,710；
7,173,026；7,109,193；7,067,511；7,064,120；7,056,913；7,049,311；7,022,699；7,015,215；
6,979,684；6,951,853；6,884,799；6,800,622；6,747,144；6,660,856；6,608,192；6,562,806；
6,977,254；6,951,853；6,909,006；6,344,451；5,880,122；4,935,362；4,764,616；4,761,412；
4,723,007, respectively; 4,723,003, respectively; 4,683,230, respectively; 4,663,453, respectively; 4,508,647, respectively; 4,464,467, respectively; 4,427,587, respectively; 4,000,304, respectively; U.S. patent application 20100203007,20100316656,20030195196. Examples of antibody-benzodiazepine dimer conjugate structures are PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB09, PB10 and PB 11:
Figure BDA0001580863800000562
Figure BDA0001580863800000571
Figure BDA0001580863800000581
Figure BDA0001580863800000591
wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)NR 3 ，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; x 4 And X 4 ' is independent CH 2 ，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；
Figure BDA0001580863800000592
Figure BDA0001580863800000593
X 1 ，X 2 ，R 1 ，R 2 And R 3 The same as defined in formulae (I) and (II). R 1 And/or R 2 A default may be used.
In another embodiment, it is preferred that two or more different cytotoxic agents are coupled to the cell binding molecule through the bridge of this patent. The two or more different cytotoxic agents may be selected from the group consisting of: tubulysin, maytansine, taxanes, CC-1065 homologues, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., Pyrrolobenzodiazepine (PBD), tolamemycin, indolophenyldiazepine, imidazobenzothiadiazole or oxazolidobenzodiazepine dimers), calicheamicin and enediyne antibiotics, actinomycin, diazoserine, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, auristatin (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PYE, auristatin TP, auristatin 2-AQ, auristatin 6-AQ, auristatin E (E), and auristatin EFP (E FP), dactinomycin, vincristine, hemimicatin, azulmicin, micatin, sultamicin, theostatin, PNU-159682 and its homologues and derivatives. Examples of conjugates comprising two or more different cytotoxic agents connected via a bridge linker are e.g. Z01, Z02, Z03, Z04, Z05, Z06, Z07, Z08, Z09, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17 and Z18:
Figure BDA0001580863800000594
Figure BDA0001580863800000601
Figure BDA0001580863800000611
Figure BDA0001580863800000621
Figure BDA0001580863800000631
Wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)NR 3 ，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or by default; x 4 And X 4 ' is independently H, CH 2 ，OH，O，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；
Figure BDA0001580863800000632
X 1 ，X 2 ，R 1 ，R 2 And R 3 The same as defined in formulae (I) and (II). R 1 And/or R 2 A default may be used.
In another example, a cell binding molecule can be coupled to a cell binding ligand or receptor through a bridge linker of the present patent. These cell-binding ligand or receptor conjugates, particularly antibody-receptor conjugates, can be used not only as targeting vehicles/guides to deliver the conjugate to malignant cells, but also to modulate or co-stimulate a desired immune response or alter signaling pathways. In immunotherapy, cells bind to ligands or receptorsPreferably to antibodies to TCR (T cell receptor) T cells or CARs (chimeric antigen receptor) T cells or B Cell Receptors (BCR), or to toxic cells. Cell binding ligands or receptors are selected from, but not limited to, folate derivatives (proteins that bind to folate receptors and are overexpressed in ovarian cancer and other malignancies) (Low P S et al, Acc. chem.2008, 41, 120-); urea derivatives of glutamate (binding to prostate specific membrane antigen, surface marker of prostate Cancer cells) (Hillier S.M et al, Cancer Res.2009, 69, 6932-6940); somatotatin (also known as Somatostatin (GHIH) or Somatostatin (SRIF)) or Somatostatin) and its homologues such as octreotide (Sandostatin) and lanreotide (Somatuline) (especially for neuroendocrine tumors, GH producing pituitary adenomas, paragangliomas, nonfunctional pituitary adenomas, pheochromocytomas) (Ginj M et al, proc.natl.acad.sci.2006, 103, 16436-16441); certain aromatic sulfonamides, specific for carbonic anhydrase IX (markers for hypoxia and renal cell carcinoma) (Neri D et al, nat. Rev. drug Discov.2011, 10, 767-777); pituitary Adenylate Cyclase Activating Peptide (PACAP) (PAC1) for pheochromocytoma and paraganglioma; vasoactive intestinal peptide (VIP/PACAP) (VPAC1, VPAC2) for lung, stomach, colon, rectal, breast, prostate, pancreatic ductal, liver and bladder cancer; cholecystokinin (CCK) (CCK1 (formerly CCK-a) and CCK2 for small cell lung cancer, medullary thyroid cancer, astrocytoma and ovarian cancer); bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH) 2 ) Gastrin Releasing Peptide (GRP) (BB1, GRP receptor subtype (BB2), BB3 and BB4) for use in renal cell carcinoma, breast, lung, stomach and prostate cancer and in neuroblastoma (Ohlsson, B et al, scand. j. gastroenterology 1999, 34(12), 1224-9; weber H C, cur. opin. endocri. dib. obes.2009, 164(1), 66-71, Gonzalez N, cur. opin. endocri. dib Obes 2008, 15(1), 58-64); neurotensin (NTR1, NTR2, NTR3) for the treatment of small cell lung cancer, neuroblastoma, pancreatic and colon cancer; substance P (NK1 receptor) for gliotic tumors; neuropeptide Y (Y1-Y6) for breast cancer; the Homing peptide comprises RGD (Arg-Gly-Asp) and NGR (Asn-Gly-Arg) and recognizesDimeric and multimeric cyclic RGD peptides (e.g., cRGDfV) of receptors (integrins) on tumor surfaces (Laakkon P, Vuorinen K. Integr Biol (Camb)2010, 2(7-8), 326-S337; Chen K, Chen X. Theranostics.2011, 1, 189-200; Garanger E et al, Anti-Cancer Agents Med. chem.7(5), 552-558; Kerr J S et al, Anticancer Research, 19(2A), 959-S968; Thumshirt G et al, Chem. Eur. J.2003, 9, 2717-S2725), Bu TAASGVRSMH, LTLRWVGLMS (chondroitin sulfate receptor 2) and F3 peptides (31 amino acid peptides binding to nucleolin receptors expressed on cell surfaces) (Zimantn S, Cancer S, 2002, 5118; Tergitz K5132; Tergin K337, Vakkorn K7-S, Biokunage P381, 31-S, 31-S, 76, 31-S, 51-S, 31-S, 31, S, cancer Res 1999, 59(12), 2869-2874; k.porkka et al, proc.nat.acad.sci.2002, 99(11), 7444-9); cell Penetrating Peptides (CPPs) (Nakase I et al, J.Control Release.2012, 159(2), 181-188); peptide hormones, such as Luteinizing Hormone Releasing Hormone (LHRH) agonists and antagonists, and gonadotropin releasing hormone (GnRH) agonists, act by targeting Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH), as well as testosterone production, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser (OtBu) -Leu-Arg-Pro-NHEt), Gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH) 2 ) Goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser (O) t Bu)-Leu-Arg-Pro-AzGly-NH 2 ) Histrelin (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), nafarelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH) 2 ) Deslorelin, abarelix (Ac-D-2 Nal-D-4-chlorophenyle-D-3- (3-pyridol) Ala-Ser- (N-Me) Tyr-D-Asn-Leu-isoproylLys-Pro-DAla-NH 2 )，Cetrorelix(Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH 2 ) Degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3- (3-pyridol) Ala-Ser-4-aminoPhe (L-hydrotyl) -D-4-aminoPhe (carba-moyl) -Leu-isoproyl Lys-Pro-D-Ala-NH 2 ) And ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3- (3-pyridol) Ala-Ser-Tyr-D- (N9, N10-diethyl) -homoArg-Leu- (N9, N10-diethyl) -homoArg-Pro-D-Ala-NH 2 )(Thundimadathil，J.，J.AminoAcids, 2012, 967347; Boccon-Gibad L. et al, Therapeutic Advances in Urology 2011, 3(3), 127-; debruyne, f., Future Oncology, 2006, 2(6), 677-; maison neuve et al proc.natl.acad.sci.2014, 111, 1-6; botos I et al, Structure 2011, 19, 447-; means T K et al, Life sci.2000, 68, 241-: C) and lipopeptides (Pam3CSK4) (kassturi S P et al, Nature 2011, 470, 543-; lane t., J R soc.med.2001, 93, 316; hotz C and Bourquin C, Oncoimmunology 1, 2012, 227-; dudek, A Z et al, clin. cancer res.2007, 13, 7119-.
Cell-binding ligands or receptors may be Ig-based and non-Ig protein molecules. Ig-based scaffolds are selected from, without limitation, derivatives of nanobodies (vhh (camelid Ig)) (muydermans s., Annu Rev biochem.2013, 82, 775-; domain antibodies (derivatives of dAb, VH or VL domains) (Holt L.J et al, Trends Biotechnol.2003, 21, 484-490); bispecific T cell engage (BiTE, bispecific diabody) (baeuuerle p.a et al, curr. opin. mol. ther.2009, 11, 22-30); double affinity retargeting antibodies (DART, bispecific secondary antibody) (Moore P.A.P et al, Blood 2011, 117(17), 4542-4551); tetravalent tandem antibodies (tandAb, dimeric bispecific antibodies) (Cochlovius B et al, Cancer Res.2000, 60(16), 4336-S4341). non-Ig based scaffolds may be selected from, but are not limited to, Anticalin (derivatives of Lipocalins) (Skerra A. FEBS J., 2008, 275(11), 2677-; adnectin (10 th FN3 (fibronectin)) (Koide A et al, J.mol.biol., 1998, 284(4), 1141-; designed ankyrin repeat proteins (DARPins) (derivatives of the Ankyrin Repeat (AR) protein) (Boersma Y.L et al, Curr Opin biotechnol.2011, 22(6), 849 857), such as DARPin C9, DARPin Ec4 and DARPin E69_ LZ3_ E01(Winkler J et al, Mol Cancer ther Ther.2009, 8(9), 2674-2683; Patricia M-K.M et al, Clin Cancer Res.2011, 17(1), 100-110; Boersma Y.L et al, J. biol. chem.2011, 286(48), 41273), 41285-41285); avimers (Domain A/Low Density Lipoprotein (LDL) receptor) (Boersma Y.L, J.biol.chem.2011, 286(48), 41273-.
Examples of antibody-cell binding ligand or receptor conjugates linked via a bridging linker are: LB01(PMSA ligand conjugate), LB02 (folate receptor conjugate), LB03 (somatostatin receptor conjugate), LB04 (octreotide, somatostatin homolog receptor conjugate), LB05 (lanreotide, somatostatin homolog receptor conjugate), LB06(CAIX receptor conjugate), LB07(CAIX receptor conjugate), LB08 (luteinizing hormone releasing hormone (LH-RH) ligand and GnRH conjugate), LB09 (luteinizing hormone releasing hormone (LH-RH) and GnRH ligand conjugate), LB10(GnRH antagonist, abarelix conjugate), LB11 (cobalamin, VB12 homolog conjugate), LB12 (gastrin releasing peptide receptor (GRPr), MBA conjugate), LB13(α v β 3 integrin receptor, cyclic RGD pentapeptide conjugate), LB14 (heterobivalent peptide ligand conjugate for VEGF receptor), LB15 (interleukin B16 (LB 16(G receptor conjugate) and TLR 17 receptor conjugate. 2 A conjugate).
Figure BDA0001580863800000661
Figure BDA0001580863800000671
Figure BDA0001580863800000681
Figure BDA0001580863800000691
Figure BDA0001580863800000701
Wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)NR 3 ，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or by default; x 4 And X 4 ' is independently H, CH 2 ，OH，O，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；m 3 And m 4 Is 0 to 5000;
Figure BDA0001580863800000702
X 1 ，X 2 ，R 1 ，R 2 and R 3 The same as defined in formulae (I) and (II). R 1 And/or R 2 A default may be used.
The drug/cytotoxic agent used in conjunction with the bridge linkers of the present invention may be any analog and/or derivative of the drug/molecule described previously. It is understood that each of the drugs/cytotoxic agents described herein may be modified and the resulting compounds still retain the relevant specificity and/or activity. It will also be appreciated by those skilled in the art that many other compounds may be substituted for the drug/cytotoxic agent described herein. Thus, the drug/cytotoxic agents of the present invention also include analogs and derivatives of these compounds.
All documents cited herein and in the examples below are incorporated by reference.
Examples
The invention is further illustrated by the following examples, the contents of which are not intended to limit the scope of the invention. In the examples, the cell lines were stored under the conditions specified in the American Standard culture Collection (ATCC), German culture Collection (DSMZ) or Shanghai cell culture Collection of Chinese academy of sciences, except for the specific instructions. Cell culture reagents were obtained from Invitrogen, unless otherwise specified. All anhydrous reagents were obtained commercially and stored in Sure-Seal bottles. Other reagents and solvents were purchased according to the highest specifications and used without further treatment. The Varian Prostar HPLC was subjected to preparative HPLC purification. NMR data were obtained at Varian Mercury 400MHz with chemical shifts in ppm, tetramethylsilane as reference (0ppm) and coupling constants (J) in Hz. Mass spectral data were obtained on a Waters XevoQTof mass spectrometer (connected to a Waters Acquity UPLC high performance liquid chromatograph and a TUV detector).
EXAMPLE 1.3- (2- (2-hydroxyethoxy) ethoxy) propionic acid tert-butyl ester (84)
Figure BDA0001580863800000711
To 350mL of anhydrous tetrahydrofuran were added 80mg (0.0025mol) of metallic sodium and diethylene glycol 83(150.1g, 1.41mol) with stirring. After complete dissolution of sodium, tert-butyl acrylate (24mL, 0.33mol) was added. The solution was stirred at room temperature for 20 hours and neutralized with 8mL of 1.0M HCl. The solvent was spun dry in vacuo and the residue was diluted with brine (250mL) and extracted with ethyl acetate (3X 125 mL). The combined organic layers were washed with water (100mL) and brine (100mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried in vacuo to yield 60.27g (78% yield) of product 84. 1 H NMR:1.41(s,9H),2.49(t,2H,J＝6.4Hz),3.59-3.72(m,10H)；ESI MS m/z-C 11 H 21 O 5 (M-H), calculated 233.15, found 233.40.
Example 2.3- (2- (2- (tosyloxy) ethoxy) propionic acid tert-butyl ester (85)
Figure BDA0001580863800000712
To a solution of 84(10.0g, 42.70mmol) in dichloromethane (50.0mL) was added pyridine (20.0 mL). A solution of methanesulfonyl chloride (7.50g, 65.81mmol) in dichloromethane (50mL) was added dropwise over 30 minutes via an addition funnel. After 5 hours, TLC analysis showed the reaction was complete. The pyridine hydrochloride formed is filtered off and the filtrate is concentrated. The residue was purified on silica gel eluting with 20% ethyl acetate in n-hexane to pure ethyl acetate to give 10.39g (76% yield) of compound 85. 1 H NMR:1.40(s,9H),3.23(s,3H),2.45(t,2H,J＝6.4Hz),3.54-3.70(m,10H)；ESI MS m/z+C 12 H 25 O 7 S (M + H), calcd 313.10, found 313.30.
Example 3.3- (2- (2-azidoethoxy) ethoxy) propionic acid tert-butyl ester (86)
Figure BDA0001580863800000713
To 50mL of DMA were added, with stirring, 3- (2- (2- (methylsulfonyloxy) ethoxy) -propionic acid tert-butyl ester 85(4.0g, 12.81mmol) and sodium azide (0.90g, 13.84 mmol). The reaction was heated to 80 ℃ and after 4 hours, TLC analysis showed the reaction was complete. The reaction was cooled to room temperature and quenched with water (25mL) and extracted with ethyl acetate (3X 35 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, concentrated in vacuo and purified on silica gel, eluting with 15% ethyl acetate in n-hexane to pure ethyl acetate to give 2.88g (87% yield) of compound 86.
1 H NMR(CDCl 3 ):1.40(s,9H),2.45(t,2H,J＝6.4Hz),3.33(t,2H,J＝5.2Hz),3.53-3.66(m,8H)；ESI MS m/z+C 11 H 22 N 3 O 7 (M + H), calcd for 260.13, found 260.20.
Example 4.3- (2- (2-azidoethoxy) ethoxy) propionic acid (87)
Figure BDA0001580863800000721
Azide 86(2.51g, 9.68mmol) was dissolved in 1, 4-dioxane (30mL) and 10mL concentrated HCl was added. The mixture was stirred for 35 minutes, diluted with EtOH (30ml) and toluene (30ml) and concentrated in vacuo. The crude product was purified on silica gel column using methanol (5% to 10%) and 1% formic acid in dichloromethane as eluent to give the title compound 87(1.63g, 83% yield). ESI MS m/z-C 7 H 12 N 3 O 4 (M-H), calculated 202.06, found 202.30.
Example 5.3- (2- (2-azidoethoxy) ethoxy) propionic acid 25-dioxopyrrolidin-1-yl ester (88)
Figure BDA0001580863800000722
