CN115300640A - 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

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CN115300640A
CN115300640A CN202210788010.0A CN202210788010A CN115300640A CN 115300640 A CN115300640 A CN 115300640A CN 202210788010 A CN202210788010 A CN 202210788010A CN 115300640 A CN115300640 A CN 115300640A
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acid
cell
drug
receptor
antibody
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赵珞博永新
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Hangzhou Dac Biotech Co Ltd
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    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
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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
The application is a divisional application of an application with the application date of 2018, 8 and 10, and the application number of 201580082582.5, and the invention name of the novel linker and the application of the novel linker 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 and conjointly couples a cell binding molecule and the micromolecule medicines, especially a cytotoxic agent, through the action of a sulfur atom pair on the cell binding molecule. 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 agents is their narrow therapeutic window, which generally does not distinguish between normal and malignant cells, and can cause toxic side effects after administration, even resulting in tolerated 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, 199-210); 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, 329-332; zhao R J, j.med.chem 2012, 55, 766-782); 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 precise targeting ability of antibodies allows targeted delivery of drugs to cancer cells, leaving normal cells substantially unaffected, and has been fully developed over the last two decades. Antibody-drug conjugates (ADCs) have gained explosive growth as a means of targeted treatment of cancer, particularly since the approval of adsitris (brentuximab vedotin) by the U.S. FDA in 2011 and Kadcyla (ado-trastuzumab emtansine) in 2013, with nearly all major pharmaceutical and biotechnology companies having concerns about (Chari, r. Et al, angelw. Chem., int. Ed.2014,53,3796-3827 siers, 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 on IgG1 antibodies, this non-selective coupling mode can lead to random cross-coupling reactions of toxic drugs in almost all regions of the antibody molecule, and in particular, to 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 subpopulations of ADCs have short circulating half-lives, low efficiency, high potential off-target toxicity, and uncertain in vivo Pharmacokinetic (PK) properties (Hamblett, k.j. Et al, clin.cancer res.2004,10,7063-7070, adem, y.t. Et al, bioconjugate chem.2014,25,656-664, boylan, n.j.bioconjugate Chem.,2013,24,1008-1016, strop. Et al, chem.biol.2013,20, 161-167. In addition, it is very challenging to maintain batch-to-batch consistency of ADCs produced by this traditional coupling method, requiring higher production quality control capability (Wakankar, a. Mabs,2011,3, 161-172).
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: introducing an unpaired cysteine on an antibody, such as Genentech's THIOMAB antibody (Junutula, j.r. et al clin. Cancer res.2010, 16,4769 Junutula, j.r. et al Nat biotechnol.2008,26,925-32, wo 7,309,300, 7,855,275, 7,521,541; or the introduction of a glutamine tag, which can be identified by transglutaminase (mTG) amplified with streptoverticillium mobaraense (Strop, p., bioconjugate chem.,2014,25,855-862 Strop. Et al, chem. Biol.2013,20,161-167 or by bacterial-derived transglutaminase (MTGase) (Dennler, p. Et al, bioconjugate. Chem.2014,25,569-us patent 20189287; introduction of thiol-L-fucose (Dennler, P. Et al, bioconjugate Chemistry 2014,25,569, okeley, N.M. et al, bioconjugate chem.2013,24, 1650); introducing unnatural amino acids by mutagenesis (Axup, j.y. Et al, proc.natl.acad.sci.2012,109,16101-16106, zimmerman, e.s., et al, bioconjugug.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, us patents 8,778,20120184001135 wo2010/081110 wo2006/069246, 2007/9312, us patents 7,332,05, 696,312, 7,638,299, wo2007/130453, us patents 7,632,492, 829,659; introduction of selenocysteine (Hofer, T. Et al Biochemistry 2009,48,12047-12057; U.S. Pat. No. 8,916,159); converting cysteine on the CXPXR consensus sequence to formylglycine (FGly) with a Formylglycine Generating Enzyme (FGE) (Drake, p.m. et al, biocononjug.chem.2014, 25,1331-1341, U.S. Pat. nos. 7,985,783, 8,097,701, 8,349,910, U.S. Pat. No. 20140141025,20100210543; or by glycoengineering with galactose or sialyltransferase (Zhou, q. Et al bioconjug. 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 in the four subtypes of IgG antibodies was well known in the sixties of the last century (Milstein C.biochem J1966, 101. 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 at the hinge region by a different number of disulfide bonds: igG 1 And IgG 4 Is 2, igG 2 4, igG 3 11 pieces of the feed. In IgG 1 In the above, the last cysteine of the light chain is linked to the 5 th cysteine of the heavy chain by disulfide bonding. 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 on the CH2 domain is the most easily reduced. Of VL, CL, VH, and CH1 domainsDisulfide bonds have a similar moderate degree of cleavable properties, whereas disulfide bonds of CH3 domains are the least susceptible to reduction (Liu, H et al, anal. Chem.,2010,82, 5219-5226).
Based on human IgG 1 The more easily cleavable interchain disulfide bonds of antibodies, many institutes and companies have adopted the strategy of chemical site-directed ligation 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 bis-alkylating reagents to form a three-carbon bridge (Badescu, g., et al, bioconju.chem.2014, 25,1124-1136,wo2013/272,wo 2014/064424), using bis-substituted heteroaryl bridges (us 2015/0105539), or via bismaleimides as bridges (WO 2014/114207). For a long time, we have also used bromomaleimide and dibromomaleimide as linkers to couple drugs and antibodies (WO 2014/009774, 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 in achieving their therapeutic effect is limited by the number of toxic small molecules that ultimately reach the tumor cells, their DAR values are preferably greater than 3 in order to increase the ADC therapeutic index (Epenetos, a.a. et al, cancer res.,1986,46,3183-3191 chari, r.v. acc. Chem. Res.,2008,41,98-107, zhao, r.y.2011j. Med. Chem.54, 3606-3623). 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. The excess reductively produced thiol, which cannot be bound by the bridging linker, can be oxidized and regenerated by an oxide such as dehydroascorbic acid (DHAA) or Cu (II) at the end of the coupling reaction, and the disulfide bond is regenerated again. 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 "open-loop" 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 lyon r.p et al, nat.biotechnol.2014, 32, 1059-62) since the latter can lose the conjugated drug by a 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 in a synergistic and targeted way; 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 the 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, wherein 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 BDA0003729824240000051
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) the thiol groups of the cell binding agent (e.g. antibody) where the disulfide bonds are reduced open, advantageously maintaining the stability of the conjugate; b) Small toxic 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 BDA0003729824240000052
Wherein:
Figure BDA0003729824240000053
is an optional single bond;
Figure BDA0003729824240000054
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 the temperature is higher than the set temperature
Figure BDA0003729824240000055
When it is a single bond, U and U' are not H, when
Figure BDA0003729824240000056
When the double bond is a bond, 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 group, a heteroaryl group, an alkoxyoxime bond or an amide bond;
R 1 and R 2 Are identical, different or default straight-chain alkyl groups having from 1 to 6 carbon atoms, branched or cyclic alkyl groups having from 3 to 6 carbon atoms, straight-chain, branched or cyclic alkenyl or alkynyl groups, or ester, ether, amide or polyethoxy (OCH) groups having from 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, most preferably 0 to 500 atoms, covalently bonded to 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 BDA0003729824240000061
wherein:
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 the same or different 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 BDA0003729824240000062
R 1 ,R 2 ,X 1 and X 2 Is as defined in 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 deriving from the reduction of disulfide bonds therein, has reacted with a bridge linker comprising a functional group Z capable of further reacting with a small drug molecule 1 And Z 2 ：
Figure BDA0003729824240000063
Wherein
Figure BDA0003729824240000064
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 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 BDA0003729824240000065
wherein
Figure BDA0003729824240000071
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 a Drug 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 conjugation thereof (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 Conjugatable MMAE, tubulysin and PBD cytotoxic Agents
FIG. 9 Synthesis of conjugated 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 a linker comprising both PBD dimer homologue and Tubulysin B homologue and an 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 linker containing both maytansine homolog and Tubulysin B homolog to 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 conjugate coupling of a linker comprising two MMAF homologs and polyethylene glycol to an antibody and a linker comprising two Tubulysin B homologs and polyethylene glycol to an antibody
FIG. 16 anti-tumor of conjugates 127,129 and 142 with T-DM1 on a human gastric tumor N87 cell modelComparison of the effects, 3mg/kg was injected intravenously at one time. 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, the animals of the control group were killed on day 37. All three compounds 127,129 and 142 outperformed T-DM1: all 6/6 animals in the group of 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 of compound 142 had no measurable tumor at day 21, and 2/6 animals had visible (measurable) tumor growth at day 48, inhibiting 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 an aliphatic hydrocarbon, straight or branched, containing from 1 to 8 carbon atoms. By "branched" is meant that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3-dimethylpentyl, 2,3, 4-trimethylpentyl, 3-methylhexyl, 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 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 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 branch of 1 to 18 carbon atomsA chain or straight chain or cyclic hydrocarbon radical comprising two monovalent radical centers resulting from the removal of two hydrogens on the same carbon or on 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 means that one or more carbon atoms (preferably 1, 2, 3 or 4 carbon atoms) on the aromatic ring are replaced by 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 ', halo, -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 a pharmaceutically acceptable salt.
"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 5 to 14, preferably 5 to 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 alkyl radical in which one of the alkyl radicals is bonded to a carbon atom, usually at a terminus 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-furylethylAnd so on.
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-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 herein and are defined as: boc, tert-butoxycarbonyl; broP, tris (dimethylamino) phosphine bromide hexafluorophosphoric acid; 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, where 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.
"pharmaceutical 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 salt" refers to derivatives of the disclosed compounds which form salts by the action of the parent compound with an acid or base. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts formed with the parent compound, for example, 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 may 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 the 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 transferring, delivering, introducing or delivering 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 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 a thiol pair on a cell binding agent to form a covalent thioether bond, 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; 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 mercapto group to form an amide, ester or thioether bond. Examples of the synthesis of these bridge linkers can be found in FIGS. 1,3,4,5,6,7, 10, 11, 12, 13, 14 and 15.
Preferably, the bridge linker has the structure shown in (I):
Figure BDA0003729824240000121
Wherein:
Figure BDA0003729824240000122
is an optional single bond;
Figure BDA0003729824240000123
is a single bond or a double bond;
when in use
Figure BDA0003729824240000124
When it is a single bond, U and U' are not H, when
Figure BDA0003729824240000125
When the double bond is a bond, U or U' can be H, but is not H at the same time;
Figure BDA0003729824240000126
is a component capable of reacting with the thiol atom pairs of 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 an interchain disulfide bond of a cell-binding agent 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 can be substituted with a thiol 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 toxic drugs to form disulfide bonds, ether bonds, estersA bond, thioether bond, thioester bond, peptide bond, hydrazone bond, carbamate bond, carbonate bond, amine bond (secondary, tertiary or quaternary), imine bond, heterocycloalkyl group, heteroaryl group, alkoxyoxime bond or amide bond;
R 1 and R 2 Are identical, different or absent straight-chain alkyl groups having from 1 to 6 carbon atoms, branched or cyclic alkyl groups having from 3 to 6 carbon atoms, straight-chain, branched or cyclic alkenyl or alkynyl groups, or ester, ether, amide or polyethoxy (OCH) groups having from 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, most preferably 0-500 atoms, covalently linked to 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 bridging linker may be 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, and the like,Hydroxamic acids, 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 form a ring with 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 BDA0003729824240000141
n-hydroxysuccinimide ester;
Figure BDA0003729824240000142
a maleimide group;
Figure BDA0003729824240000143
a disulfide;
Figure BDA0003729824240000144
a haloacetyl group;
Figure BDA0003729824240000145
acyl halide;
Figure BDA0003729824240000146
a vinyl sulfonyl group;
Figure BDA0003729824240000147
an acryloyl group;
Figure BDA0003729824240000148
2- (p-toluenesulfonyloxy) acetyl;
Figure BDA0003729824240000149
2- (methylsulfonyloxy) acetyl;
Figure BDA00037298242400001410
2- (nitrophenoxy) acetyl;
Figure BDA00037298242400001411
2- (dinitrophenoxy) acetyl;
Figure BDA00037298242400001412
2- (fluorophenoxy) acetyl;
Figure BDA00037298242400001413
2- (difluorophenoxy) acetyl;
Figure BDA00037298242400001414
2- (trifluoromethanesulfonyloxy) acetyl;
Figure BDA00037298242400001415
a ketone or an aldehyde;
Figure BDA00037298242400001416
2- (pentafluorophenoxy) acetyl;
Figure BDA00037298242400001417
oxadiazole phenyl methane sulfonate (ODA);
Figure BDA00037298242400001418
An acid anhydride;
Figure BDA00037298242400001419
an alkoxyamine;
Figure BDA00037298242400001420
nitrine;
Figure BDA00037298242400001421
alkynyl or
Figure BDA00037298242400001422
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 straight-chain alkyl or polyethoxy (OCH) radical having 1 to 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 terminal of the other component as shown in (Ia):
Figure BDA0003729824240000151
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 the same or independent 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-anhydride or anhydride formed 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-oxytriazololylalkyl phosphate (PyBOP), diethyl pyrocarbonate (DEPC), N, N, N ', N ' -tetramethylchloroamidine hexafluorophosphate, 2- (7-oxybenzotriazole) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU), 1- [ (dimethylamine) (morpholinyl) methylenecarbodiimide), N ' -methylenecarbodiimide, N ' -tetramethyluronium hexafluorophosphate ]-1[1,2,3]Triazolo [4,5-b]1-pyridine-3-oxidophorophosphate (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) thioureaOnium hexafluorophosphate, 2- (2-pyridon-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TPTU), sulfur- (1-oxo-2-pyridyl) -N, N, N ', N' -tetramethylthiourea hexafluorophosphate, oxygen- [ (ethoxycarbonyl) cyanomethylamine]N, N, N ', N ' -tetramethylthiourea Hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate (COMU), (benzotriazol-1-yloxy) pyrrolidine carbohexafluorophosphate (HBPyU), N-benzyl-N ' -cyclohexylcarbodiimide (or loaded on a polymer), dipyrrolidinyl (N-succinimidyloxy) carbenium 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, 3-tetramethylurea tetrafluoroborate (TCTU), tris (dimethylamino) phosphine hexafluorophosphate (BroP), 1-N-propylphosphoric anhydride (CA),
Figure BDA0003729824240000161
) 2-isocyanoethylmorpholine (MEI), N' -tetramethylurea-oxy- (N-succinimidyl) Hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy- [ (ethoxycarbonyl) cyanomethylamine]N, N, N ', N' -tetramethylthionurebetrafluoroborate (TOTU), 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl-1, 3-tetramethyluronium tetrafluoroborate (TSTU), N, N, N ', 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 linkers 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 BDA0003729824240000162
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-Thiooctanoic acid (SPP), 4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid (MCC), (4-acetyl) aminobenzoic acid (SIAB), 4-Thiobutyric 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, are set forth in part herein.
Examples of linkers comprising these components are:
Figure BDA0003729824240000163
(containing 6-Maleimidohexanoic acid MC)
Figure BDA0003729824240000164
(including 3-Maleimidopropionic acid MP)
Figure BDA0003729824240000171
(including p-aminobenzyloxycarbonyl group PAB)
Figure BDA0003729824240000172
Figure BDA0003729824240000173
Figure BDA0003729824240000174
(containing Ethylmaleimide ME)
Figure BDA0003729824240000175
(including valine-citrulline)
Figure BDA0003729824240000176
(comprising 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid MCC)
Figure BDA0003729824240000177
Figure BDA0003729824240000178
(comprising (4-acetyl) aminobenzoic acid)
Figure BDA0003729824240000179
(containing 4-thio-2-hydroxysulfonylbutanoic acid, 2-thio-SPDB)
Preferred conjugates are of formula (II):
Figure BDA00037298242400001710
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 BDA0003729824240000181
R 1 ，R 2 ，X 1 And X 2 The definition of (A) is the same as that of the structural formula (I).
Drug, as described in more detail below 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 dimers).
For the synthesis of conjugate conjugates, the cell binding agent can 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 may 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 cell-binding agent-drug conjugates linked by disulfide bonds, Is achieved by disulfide-bond exchange between a disulfide bond in the modified cell-binding agent and a free thiol-containing drug; 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; the synthesis of conjugates containing acid-labile hydrazones in the molecule can be achieved by reacting a hydrazide group with a carbonyl group in a linker, as is well known in the art (see p. Hamann et al, cancer res.1993, 53, 3336-334, b. Laguzza et al, j. Med. Chem.,1959, 32, 548-555, p. Trail et al, cancer res.,1997, 57, 100-105; synthesis of conjugates containing triazole groups can be achieved by click chemistry reactions (dipolar cycloaddition) of 1-alkynes on one linker with drugs containing azide groups (Lutz, J-f. Et al, adv. Drug del.rev.2008, 60, 958-970 slettten, e.m. Et al, acc. Chem. Research 2011, 44, 666-676).