To a solution of compound 87(1.60g, 7.87mmol) in dichloromethane (30mL) was added NHS (1.08g, 9.39mmol) and EDC (3.60g, 18.75mmol) with stirring. After 8 h, TLC analysis showed the reaction was complete and the reaction mixture was concentrated and purified on silica gel using ethyl acetate (5% to 10%) in dichloromethane as eluent to give the title compound 88(1.93g, yield 82%). ESI MS m/z + C 11 H 17 N 4 O 6 (M + H), calcd. 301.11, found 301.20.
EXAMPLE 6 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (3- (2- (2-azidoethoxy) ethoxy) propionamido) -4-hydroxyphenyl) -2-methylpentanoic acid (94)
Figure BDA0001580863800000723
In the reaction of (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidin-4-yl) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3-amino-4-hydroxyphenyl) -2-methylpentanoic acid, 93(Huang Y.et al, Med Chem. #44,249 th ACS National Meeting, Denver, CO, Mar.22-26,2015; WO2014009774) (100mg,0.131mmol) DMA (10ml) and NaH 2 PO 4 88(80.0mg, 0.266mmol) was added to a mixture of buffers (5ml, 1.0M, pH7.5) in four portions over two hours. The mixture was stirred overnight, concentrated and purified by C-18 preparative HPLC (3.0X 25cm) eluting with 80% to 10% water/methanol over 45 min (25ml/min) to give the title compound (101.5mg, 82% yield). LC-MS (ESI) m/z C 45 H 70 N 9 O 11 S[M+H] + Calculated 944.48, found 944.70.
EXAMPLE 7 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methyl- -2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (3- (2- (2-aminoethoxy) ethoxy) propionamido) -4-hydroxyphenyl) -2-methylpentanoic acid (95)
Figure BDA0001580863800000731
Pd/C (25mg, 10% Pd, 50% wet) was added to a solution of compound 94(100.0mg, 0.106mmol) containing 0.1% HCl in methanol (25ml) in a hydrogenation reactor. After evacuating the air from the vessel, 35psi H was introduced 2 . After shaking the mixture for 4 h, it was filtered through celite, the filtrate was concentrated and purified on C-18 preparative HPLC (3.0x 25cm) eluting with 85% to 15% water/methanol (25ml/min) over 45 min to give the title compound (77.5mg, 79% yield). LC-MS (ESI) m/z C 45 H 72 N 7 O 11 S[M+H] + Calculated 918.49, found 918.60.
Example 8.4- (benzyloxy) -3-methoxybenzoic acid
Figure BDA0001580863800000732
To a mixture of 4-hydroxy-3-methoxybenzoic acid (50.0g, 297.5mmol) in ethanol (350ml) and NaOH solution (2.0M, 350ml) was added benzyl bromide (140.0g, 823.5 mmol). The mixture was stirred at 65 ℃ for 8 h, concentrated and then co-concentrated with water (2X 400ml) to-400 ml, acidified to pH 3.0 with 6M HCl, the solid collected by filtration, crystallized from EtOH and dried under vacuum at 45 ℃ to give the title compound (63.6g, 83% yield). ESI MS M/z +281.2(M + Na).
Example 9.4- (benzyloxy) -5-methoxy-2-nitrobenzoic acid
Figure BDA0001580863800000733
To a mixture of 4- (benzyloxy) -3-methoxybenzoic acid (63.5g, 246.0mmol) in dichloromethane (400ml) and HOAc (100ml) was added fuming nitric acid (25.0ml, 528.5 mmol). The mixture was stirred for 6 hours, concentrated and crystallized from EtOH, dried at 45 ℃ in vacuo to give the title compound (63.3g, 85% yield). ESI MS M/z +326.1(M + Na).
EXAMPLE 10 (2S, 4R) -4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride
Figure BDA0001580863800000741
Thionyl chloride (17mL, 231mmol) was added dropwise to a solution of trans-4-hydroxy-L-proline (15.0g, 114.3mmol) in anhydrous methanol (250mL) at 0-4 ℃. The resulting mixture was stirred at room temperature overnight, concentrated, and crystallized from ethanol/n-hexane to give the title compound (18.0g, 87% yield). ESI MS M/z +168.2(M + Na).
EXAMPLE 11 (2S, 4R) -2-hydroxypyrrolidine-12-dicarboxylic acid 1-tert-butyl 2-methyl ester
Figure BDA0001580863800000742
To a mixture of methyl trans-4-hydroxy-L-proline (18.0g, 107.0mmol) in MeOH (150ml) and sodium bicarbonate solution (2.0M, 350ml) was added (BOC) 2 O (30.0g, 137.6mmol) was added in three portions over 4 hours. After stirring for a further 4 hours, the reaction was concentrated to about 350mL and extracted with ethyl acetate (4X 80 mL).The combined organic layers were washed with brine (100mL), dried (magnesium sulfate), filtered, concentrated and passed through SiO 2 Purification by column (1:1 n-hexane/ethyl acetate) gave the title compound (22.54g, 86% yield). ESI MS M/z +268.2(M + Na).
EXAMPLE 12 (S) -1-methyl-4-oxopyrrolidine-12-dicarboxylic acid 1-tert-butyl ester
Figure BDA0001580863800000743
The title compound was prepared by Dess-Martin oxidation as follows: franco Manfre et al.J.org.chem.1992,57, 2060-. Alternatively, the Swern oxidation can be carried out as follows: will cool to-78 ℃ (COCl) 2 To a solution of (13.0mL, 74.38mmol) in dichloromethane (350mL) was added anhydrous DMSO (26.0 mL). After stirring at-78 ℃ for 15 minutes, a solution of (2S, 4R) -1-tert-butyl 2-methyl-4-hydroxypyrrolidine-12-dicarboxylate (8.0g, 32.63mmol) in dichloromethane (100ml) was added. After stirring for 2 h, triethylamine (50ml, 180.3mmol) was added dropwise and the solution was warmed to room temperature. Using NaH for the mixture 2 PO 4 The (400ml, 1.0M) solution was diluted and the two phases were separated. The aqueous phase was extracted with dichloromethane (2X 60ml) and the organic layers were combined, dried over magnesium sulphate, filtered, concentrated and passed over SiO 2 Purification by column (7:3 n-hexane/ethyl acetate) gave the title compound (6.73g, 85% yield). ESI MS M/z +266.2(M + Na).
EXAMPLE 13 (S) -1-methyl-4-methylenepyrrolidine-12-dicarboxylic acid 1-tert-butyl 2-methyl ester
Figure BDA0001580863800000751
To a solution of methyltriphenylphosphonium bromide (19.62g, 55.11mmol) in tetrahydrofuran (150mL) at 0 deg.C was added dropwise a solution of potassium tert-butoxide (6.20g, 55.30mmol) in anhydrous tetrahydrofuran (80 mL). After stirring for 2h, to the resulting yellow ylide emulsion was added a solution of (S) -1-tert-butyl 2-methyl 4-oxopyrrolidine-12-dicarboxylic acid ester (6.70g, 27.55mmol) in tetrahydrofuran (40 mL). Stirring at room temperatureAfter stirring for 1 hour, the reaction mixture was concentrated, diluted with ethyl acetate (200mL), washed with water (150mL), brine (150mL), dried over magnesium sulfate and concentrated in SiO 2 Purification on a column (9:1 n-hexane/ethyl acetate) gave the title compound (5.77g, 87% yield). EIMS M/z +264(M + Na).
EXAMPLE 14 (S) -4-methylpyrrolidine-2-carboxylic acid methyl ester
Figure BDA0001580863800000752
To a solution of (S) -1-methyl-4-methylenepyrrolidine-12-dicarboxylic acid 1-tert-butyl ester (5.70g, 23.63mmol) in ethyl acetate (40ml) was added concentrated HCl (10 ml). The mixture was stirred for 1 hour, diluted with toluene (50ml), concentrated and crystallized from ethanol/n-hexane to give the title compound (3.85g, hydrochloride, 92% yield). EIMS M/z +142.2(M + H).
Example 15 (S) -1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carboxylic acid methyl ester
Figure BDA0001580863800000753
A catalytic amount of DMF (30 μ l) was added to a solution of 4- (benzyloxy) -5-methoxy-2-nitrobenzoic acid (2.70g, 8.91mmol) and oxalyl chloride (2.0mL, 22.50mmol) in anhydrous dichloromethane (70mL), and the resulting mixture was stirred at room temperature for 2 hours. Excess dichloromethane and oxalyl chloride were removed in vacuo. The acid chloride was resuspended in fresh dichloromethane (70mL) and added dropwise to Et 4-methylene-L-proline methyl ester hydrochloride (1.58g, 8.91mmol) under argon at 0 deg.C 3 N (6mL) solution. The reaction mixture was allowed to warm to room temperature and stirring was continued for 8 hours. Removal of dichloromethane and Et 3 After N, the residue is taken up in H 2 Partition between O and ethyl acetate (70/70 mL). The aqueous layer was further extracted with ethyl acetate (2X 60 mL). The organic layers were combined, washed with brine (40mL), dried over magnesium sulfate, and concentrated. The residue was purified by flash chromatography on silica gel (2:8 n-hexane/ethyl acetate) to give (S) -1- (4- (benzyloxy)Methyl 5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carboxylate (2.88g, 76.1% yield). EIMS M/z 449.1([ M ]] + +Na)。
Example 16 (S) -1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carbaldehyde
Figure BDA0001580863800000761
A solution of (S) -methyl 1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carboxylate (2.80g, 6.57mmol) in anhydrous dichloromethane (60mL) was stirred vigorously at-78 ℃ and DIBAL-H (10mL of a 1M solution in dichloromethane) was added dropwise under an argon atmosphere. After the mixture was stirred for another 90 minutes, the excess reagent was decomposed by the addition of methanol (2mL) and 5% HCl (10 mL). The resulting mixture was warmed to 0 ℃. After separation of layers, the aqueous layer was further extracted with dichloromethane (3X 50 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated to give (S) -1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carbaldehyde (2.19g, 84% yield). EIMS M/z 419.1([ M ]] + +Na)。
Example 17 (S) -8- (benzyloxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-a-aza-5 (11aH) -one
Figure BDA0001580863800000762
(S) -1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carbaldehyde (2.18g, 5.50mmol) and Na 2 S 2 O 4 (8.0g, 45.97mmol) in tetrahydrofuran (60ml) and H 2 The mixture in O (40ml) was stirred at room temperature for 20 hours. The solvent was removed under high vacuum, the residue was resuspended in MeOH (60mL), and HCl (6M) was added dropwise to pH-2. The resulting mixture was stirred at room temperature for one hour, most of the MeOH was removed and diluted with ethyl acetate (100 mL). The ethyl acetate solution was washed with saturated sodium chloride solution, NaHCO 3 The aqueous solution and the brine are washed,dried over magnesium sulfate and concentrated. Chromatography on flash silica gel (97:3 CHCl) 3 MeOH) gave (S) -8- (benzyloxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-aza-5 (11aH) -one (1.52g, 80%). EIMS M/z 372.1([ M ]] + +Na)。
EXAMPLE 18 (S) -8-hydroxy-7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-a-aza-5 (11aH) -one
Figure BDA0001580863800000763
To a solution of (S) -8- (benzyloxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-aza-5 (11aH) -one (1.50g,4.32mmol) in dichloromethane (70ml) at 0 ℃ was added 25ml CH 2 SO 3 H. The mixture was stirred at 0 ℃ for 10 min, then at room temperature for 2 h, diluted with dichloromethane and diluted with cold 1.0M NaHCO 3 The pH was adjusted to 4 and filtered. The aqueous layer was extracted with dichloromethane (3X 60ml) and the organic layers were combined, dried over sodium sulfate, filtered, concentrated and concentrated in SiO 2 Purifying on a chromatographic column with CH 3 OH/dichloromethane (1:15) gave 811mg (73% yield) of the title product. EIMS M/z 281.1([ M ]] + +Na)。
EXAMPLE 19 (11aS, 11a 'S) -88' - (pentane-15-diylbis (oxy)) bis (7-methoxy-2-methylene-23-dihydro-1H-benzo e pyrrolo 12-a 14 diazepin-5 (11aH) -one) (97)
Figure BDA0001580863800000771
To the stirred Cs 2 CO 3 To a suspension (8ml) of (0.761g, 2.33mmol) in butanone was added (S) -8-hydroxy-7-methoxy-2-methylene-23-dihydro-1H-benzo e pyrrolo 12- α 14 diaza-5 (11aH) -one (401mg, 1.55mmol) and 1, 5-diiodopentane (240mg, 0.740 mmol). The mixture was stirred at room temperature overnight, concentrated, and concentrated in SiO 2 Purification on a chromatographic column eluting with ethyl acetate/dichloromethane (1:10) gave 337mg (78% yield) of the title product. EIMS m/z 607.2([M] + +Na)。
EXAMPLE 20 (S) -7-methoxy-8- ((5- ((S) -7-methoxy-2-methylene-5-oxo-2, 3,5,10,11,11 a-hexahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -2-methylene-23-dihydro-1H-benzo e-pyrrolo 12, 2-a 14 diazepin-5 (11aH) -one (98)
Figure BDA0001580863800000772
To a solution of (11aS, 11a 'S) -88' - (pentane-15-diylbis (oxy)) bis (7-methoxy-2-methylene-23-dihydro-1H-benzo e pyrrolo 12a 14 diazepin-5 (11aH) -one) (150mg, 0.256mmol) in anhydrous dichloromethane (1mL) and anhydrous ethanol (1.5mL) at 0 deg.C was added a solution of sodium borohydride in methoxyethyl ether (85. mu.L, 0.5M,0.042 mmol). After 5 minutes the ice bath was removed and the mixture was stirred at room temperature for 3 hours, then cooled to 0 ℃, quenched with saturated ammonium chloride, diluted with dichloromethane and separated. The organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered through celite and concentrated. The residue was purified by reverse phase HPLC (C18 column, acetonitrile/water). The corresponding fractions were extracted with dichloromethane and then concentrated to give the title compound (98), (S) -7-methoxy-8- ((5- (((S) -7-methoxy-2-methylene-5-oxo-8H-xanthen-9-yl) 2,3,5,10,11,11a hexahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-a 14 diaza-5 (11aH) -one (64.7mg, 43%), MS M/z +609.2(M + Na),625.3(M + K),627.2(M + Na + H) 2 O); (11aS, 11a 'S) -88' - (pentane-15-diylbis (oxy)) bis (7-methoxy-2-methylene-2, 3, 11,11 a-tetrahydro-1H-benzo e pyrrolo 12-a 14 diazepin-5 (10H) -one), (99) (16.5mg, 11.1%) MS M/z +611.2(M + Na),627.2(M + K),629.2(M + Na + H) -one 2 O); and unreacted starting material (10.2mg, 6.8%), MS M/z +607.2(M + Na),625.2(M + Na + H) 2 O)。
EXAMPLE 21 (S) -8- ((5- ((S) -10- (3- (2- (2-azidoethoxy) ethoxy) propionyl) -7-methoxy-2-methylene-5-oxo-2, 2,3,5,10,11,11a hexahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-a 14 diaza-5 (11aH) -one (99)
Figure BDA0001580863800000781
To a mixture of compound 98(60.0mg, 0.102mmol) and compound 88(40.5mg, 0.134mmol) in dichloromethane (5ml) was added EDC (100.5mg, 0.520 mmol). Stirred at room temperature overnight, concentrated and concentrated in SiO 2 Purification on a chromatographic column eluting with ethyl acetate/dichloromethane (1:6) gave 63.1mg (81% yield) of the title product 99. ESI MSm/z + C 40 H 50 N 7 O 9 (M + H), calculated 772.36, found 772.30.
EXAMPLE 22 (S) -8- ((5- ((S) -10- (3- (2- (2-aminoethoxy) ethoxy) propionyl) -7-methoxy-2-methylene-5-oxo-2, 2,3,5,10,11,11a hexahydro-1H-benzo e pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e pyrrolo 12-a 14 diaza-5 (11aH) -one (100)
Figure BDA0001580863800000782
To a mixture of compound 99(60mg, 0.078mmol) in tetrahydrofuran (5ml) and NaH 2 PO 4 PPh was added to a mixture of buffers (50mM, pH5.0, 1ml) 3 (70mg, 0.267 mmol). The mixture was stirred at room temperature overnight, concentrated and purified by C-18 preparative chromatography on water/CH 3 CN (from 90% water to 35% water in 35 minutes) to yield, after drying on a high vacuum pump, 45.1mg (79% yield) of the title product 100. ESI MS m/z + C 40 H 52 N 5 O 9 (M + H), calculated 746.37, found 746.50.
EXAMPLE 23 (S) -2- (hydroxymethyl) pyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0001580863800000791
Boc-L-proline (10.0g, 46.4mmol) was dissolved in 50mL tetrahydrofuran, cooled to 0 deg.C and BH was carefully added thereto 3 Tetrahydrofuran solution (1.0M, 46.4 mL). The mixture was stirred at 0 ℃ for 1.5 hours, then poured into ice water and extracted with ethyl acetate. The organic layer was washed with brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound as a white solid (8.50g, 91% yield). 1 H NMR(500MHz,CDCl 3 )δ3.94(dd,J＝4.9,2.7Hz,2H),3.60(ddd,J＝18.7,11.9,9.3Hz,2H),3.49–3.37(m,1H),3.34–3.23(m,1H),2.06–1.91(m,1H),1.89–1.69(m,2H),1.65–1.51(m,1H),1.49–1.40(m,9H)。
EXAMPLE 24 (S) -2-formylpyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0001580863800000792
To a solution of tert-butyl (S) -2- (hydroxymethyl) pyrrolidine-1-carboxylate (13.0g, 64.6mmol) in dimethyl sulfoxide (90mL) was added triethylamine (40mL), after stirring for 15min the mixture was cooled on an ice bath and sulfur trioxide-pyridine complex (35.98g, 226mmol) was added portionwise over 40 min. The reaction was warmed to room temperature and stirred for 2.5 hours. After addition of ice (250g), the mixture was extracted with dichloromethane (150 mL. times.3). The organic phase was washed with 50% citric acid solution (150mL), water (150mL), saturated sodium bicarbonate solution (150mL) and brine (150mL), dried over anhydrous sodium sulfate, and the solvent was removed in vacuo to give the title compound (10.4g, 81% yield) as a thick oil which was used without further purification. 1 H NMR(500MHz,CDCl 3 )δ9.45(s,1H),4.04(s,1H),3.53(dd,J＝14.4,8.0Hz,2H),2.00–1.82(m,4H),1.44(d,J＝22.6Hz,9H)。
EXAMPLE 25 (4R, 5S) -4-methyl-5-phenyl-3-propionyloxyoxazolidin-2-one
Figure BDA0001580863800000793
Under the protection of nitrogen at-78 ℃, n-hexane of n-butyllithium is addedThe solution (21.6mL, 2.2M, 47.43mmol) was added dropwise to a stirred solution of 4-methyl-5-phenyloxazolidine-2-one (8.0g, 45.17mmol) in tetrahydrofuran (100 mL). The reaction was held at-78 ℃ for 1 h, then propionyl chloride (4.4mL, 50.59mmol) was added slowly. The reaction mixture was warmed to-50 ℃, stirred for 2 hours, and then quenched by addition of saturated ammonium chloride solution (100 mL). The organic solvent was removed in vacuo and the resulting solution was extracted with ethyl acetate (3X 100 mL). The organic layers were combined, washed with saturated sodium bicarbonate solution (100mL) and brine (100mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography (20% ethyl acetate/n-hexane) to give the title compound as a thick oil (10.5g, 98% yield). 1 H NMR(500MHz,CDCl 3 )δ7.45–7.34(m,3H),7.30(d,J＝7.0Hz,2H),5.67(d,J＝7.3Hz,1H),4.82–4.70(m,1H),2.97(dd,J＝19.0,7.4Hz,2H),1.19(t,J＝7.4Hz,3H),0.90(d,J＝6.6Hz,3H)。
EXAMPLE 26 tert-butyl (S) -2- ((1R, 2R) -1-hydroxy-2-methyl-3- ((4R, 5S) -4-methyl-2-oxo-5-phenyloxazolidin-3-yl) -3-oxopropyl) pyrrolidine-1-carboxylate
Figure BDA0001580863800000801
To a solution of (4R, 5S) -4-methyl-5-phenyl-3-propionyloxy-oxazolidin-2-one (9.40g, 40.4mmol) in dichloromethane (60mL) at 0 deg.C was added Et 3 N (6.45mL, 46.64mmol) followed by the addition of a 1M solution of dibutylborotrifluoromethane sulfonate in dichloromethane (42mL, 42 mmol). The mixture was stirred at 0 ℃ for 45 minutes, then cooled to-70 ℃ and a solution of tert-butyl (S) -2-formylpyrrolidine-1-carboxylate (4.58g, 22.97mmol) in dichloromethane (40mL) was slowly added (over 30 minutes). The reaction was stirred at-70 ℃ for 2 hours, 0 ℃ for 1 hour, room temperature for 15 minutes, then quenched with phosphate buffered saline ( pH 7,38 mL). MeOH-30% H was added at 10 deg.C 2 O 2 (2:1,100mL), stirred for 20 min, water (100mL) was added and the mixture was concentrated in vacuo. More water (200mL) was added to the residue and extracted with ethyl acetate (3X 100 mL). Subjecting the organic layer to 1N KHSO 4 (100mL), sodium bicarbonate solution (100mL) and brine (100mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (10% -50% ethyl acetate/hexanes) to give the title compound as a white solid (7.10g, 71% yield). 1 H NMR(500MHz,CDCl 3 )δ7.39(dt,J＝23.4,7.1Hz,3H),7.30(d,J＝7.5Hz,2H),5.67(d,J＝7.1Hz,1H),4.84–4.67(m,1H),4.08–3.93(m,3H),3.92–3.84(m,1H),3.50(d,J＝9.0Hz,1H),3.24(d,J＝6.7Hz,1H),2.15(s,1H),1.89(dd,J＝22.4,14.8Hz,3H),1.48(d,J＝21.5Hz,9H),1.33(d,J＝6.9Hz,3H),0.88(d,J＝6.4Hz,3H)。
EXAMPLE 27 tert-butyl (S) -2- ((1R, 2R) -1-methoxy-2-methyl-3- ((4R, 5S) -4-methyl-2-oxo-5-phenyloxazolidin-3-yl) -3-oxopropyl) pyrrolidine-1-carboxylate
Figure BDA0001580863800000802
To (S) -tert-butyl 2- ((1R, 2R) -1-hydroxy-2-methyl-3- ((4R, 5S) -4-methyl-2-oxo-5-phenyloxazolidin-3 yl) -3-oxopropyl) pyrrolidine-1-carboxylate (5.1g, 11.9mmol) and molecular sieves (molecular sieves) (1R, 2R) -1-hydroxy-2-methyl-3- ((4R, 5S) -4-methyl-2-oxo-5-phenyloxazolidin-3 yl) under nitrogen protection
Figure BDA0001580863800000803
5g) To the mixture of (1) was added anhydrous dichloroethane (30 mL). The mixture was stirred at room temperature for 20 minutes and cooled to 0 ℃. Proton sponge (6.62g, 30.9mmol) was added followed by trimethyloxonium tetrafluoroborate (4.40g, 29.7 mmol). Stirring was continued at 0 ℃ for 2 hours and at room temperature for 48 hours. The reaction mixture was filtered, the filtrate concentrated and purified by column chromatography (20-70% ethyl acetate/n-hexane) to give the title compound as a colorless solid (1.80g, 35% yield). 1 H NMR(500MHz,CDCl 3 )δ7.46–7.27(m,5H),5.65(s,1H),4.69(s,1H),3.92(s,1H),3.83(s,1H),3.48(s,3H),3.17(s,2H),2.02–1.68(m,5H),1.48(d,J＝22.3Hz,9H),1.32(t,J＝6.0Hz,3H),0.91–0.84(m,3H)。
EXAMPLE 28 (2R, 3R) -3- ((S) -1- (tert-Butoxycarbonyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoic acid
Figure BDA0001580863800000811
To (S) -tert-butyl 2- ((1R, 2R) -1-methoxy-2-methyl-3- ((4R, 5S) -4-methyl-2-oxo-5-phenyloxazolidin-3-yl) -3-oxopropyl) pyrrolidine-1-carboxylate (1.80g, 4.03mmol) in tetrahydrofuran (30mL) and H at 0 deg.C 2 To a solution in O (7.5mL) was added 30% H 2 O 2 (1.44mL, 14.4mmol) (5 min), followed by the addition of a solution of LiOH (0.27g, 6.45mmol) in water (5 mL). After stirring at 0 ℃ for 3 hours, 1N sodium sulfite (15.7mL) was added and the mixture was allowed to warm to room temperature and stirred overnight. The tetrahydrofuran was removed in vacuo and the aqueous phase was washed with dichloromethane (3X 50mL) to remove the oxazolidinone. The aqueous phase was acidified to pH 3 with 1N HCl and extracted with ethyl acetate (3X 50 mL). The organic layer was washed with brine (50mL), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound as a colorless oil (1.15g, 98% yield). 1 H NMR(500MHz,CDCl 3 )δ3.99–3.74(m,2H),3.44(d,J＝2.6Hz,3H),3.23(s,1H),2.60–2.45(m,1H),1.92(tt,J＝56.0,31.5Hz,3H),1.79–1.69(m,1H),1.58–1.39(m,9H),1.30–1.24(m,3H)。
EXAMPLE 29 Ethyl (4S, 5S) -4- ((tert-butoxycarbonyl) amino) -5-methyl-3-oxoheptanoate
Figure BDA0001580863800000812
To an ice-cold solution of (2S, 3S) -2- ((tert-butoxycarbonyl) amino) -3-methyl-pentanoic acid (4.55g, 19.67mmol) in tetrahydrofuran (20mL) was added 1, 1' -carbonyldiimidazole (3.51g, 21.63 mmol). After the evolution of gas had ceased, the resulting mixture was stirred at room temperature for 3.5 hours. A freshly prepared solution of isopropylmagnesium bromide in tetrahydrofuran (123mmol, 30mL) was added dropwise to a pre-cooled (0 ℃ C.) ethyl hydrogen malonate (6.50g, 49.2mmol) maintaining the internal temperature below 5 ℃. The mixture was stirred at room temperature for 1.5 hours, then the magnesium enol solution was cooled in an ice-water bath, followed by gradual addition of the imidazolide solution over 1 hour via a double-ended needle. Will be provided withThe resulting mixture was stirred at 0 ℃ for 30 minutes and then at room temperature for 64 hours. The reaction mixture was quenched by the addition of 10% aqueous citric acid (5mL) and acidified to pH 3 with another 10% aqueous citric acid (110 mL). The mixture was extracted with ethyl acetate (150 mL. times.3). The organic extracts were washed with water (50mL), saturated aqueous sodium bicarbonate (50mL) and saturated aqueous sodium chloride (50mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography using ethyl acetate/n-hexane (1: 4) as an eluent to give the title compound (5.50g, yield 93%). 1 H NMR(500MHz,CDCl 3 )δ5.04(d,J＝7.8Hz,1H),4.20(p,J＝7.0Hz,3H),3.52(t,J＝10.7Hz,2H),1.96(d,J＝3.7Hz,1H),1.69(s,2H),1.44(s,9H),1.28(dd,J＝7.1,2.9Hz,3H),0.98(t,J＝6.9Hz,3H),0.92–0.86(m,3H)。
EXAMPLE 30 ethyl (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoate
Figure BDA0001580863800000821
To a solution of ethyl (4S, 5S) -4- ((tert-butoxycarbonyl) amino) -5-methyl-3-oxoheptanoate (5.90g, 19.83mmol) in ethanol (6mL) at-60 deg.C was added sodium borohydride (3.77g, 99.2 mmol). The reaction mixture was stirred at-55 ℃ or lower for 5.5 hours, then quenched with 10% aqueous citric acid (100mL), adjusted to pH 2, and extracted with ethyl acetate (100 mL. times.3). The organic extracts were washed with saturated aqueous sodium chloride (100mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (10-50% ethyl acetate/n-hexane) to give the pure diastereomers (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid ethyl ester (2.20g, 37% yield) and a mixture of (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid ethyl ester and (3S, 4S, 5S) - ((tert-butoxycarbonyl) -amino) -3-hydroxy-5-methyl-heptanoic acid ethyl ester (2.0g, 34% yield, about 9:1 ratio). 1 H NMR(500MHz,CDCl 3 )δ4.41(d,J＝9.3Hz,1H),4.17(tt,J＝7.1,3.6Hz,2H),4.00(t,J＝6.9Hz,1H),3.55(dd,J＝11.7,9.3Hz,1H),2.56–2.51(m,2H),2.44(dd,J＝16.4,9.0Hz,1H),1.79(d,J＝3.8Hz,1H),1.60–1.53(m,1H),1.43(s,9H),1.27(dd,J＝9.3,5.0Hz,3H),1.03–0.91(m,7H)。
EXAMPLE 31 (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid
Figure BDA0001580863800000822
To a solution of (2R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid ethyl ester (2.20g, 7.20mmol) in ethanol (22mL) was added a 1N aqueous sodium hydroxide solution (7.57mL, 7.57 mmol). The mixture was stirred at 0 ℃ for 30 minutes and then at room temperature for 2 hours. The resulting solution was acidified to pH4 by addition of 1N aqueous hydrochloric acid solution, and then extracted with ethyl acetate (50 mL. times.3). The organic extracts were washed with 1N aqueous potassium hydrogen sulfate (50mL) and saturated aqueous sodium chloride (50mL), dried over sodium sulfate, and concentrated in vacuo to give the title compound (1.90g, 95% yield).
1 H NMR(500MHz,CDCl 3 )δ4.50(d,J＝8.7Hz,1H),4.07(d,J＝5.5Hz,1H),3.59(d,J＝8.3Hz,1H),2.56–2.45(m,2H),1.76–1.65(m,1H),1.56(d,J＝7.1Hz,1H),1.45(s,9H),1.26(t,J＝7.1Hz,3H),0.93(dd,J＝14.4,7.1Hz,6H)。
EXAMPLE 32 (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) (methyl) amino) -3-methoxy-5-methylheptanoic acid
Figure BDA0001580863800000831
To a solution of (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid (1.90g, 6.9mmol) in tetrahydrofuran (40mL) was added sodium hydride (60 wt%, 1.93g, 48.3 mmol). After stirring for 1 hour, iodomethane (6.6mL, 103.5mmol) was added. Stirring was continued for 40 hours at 0 deg.C, then saturated aqueous sodium bicarbonate (50mL) and water (100mL) were added. The mixture was washed with diethyl ether (50 mL. times.2), and the aqueous layer was acidified to pH3 with 1N aqueous potassium hydrogensulfate solution, followed by extraction with ethyl acetate (50 mL. times.3). The combined organic extracts were diluted with 5%Aqueous sodium thiosulfate (50mL) and saturated aqueous sodium chloride (50mL) were washed, dried over sodium sulfate, and concentrated in vacuo to give the title compound (1.00g, 48% yield). 1 H NMR(500MHz,CDCl 3 )δ3.95(d,J＝75.4Hz,2H),3.42(d,J＝4.4Hz,3H),2.71(s,3H),2.62(s,1H),2.56–2.47(m,2H),1.79(s,1H),1.47(s,1H),1.45(d,J＝3.3Hz,9H),1.13–1.05(m,1H),0.96(d,J＝6.7Hz,3H),0.89(td,J＝7.2,2.5Hz,3H).
EXAMPLE 33 general procedure for removal of Boc functionality with trifluoroacetic acid
To a solution of N-Boc amino acid (1.0mmol) in dichloromethane (2.5mL) was added trifluoroacetic acid (1.0 mL). After stirring at room temperature for 1-3 hours, the reaction mixture was concentrated in vacuo. Azeotropic concentration with toluene yielded the deprotected product, which was used without any further purification.
EXAMPLE 34 tert-butyl (S) -2- ((1R, 2R) -1-methoxy-3- (((S) -1-methoxy-1-oxo-3-phenylpropan-2-yl) amino) -2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate
Figure BDA0001580863800000832
To a solution of (2R, 3R) -3- ((S) -1- (tert-butoxycarbonyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropionic acid (100mg, 0.347mmol) and L-phenylalanine (107.8mg, 0.500mmol) in DMF (5mL) was added diethyl cyanophosphonate (75.6. mu.L, 0.451mmol), followed by Et 3 N (131. mu.L, 0.94 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80mL), washed with 1N aqueous potassium hydrogen sulfate (40mL), water (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated aqueous sodium chloride (40mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give the title compound (130mg, 83% yield) as a white solid. 1 H NMR(500MHz,CDCl 3 )δ7.28(dd,J＝7.9,6.5Hz,2H),7.23(t,J＝7.3Hz,1H),7.16(s,2H),4.81(s,1H),3.98–3.56(m,5H),3.50(s,1H),3.37(d,J＝2.9Hz,3H),3.17(dd,J＝13.9,5.4Hz,2H),3.04(dd,J＝14.0,7.7Hz,1H),2.34(s,1H),1.81–1.69(m,2H),1.65(s,3H),1.51–1.40(m,9H),1.16(d,J＝7.0Hz,3H)。
EXAMPLE 35 (S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((tert-butoxycarbonyl) - (methyl) amino) 3-methoxy-5-methylheptyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropionic acid
Figure BDA0001580863800000841
To a mixed solution of (S) -2- ((1R, 2R) -1-methoxy-3- (((S) -1-methoxy-1-oxo-3-phenylpropan-2-yl) (0.29mmol)) and (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) (methyl) amino) -3-methoxy-5- (4-methoxyphenylamino) -2-methyl-3-oxopropyl) pyrrolidine-1-carboxylic acid tert-butyl ester (96.6mg, 0.318mmol) in DMF (5mL) was added diethyl cyanophosphonate (58. mu.L, 0.347mmol) followed by Et 3 N (109. mu.L, 0.78 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80mL), washed with 1N aqueous potassium hydrogen sulfate (40mL), water (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated aqueous sodium chloride (40mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give the title compound (150mg, 81% yield) as a white solid. LC-MS (ESI) m/z C 34 H 55 N 3 O 8 [M+H] + Calculated 634.40, found 634.40.
EXAMPLE 36 (S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2-amino) -N, 3-dimethylbutyrylamino) -3-methoxy-5-methylheptyl) -pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropan
Figure BDA0001580863800000842
To ((S) -1- ((3R, 4S, 5S) -4- ((tert-butoxycarbonyl) - (methyl) amino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropionylamino) -Methyl 3-phenylpropionate (0.118mmol) and Boc-Val-OH (51.8mg, 0.236mmol) in DCM (5mL) was added bromo-tris (dimethylamino) -phosphonium hexafluorophosphate (BroP, 70.1mg, 0.184mmol) followed by diisopropylethylamine (70. mu.L, 0.425 mmol). The mixture was protected from light and stirred at 0 ℃ for 30 minutes, then at room temperature for 2 days. The reaction mixture was diluted with ethyl acetate (80mL), washed with 1N potassium hydrogen sulfate water (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated aqueous sodium chloride (40mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (20-100% ethyl acetate/n-hexane) to give the title compound (67mg, 77% yield) as a white solid. LC-MS (ESI) m/z C 39 H 64 N 4 O 9 [M+H] + Calculated 733.47, found 733.46.
EXAMPLE 37 preparation of the compound Boc-N-Me-Val-OH
Figure BDA0001580863800000851
To a solution of Boc-L-Val-OH (2.00g, 9.2mmol) and iodomethane (5.74mL, 92mmol) in dry tetrahydrofuran (40mL) was added sodium hydride (3.68g, 92mmol) at 0 ℃. The reaction mixture was stirred at 0 ℃ for 1.5 hours, then warmed to room temperature and stirred for 24 hours. The reaction was quenched with ice water (50mL), water (100mL) was added, the mixture was washed with ethyl acetate (50 mL. times.3), and the aqueous solution was acidified to pH 3 and extracted with ethyl acetate (50 mL. times.3). The combined organic phases were dried over sodium sulfate and concentrated to afford Boc-N-Me-Val-OH as a white solid (2.00g, 94% yield). 1 H NMR(500MHz,CDCl 3 )δ4.10(d,J＝10.0Hz,1H),2.87(s,3H),2.37–2.13(m,1H),1.44(d,J＝26.7Hz,9H),1.02(d,J＝6.5Hz,3H),0.90(t,J＝8.6Hz,3H)。
EXAMPLE 38 methyl (S) -2- ((2R, 3R) -3- ((S) -1- ((6S, 9S, 12S, 13R) -12- ((S) -sec-butyl) diisopropyl-13-methoxy-2, 2,5,11 tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triaza-decan-15-yl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide-3-phenylpropionate
Figure BDA0001580863800000852
To a solution of (S) -1- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- (tert-butoxycarbonyl) amino) -N, 3-dimethylbutyrylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid methyl ester (0.091mmol) and Boc-N-Me-Val-OH (127mg, 0.548mmol) in DMF (5mL) was added diethyl cyanophosphonate (18.2. mu.L, 0.114mmol) followed by 4-methylmorpholine (59. mu.L, 0.548 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80mL), washed with 1N aqueous potassium hydrogen sulfate (40mL), water (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated aqueous sodium chloride (40mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (20-100% ethyl acetate/n-hexane) to give the title compound (30mg, 39% yield) as a white solid. LC-MS (ESI) m/z C 45 H 75 N 5 O 10 [M+H] + Calculated 846.55, found 846.56.
EXAMPLE 39 (S) -2- ((2R, 3R) -3- ((S) -1- ((6S, 9S, 12S, 13R) -12- ((S) -sec-butyl) 9 diisopropyl-13-methoxy-2, 2,5, 11-tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triazadec-15-yl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropionic acid