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 may also be condensed with the carboxyl group on the linker of formula (I) to form an ester by the action of a condensing agent such as EDC or DCC. The drug-modified bridge linker is then coupled to a cell binding molecule. The amino-containing drug can react with the carboxylic acid ester 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 BDA0003729824240000191
wherein
Figure BDA0003729824240000192
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 water-miscible polar organic solvent, 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., 1-500mM. While dissolving a cell-binding agent, such as an antibody, in a buffer solution having a pH of 5 to 9.5, preferably 6 to 8.5 at a concentration of 1 to 35mg/ml, and reacting with 1 to 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 solution of the cell-binding agent and incubated at a temperature of 4 to 45 deg.C, preferably room temperature, and the progress of the reaction is monitored by measuring the decrease in absorbance of the solution 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 degree to which the cell-binding agent is modified can be determined by measuring the UV absorbance of the nitro-pyrithione, dinitro-pyridyldithione, pyrithione, formamide pyridyldithione, and dimethylamide pyridyldithione groups produced by the reaction. If the conjugate does not have a 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 which linker to use based on the reactivity of its functional groups.
Modified cytotoxic drugs
The cytotoxic drug structure modified with the bridge linker of the invention is preferably (IV):
Figure BDA0003729824240000211
wherein
Figure BDA0003729824240000212
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 a drug molecule and a linker with the structure of 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 (B) 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, sulfhydryl containing drugs may be reacted with R in neutral pH buffers 1 Or R 2 Maleimide reaction on the component to form 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 in formula (IV). The hydroxyl-containing drugs can be reacted with R containing halogen, p-toluenesulfonic acid or methanesulfonic acid ester under weak base conditions 1 Or R 2 Reaction of the components to give R bound by ether bonds 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 a thioether bond 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 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 a modified drug comprising a thioether bond as 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, binding to specific antigens or having a 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, 155-158); 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.1985 260932-937); carbohydrate binding proteins or lipoproteins, such as lectins; a cellular nutrient-delivery molecule; small molecule inhibitors, e.g. prostate specific Sex 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, nano,2008,19,1309-12, medarova, et al, nat. Med.2007,13,372-7, javier, et al, bioconjugate chem.2008, 1309-12), liposomes (poimed, et al, curr.phar.2004, 10, 2981-9) and virus coat (virus 2009, biochem, 2009, 71, nanoseal, 2009, 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. Pat. No. 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 BDA0003729824240000221
G; milstein, C.Nature 1975, 256. 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, mice, rats, hamsters 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 carrying out the monoclonal antibodies of the invention can be identified 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 such as Cu, mn, fe or Zn or/and heavy metal salts, 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), as described in U.S. Pat. nos. 4,341,761;4,399,121;4,427,783;4,444,887;4,451,570;4,466,917;4,472,500;4,491,632;4,493,890. Monoclonal antibodies can also be prepared by anti-receptor peptides or peptides containing a terminal carboxyl group, as is well known in the art, see the references Niman et al, proc.natl.acad.sci.usa,1983, 80; geysen et al, proc.Natl.Acad.Sci.USA,1985, 82; 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; little et al, biotechnol adv.1994,12 (3): 539-55; clackson et al, nature 1991, 352; huse et al, science 1989, 246.
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; 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), reference is made to 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 as a "chimeric antibody" that is significantly less immunogenic in humans than the mouse monoclonal antibody (Kipriyanov et al, molBiotechnol.2004,26, 39-60 Houdebine, curropinBiotechnol.2002, 13. 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, 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 (Szardnngs, 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 that are linked to drug molecules via the bridging linker of this patent on conjugates for the treatment of cancer, autoimmune and/or infectious diseases 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), adematumab (anti-EpCAM, CD 326), afimumab (anti-TNF- α), afutuzumab (anti-CD 20), alemizumab (anti-VEGFR 2), ALD518 (anti-IL-6), alemtuzumab (Campath, mabampath, anti-CD 52), altumomab (anti-CEA), antatumomab (anti-TAG-72), antrukizumab (IMA-638, anti-IL-13), apolizumab (anti-HLA-DR), azithromycin (anti-CEA), aselizumab (anti-L-selectin CD 62L), atilizumab (tocilizumab, actermra, roActemra, anti-IL-6 receptor), atorolimumab (anti-Rhesus factor), bapineuzumab (anti-beta amyloid), basiliximab (simulct, anti-CD 25 (alpha chain of IL-2 receptor)), bavituximab (anti-phosphatidylserine), bectmomab (Lympho)Scan, anti-CD 22), belimumab (BenlystSub>A, lymphostat-B, anti-BAFF), benralizumab (anti-CD 125), bertelimumab (anti-CCL 11 (eotaxin-1)), besillesomab (Scintiun, anti-CEA-associated antigen), bevacizumab (Avastin, anti-VEGF-A), biciromab (FibriScint, anti-fibrin II betSub>A chain), bivatuzumab (anti-CD 44v 6), blinatumomab (BiTE, anti-CD 19), brentuximab (cAC 10, anti-CD 30 TNRSF 8), briakinumab (anti-IL-12, IL-23), canakinumab (Ilaris, anti-IL-1), cantuzumab (C242, anti-Canag), catmaprobrobrobrobab, cataxolob (Camovab, anti-Cizimab (anti-CD 3-C49), anti-TNF α 49), cetuximab (erbitux, IMC-C225, anti-EGFR), citatuzumab bogotox (anti-EpCAM), cixuumumab (anti-IGF-1), clenoliximab (anti-CD 4), clivatuzu-MAb (anti-MUC 1), conatumumab (anti-TRAIL-R2), CR6261 (anti-influenzSub>A A hemagglutinin), dacetuzumab (anti-CD 40), daclizumab (Zenapax, anti-CD 25 (IL-2 receptor alphSub>A chain)), daratumKL (anti-CD 38 (cyclic ADP ribosylhydrolase), denosumab (ProliSub>A, anti-RANU), detumomab (anti-B lymphomSub>A cells), dorlimaab, dorlixizumab, proremaximab, proremab (anti-GD 3 ganglioside), eculizumab (anti-C5), edomumab (anti-Edodex), edodex-CAM 1, anti-endotoxin-CAM 17), efalizumab (Raptiva, anti-LFA-1 (CD 11 a)), efanguumab (Mycogarb, anti-Hsp 90), elotuzumab (anti-SLAMF 7), elisilimomab (anti-IL-6), enlimomab mab (anti-ICAM-1 (CD 54)), epitumomab (anti-epilialin), epitalizumab (anti-CD 22), erlizumab (anti-ITGB 2 (CD 18)), ertumaxomab (Rexoun, anti-HER 2/neu, CD 3), epitalizumab (Abegrin, anti-integrin. Alpha. V. Beta.3), exbivirumab (anti-hepatitis B surface antigen), fanolisomab (NeutroSpec, anti-CD 15), faralimob (anti-interferon receptor), farletuzumab (anti-folate receptor 1), felvizumab (anti-respiratory syncytial virus), fezakinumab (anti-IL-22), figitumumab (anti-IGF-1 receptor), fontaolizumab (anti-IFN-. Gamma.), foravirmamab (anti-rabies glycoprotein), fresolimumab (anti-TGF-. Beta.), galiximab (anti-CD 80), gantenerumab (anti-beta amyloid), gavilimomab (anti-CD 147 (baigin)), gemtuzumab (anti-CD 33), girentiximab (anti-carbonic anhydrase 9), glembatuzumab (CR 011, anti-GPNMB), golmizumab (Simponin, anti-TNF-. Alpha.), gomiliximab (anti-CD 23 (IgE receptor for IgE), gomilizumab (anti-CD 23 (IgE) In vivo)), ibalizumab (anti-CD 4), ibritumomab (anti-CD 20), igovomab (Indamaginis-125, anti-CA-125), imciroma (Myoscint, anti-cardiac myosin), infliximab (Remicade, anti-TNF-. Alpha.), intetumumab (anti-CD 51), inolimomab (anti-CD 25 (IL-2 receptor. Alpha. Chain), ebizumab (anti-CD 22), iplilimumab (anti-CD 152), iratumumab (anti-CD 30 (TNFRSF 8)), keliximab (anti-CD 4), labetuzumab (CEA-Cide, anti-CEA), lebrikizumab (anti-IL-13), lemalesomab (anti-NCA-90 (anti-granulocyte antigen)), lerdeluumab (anti-TRAIL 2), letumumab (anti-R2), libizumab (anti-hepatob (anti-HBeAg), lintuzumab (anti-CD 33), lumimumab (anti-CD 40), lumimumab (anti-CD 23 (IgE receptor), mapatumumab (anti-TRAIL-R1), macimomab (anti-T-cell receptor), matuzumab (anti-EGFR), mepolizumab (Bosatria, anti-IL-5), metelimumab (anti-TGF beta 1), milatuzumab (anti-CD 74), minretemomab (anti-TAG-72), mitumomab (BEC-2, anti-GD 3 ganglioside), morolimumab (anti-rhesus factor), motavizumab (Numax, anti-respiratory virus), muromonab-CD3 (Orthoclone OKT3, anti-CD 3), nacolomab (anti-C242), naptolimumab (anti-5T 4), natalizumab (anti-4), nebuzumab (anti-endotoxin), necitumumab (anti-EGFR), nerelimomab (anti-TNF- α), nimituzumab (theramiam, theraloc, anti-EGFR), nofetumab, ocrelizumab (anti-CD 20), orilimumab (Afolimomab, anti-LFA-1 (CD 11 Sub>A)), ofatumumab (ArzerrSub>A, anti-CD 20), olaratumab (anti-PDGF-R α), omalizumab (Xolair, anti-IgE Fc region) Oportuzumab (anti-EpCAM), oregoomab (OvaRex, anti-CA-125), otelixixizumab (anti-CD 3), palibaximab (anti-lipoteichoic acid), palivizumab (Synagis, abboagis, anti-respiratory syncytial Synagis), panini (velicix, ABX-EGF, anti-EGFR), panobacumab (anti-Pseudomonas aeruginosSub>A), palivizumab (anti-IL-4), pemtumumab (Theragyn, anti-MUC 1), pertuzumab (Omnitarg, 2C4, anti-HER 2/neu), pexelizumab (anti-C5), pintumomab (anti-adenocarcinomSub>A antigen), priliximab (anti-D4), pritumumab (anti-vimentin), PRO140 (anti-CCR 5) Racotumob (1E 10, anti-N-glycolylneuraminic acid (NeuGc, NGNA) -ganglioside GM 3)), rafivirumab (anti-rabies virus glycoprotein), ramucerumab (anti-VEGFR 2), ranibiumab (Luceis, anti-VEGF-A-VEGF ) Raxibacumab (anti-anthrax toxin, protective antigen), regavirumab (anti-cytomegalovirus glycoprotein B), reslizumab (anti-IL-5), rilutummab (anti-HGF), rituximab (MabThera, rituxanmab, anti-CD 20), robatatuzumab (anti-IGF-1 receptor), rontalizumab (anti-IFN-. Alpha.), rovelizumab (LeukAr-rest, anti-CD 11, CD 18), ruplizumab (antva, anti-CD 154 (CD 40L)), satumomab (anti-TAG-72), sevirumab (anti-cytomegalovirus), sibrotuzumab (anti-FAP), sifaramumab (anti-IFN-. Alpha.), sirtumab (anti-IL-6), siplizumab (anti-CD 2), smartMI 95 (anti-CD 33), anti-beta protein (amyloid beta protein), sonepcizumab (anti-sphingosine-1-phosphate), sontuzumab (anti-epidialin), stamulumab (anti-myostatin), suleumab (Leukoscan, anti-NCA-90 (granulocyte antigen)), tacatuzumab (anti-. Alpha.fetoprotein), tadocuzumab (anti-integrin. Alpha.IIb. Beta.3), talizumab (anti-IgE), tanezumab (anti-NGF), taplitmumab (anti-CD 19), tefibuzumab (Aurexis, (anti-aggregative factor A)), telimomab, tenatumomab (anti-tenascin C), teneliximab (anti-CD 40), teplizumab (anti-CD 3), TGN1412 (anti-CD 28), tigliomab (Tremelilimumab, anti-CTLA-4), tigagazumab (anti-CTLA-650-IL-13), tocilizumab (Atlizumab, actemra, roacttemra, IL-6 receptor), totalizumab (anti-CD 154 (CD 40L)), tositumumab (anti-CD 20), trastuzumab (herceptin, anti-HER 2/neu), tremelimumab (anti-CTLA-4), tucotuzumab celloulin (anti-EpCAM), tuvirumab (anti-hepatitis B virus), urtoxuzumab (anti-E.coli), ustekinumab (Stelara, anti-IL-12, IL-23), vapaliximab (anti-AOC 3 (VAP-1)), vidolizumab (anti-integrin. Alpha.4. Beta.7), vituzumab (anti-CD 20), vepalimomab (anti-AOC 3 (VAP-1)), visilizumab (Nuvion, anti-CD 3), vitaxin (anti-angiointegrin avb 3), volociximab (anti-integrin. Alpha.5. Beta.1), votumumab (HumaSPECT, anti-tumor antigen CTAA 16.88), zalutumumab (HuMax-EGFR, zanolimumab (HuMax-CD 4, anti-CD 4), ziralimumab (anti-CD 147 (bain)), zolinmomab (anti-CD 5), enscip
Figure BDA0003729824240000271
Alefacept
Figure BDA0003729824240000272
Abatacept
Figure BDA0003729824240000273
Rilonacept (Arcalast), 14F7 (anti-IRP-2 (ferromodulin 2)), 14G2a (anti-GD 2 ganglioside, from Nat. Cancer Inst., treatment of melanoma and solid tumors), J591 (anti-PSMA, from Weill Cornell medical college, treatment of prostate Cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma-associated antigen), sorin Radiofarci SRL (from Milan Italy, treatment of melanoma), COL-1 (anti-CEACAM 3, CGMCC 1, from Nat Cancer Inst., treatment of colorectal Cancer and gastric Cancer), CYT-356 (anti-IRP-2 (IRM 2)), CYT-2 a (anti-GD 2 ganglioside, treatment of melanoma, anti-PSMA, from Weill Cornell medical institute of Weill, treatment of prostate Cancer), and anti-Cancer therapy
Figure BDA0003729824240000274
For the treatment of prostate cancer), HNK20 (OraVax Inc. for the treatment of respiratory syncytial virus infection), immuRAIT (derived from Immunodics, for the treatment of NHL), lym-1 (anti-HLA-DR 10, peregrine Pharm), MAK-195F (anti-TNF (tumor necrosis factor, TNFA, TNF- α, TNFSF2, derived from Abbott/Knell, for the treatment of septic shock), MEDI-500 (T10B 9, anti-CD 3, TR α β (T cell receptor α/β), derived from ImMedmmunone, for graft versus host disease), RING SCAN (anti-TAG 72 (tumor associated glycoprotein 72), derived from Neoprene Corp, for breast cancer, colon and rectal cancer), avicidin (anti-EPCAM (epithelial cell adhesion molecule)), anti-tactd 1 (tumor associated calcium signaling 1), anti-GA 733-2 (gastrointestinal tumor associated Protein 2), anti-EGP-2 (epithelial glycoprotein 2), anti-KSA, KS1/4 antigen, M4S, tumor antigen 17-1a, cd326 (from NeoRx, for treating colon cancer, ovarian cancer, prostate cancer and NHL), mpho cid (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 (CD 13), annexin A1, B7-H3 (CD 276, 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 diseases, malignant ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, merk cell cancer, and liquid tumors, multiple myeloma), CD66e (various cancers), CD70 (metastatic renal cell cancer and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (various cancers), mucin (various cancers), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen, CEA, CD66 e) (breast, colorectal cancer and lung cancer), DLL4, EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD 184, hematologic tumors, solid tumors), endoglin (CD 105, 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 receptor, T cell leukemia and lymphoma), 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, ovarian, cervical, bronchial 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, melanoma), PLAP-like testicular alkaline phosphatase (ovarian, testicular cancer), PSMA (prostate cancer), PSA (prostate cancer), ROBO4, TAG 72 (tumor-associated glycoprotein 72), AML, gastric, colorectal, ovarian cancer), T-cell transmembrane proteins (various cancers), tie (CD 202B), 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 (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD 106, melanoma), VEGF-a, VEGF-2 (CD 309) (various cancers). Other tumors are associated, and the antigens recognized by antibodies have been summarized and reviewed (Gerber, et al, mAbs 2009,1, 247-253, novellino et al, cancer Immunol Immunother.2005, 54 (3), 187-207, franke, et al, cancer Biotherradiopharmarm.2000, 15, 459-76).
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, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49B, CD49C, CD51, CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79B, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158, CD163, CD166,. CD168, CD174, CD180, CD184, CDw186, CD194, CD195, CD200, CD200a, CD200B, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326,4-1BB,5AC,5T4 (Trophoblast glycoprotein, TPBG, WNT-activation inhibitor 1 or WAIF 1), 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 (VAP-1), B7-H3, bacillus anthracis, BAFF (B cell activating factor), B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, canAg, canine lupus erythematosus IL31, carbonic anhydrase IX, cardiac myosin, 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 (CCK 2R), CLDN18 (Claudin-18), clusterin A, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD 115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD 184), C-X-C chemokine receptor 4, cyclic ADP ribonuclease, cyclin B1, CYP1B1, cytomegalovirus glycoprotein B, dabigatran, DLL4 (DeltaLigh-like 4), DPP4 (dipeptidyl-peptidase 4), 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 (CD 105), endothelin B receptor, endotoxin, epCAM (epithelial cell adhesion molecule), ephA2, episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS 2 ETS 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-associated antigen 1, respiratory syncytial virus F protein, frizzled receptor, fucose GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, globoH, glypican 3, N-glycolylneuraminic acid, GM3, 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 protein, 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, IGER, IL-1, IL-2R (interleukin 2 receptor), IL-4, IL-5, IL-6, I-6R (Interleukin 6 receptor), I-9, I-10, I-12, I-13, I-17, I-17A, I-20, I-22, I-23, I31RA, IGF2 (insulin-like growth factor 2), integrins (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, α v β 5), interferon γ -inducing proteins, ITGA2, ITGB2, KIR2D, CK, e, egumain, ewis-Y antigen, FA-1 (lymphocyte function-associated antigen 1, CD11A), HRH, INGO-1, lipoteichoic acid, IV1A, MP2, TA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE-1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or glycosylinhibitory factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSN (mesothelin), MUC1 (mucin 1, cell surface associated (MUC 1) or Polymorphic Epithelial Mucin (PEM)), MUC 1-KH, MUC16 (CA 125), MCP1 (monocyte chemotactic protein 1), melanA/MART1, M-IAP, MPG, MS4A1, MYCN, myelin-associated glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), nectin-4 (ASG-22 ME), NGF, neuronal apoptosis regulating protease 1, NOGO-A, notch receptor, nucleolin, neu oncogene product, NY-BR-1, NY-ESO-1, ox-40, oxldl (oxidized low density lipoprotein), OY-TES1, P21, 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-ra (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, polysialic acid, protease 3 (PR 1), prostate cancer, PS (phosphatidylserine), prostate cancer cell, pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD 240), rhesuus factor, RANKL, rhoC, ras mutations, 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, STn, TAG-72 (tumor associated glycoprotein), survivin, T cell receptor, TEM T cell transmembrane protein, TEM1 (tumor vascular endothelial marker 1), TENB2, tenascosin C (TN-C), TGF-alpha, TGF-beta (transforming growth factor beta), TGF- β 1, TGF- β 2 (transforming growth factor 2), tie (CD 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 (DR 5)), tumor-associated calcium signal sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1 (CD 106), VEGF, VEGF-A, VEGF-2 (CD 309), VEGFR-1, VEGFR2, vimentin, WTRP 1, XA 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. Cancers of interest 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 cancer, carcinoid tumors, gastrointestinal cancer, unknown small cell cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewing family tumor (PNET), intracranial germ cell tumors, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach cancer), extragonadal germ cell tumors, peritrophoblastoma, head and neck cancer, hypopharynx cancer, islet cell cancer, renal cancer (renal cell cancer), cancer, leukemia (acute lymphocyte, acute myeloid lineage, chronic lymphocytic, chronic granulocytic, hairy cells), lip and oral cancers, liver cancer, lung cancer (non-small cell, small cell), lymphomas (aids-related, central nervous system, cutaneous T-cell, hodgkin's disease, non-hodgkin's disease), malignant mesothelioma, melanoma, merkel cell carcinoma, metastatic squamous neck and occult primary cancer, multiple myeloma and other plasma cell tumors, mycosis fungoides, myelodysplastic syndrome, myelodysplastic disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low malignancy), pancreatic cancer (exocrine, islet cell carcinoma), paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasma cell tumors, prostate rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (migrating cells), salivary gland cancer, sezary syndrome, skin cancer (cutaneous T-cell lymphoma, kaposi's sarcoma, melanoma), small bowel tumor, soft tissue sarcoma, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, cancer of the urethra, uterine cancer, unusual juvenile cancers, vaginal tumors, vulval tumors and wilms' tumors.
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, achlorhydrada 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, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, crohn's disease (idiopathic inflammatory bowel disease), cushing syndrome, cutaneous leukocytosis vasculitis, dego's disease, dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, dressler syndrome, discoid erythema, eczema, endometriosis, adnexitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa, erythema nodosum, idiopathic mixed cryoglobulinemia, erwinia syndrome, fibrodysplastic ossification, fibromyalgia, fibrosing myositis, fibrosing blepharitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, graves ' disease, guillain-Barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, hemolytic anemia, henoch-Schonlein purpura, hepatitis gestationis, hidradenitis suppurativa, sheese syndrome (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), renal disease (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, scleroderma, linear IgA disease (LAD), lou Gehrig's disease (also known as amyotrophic lateral sclerosis), lupus hepatitis, lupus erythematosus, majeed syndrome, meniere's disease, microscopic polyarteritis, miller-Fisher syndrome, mixed connective tissue disease, hard blotch, mueller-Habeneman disease, mcClolith syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic disease), neuromyotonia nervosa, blepharoid pemphigus, opsoclonus mydonnus syndrome, ord thyroiditis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disease associated with streptococci), paraeoplasic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, parry Romberg syndrome, parsonnage-Turner syndrome, pars plana inflammation, 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, reit's syndrome, restless leg syndrome, post-nerve fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, schmidt's syndrome, schnitzler's syndrome, schnithler's syndrome, scleritis, scleroderma, sjogren's syndrome, spondyloarthropathies, hyperviscosity, still's disease, stiff person's syndrome, subacute bacterial endocarditis, suzak's syndrome, sweet's syndrome, chorea minor, sympathetic anemia, takayasu's 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 conjugate 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 antibodies IgM, igG, anti-celiac disease antibodies, anti-phospholipid antibodies 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-P62 antibodies, anti-sp 100 antibodies, anti-mitochondrial (M) antibodies, anti-topoisomerase (anti-MCV) antibodies, anti-neutrophil 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 chemokine receptor, a major histocompatibility protein, a lectin (type C, type S or type I) or a complement control 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, argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesia disease, bacillus cereus infection, bacterial pneumonia, bacterial vaginitis, bacteroides infection, saccharomycosis parvum, ascariasis, BK virus infection, black knot disease, human blastocyst protozoa infection, blastomycosis, vibrio hemorrhagic fever, borrelia infection, botulism (and infantile botulism), brazilian hemorrhagic fever, brucella disease, burkholderia infection, brucella ulcer, calicivirus infection (norovirus and Sabovirus), campylobacteriosis, candidiasis (candidiasis, thrush), candidiasis of cats, cellulitis, chagas disease (trypanosomiasis), ascomycetes, chicken pox, chlamydia infection with pneumonia, cholera, glioblastoma, clonorchis sinensis, clostridium difficile infection, coccidioidomycosis, colorado tick fever, common cold (acute viral nasopharyngitis, acute rhinitis), creutzfeldt-jakob disease, crimean-congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, skin larva migration, cyclosporinosis, enterobacteriaceae infection, enterovirus infection, epidemic typhus, infectious erythema (fifth disease), acute eruption, fascioliasis, dyshepatis, fatal familial insomnia, filariasis, clostridium perfringens food poisoning, free living amoeba infection, clostridial infection, gas gangrene (clostridial muscular necrosis), geotrichiosis, gerstman-straussler-scheinker disease syndrome, giardiasis, melilotis equi, gonorrhea, granulomatous diarrhea (fifth disease), group a streptococcal infection, group B streptococcal infection, haemophilus influenzae infection, hand-foot-and-mouth disease (HFMD), hantavirus lung syndrome, helicobacter pylori infection, hemolytic uremic syndrome, nephrotic 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 papillomavirus infection, human parainfluenza virus infection, membranous tapeworm disease, epstein-barr virus infectious mononucleosis (monotype), influenza, isosporosis, kawasaki disease, keratitis, king-Geobacillus infection, kuru, lassa fever, legionnaires disease (refuge legionnaires 'disease), legionnaires disease (Pontiacre fever), leishmaniasis, lyme disease, lymphofilariasis (elephantiasis), lymphocytic choriomeningitis, malaria, marburg hemorrhagic fever, measles, melioidosis (Huichthy's disease), meningitis, meningococcosis, posterior zoiasis, microsporidiosis, molluscum contagiosum, mumps, mouse typhus (endemic typhus), mycoplasmal pneumonia, foot fungus swelling, myiasis, neonatal conjunctivitis (neonatal ophthalmopathy), variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardia disease, onchocerciasis (Heanoomycosis), paracoccidioidomycosis (southern Meyeriana), paragonimiasis, pasteurellosis, head lice, body lice, pubic lice, pelvic inflammatory disease, whooping cough, plague, pneumococcal infections, pneumocystic pneumonia, poliomyelitis, prevotella infections, primary amoeba meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, mouse bite fever, respiratory tract virus infections, nosemospore disease, rhinovirus infections, rickettsia infections, rickettsia, rifles, rift valley fever, rocky mountain spotted fever, rotavirus infections, rubella, salmonella, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, syncytial disease, septicemia, shigellasis (Bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporothrix, staphylococcal food poisoning, infection with staphylococcus aureus, strongyloides stercoralis, syphilis, tapeworm disease, tetanus, tinea barbae (Barber itch), tinea capitis, tinea corporis, tinea cruris, tinea manuum, tinea pedis (tinea pedis), onychomycosis (onychomycosis), tinea versicolor, toxocariasis (eye larva migration), toxoplasmosis (visceral larva migration), toxoplasmosis, trichinosis, trichomoniasis (trichuria infection), tuberculosis, tularemia, ureaplasma infection, venezuelan equine encephalitis, venezuelan hemorrhagic fever, viral pneumonia, west nile fever, yersinia alba (tinea alba), 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, anaplasma, bacillus anthracis, vibrio haemolyticus, hunningvirus, ascaris, aspergillus, astroviridae, babesia, bacillus cereus, various bacteria, bacteroides, escherichia coli, ascaris, BK virus, oesophaga, human blastomyceliophthora, blastomyces dermatitidis, marulovirus, borrelia, clostridium botulinum, sinomenii, brucella, typically Burkholderia cepacia and other Burkholderia species, mycobacteria ulcerobacter, calicidae, campylobacter, typically Candida albicans and other Candida species, bartonella henselae, group A streptococci and staphylococci, trypanosoma cruzi, haemophilus ducreyi, VZV, chlamydia trachomatis, colorado tick fever virus, rhinovirus, coronavirus, CJD prion, crimeria-Congo hemorrhagic fever virus, cryptococcus neoformans, cryptosporidium, harpagophytum brasiliensis, various parasites, cyclosporidium, cestode, cytomegalovirus, dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) -flavivirus, bifidobacterium fragilis, corynebacterium diphtheriae, cestode, heteroclada, ebola, echinococcus, enterovirus, pechholtzia, parvovirus B19, human herpesvirus 6 and human herpesvirus 7, fasciola gingivalis, fasciola hepatica and Megastillus megalosa, FFI prion, from the order of the filariales superfamily, clostridium perfringens, clostridium, other clostridium species, geotrichum candidum, GSS prion, giardia intestinalis, burkholderia, microsporum spinosum and candida gram, gonococcus, klebsiella granulomatosa, streptococcus pyogenes, streptococcus agalactiae, haemophilus influenzae, enteroviruses, mainly coxsackie a virus 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 haemophilus thermoaminogenes for Chlamydomonas carcinoma, human bocavirus, ehrlichia, hemophilophilus aparinus, human metapneumovirus, ehrlichia chalcones, human papilloma virus, human parainfluenza virus, taenia miniata and Delavayi taenia, ebavirus, orthomyxoviridae family, isospora beijerinckii, chryseobacterium, klebsiella pneumoniae, klebsiella, legionella pneumophila, leishmania, mycobacterium leprae and Mycobacterium tuberculosis, leptospira, listeria monocytogenes, borrelia and other Borrelia species, trichinella and filarial worms, lymphocytic choriomeningitis virus (LCMV) Plasmodium, marburg virus, measles virus, burkholderia pseudomallei, neisseria meningitidis, retrotransverson schistosoma, microsporiales, molluscum Contagiosum Virus (MCV), mumps virus, rickettsia typhi, mycoplasma pneumoniae, multiple bacterial and fungal parasitic dipteran larvae, chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prions, nocardia and other Nocardia species, spanish, pectioideae traria, simania and other subgenera, pasteurella, head lice, human body lice, yersinia pestis, streptococcus pneumoniae, pneumococci, poliovirus, prevotella, neisseria, JC virus, chlamydia psittaci, coxix burgerii, rabies virus, S.unicinctus and Spirobacterium, respiratory syncytial virus, nosema bacteria, rhinovirus, rickettsia, and any combination thereof, consisting of rickettsia minor, rift valley fever virus, rickettsia, rotavirus, rubella, salmonella, SARS coronavirus, human sarcoptidosis, schistosoma, somatocyte, shigella, varicella zoster virus, togaku or smallpox, schericonella anserina, staphylococcus aureus, streptococcus pyogenes, strongyloides, treponema pallidum, taenia, tetanus, tinea genus uranium, tinea genus, epidermophyton floccosum, trichophyton rubrum, trichophyton mentagrophytes, trichophyton rubrum, venez Exophyton 40692, trichophyton genus, genus Cytospora, toxoplasma or toxoplasma gondii, toxoplasma gondii, trichinella spiralis, trichostoma vaginalis, trichuris, mycobacterium tuberculosis, turra, urea and equine encephalitis virus, venezuelan equine encephalitis virus, vibrio cholerae, guaraeto virus, west Nile virus, beigelii filariosis, yersinia pseudotuberculosis, yersinia enterocolitica, yellow fever virus, mucor order (mucormycosis) and entomomycetales order (entomophthora mycosis), pseudomonas aeruginosa of Mucor order, campylobacter (Vibrio), aeromonas, eisenia, yersinia, shigella, salmonella typhi, jaas, spirochaira perna, borrelia burgdorferi, spirosoma, pneumocystis, brucella, mycoplasma, rickettsia rickettsii, tsutsutsugamsii, chlamydia, pathogenic fungi (aspergillus fumigatus, candida albicans, histoplasma capsulatum), protozoa (entamoeba histolytica, tenas trichomonas, hominis trichomonas, trypanosoma gambiae, trypanosoma rhodesiense, leishmania rozei, leishmania tropicalis, leishmania brasiliensis, pneumocystis pneumonia, plasmodium vivax, plasmodium falciparum, plasmodium malariae) or helminths (schistosoma japonicum, schistosoma mansoni, schistosoma japonicum 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 virus 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 inflammation).
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 serum-containing medium at about 37 ℃ for about 30 minutes to about 48 hours, to which medium the conjugate of the invention is added at a concentration ranging from about 1pM to 0.1mM. 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 linkers of this patent may 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, 100 mg/mL) can be added. The intravenous dose will be about 50. Mu.g to 20mg/kg (body weight) per week (10. Mu.g to 200mg/kg per injection). The patient can receive the second course of treatment 4-20 weeks after treatment. The specific clinical protocol 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 race, the patient's disease state of 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 of the 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 disorder, the overall health 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 intended route of delivery of the drug, as well as the rate of administration (bolus or continuous infusion) and the dosing 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 a powder, nasal drops or aerosol; or dermally, such as by use of topical ointments, creams, lotions, gels or sprays, or by transdermal patches.