Figure BDA0001580863800000861
To a solution of (S) -2- ((2R, 3R) -3- ((S) -1- ((6S, 9S, 12S, 13R) -12- ((S) -sec-butyl) diisopropyl-13-methoxy-2, 2,5, 11-tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triazadecano 15-acyl) pyrrolidin-2-yl) -3- (1-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid methyl ester (30mg, 0.035mmol) in tetrahydrofuran (1.0ml) was added an aqueous LiOH solution (1.0M, 0.8ml) and the mixture was stirred at room temperature for 35 minutes with 0.5M H 3 PO 4 Neutralized to pH 6, concentrated and concentrated in SiO 2 Purification on column with CH 3 OH/Dichloromethanealkane/HOAc (1:10:0.01) to give the title compound (25.0mg, 85% yield). LC-MS (ESI) m/z C 44 H 74 N 5 O 10 [M+H] + Calculated 832.54, found 832.60.
EXAMPLE 40 (S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -N, 3-dimethyl-2- (S) -3-methyl-2- (methylamino) butyrylamino) -3-methoxy-5-methylheptanoyl) -pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid (101)
Figure BDA0001580863800000862
A mixture of (S) -2- ((2R, 3R) -3- ((S) -1- ((6S, 9S, 12S, 13R) -12- ((S) -sec-butyl) -69-diisopropyl-13 methoxy-2, 2,5, 11-tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triaza-decan-15-yl) pyrrolidin-2-yl) -3-di-2-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid (25mg, 0.030mmol), concentrated HCl (0.3ml) and 1, 4-dioxane (0.9ml) was stirred at room temperature for 35 minutes and then diluted with EtOH (1.0ml) and toluene (1.0ml), after concentration, it was diluted with ethanol/toluene (2:1) and concentrated to give the title compound as a white solid (22mg, 100% yield) for next step without further purification. LC-MS (ESI) m/z C 39 H 66 N 5 O 8 [M+H] + Calculated 732.48, found 732.60.
EXAMPLE 41 (2S) -2- ((2R, 3R) -3- ((2S) -1- ((11S, 14S, 17S) -1-azido-17- ((R) -sec-butyl) -11, 14 diisopropyl-18-methoxy-10, 16-dimethyl-9, 12, 15-trioxo-36-dioxa-10, 13, 16-triaza-icosa-20-oyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid
Figure BDA0001580863800000871
To a crude product of Compound 101 (22mg, 0.030mmol) in DMA (0.8ml) and NaH 2 PO 4 To a mixed solution of buffer (0.7ml, 1.0M, pH 7.5) was added compound 88(18.0mg,0.060mmol) was added in four portions over 2 hours. The mixture was stirred overnight, concentrated and concentrated on SiO 2 Purification on column with CH 3 OH/dichloromethane/HOAc (1:8:0.01) gave the title compound (22.5mg, 82% yield). LC-MS (ESI) m/z C 46 H 77 N 8 O 11 [M+H] + Calculated 917.56, found 917.60.
EXAMPLE 42 (2S) -2- ((2R, 3R) -3- ((2S) -1- ((11S, 14S, 17S) -1-amino-17- ((R) -sec-butyl) -11, 14 diisopropyl-18-methoxy-10, 16-dimethyl-9, 12, 15-trioxo-36-dioxa-10, 13, 16-triazicosan- -20 acyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid (103)
Figure BDA0001580863800000872
To a solution of compound 102(22.0mg, 0.024mmol) in methanol (5ml) was added Pd/C (5mg, 10% Pd, 50% wet) in a hydrogenation reactor. After evacuating the air from the vessel, 25psi of H was introduced 2 . The mixture was shaken for 4 h, filtered through celite, and concentrated to give the crude title product (-20 mg,. -92% yield) which was used in the next step without further purification. ESI MS m/z + C 46 H 79 N 6 O 11 (M + H), calculated 891.57, found 891.60.
Example 43.2, 3-Dibromosuccinic anhydride (70)
Figure BDA0001580863800000873
To a solution of 2.3-dibromosuccinic acid (10.00g,36.51mmol) in dry dichloromethane (100ml) was added phosphorus pentoxide (12.21g,85.84mmol) at 0 ℃. The mixture was stirred at 0 ℃ for 2 hours and then at room temperature for 5 hours, over short SiO 2 The column was filtered and the column was washed with ethyl acetate/dichloromethane (1: 6). The filtrates were combined, concentrated and the product was solidified in ethyl acetate/n-hexane (6.63g, 71% yield). ESI MS m/z + C 4 H 2 Br 2 O 3 (M + H), calculated 256.85, found 256.70.
Example 44.23-dibromo-4- ((2- (2- (3- ((S) -7-methoxy-8- ((5- (((S) -7-methoxy-2-methylene-5-oxo-2, 3, 5, 11 a-tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -2-methylene 5-oxo-2, 3, 11, 11 a-tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-10 (5H) -yl) -3-oxopropoxy) ethoxy) ethyl) amino) -4-oxobutanoic acid (124)
Figure BDA0001580863800000881
To a solution of compound 100(40.0mg, 0.068mmol) in DCM (4ml) was added DIPEA (12uL, 0.069mmol) and 2, 3-dibromosuccinic anhydride (38.0mg, 0.148mmol) at 0 deg.C. The mixture was stirred at 0 ℃ for 2 hours and then at room temperature for 5 hours. The mixture was concentrated and concentrated in SiO 2 Purification on column with CH 3 OH/dichloromethane/HOAc (1:6:0.01) gave the title compound (56.5mg, 83% yield). LC-MS (ESI) m/z C 44 H 53 Br 2 N 5 O 12 [M+H] + Calculated 1002.21, found 1002.40(M + H), 1004.40(M +2+ H).
EXAMPLE 45 ethyl (2- (2- (3- ((S) -7-methoxy-8- ((5- (((S) -7-methoxy-8- -7-methoxy-2-methylene-5-oxo-2, 3, 5, 11 a-tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14-diaza-8-yl) oxy) pentyl) oxy) -2-methylene-5-oxo-2, 3, 11, 11 a-tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-10 (5H) -yl) -3-oxopropoxy) ethoxy) ethyl) amino) -4-oxobutanoate (125).
Figure BDA0001580863800000882
To a solution of compound 125(55.0mg, 0.054mmol) in dichloromethane (3ml) were added NHS (10.0mg, 0.086mmol) and EDC (30.5mg, 0.158 mmol). The mixture was stirred at room temperature overnight, concentrated and concentrated on SiO 2 Purification on a column eluting with ethyl acetate/dichloromethane (1:5) gave the title compound (50.5mg,85% yield). LC-MS (ESI) m/z C 48 H 56 Br 2 N 6 O 14 [M+H] + Calculated 1099.22, found 1099.40(M + H),1101.40(M +2+ H),1119.50(M +2+ H) 2 O)。
EXAMPLE 46 (2S, 2R, 3R) -3- ((2S) -1- ((13S, 26S, 29S, 32S) -12, 13-dibromo-32- ((R) butyl) -2629 diisopropyl 33 methoxy-1- ((S) -7-methoxy-8- ((5- (((S) -7-methoxy-2-methylene-5-oxo-2, 3, 5, 11a tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -2-methylene-5-oxo-2, 3, 11, 11a tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-10 (5H) -yl) -2531 dimethyl 1, 11, 14, 24, 27, 30-hexanoxy-4, 7, 18, 21-tetraoxa-10, 15, 25, 28, 31-pentaaza-thirty-five-35 acyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide-3-phenylpropionic acid (126)
Figure BDA0001580863800000891
DMA (1ml) and NaH in Compound 103 (. about.20 mg, 0.022mmol) 2 PO 4 To a mixture of the buffer (0.6ml, 0.15M, pH7.5) was added compound 125(30.0mg, 0.027 mmol). After stirring for 7 h, concentration and purification by C-18HPLC (. PHI.2.0 cm. times.25 cm), eluting with water/acetonitrile (10ml/min flow rate, from 90% water to 15% water in 50 min) the title product 126 was obtained after drying on high vacuum pump (26.1mg, 63% yield). ESI MS m/z + C 90 H 130 Br 2 N 11 O 22 (M + H), calculated 1874.77, found 1874.50.
EXAMPLE 47 conjugation of Compound 126 to an antibody preparation 127
Figure BDA0001580863800000892
To 2.0mL of a 10mg/mL solution of herceptin pH7.0-8.0, 0.70-2.0mL of 100mM NaH was added 2 PO 4 pH6.5-7.5, TCEP (28. mu.L, 20mM in water) and Compound 126 (14. mu.L, 20mM in DMA). The mixture was allowed to stand at room temperatureIncubate for 4-16 hours, then add DHAA (135. mu.L, 50 mM). After overnight incubation at room temperature, the mixture was purified on a G-25 column using 100mM NaH 2 PO 4 And eluting with 50mM NaCl pH 6.0-7.5 buffer solution to obtain 16.5-17.7 mg of conjugate 127 (in 13.1-15.0 ml buffer solution, 82% -88% yield). The drug/antibody ratio (DAR) (PBD dimer and MMAF combination on each antibody) was determined to be 3.85 by UPLC-QTof mass spectrometry. The monomer content of the SEC HPLC (Tosoh Bioscience, Tskgel G3000SW,7.8mm ID x 30cm,0.5ml/min,100min) analysis was 96-99%, and the SDS-PAGE gel was detected as a single band.
EXAMPLE 48 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentyl) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (12, 13-dibromo 24- ((S) -7-methoxy-8- ((5- (((S) -7-methoxy-2-methylene-5-oxo-2, 3, 5, 11 a-tetrahydro-1H-benzo e-pyrrolo 12-. alpha.14 diaza-8-yl) oxy) pentyl) oxy) -2-methylene-5- oxo 3, 11, 11a tetrahydro-1H-benzo e pyrrolo 12- α 14 diaza-10 (5H) -yl) -11, 14, 24-trioxo-4, 7, 18, 21-tetraoxa-10, 15-diazatetracarboxamido) -4-hydroxyphenyl) -2-methylpentanoic acid (128)
Figure BDA0001580863800000901
Compound 95(20mg, 0.021mmol) was added to DMA (1ml) and NaH 2 PO 4 To a mixture of the buffer (0.6ml, 0.15M, pH7.5) was added compound 125(30.0mg, 0.027 mmol). The mixture was stirred for 8 h, concentrated, and purified by C-18 preparative chromatography (. phi.2.0 cm. times.25 cm), eluting with water/acetonitrile (flow rate 10 ml/min, from 90% water to 20% water over 50 min) to give the title product 128(26.6mg, 64% yield) after drying on high vacuum pump. ESI MS m/z + C 89 H 123 Br 2 N 12 O 22 S (M + H), calcd. 1901.69, found 1901.90.
EXAMPLE 49 conjugation of Compound 128 with an antibody preparation 129
Figure BDA0001580863800000902
To 2.0mL of 10mg/mL herceptin solution of pH7.0-8.0 was added 0.70-2.0mL of 100mM NaH 2 PO 4 pH6.5-7.5 buffer, TCEP (28. mu.L, 20mM in water) and Compound 128 (14. mu.L, 20mM in DMA). The mixture was incubated at room temperature for 4-16 hours, followed by the addition of DHAA (135. mu.L, 50 mM). After overnight incubation at room temperature, the mixture was purified on a G-25 column using 100mM NaH 2 PO 4 And eluting with 50mM NaCl pH 6.0-7.5 buffer solution to obtain 16.4-17.6 mg of conjugate 129 (in 13.4-15.1 ml buffer solution, the yield is 82% -88%). The drug/antibody ratio (DAR) (PBD dimer and Tubulysin homolog combination on each antibody) was determined to be 3.9 by UPLC-QTof mass spectrometry. SEC HPLC (Tosoh Bioscience, Tskgel G3000SW,7.8mm ID x 30cm,0.5ml/min,100min) analyzed its monomer content at 96-99%, and SDS-PAGE gel measurements showed a single band.
Example 50 bis (25-dioxopyrrolidin-1-yl) 23-dibromosuccinate (9)
Figure BDA0001580863800000903
A solution of 2, 3-dibromosuccinic acid (5.0g, 18.25mmol), N-hydroxysuccinimide (NHS) (5.01g, 43.56mmol) and EDC (12.02g, 62.60mmol) in dichloromethane (100ml) was stirred at room temperature overnight, concentrated and concentrated in SiO 2 Purification on a column eluting with ethyl acetate/dichloromethane (1:6) gave the title compound (6.74g, 79% yield). LC-MS (ESI) m/z C 12 H 11 Br 2 N 2 O 8 Calculated value [ M + H] + 468.88，[M+H+2] + 470.88, found 468.70, 470.70.
EXAMPLE 51 (R, R, S, S, R, 4R, 4'R) -55' - ((((12, 13-dibromo-11, 14-dioxo-4, 7, 18, 21-tetraoxa-10, 15-diazidotetracos-1, 24-diacyl) bis (azanediyl)) bis (4-hydroxy-31-phenylene)) -bis (4- (2- ((1R, 3R) -1-acetoxy ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) -pentanoyl) -4-methylpentyl) thiazole-4-carboxamido) -2-methylpentanoic acid) (141)
Figure BDA0001580863800000911
DMA (1ml) and NaH in Compound 95(40mg, 0.042mmol) 2 PO 4 To a mixture of buffer (0.6ml, 0.15M, pH7.5) was added bis (2, 5-dioxopyrrolidin-1-yl) -2, 3-dibromosuccinate (9) (18.0mg, 0.038 mmol). The mixture was stirred for 8 hours, concentrated and purified by C-18 preparative chromatography (. phi.2.0 cm. times.25 cm), eluting with water/acetonitrile (10ml/min flow rate, from 90% water to 20% water over 90 minutes) to give the title product 141(38.5mg, 49% yield) after drying on high vacuum pump. ESI MS m/z + C 94 H 143 Br 2 N 14 O 24 S 2 (M + H), calculated 2073.81, found 2073.60.
EXAMPLE 52 conjugation of Compound 141 to an antibody preparation 142
Figure BDA0001580863800000912
To 2.0mL of 10mg/mL herceptin solution of pH7.0-8.0 was added 0.70-2.0mL of 100mM NaH 2 PO 4 pH6.5-7.5 buffer, TCEP (28. mu.L, 20mM in water) and Compound 141 (14. mu.L, 20mM in DMA). The mixture was incubated at room temperature for 4-16 hours, followed by the addition of DHAA (135. mu.L, 50 mM). After overnight incubation at room temperature, the mixture was purified on a G-25 column using 100mM NaH 2 PO 4 And eluting with 50mM NaCl pH 6.0-7.5 buffer solution to obtain 16.4-17.6 mg of conjugate 142 (in 13.4-15.2 ml buffer solution, 82% -88% yield). The drug/antibody ratio (DAR) was determined to be 3.9 by UPLC-QTof mass spectrometry. The monomer content of the SEC HPLC (Tosoh Bioscience, TskgelG3000SW,7.8mm ID x 30cm,0.5ml/min,100min) was 96-99%, and the SDS-PAGE gel showed a single band.
Example 53 in vitro cytotoxicity assessment of conjugates 127, 129 and 142 (comparative T-DM 1):
cell lines used in cytotoxicity experiments included HL-60, a human promyelocytic leukemia cell line, NCI-N87, a human gastric cancer cell line, BT-474, which is a human invasive ductal cancer cell line, and SKOV3, which is a human ovarian cancer cell line. For HL-60, NCI-N87 and BT-474 cells, these cells were grown in RPMI-1640 medium with 10% FBS. For SKOV3 cells, cells were grown in McCoy 5A medium containing 10% FBS. To run the assay, cells (180 μ Ι, 6000 cells) were added to 96-well plates and incubated at 37 ℃ in an environment of 5% carbon dioxide for 24 hours, after which the cells were treated with different concentrations of compound (20 μ Ι) in a total volume of 0.2 mL. Control wells contained cells and media, with no test compound. After the plate was incubated at 37 ℃ for 120 hours in an atmosphere of 5% carbon dioxide, MTT (5mg/mL) was added thereto, and the plate was incubated at 37 ℃ for 1.5 hours. After careful removal of the medium DMSO (180. mu.L) was added, shaken for 15 min and the absorbance measured at 490nm and 570nm using a 620nm reference filter. The inhibition rate was calculated according to the following formula:
Inhibition [% 1- (assay value-blank value)/(control value-blank value) ] × 100%
Cytotoxicity results:
Figure BDA0001580863800000921
the specificity of conjugate 127 for N87 cells exceeded 889 (IC) 50 >8/IC 5 0 ═ 0.009), and over 800 for SK-OV-3 cells; the specificity of conjugate 129 on N87 cells exceeded 666 (IC) 50 >8/IC 50 0.012), over 533 for SK-OV-3 cells; the specificity of the conjugate 142 to N87 cells exceeded 155 (IC) 50 >15/IC 50 0.097), over 180 for SK-OV-3 cells; the specificity of the conjugate T-DM1 on N87 cells exceeded 57 (IC) 50 >15/IC 50 0.263) for SK-OV-3 cells over 80.
The three novel conjugates 127, 129 and 142 were significantly more effective than the marketed conjugate T-DM 1.
Example 54 in vivo antitumor Activity
Tumor differentiation in human gastric cancer N-87 cell lineConjugates 127, 129 and 142 were evaluated for therapeutic efficacy in vivo with T-DM1 in a xenograft model. Five-week-old female BALB/c nude mice (30 animals) were inoculated subcutaneously with N-87 cancer cells (5X 10) in 0.1mL serum-free medium in the area under the right shoulder 6 Individual cells/mouse). The tumor grows for 8 days to reach 133mm 3 Average size of (d). The animals were then randomly divided into 5 groups (6 animals per group). The first group of mice served as a control group and were injected with phosphate buffered saline solution. The remaining three groups were dosed at 3mg/kg intravenously with conjugates 127, 129, 142 and T-DM1, respectively. The three-dimensional size of the tumor was measured every 4 days and the formula was used: tumor volume was calculated as 1/2 (length × width × height). The body weight of the animals was also measured. Mice were sacrificed when either of the following criteria was met: (1) weight loss greater than 20% of pretreatment weight and (2) tumor volume greater than 1500mm 3 (3) inability to eat and drink water in case of illness, or (4) skin necrosis. If no tumor can be palpated, the mouse is considered tumor-free.
The results are plotted in fig. 16. All four conjugates did not cause weight loss in the animals. The animals in the control group had a tumor volume of more than 1500mm 3 While the illness was killed on day 37. All 6/6 animals in the group with compounds 127 and 129 had no measurable tumor at all from day 13 to day 60 (end of experiment). All 6/6 animals in the group with compound 142 had no measurable tumor at day 21 and 2/6 animals had visible (measurable) tumor growth at day 48. In contrast, T-DM1 at a dose of 3mg/kg failed to completely eradicate the tumor, inhibiting tumor growth for only about 28 days.