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 linked, 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, e.g., WO05058367A2 and U.S. Pat. No. 4,956,303, et al, 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, mechlorethamine, benzene mustard cholesterol, prednimustine, thiotepa, tricresyl, 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 oxazolidinobenzodiazepine); nitrosoureas (carmustine, lomustine, fustin chloride, fotemustine, nimustine, lamustine); alkyl sulfonates (busulfan, endosulfan and sulfur); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines, such as chromanone, carotenone, metoclopramide and urotropin; 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 (DM 1, 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, e.g. antifolates, DHFR inhibitors Formulations (methotrexate, trexate, denopterin, 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 florolinic acid, e) hormone therapy agents, such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (gostatulin acetate); anti-androgens (bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 homologues (CB 1093, 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 245634), bafetinib (INNO-406), bosutinib (SKI-606), cabotinib, vesukib, iniparib, ruxolitinib, CYT387, axitinib, tivozab, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, isnext; g) Antibiotics, such as enediynes antibiotics (calicheamicins, in particular calicheamicin γ 1, δ 1, α 1 and β 1 (see j.med.chem.1996,39 (11), 2103-2117: 183-186), dynein, including dynein a and deoxymithramycin, esperamicin, cadamycin, C-1027, maduropeptin and neocarzinostain chromophores and related chromoprotenedialkyne antibiotic chromophores), aclacinomysins, actinomycin, amrinomycin, azaserine, bleomycin, carnomycin, clarithromycin, carminomycin, carcinomycin, doxorubicin, morpholino doxorubicin, 2-pyrroline doxorubicin and deoxydaunorubicin, epirubicin, doxorubicin, idarubicin, maccomycin, mycophenolic acid, lopithromycin, pellomycin, puromycin, triiron doxorubicin, streptozotocin, tubercidin, ubenimex, staudine, zorubicin; h) Others, such as polyketides (annonaceous acetogenins), in particular bullatacin and bullatacinone; gemcitabine, epoxygenases (e.g., capeline), bortezomib, thalidomide, lenalidomide, pomidomide, tosedostat, zybrestat, PLX4032, STA-9090, stimuvax, allovivin-7, xegeva, provenge, yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., staurosporins), actinomycins (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, pelomycin), anthracyclines (e.g., daunorubicin), doxorubicin (e.g., 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 (MGCD 0103), belinostat, PCI-24781, entinostat, SB939, resminostat, givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide a; anti-adrenal agents, e.g. aminoglutethimide, mitotane, trilostane, acetoglucuronolactone, aldphosphoramide, aminolevulinic acid, amsacrine, arabinoside, bestraucil, bisantrene, edaraxate, defofamine, meclocin, diazaquinone, eflornithine (DFMO), elfomitine, illimmonium, ethyl acetateGluconic acid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, diamminenitracridine, pentostatin, mechlorethamine, pirarubicin, podophyllic acid, 2-ethylhydrazine, procarbazine;
Figure BDA0003729824240000411
piperazinedione propanes; rhizomycin; (xi) zolo; spiro germanium; geobacillus azavor; a tri-imine quinone; trichlorotriethylamine; trichothecenes (in particular T-2 toxin, verrucosin A, bacosporin A and anguidine), polyurethanes, siRNA, 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 (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylgestomycin; b) Amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) Ansamycin: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) Cephem: cephem (loracarbef), cephalosporins, chlorammonicillin, cephradine, cefadroxil, cephalonine, ceftazidime, cephalothin or cephalotaxin, cephalexin, cefalexin, cefamandole, cefapirin, hydroxylamine azole cephalosporin, fluxazole cephalosporin, sporoximone, oxazoline cephalosporin, cefbuperazone, cefcapene, cefixime, cefpodoxime, cefixime, cefprozil, cefetamet, ceftezole, cefuroxime, cefdinir, cefditoren, cefpirazole, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, tiamidozole cephalosporin, cefozopran, cephalexin, cefimidazole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, cefbutramine, cefotaxime, ceftizoxime, ceftriaxone, cefuroxime, ceftizoxime, cephamycin (cefoxitin, cefotetan, cefmetazole), cefixime (flomoxef, cephalosporin); 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, freramycin, josamycin, ketolide (telithromycin, seithromycin), midecamycin, mickamycin, oleandomycin, rifamycin (isoniazid, rifampin, rifabutin, rifapentine), ropiniromycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK 506), 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, carnotrufloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosloxacin, 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: apaviralol, maraviroc, vicrivroc, gp41 (enfuvirtide), PRO 140, cd4 (abalizumab); b) Integrase inhibitors: raltegravir, elvite-gravir, globoid dnan a; c) Maturation inhibitor (b): bevirimat, vivecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: abacavir, aximavir, adefovir, armocivir, abciximab, brivudine, cidofovir, clevudine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, flurocilin (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 (3 TC), 1-nucleosides (e.g., β -1-thymidine and β -1-2' -deoxycytidine), penciclovir, racivir, ribavirin, fuvirdine, d4T, talibarine (viraine), temimidine, tenofovir, triflurovir, valacyclovir, valaciclovir, ganciclovir, azavudine (AZT), zivudine (AZT); f) non-nucleosides: 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, canola, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, priconalide, 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 may be used 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, coligen 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 (porphine, 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), fluoProbes (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 with the linkers of the present invention are: allophycocyanin (APC), annatto protein, APC-Cy7 conjugate, 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 conjugate, 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, tau-380-NHS, setau-405-maleimide, seta-405-NHS, seta-680-NHS, seta-PerCP-680, seta-NHS, seta-647-425, redaX-Tris-425.
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 linkers of the invention for the study of cells, 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, pH 7.2), 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) Y66H mutation), GFP (Y66W mutation), GFPuv, hcRed1, J-Red, katusha, kusabra Orange (monomer, MBL), mC, mCFFP, mCBL, bamH, monomer (tage, eagle, knot, kemrel), kemrel-monomer (Kemrel), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer, tsien laboratories), mStrawberry, mTFP1, mTurquose 2, P3 (phycobilisome complex), polymethyleneflavin-chlorophyll-protein complex (PerCP), R-phytoerythrin (RPE), T-Sapphire, tagCFP (dimer, evagen), tagGFP (dimer, evagen), tagFP (dimer, evagen), tagYFP (dimer, evagen), topalto (tandem dimer), topaz, turboFP602 (dimer, evagen 635), turboFPP (dimer, evagen), turboGFP (dimer, evagen), turboRFP (dimer, evagen), venus, wild type YPeS, zonet 1 (CloGFP), zsYellow1 (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 from about 10Da to about 200kDa, preferably a molecular weight of from about 88Da to about 40kDa; having two branches, each branch having a molecular weight of about 88Da to about 40kDa; more preferably, there are two branches, each of about 88Da to about 20kDa. In one embodiment, the polyalkylene glycol is polyethylene glycol having a molecular weight of about 10kDa, about 20kDa, or about 40kDa. 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;5622986;5643575;5728560;5730990;5738846;5811076;5824701;5840900;5880131;5900402;5902588;5919455;5951974;5965119;5965566;5969040;5981709;6011042;6042822;6113906;6127355;6132713;6,177,087 and 6,180,095. The structures of antibody-polyalkylene glycol conjugates linked by a bridging linker are as Pg01:
Figure BDA0003729824240000461
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 BDA0003729824240000462
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 of the formula(I) R as defined in (1) 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), tomimemycin, indolophenyldiazepine, imidazobenzothiadiazole or oxazolidonodiazepine dimers), calicheamicin and enediyne antibiotics, actinomycin, azathricin, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, auristatin (e.g., MMAE, MMAF, auristatin TP, auristatin 2-AQ,6-AQ, EB (AEB) and EFP (AEFP)), duocarmycin, thiotepa, vincristine, hemistalin, azunmide, iminostatin, pyaminin, thiostatin, subactin, theobacterin, theobacteramicin, theostemine, theobromamine, picloramine-1592, 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.; mol. Pharmaceuticals, 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.; mol. Pharmaceuticals, 2012,9,168-175.Friestad, 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.; et al eur. Pat. Appl.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.off.de 10254439; DE 10241152; DE 10008089.Leung, d; et al, WO 2002077036.Reichenbach, H.; et al, ger.Offen.DE 19638870; wolfgang, r.; US 20120120129779, 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 BDA0003729824240000481
Figure BDA0003729824240000491
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 of one another, H, na, K, ca, mg, NH 4 ，NR 1 R 2 R 3 (ii) a n is 1 to 30;
Figure BDA0003729824240000492
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 1992,256,1172-1178; proc.natl.acad.sci usa.1993,90,5881-5888, and U.S. Pat. No. 4,970,198;5,053,394;5,108,912;5,264,586;5,384,412;5,606,040;5,712,374;5,714,586;5,739,116;5,770,701;5,770,710;5,773,001;5,877,296;6,015,562;6,124,310;8,153,768. Examples of structures of antibody-calicheamicin homologs linked by bridge linkers are C01:
Figure BDA0003729824240000493
wherein the mAb is an antibody; n is 1 to 30;
Figure BDA0003729824240000501
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;4,361,650;4,307,016;4,294,757;4,294,757;4,371,533;4,424,219;4,331,598;4,450,254;4,364,866;4,313,946;4,315,929;4,362,663;4,322,348;4,371,533;4,424,219;5,208,020;5,416,064;5,208,020;5,416,064;6,333.410;6,441,163;6,716,821,7,276,497;7,301,019;7,303,749;7,368,565;7,411,063;7,851,432 and 8,163,888. An example of an antibody-maytansine conjugate linked by a bridging linker is the M01:
Figure BDA0003729824240000502
Wherein the mAb is an antibody; n is 1 to 30;
Figure BDA0003729824240000503
X 1 ，X 2 ，R 1 ，R 2 and R 3 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: k C.Nicolaou et al, J.am.chem.Soc.1995,117,2409-2420; ojima et al, j.med.chem.1996,39:3889-3896;1997,40,267-278;2002,45,5620-5623; ojima et al, proc.natl.acad.sci.,1999, 96; kim et al, bull, korean chem, soc, 1999,20,1389-1390; miller, et al.j.med.chem.,2004,47,4802-4805; U.S. Pat. nos. 5,475,011 5,728,849,5,811,452;6,340,701;6,372,738;6,391,913,6.436,931;6,589,979;6,596,757;6,706,708;7,008,942;7,186,851;7,217,819;7,276,499;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 Tx03:
Figure BDA0003729824240000511
wherein the mAb is an antibody; n is 1 to 30;
Figure BDA0003729824240000512
X 1 ，X 2 ，R 1 ，R 2 and R 3 The same as defined in formulae (I) and (II).
CC-1065 homologs and the duocarmycin homologs are also preferred cytotoxic agents linked to the bridge linkers of this patent. Examples of CC-1065 homologs and duocarmycin homologs and their synthesis can be found in: warpheoski, et al, j.med.chem.31:590-603 (1988), d.boger et al, j.org.chem;66;6654-6661,2001; <xnotran> 4169888,4391904,4671958,4816567,4912227,4923990,4952394,4975278,4978757,4994578,5037993,5070092,5084468,5101038,5117006,5137877,5138059,5147786,5187186,5223409,5225539,5288514,5324483,5332740,5332837,5334528,5403484,5427908,5475092,5495009,5530101,5545806,5547667,5569825,5571698,5573922,5580717,5585089,5585499,5587161,5595499,5606017,5622929,5625126,5629430,5633425,5641780,5660829,5661016,5686237,5693762,5703080,5712374,5714586,5739116,5739350,5770429,5773001,5773435,5786377 5786486,5789650,5814318,5846545,5874299,5877296,5877397,5885793,5939598,5962216,5969108,5985908,6060608,6066742,6075181,6103236,6114598,6130237,6132722,6143901,6150584,6162963,6172197,6180370,6194612,6214345,6262271,6281354,6310209,6329497,6342480,6486326,6512101,6521404,6534660,6544731,6548530,6555313,6555693,6566336,6,586,618,6593081,6630579,6,756,397,6759509,6762179,6884869,6897034,6946455,7,049,316,7087600,7091186,7115573,7129261,7214663,7223837,7304032,7329507,7,329,760,7,388,026,7,655,660,7,655,661,7,906,545, 8,012,978. </xnotran> Examples of antibody-CC 1065 homolog structures linked by bridging linkers are as in CC01, CC02 and CC03:
Figure BDA0003729824240000521
Wherein the mAb is an antibody; n is 1 to 30; 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;
Figure BDA0003729824240000531
X 1 ，X 2 ，R 1 ，R 2 ，M 1 and M 2 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; pieteersz, C.A., E., et al, cancer Res.1988,48,926-9311; trouet, et al, 1982,79,626-629; z.brich et al, j.controlled Release,1992,19,245-258; chen et al, syn.comm.,2003,33,2377-2390; king et al, bioconj. Chem.,1999,10,279-288; 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.1995,3,1305-1312; scott et al, bioorg.Med.l chem.Lett.1996, 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; U.S. Pat. nos. 5,106,951;5,122,368;5,146,064;5,177,016;5,208,323;5,824,805;6,146,658;6,214,345;7569358;7,803,903;8,084,586;8,053,205. Examples of antibody-doxorubicin homolog structures linked by bridge linkers are Da01, da02, da03 and Da04:
Figure BDA0003729824240000532
Figure BDA0003729824240000541
Wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independently H, O, NH, NHC (O), NHC (O) NH, C (O), R 1 Or OC (O);
Figure BDA0003729824240000542
X 1 ，X 2 ，R 1 and R 2 As defined in formulae (I) and (II).
Auristatins and dolastatins are preferred cytotoxic agents linked to a bridge linker. Auristatins (e.g., polyhauristatin 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; U.S. patent applications 11134826,20060074008,2006022925, U.S. Pat. Nos. 4414205,4753894,4764368,4816444,4879278,4943628,4978744,5122368,5165923,5169774,5286637,5410024,5521284,5530097,5554725,5585089,5599902,5629197, 565483, 5654399, 56149, 5665860,5708146,5714586,5741892,5767236,5767237,5780588,5821337,5840699,5965537, 4934,6033876,6034065,6048720,6054297,6054561,6124431, 43721,6162930, 1436245, 6239104,6323315,6342219, 63426342221, 644213, 6564416911, 6656644164416441644164415641569, 60563756375637569, 70989, 7098417941989, 7098417992989, 70989, 79417992989, 70989, 6497989, 79417992989, 64974156989, 64794156989, 644156989, 6441563756989, 605637563756989, 60563756989, 6056375637569, 605637563756989, 6056989, 5637569, 56375637563756375637569, 569, 56989, 5637563756375637563756989, 563756375637563756989, 563756989, 56375637569, and 5637563756989. Examples of conjugate structures in which antibody-auristatin is linked by a bridging linker are as exemplified by Au01, au02, au03, au04, and Au05:
Figure BDA0003729824240000551
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 of one another, H, na, K, ca, mg, NH 4 ，NR 1 R 2 R 3 ；
Figure BDA0003729824240000561
X 1 ，X 2 ，R 1 ，R 2 And R 3 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: U.S. Pat. nos. 8,163,736;8,153,627;8,034,808;7,834,005;7,741,319;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;4,723,003;4,683,230;4,663,453;4,508,647;4,464,467;4,427,587;4,000,304; 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 PB11:
Figure BDA0003729824240000562
Figure BDA0003729824240000571
Figure BDA0003729824240000581
Figure BDA0003729824240000591
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 is a group of 1 And M 2 Independently H, na, K, ca, mg, NH 4 ，NR 1 R 2 R 3 ；
Figure BDA0003729824240000592
Figure BDA0003729824240000593
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 homologs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD), tomimecin, indolophenyldiazepine, imidazobenzothiadiazole or oxazolidonodiazepine dimers), calicheamicin and enediyne antibiotics, actinomycin, diazoserin, 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 EB (AEB) and Auristatin EFP (AEFP)), dactinomycin, thiotepa, vincristine, hemidamulin, nanazine, micin, sulosin, sulbacteramicin, theostatin, PNamicine-159amide, and derivatives thereof. Examples of conjugates comprising two or more different cytotoxic agents connected via a bridge linker are as Z01, Z02, Z03, Z04, Z05, Z06, Z07, Z08, Z09, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17 and Z18:
Figure BDA0003729824240000594
Figure BDA0003729824240000601
Figure BDA0003729824240000611
Figure BDA0003729824240000621
Figure BDA0003729824240000631
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 BDA0003729824240000632
X 1 ，X 2 ，R 1 ，R 2 And R 3 As defined in formulae (I) and (II). R is 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 conjugates of cell-bound ligands or receptors, particularly antibody-receptor conjugates, can be used not only as targeting vectors to deliver the conjugate to malignant cells, but also to modulate or co-stimulate a desired immune response or alter signaling pathways. In immunotherapy, the cell binding ligand or receptor is preferably coupled to an antibody to a TCR (T cell receptor) T cell or CAR (chimeric antigen receptor) T cell or B Cell Receptor (BCR), or to a toxic cell. 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 cancers and other malignancies) (Low P S et al, acc. Chem.2008, 41, 120-129); urea glutamate derivatives (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 Growth Hormone Inhibitory Hormone (GHIH) or growth hormone release inhibitory factor (SRIF)) or Somatostatin) and its homologues such as octreotide (Sandostatin) and lanreotide (Somatuline) (particularly 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 of hypoxia and renal cell carcinoma) (Neri D et al, nat. Rev. Drug discov.2011, 10, 767-777); pituitary Adenylate Cyclase Activating Peptide (PACAP) (PAC 1) for pheochromocytoma and paraganglioma; vasoactive intestinal peptide (VIP/PACAP) (VPAC 1, VPAC 2) for lung, stomach, colon, rectal, breast, prostate, pancreatic ductal, liver and bladder cancer; cholecystokinin (CCK) (CCK 1 (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) (BB 1, GRP receptor subtype (BB 2), BB3 and BB 4) is used in renal, breast, lung, stomach and prostate cancer as well as neuroblastoma (Ohlsson, B et al, scan.j. gastroenterology 1999, 34 (12), 1224-9; weber H C, cur. Opin. Endocri. Diab. Obes.2009, 164 (1), 66-71, gonzalez n, cur. Opin. Endocri. Diab Obes 2008, 15 (1), 58-64); neurotensin (NTR 1, NTR2, NTR 3) for the treatment of small cell lung cancer, neuroblastoma, pancreatic cancer and colon cancer; substance P (NK 1 receptor) for glioblastomas; neuropeptide Y (Y1-Y6) for breast cancer; homing peptides include RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (e.g., cRGDfV) that recognize receptors (integrins) on the surface of tumors (Laakkonen P, vuorinen K. Integr Biol (Camb) 2010,2 (7-8), 326-337; 2717-2725), TAASGVRSEH, LTLRWVGLMS (chondroitin sulfated proteoglycan NG2 receptor) and F3 peptide (31 amino acid peptide that binds to cell surface-expressed nucleolin receptor) (Zitzmann S., cancer Res 2002, 62 (18), 5139-5143; 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 ) Himalarelin (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), nafarelin (Pyr-His-Trp-Ser-Tyr-D-Tr)p-Leu-Arg-Pro-Gly-NH 2 ) Delorelin, abarelix (Ac-D-2 Nal-D-4-chlorophenylene-D-3- (3-pyridyl) Ala-Ser- (N-Me) Tyr-D-Asn-Leu-isopropylLys-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-2 Nal-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-2 Nal-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 ) (thundmadathil, j., j.amino Acids,2012, 967347; boccon-Gibad L. Et al, therapeutic Advances in Urology 2011,3 (3), 127-140; debruyne, f., future Oncology,2006,2 (6), 677-696) and Pattern Recognition Receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins and Nodlike receptors (NLRs) (Fukata, M et al, semin. Immunol.2009, 21, 242-253; maison neuve et al proc.natl.acad.sci.2014, 111,1-6; botos I et al, structure 2011, 19, 447-459; means T K et al, life sci.2000, 68, 241-258) etc. range from small (imiquimod, guanisine and adenosine homologues) to large and complex biological macromolecules such as Lipopolysaccharide (LPS), nucleic acids (CpG DNA, poly i: c) And lipopeptides (Pam 3CSK 4) (kassuri S P et al, nature 2011, 470, 543-547; lane t, J R soc.med.2001, 93, 316; hotz C and Bourquin C, oncoimmunology 1, 2012, 227-228; dudek, A Z et al, clin. Cancer Res.2007, 13, 7119-7125).