Claims (37)

1. A bridge connector of the structure shown in (I):
Figure FDA0003621222910000011
wherein:
-is an optional single bond;
Figure FDA0003621222910000013
is a single bond or a double bond;
when in use
Figure FDA0003621222910000014
When it is a single bond, U and U' are not H, when
Figure FDA0003621222910000015
When the double bond is a bond, U or U' can be H, but is not H at the same time;
Figure FDA0003621222910000012
is a component capable of reacting with a thiol group pair on a cell binding agent, including a 2, 3-disubstituted succinic acid group, a 2-monosubstituted or 2, 3-disubstituted fumaric acid group, a 2-monosubstituted or 2, 3-disubstituted maleic acid group; the sulfhydryl pair is generated by reducing interchain disulfide bond of the cell binding agent by a reducing agent, wherein the reducing agent comprises dithiothreitol, dithioerythritol, L-glutathione and tris (2-carboxyethyl) phosphine, or/and beta-mercaptoethanol;
U and U' are the same or different leaving groups which may be substituted with a thiol group, including: halogen, methanesulfonyl, p-toluenesulfonyl, trifluoromethylsulfonyl, trifluoromethylsulfonate, nitrophenol, N-hydroxysuccinimide, phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl-5-phenylisoxazole-3' -sulfonate, or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate;
R 1 and R 2 Is the same, different or default straight-chain alkyl group with 1 to 6 carbon atoms, branched chain or cycloalkyl group with 3 to 6 carbon atoms, straight chain, branched chain or cycloalkenyl or alkynyl, or ester group, ether group, amide group or polyethoxy (OCH) group with 1 to 6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to about 1000, or a combination of these groups;
X 1 and X 2 Independently selected from NH, N (R) 3 ) O, S or CH 2 ；R 3 Is H, a straight chain alkyl group of 1 to 6 carbon atoms, a branched or cyclic alkyl group of 3 to 6 carbon atoms, a straight chain, branched or cyclic alkenyl or alkynyl group, or an ester, ether, amide or polyethoxy (OCH) group of 1 to 6 carbon atoms 2 CH 2 ) p Wherein p is an integer from 0 to 1000, or a combination of these groups;
Z 1 And Z 2 Are the same or different functional groups capable of reacting with cytotoxic drugs and have the following structure:
Figure FDA0003621222910000021
n-hydroxysuccinimide ester;
Figure FDA0003621222910000022
a maleimide group;
Figure FDA0003621222910000023
a disulfide;
Figure FDA0003621222910000024
a haloacetyl group;
Figure FDA0003621222910000025
an acid halide or ester;
Figure FDA0003621222910000026
a vinyl sulfonyl group;
Figure FDA0003621222910000027
an acryloyl group;
Figure FDA0003621222910000028
2- (p-toluenesulfonyloxy) acetyl;
Figure FDA0003621222910000029
2- (methylsulfonyloxy) acetyl;
Figure FDA00036212229100000210
2- (nitrophenoxy) acetyl;
Figure FDA00036212229100000211
2- (dinitrophenoxy) acetyl;
Figure FDA00036212229100000212
(fluorophenoxy) acetyl;
Figure FDA00036212229100000213
2- (difluorophenoxy) acetyl;
Figure FDA00036212229100000214
2- (pentafluorophenoxy) acetyl;
Figure FDA00036212229100000215
a ketone or an aldehyde;
Figure FDA00036212229100000216
2- (trifluoromethanesulfonyloxy) acetyl;
Figure FDA00036212229100000217
oxadiazole phenyl methane sulfonate (ODA);
Figure FDA00036212229100000218
an acid anhydride;
Figure FDA00036212229100000219
an alkoxyamino group;
Figure FDA00036212229100000220
nitrine;
Figure FDA00036212229100000221
alkynes or
Figure FDA00036212229100000222
Hydrazine; wherein, X 1 Is F, Cl, Br, I or a leaving group; x 2 Is O, NH, N (R) 1 ) Or CH 2 ；R 5 And R 3 Is R 1 Aryl, heteroaryl or one or more H independently by-R 1 Halogen, -OR 1 、-SR 1 、-NR 1 R 2 、-NO 2 、-S(O)R 1 、-S(O) 2 R 1 or-COOR 1 Substituted aryl; leaving groups include nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethylsulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, self-anhydrides, or anhydrides generated with other anhydrides, or polypeptide condensation reactions or Mitsunobu reaction intermediates.
2. A bridge linker according to claim 1, wherein the 2, 3-disubstituted succinic acid group, 2-monosubstituted or 2, 3-disubstituted fumaric acid group, 2-monosubstituted or 2, 3-disubstituted maleic acid group is produced by direct condensation of 2, 3-disubstituted succinic acid, 2-monosubstituted or 2, 3-disubstituted fumaric acid, 2-monosubstituted or 2, 3-disubstituted maleic acid or derivatives thereof and amine, alcohol or mercapto groups on the terminal ends of other components as shown in (Ia):
Figure FDA0003621222910000031
wherein X in formula (Ia) is X in claim 1 1 Or X 2 Including NH, N (R) 3 ) O, or S; r is R in claim 1 1 And/or R 2 ；R 1 ，R 2 And R 3 The definition of (a) is also the same as in claim 1;
Lv 1 and Lv 2 Identical or independent of each other are-OH, F, Cl, Br, I, nitroPhenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3' -sulfonic acid, self-anhydrides or anhydrides formed with other anhydrides including acetic anhydride or formic anhydride; or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate; the condensing agent comprises: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N, N ' -diisopropylcarbodiimide, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, carbonyldiimidazole, O-benzotriazol-N, N, N ', N ' -tetramethyluronium tetrafluoroborate, O-benzotriazol-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinophosphate hexafluorophosphate, diethyl pyrocarbonate, N, N ', N ' -tetramethylformamidinium hexafluorophosphate, 2- (7-oxybenzotriazole) -N, n, N ', N' -tetramethyluronium hexafluorophosphate, 1- [ (dimethylamine) (morpholinyl) methylene ]-1[1,2,3]Triazolo [4,5-b]1-pyridine-3-oxohexafluorophosphate, 2-chloro-1, 3-dimethylimidazolium hexafluorophosphate, chlorotriapyrrolidinylphosphonium hexafluorophosphate, bis (tetramethylene) fluorocarboxamide, N, N, N ', N' -tetramethyl-thio- (1-oxo-2-pyridyl) thiouronium hexafluorophosphate, 2- (2-pyridone-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate, thio- (1-oxo-2-pyridyl) -N, N, N ', N' -tetramethylthiouronium hexafluorophosphate, oxy- [ (ethoxycarbonyl) cyanomethylamine]-N, N, N ', N ' -tetramethylthiourea hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate, (benzotriazol-1-yloxy) dipyrrolidine carbahexafluorophosphate, N-benzyl-N ' -cyclohexylcarbodiimide (or supported on a polymer), dipyrrolidine alkyl (N-succinimidyloxy) carbonium hexafluorophosphate, 1- (chloro-1-pyrrolidinylmethylene) pyrrolidine hexafluorophosphate, 2-chloro-1, 3-dimethylimidazole tetrafluoroborate, (benzotriazol-1-yloxy) dipiperidine carbahexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium tetrafluoroborate, tris bromide(dimethylamino) phosphinohexafluorophosphoric acid, 1-N-propylphosphoric anhydride, 2-isocyanoethylmorpholine, N, N, N ', N' -tetramethylurea-oxy- (N-succinimidyl) hexafluorophosphate, 2-bromo-1-ethylpyridinium tetrafluoroborate, oxy- [ (ethoxycarbonyl) cyanomethylamine ]-N, N '-tetramethylthionurebetrafluoroborate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate, N' -tetramethyl-oxy- (3, 4-dihydro-4-oxo-1, 2, 3-benzotriazin-3-yl) uronium tetrafluoroborate, azodipiperidine, bis (4-chlorobenzyl) azodicarboxylate, di-tert-butyl azodicarboxylate, diisopropyl azodicarboxylate, diethyl azodicarboxylate.
3. The bridge connector of claim 1, wherein said R 1 And/or R 2 May be comprised of a linker component of one or more of the following: 6-maleimidocaproic acid, 3-maleimidopropionic acid, valine-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, 4-thiovaleric acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, 4-thiobutyric acid, ethylmaleimide, 4-thio-2-hydroxysulfobutyric acid, pyridinedithiol, alkoxyamino, vinyloxy, 4-methyl-4-dithio-valeric acid, azide, alkyne, dithio, peptide, and/or (4-acetyl) aminobenzoate.
4. A bridge connector according to claim 1, wherein connector component R 1 ，R 2 And/or R 3 A peptide comprising 1-20 natural or unnatural amino acid units, aminobenzyl units, 6-maleimidocaproyl units, disulfides, thioether units, hydrazone units, triazole units, or alkoxyoxime units.
5. A bridge connector according to claim 1, wherein connector component R 1 And/or R 2 Can be cleaved by proteolytic enzymes.
6. A cell-binding agent-drug conjugate having the structure of formula (II):
Figure FDA0003621222910000041
which is prepared by mixing Cb and Drug 1 And Drug 2 Connected via a bridge linker of structure (I) according to claim 1, wherein:
Figure FDA0003621222910000051
is a double bond;
cb is a cell binding agent, linked to Drug via a pair of sulfhydryl groups 1 And Drug 2 (ii) a The sulfydryl is generated by reducing a cell binding agent interchain disulfide bond by a reducing agent, and the reducing agent comprises dithiothreitol, dithioerythritol, L-glutathione and tris (2-carboxyethyl) phosphine, or/and beta-mercaptoethanol;
Drug 1 and Drug 2 (ii) are the same or different cytotoxic agents that are linked to the cell binding agent by a bridging linker with an alkyl, alkylene, alkenylene, alkynylene, ether, polyalkoxy, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, carbamate, amino acid, peptide, acyloxyamine, hydroxamic acid, disulfide bond, thioether, thioester, carbonate, heterocycle, heteroalkyl, heteroaryl or alkoxyoxime bond, and combinations thereof;
n is 1 to 30.
7. The cell binding agent-Drug conjugate of claim 6, wherein the Drug 1 And Drug 2 Identical or each independently selected from:
1) chemotherapeutic agents: a) alkylating agent: chlorpheniramine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, dimethoxyamine hydrochloride, mechlorethamine oxide, amlodipine hydrochloride, mycophenolic acid, dulcitol, pipobroman, neomechlorethamine, benzene mustard cholesterol, prednimustine, thiotepa, triamcinolone pair, uracil; CC-1065; (ii) a duocarmycin; benzodiazepine dimers; aA nitrourea; an alkyl sulfonate; a triazene; a platinum-containing compound; aziridines; ethyleneimine and methyl melamine; b) plant alkaloid: a vinca alkaloid; taxoids and their analogs; maytansine alkaloids and analogues thereof; cryptophycin; epothilone, juncecrogol, discodermolide, bryozoalactone, dolastatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictyin, spongistatin; c) DNA topoisomerase inhibitors: etoposide tinib; mitomycin; d) an antimetabolite: antifolates, DHFR inhibitors; an IMP dehydrogenase inhibitor; a ribonucleotide reductase inhibitor; pyrimidine analogs, uracil analogs; a cytosine analogue; a purine analog; a folic acid supplement; e) hormone therapy agent: receptor antagonists, antiestrogens, LHRH agonists; anti-androgens; retinoids, vitamin D3 analogs; a photodynamic therapy agent; a cytokine; f) kinase inhibitors: BIBW 2992, imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080, mubritinib, ponatinib, bafetinib, bosutinib, cabozantinib, vismodegib, ininib, ruxolitinib, CYT387, tivozanib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, isnext; g) antibiotics: enediynes antibiotics, aclacinomysins, actinomycin, amtricin, azaserine, bleomycin, carnomycin, clarithromycin, carminomycin, pheochromomycin, doxorubicin, morpholino doxorubicin, 2-pyrroline doxorubicin and doxorubicin, epirubicin, doxorubicin, idarubicin, marcomycin, mycin, mycophenolic acid, lophenycin, pellomycin, puromycin, triiron doxorubicin, streptozotocin, tubercidin, ubenimex, setastin, doxorubicin; h) and others: a polyketide compound; gemcitabine, oxigenin, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovivin-7, Xegeva, Proveng, Yervoy, prenylation inhibitors, dopaminergic neurons Toxins, actinomycin, bleomycin, anthracyclines, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors, Ca 2+ Inhibitors of atpase, inhibitors of histone deacetylase; celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide a; anti-adrenal agents;
Figure FDA0003621222910000061
piperazinedione propane; rhizomycin; (iv) Wenzuo; spiro germanium; geobacillus azavor; a tri-imine quinone; trichlorotriethylamine; trichothecenes, polyurethanes, siRNA and antisense drugs;
2) autoimmune disease drugs: cyclosporine, cyclosporin a, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids, DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus;
3) anti-infectious disease drugs: a) aminoglycosides: amikacin, aspartame, gentamicin, hygromycin B, kanamycin, neomycin, netilmicin, spectinomycin, streptomycin, tobramycin, and metysomycin; b) amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycin: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephem: cephem, cephalosporacetonitrile, chloraminobenzylpenicillin, cephradine, cefadriamine, ceftaroline, ceftazidime, cephalothin or cephalotaxin, cephalexin, cefalexin, cefamandole, cefapirin, azaconazole cephalosporin, fluxazole cephalosporin, sporocetone, azolin cephalosporin, cefbuperazone, cefcapene, cefdaxime, ceftazidime, cefixime, cefoxitin, cefprozil, cefalomide, ceftezole, cefuroxime, cefdinir, cefditoren, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, cefotetan, cefotaxime, thienazole cephalosporin, cefozopran, cefpirome, cefpodoxime, cefquinome, cefsulodin, cefteram, ceftibuten, ceftizoxime, cephapirin, ceftriaxone, ceftizoxime, cephamycin, oxacephem; f) glycopeptide: bleomycin, vancomycin, teicoplanin, ramoplanin, g) glycylcycline: tigecycline, h) beta-lactamase inhibitors: penam, oxapenam; i) lincosamide: clindamycin, lincomycin; j) lipopeptides: daptomycin, a54145, a calcium dependent antibiotic; k) macrolides: azithromycin, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolide, midecamycin, mickamycin, oleandomycin, rifamycin, lopimycin, roxithromycin, spectinomycin, spiramycin, tacrolimus, acearubamycin, telithromycin; l) monocyclic amines: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin, amoxicillin, azlocillin, benzylpenicillin, benzathine penicillin benzyl penicillin, benzathine penicillin phenoxymethyl penicillin, cloxacillin, procaine penicillin, mezlocillin, methicillin, nafcillin, oxacillin, acemethicillin, penicillin, nafcillin, phenoxymethyl penicillin, piperacillin, ampicillin, sulfocillin, temocillin, ticarcillin; o) a polypeptide: bacitracin, colistin, polymyxin B; p) quinolones: alatrefloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, gatifloxacin, gemifloxacin, grepafloxacin, carnotrufloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosloxacin; q) streptogramins: pristinamycin, quinupristin/dalfopristin; r) sulfonamides: sulfonamides: sulfonamides, sulfadiazine, sulfasalazine, sulfisoxazole, tamoxifen, trimethoprim-sulfamethoxazole; s) steroid antibacterial drugs: such as fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, cimeticycline, demeclocycline, ramoxiline, mecycline, methacycline, minocycline, oxytetracycline, penicillin V kalipecycline, pyrrolidine methyltetracycline, tetracycline, glycylcycline: u) other types of antibiotics: annonacin, arsine, bactoprenol inhibitor, DANAL/AR inhibitor, dictyostatin, discodermolide, juncecrogol, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimulide, metronidazole, mupirocin, NAM synthesis inhibitor, nitrofurantoin, paclitaxel, placido, pyrazinamide, quinupristin/dalfopristin, rifampin, tazobactam tinidazole, echinaconine;