Cell-binding ligands or receptors can be Ig-based and non-Ig protein molecules. Ig-based scaffolds are selected from, without limitation, nanobodies (derivatives of VHH (camelid Ig)) (muydermans s., annu Rev biochem.2013, 82, 775-797); domain antibodies (dAb, derivatives of 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); dual affinity retargeted 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), 4336-4341). non-Ig-based scaffolds may be selected from, but are not limited to, antincalin (derivatives of Lipocalins) (Skerra a. Febs j.,2008, 275 (11), 2677-2683, beste G et al, proc.nat. Acad.1999, 96 (5), 1898-1903, skerra a. Biochim biophysis acta,2000, 1482 (1-2), 337-350 Skerra, a.curr Opin biotechnol.2007, 18 (4), 295-304 Skerra a. Febs j.2008, 275 (11), 2677-2683; adnectin (No. 10 FN3 (fibronectin)) (Koide a et al, j.mol. Biol,1998, 284 (4), 1141-1151; designed ankyrin repeat proteins (DARPins) (derivatives of Ankyrin Repeat (AR) proteins) (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.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; avimers (Domain A/Low Density Lipoprotein (LDL) receptor) (Boersma Y.L, J.biol.chem.2011, 286 (48), 41273-41285, silverman J et al, nat.Biotechnol 2005, 23 (12), 1556-1561.
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 (grlb), MBA conjugate), α v β 3 integrin receptor, cyclic RGD pentapeptide conjugate), LB14 (heterobivalent peptide ligand conjugate for VEGF receptor), LB15 (neuro interleukin B conjugate), LB16 (G protein receptor bombesin conjugate) and LB17 (tols T-like receptor T17 (tols receptor T-like conjugate)LR 2 A conjugate).
Figure BDA0003729824240000661
Figure BDA0003729824240000671
Figure BDA0003729824240000681
Figure BDA0003729824240000691
Figure BDA0003729824240000701
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 is a group of 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 BDA0003729824240000702
X 1 ，X 2 ，R 1 ，R 2 and R 3 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 will be appreciated that each of the drugs/cytotoxic agents described herein may be modified, the resulting compounds still retaining 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 (0 ppm) 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 BDA0003729824240000711
To 350mL of anhydrous tetrahydrofuran were added 80mg (0.0025 mol) of metallic sodium and diethylene glycol 83 (150.1g, 1.41mol) with stirring. After the sodium was completely dissolved, 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, the residue diluted with brine (250 mL) and ethyl acetateEster (3X 125 mL) was extracted. The combined organic layers were washed with water (100 mL) and brine (100 mL), dried over sodium sulfate, and the solvent was removed. The colorless oil obtained 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 BDA0003729824240000712
To a solution of 84 (10.0 g, 42.70mmol) in dichloromethane (50.0 mL) was added pyridine (20.0 mL). A solution of methanesulfonyl chloride (7.50g, 65.81mmol) in dichloromethane (50 mL) 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), calculated 313.10, found 313.30.
Example 3.3- (2- (2-azidoethoxy) ethoxy) propionic acid tert-butyl ester (86)
Figure BDA0003729824240000713
To 50mL of DMA were added, with stirring, 3- (2- (2- (methanesulfonyloxy) ethoxy) -propionic acid tert-butyl ester 85 (4.0 g, 12.81mmol) and sodium azide (0.90g, 13.84mmol). 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 (25 mL) 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 with 15% ethyl acetate in n-hexaneElution to pure ethyl acetate gave 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), calculated 260.13, found 260.20.
Example 4.3- (2- (2-azidoethoxy) ethoxy) propionic acid (87)
Figure BDA0003729824240000721
Azide 86 (2.51g, 9.68mmol) was dissolved in 1, 4-dioxane (30 mL) and 10mL concentrated HCl was added. The mixture was stirred for 35 minutes, diluted with EtOH (30 ml) and toluene (30 ml) and concentrated in vacuo. The crude product was purified on a 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 BDA0003729824240000722
To a solution of compound 87 (1.60g, 7.87mmol) in dichloromethane (30 mL) 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, 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 (1.93 g, 82% yield). ESI MS m/z + C 11 H 17 N 4 O 6 (M + H), calcd for 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) propanamido) -4-hydroxyphenyl) -2-methylpentanoic acid (94)
Figure BDA0003729824240000723
In the case 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-formylamino) -5- (3-amino-4-hydroxyphenyl) -2-methylpentanoic acid, 93 (Huang Y.et al, med Chem. #44,249, et al th ACS National Meeting, denver, CO, mar.22-26, 2015; WO 2014009774) (100mg, 0.131mmol) DMA (10 ml) and NaH 2 PO 4 88 (80.0 mg, 0.266mmol) was added to a mixture of buffers (5 ml,1.0M, pH 7.5) and added in four portions over two hours. The mixture was stirred overnight, concentrated and purified by C-18 preparative HPLC (3.0X 25 cm) eluting with 80% to 10% water/methanol (over 45 min, 25 ml/min) to give the title compound (101.5 mg, 82% yield). LC-MS (ESI) m/z C 45 H 70 N 9 O 11 S[M+H] + Calculated 944.48 and 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) propanamido) -4-hydroxyphenyl) -2-methylpentanoic acid (95)
Figure BDA0003729824240000731
In the hydrogenation reactor, pd/C (25mg, 10% Pd,50% wet) was added to a solution of compound 94 (100.0 mg, 0.106mmol) containing 0.1% HCl in methanol (25 ml). 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 (25 ml/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 BDA0003729824240000732
To a mixture of 4-hydroxy-3-methoxybenzoic acid (50.0 g,297.5 mmol) in ethanol (350 ml) and NaOH solution (2.0M, 350ml) was added benzyl bromide (140.0 g,823.5 mmol). The mixture was stirred at 65 ℃ for 8 h, concentrated and then co-concentrated with water (2X 400 ml) 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 (63.6 g, 83% yield). ESI MS M/z +281.2 (M + Na).
Example 9.4- (benzyloxy) -5-methoxy-2-nitrobenzoic acid
Figure BDA0003729824240000733
To a mixture of 4- (benzyloxy) -3-methoxybenzoic acid (63.5g, 246.0 mmol) in dichloromethane (400 ml) and HOAc (100 ml) was added fuming nitric acid (25.0 ml,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 (63.3 g,85% yield). ESI MS M/z +326.1 (M + Na).
Example 10 (2S, 4R) -4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride
Figure BDA0003729824240000741
Thionyl chloride (17mL, 231mmol) was added dropwise to a solution of trans-4-hydroxy-L-proline (15.0 g,114.3 mmol) in anhydrous methanol (250 mL) at 0-4 ℃. The resulting mixture was stirred at room temperature overnight, concentrated, and crystallized from ethanol/n-hexane to give the title compound (18.0 g,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 BDA0003729824240000742
To a mixture of methyl trans-4-hydroxy-L-proline (18.0g, 107.0mmol) in MeOH (150 ml) and sodium bicarbonate solution (2.0M, 350ml) was added (BOC) 2 O (30.0 g,137.6 mmol) 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 (100 mL), dried (magnesium sulfate), filtered, concentrated and passed through SiO 2 Column (1. ESI MS M/z +268.2 (M + Na).
EXAMPLE 12 (S) -1-methyl-4-oxopyrrolidine-12-dicarboxylic acid 1-tert-butyl ester
Figure BDA0003729824240000743
The title compound was prepared by Dess-Martin oxidation as follows: franco Manfre et al.J.org.chem.1992,57,2060-2065. Alternatively, the Swern oxidation can be carried out as follows: will cool to-78 ℃ (COCl) 2 To a solution of (13.0 mL, 74.38mmol) in dichloromethane (350 mL) 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-dicarboxylic acid ester (8.0 g, 32.63mmol) in methylene chloride (100 ml) was added. After stirring for 2 hours, triethylamine (50ml, 180.3mmol) was added dropwise and the solution was warmed to room temperature. Using NaH as mixture 2 PO 4 (400ml, 1.0M) the solution was diluted and the two phases were separated. The aqueous phase was extracted with dichloromethane (2X 60 ml) and the organic layers were combined, dried over magnesium sulphate, filtered, concentrated and passed over SiO 2 Purification by column (7. 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 BDA0003729824240000751
To a solution of methyltriphenylphosphonium bromide (19.62g, 55.11mmol) in tetrahydrofuran (150 mL) 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). After stirring at room temperature for 1 hour, the reaction mixture was concentrated, diluted with ethyl acetate (200 mL), washed with water (150 mL), brine (150 mL), dried over magnesium sulfate, and concentrated on SiO 2 Purification on a chromatography column (9. EIMS M/z +264 (M + Na).
EXAMPLE 14 (S) -4-methylpyrrolidine-2-carboxylic acid methyl ester
Figure BDA0003729824240000752
To a solution of (S) -1-methyl-4-methylenepyrrolidine-12-dicarboxylic acid 1-tert-butyl ester (5.70g, 23.63mmol) in ethyl acetate (40 ml) was added concentrated HCl (10 ml). The mixture was stirred for 1 h, diluted with toluene (50 ml), concentrated and crystallized from ethanol/n-hexane to give the title compound (3.85 g, 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 BDA0003729824240000753
A catalytic amount of DMF (30. Mu.l) was added to a solution of 4- (benzyloxy) -5-methoxy-2-nitrobenzoic acid (2.70g, 8.91mmol) and oxalyl chloride (2.0 mL, 22.50mmol) in anhydrous dichloromethane (70 mL), and the resulting mixture was stirred at room temperature for 2 hours. Excess dichloromethane and oxalyl chloride were removed in vacuo. Resuspending the acid chlorideIn fresh dichloromethane (70 mL) and added dropwise to 4-methylene-L-proline methyl ester hydrochloride (1.58g, 8.91mmol) in Et at 0 deg.C under argon 3 N (6 mL) 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 (40 mL), dried over magnesium sulfate, and concentrated. The residue was purified by flash chromatography on silica gel (2. EIMS M/z 449.1 ([ M ]] + +Na)。
Example 16 (S) -1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carbaldehyde
Figure BDA0003729824240000761
A solution of (S) -methyl 1- (4- (benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carboxylate (2.80g, 6.57mmol) in anhydrous dichloromethane (60 mL) was stirred vigorously at-78 deg.C, and DIBAL-H (10mL of 1M in dichloromethane) was added dropwise under an argon atmosphere. After stirring the mixture for an additional 90 minutes, the excess reagent was broken down by the addition of methanol (2 mL) 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 (2.19 g, 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 (11 aH) -one
Figure BDA0003729824240000762
Reacting (S) -1- (4-(benzyloxy) -5-methoxy-2-nitrobenzoyl) -4-methylenepyrrolidine-2-carbaldehyde (2.18g, 5.50mmol) and Na 2 S 2 O 4 (8.0 g, 45.97mmol) in tetrahydrofuran (60 ml) and H 2 The mixture in O (40 ml) was stirred at room temperature for 20 hours. The solvent was removed under high vacuum, the residue was resuspended in MeOH (60 mL), 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 brine were washed, dried over magnesium sulfate and concentrated. Chromatography on flash silica gel (97 3 MeOH) gave (S) -8- (benzyloxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-aza-5 (11 aH) -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 (11 aH) -one
Figure BDA0003729824240000763
To a solution of (S) -8- (benzyloxy) -7-methoxy-2-methylene-23-dihydro-1H-benzo e-pyrrolo 12-aza-5 (11 aH) -one (1.50g, 4.32mmol) in dichloromethane (70 ml) 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 60 ml) 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. 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 (11 aH) -one) (97)
Figure BDA0003729824240000771
To the stirred Cs 2 CO 3 To a suspension (8 ml) of (0.761g, 2.33mmol) in butanone was added (S) -8-hydroxy-7-methoxy-2-methylene-23-dihydro-1H-benzo-e-pyrrolo 12-. Alpha.14-diaza-5 (11 aH) -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 chromatography column eluting with ethyl acetate/dichloromethane (1. 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 (11 aH) -one (98)
Figure BDA0003729824240000772
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 (11 aH) -one) (150mg, 0.256 mmol) in anhydrous dichloromethane (1 mL) and anhydrous ethanol (1.5 mL) at 0 deg.C was added a solution of sodium borohydride in methoxyethyl ether (85. Mu.l, 0.5M, 0.042mmol). 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-. Alpha.14 diaza-5 (11 aH) -one (64.7 mg, 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) 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, 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 (11 aH) -one (99)
Figure BDA0003729824240000781
To a mixture of compound 98 (60.0 mg, 0.102mmol) and compound 88 (40.5 mg, 0.134mmol) in dichloromethane (5 ml) was added EDC (100.5 mg, 0.520mmol). Stirred at room temperature overnight, concentrated and concentrated in SiO 2 Purification on a chromatographic column eluting with ethyl acetate/dichloromethane (1. ESI MS m/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, 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 (11 aH) -one (100)
Figure BDA0003729824240000782
To compound 99 (60mg, 0.078mmol) in tetrahydrofuran (5 ml) and NaH 2 PO 4 PPh was added to a mixture of buffers (50mM, pH5.0, 1ml) 3 (70mg, 0.267mmol). The mixture was stirred at room temperature overnight, concentrated, and purified by C-18 preparative chromatographyUsing water/CH 3 CN (from 90% water to 35% water over 35 minutes) to yield 45.1mg (79% yield) of the title product 100 after drying on a high vacuum pump. 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 BDA0003729824240000791
Boc-L-proline (10.0 g,46.4 mmol) was dissolved in 50mL tetrahydrofuran, cooled to 0 deg.C, and BH was carefully added thereto 3 In tetrahydrofuran solution (1.0M, 46.4mL). 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 (50 mL), 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 BDA0003729824240000792
To a solution of tert-butyl (S) -2- (hydroxymethyl) pyrrolidine-1-carboxylate (13.0 g,64.6 mmol) in dimethyl sulfoxide (90 mL) was added triethylamine (40 mL), and 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 (250 g), the mixture was extracted with dichloromethane (150 mL. Times.3). The organic phase was washed with 50% citric acid solution (150 mL), water (150 mL), saturated sodium bicarbonate solution (150 mL) and brine (150 mL), dried over anhydrous sodium sulfate, and the solvent was removed in vacuo to give the title compound (10.4 g,81% yield) as a thick oil without Further purified and used directly. 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 BDA0003729824240000793
A solution of n-butyllithium in n-hexane (21.6 mL,2.2M, 47.43mmol) was added dropwise to a stirred solution of 4-methyl-5-phenyloxazolidin-2-one (8.0 g, 45.17mmol) in tetrahydrofuran (100 mL) at-78 ℃ under nitrogen blanket. The reaction was held at-78 ℃ for 1 hour, 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 the 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 (100 mL) and brine (100 mL), 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 BDA0003729824240000801
At 0 ο To a solution of (4R, 5S) -4-methyl-5-phenyl-3-propionyloxyoxazolidin-2-one (9.40g, 40.4 mmol) in dichloromethane (60 mL) was added Et 3 N (6.45mL, 46.64mmol) and then 1M dibutylboron triflate in dichloromethane (42mL, 42mmol) was added. Mixing the componentsThe 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 (40 mL) was added slowly (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, 38mL). Adding MeOH-30% at 10 ℃ 2 O 2 (2, 1,100ml), stirred for 20 minutes, then water (100 mL) was added and the mixture was concentrated in vacuo. More water (200 mL) was added to the residue and extracted with ethyl acetate (3X 100 mL). The organic layer was washed with 1N KHSO 4 (100 mL), sodium bicarbonate solution (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (10% -50% ethyl acetate/n-hexane) 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 BDA0003729824240000802
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 (1.9 mmol) under nitrogen
Figure BDA0003729824240000803
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, 30.9 mmol) was added followed by trimethyl oxonium tetrafluoroborate (4.40g, 29.7 mmol). Stirring at 0 deg.C for 2 hr, stirring at room temperature for 48 hrThen (c) is performed. 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 BDA0003729824240000811
To 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 (1.80g, 4.03mmol) at 0 ℃ in tetrahydrofuran (30 mL) and H 2 Adding 30% H to the solution in O (7.5 mL) 2 O 2 (1.44mL, 14.4 mmol) (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 (15.7 mL) 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 50 mL) 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 (50 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound as a colorless oil (1.15 g, 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 (4S, 5S) -4- ((tert-butoxycarbonyl) amino) -5-methyl-3-oxoheptanoic acid ethyl ester
Figure BDA0003729824240000812
To (2S, 3S) -2- ((tert-butyl)Butoxycarbonyl) amino) -3-methyl-pentanoic acid (4.55g, 19.67mmol) in tetrahydrofuran (20 mL) in ice was added 1,1' -carbonyldiimidazole (3.51g, 21.63mmol). After the evolution of gas had ceased, the resulting mixture was stirred at room temperature for 3.5 hours. A solution of freshly prepared isopropyl magnesium bromide in tetrahydrofuran (123mmol, 30mL) was added dropwise to pre-cooled (0 ℃ C.) ethyl hydrogen malonate (6.50g, 49.2 mmol) maintaining the internal temperature below 5 ℃. The mixture was stirred at room temperature for 1.5 hours, then the magnesium enolate solution was cooled in an ice-water bath, followed by gradual addition of the imidazolide solution over 1 hour via a double-ended needle. The 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 (5 mL) 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 (50 mL), saturated aqueous sodium bicarbonate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography using ethyl acetate/n-hexane (1. 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 (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid ethyl ester
Figure BDA0003729824240000821
To a solution of ethyl (4S, 5S) -4- ((tert-butoxycarbonyl) amino) -5-methyl-3-oxoheptanoate (5.90g, 19.83mmol) in ethanol (6 mL) at-60 ℃ was added sodium borohydride (3.77g, 99.2mmol). The reaction mixture was stirred at-55 ℃ or lower for 5.5 hours, then quenched with 10% aqueous citric acid (100 mL), adjusted to pH 2, and extracted with ethyl acetate (100 mL. Times.3). The organic extracts were washed with saturated aqueous sodium chloride (100 mL), dried over sodium sulfate and concentrated in vacuo. Column chromatography (1)0-50% ethyl acetate/n-hexane) to obtain pure diastereomers (3r, 4s, 5s) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid ethyl ester (2.20 g, 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 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 BDA0003729824240000822
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 (22 mL) was added a 1N aqueous sodium hydroxide solution (7.57mL, 7.57mmol). 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 adding 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 (50 mL) and saturated aqueous sodium chloride (50 mL), 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 BDA0003729824240000831
To a solution of (3R, 4S, 5S) -4- ((tert-butoxycarbonyl) amino) -3-hydroxy-5-methyl-heptanoic acid (1.90g, 6.9mmol) in tetrahydrofuran (40 mL) was added sodium hydride (60 wt%,1.93g, 48.3mmol). After stirring for 1 hour, iodomethane (6.6 mL,103.5 mmol) was added. Stirring was continued for 40 h at 0 deg.C, then saturated aqueous sodium bicarbonate (50 mL) and water (100 mL) were added. The mixture was washed with diethyl ether (50 mL. Times.2), and the aqueous layer was acidified to pH3 with a 1N aqueous solution of potassium hydrogensulfate, followed by extraction with ethyl acetate (50 mL. Times.3). The combined organic extracts were washed with 5% aqueous sodium thiosulfate (50 mL) and saturated aqueous sodium chloride (50 mL), dried over sodium sulfate, and concentrated in vacuo to give the title compound (1.00 g, 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 Boc functional group removal with trifluoroacetic acid
To a solution of N-Boc amino acid (1.0 mmol) in dichloromethane (2.5 mL) 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-phenylprop-2-yl) amino) -2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate
Figure BDA0003729824240000832
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 (5 mL) was added diethyl cyanophosphonate (75.6. Mu.L, 0.451 mmol), 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 is treated with ethyl acetateThe ester (80 mL) was diluted, washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium bicarbonate (40 mL) and saturated aqueous sodium chloride (40 mL), 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 BDA0003729824240000841
To a mixed solution of (S) -2- ((1R, 2R) -1-methoxy-3- (((S) -1-methoxy-1-oxo-3-phenylpropan-2-yl) (0.29 mmol)) 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 (5 mL) was added diethyl cyanophosphonate (58. Mu.L, 0.347 mmol), 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 (80 mL), washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium bicarbonate (40 mL), and saturated aqueous sodium chloride (40 mL), 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 value 634.40, found 634.40.
EXAMPLE 36 (S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2-amino) -N, 3-dimethylbutyramido) -3-methoxy-5-methylheptyl) -pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropan
Figure BDA0003729824240000842
To a solution of ((S) -1- ((3R, 4S, 5S) -4- ((tert-butoxycarbonyl) - (methyl) amino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid methyl ester (0.118 mmol) and Boc-Val-OH (51.8mg, 0.236 mmol) in DCM (5 mL) 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 (80 mL), washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium bicarbonate (40 mL), and saturated aqueous sodium chloride (40 mL), 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 and found 733.46.
EXAMPLE 37 preparation of the compound Boc-N-Me-Val-OH
Figure BDA0003729824240000851
To a solution of Boc-L-Val-OH (2.00g, 9.2mmol) and iodomethane (5.74mL, 92mmol) in anhydrous tetrahydrofuran (40 mL) 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 (50 mL), water (100 mL) 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, 5,11 tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triazadec-15-acyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropionate
Figure BDA0003729824240000852
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 (0.091 mmol) and Boc-N-Me-Val-OH (127mg, 0.548mmol) in DMF (5 mL) was added diethyl cyanophosphonate (18.2. Mu.L, 0.114 mmol) 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 (80 mL), washed with 1N aqueous potassium hydrogensulfate (40 mL), water (40 mL), saturated aqueous sodium bicarbonate (40 mL), and saturated aqueous sodium chloride (40 mL), 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, 5, 11-tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triazadecano-15-acyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid
Figure BDA0003729824240000861
To a solution of (S) -2- ((2R, 3R) -3- ((S) -1- ((6S, 9S,12S, 13R) -12- ((S) -sec-butyl) diisopropyl-13-methoxy-2, 5, 11-tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triazacyclo-15-acyl) pyrrolidin-2-yl) -3- (1-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid methyl ester (30mg, 0.035mmol) in tetrahydrofuran (1.0 ml) was added an aqueous LiOH solution (1.0M, 0.8ml), the mixture was stirred at room temperature for 35 minutes, and 0.5M H 3 PO 4 Neutralized to pH 6, concentrated and concentrated in SiO 2 Purification on column with CH 3 OH/dichloromethane/HOAc (1. LC-MS (ESI) m/z C 44 H 74 N 5 O 10 [M+H] + Calcd for 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 BDA0003729824240000862
A mixture of (S) -2- ((2R, 3R) -3- ((S) -1- ((6S, 9S,12S, 13R) -12- ((S) -sec-butyl) -69-diisopropyl-13 methoxy-2, 5, 11-tetramethyl-4, 7, 10-trioxo-3-oxa-5, 8, 11-triaza-decan-15-yl) pyrrolidin-2-yl) -3-2-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid (25mg, 0.030mmol), concentrated HCl (0.3 ml) and 1, 4-dioxane (0.9 ml) was stirred at room temperature for 35 minutes, then diluted with EtOH (1.0 ml) and toluene (1.0 ml), and after concentration diluted with ethanol/toluene (2 1) and concentrated to give the title compound as a white solid (22 mg,. About.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 and 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-eicosa-20-oyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid
Figure BDA0003729824240000871
To a crude product (22mg, 0.030mmol) of Compound 101, DMA (0.8 ml) and NaH 2 PO 4 Compound 88 (18.0mg, 0.060mmol) was added to a mixed solution of a buffer (0.7ml, 1.0M, pH 7.5) 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. 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-triaza-icosa-20 acyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid (103)
Figure BDA0003729824240000872
In the hydrogenation reactor, pd/C (5mg, 10% Pd,50% wet) was added to a solution of compound 102 (22.0 mg, 0.024mmol) in methanol (5 ml). 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 BDA0003729824240000873
To a solution of 2.3-dibromosuccinic acid (10.00g, 36.51mmol) in anhydrous dichloromethane (100 ml) 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, through short SiO 2 The column was filtered and the column was washed with ethyl acetate/dichloromethane (1. 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), calcd.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 BDA0003729824240000881
DIPEA (12uL, 0.069mmol) and 2, 3-dibromosuccinic anhydride (38.0mg, 0.148mmol) were added to a solution of compound 100 (40.0mg, 0.068mmol) in DCM (4 ml) at 0 ℃. 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. LC-MS (ESI) m/z C 44 H 53 Br 2 N 5 O 12 [M+H] + Calcd for 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 BDA0003729824240000882
To a solution of compound 125 (55.0mg, 0.054mmol) in dichloromethane (3 ml) were added NHS (10.0mg, 0.086mmol) and EDC (30.5mg, 0.158mmol). The mixture was stirred at room temperature overnight, concentrated and concentrated on SiO 2 Purification on column eluting with ethyl acetate/dichloromethane (1). LC-MS (ESI) m/z C 48 H 56 Br 2 N 6 O 14 [M+H] + Calcd 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- α 14 diaza-8-yl) oxy) pentyl) oxy) -2-methylene-5-oxo-2, 3, 11, 11a tetrahydro-1H-benzo e-pyrrolo 12- α 14 diaza-10 (5H) -yl) -2531 dimethyl 1, 11, 14, 24, 27, 30-hexyloxy-4, 7, 18, 21-tetraoxa-10, 15, 25, 28, 31-pentaaza-thirty-penta-35 acyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropionic acid (126)
Figure BDA0003729824240000891
DMA (1 ml) and NaH in Compound 103 (. About.20mg, 0.022mmol) 2 PO 4 Compound 125 (30.0mg, 0.027mmol) was added to a mixture of the buffer (0.6ml, 0.15M, pH 7.5). After stirring for 7 h, concentration and purification by C-18HPLC (. PHI.2.0cm. Times.25cm), eluting with water/acetonitrile (10 ml/min flow rate, from 90% water to 15% water in 50 min), the title product 126 was obtained after drying on high vacuum pump (26.1 mg, 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 BDA0003729824240000892
To 2.0mL of 10mg/ml of a solution of herceptin of pH7.0 to 8.0, 0.70 to 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 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 50mM NaCl pH 6.0-7.5 to obtain 16.5-17.7 mg of conjugate 127 (in 13.1-15.0 ml buffer, 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. SEC HPLC (Tosoh Bioscience, tskgel G3000SW,7.8mm ID x 30cm,0.5ml/min,100 min) analysis gave a monomer content of 96-99%, with the SDS-PAGE gels detected as single bands.
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-2, 3, 11, 11a tetrahydro-1H-benzo-e-pyrrolo 12-. Alpha.14 diaza-10 (5H) -yl) -11, 14, 24-trioxo-4, 7, 18, 21-tetraoxa-10, 15-diaza tetracarboxamido) -4-hydroxyphenyl) -2-methylpentanoic acid (128)
Figure BDA0003729824240000901
Compound 95 (20mg, 0.021mmol) was added to DMA (1 ml) and NaH 2 PO 4 Compound 125 (30.0mg, 0.027mmol) was added to a mixture of the buffer (0.6ml, 0.15M, pH 7.5). The mixture was stirred for 8 hours and concentratedAnd purified by C-18 preparative chromatography (. Phi.2.0cm. Times.25cm), eluting with water/acetonitrile (flow rate 10 ml/min, from 90% water to 20% water over 50 min), to give the title product 128 (26.6 mg, 64% yield) after drying on a 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 to an antibody preparation 129
Figure BDA0003729824240000902
To 2.0mL of 10mg/ml of the Herceptin solution pH7.0-8.0, 0.70-2.0mL of 100mM NaH was added 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 incubation overnight at room temperature, the mixture was purified on a G-25 column with 100mM NaH 2 PO 4 50mM NaCl pH6.0-7.5 to obtain 16.4-17.6 mg of conjugate 129 (in 13.4-15.1 ml of buffer, 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,100 min) analysis of its monomer content 96-99%, SDS-PAGE gel measurement shows a single band.
Example 50 bis (25-dioxopyrrolidin-1-yl) 23-dibromosuccinate (9)
Figure BDA0003729824240000903
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 (100 ml) was stirred at room temperature overnight, concentrated and concentrated in SiO 2 Purification on a column eluting with ethyl acetate/dichloromethane (1). LC-MS (E)SI)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-tetracosanodiol-1, 24-diacyl) bis (azanediyl)) bis (4-hydroxy-31-phenylene)) -bis (4- (2- ((1R, 3R) -1-acetoxy ((S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) -4-methylpentyl) thiazole-4-carboxamido) -2-methylpentanoic acid) (141)
Figure BDA0003729824240000911
DMA (1 ml) and NaH in Compound 95 (40mg, 0.042mmol) 2 PO 4 Di (2, 5-dioxopyrrolidin-1-yl) -2, 3-dibromosuccinate (9) (18.0 mg, 0.038mmol) was added to a mixed solution of a buffer (0.6 ml,0.15M, pH 7.5). The mixture was stirred for 8 h, concentrated and purified by C-18 preparative chromatography (. Phi.2.0 cm x 25cm) eluting with water/acetonitrile (flow rate of 10ml/min, from 90% water to 20% water over 90 min) to afford the title product 141 (38.5 mg,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), calcd for 2073.81, found 2073.60.
EXAMPLE 52 conjugation of Compound 141 to an antibody preparation 142
Figure BDA0003729824240000912
To 2.0mL of 10mg/ml of the herceptin solution having a pH of 7.0 to 8.0, 0.70 to 2.0mL of 100mM NaH was added 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 Elution with a 50mM NaCl buffer solution having a pH of 6.0 to 7.5 gives 16.4 to 17.6mg of conjugate 142 (in 13.4 to 15.2ml of buffer solution, 82% to 88% yield). The drug/antibody ratio (DAR) 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,100 min) analysis showed a monomer content of 96-99%, and SDS-PAGE gels showed single bands.
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, a human invasive ductal cancer cell line, and SKOV3, a human ovarian cancer cell line. For HL-60, NCI-N87 and BT-474 cells, these cells were grown in RPMI-1640 medium containing 10% FBS. For SKOV3 cells, the cells were grown in McCoy 5A medium containing 10% by weight 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 (5 mg/mL) was added thereto and 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- (analytical value-blank control value)/(control value-blank control value) ] × 100%
Cytotoxicity results:
Figure BDA0003729824240000921
the specificity of conjugate 127 for N87 cells exceeded 889 (IC) 50 >8/IC 5 0= 0.009), and more than 800 for SK-OV-3 cells; the specificity of conjugate 129 on N87 cells exceeded 666 (IC) 50 >8/IC 50 ＝0.012)，More than 533 for SK-OV-3 cells; the specificity of the conjugate 142 for 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-DM1.
Example 54 in vivo antitumor Activity
The in vivo efficacy of conjugates 127, 129 and 142 with T-DM1 was evaluated on a human gastric carcinoma N-87 cell line tumor xenograft model. Five-week-old female BALB/c nude mice (30 animals) were inoculated subcutaneously with 0.1mL of N-87 cancer cells (5X 10) in serum-free medium 6 Individual cells/mouse). The tumor grows for 8 days to reach 133mm 3 Average size of (a). 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 =1/2 (length × width × height) tumor volume was calculated. 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. Animals in the control group had a tumor volume greater than 1500mm 3 While the illness was killed on day 37. All 6/6 animals in the group of compounds 127 and 129 had no measurable tumor at all from day 13 to day 60 (end of experiment). No measurable tumor was observed on day 21 in all 6/6 animals of the group with compound 142, and tumor growth was (measurable) observed in 2/6 animals on 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 (33)

1. A bridge connector of the structure shown in (I):
Figure FDA0003729824230000011
wherein:
-is an optional single bond;
Figure FDA0003729824230000012
is a single bond or a double bond;
when the temperature is higher than the set temperature
Figure FDA0003729824230000013
When it is a single bond, U and U' are not H, when
Figure FDA0003729824230000014
When the double bond is one, U or U' can be H, but is not H at the same time;
Figure FDA0003729824230000015
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;
R 1 and R 2 Are identical, different or default straight-chain alkyl groups having from 1 to 6 carbon atoms, branched or cyclic alkyl groups having from 3 to 6 carbon atoms, straight-chain, branched or cyclic alkenyl or alkynyl groups, or ester, ether, amide or polyethoxy (OCH) groups having from 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, most preferably 0 to 500 atoms, covalently bonded to 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.
Z 1 And Z 2 Are the same or different functional groups capable of reacting with cytotoxic drugs to form disulfide, ether, ester, thioether, thioester, peptide, hydrazone, carbamate, carbonate, amine (secondary, tertiary or quaternary), imine, heterocycloalkyl, heteroaryl, alkoxyoxime or amide bonds having the following structure:
Figure FDA0003729824230000021
n-hydroxysuccinimidylAn amine ester;
Figure FDA0003729824230000022
a maleimide group;
Figure FDA0003729824230000023
a disulfide;
Figure FDA0003729824230000024
a haloacetyl group;
Figure FDA0003729824230000025
an acid halide or ester;
Figure FDA0003729824230000026
a vinyl sulfonyl group;
Figure FDA0003729824230000027
an acryloyl group;
Figure FDA0003729824230000028
2- (p-toluenesulfonyloxy) acetyl;
Figure FDA0003729824230000029
2- (methylsulfonyloxy) acetyl;
Figure FDA00037298242300000210
2- (nitrophenoxy) acetyl;
Figure FDA00037298242300000211
2- (dinitrophenoxy) acetyl;
Figure FDA00037298242300000212
2- (fluorophenoxy) acetyl;
Figure FDA00037298242300000213
2- (difluorophenoxy) ethaneAn acyl group;
Figure FDA00037298242300000214
2- (pentafluorophenoxy) acetyl;
Figure FDA00037298242300000215
a ketone or an aldehyde;
Figure FDA00037298242300000216
2- (trifluoromethanesulfonyloxy) acetyl;
Figure FDA00037298242300000217
oxadiazole phenyl methane sulfonate (ODA);
Figure FDA00037298242300000218
An acid anhydride;
Figure FDA00037298242300000219
an alkoxyamino group;
Figure FDA00037298242300000220
nitrine;
Figure FDA00037298242300000221
alkyne or
Figure FDA00037298242300000222
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 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 groups; leaving groups include nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenolPhenol, pentachlorophenol, trifluoromethylsulfonic 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 such as acetic anhydride, formic anhydride, or polypeptide condensation or Mitsunobu reaction intermediates.