4) Antiviral drugs: a) entry/fusion inhibitors: apavir, maraviroc, vicrivroc, gp 41; b) integrase inhibitors: raltegravir, elvite-gravir, globoid dna a; c) maturation inhibitors: bevirimat, vivocon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) nucleosides and nucleotides: abacavir, adefovir, amoxicillin, abciximab, brivudine, cidofovir, cladribine, dexamethasone, didanosine, elvucitabine, emtricitabine, entecavir, famciclovir, fluxacillin, 3 ' -fluoro-substituted 2 ', 3 ' -deoxynucleoside analogs, fomivirsen, 9-guanine, iodoglycoside, lamivudine, 1-nucleoside, penciclovir, racivir, ribavirin, diliptin, stavudine, talribavirin, telivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine; f) non-nucleoside: amantadine, atitidine, carboplatin, diarylpyrimidine, delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, polyethylene glycol interferon, lovirmine, lodenosine, methylindazizone, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod, aceticamide adamantane; g) protease inhibitors: amprenavir atazanavir, boceprevir, daronavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir, tipranavir; h) other types of antiviral drugs: antioxidase, arbidol, kalarotide, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate, foscarnet, griffine, taribavirin, hydroxyurea, KP-1461, miltefosine, priconaril, anabolic inhibitor, ribavirin, seliciclib;
5) A radioisotope selected from 3 H， 11 C， 14 C， 18 F， 32 P， 35 S， 64 Cu， 68 Ga， 86 Y， 99 Tc， 111 In， 123 I， 124 I，125I， 131 I， 133 Xe， 177 Lu， 211 At and 213 Bi；
6) a chromonic molecule that can absorb a light, including ultraviolet, fluorescent, infrared, near infrared, or visible light; yellow pigments, red blood cells, iridescent pigments, white blood cells, melanin and blue-green pigments, fluorescent molecules, visual light transduction molecules, photonic molecules, luminescent molecules and fluorescein compounds; a non-protein organic fluorophore; (ii) a cyanine derivative; squaric acid derivatives and ring-substituted squaric acids; a naphthalene derivative; coumarin derivatives; oxadiazole derivatives; an anthracene derivative; a pyrene derivative; an oxazine derivative; an acridine derivative; arylmethylamine derivatives and tetrapyrrole derivatives; analogs and derivatives of the following fluorescent compounds: CF dyes, DRAQ and CyTRAK probes, BODIPY, Alexa Fluor, Dylight Fluor, Atto, Tracy, FluProbes, Abberior dyes, DY and Megasstokes dyes, Sulfo Cy dyes, HiLyte Fluor, Seta, Setau and Square dyes, SureLight dyes, APCXL, RPE, BPE, allophycocyanin, annopalin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, hydroxycoumarin, lisamine rhodamine B, lucifer yellow, Me-methoxycoumarin, NBD, Pacific Blue or, Pacific ange, PE-780-Cy 5 conjugates, PE-Cy-R613, NHTata protein, Seta-S-555, Seta-Sedbs-670, Sedi-Sedco-S-555, Sedbla-555, Sedi-S-670, Secale-S-555, Setau-380-NHS, Setau-405-maleimide, Setau-405-NHS, Setau-425-NHS, Setau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine, 7-AAD, acridine orange, chromomycin A3, CyTRAK orange, DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, Hoechst33342, LDS, mithramycin, propidium iodide 751, SYTOX blue, SYTOX green, SYTOX orange, thiazole orange, TO-PRO, cyanine dye monomers, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1;
7) Pharmaceutically acceptable salts, acids or derivatives thereof of the above drugs.
8. The cell binding agent-drug conjugate of claim 7, wherein the CC-1065 comprises the aldorexin, kazelaixin, and bizelaixin synthetic analogs thereof; the duocarmycin comprises synthetic analogues KW-2189 and CBI-TMI; the benzodiazepine dimer comprises a dimer of pyrrolobenzodiazepine, tolamectin, indolophenyldiazepine, imidazobenzothiadiazole or oxazolidinobenzodiazepine; the nitrosourea comprises carmustine, lomustine, fustin chloride, fotemustine, nimustine or lamustine; the alkyl sulfonate comprises busulfan, endosulfan or sulfur; the triazenes include dacarbazine; the platinum-containing compound comprises carboplatin, cisplatin or oxaliplatin; the aziridines include chromanone, caronone, metoclopramide or ulidopa; the ethyleneimine and the methyl melamine include hexamethylmelamine, triethylenetriamine, triethylphosphoramide, triethylenethiophosphoramide, or trimethylolmethylamine; the vinca alkaloid comprises vincristine, vinblastine, vindesine, vinorelbine or catharanthine; the taxoids and analogues thereof include paclitaxel, docetaxel; the maytansinoids and analogues thereof include DM1, DM2, DM3, DM4, maytansine or ansamycin; the cryptophycin comprises cryptophycin 1 and cryptophycin 8; the etoposide tinib comprises 9-amino camptothecin, clinostat, daunomycin, etoposide phosphate, irinotecan, mitoxantrone, norflurazon, retinoic acid, teniposide, topotecan and 9-nitrocamptothecin; the mitomycin comprises mitomycin C; the DHFR inhibitor comprises methotrexate, trametes, denopterin, pteropterin, aminopterin or other folic acid analogs; the IMP dehydrogenase inhibitor comprises mycophenolic acid, thiazolorubiline, ribavirin or EICAR; the ribonucleotide reductase inhibitor comprises hydroxyurea or deferoxamine; the uracil analogs include ancitabine, azacitidine, 6-azauracil, capecitabine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed or Tomudex; the cytosine analogue comprises cytarabine, cytosine arabinoside or fludarabine; the purine analogues comprise azathioprine, fludarabine, mercaptopurine, thiamine or thioguanine; the folic acid supplement comprises florolinic acid; the antiestrogen comprises megestrol, raloxifene or tamoxifen; the LHRH agonists include gostatin, leuprolide acetate; the antiandrogen comprises bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane or other androgen inhibitors; said vitamin D3 analogs include CB1093, EB1089KH1060, cholecalciferol or ergocalciferol; the photodynamic therapy agent comprises verteporfin, phthalocyanine, photosensitizer Pc4 or demethoxy-hypocrellin A; the cytokine comprises interferon-alpha, interferon-gamma, tumor necrosis factor or human protein containing TNF; the enediyne antibiotics comprise calicheamicin or dactinomycin, the calicheamicin comprises calicheamicin gamma 1, delta 1, alpha 1 or beta 1, and the dactinomycin comprises dactinomycin A, deoxymithramycin, epothilones, cadomycin, C-1027, maduropeptin and a neocarcinomycin chromophore or a related chromoprotein enediyne antibiotic chromophore; said polyketide comprises annonaceous acetogenins, said annonaceous acetogenins comprising bullatacin or bullatacinone; the epoxygenase comprises a capeline and a capeline; the prenylation inhibitors include, for example, lovastatin; the dopaminergic neurotoxin comprises staurosporine, the actinomycin comprises actinomycin D or dactinomycin, the bleomycin comprises bleomycin A2, bleomycin B2 or pelomycin, the anthracycline comprises daunorubicin, the doxorubicin comprises adriamycin, the MDR inhibitor comprises verapamil, the Ca2+ ATPase inhibitor comprises thapsigargin, the histone deacetylase inhibitor comprises vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat, Belinostat, PCI-24781, entinostat, SB939, reminiostat, Givinostat, AR-42, CUDC-101, sulforaphane or trichostatin A; the adrenal drugs include aminoglutethimide, mitotane, trilostane, acetoglucuronolactone, aldphosphoramide, aminolevulinic acid, amsacrine, arabinoside, bestraucil, bisantrene, edaxate, defofamine, meclocine, diazaquinone, eflornithine (DFMO), eformitine, etinaminium, ethylgluconic acid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, diaminenitracridine, pentostatin, mechlorethamine, pirarubicin, podophyllic acid, 2-ethyl hydrazine, procarbazine; the trichothecene comprises T-2 toxin, verrucomicin A, bacillocin A or anguidine;
The corticosteroid comprises amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucolonide, dexamethasone, triamcinolone acetonide or beclomethasone dipropionate;
the gentamicin comprises netilmicin, sisomicin or isepamicin; the kanamycin comprises amikacin, arbekacin, aminodeoxykanamycin, dibekacin or tobramycin; the neomycin comprises framycetin, paromomycin or ribostamycin; the cephem comprises loracarbef; the cephamycin comprises cefoxitin, cefotetan or cefmetazole, and the oxygen (carbon) cephem comprises flomoxef or cefprozil; the vancomycin comprises oritavancin or telavancin; the teicoplanin comprises dalbavancin; the penam comprises sulbactam or tazobactam; the oxapenam comprises clavulanic acid; the ketolide comprises telithromycin or cerubicin; said rifamycin comprises isoniazid, rifampin, rifabutin or rifapentine; the ampicillin comprises pivampicillin, siloxacillin, bacampicillin, ampicillin or adriamycin; the glycylcyclines include tigecycline: the DANAL/AR inhibitor comprises cycloserine; the NAM synthesis inhibitor comprises fosfomycin;
The 3 ' -fluoro-substituted 2 ', 3 ' -deoxynucleoside analogs include 3, 3 ' -fluoro-2 ', 3 ' -dideoxythymidine and 3 ' -fluoro-2 ', 3 ' -dideoxyguanosine; the 1-nucleoside includes beta-1-thymidine and beta-1-2' -deoxycytidine;
the xanthene derivative comprises fluorescein, rhodamine, Oregon green, eosin or Texas red; the cyanine derivative comprises a cyanine, an indocarbocyanine, an oxacyanine, a thiocyanine or a merocyanine; the squaric acid derivatives and ring-substituted squaric acids include Seta, SeTau or Square dyes; the naphthalene derivative comprises dansyl or sodium fluosilicate derivative; the oxadiazole derivative comprises pyridyl oxazole, nitrobenzoxazole or benzooxadiazole; the anthracene derivative comprises DRAQ5, DRAQ7 or CyTRAK orange; the pyrene derivative comprises cascade blue; the oxazine derivative comprises nile red, nile blue, cresyl violet or oxazine 170; the acridine derivative comprises flavonol flavin, acridine orange or acridine yellow; the arylmethylamine derivative comprises auramine, crystal violet or malachite green; the tetrapyrrole derivatives include porphin, phthalocyanine or bilirubin;
the fluorescent compound is selected from: DCFH, DHR, Fluo-3, Fluo-4, Indo-1, SNARF, allophycocyanin, AmCyan1, AsRed2, Cirsium green, Azurite, B-phycoerythrin, Cerulean, CyPet, DsRed monomer, DsRed2, EBFP, EBFP2, ECFP, EGFP, Emerald, EYFP, S65A, S65C, S65L, Y66H or Y66W mutated GFP, GFPuv, HcRed1, J-Red, Katusha, sabira Orange, mCFFP, mChehery, mKeima-Red, KOmOrange, mRed, mRasperry, mRaspry, mRFP1, Trawb 1, mThio 2, MBerberry, Tarborq-34, TarbykurbsGFP, TarbsGFP-GFP, TarbsGFP-11, Tarboryfp-11, TarbsGFP, Tarboryfp-T, Tarboryfp 602, Tarboryfp, TarbsGFP, Tarboryfp-T-11, Tarboryfp, Tarborgfp, Tarborf-T-S11, TarborfYf, TarborfyGFP, TarbsGFP, TarbsF-T type GFP, TarbsGFP, TarbsF-T type F-type F, TarbsF-type F-T type F-type F, TarbsF-type F, TarbsF, and TarbsF-type F.
9. The cell binding agent-Drug conjugate of claim 6, wherein the Drug 1 And Drug 2 Are chromonic molecules.
10. The cell binding agent-Drug conjugate of claim 6, wherein the Drug 1 And Drug 2 Is a polyalkylene glycol or polyalkylene glycol homologue including polyethylene glycol, polypropylene glycol and copolymers of ethylene oxide and propylene oxide having a molecular weight of between 10Da and 200 kDa.
11. The cell binding agent-Drug conjugate of claim 6, wherein the Drug 1 And Drug 2 Is a cell-bound ligand, receptor or receptor analog.
12. The cell-binding agent-drug conjugate of claim 11, wherein the cell-binding ligand or receptor is selected from the group consisting of a folate derivative; a glutamic acid urea derivative; somastatin and homologs thereof; an aromatic sulfonamide; pituitary adenylate cyclase activating peptide; vasoactive intestinal peptide; cholecystokinin; bombesin/gastrin releasing peptide; neurotensin; substance P; neuropeptide Y; a Homing peptide; a cell penetrating peptide; peptide hormones: luteinizing hormone releasing hormone agonists and antagonists, and gonadotropin releasing hormone agonists, which act by targeting follicle stimulating hormone and luteinizing hormone and testosterone production; pattern recognition receptors: toll-like receptors, C-type lectins and Nodlike receptors; a nanobody; a domain antibody; bispecific T cell engage; dual affinity retargeting antibodies; tetravalent tandem antibodies; anticalin; an Adnectin; designed ankyrin repeat proteins; avimer; EGF receptors and VEGF receptors.
13. The cell binding agent-drug conjugate of claim 12, wherein the neurotensin comprises NTR1, NTR2, or NTR 3; substance P (NK1 receptor); the neuropeptide Y comprises neuropeptide Y1-Y6; the Homing peptide comprises RGD, NGR, dimeric and multimeric cyclic RGD peptides, the dimeric and multimeric cyclic RGD peptides comprise cRGDfV; a cell penetrating peptide; the peptide hormones include buserelin, Gonadorelin, goserelin, histrelin, nafarelin, deslorelin, abarelix, Cetrorelix, degarelix, or ganirelix.
14. The cell binding agent-Drug conjugate of claim 6, wherein the Drug 1 And Drug 2 Independently of tubulysin, calicheamicin, auristatin, maytansinoids, CC-1065 analogs, daunorubicin and doxorubicin compounds, taxanes, cryptophycin, epothilones, benzodiazepine dimers including pyrrolobenzodiazepine, tomimemycin, apramycin, indolophenodiazepine, imidazobenzothiadiazole, or oxazolidinobenzodiazepine dimers, calicheamicin and enediyne antibiotics, actinomycin, azathricin, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin/auri, including MMAE, MMAF, PYE, auritp, auristatin 2-AQ, auristatin 6-AQ, auristatin EB or auristatin, doxorubicin, auristatin, vincristine, hemistalin, azumamide, micronin, radiosubin, alternactin, microsclerominin, theonelamide, esperamicin, siRNA, nucleolytic enzymes and/or pharmaceutically acceptable salts, acids and/or homologues and derivatives of the above molecules.
15. The cell-binding agent-drug conjugate of claim 6, wherein the cell-binding agent is selected from the group consisting of antibodies, proteins, vitamins, peptides, polymeric micelles, liposomes, lipoprotein-based drug carriers, nanoparticle drug carriers, dendrimers, and coated with cell-binding ligands or combinations thereof.
16. The cell-binding agent-drug conjugate of claim 6, wherein the cell-binding agent comprises an intact antibody; a single chain antibody; a monoclonal antibody, a single chain monoclonal antibody or a monoclonal antibody fragment binding to a target cell, a chimeric antibody fragment binding to a target cell, a single domain antibody fragment binding to a target cell, a surface modified antibody, a single chain surface modified antibody, or a surface modified antibody fragment binding to a target cell, a humanized antibody, a single chain humanized antibody, or a humanized antibody fragment binding to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule or a large molecular weight protein.
17. The cell-binding agent-drug conjugate of claim 6, wherein the cell-binding agent comprises an agent capable of targeting the following cells: a tumor cell, a virus-infected cell, a microorganism-infected cell, a parasite-infected cell, a cell of an autoimmune disease, an activated tumor cell, a bone marrow cell, an activated T cell, a B cell, or a melanocyte.