2. A cell-binding agent-drug conjugate having the structure of formula (II):
Figure FDA0003729824230000031
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 Are the same or different cytotoxic agents that are linked to the cell binding agent by a bridge 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 linkage, and combinations thereof.
n is 1 to 30; "-",
Figure FDA0003729824230000032
R 1 ，R 2 ，X 1 and X 2 Is as defined in claim 1.
3. A compound having the structure of formula (III):
Figure FDA0003729824230000033
wherein the molar ratio of the molar ratio to the molar ratio of the molar ratio to the molar ratio,
Figure FDA0003729824230000034
Cb,Z 1 ,Z 2 ,n,R 1 ,R 2 ,X 1 and X 2 As defined in claims 1 and 2.
4. A compound having the structure of formula (IV):
Figure FDA0003729824230000035
wherein "-",
Figure FDA0003729824230000036
U,U’,Drug 1 ,Drug 2 ,R 1 ,R 2 ,X 1 and X 2 As defined in claims 1 and 2.
5. In claim 1, the bridge linker having the structure of formula (I), wherein the 2, 3-disubstituted succinic acid group, the 2-monosubstituted or 2, 3-disubstituted fumaric acid group, the 2-monosubstituted or 2, 3-disubstituted maleic acid group is produced by direct condensation of the 2, 3-disubstituted succinic acid, the 2-monosubstituted or 2, 3-disubstituted fumaric acid, the 2-monosubstituted or 2, 3-disubstituted maleic acid or a derivative thereof and an amine (primary or secondary amine), alcohol or mercapto group at the terminal of the other component, as shown in (Ia):
Figure FDA0003729824230000041
Wherein X in formula (Ia) is X in claim 1 1 Or X 2 Is NH, N (R) 3 ) O, or S; r is as in claim 1R of (A) to (B) 1 And/or R 2 ；R 1 ，R 2 And R 3 Is also defined in claim 1.
Lv1 and Lv2 are identical or independent of one another OH, F, cl, br, I, 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, an anhydride formed from an anhydride with another acid such as 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 (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-oxytriazololylalkyl phosphate (PyBOP), diethyl pyrocarbonate (DEPC), N, N, N ', N ' -tetramethylchloroamidine hexafluorophosphate, 2- (7-oxybenzotriazole) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU), 1- [ (dimethylamine) (morpholinyl) methylenecarbodiimide), N ' -methylenecarbodiimide, N ' -tetramethyluronium hexafluorophosphate ]-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-pyridon-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TPTU), thio- (1-oxo-2-pyridinyl) -N, N, N ', N' -tetramethylthiouronium hexafluorophosphate, oxy- [ (ethoxycarbonyl) cyanomethylamine]-N, N '-tetramethylthiourea hexafluorophosphate (hotsu), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholine-carbonium hexafluorophosphate (COMU), (benzotriazol-1-yloxy) dipyrrolidine carbohexafluorophosphate (HBPyU), N-benzyl-N' -cyclohexylcarbodiimide (or supportedOn the polymer), dipyrrolidinyl (N-succinimidyloxy) carbonium hexafluorophosphate (HSPyU), 1- (chloro-1-pyrrolidinylmethylene) pyrrolidinium hexafluorophosphate (PyClU), 2-chloro-1, 3-dimethylimidazolium tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipiperidino hexafluorophosphate (HBPipU), 6-chlorobenzotriazole-1, 3-tetramethyluronium tetrafluoroborate (TCTU), tris (dimethylamino) phosphonium bromide hexafluorophosphate (BroP), 1-N-propylphosphoric anhydride (PPACA,
Figure FDA0003729824230000042
) 2-isocyanoethylmorpholine (MEI), N' -tetramethylurea-oxy- (N-succinimidyl) Hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy- [ (ethoxycarbonyl) cyanomethylamine]N, N, N ', N' -tetramethylthionurebetrafluoroborate (TOTU), 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl-1, 3-tetramethyluronium tetrafluoroborate (TSTU), N, N, N ', 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).
6. In claims 2 and 4, drug in formulas (II) and (IV) 1 And Drug 2 Identical or each independently selected from:
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, mechlorethamine, benzene mustard cholesterol, prednimustine, thiotepa, tricresyl, uracil; CC-1065 (including its aldorexin, kazelaixin and bizelaixin synthetic analogs); duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI); benzodiazepine dimers (e.g. Pyrrolobenzodiazepine (PBD) or tomimecin, indolonobenzodiazepine, imidazobenzothiadiazole or oxazole) Dimers of the alkylbenzodiazepines); nitrosoureas (carmustine, lomustine, fustin chloride, fotemustine, nimustine, lamustine); 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 alkaloids such as vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, catharanthine); taxoids (paclitaxel, docetaxel) and their analogs; maytansinoids (DM 1, DM2, DM3, DM4, maytansine and ansamycin) and analogs thereof; cryptophycins (especially cryptophycin 1 and cryptophycin 8); epothilone, sarcodictol, discodermolide, bryostatin, dolastatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictyin, spongistatin; c) DNA topoisomerase inhibitors, such as etoposide tinib (9-aminocamptothecin, camptothecin, clinatot, daunomycin, etoposide phosphate, irinotecan, mitoxantrone, nosaline, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycin (mitomycin C); d) Antimetabolites, such as antifolates, DHFR inhibitors (methotrexate, trametes, denormaldehyde, pteropterin, aminopterin (4-aminobenzoic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazolufrin, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine analogues, uracil analogues (ancitabine, azacitidine, 6-azauracil, capecitabine (hiloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (Tomudex), cytosine analogues (cytarabine, cytosine arabinoside, fludarabine), purine analogues (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine), folic acid supplements, such as florolinic acid (ii) a e) Hormonal therapy agents such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (gostadrine, leuprolide acetate); anti-androgens (bicalutamide, flutamide, carrousel, betaandrosterone propionate, epiandrosterone, goserelin, leuprorelin, metulidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 analogues (CB 1093, 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 245634), bafetinib (INNO-406), bosutinib (SKI-606), cabotinib, vesukib, iniparib, ruxolitinib, CYT387, axitinib, tivozab, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, isnext; g) Antibiotics, such as enediynes antibiotics (calicheamicins, in particular calicheamicin γ 1, δ 1, α 1 and β 1 (see J.Med.chem.1996,39 (11), 2103-2117, angew Chem Intl.Ed.Engl.1994, 33-186), dynemycins, including dynemycin A and deoxymithramycin, esperamycin, cadomycin, C-1027, maduropeptin and neocarcinomycin chromophore and related chromoprotein enediynin antibiotics chromophores), acacinomyins, actinomycin, azaserine, bleomycin, carnomycin, clarithromycin, carminomycin, carvacrol, doxorubicin, morpholino doxorubicin, 2-pyrroline doxorubicin and doxorubicin, epirubicin, aclarubicin, idarubicin, marcomycin, mycophenolic acid, lophenicol, pelomycin, pellomycin, puromycin, triiron doxorubicin, streptozotocin, tubercidin, ubenimex, setastin, zorubicin (ii) a h) Others, such as polyketides (annonaceous acetogenins), in particular bullatacin and bullatacinone; gemcitabine, epoxygenases (e.g., capeline), bortezomib, thalidomide, lenalidomide, pomidomide, tosedostat, zybrestat, PLX4032, STA-9090, stimuvax, allovivin-7, xegeva, provenge, yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., staurosporins), actinomycins (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, pelomycin), anthracyclines (e.g., daunorubicin), doxorubicin (e.g., 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 (MGCD 0103), belinostat, PCI-24781, entinostat, SB939, resminostat, 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 FDA0003729824230000061
Piperazinedione propane; rhizomycin; (iv) Wenzuo; spiro germanium; geobacillus azavor; a tri-imine quinone; trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucomicin A, bacillocin A and anguidine), polyurethanes, siRNA and antisense drugs.
2) Autoimmune disease drugs: cyclosporine, cyclosporin a, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluconazole, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus.
3) Anti-infectious disease drugs: a) Aminoglycosides: amikacin, astemicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylgestomycin; b) Amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) Ansamycin: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) Cephem: cephem (loracarbef), cephalosporins, chlorammonicillin, cephradine, cefadroxil, cephalonine, ceftazidime, cephalothin or cephalotaxin, cephalexin, cefalexin, cefamandole, cefapirin, hydroxylamine azole cephalosporin, fluxazole cephalosporin, sporoximone, oxazoline cephalosporin, cefbuperazone, cefcapene, cefixime, cefpodoxime, cefixime, cefprozil, cefetamet, ceftezole, cefuroxime, cefdinir, cefditoren, cefpirazole, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, tiamidozole cephalosporin, cefozopran, cephalexin, cefimidazole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, cefbutam, cefotiarin, ceftizoxime, ceftriaxone, cefuroxime, ceftizoxime, cephamycin (cefoxitin, cefotetan, cefcyanazole), cefixime (flomoxef, cephalosporine); 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, freramycin, josamycin, ketolide (telithromycin, seithromycin), midecamycin, mickamycin, oleandomycin, rifamycin (isoniazid, rifampin, rifabutin, rifapentine), ropiniromycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK 506), oleandomycin acetate, telithromycin; l) monocyclic amines: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, herocillin, bacampicillin, ampicillin, doxorubicin), oxacillin, azlocillin, benzylpenicillin, benzathine phenoxymethylpenicillin, cloxacillin, procaine penicillin (metilin), mezlocillin, methicillin, nafcillin, oxacillin, acemethicillin, penicillin, nafcillin, 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, carnotrexacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) streptogramins: pristinamycin, quinupristin/dalfopristin, r) sulfonamides: sulfonamide drugs: sulfonamides, sulfadiazine, sulfasalazine, sulfisoxazole, tamoxifen, trimethoprim-sulfamethoxazole (sulfamethoxazole); s) steroid antibacterial agents: 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: annonacin, arsinamine, bactoprenol inhibitor (bacilli), DANAL/AR inhibitor (cycloserine), dictyostatin, discodermolide, junceella alcohol, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimnalide, metronidazole, mupirocin, NAM synthesis inhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, placido, pyrazinamide, quinupristin/dalfopristin, rifampin (rifampin), tazobactam tinidazole, echinaconine.
4) Antiviral drugs: a) Entry/fusion inhibitors: apavirlo, 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, aximavir, adefovir, armocivir, abciximab, brivudine, cidofovir, clevudine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, flurocidine (5-FU), 3 '-fluoro-substituted 2',3 '-deoxynucleoside analogs (such as 3,3' -fluoro-2 ',3' -dideoxythymidine (FLT) and 3 '-fluoro-2', 3 '-dideoxyguanosine (FLG), fomivirsen, 9-guanine, idoxuridine, lamivudine (3 TC), 1-nucleosides (e.g., β -1-thymidine and β -1-2' -deoxycytidine), penciclovir, racivir, ribavirin, digividine, stavudine (d 4T), talibarine (viradine), telbivudine, tenofovir, trifluzumab, ganciclovir (azzid), zivudine (azx, zivudine: amantadine, atitidine, carboplatin, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, peginterferon, lovirdine, lodenosine, methidazone, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod (R-848), acetimidamantadine; g) Protease inhibitors: amprenavir atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pratensairi, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other types of antiviral drugs: antioxidase, arbidol, canola, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, priconalide, anabolic inhibitor, ribavirin, seliciclib.
5) A radioisotope (radionuclide) 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 or 213 Bi。
6) A chromonic molecule capable of absorbing a light, such as ultraviolet, fluorescent, infrared, near infrared, or visible light; yellow pigment, red blood cell, iridescent pigment, white blood cell, melanin and blue-green pigment, fluorescent molecule (fluorescent chemical substance which absorbs light and emits light), visual light transduction molecule, photon molecule, luminescence molecule and fluorescein compound; 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 (porphine, phthalocyanine, bilirubin); analogs and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioS-tatus), BODIPY (Invitrogen), alexa Fluor (Invitrogen), dyLight Fluor (Thermoscientific, pierce), atto and Tracy (Sigma Aldrich), fluoProbes (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, phyobisomes) (Columbia Biosciences), APCXL, RPE, BPE (Phyco-Bioh), allophycocyanin (Biotecp), BODIPC-7 APC, bioCy-Cy-7, blue Cy-Cy 3, bluey-Cy-3, 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, setau-380-NHS, setau-405-maleimide, setau-405-NHS, setau-425-NHS, setau-647-NHS, tex Red, TRITC, truda, rhodamine, X-AAD, 7-AAD (7-Aminomycin), acridine-CG, acridine-3A-selective amino-CG, 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, pH 7.2), 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), GFv, hcRed1, J-Red, katusha, kusabira Orange (monomer, MBL), mFP, mCheerz (monomer, MBL), kmFP, kjemFP, kemIressa, monomer (monomer, kemIrmor), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer, tsien laboratories), mStrawberry, mTFP1, mTurquose 2, P3 (phycobilisome complex), polymethyleneflavin-chlorophyll-protein complex (PerCP), R-phytoerythrin (RPE), T-Sapphire, tagCFP (dimer, evagen), tagGFP (dimer, evagen), tagFP (dimer, evagen), tagYFP (dimer, evagen), topalto (tandem dimer), topaz, turboFP602 (dimer, evagen 635), turboFPP (dimer, evagen), turboGFP (dimer, evagen), turboRFP (dimer, evagen), venus, wild type YPeS, zonet 1 (CloGFP), zsYellow1 (tetramer, clontech).
7) Pharmaceutically acceptable salts, acids or derivatives thereof of the above drugs.
7. In claims 2 and 4, drug in formulas (II) and (IV) 1 And Drug 2 For chromogenic molecules, the conjugates of formulae (II) and (IV) can be used to detect, monitor or study the function of a cell-binding molecule, the interaction with a target cell, and/or the interaction of the conjugate with a target, particularly a target cell.
8. In the context of claims 2 and 4,drug in formulas (II) and (IV) 1 And Drug 2 Is a polyalkylene glycol or polyalkylene glycol homologue, which when administered to a mammal increases the half-life of the cell-binding agent. Polyalkylene glycols include polyethylene glycol (PEG), polypropylene glycol and copolymers of ethylene oxide and propylene oxide having a molecular weight of between 10Da and 200 kDa.
9. In claims 2 and 4, the Drug in formulas (II) and (IV) 1 And Drug 2 Is a cell-bound ligand or receptor analog. Conjugates of these cell-binding ligands or receptors can be used not only as targeting vectors/guides to deliver the conjugates to malignant cells, but also to modulate or co-stimulate a desired immune response or alter signaling pathways.
10. A Drug on a conjugate as claimed in claims 2 and 4 1 And Drug 2 Preferably, the therapeutic agent is selected from the group consisting of tubulysin, calicheamicin, auristatin, maytansinoids, CC-1065 analogs, daunorubicin and doxorubicin compounds, taxanes (taxanes), cryptophycin, epothilones, benzodiazepines dimers (e.g., pyrrolobenzodiazepine (PBD), tolmetycin, antromycin, indolophenyldiazepine, imidazobenzothiadiazepine or oxazolidobenzodiazepine dimers), calicheamicins and enediynes antibiotics, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin/auricle (e.g., MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, auristatin 6-AQ, auristatin (AEB) and Auristatin EFP (AEFP)), doxycycline, vincristine, hemistatin, zeamine, auristatin 6-AQ, auristatin, theocin, derivatives thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable derivatives thereof.
11. The cell binding agent/molecule of claims 2 and 3 selected from the group consisting of antibodies, proteins, vitamins (e.g., folic acid), peptides, polymeric micelles, liposomes, lipoprotein-based drug carriers, nanoparticle drug carriers, dendrimers, and coated with cell binding ligands or combinations thereof.
12. The cell binding agent/molecule of claims 2, 3 and 11 is preferably an antibody, a whole antibody (polyclonal antibody, monoclonal antibody, dimer, multimer, multispecific antibody, e.g. bispecific or trispecific antibody); a single chain antibody; an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody or a monoclonal antibody fragment that binds to a target cell, a chimeric antibody fragment that binds to a target cell, a single domain antibody fragment that binds to a target cell, a surface modified antibody, a single chain surface modified antibody, or a surface modified antibody fragment that binds to a target cell, a humanized antibody, a single chain humanized antibody, or a humanized antibody fragment that binds to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient-transporting molecule or a large molecular weight protein.
13. The cell-binding agent/molecule of claims 2, 3 and 11 may be any 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.
14. The cell binding agent/molecule of claims 2, 3 and 11 may be any drug/molecule capable of acting against one of the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11B, CD11C, CD12w, CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49B, CD49C, CD51, CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79a, CD79B, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158, CD163, CD166,. CD168, CD174, CD180, CD184, CDw186, CD194, CD195, CD200, CD200a, CD200B, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326,4-1BB,5AC,5T4 (Trophoblast glycoprotein, TPBG,5T4, WNT-activation inhibitor 1 or WAIF 1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, alpha v beta 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 activating factor), B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, canAg Canine lupus erythematosus IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (C-C fragment chemokine 11), CCR4 (C-C chemokine receptor 4, CD194), CCR5, CD3E (ε), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), ch4D5, cholecystokinin 2 (CCK 2R), CLDN18 (Claudin-18), clusterin A, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD 115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD 184), C-X-C chemokine receptor 4, cyclic ADP ribonuclease, cyclin B1, CYP1B1, cytomegalovirus glycoprotein B, dabigatran, DLL4 (delta-like ligand 4), DPP4 (dipeptide-peptidase 4), 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 (CD 105), endothelin B receptor, endotoxin, epCAM (epithelial cell adhesion molecule), ephA2, episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS 2 ETS fusion gene), escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FC1, alpha fetoprotein GR, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, fos-associated antigen 1, respiratory syncytial virus F protein, frizzled receptor, fucose GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, globoH, glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), CY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase C receptor, heat stable enterotoxin receptor (hSTAR), heat shock protein, 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, HPVE6/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 of IgE, IGHE, IL-1, IL-2R (interleukin 2 receptor), IL-4, IL-5, IL-6R (interleukin 6 receptor), i-9, I-10, I-12, I-13, I-17, I-17A, I-20, I-22, I-23, I31RA, IGF2 (insulin-like growth factor 2), integrins (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, α v β 5), interferon γ -inducing proteins, ITGA2, ITGB2, KIR2D, CK, e, egumain, ewis-Y antigen, FA-1 (lymphocyte function-related antigen 1, CDCDCD1A), HRH, GO IN-1, lipoteichoic acid, IV1A, MP2, TA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-3, MAGE-1, MAGE-4, MCP-1, macrophage-inhibiting factors, or Glycosylsuppressors (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSN (mesothelin), MUC1 (mucin 1, cell surface associated (MUC 1) or Polymorphic Epithelial Mucin (PEM)), MUC 1-KH, MUC16 (CA 125), MCP1 (monocyte chemotactic protein 1), melanA/MART1, M-IAP, MPG, MS4A1, MYCN, myelin-associated glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), nectin-4 (ASG-22 ME), NGF, neuronal apoptosis regulating protease 1, NOGO-A, notch receptor, nucleolin, neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, oxLDL (oxidized low density lipoprotein), OY-TES1, P21, 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 α (alpha platelet-derived growth factor receptor), PDGFR- β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate co-transporter, PMEL 17, polysialic acid, proteinase 3 (PR 1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD 240), rhesus factor, RANKL, rhoC, ras mutations, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoints, 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, STn, TAG-72 (tumor-associated glycoprotein), survivin, T-cell receptor, T-cell transmembrane protein, transmembrane 1 (tumor vascular endothelial marker 1), TENB2, tenscin C (TGF-C), TGF-alpha, TGF-beta (transforming growth factor beta), TGF-beta 1, TGF-beta 2 (transforming growth factor 2), tie (CD 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 (DR 5)), tumor-associated calcium signal sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1 (CD 106), VEGF, VEGF-A, VEGF-2 (CD 309), VEGFR-1, VEGFR2, vimentin, WTR1, XAGE 1, sub>A cell expressing any insulin growth factor receptor, or any epidermal growth factor receptor.
15. The tumor cell of claim 13, wherein the tumor cell is selected from the group consisting of lymphoma cells, myeloma cells, renal cell carcinoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colon cancer cells, gastric cancer cells, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer cells, testicular cancer cells, and any uncontrolled, accelerated growth, differentiation carcinogenic cell.
16. Linker component R according to claims 1,2,3 and 4 1 And/or R 2 May be comprised of one or more linker components: 6-Maleimidohexanoic acid (MC), 3-Maleimidopropionic acid (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-Thiooctanoic acid (SPP), 4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid (MCC), 4-Thiooctanoic acid (SPDB), ethylmaleimide (ME), 4-thio-2-hydroxysulfobutanoic acid (2-Sulfo-SPDB), pyridinedithiol (PySS), alkoxyamino (AOA), ethyleneoxy (EO), 4-methyl-4-dithio-pentanoic acid (MPDP), azido (N-maleimidopropanoic acid (MP), valine-citrulline (val-cit), alanine-phenylalanine-phe (ala-phe), N-carbobenzoxy (2-Sulfo-SPDB), pyridine dithio-1-carboxylic acid (MPDP), and N-hydroxy-methyl-2-thiopropionic acid (MPDP) 3 ) An alkyne, a dithio, a peptide, and/or a (4-acetyl) aminobenzoate (SIAB).
17. The method of claim 2, wherein the Drug 1 And Drug 2 Preferred conjugates T01, T02, T03, T04, T05, T06 and T07 which are Tubulysin homologs having the general structural formula (II):
Figure FDA0003729824230000141
Figure FDA0003729824230000151
Wherein the mAb is an antibody; z 3 And Z 3 ' is independent H, OP (O) (OM) 1 )(OM 2 )，OCH 2 OP(O)(OM 1 )(OM 2 )，OSO 3 M 1 ，R 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 of one another, H, na, K, ca, mg, NH 4 ，NR 1 R 2 R 3 (ii) a n is 1 to 30; "-",
Figure FDA0003729824230000152
X 1 ，X 2 ，R 1 ，R 2 and R 3 The definitions are the same as in claims 1 and 2.
18. In claim 2, drug 1 And Drug 2 A preferred conjugate C01 having the general structural formula (II):
Figure FDA0003729824230000161
wherein the mAb is an antibody; n is 1 to 30; "-",
Figure FDA0003729824230000162
X 1 ，X 2 ，R 1 ，R 2 and R 3 The definitions are the same as in claims 1 and 2.
19. The method of claim 2, wherein the Drug 1 And Drug 2 A preferred conjugate M01, which is a maytansinoid homologue, having the general structural formula (II):
Figure FDA0003729824230000163
wherein the mAb is an antibody; n is 1 to 30; "-",
Figure FDA0003729824230000164
X 1 ，X 2 ，R 1 ，R 2 and R 3 The definitions are the same as in claims 1 and 2.
20. In claim 2, drug 1 And Drug 2 Preferred conjugates Tx01, tx02 and Tx03 having the general structural formula (II) are taxane homologs:
Figure FDA0003729824230000165
Figure FDA0003729824230000171
wherein the mAb is an antibody; n is 1 to 30; "-",
Figure FDA0003729824230000172
X 1 ，X 2 ，R 1 and R 2 The definitions are the same as in claims 1 and 2.
21. In claim 2, drug 1 And Drug 2 Preferred conjugates CC01, CC02, CC03, which are analogs of CC-1065 and/or a duocarmycin analog, having the general structural formula (II):
Figure FDA0003729824230000173
Figure FDA0003729824230000181
Wherein the mAb is an antibody; n is 1 to 30; 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; "-",
Figure FDA0003729824230000182
X 1 ，X 2 ，R 1 ，R 2 ，M 1 and M 2 The definitions are the same as in claims 1 and 2.
22. In claim 2, drug 1 And Drug 2 Preferred conjugates Da01, da02, da03 and Da04 having the general structural formula (II) are daunorubicin/doxorubicin homologues:
Figure FDA0003729824230000183
Figure FDA0003729824230000191
wherein the mAb is an antibody; n is 1 to 30; x 3 And X 3 ' is independently H, O, NH, NHC (O), NHC (O) NH, C (O), R 1 Or OC (O); "-",
Figure FDA0003729824230000192
X 1 ，X 2 ，R 1 and R 2 As defined in claims 1 and 2.
23. The method of claim 2, wherein the Drug 1 And Drug 2 Preferred conjugates of the general structural formula (II) Au01, au02, au03, au04 and Au05, which are auristatin homologs:
Figure FDA0003729824230000193
Figure FDA0003729824230000201
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 FDA0003729824230000202
X 1 ，X 2 ，R 1 ，R 2 And R 3 As defined in claims 1 and 2.
24. The method of claim 2, wherein the Drug 1 And Drug 2 Preferred conjugates of the benzodiazepine dimer homologue having the general structural formula (II) PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB09, PB10 and PB11:
Figure FDA0003729824230000203
Figure FDA0003729824230000211
Figure FDA0003729824230000221
Figure FDA0003729824230000231
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 FDA0003729824230000232
X 1 ，X 2 ，R 1 ，R 2 And R 3 As defined in claims 1 and 2.R is 1 And/or R 2 A default may be used.
25. The method of claim 2, wherein the Drug 1 And Drug 2 Preferably two or more different cytotoxic agents. May be selected from the following combinations of structures: tubulysin, maytansine, taxanes, CC-1065 homologs, daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD), tolamemycin, indolophenyldiazepine, imidazobenzothiadiazepine 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 EB (AEB) and Auristatin EFP (AEFP)), dactinomycin, thioteparin, vincristine, hemidamycin, azulmicin, sultamicin, sultamide, and their derivatives, such as puromycin-682 and 1597 . More preferred conjugates comprising two or more different cytotoxic agents such as Z01, Z02, Z03, Z04, Z05, Z06, Z07, Z08, Z09, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17 and Z18:
Figure FDA0003729824230000241
Figure FDA0003729824230000251
Figure FDA0003729824230000261
Figure FDA0003729824230000271
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 is a group of 1 And M 2 Independently of one another, H, na, K, ca, mg, NH 4 ，NR 1 R 2 R 3 ；“-”，
Figure FDA0003729824230000272
X 1 ，X 2 ，R 1 ，R 2 And R 3 The definitions are the same as in claims 1 and 2. R 1 And/or R 2 A default may be used.
26. The method of claim 2, wherein the Drug 1 And Drug 2 Being polyalkylene glycol homologues, preferred conjugates of formula (II) such as Pg01:
Figure FDA0003729824230000273
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 FDA0003729824230000281
X 1 ，X 2 ，R 1 ，R 2 and R 3 As defined in claims 1 and 2; r is 4 Is OH, H, R 1 Or R as defined in claim 1 3 。
27. The method of claim 2, wherein the Drug 1 And Drug 2 For cell-bound ligands or receptor analogs, preferred conjugates having the structural formula (II) 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), α v β 3 integrin receptor, cyclic RGD pentapeptide conjugate), LB14 (heterobivalent peptide ligand conjugate of VEGF receptor), LB15 (neurointerleukin B conjugate), LB16 (G protein receptor bombesin conjugate) and LB17 (Toll-like receptor class receptor) 2 Conjugate):
Figure FDA0003729824230000282
Figure FDA0003729824230000291
Figure FDA0003729824230000301
Figure FDA0003729824230000311
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 is a group of 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 FDA0003729824230000321
X 1 ，X 2 ，R 1 ，R 2 and R 3 The definitions are the same as in claims 1 and 2. R is 1 And/or R 2 A default may be used.
28. A pharmaceutical composition for the treatment or prevention of cancer, autoimmune disease or infectious disease comprising a therapeutically effective dose of the conjugate of claims 2, 17,18, 19, 20, 21, 22, 23, 24, 25, 26 and/or 27, and a pharmaceutically acceptable salt, carrier, diluent or excipient thereof, or a combination thereof.
29. The conjugate of claim 2, 17,18, 19, 20, 21, 22, 23, 24, 25, 26 and/or 27, having in vitro, in vivo or ex vivo activity.
30. The conjugate of claims 2, 17,18, 19, 20, 21, 22, 23, 24, 25, 26 and/or 27, wherein linker component R 1 ，R 2 ，R 3 And/or R 4 A peptide comprising one of 1 to 20 natural or unnatural amino acid units, an aminobenzyl unit, a 6-maleimidocaproyl unit, a disulfide, a thioether unit, a hydrazone unit, a triazole unit, or an alkoxyoxime unit.
31. The linker component R of claims 2, 17,18, 19, 20, 21, 22, 23, 24, 25, 26 and/or 27 1 And/or R 2 Can be cleaved by proteolytic enzymes.
32. A pharmaceutical composition comprising a therapeutically effective amount of a conjugate of claims 2, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and/or 28 for simultaneous use with other therapeutic agents such as chemotherapeutic agents, radiation therapy, immunotherapeutic agents, autoimmune disease drugs, anti-infective drugs or other conjugates, synergistically effective to treat or prevent cancer, autoimmune disease or infectious disease.
33. The synergist according to claim 32 is preferably selected from one or more of the following drugs: abirapu, abiraterone acetate
Figure FDA0003729824230000322
Acetaminophen/hydrocodone, adalimumab, afatinib maleate
Figure FDA0003729824230000323
Alemtuzumab
Figure FDA0003729824230000324
Aliretin A acid
Figure FDA0003729824230000325
ado-trastuzumab emtansine (Kadcyla) TM ) Mixed salt of amphetamine (amphetamine/dexamphetamine), anastrozole
Figure FDA0003729824230000326
Aripiprazole, atazanavir, atezolizumab (MPDL 3280A), atorvastatin, axitinib
Figure FDA0003729824230000327
Berlin (Beleodaq) TM ) Bevacizumab
Figure FDA0003729824230000328
Cabazitaxel
Figure FDA0003729824230000329
Cabotinib (Cometriq) TM ) Bexarotene
Figure FDA00037298242300003210
blinatumomab(Blincyto TM ) Bortezomib
Figure FDA00037298242300003211
Bosutinib
Figure FDA00037298242300003212
brentuximab vedotin
Figure FDA00037298242300003213
Budesonide, budesonide/formoterol, buprenorphine, capecitabine, carfilzomib
Figure FDA00037298242300003214
Celecoxib, ceritinib (LDK 378/Zykadia), cetuximab
Figure FDA00037298242300003215
Ciclosporin, cinacalcet, crizotinib
Figure FDA00037298242300003216
Dabigatran, dabrafenib
Figure FDA00037298242300003217
Alfa darbetin, darunavir, imatinib mesylate
Figure FDA0003729824230000331
Dasatinib
Figure FDA0003729824230000332
di-Ni interleukin
Figure FDA0003729824230000333
Dinosame
Figure FDA0003729824230000334
Divalproex sodium, dexlansoprazole, dexmethylphenidate, dinutuximab (Unituxin) TM ) Doxycycline, duloxetine, emtricitabine/rilpivirine/tenofovir disoproxil fumarate, emtricitabine/tenofovir/efavirenz, enoxaparin, enzalutamide
Figure FDA0003729824230000335
Afavastin, erlotinib
Figure FDA0003729824230000336
Esomeprazole, eszopiclone, etanercept, everolimus
Figure FDA0003729824230000337
Exemestane
Figure FDA0003729824230000338
Everolimus
Figure FDA0003729824230000339
Ezetimibe, ezetimibe/simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone/salmeterol, fulvestrant
Figure FDA00037298242300003310
Gefitinib
Figure FDA00037298242300003311
Glatiramer, goserelin acetate (Zoladex), imatinib (Gleevec), ibritumomab tiuxetan
Figure FDA00037298242300003312
Ibrutinib (Imbruvica) TM ) Adalaris, ai dallas
Figure FDA00037298242300003313
Infliximab, insulin aspart, insulin detemir, insulin glargine, insulin lispro, interferon beta 1a, interferon beta 1b, lapatinib
Figure FDA00037298242300003314
Ipilimumab
Figure FDA00037298242300003315
Ipratropium bromide/salbutamol, orcipretatide acetate (A)
Figure FDA00037298242300003316
Depot), lenalidomide
Figure FDA00037298242300003317
Levatinib mesylate (Lenvima) TM ) Letrozole
Figure FDA00037298242300003318
Levothyroxine, lidocaine, linezolid, lidocaineRalutide, MEDI4736 (AstraZeneca, celgene), memantine, methylphenidate, metoprolol, modafinil, mometasone, nilotinib
Figure FDA00037298242300003319
Nivolumab (nivolumab)
Figure FDA00037298242300003320
Olympic single antibody
Figure FDA00037298242300003321
Orbiuzumab (Gazyva) TM ) Olaparib (Lynparza) TM ) Olmesartan, olmesartan/hydrochlorothiazide, omalizumab, omega-3 fatty acid ethyl ester, oseltamivir, oxycodone, palbociclib
Figure FDA00037298242300003322
Palivizumab panitumumab
Figure FDA00037298242300003323
Panobinostat
Figure FDA00037298242300003324
Pazopanib
Figure FDA00037298242300003325
Pembrolizumab
Figure FDA00037298242300003326
Pemetrexed (Alimta), pertuzumab (Perjeta) TM ) Pneumococcal conjugate vaccine, pomalidomide
Figure FDA00037298242300003327
Pregabalin, quetiapine, rabeprazole radium chloride 223
Figure FDA00037298242300003328
Raloxifene, latiravir, ramucimumab
Figure FDA00037298242300003329
Ralizumab, regorafenib
Figure FDA00037298242300003330
Rituximab
Figure FDA00037298242300003331
Rivaroxaban, romidepsin
Figure FDA00037298242300003332
Rosuvastatin, ruxotinib phosphate (Jakafi) TM ) Salbutamol, sevelamine, sildenafil, siltiximab (Sylvant) TM ) Sitagliptin, sitagliptin/metformin, solifenacin, sorafenib
Figure FDA00037298242300003333
Sunitinib
Figure FDA00037298242300003334
Tadalafil, tamoxifen, telaprevir, temsirolimus, tenofovir/emtricitabine, testosterone gel, thalidomide (Immunopren, talidex), tiotropium bromide, toremifene
Figure FDA00037298242300003335
Trametinib
Figure FDA00037298242300003336
Trastuzumab, tretinoin
Figure FDA00037298242300003337
Ultexan, valsartan, vandetanib
Figure FDA00037298242300003338
Verofini
Figure FDA00037298242300003339
Vorinostat
Figure FDA00037298242300003340
Abibercept (Abbercept)
Figure FDA00037298242300003341
Zostavax and homologs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients thereof, or combinations thereof.
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