18. The cell-binding agent-drug conjugate of claim 17, wherein the tumor cell is selected from a lymphoma cell, a myeloma cell, a renal cell carcinoma cell, a breast cancer cell, a prostate cancer cell, an ovarian cancer cell, a colon cancer cell, a gastric cancer cell, a squamous cell carcinoma, a small-cell lung cancer, a non-small-cell lung cancer cell, a testicular cancer cell, or any uncontrolled, accelerated growth, differentiation-causing cancer cell.
19. The cell-binding agent-drug conjugate of claim 6, wherein the cell-binding agent comprises a drug/molecule capable of resisting one of the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD14, CD14, CD14, CD14, CD 6856854, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD 6856854, CD14, CD14, CD14, CD14, CD14, CD14, CD14 6856856854, CD14, CD14 6856854, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD14, CD 685, CD276(B7-H3), CD303, CD304, CD309, CD326,4-1BB,5AC,5T4, adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, α v β 6, aminopeptidase N, amyloid β, androgen receptor, pro-angiogenic protein factor 2, pro-angiogenic protein factor 3, annexin A1, anthrax toxin protective antigen, anti-metastatic protein receptor, AOC3, B7-H3, Bacillus anthracis, BAFF, B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125, CA-IX, CALLA, 737Ag, canine lupus erythematosus IL 6, carbonic anhydrase IX, cardiac myoglobin, CCL11, CCR4, CCR5, CD3E (epsilon) CEA 3884, ACA 3, CALA 38Ag, CA 5, CA 385-CSF, CACSF 385, CACSF 465, CRF 5, CADE, CACSF 465, CADE-K3, CADE-K5, CADE-K3, CADE-K4653, CADE-K3, CADE 4653, CADE-K3, CADE 4653, CADE-2, CTLA4, CTAA16.88 tumor antigen, CXCR4, C-X-C chemokine receptor 4, cyclic ADP ribonuclease, cyclin B1, CYP1B1, cytomegalovirus glycoprotein B, Dabigatran, DLL4, DPP4, DR5, Escherichia coli shiga toxin type-1, Escherichia coli shiga toxin type-2, ED-B, EGFL7, EGFR, EGFRII, EGFRvIII, endoglin, endothelin B receptor, endotoxin, EpCAM, EphA2, Episialin, ERBB2, ERBB3, ERG, Escherichia coli, ETV6-AML, FA, FCGR1, fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR folic acid, folate receptor alpha, folate hydrolase, Fos-related antigen 1, respiratory tract virus F protein, frizzled ganglioside 1, fucosyl ganglioside 2, Globicoside 5828, Globica GD 26, Globica-3, acetyl neuraminic acid, GD3, Glycine type neuraminic acid, IgG 6326, IgG 3, GM3, GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB, GUCY2C, guanylate cyclase C, intestinal guanylate cyclase, guanylate cyclase C receptor, thermostable enterotoxin receptor, heat shock proteins, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2, HER2/neu, HER3, IgG4, HGF/SF, HHGFR, HIV-1, histone complex, HLA-DR, HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1, IGF1R, IGHE, IFN-gamma, influenza hemagglutinin, IgE, IgE Fc region, IL-1, IL-2R, IL-4, IL-5, IL-6R, IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2, integrins including α 4, α IIb β 3, α v β 3, α 4 β 7, α 5 β 1, α 6 β 4, α 7 β 7, α ll β 3, α 5 β 5 or α v β 5, interferon γ inducible proteins, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1, LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, T1, MAGE-1, MIF-464, MUMA 84, MUMA-84, MUGE-11, MUMA-11, My-Y antigen, MAG-1, MAG-3, MAGE-11, MAG-11, MAGE-11, MAG-7, MAG-11, MAG-7, MAG-11, MAG-7, MAGE, MAG-7, MAG-7, MAG-7, MAG-7, MAG-7, MAG-7, MAG, NCA-90, Nectin-4, NGF, neuronal apoptosis regulating protease 1, NOGO-A, Notch receptors, nucleolin, Neu oncogene products, NY-BR-1, NY-ESO-1, OX-40, OxLDL, OY-TES1, P21, A P53 non-mutant, P97, PAP, anti- (N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1, PDGF-R α, PDGFR- β, PDL-1, PLAC1, PLAP testis alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate co-transporter, PMEL 17, polysialic acid, protease 3, prostate cancer, PS, prostate cancer cells, Pseudomonas aeruginosA, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD, Rheus factor, KL, RhoC, RalsA mutation, Rals 5, Ras, syncytial 4, RON sarcomA, SART3, Sclerostin, SLAMF7, Selectin P, SDC1, systemic lupus erythematosus (a), somatomedin C, SIP, somatostatin, sperm protein 17, SSX2, STEAP1, STEAP2, STn, TAG-72, survivin, T cell receptor, T cell transmembrane protein, TEM1, TENB2, Tenascin C, TGF- α, TGF- β, TGF- β 1, TGF- β 2, Tie, Tie2, TIM-1, Tn, TNF, TNF- α, TNFRSF8, TNFRSF10B, TNFRSF13B, TPBG, TRAIL-R1, TRAILR2, tumor associated calcium signaling sensor 2, tumor specific glycosylated MU686C 8, TWEAK receptor, TYRP1, TRP-2, tyrosinase, VEGF-1, VCAM-1, VEGF-A, VEGF-352, VEGFR 3527, VEGF-3, VEGF-binding factor, VEGF-receptor expression of any of epidermal growth factor, VEGF receptor, growth factor, VEGF receptor of epidermal growth factor, VEGF 11, VEGF receptor expression of any of epidermal growth factor receptor, VEGF receptor of growth factor receptor.
20. A compound having the structure of formula (III):
Figure FDA0003621222910000151
which is formed by reacting Cb with a bridge linker of the structure of claim 1 as shown in (I),
Figure FDA0003621222910000153
is a double bond, Cb is a cell binding agent, Cb is as defined in any one of claims 6 and 15 to 19, and n is 1 to 30.
21. A compound having the structure of formula (IV):
Figure FDA0003621222910000152
it is prepared from Drug 1 And Drug 2 Formed by reaction with a bridge linker of structure (I) according to claim 1, wherein Drug 1 And Drug 2 Being the same or different cytotoxic agent, said Drug 1 And Drug 2 As defined in any one of claims 6 to 14.
22. A cell binding agent-drug conjugate, wherein the conjugate is selected from the group consisting of T01, T02, T03, T04, T05, T06, and T07:
Figure FDA0003621222910000161
Figure FDA0003621222910000171
wherein the mAb is an antibody; z 3 And Z 3 ' is independently H, OP (O) (OM) 1 )(OM 2 )，OCH 2 OP(O)(OM 1 )(OM 2 )，OSO 3 M 1 ，R 1 Or O-glycoside, NH-glycoside, S-glycoside or CH 2 -a glycoside; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 (ii) a n is
Figure FDA0003621222910000172
“-”，X 1 ，X 2 ，R 1 ，R 2 And R 3 The definition is the same as that of claim 1 or 6,
Figure FDA0003621222910000173
is a double bond.
23. A cell-binding agent-drug conjugate, wherein the conjugate comprises C01:
Figure FDA0003621222910000181
wherein the mAb is an antibody; n is
Figure FDA0003621222910000184
“-”，X 1 ，X 2 ，R 1 ，R 2 And R 3 The definitions are the same as in claim 1 or 6,
Figure FDA0003621222910000185
is a double bond.
24. A cell-binding agent-drug conjugate, wherein the conjugate comprises M01:
Figure FDA0003621222910000182
Wherein the mAb is an antibody; n is
Figure FDA0003621222910000186
“-”，X 1 ，X 2 ，R 1 ，R 2 And R 3 The definitions are the same as in claim 1 or 6,
Figure FDA0003621222910000187
is a double bond.
25. A cell binding agent-drug conjugate, wherein the conjugate is selected from the group consisting of Tx01, Tx02, and Tx 03:
Figure FDA0003621222910000183
Figure FDA0003621222910000191
wherein the mAb is an antibody; n is
Figure FDA0003621222910000193
“-”，X 1 ，X 2 ，R 1 And R 2 The definitions are the same as in claim 1 or 6,
Figure FDA0003621222910000194
is a double bond.
26. A cell-binding agent-drug conjugate, wherein the conjugate is selected from the group consisting of CC01, CC02, and CC 03:
Figure FDA0003621222910000192
Figure FDA0003621222910000201
wherein the mAb is an antibody; n is
Figure FDA0003621222910000203
Z 4 And Z 4 ' is H, PO (OM) 1 )(OM 2 )，SO 3 M 1 ，CH 2 PO(OM 1 )(OM 2 )，CH 3 N(CH 2 CH 2 ) 2 NC(O)-，O(CH 2 CH 2 ) 2 NC(O)-，R 1 Or a glycoside; x 3 And X 3 ' is O, NH, NHC (O), OC (O), -C (O) O, R 1 Or by default; "-", X 1 ，X 2 ，R 1 ，R 2 ，M 1 And M 2 The definitions are the same as in claim 1 or 6,
Figure FDA0003621222910000204
is a double bond.
27. A cell-binding agent-drug conjugate, wherein the conjugate is selected from Da01, Da02, Da03 and Da 04:
Figure FDA0003621222910000202
Figure FDA0003621222910000211
wherein the mAb is an antibody; n is
Figure FDA0003621222910000212
X 3 And X 3 ' is independently H, O, NH, NHC (O) NH, C (O), R 1 Or OC (O); "-", X 1 ，X 2 ，R 1 And R 2 As defined in claim 1 or 6,
Figure FDA0003621222910000213
is a double bond.
28. A cell-binding agent-drug conjugate, wherein the conjugate is selected from the group consisting of Au01, Au02, Au03, Au04 and Au 05:
Figure FDA0003621222910000221
wherein the mAb is an antibody; n is
Figure FDA0003621222910000222
X 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)R 1 Or by default; x 4 And X 4 ' is independent CH 2 ，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; z 3 And Z 3 ' is independently H, R 1 ，OP(O)(OM 1 )(OM 2 )，OCH 2 OP(O)(OM 1 )(OM 2 )，NHR 1 ，OSO 3 M 1 Or O-glycoside, NH-glycoside, S-glycoside or CH 2 -a glycoside; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；“-”，X 1 ，X 2 ，R 1 ，R 2 And R 3 As defined in claim 1 or 6,
Figure FDA0003621222910000232
is a double bond.
29. A cell binding agent-drug conjugate, wherein the conjugate is selected from PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB09, PB10, and PB 11:
Figure FDA0003621222910000231
Figure FDA0003621222910000241
Figure FDA0003621222910000251
wherein the mAb is an antibody; n is
Figure FDA0003621222910000252
X 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)NR 3 ，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; x 4 And X 4 ' is independent CH 2 ，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；“-”，X 1 ，X 2 ，R 1 ，R 2 And R 3 As defined in claim 1 or 6; r 1 And/or R 2 It is possible to default to the default,
Figure FDA0003621222910000262
is a double bond.
30. A cell binding agent-drug conjugate, wherein the conjugate is selected from the group consisting of Z01, Z02, Z03, Z04, Z05, Z06, Z07, Z08, Z09, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17 and Z18:
Figure FDA0003621222910000261
Figure FDA0003621222910000271
Figure FDA0003621222910000281
Figure FDA0003621222910000291
wherein the mAb is an antibody; n is
Figure FDA0003621222910000292
X 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)NR 3 ，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or by default; x 4 And X 4 ' is independently H, CH 2 ，OH，O，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；“-”，
Figure FDA0003621222910000303
X 1 ，X 2 ，R 1 ，R 2 And R 3 The definitions are the same as in claim 1 or 6; r 1 And/or R 2 A default may be used.
31. A cell-binding agent-drug conjugate, wherein the conjugate comprises Pg 01:
Figure FDA0003621222910000301
wherein the mAb is an antibody; n is
Figure FDA0003621222910000304
R 'and R' are independently H or CH 3 ；m 3 And m 4 Independently is
Figure FDA0003621222910000306
“-”，X 1 ，X 2 ，R 1 ，R 2 And R 3 Is as defined in claim 1 or 6,
Figure FDA0003621222910000305
is a double bond; r is 4 Is OH, H, R 1 Or R as defined in claim 1 3 。
32. A cell-binding agent-drug conjugate, wherein the conjugate is selected from the group consisting of: LB01, LB02, LB03, LB04, LB05, LB06, LB07, LB08, LB09, LB10, LB11, LB12, LB13, LB14, LB15, LB16, and LB 17:
Figure FDA0003621222910000302
Figure FDA0003621222910000311
Figure FDA0003621222910000321
Figure FDA0003621222910000331
Figure FDA0003621222910000341
wherein the mAb is an antibody; n is
Figure FDA0003621222910000343
X 3 And X 3 ' is independent CH 2 ，O，NH，NHC(O)，NHC(O)NH，C(O)，OC(O)，OC(O)NR 3 ，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or by default; x 4 And X 4 ' is independently H, CH 2 ，OH，O，C(O)，C(O)NH，C(O)N(R 1 )，R 1 ，NHR 1 ，NR 1 ，C(O)R 1 Or C (O) O; m 1 And M 2 Independently H, Na, K, Ca, Mg, NH 4 ，NR 1 R 2 R 3 ；m 3 And m 4 Is 0 to 5000; "-",
Figure FDA0003621222910000342
X 1 ，X 2 ，R 1 ，R 2 and R 3 The definitions are the same as in claim 1 or 6; r 1 And/or R 2 A default may be used.
33. A pharmaceutical composition for the treatment or prevention of cancer, autoimmune disease or infectious disease comprising a therapeutically effective dose of the cell-binding agent-drug conjugate of any one or more of claims 6-20, 22-32, and a pharmaceutically acceptable salt, carrier, diluent, or excipient thereof, or a combination thereof.
34. The pharmaceutical composition of claim 33, wherein the cell-binding agent-drug conjugate has in vitro, in vivo, or ex vivo activity.
35. The pharmaceutical composition of claim 33, further comprising an additional therapeutic agent.
36. The pharmaceutical composition of claim 35, wherein the additional therapeutic agent comprises a chemotherapeutic drug, radiation therapy, immunotherapeutic agent, autoimmune disease drug, anti-infective drug, or other conjugate.
37. The pharmaceutical composition of claim 35, wherein the additional therapeutic agent is selected from one or more of the following drugs: (iii) Adiraplet, Abiraterone acetate, acetaminophen/hydrocodone, Adamantide, Afatinib maleate, alemtuzumab, Alivelac, trastuzumab, amphetamine/dexamphetamine, anastrozole, Aripiprazole, Atezolizumab, atorvastatin, axitinib, Berlin, bevacizumab, cabazitaxel, Caboptidine, Bexarotene, blinatumomab, Bortezomib, Bosutinib, Brentuximab vedotti, budesonide/Fomitoterol, buprenorphine, capecitabine, carfilzomib, celecoxib, ceritinib, cetuximab, cyclosporine, cinacalcetearecavir, crizotinib, dabigatran, Defarantin, Difinavir, Darunavir, imatinib, Imatinib mesylate, Metronine, Dinetrin, Dermatoprazine, Dinetrin, Metrex, D-L, D-N, D-L, D-N, D-E, D-E, doxycycline, duloxetine, emtricitabine/rilpivirine/tenofovir disoproxil fumarate, emtricitabine/tenofovir/efavirenz, enoxaparin, enzalutamide, alfapertine, erlotinib, esomeprazole, ezetimibe/simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone/salmeterol, fulvestrant, gefitinib, glatiramer, goserelin acetate, imatinib, temozolob, ibrutinib, insulin aspart, insulin detemir, insulin glargine, insulin lisine, interferon beta 1a, interferon beta 1b, lapatinib, Ipilimumab, ipratropium bromide/salbutamol, glatiramer acetate, lenalidomide, ledum, lexolyxole sulfonate, letrozole, levothyroxine, lidocaine, linezolid, liraglutide, MEDI4736, memantine, methylphenidate, metoprolol, modafinil, mometasone, nilotinib, Nivolumab, ofatumumab, orlistat, pazopanib, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, quetiapine, reberidine chloride 223, raloxifene, latilazerivir, ramucirumab, ranibizumab, regorab, rituximab, rivaroxaban, romidepsin, rosuvastatin, ruxolitinib phosphate, albuterol, suviamine, sildenafil, siltiximab, sitagliptin/metformin, filgrafenacin, sorafenib, sunitinib, tadalafilgra, temozine, temozoxib, tiprexiprir, tricitabine, temozoxib, fosinopril/tenofovir, testosterone gel, thalidomide, tiotropium bromide, toremifene, tremetinib, Tretinoin, eculizumab, valsartan, vandetanib, vemurafenib, vorinostat, aflibercept, Zostavax and its homologs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients, or combinations thereof.
CN201580082582.5A 2015-08-10 2015-08-10 Novel linkers and their use for specific coupling of drugs and biomolecules Active CN108289964B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210788010.0A CN115300640A (en) 2015-08-10 2015-08-10 Novel linkers and their use for specific coupling of drugs and biomolecules

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2015/056083 WO2015155753A2 (en) 2015-08-10 2015-08-10 Novel linkers and their uses in specific conjugation of drugs to a biological molecule

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202210788010.0A Division CN115300640A (en) 2015-08-10 2015-08-10 Novel linkers and their use for specific coupling of drugs and biomolecules

Publications (2)

Publication Number Publication Date
CN108289964A CN108289964A (en) 2018-07-17
CN108289964B true CN108289964B (en) 2022-08-12

Family

ID=54288492

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202210788010.0A Pending CN115300640A (en) 2015-08-10 2015-08-10 Novel linkers and their use for specific coupling of drugs and biomolecules
CN201580082582.5A Active CN108289964B (en) 2015-08-10 2015-08-10 Novel linkers and their use for specific coupling of drugs and biomolecules

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202210788010.0A Pending CN115300640A (en) 2015-08-10 2015-08-10 Novel linkers and their use for specific coupling of drugs and biomolecules

Country Status (4)

Country Link
JP (1) JP6817288B2 (en)
CN (2) CN115300640A (en)
CA (1) CA2991975C (en)
WO (1) WO2015155753A2 (en)

Families Citing this family (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7330515B2 (en) * 2015-08-10 2023-08-22 ハンジョウ ディーエーシー バイオテック シーオー．，エルティディ． Novel conjugates and their use in specific conjugation of biomolecules and drugs
MA43345A (en) 2015-10-02 2018-08-08 Hoffmann La Roche PYRROLOBENZODIAZEPINE ANTIBODY-DRUG CONJUGATES AND METHODS OF USE
MA44334A (en) 2015-10-29 2018-09-05 Novartis Ag ANTIBODY CONJUGATES INCLUDING A TOLL-TYPE RECEPTOR AGONIST
AU2016202632B2 (en) 2016-02-04 2021-09-09 Hangzhou Dac Biotech Co, Ltd Specific conjugation linkers, specific immunoconjugates thereof, methods of making and uses such conjugates thereof
CN106397226A (en) * 2016-04-19 2017-02-15 福安药业集团宁波天衡制药有限公司 Novel preparation method for tamoxifen
BR112018071465A2 (en) * 2016-04-20 2019-02-05 Hangzhou Dac Biotech Co Ltd amanita toxin derivatives and their conjugation to a cell binding molecule
WO2018009916A1 (en) 2016-07-07 2018-01-11 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
KR20220147720A (en) * 2016-11-14 2022-11-03 항저우 디에이씨 바이오테크 씨오, 엘티디 Conjugation linkers, cell binding molecule-drug conjugates containing the likers, methods of making and uses such conjugates with the linkers
NZ755835A (en) 2017-01-17 2023-12-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
EP3601346A1 (en) * 2017-03-29 2020-02-05 H. Hoffnabb-La Roche Ag Bispecific antigen binding molecule for a costimulatory tnf receptor
CN116999573A (en) 2017-09-29 2023-11-07 第一三共株式会社 Antibody-drug conjugate, preparation method thereof, pharmaceutical composition and application thereof in preparation of drugs for treating tumors
RU2020117698A (en) 2017-12-01 2022-01-04 Эббви Инк. GLUCOCORTICOID RECEPTOR AGONIST AND ITS IMMUNOCONJUGATES
WO2019109415A1 (en) * 2017-12-04 2019-06-13 清华大学 Compound for targeted degradation of hmgcr and application thereof
CN109956972A (en) * 2017-12-22 2019-07-02 四川科瑞德凯华制药有限公司 Phosphinothioylidynetrisaziridine crystal form I and its preparation method and application
MX2020006192A (en) * 2017-12-31 2020-08-20 Hangzhou Dac Biotech Co Ltd A conjugate of a tubulysin analog with branched linkers.
CN108586556B (en) * 2018-01-17 2021-04-20 浙江海正药业股份有限公司 Crystal form of pentostatin, preparation method and application thereof
US20210309679A1 (en) * 2018-07-23 2021-10-07 Guangzhou Othrotx Co., Ltd. Bisphosphonate drug conjugates
WO2020033200A1 (en) * 2018-08-09 2020-02-13 Siemens Healthcare Diagnostics Inc. Composition of a bilirubin stock and a method of preparation thereof
AU2018445278B2 (en) * 2018-10-12 2022-07-28 Hangzhou Dac Biotech Co., Ltd Conjugation linkers containing 2,3-diaminosuccinyl group
WO2020081493A1 (en) 2018-10-16 2020-04-23 Molecular Templates, Inc. Pd-l1 binding proteins
AU2019379418A1 (en) * 2018-11-14 2021-06-03 Daiichi Sankyo Company, Limited Anti-CDH6 antibody-pyrrolobenzodiazepine derivative conjugate
BR112021010982A2 (en) * 2018-12-07 2021-08-31 Oxford University Innovation Limited BINDERS
WO2020190725A1 (en) 2019-03-15 2020-09-24 Bolt Biotherapeutics, Inc. Immunoconjugates targeting her2
EP3952916A1 (en) * 2019-04-11 2022-02-16 Sony Group Corporation Programmable polymeric drugs
US20220168435A1 (en) * 2019-04-11 2022-06-02 Sony Group Corporation Programmable polymeric drugs
SG11202111402PA (en) 2019-05-14 2021-11-29 Nuvation Bio Inc Anti-cancer nuclear hormone receptor-targeting compounds
CN110256313B (en) * 2019-05-15 2021-01-29 江苏省原子医学研究所 Photosensitizer prodrug compound and preparation method and application thereof
US20230115871A1 (en) * 2019-06-24 2023-04-13 Hangzhou Dac Biotech Co., Ltd A conjugate of a cytotoxic agent to a cell binding molecule with branched linkers
KR20220029725A (en) 2019-06-29 2022-03-08 항저우 디에이씨 바이오테크 씨오, 엘티디 Cell-binding molecule-tubulin derivative conjugate and method for preparing same
CN112341521A (en) * 2019-08-09 2021-02-09 上海翰森生物医药科技有限公司 Small molecule active compound and antibody conjugate thereof, preparation method and medical application thereof
CN110508029B (en) * 2019-09-04 2022-01-28 武玉香 Immunoaffinity column for simultaneously extracting florfenicol, chloramphenicol and thiamphenicol
WO2021055816A1 (en) 2019-09-18 2021-03-25 Molecular Templates, Inc. Pd-l1 binding molecules comprising shiga toxin a subunit scaffolds
US11952349B2 (en) 2019-11-13 2024-04-09 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds
CN115569133B (en) * 2020-02-26 2024-04-26 上海科技大学 Use of methylindigoindigo against coronaviruses
CN111393346B (en) * 2020-06-02 2020-08-25 凯莱英生命科学技术(天津)有限公司 Synthesis method of N-Boc-Dolaprine and Boc-Dap DCHA
WO2022051591A2 (en) 2020-09-04 2022-03-10 Novarock Biotherapeutics, Ltd. Nectin-4 antibodies and uses thereof
CN112285361B (en) * 2020-09-27 2023-12-05 中国人民解放军空军军医大学 Agent for eliminating interference of anti-CD 38 monoclonal antibody medicine against human globulin detection
CN114478420A (en) * 2020-11-13 2022-05-13 北京大学 Multi-specific biological coupling connecting arm and synthetic method thereof
CN114605367B (en) * 2020-12-03 2023-06-27 中国人民解放军军事科学院军事医学研究院 Coumarin-containing linker and antibody-conjugated drug containing the same
CN112535678A (en) * 2020-12-28 2021-03-23 烟台大学 Trituzumab-linked maytansine nanoparticle composition
CN112675313A (en) * 2020-12-28 2021-04-20 烟台大学 Trituzumab-trastuzumab-fragment-linked maytansine nanoparticle composition
CN112675305B (en) * 2021-01-22 2023-05-23 中国科学院深圳先进技术研究院 Amphiphilic molecule self-assembled nano-drug for tumor treatment and preparation method and application thereof
CA3213295A1 (en) 2021-03-17 2022-09-22 Molecular Templates, Inc. Pd-l1 binding proteins comprising shiga toxin a subunit scaffolds and cd8+ t cell antigens
BR112023019420A2 (en) 2021-03-23 2023-10-24 Nuvation Bio Inc ANTI-CANCER NUCLEAR HORMONE RECEPTOR TARGETING COMPOUNDS
CN114106088A (en) * 2021-04-28 2022-03-01 联宁（苏州）生物制药有限公司 Bromomethylpyrazine-based drug conjugates and ADCs
WO2022078524A2 (en) * 2021-11-03 2022-04-21 Hangzhou Dac Biotech Co., Ltd. Specific conjugation of an antibody
CN114533669B (en) * 2022-01-18 2022-11-04 武汉大学 Oral care composition and preparation method and application thereof
WO2024073106A1 (en) * 2022-09-29 2024-04-04 Tract Pharmaceuticals, Inc. Compounds and compositions useful as inhibitors of taps
CN116754760B (en) * 2023-06-14 2024-01-26 之江实验室 Method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of antibody

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014197854A1 (en) * 2013-06-06 2014-12-11 Igenica Biotherapeutics, Inc. Novel linkers for antibody-drug conjugates and related compounds, compositions, and methods of use

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL45594A (en) * 1973-11-12 1977-12-30 Stauffer Chemical Co Bis-substituted succinamides and their use as herbicides
EP2480230A4 (en) * 2009-09-24 2015-06-10 Seattle Genetics Inc Dr5 ligand drug conjugates
AU2012348017A1 (en) * 2011-12-05 2014-07-03 Igenica Biotherapeutics, Inc. Antibody-drug conjugates and related compounds, compositions, and methods
EP2822597A1 (en) * 2012-03-09 2015-01-14 UCL Business Plc. Chemical modification of antibodies
HUE048574T2 (en) * 2012-07-12 2020-08-28 Hangzhou Dac Biotech Co Ltd Conjugates of cell binding molecules with cytotoxic agents
JP6133431B2 (en) * 2012-11-24 2017-05-24 ハンジョウ ディーエーシー バイオテック シーオー．，エルティディ．Ｈａｎｇｚｈｏｕ Ｄａｃ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． Use of hydrophilic conjugates and conjugation reactions between drug molecules and cell binding molecules
MX2016009332A (en) * 2014-01-29 2016-12-20 Shanghai hengrui pharmaceutical co ltd Ligand-cytotoxicity drug conjugate, preparation method therefor, and uses thereof.
CN108811499B (en) * 2015-07-04 2021-09-24 杭州多禧生物科技有限公司 Specific conjugation of cell binding molecules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014197854A1 (en) * 2013-06-06 2014-12-11 Igenica Biotherapeutics, Inc. Novel linkers for antibody-drug conjugates and related compounds, compositions, and methods of use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
《Functional native disulfide bridging enables delivery of a potent, stable and targeted antibody–drug conjugate (ADC)》;Joao P.M. Nunes等;《CHEMICAL COMMUNICATIONS》;20150101;第51卷(第53期);摘要，scheme 1，Fig 2-3 *

Also Published As

Publication number Publication date
WO2015155753A2 (en) 2015-10-15
CN115300640A (en) 2022-11-08
CN108289964A (en) 2018-07-17
WO2015155753A3 (en) 2016-06-30
CA2991975A1 (en) 2015-10-15
CA2991975C (en) 2021-04-06
JP2018532695A (en) 2018-11-08
JP6817288B2 (en) 2021-01-20

Similar Documents

Publication Publication Date Title
CN108289964B (en) Novel linkers and their use for specific coupling of drugs and biomolecules
CN110099682B (en) Coupled connector, cell binding molecule-drug conjugate containing same, and preparation and application thereof
US11779663B2 (en) Specific conjugation linkers, specific immunoconjugates thereof, methods of making and uses such conjugates thereof
CN108811499B (en) Specific conjugation of cell binding molecules
CN108449940B (en) Conjugated bridge linkers to cell binding molecules
US10293055B2 (en) Acetylenedicarboxyl linkers and their uses in specific conjugation of a cell-binding molecule
CN110621673A (en) Double-stranded linked cytotoxic drug conjugates
US10292961B2 (en) Disulfur bridge linkers for conjugation of a cell-binding molecule
JP2024023191A (en) Bridged pyrrolobenzodiazepine dimer (PBD) derivatives and conjugates thereof
CN117980327A (en) Specific coupling of antibodies
CN115181164A (en) Derivatives of amatoxins and their coupling to cell binding molecules
CN115443134A (en) Conjugates of cell binding molecules and camptothecin analogs
CN113195487A (en) 2, 3-diaminosuccinyl conjugate linkers
CN113423430A (en) Amanitoxin conjugates containing branched linkers
CN114040781A (en) Formulation of conjugate of TUBULYSIN derivative and cell binding molecule
JP2023159139A (en) Novel linkers and their uses in specific conjugation of drugs to biological molecule
NZ744940B2 (en) Conjugation linkers, antibody-drug conjugates thereof, and methods of synthesis and use of such conjugates

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right

Effective date of registration: 20190428

Address after: 12 Zhongzi Science and Technology Park, 260 No. 6 Street, Hangzhou Economic and Technological Development Zone, Hangzhou City, Zhejiang Province

Applicant after: HANGZHOU DAC BIOTECH Co.,Ltd.

Address before: 215123 Room 1108, Building 99, Xinghu Street, Suzhou Industrial Park, Jiangsu Province

Applicant before: SUZHOU M-CONJ BIOTECH Co.,Ltd.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant