NZ744940B2 - Conjugation linkers, antibody-drug conjugates thereof, and methods of synthesis and use of such conjugates - Google Patents

Conjugation linkers, antibody-drug conjugates thereof, and methods of synthesis and use of such conjugates

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Publication number
NZ744940B2
NZ744940B2 NZ744940A NZ74494016A NZ744940B2 NZ 744940 B2 NZ744940 B2 NZ 744940B2 NZ 744940 A NZ744940 A NZ 744940A NZ 74494016 A NZ74494016 A NZ 74494016A NZ 744940 B2 NZ744940 B2 NZ 744940B2
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New Zealand
Prior art keywords
acid
cell
mmol
receptor
inhibitors
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NZ744940A
Other versions
NZ744940A (en
Inventor
Minjun Cao
Shun Gai
Huihui Guo
Zhixiang Guo
Yuanyuan Huang
Junxiang Jia
Wenjun Li
Chen Lin
Qianqian Tong
Hongsheng Xie
Original Assignee
Hangzhou Dac Biotech Co Ltd
Filing date
Publication date
Application filed by Hangzhou Dac Biotech Co Ltd filed Critical Hangzhou Dac Biotech Co Ltd
Priority claimed from PCT/IB2016/050580 external-priority patent/WO2016059622A2/en
Publication of NZ744940A publication Critical patent/NZ744940A/en
Publication of NZ744940B2 publication Critical patent/NZ744940B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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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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    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
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    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
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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
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
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Abstract

Disclosed is an acetylenedicarbonyl linker for conjugation of a cytotoxic agent, a drug or a functional molecule to a cell-binding agent/molecule. Also disclosed are methods of making such linkers, and of using such linkers in making homogeneous conjugates. Further disclosed is application of the conjugates in the treatment of cancers, infectious diseases and autoimmune disorders. njugates in the treatment of cancers, infectious diseases and autoimmune disorders.

Description

Disclosed is an acetylenedicarbonyl linker for conjugation of a cytotoxic agent, a drug or a functional le to a cell-binding agent/molecule. Also disclosed are methods of making such linkers, and of using such linkers in making homogeneous conjugates. Further disclosed is application of the conjugates in the treatment of cancers, infectious diseases and autoimmune disorders.
NZ 744940 CONJUGATION LINKERS, ANTIBODY-DRUG CONJUGATES THEREOF, AND METHODS OF SYNTHESIS AND USE OF SUCH CONJUGATES FIELD OF THE INVENTION The present ion relates to linkers used for the conjugation of compounds, in particular, cytotoxic agents to specific pairs of sulfur atoms of a cell-binding molecule. The present ion also relates to methods of making cell-binding agent-drug (cytotoxic agent) conjugates in a specific manner comprising either modification of drugs with these linkers first, followed by reaction with prepared cell-binding agents; or modification of cell-binding agents with these linkers first, followed by reaction with drugs.
BACKGROUND OF THE INVENTION The targeted delivery of highly active or potent pharmaceutical drugs by antibodies or other biological ligands to specific sites of disease in human body is an exciting approach in cancer ent that has gained significant st over the past few years. In ular, since US FDA approvals of is (brentuximab vedotin) in 2011 and Kadcyla (ado-trastuzumab emtansine) in 2013, almost every major pharmaceutical and biotech company has adopted the applications of antibody-drug ate (ADC) for ed treatment of s (Chari, R. et al, Angew. Chem., Int. Ed. 2014, 53, 3796−3827; Sievers, E. L. et al. Annu Rev Med. 2013, 64, -29; Mehrling, T. Future Oncol, 2015, 11, 549). In conjugated form, the cytotoxic agents exhibit more selective eutic activity, sparing non-target cells from many of the toxic effects and improving the safety profile (Van den Mooter, T. et al Expert Opin Biol Ther. 2015, , 749-60).
The big challenge of chemotherapeutic drugs is their narrow therapeutic windows due to they normally cannot discriminate between normal and malignant cells, thus causes side effects which limit the ted doses below the clinically effective ones. In contrast, immunotherapy , normally in the form of monoclonal antibodies (mAb) can specifically bind to certain proteins or molecules of malignant cells , leaving normal cells unharmed, and thus has less side effects and wider eutic windows than chemotherapy. Monoclonal ant ibodies (mAb) can target against malignant cells by several mechanisms, such as, 1). Making the cancer cell more visible to the immune system (Villaruz, L. C. et al, 2014, Transl Lung Cancer Res, 3, 2-14;Camacho, L.
H. 2015 Cancer Med 4, 661-72); 2). Blocking growth s (Dillman, R. O. 2011, Cancer Biother Radiopharm, 26, 1-64; Ferris, R. L. et al 2010, J Clin Oncol, 28, 4390-9); 3). Stopping new blood vessels from forming (Arrillaga-Romany, I., et al, 2014, Expert Opin Investig Drugs, 23, 199-210); 4). Delivering radiation to cancer cells (Chapuy, B. et al, 2007, Biotechnol J. 2, 1435-43); 5). Delivering chemotherapy drug to cancer cells (Chari R. J.2008 Acc Chem Res. 41, 98-107; Mullard A. 2013, Nature Reviews Drug Discovery 12, 329-32; Zhao, R. J. 2012, J.
Med. Chem., 55, ); and 6). Delivering enzyme to cancer cells (Francis R. J. et al, 2002, Br. J. Cancer 87, 600-7).One of these above strategies, delivering chemotherapy to cancer cells called dy-drug conjugates (ADCs), which enables to target and deliver drugs to cancer cells leaving normal cells largely unaffected by the exquisite targeting ability of antibodies, has undergone intense tation in the last two decades. Currently there are more than 50 ADC drugs in the clinic trials according to www.clinictrails.gov.
The first-generation ADCs, including Kadcyla and Adcetris, are produced through nonselective ation of native lysine amines or inter chain cysteine thiols on an antibody respectively to a cytotoxic drug. Since there are over 50 e-exposed lysines and 8 hinge cysteine es in IgGl antibodies, this nonselective conjugation results in randomly crosslinkage of cytotoxic drugs to practically all areas of the antibody molecule, particularly having a diverse tion of ADCs with a wide distribution of drugs per antibody (DAR) (Wang, L., et al. 2005 Protein Sci. 14, 2436; Hamblett, K. J., et al.2004 Clin. Cancer Res. 10, 7063).Thus some of the undesired ADC subpopulation could lead to shorter circulation half-life, lower efficacy, potentially increased off-target ty and a wide range of in vivo pharmacokinetic(PK) properties (Hamblett, K. J. et al, Clin. Cancer Res. 2004, 10, 7063-70; Adem, Y. T. et al, Bioconjugate Chem. 2014, 25, 656-664; Boylan, N. J. Bioconjugate Chem.,2013, 24, 1008-1016; Strop, P., et al 2013 Chem. Biol. 20, ). In addition, with this classical conjugation, the batch-to-batch consistency in ADC production can be challenging and may require diligent cturing capabilities (Wakankar, A. mAbs, 2011, 3, 161-172).
Therefore, biotechnology companies and academic utions are highly focusing on establishing novel reliable methods for site-specific ADC ation. So far, there are several approaches developed in recent years for site selective ADC preparation (Panowski, S, 2014, mAbs 6, 34). They include incorporation of unpaired cysteines, e.g. engineered ve cysteine residues, called THIOMAB from Genentech ula, J. R., et al 2010 Clin. Cancer Res. 16, 4769; Junutula, J. R., et al 2008 Nat hnol. 26, 925-32; US Patents 8,309,300; 7,855,275; 7,521,541; 7,723,485, WG2008/141044), genetically introduced glutamine tag with Strep to verticillium mobaraense transglutaminase (mTG) (Strop, P., Bioconjugate Chem., 2014, 25, 855-862; Strop, P., et ah, 2013, Chem. Biol. 20, 161-167; US Patent 8,871,908 for Rinat-Pfizer) or with Microbial transglutaminase (MTGase) (Dennler, P., et al, 2014, Bioconjug. Chem. 25, 569-578. US pat appl 89287 for Innate Pharma; US Pat 7,893,019 for Bio-Ker S.r.l. (IT)), incorporation of thiolfucose (Okeley, N. M., et al 2013 Bioconjugate Chem. 24, 1650), incorporation of unnatural amino acids through mutagenesis (Axup, J.Y., et ah, 2012, Proc. Natl. Acad. Sci. 109, 16101-16106; Zimmerman, E.S., et ah, 2014, jug. Chem. 25, 351-361; Wu, P., et al, 2009 Proc. Natl. Acad. Sci. 106, 3000-5; Rabuka, D., et al, 2012 Nat. Protoc. 7, 1052-67; US Pattent 8,778,631 and US Pat Appl. 20100184135, W02010/081110 for Sutro Biopharma; W02006/069246, 2007/059312, US Patents 7,332,571, 7,696,312, and 299 for Ambrx; W02007/130453, US patents 7,632,492 and 7,829,659 for Allozyne), Incorporation of selenocysteine into antibodies , T., et al 2009, Biochemistry 48, 12057; US Patent 8,916,159 for US National Cancer Institute), Conversion of cysteines located in the CXPXR consensus sequence to formylglycine (FGly) with formylglycine generating enzyme (FGE) (Drake, P.M., et ah, 2014, Bioconjug.
Chem. 25, 1331-1341. o, I. S. et al 7,985,783; 8,097,701; 8,349,910, and US Pat Appl 20140141025, 20100210543 for Redwood ence), and through glycoengineeringly uction of sialic acid with the use of galactosyl- and sialytransferases (Zhou, Q., et al 2014, Bioconjug.Chem.,25, 510-520, US Pat Appl 20140294867for Sanofi-Genzyme). These above methods have produced nearly homogeneous product es, but they are ed antibodyengineering ses and reoptimization of cell culture conditions. Moreover, expression yields for genetic encoding of an unnatural amino acid were typically not promisingly high enough (Tian, F., et al, 2014, Proc. Natl. Acad. Sci. U. S. A. Ill, 1766-71) which has significant impact on the cost of goods of the ADC. In addition, it has been known that ADCs obtained by conjugation to cysteine side chains often display limited stability in circulation, leading to premature disconnection of the cytotoxic payload before the tumor site is reached (Junutula, J. R., et al 2008, Nat. Biotechnol. 26, 925-32).
The disulfide bond structures of the four subclasses of IgG antibodies were known in the 1960s (Milstein C. Biochem J 1966, 101: 338-51; Pink JR, Milstein C. Nature 1967, 214: 92-4; Frangione B, Milstein C. Nature 1967, 216: ; Pink JR, Milstein C. Nature 1967, 216: 941-42; Frangione B, et al. Biochem J. 1968, 106,15-21; Frangione B, Milstein C. J Mol Biol 1968, 33: 893-906; Edelman GM, et al. Proc Natl Acad Sci USA 1969; 63: 78-85; Frangione B, et al. Nature 196, 221: 145-8, Spiegelberg, H. F. et al Biochemistry, 1975, 10, 2157-63). Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. Among the four subclasses of IgG antibodies, IgGi, IgGi, IgG-, and IgG4, each IgG ns a total of 12 chain disulfide bonds; each disulfide bond is ated with an individual IgG domain. The two heavy chains are ted in the hinge region by a variable number of disulfide bonds: 2 for IgGi and IgG4, 4 for IgGi and 11 for IgG-,. The light chain of the IgGi is connected to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the fifth cysteine residue of the heavy chain. But, for IgGi, IgGsand IgG4, the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine e of the heavy chain (Liu, H. and May, K., 2012, mAbs 4, 17-23). On the ranks of the susceptibility of disulfide bonds in human IgGl antibodies by experimental reduction, ential alkylation, and LC-MS analysis (Liu, H, et al, Anal. Chem., 2010, 82, 5219-26), inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds, and the ide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy . The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, ide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CHI domains had similar and moderate susceptibility, while disulfide bonds in the CHS domain were the least tible to reduction(Liu, H, et al Anal. Chem., 2010, 82, 5219-5226).
Based on the greater susceptibility of inter chain vs. intra chain disulfide bonds in human IgGl antibodies, several institutions and ies adopted the chemically specific conjugation strategy through rebridging reduced inter chain disulfide bonds of a native antibody, such as, using bromo or dibromo-maleimides, called next tion maleimides (NGMs) (Schumacher, F.F., et al 2014, Org. Biomol. Chem. 12, 7261-69; UCL Cancer Institute), applying bis-alkylating reagents via a three-carbon bridge (Badescu, G., et ah, 2014, Bioconjug.
Chem. 25, 1124-1136., WO2013/190272, WO2014/064424 for PolyTherics Ltd), with disubstituted heteroaryl bridge (US Pat Appl. 2015/0105539 for Concortis Biosystem), or h di-maleimide as a bridge 4/114207). We have also used bromo maleimide and dibromomaleimide linkers to ate both drugs and antibodies for a quite while (WO2014/009774, ). However, these above bridge linkers were designed in the way to conjugate only one cytotoxic agents to a pair of disulfide bonds, and therefore at most of time they only produced ADCs at DAR less than 2 (drugs per antibody), due to limited numbers (about two pairs) of reduced disulfide bonds are more accessible for conjugation.
As one of the major issues for ADCs is the limited numbers or amount of cytotoxic compound that ultimately reaches the tumor, and thus the ble DAR over 3 is much important factor for improvement of ADC therapeutical index (Epenetos, A. A. et al, Cancer Res., 1986, 46, 3183-91; Chari, R. V. Acc. Chem. Res., 2008, 41, 98-107, Zhao, R. Y. et al, 2011, J. Med. Chem. 54, 3606-23), we therefore sed novel disulfur bridge linkers (PCT/IB2015/ ) that not only are able to conjugate two or more drugs per linker for achieving higher DARs (>4), but also can selectively rebridge pairs of reduced inter chain disulfide bonds on surface of antibody, which are generated by overloaded TCEP or DTT reduction agents. And the over reduced pairs of thiol groups that are inaccessibly reached by the bridge linkers can be recoupled (regenerated) by an oxide, e.g. dehydroascorbic acid (DHAAJ or Cu(II), to form back disulfide bonds at the end of conjugation. In principal, this rebridging back of reduced disulfide bonds results in more stable or longer half-life of ADCs in ison with traditional thiol linked ADCs.
It has been reported that the "ring-opened" succinimide ring linker bearing hioether bond has improved in vitro stability, ed PK exposure, and ed efficacy as compared to the mono-thiol-maleimide-conjugated ADCs (Turney, L. N, et al, 2014, Bioconjug. Chem. 25, 1871-80; Lyon, R. P, et al. 2014, Nat. Biotechnol. 32, 1059-62), due to the latter is prone to payload loss via a retro-Michael type reaction of the maleimide conjugation (Shen, B. Q, et al, 2012, Nat Biotechnol. 30, 184-9; Turney, L. N, et al, 2014 Bioconjug Chem. 25, 1871-80). In this patent application, we extend the scopes of our r patent application. The bridge linkers of this invention containing a 2,3-disubstituted succinic group, or substituted, or 2,3-disubstituted fumaric or maleic (trails (E)~ or cis (Z)-- butenedioic) group, or acetylenedicarboxyl group have less payload loss as compared to their rolyzed bromo or dibromo-maleimide linkers which were tested in our lab. In other words, the methods of this ion can be used for the immunoconjugates that carry a combination of drugs and functional molecules such as PEGs, which can be used to shield the hydrolysis of conjugated drugs by hydrolases in the blood circulation as well as inhibit the degradation of the antibody by proteinases. Thus the major advantages of this patent for immunoconjugates include: prolonged the half lives of the ates during the targeted delivery; ated in steps of two or more different function molecules/drugs that act in different phases of the cell cycle to se the number of target cells exposed to the particular pharmaceutical drugs or effectors; minimized exposure to non-target cells, tissues or organs through conjugation of the function molecules; precisely controlled over drug payloads and drug ratios at the specific sites leading to homogenous final products. In short, the bridge linkers of the invention can stabilize ADCs as well as make homogeneous production of ADCs in a simple manner.
SUMMARY OF THE INVENTION The present invention provides linkers ning a 2,3-disubstituted succinic group, or 2- monosubstituted, or 2,3-disubstitutedfumaric or maleic (trans (E) - or cis (Z)-butenedioic) group, or acetylenedicarboxyl group to link a drug and/or a on le to a cell-binding agent (e.g., an antibody). The preferred formula of the cell-binding molecule-linker-dmg conjugates SN ^Drugi) Cb- ^S^L^fR2) can be represented as: m2 n, wherein Cb is a inding agent, L is a linker containing succinic, fumaric or maleic group; Dmgl is a drug molecule; mi, m2 and n are an integer from 1 to 30; and two S (sulfur) elements from Cb bridgely link to L, which covalently connects a drug, Drugi and a function molecule, R2, which is for stabilizing the conjugated "Dmgl" during the circulation. The advantages in applying the linker in the cell molecule-drug conjugate are: a). Retaining the stability of the conjugates by covalently cross-linking (rebridging ) the pairs of reduced disulfur atoms of the cell-binding agents, particularly of antibodies; b). Enabling conjugation of the xic agents/drugs to specific sites of a cell-binding molecule, e.g. the inter chain disulfide bond sites of IgG dies, resulting in homogeneous tion of ADCs.
In one aspect of the present invention, the linker is represented by Formula (I) O O (Zj—R^Xt^U-7 II X2-fR2)m2 V 'mi u \ U' (I) Wherein ---- represents an al single bond. == represents either a single bond, or a double bond, or a triple bond.
It ed that when == represents a single bond, both U and U’ are not H; when == represents a double bond, either U or U’ can be H, but they are not H at the same time; when == represents a triple bond, both U and U’ can be absent.
U and U’ ent the same or different leaving group that can be substituted by a thiol.
Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS), yl; ophenoxyl; luorophenoxyl, tetrafluorophenoxyl, orophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, IH-imidazole-l-yl, chlorophenoxyl, dichlorophenoxyl, orophenoxyl, tetrachlorophenoxyl, N-(benzotriazolyl )oxyl, 2-ethylphenylisoxazolium-yl, phenyloxadiazol-yl (ODA), oxadiazol-yl, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions.
Zi is a function group that enables to react with a cytotoxic drug, to form a disulfide, ether, ester, thioether, ter, peptide, hydrazone, carbamate, carbonate, amine (secondary, tertiary, or quarter), imine, cycloheteroalkyane, heteroaromatic, alkyloxime or amide bond; Ri can be absent, or can be selected from Ci-Cg of alkyl; Ci-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; Cs-Cg of aryl, Ar-alkyl, cyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 carbon atoms of esters, ether, or amide; or polyethyleneoxy unit of formula (OCHiCt^p or H(CH3))p, wherein p is an r from 0 to about 1000, or combination of above groups thereof.
Additionally Ri is a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which covalently connects to Xi and Zi. The atoms used in forming the Ri may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination above thereof.
Xi and X2 are independently selected from NH; NHNH; N(R3); N(R3)N(R3-); O; S; Ci-C8 of alkyl; Ci-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; Cs-Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 amino acids; n R3 and R3’ are independently H;Ci-Cg of alkyl; Ci-Cg of hetero-alkyl, alkylcycloalkyl, heterocycloalkyl; C3-Cg of aryl, Ar-alkyl, cyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 carbon atoms of esters, ether, or amide; or polyethyleneoxy unit of formula (OCHiCt^p or (OCH2CH(CH3))p, wherein p is an r from 0 to about 1000, or combination above thereof.
R2is ndently selected from OH, H, NH2, SH, NHNH2, N(R3)(R3-), N(R3)NH(R30, polyethyleneoxy unit of formula HijpOR-, or (OCH2CH(CH3))pOR3 or NH(CH2CH20)PR3, or NH(CH2CH(CH3)0)PR3, or N[(CH2CH20)pR3][(CH2CH20VR3 ], or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; Ci-Cg of alkyl; Ci-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, lkyl, heteroalkylcycloalkyl, alkylcarbonyl, aryl; Wherein R3 and R3’ are independently H; Ci-Cg of alkyl; Ci-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-Cg of aryl, Ar-alkyl, heterocyclic, yclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 carbon atoms of esters, ether, or amide; or 1-8 amino acids; or polyethyleneoxy unit of formula (OCHiCHi^ or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or combination above thereof. mi and m2 are independently an integer from 1 to 30, preferably from 1 to 10.
In another aspect, this invention provides a cell-binding agent-drug conjugate of Formula (II), in which the cell-binding agent, Cb, and the drug, Dmgl, has reacted at the ends of the bridge linker: (Drug!—Rx-j-] X, sx ' ‘'in | Cb ' Ill 2 O n Wherein: Cb represents a cell-binding agent, preferably an antibody; Inside the bracket (square parentheses) are the linker-drug components that are conjugated to pairs of thiols of the cell-binding molecule. The thiols are preferred pairs of sulfur atoms reduced from the inter chain disulfide bonds of the cell-binding agent by a reduction agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris (2- carboxyethyl) phosphine (TCEP), aptoethylamine (P-MEA), or/and beta mercaptoethanol (P-ME, 2-ME).
Dmgi represents a cytotoxic agent or a drug, which is linked to the cell-binding agent via the bridge linker of the patent h Ri that can be containing an Ci-Cg of alkane; Ci-Cg of alkylene, alkenylene, alkynylene, aromatic, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, amine, urethanes, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide, thioether, thioester, carbamate, ate, heterocyclic ring, heteroalkyl, heteroaromatic, or alkoxime; or combination above thereof. "==" represents either single bond or double bond.
Inside the square bracket are agents that are ated to a inding molecule through a pair of sulfur atoms on the cell-binding molecule. n is 1 ~ 30; Ri, Ri, mi, m2, Xi and X2 are described the same previously in Formula (I).
In a r aspect, the present invention provides a modified cell-binding agent of Formula (III), in which the cell-binding agent, Cb, through its pair of thiols generated with reduction of disulfide bonds, has reacted with the bridge linker, which has Zi, the function groups capable of ng with a drug: (Zi-Rr)-] X| sx ' 'mi Cb (R2}-x2 S'' ' m2 O n (III) Wherein "---- ", Zi, Z2, n, Ri, R2, mi, m2, Xi, and X2 are defined the same as in Formula (I). "==" and Cb are defined the same as in Formula (II).
In an even further aspect, the present invention provides a modified drug of Formula (IV), in which the drug, Drugi, has d with the linker of Formula (I), which still has the 2,3- disubstituted succinic group, or 2-monosubstituted, or 2,3-disubstitutedfumaric or maleic (trans (E)- or cis tenedioic) group, or acetylenedicarboxyl group capable of reacting with a pair of thiols of the cell-binding agent: O O (Drugl— IUx2TR>) \ /m, u/ \ m2 U' (IV) Wherein "---- U, U\ Ri, R2, mi, m2, Xi, and X2 are defined the same as in Formula (I). Drugi and Cb are defined the same as in Formula (II).
The t invention further s to a method of making a cell-binding le-drug conjugate of Formula (II), wherein the drugs, "Drugi"is linked to a cell-binding agent via the bridge linker.
The present invention also relates to a method of making a modified inding molecule of Formula (III), wherein the cell-binding molecule is reacted with the bridge linker of Formula (I).
The present invention also relates to a method of making a modified drug of formula (IV), wherein the drug is reacted with the bridge linker of Formula (I).
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the sis of a bridge linker containing a drug and a polyethylene glycol, and the application of this linker in the conjugation of the two different molecules to an antibody via amide bonds.
Figure 2 shows the synthesis of bridge linkers containing a drug and a polyethylene glycol, and the application of the linkers in the conjugation of the two different molecules to an antibody via amide bonds.
Figure 3 shows the synthesis of bridge linkers containing a drug and a polyethylene glycol, and the ation of the linkers in the conjugation of the two different molecules to an antibody via amide bonds.
Figure 4 shows the synthesis of bridge s containing a drug and a polyethylene glycol, and the application of the s in the conjugation of the two different molecules to an antibody via oxime linkage.
Figure 5 shows the synthesis of bridge linkers containing a drug and a hylene , and the application of the linkers in the conjugation of the two different molecules to an antibody via hydrazone e.
Figure 6 shows the sis of bridge linkers containing a drug and a polyethylene glycol, and the application of the linkers in the conjugation of the two different molecules to an antibody via amide bonds.
Figure 7 shows the synthesis of bridge linkers containing a drug, a polyethylene glycol or short alkyl acid, and the conjugation of the drug via these linkers to an antibody.
Figure 8 shows the synthesis of bridge linkers containing a drug, a polyethylene glycol or peptides, and the application in the ation of the bridge linker-drug molecules to an antibody via hinder amide linkage.
Figure 9 shows the synthesis of bridge linkers containing peptides or polyethylene s, and the application of these linkers in the ation of the bridge linker-drug molecules to an antibody via amide linkage.
Figure 10 shows the synthesis of the components of Tubulysin analogs.
Figure 11 shows the synthesis of the ents of bridge linkers containing polyethylene glycol.
Figure 12 shows the synthesis of the linkers of this patent application.
Figure 13 shows the synthesis the conjugates of dy-tubulysin analogs via a bridge linker of this patent application.
Figure 14 shows the synthesis the conjugates of antibody-tubulysin analogs via a bridgelinker of this patent application.
Figure 15 shows the synthesis of the components of MMAF analogs.
Figure 16 shows the synthesis of a conjugate of antibody- PBD dimer analog via the bridge- linker.
Figure 17 shows the synthesis of conjugates of antibody- PBD dimer analogs via the bridgelinkers of this patent application.
Figure 18 shows the synthesis of conjugates of antibody- MMAF analogs via the bridge linkers of this patent application.
Figure 19 shows the synthesis of conjugates of antibody- MMAF analogs via the bridge linkers of this patent application.
Figure 20 shows the synthesis of conjugates of antibody- tubulysin s via the bridge s of this patent application.
Figure 21 shows the synthesis of the conjugates of Maytansinoid analogs via the bridge linkers of this patent application.
Figure 22 shows the synthesis of the conjugates of MMAF analogs, sin analogs and amatoxin analogs via the bridge linkers of this patent application.
Figure 23 shows the sis of the conjugates of MMAF analogs, tubulysin analogs and amatoxin analogs via the bridge linkers of this patent application.
Figure 24 shows the synthesis of the conjugates of amatoxin analogs via the bridge linkers of this patent application.
Figure 25 shows the synthesis of the conjugates of MMAF analogs and tubulysin analogs via the bridge s of this patent application.
Figure 26 shows the synthesis of the conjugates containing both PBD dimer analogs and polyethylene glycols, both PBD dimer analogs and MMAF analogs, and both PBD dimer analog and Tubulysin analogs, to an antibody via the bridge linkers of this patent.
Figure 27 shows the synthesis of the conjugates containing both PBD dimer analogs and Tubulysin analogs, both Tubulysin analogs and hylene glycols, and both MMAF analogs and polyethylene glycols, to an antibody via the bridge linkers of this patent application.
Figure 28 shows the synthesis of the ates containing both MMAF analogs and polyethylene glycols, and Tubulysin B s via cyclic linkage of the bridge linkers of this patent ation.
Figure 29 shows the synthesis of the ates of tubulysin analogs via the bridge linkers of this patent application.
Figure 30 shows the synthesis of the ates of sin s via the bridge linkers of this patent application.
Figure 31 shows the synthesis of the conjugates of MMAF analogs via the bridge linkers of this patent application.
Figure 32 shows the synthesis of the conjugates of MMAF analogs via the bridge linkers of this patent application.
Figure 33 shows the synthesis of the conjugates of MMAF analogs and tubulysin analogs via the bridge linkers of this patent application.
Figure 34 shows the synthesis of the conjugates of tubulysin analogs via the bridge linkers of this patent application.
Figure 35 shows the SDS-PAGE gel containing reduce agent DTT in the development. Lane 1 is biomarker, Lane 2 is conjugate 201, Lane 3 is conjugate 206, Lane 4 is conjugate 354, Lane 5 is Tubulysin analog conjugate through the traditional maleimidopropanoyl linker that we disclosed previously (Huang Y. et al, Med Chem. #44, 249th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; W02014009774). The conjugates 201, 206 and 354 via the bridge linkers of this patent application have the major band of 75KD which indicates that the heavy chain and the light chain of the mAb were crossly linked with the bridge linkers. In comparison, the traditional ido linker has no 75KD band, and the 50KD band is the single heavy chain of the antibody.
Figure 36 shows the comparison of the anti-tumor effect of conjugate compounds 196a, 201, 206, 354, 358, 377, 385 and 407 with T-DM1 using human gastric tumor N87 cell model at dosing of 6 mg/kg, i.v., one injection. Top figure indicates that all the 9 conjugates did not cause the animal body weight loss. The bottom figure shows that conjugate compounds 196a, 201, 206, 277, 354, and 407 had better antitumor ty than T-DM1. In particular, compounds 201 and 407 were able to eliminate the tumors until the end of experiment.
DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS "Alkyl" refers to an aliphatic arbon group or univalent groups derived from alkane by removal of one or two en atoms from carbon atoms. It may be straight or branched having 1 to 8 carbon atoms in the chain. "Branched" means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, z'-propyl, n-butyl, /-butyl, yl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, 3,3-dimethylpentyl, trimethylpentyl, 3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, methylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3- methylheptyl, n-hcptyl, tyl, l, and isooctyl. A Ci-Cg alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-Cg alkyl,(Ci-Cg alkyl), -aryl, -C(0)R', -OC(0)R', -C(0)OR', -C(0)NH2, -C(0)NHR', -C(0)N(R')2, -NHC(0)R', - SR', -S(0)2R', -S(0)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-Cg alkyl and aryl.
"Halogen" refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom.
"Heteroalkyl" refers to C2-Cg alkyl in which one to four carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. cycle" refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms ed as a bicycle [5,6] or [6,6] system. entative Cs-Cg carbocycles e, but are 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.
A "Cs-Cg carbocycle" refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated nonaromatic carbocyclic ring. A 0,-Cg carbocycle group can be tituted or substituted with one or more groups including, but not limited to, -Ci-Cg alkyl,(Ci-Cg alkyl), -aryl, -C(0)R', - 0C(0)R', -C(0)0R', -C(0)NH2, -C(0)NHR', -C(0)N(R')2, )R', -SR', -S(0)R',-S(0)2R', - OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-Cg alkyl and aryl.
"Alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbutenyl, n-pentenyl, hexylenyl, heptenyl, octenyl.
"Alkynyl" refers to an aliphatic hydrocarbon group containing a -carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain. ary alkynyl groups include ethynyl, propynyl, n-butynyl, nyl, 3-methylbutynyl, 5-pentynyl, ynyl, hexylynyl, heptynyl, and octynyl.
"Alkylene" refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two lent radical centers derived by the removal of two en atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene ls include, but are not limited to: methylene (-CH2-), 1,2-ethyl (-CH2CH2-), opyl (- CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like. ylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (-CH=CH-).
"Alkynylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene, gyl and 4-pentynyl.
"Aryl" or Ar refers to an aromatic or hetero aromatic group, composed of one or several WO 59622 2016/050580 rings, comprising three to en carbon atoms, preferentially six to ten carbon atoms. The term of "hetero aromatic group" refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N. The term aryl or Ar also refers to an aromatic group, wherein one or several H atoms are replaced independently by -R’, -halogen, -OR’, or -SR’, -NR’R", -N=NR’, -N=R’, -NR’R 9 9 N02, -S(0)R’, -S(0)2R’, -S(0)20R’, -0S(0)20R’, -PR’R", -P(0)R’R", -P(OR’)(OR"), - R’)(0R") or -0P(0)(0R’)(0R") wherein R’, R9 9 are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts.
"Heterocycle" refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also bed in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, the disclosure of which is hereby incorporated by reference. Preferred nonaromatic heterocyclic include, but are not limited to epoxy, aziridinyl, thiiranyl, idinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, linyl, thiazolidinyl, tetrahydrothiopyranyl, nyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused systems resulting from the condensation with a phenyl group.
The term "heteroaryl" or aromatic heterocycles refers to a 3 to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi-, or multi-cyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl, l, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, l, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, lyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-A-oxide, as well as the fused systems resulting from the condensation with a phenyl group.
"Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocyclic" and the like refer also to the corresponding "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene", ocyclene" and the likes which are formed by the removal of two hydrogen atoms.
"Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a al or sp carbon atom, is replaced with an aryl radical. Typical a arylalkyl groups include, but are not d to, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, 2- ophenylethan-l-yl and the like.
"Heteroarylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, lly a al or sp carbon atom, is replaced with a heteroaryl a l. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl and the like.
Examples of a "hydroxyl protecting group" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, ydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, /-butyldimcthylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate , pivaloate, benzoate, methanesulfonate and p-tolucncsulfonatc.
"Leaving group" refers to a onal group that can be tuted by another functional group. Such leaving groups are well known in the art, and es include, but are not limited to, a halide (e.g., chloride, bromide, and iodide), methanesulfonyl (mcsyl), p-tolucncsulfonyl (tosyl), trifluoromethylsulfonyl ate), and trifluoromethylsulfonate.
The following abbreviations may be used herein and have the indicated definitions: Boc, tertbutoxy carbonyl; BroP, bromotrispyrrolidinophosphonium hexafluorophosphate; GDI, 1,1'- carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl-aluminium hydride; DIPEA, diisopropylethylamine; DEPC, diethyl phosphorocyanidate; DMA, N,N- dimethyl acetamide; DMAP, 4-(N, N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDC, l-(3-dimethylaminopropyl) ethylcarbodiimide hydrochloride; ESTMS, electrospray mass spectrometry; HATU, 0-(7- azabenzotriazol-l-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate; HOBt, 1- ybenzotriazole; HPLC, high re liquid chromatography; NHS, N- Hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphatebuffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size-exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TEA, trifluoroacetic acid; THE, tetrahydrofuran; Val, valine.
The "amino acid(s)" can be l and/or unnatural amino acids, preferably alpha-amino acids. Natural amino acids are those encoded by the genetic code, which are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, cine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine. The unnatural amino acids are derived forms of proteinogenic amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma- aminobutyric acid (the neurotransmitter), ornithine, line, beta alanine nopropanoic acid), gamma-carboxyglutamate, selenocysteine nt in many noneukaryotes as well as most eukaryotes, but not coded directly by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formylmethionine (which is often the initial amino acid of proteins in bacteria, mitochondria, and chloroplasts), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimetics. Analogs are compounds having the same general H2N(R)CHC02H structure of a natural amino acid, except that the R group is not one found among the natural amino acids. Examples of analogs include homoserine, norleucine, methionine-sulfoxide, and methionine methyl sulfonium. Preferably, an amino acid mimetic is a compound that has a structure different from the l al ure of an alpha-amino acid but ons in a manner similar to one. The term "unnatural amino acid" is intended to represent the "D" stereochemical form, the l amino acids being of the "L" form. When 1-8 amino acids are used in this patent application, amino acid sequence is then preferably a cleavage recognition sequence for a protease. Many cleavage recognition sequences are known in the art.
See, e.g., Matayoshi et al. e 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. l. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); the disclosures of which are incorporated herein by nce. In particular, the sequence is selected from the group consisting of Val-Cit, Ala- Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, a-Asn, Lys, Cit, Ser, and Gin.
"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an e, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
"Pharmaceutically acceptable solvate" or "solvate" refer to an ation of one or more solvent molecules and a sed compound. es of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
"Pharmaceutically acceptable ent" includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, 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 for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the itions as suitable therapeutic combinations.
As used herein, "pharmaceutical salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include the conventional xic salts or the quaternary ammonium salts of the parent compound formed, for e, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from nic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, esulfonic, glucuronic, glutamic, c, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.
The pharmaceutical salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional al methods. Generally, such salts can be ed via reaction the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, ueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are red. Lists of suitable salts are found in Remington's ceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
"Administering" or "administration" refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a t. Such modes include oral administration, topical t, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device.
The novel conjugates disclosed herein use the bridge linkers. Examples of some suitable s and their synthesis are shown in Figures 1 to 34.
THE BRIDGE LINKERS The synthetic routes to e bridge linkers as well as the preparation of the conjugates of drugs to a cell binding molecules of the present invention are shown in Figures 1-34. The bridge s possess two elements: a) A tuent that is a 2,3-disubstituted succinic group; or 2- monosubstituted, or 2,3-disubstitutedfumaric group; or 2-monosubstituted, or 2,3- disubstitutedmaleic group; or acetylenedicarboxyl group, which can react to a pair of thiols to form covalent thioether bonds, and b) A group, such as but not limited to, a disulfide, maleimide, haloacetyl, de, ketone, azide, amine, alkoxyamine, ide, ethenesulfonyl, acyl halide(acid halide), acryl (acryloyl), and/or acid anhydride group, capable of reaction with a drug.
The bridge substituents of 2,3-disubstituted succinic group; or 2-monosubstituted, or 2,3- disubstituted fumaric group; or 2-monosubstituted, or 2,3-disubstitutedmaleic group; or acetylenedicarboxyl group; can be introduced by direct condensation of these 2,3- disubstitutedsuccinic acid, or 2-monosubstitutedor 2,3-disubstitutedfumaric or maleic, or acetylenedicarboxyl groups with an amine, an alcohol, or a thiol group to form amide, ester or thioester bonds. The synthesis of these bridge linkers and their application for antibody conjugation are exampled in the Figures 1-34.
Preferably, the bridge linkers are compounds of the a (I) below: o o (Zj—R^Xt^U-7 II X2-fR2)m2 V 'mi u \ U' (I) Wherein "---- " represents an optional single bond; "==" represents either a single bond, or a double bond, or a triple bond.
It provided that when == represents a single bond, both U and U’ are not H; when == represents a double bond, either U or U’ can be H, but are not H at the same time; when == represents a triple bond, both U and U’ can be absent.
O O ^-7 II s Wherein the ent: u \ U' , which is 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstitutedfumaric group, or 2-monosubstituted or 2,3- disubstitutedmaleic group, or acetylenedicarboxyl group, is capable of reacting with a pair of thiols of the cell-binding agent; The pair of thiols are preferred pairs of sulfur atoms reduced from the inter chain disulfide bonds of the cell-binding agent by a ng agent, such as dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine (P-MEA), or/and beta mercaptoethanol (P-ME, 2- U and esent the same or ent leaving group that can be substituted by a thiol. Such leaving groups are, but are not limited to, a halide (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl,N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, dazole- l-yl,chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxyl, 2-ethyl phenylisoxazolium-3'-sulfonyl, phenyloxadiazole-sulfonyl (-sulfone-ODA), oxadiazol-yl, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions.
Zi and Z2 are the same or different a function group that enables to react with a cytotoxic drug, to form a disulfide, her, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, ry, or quarter), imine, eteroalkyane, heteroaromatic, me or amide bond; Ri can be absent, or can be ed from Ci-Cg of alkyl; Ci-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; Cs-Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, arbonyl, heteroaryl; or 1-8 carbon atoms of esters, ether, or amide; or polyethyleneoxy unit of formula (OCHiCt^p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or combination f.
Additionally Ri is a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, which ntly connects to Xi and Zi. The atoms used in forming the Ri may be combined in all chemically relevant ways, such as forming alkane, alkenylene, alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, , ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination thereof.
Xi and X2are independently selected from NH; NHNH; N(R3); N(R3)N(R3-); O; S; B(R3); Si(R3)N(R3’); P(0)(R3); Ci-Cg of alkyl; C2-C8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; Cs-Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 amino acids; Wherein R3 and R3’ are independently H; Ci-Cg of alkyl; C2-C8 of heteroalkyl, ycloalkyl, heterocycloalkyl; C3-Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 carbon atoms of esters, ether, or amide; or polyethyleneoxy unit of formula (OCH2CH2)por (OCH2CH(CH3))p, wherein p is an r from 0 to about 1000, or combination f. In addition, Xi and X3 can be independently absent.
R2is independently ed from OH, H, NH2, SH, NHNH2, N(R3)(R3-); N(R3)NH(R3-); polyethyleneoxy unit of formula (OCH2CH2)pOR3i or( OCH 2CH(C H3))pOR3 or NH(CH2CH20)pR3, or NH(CH2CH(CH3)0)PR3, or N[(CH2CH20)PR3][ (CH2CH20)p R3 ],or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; Ci-Cg of alkyl; C2-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; Cs-Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, lkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; Wherein R3 and R3’ are independently H; Ci-Cg of alkyl; C2-C8 of heteroalkyl, ycloalkyl, heterocycloalkyl; C3-C8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, aryl; or 1-8 carbon atoms of esters, ether, or amide; or 1-8 amino acids; or polyethyleneoxy unit of formula t^p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or ation thereof. mi and m2 are independently an integer from 1 to 30, preferably from 1 to 10.
In another embodiment, Ri, R3 and R3’, can be respectively a chain of atoms selected from C, N, O, S, Si, and P which covalently connects the cell-surface binding molecule and/or the conjugated drug. The atoms used in forming the bridge linker may be combined in all chemically relevant ways, such as g alkylene, alkenylene, and alkynylene, ethers, yalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, lamines, urethanes, amino acids, acyloxylamines, hydroxamic acids, and many others. In addition, it is to be understood that the atoms forming the linker (L) may be either saturated or unsaturated, or may be radicals, or may be cyclized upon each other to form divalent cyclic structures, including lkanes, cyclic ethers, cyclic amines, arylenes, arylenes, and the like in the linker.
Examples of the functional group, Zi, which s linkage of a cytotoxic drug, include groups that enable linkage via a disulfide, thioether, thioester, peptide, hydrazone, ester, ate, carbonate, alkoxime or an amide bond. Such functional groups include, but are not limited to, thiol, ide, amino, carboxyl, aldehydes, ketone, maleimido, haloacetyl, hydrazines, amino, and/or hydroxy.
Examples of the functional group, Zi, that enables reaction with the terminal of amine of a drug/ cytotoxic agent, can be, but not limited to, N-hydroxysuccinimidc esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, carboxylic acid chlorides or carboxylic acid anhydride; With the terminal of thiol, can be, as but not limited to, pyridyldisulfides, nitropyridyldisulfides, maleimides, haloacetates, methylsulfonephenyloxadiazole (ODA), carboxylic acid chlorides and carboxylic acid anhydride; With the al of ketone or de, can be, as but not limited to, amines, alkoxyamines, hydrazines, acyloxylamine, or hydrazide; With the terminal of azide, can be, as but not limited to, alkyne. Examples of these function groups are displayed below: o o o o N-hydroxysuccini mide ester; O maleimide; O O s < X' R5‘ X disulfide; haloacetyl; acyl halide(acid halide), S-X2'-i o"-w o II V Ts-'O^^^X 'Ar, o ethenesulfonyl; acryl (acryloyl); ^ 2 Ms'°^X2'A (tosyloxy)acetyl; 2-(mesyloxy)acetyl; (nitrophenoxy)acetyl; o2n 2-(dinitrophenoxy)acetyl; 2-(fluorophenoxy)-acetyl; F (difluorophenoxy) - acetyl; '°^x2A 2-(((trifluoromethyl)-sulfonyl)oxy)acetyl; F^°^xA ketone, or aldehyde, F F tafluorophenoxy) acetyl; N-N &xA)2 , methylsulfonephenyloxadiazole (ODA); o R&o^xAO acid anhydride, alky loxy amino; Nj----- S5 azido, r3 alkynyl, orh2nhn hydrazide. Wherein Xi’ is F, Cl, Br, I or Lvy X2’ is O, NH, N(Ri), or CH2; R3 and R5 are independently H, Ri, aromatic, heteroaromatic, or ic group wherein one or several H atoms are replaced independently by -Ri, -halogen, -ORi, -SRi, -NRiR2, - N02, i,-S(0)2Riior -COORi; Lvs is a leaving group selected from nitrophenol; N- hydroxysuccinimide (NHS); phenol; ophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; uorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethylphenylisoxazolium-3'- sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, WO 59622 formyl anhydride; or an intermediate molecule generated with a condensation reagent for e coupling reactions or for Mitsunobu reactions.
In preferred embodiments, Ri, R2, R3 and R3’, are linear alkyl having from 1-6 carbon atoms, or polyethyleneoxy unit of formula Hi^p = 1-100.
The key step of synthesis of the bridge linker containing 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstitutedfumaric group, or 2-monosubstituted or 2,3- disubstitutedmaleic group, or acetylenedicarboxyl group, is the condensation of the 2,3- disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstitutedfumaricacid,or 2- monosubstituted or 2,3-disubstitutedmaleic acid, or acetylenedicarboxyl group, or their acid derivatives, with the other components containing an amine (1° or 2° amines), alcohol, or thiol on their terminal, as shown in the following scheme (la) and (lb): o o o o Lvj—^ II + X,---- R'-/ /-Ri-x,-!! II /-----\ Lv2 /----\ ■Lv2 u U' u U’ *2—*2-/ o VU-x2-r2-| u/ U' (la) o o o o Lvr-U ^-U-Lv, + X2-R2-/ Lvj—^ u/ /—\ U' u U' o o *2—R2-/ ^R1-X1JJ II x2-R2-| u/—\U' (lb) Wherein Xi, X2. R|. and R2 are described the same in Formula (I).
Lvi and Lv2 are the same or independently OH; F; Cl; Br; I; nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole;dichlorophenol;tetrachlorophenol;lhydroxybenzotriazole ; tosylate; mesylate; 2-ethylphenylisoxazolium-3'-sulfonate,anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl ide; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions, e.g. condensation reagents are: EDC Dimethylaminopropyl)-N'- arbodiimide), DCC lohexyl-carbodiimide), N,N'-Diisopropylcarbodiimide (DIC), NCyclohexyl-N'- (2-morpholino-ethyl)carbodiimide metho-p-toluenesulfonate (CMC,or CME-CDI), arbonyldiimi-dazole (CDI), TBTU (0-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluroninm tetrafluoroborate), N,N,N',N'-Tetramethyl(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU), (Benzotriazol-l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-l-yloxy)tripyrrolidinophosphonium uorophosphate (PyBOP), Diethyl cyanophosphonate (DEPC), Chloro-N,N,N',N'-tetramethylformamidiniumhexafluorophosphate, l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate , 1 -[(Dimethylami-no)(morpholino)methylene]-1H-[ 1,2,3]triazolo[4,5-b]pyridine- 1-ium 3-oxide hexafluorophosphate (HDMA), 2-Chloro-l,3-dimethyl-imidazolidinium hexafluorophosphate (CIP), Chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), Fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N',N'- Tetramethyl-S-(l-oxidopyridyl)thiuronium hexafluorophosphate, 0-(2-Oxo-l(2H)pyridyl)- N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(l-Oxidopyridyl)-N,N,N',N'- tetramethylthiuronium tetrafluoroborate, 0-[(Ethoxycarbonyl)-cyanomethylenamino]-N,N,N',N'- tetramethyluronium hexafluorophosphate (HOTU), (l-Cyanoethoxyoxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), O-(Benzotriazol-l-yl)- ,N'-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), N-Benzyl-N'-cyclohexylcarbodiimide (with, or without polymer-bound), olidino(N-succinimidyl-oxy)carbenium hexafluoro-phosphate (HSPyU), Chlorodipyrrolidinocarbenium hexafluorophosphate ), 2- Chloro-l,3-dimethylimidazolidinium tetrafluoroborate(CIB), (Benzotriazol-l-yloxy)dipiperidinocarbenium hexafluorophosphate (HBPipU), 0-(6-Chlorobenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TCTU), Bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, T3P®), 2-Morpholinoethyl isocyanide (MEI), N,N,N',N'-Tetramethyl(N-succinimidyl)uronium hexafluorophosphate , 2-Bromo-l-ethyl-pyridinium tetrafluoroborate (BEP), 0-[(Fthoxycarbonyl)cyanomethylenamino ]-N,N,N',N'-tetra-methyluronium tetrafluoroborate (TOTU), -Dimethoxyl ,3,5-triazinyl)methylmorpholiniumchloride (MMTM, DMTMM), N,N,N',N'-Tetramethyl- 0-(N-succinimidyl)uronium tetrafluoroborate (TSTU), 0-(3,4-Dihydrooxo-1,2,3-benzotriazin- 3-yl)-N,N,N',N'-tetramethyluronium tetrafluoro-borate (TDBTU), 1,1 '-(Azodicarbonyl)- dipiperidine (ADD), Di-(4-chlorobenzyl)azodicarboxylate (DCAD), t-butyl azodicarboxylate (DEAD),Diisopropyl azodicarboxylate (DIAD), Diethyl arboxylate (DEAD).In on, Lvl and Lv2 can be an anhydride, formed by acid themselves or formed with other Ci-Cg acid anhydrides.
The detail examples of the synthesis of the bridge linkers are shown in the figures 1-33.
Normally the bridge substituents of 2,3-disubstituted succinic group, or substituted or 2,3- disubstitutedfumaric group, or substituted or 2,3-disubstitutedmaleic group, or acetylenedicarboxyl group, can be condensated with linker components containing function groups capable to react to drugs of desired conjugation.
WO 59622 CELL-BINDING AGENT-DRUG CONJUGATES The conjugates of the present invention can be represented by the following formula, >8 /Drug, x , wherein Cb is a cell-binding agent, L is linker containing succinic, fumaric or maleic group, or acetylenedicarboxyl group, Drugi is a drug molecule, n is an r from 1 to 30, and two S (sulfur) elements from Cb bridgely link to L, which covalently connects a drug and a functional le R2.
The bridge linker L may be composed of one or more linker ents. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citmlline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoate ("SPP"), 4-(N-maleimidomethyl)-cyclohexane-l carboxylate ("MCC"), (4-acetyl)aminobenzoate ("SIAB"), 4-thio-butyrate (SPDB), 4-thiohydroxysulfonylbutyrate fo-SPDB), ethyleneoxy -CH2CH2O— as one or more repeating units ("EO" or "PEG"). Additional linker components are known in the art and some are described herein. e structures of these components containing linkers are: O ; (MC, 6-maleimidocaproyl containing) N, s o (MP, maleimidopropanoyl containing) VHNtju> HV o H (PAB, p-aminobenzyloxycarbonyl containing) O o o S^vTVsA ^ , O O o O r,fi iEnh o h Ujx V 1 O I ____ _ c O o ii\^y (ME, maleimidoethyl containing).
. H O n O H < O Q h2n N H H H HN ',l1 *12JN H i i v (valine-citmlline containing) o O I V-f-acio-B-SA O o (MCC, aleimidomethyl)cyclohexane-l carboxylate containing) HL" i/w-5*. v" O o ^ NaOtij|a. ((4-acetyl)aminobenzoate containing) 9 (4-thiohydroxysulfonyl- /Xs/Jv^y\ butyrate, 2-sulfo-SPDB), (PAB), iO 4-thio-pentanoate (SPP), O 4-thio-butyrate (SPDB), O 4-(N-maleimidomethyl)cyclo-hexane-1 - r SOE carboxylate (MCC), O maleimidoethyl (ME), o 4-thio ysulfonyl-butyrate (2-Sulfo-SPDB), aryl-thiol (PySS), i_o-0>-NsA (4-acetyl)amino-benzoate , oxylbenzylthio, aminobenzylthio, HN^ 3^ s dioxylbenzylthio, diaminobenzylthio, J amino- nzylthio, alkoxy amino (AOA), ethyleneoxy(EO), V'S'W*- /—N N dithio, ^ 4-methyldithio-pentanoic (MPDP), 'S==V O O H Y^Y ^rV H o H triazole, o alkylsulfonyl, o alkylsulfonamide, o sulfon- O O S-M n H bisamide, OH Phosphondiamide, OH alkylpho mide, O O OH phosphinic acid, OH N-methylphosphonamidic acid, I " i M H v-n-p-n-^ HN c OH N,N’-dimethylphosphonamidic acid, N,N’- N—N. I V % dimethylphosphondiamide, hydrazine, acetimidamide, O O S5- ^^-N-N-^jS5 A N-O^/ y oxime, vAA acetylacetohydrazide, / ^ X thyl-amine, y aminoethyl-aminoethyl-amine.
Preferably, the conjugates have the following Formula (II): ( Drug] RjA—} Xi SX ' ' m | Cb (r2}-X2 V m2 O n Wherein: Cb represents a cell-binding agent, preferably an antibody, which conjugates to a Drug, Dmgi and a function molecule, R2, via a pair of sulfur atoms (thiols).
Inside the bracket (square heses) are the linker-drug components that are conjugated to pairs of thiols of the cell-binding agent/molecule. The thiols are preferred pairs of sulfur atoms reduced from the inter chain disulfide bonds of the cell-binding agent by a ion agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris (2- carboxyethyl) phosphine (TCEP), aptoethylamine (P-MEA), or/and beta mercaptoethanol (P-ME, 2-ME).
Dmgi represents a cytotoxic agent, linked to a inding agent via the bridge linker of this patent through an alkyl, alkylene, alkenylene, alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, nes, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide, thioether, thioester, ate, carbonate, heterocyclic ring, heteroalkyl, heteroaromatic, or alkoxime bond, or combination above thereof. "==" representseither single bond or double bond. n is 1 ~ 30; Ri, R2, mi, m2, Xi and X2 are described the same previously in Formula (I).
As described in more detail below, Dmgi can be any of many small molecule drugs, including, but not limited to, tubulysins, calicheamicins, auristatins, maytansinoids, CC-1065 s, morpholines doxorubicins, taxanes, cryptophycins, amatoxins (amanitins), epothilones, geldanamycins, duocarmycins, daunomycins, methotrexates, vindesines, vincristines, and benzodiazepine dimers (e.g., dimmers of obenzodiazepine (PBD) or tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines).
To synthesize the conjugate, the cell-binding agent can be first modified with the bridge s of the present invention through reduction of disulfide bonds of the cell-binding molecule.
The yielded a pair of free thiols can react to the bridge linker of a (I) at pH 5-9 aqueous media with or without addition of 0-30% of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THE, panol, dioxane, propylene glycol, or ethylene diol, to uce the reactive group of Zi containing disulfide, maleimido, haloacetyl, azide, 1-yne, ketone, aldehyde, alkoxyamino, or hydrazide groups, as well a function le group R2, which is preferably a polyethylene glycol, carboxylic acid, or carboxylic amide, or ester, or other tives. Then a reactive group of a cytotoxic agent reacts to the ed cell-binding molecule accordingly. For example, synthesis of the cell-binding agentdmg conjugates linked via disulfide bonds is achieved by a disulfide exchange n the disulfide bond in the modified cell-binding agent and a drug containing a free thiol group.
Synthesis of the cell-binding agent-drug conjugates linked via thioether is achieved by reaction of the maleimido or haloacetyl or ethylsulfonyl modified inding agent and a drug containing a free thiol group. Synthesis of conjugates bearing an acid labile hydrazone can be achieved by reaction of a carbonyl group with the hydrazide moiety in the linker, by methods known in the art (see, for example, P. Hamann et ah, Cancer Res. 53, 3336-34, 1993; B. Laguzza et ah, J. Med.
Chem., 32; 548-55, 1959; P. Trail et ah, Cancer Res., 57; 100-5, 1997). Synthesis of conjugates bearing triazole linkage can be ed by on of a 1-yne group of the drug with the azido moiety in the linker, through the click chemistry (Huisgen cycloaddition) (Lutz, J-F. et al, 2008, Adv. Drug Del. Rev. 60, 958-70; Sletten, E. M. et al 2011, AccChem. Research 44, 666-76).
Alternatively, the drug can react with the bridge linkers of the present invention that have conjugated to a cell-binding molecule to give a modified cell-binding molecule linker of Formula (III) bearing functionalities. For e, a thiol-containing drug can react with the modified nding molecule bridge linker of Formula (III) bearing a maleimido, or a haloacetyl, or an ethylsulfonyl substituent at pH 5.5-9.0 in aqueous buffer to give a cell-binding molecule-drug conjugate via a thioether linkage. A thiol-containing drug can undergo disulfide ge with a modified bridge linker of Formula (III) bearing a pyridyldithio moiety to give a conjugate a disulfide bond linkage. A drug bearing a hydroxyl group or a thiol group can be reacted with a ed bridge linker of Formula (III) bearing a n, ularly the alpha halide of carboxylates, in the presence of a mild base, e.g. pH 8.0-9.5, to give a modified drug bearing an ether or thiol ether link. A hydroxyl group containing drug can be condensed with a bridge cross linker of Formula (I) g a carboxyl group, in the presence of a dehydrating agent, such as EDC or DCC, to give ester linkage, then the subject drug modified bridge linker undergoes the conjugation with a cell-binding molecule. A drug containing an amino group can condensate with a carboxyl ester of NHS, imidazole, henol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol;tetrachlorophenol; 1-hydroxyben-zotriazole; tosylate; mesylate; 2-ethylphenylisoxazolium-3'-sulfonate on the cell-binding molecule-bridge linker of Formula (III) to give a conjugate via amide bond e.
The ate may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, and ion exchange or by is. In some cases, a small molecule as a cell-binding agent (e.g. folic acid, melanocyte stimulating hormone, EOF etc) ated with a small molecular drugs can be purified by chromatography such as by HPLC, medium pressure column chromatography or ion ge chromatography.
MODIFIED CELL-BINDING AGENTS/MOLECULES The cell-binding agent modified by reaction with linkers of the present invention is preferably represented by the Formula (III): (zt-rA-iX| sx ' ‘'in | Cb (RihXj ' m2 O n Wherein == represents either a single bond, or a double bond.
Cb, Zi, Z2, n, Ri, R2, mi, m2, Xi, and X2 are defined the same as in Formula (I) and (II).
In preferred embodiments, Zi is a disulfide substituent, maleimido, haloacetyl, alkoxyamine, azido, ketone, aldehyde, ine, alkyne, an /V-hydroxysuccinimide ester, or a carboxyl ester formed with phenol; ophenol; pentafluorophenol; tetrafluoro-phenol; rophenol; monofluorophenol; pentachlorophenol; triflate; ole; dichlorophenol;tetrachlorophenol;lhydroxybenzotriazole ; tosylate; mesylate; 2-ethylphenylisoxa-zolium-3'-sulfonate. Zi can then react with a cytotoxic agent h disulfide, thioether, hydrazone, amide, me, carbamate, ester, ether bond or hetero-aromatic ring. The modified cell-binding agent can be prepared via a reaction of the cell-binding agent with the bridge linkers of Formula (I) as described in Formula (II) above.
In order to achieve a higher conjugation reaction yield of substituted succinic group, or 2-monosubstituted or 2,3-disubstitutedfumaric group, or substituted or 2,3- disubstitutedmaleic group, or acetylenedicarboxyl group, on the bridge s of the Formula (I) with a pair of free thiols on the cell-binding molecule, preferably on an antibody, a small percentage of organic vent may be required to add to the reaction mixture, as well in the solution after the reaction to maintain lity of the Formula (III) in s solution. To modify the cell-binding agents, the cross-linking reagent (bridge linker) of Formula (I) can be first dissolved in a polar organic solvent that is miscible with water, for example different alcohols, such as methanol, ethanol, and propanol, acetone, acetonitrile, tetrahydrofuran (THE), 1,4- dioxane, dimethyl formamide (DMF), dimethyl acetamide (DMA), or dimethylsulfoxide (DMSO) at a high concentration, for example 1-500 mM. Meanwhile, the cell-binding molecule, such as antibody dissolved in an aqueous buffer pH 5-9.5, preferably pH 6-8.5, at 1-35 mg/ml concentration was d with 1-20 equivalent of TCEP or DTT for 20 min to 48 hour. After the reduction, DTT can be removed by SEC chromatographic purification. TCEP can be optionally removed by SEC chromatography too, or staying in the reaction mixture for the next step reaction without cation. Furthermore, the reduction of antibodies or the other cell-binding agents WO 59622 with TCEP can be performed with a bridge linker of Formula (I), for which the cross-linking conjugation for the cell-binding molecules can be achieved simultaneously along with the TCEP reduction.
The aqueous solutions for the modification of inding agents are buffered between pH 6 and 9, preferably between 6.5 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges. Typical buffers include phosphate, triethanolamine HC1, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, sucrose and salts, for examples, NaCl and KC1. After the addition of the bridge linker of Formula (I) into the solution containing the d cell-binding molecules, the reaction mixture is incubated at a temperature of from 4 °C to 45 °C, preferably at ambient temperature. The progress of the reaction can be monitored by measuring the decrease in the absorption at a certain UV wavelength, such as at 254 nm, or increase in the absorption at a certain UV wavelength, such as 280 nm, or the other appropriate wavelength. After the reaction is complete, isolation of the modified cell-binding agent can be performed in a routine way, using for example gel filtration chromatography, or adsorptive tography.
The extent of modification can be assessed by ing the absorbance of the nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxylamidopyridine ne and dicarboxylamidopyridine ne group released via UV a. For the conjugation without a chromophore group, the modification or conjugation reaction can be monitored by LC-MS, preferably by TOF mass spectrometry, or ry electrophoresis-mass ometry (CE-MS). The bridge linkers described herein have e functional groups that can react with any drugs, preferably cytotoxic agents that possess a suitable tuent. For examples, the modified cell-binding molecules bearing an amino or hydroxyl substituent can react with drugs g an N-hydroxysuccinimide (NHS) ester, the modified cell-binding molecules bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group.
Additionally, the modified cell-binding molecules bearing a carbonyl (ketone or aldehyde) substituent can react with drugs bearing a hydrazide or an alkoxyamine. One skilled in the art can readily ine which linker to use based on the known reactivity of the available functional group on the linkers.
MODIFIED CYTOTOXIC DRUGS The cytotoxic drugs modified by reaction with cross-linkers of the present invention are preferably represented by the Formula (IV): o o ( Drug!—R,4-Xi--il II -(r2) \ Jmi u/ \ U m2 Wherein "==" represents either a single bond, or a double bond, or a triple bond.
"---- ", U, U\ Dmgi, Dmg2, Ri, Ri, mi, m2, Xi, and X2 are defined the same as in Formula (I) and (II).
The modified drugs can be prepared via reaction of the drug with the s of the Formula (I) to give a ed drug of Formula (IV) bearing functionality of an 2,3-disubstituted succinic group, or 2-monosubstituted or 2,3-disubstitutedfumaric group, or substituted or 2,3- titutedmaleic group, or acetylenedicarboxyl group. But for drugs containing a thiol, or the drugs undergoing to conjugation of a inding molecule via the bridge linkers through thioether, thioester or disulfide bond, it is therefore preferred that the Drugi may be sized to connect to Ri in a piece of ents via the linkage of thioether, thioester or disulfide bond first. Then the sized i component is assembled to2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstitutedfumaric acid, or 2-monosubstituted or 2,3- disubstitutedmaleic acid, or acetylenedicarboxyl group, to form the bridge linker modified drugs of Formula (IV).
For examples of the synthesis, a thiol-containing drug can be reacted with the linker of components Ribearing a maleimido substituent at neutral pH in aqueous buffer to give a Ri-Dmgi compartment bearing thioether linkage, and ing by condensation with either 2,3- tituted succinic acid, or 2-monosubstituted or substitutedfumaric acid, or 2- monosubstituted or 2,3-disubstitutedmaleic acid, or acetylenedicarboxyl group, to give a modified drug of Formula (IV) bearing thioether linkage. A drug bearing a hydroxyl group can be reacted with a linker component Ri bearing a halogen, or a tosylate, or a mesylate, in the presence of a mild base, to give a Ri-Drugi compartment bearing ether linkage, and following by condensation with 2,3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstitutedfumaric acid, or 2- monosubstituted or 2,3-disubstitutedmaleic acid, or acetylenedicarboxyl group, to give a modified drug of Formula (IV) bearing thioether linkage. A hydroxyl group containing drug can be condensed with a linker of Formula (I) bearing a carboxyl group, in the presence of a ating agent, such as EDC or dicyclohexylcarbodiimide (DCC), to give a modified drug of Formula (IV) via ester linkage. A drug bearing a thiol group can also react the linker of components Ri bearing a maleimido or a vinylsulfonyl, or a haloacetyl group, give a Ri-Dmgi compartment bearing thioether linkage, and following by condensation with a compartment of substituted succinic acid, or 2-monosubstituted or 2,3-disubstitutedfumaric acid, or 2-monosubstituted or 2,3- disubstitutedmaleic acid, or acetylenedicarboxyl group, to give a modified drug of Formula (IV) bearing her linkage. An amino group containing drug can similarly undergo condensation with a carboxyl group on the bridge linker of Formula (I) to give a modified drug of Formula (IV) bearing amide bonds. The modified drug can be purified by standard s such as column chromatography over silica gel or alumina, crystallization, preparatory thin layer chromatography, ion ge chromatography, or HPLC.
CELL-BINDING AGENTS The cell-binding molecule, Cb, that comprises the conjugates and the modified cell-binding agents of the present invention may be of any kind presently known, or that become known, molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically ed.
The cell binding agents include, but are not limited to, large lar weight proteins such as, for example, full-length dies (polyclonal antibodies, monoclonal antibodies, , multimers, multispecific antibodies (e.g., bispecific antibodies); single chain antibodies; fragments of antibodies such as Fab, Fab', F(ab')2, Fv, [Parham, J. Immunol. 131, 2895-902 (1983)], fragments produced by a Fab expression library, diotypic (anti-id) antibodies, CDR's, diabody, triabody, tetrabody, miniantibody, small immune proteins (SIP), and epitopebinding fragments of any of the above which immune-specifically bind to cancer cell antigens, viral antigens, microbial antigens or a protein generated by the immune system that is capable of recognizing, binding to a specific antigen or exhibiting the desired biological activity (Miller et al (2003) J. of Immunology 170: 4854-61); interferons (such as type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon-gamma ); es such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such as androgens and ens, cyte-stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors (EGF), granulocyte-macrophage -stimulating factor (GM-CSF), transforming growth factors (TGF), such as TGFa, TGFp, insulin and insulin like growth factors (IGF-I, IGF-II) G-CSF, MCSF and GM-CSF [Burgess, Immunology Today, 5, 155-8 (1984)]; vaccinia growth factors (VGF); fibroblast growth factors (FGFs); smaller molecular weight proteins, poly-peptide, es and peptide hormones, such as bombesin, gastrin, gastrin-releasing peptide; plateletderived growth factors; interleukin and cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL- 6), leukemia tory factors, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins, such as folate; apoproteins and glycoproteins, such as transferrin [O'Keefe et al, 260 J.
Biol. Chem. 932-7 (1985)]; sugar-binding proteins or lipoproteins, such as lectins; cell nutrienttransport les; and small lar inhibitors, such as te-specific membrane antigen (PSMA) inhibitors and small molecular tyrosine kinase inhibitors (TKI), 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); rticles (Liong, et al, ACS Nano, 2008, 2, 2; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12); liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9); viral capsides (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93).
In general, a monoclonal antibody is preferred as a cell-surface binding agent if an appropriate one is available. And the antibody may be murine, human, humanized, chimeric, or derived from other species.
Production of antibodies used in the present invention involves in vivo or in vitro procedures or combinations thereof. Methods for producing onal anti-receptor e antibodies are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonal antibody is typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired n (Kohler, G.; Milstein, C. . Nature 256: 495-7). The detailed procedures are described in "Antibodies—A Laboratory Manual", Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988), which is incorporated herein by reference. Particularly monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with the n of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact specified receptors or inhibit receptor ty on target cells.
A monoclonal antibody used in the present invention can be produced by initiating a monoclonal hybridoma culture sing a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate n icity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the dy molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques, such as using protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusive chromatographies (particularly by ty for the specific antigen after protein A, and sizing column chromatography); centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
Media useful for the preparation of these compositions are both well-known in the art and commercially available and e synthetic culture media. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et ah, Virol. 8, 396 (1959)) supplemented with 4.5 gm/1 e, 0-20 mM glutamine, 0-20% fetal calf serum, several ppm amount of heavy metals, such as Cu, Mu, Fe, or Zn, etc, or/and the other heavy metals added in their salt forms, and with an anti-foaming agent, such as polyoxyethylene-polyoxypropylene block mer.
In addition, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with an oncovirus, such as Epstein-Barr vims (EBV, also called human herpesvims 4 (HHV-4)) or Kaposi's sarcoma-associated herpesvims (KSHV). See, 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. A monoclonal antibody may also be produced via an anti-receptor e or peptides containing the carboxyl terminal as described nown in the art. See Niman et ah, Proc. Natl. Acad. Sci. USA, 80: 4949-53 (1983); Geysen et ah, Proc. Natl. Acad. Sci. USA, 82: 178-82 (1985); Lei et al.
Biochemistry : 6675-88, (1995). Typically, the anti-receptor e or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen for producing anti­ receptor peptide monoclonal antibodies.
There are also a number of other well-known techniques for making monoclonal antibodies as binding molecules in this invention. Particularly useful are methods of making fully human antibodies. One method is phage display technology which can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity ment. Phage display has been thoroughly described in the literature and the ction and screening of phage display libraries are well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13 ; Little et al, Biotechnol Adv. 12(3): 539-55 (1994); Clackson et ah, Nature 352: 264-8 ; Huse et ah, Science 246: 1275-81 (1989).
Monoclonal antibodies derived by hybridoma technique from another species than human, such as mouse, can be humanized to avoid human anti-mouse antibodies when infused into humans. Among the more common methods of humanization of antibodies are complementaritydetermining region grafting and resurfacing. These methods have been extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 492; Liu et al, Immunol Rev. 222: 9-27 (2008); o et al, Front Biosci. 13: 1619-33 (2008); Lazar et al, Mol Immunol. 44(8): 8 (2007); Li et al, WO 59622 Proc. Natl. Acad. Sci. USA. ): 3557-62 (2006) each incorporated herein by reference.
Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, s, or other mammals, carrying large portions of the human immunoglobulin heavy and light chains, with an immunogen. Examples of such mice are: the Xenomouse. (Abgenix/Amgen), the HuMAb-Mouse ex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. Nos. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 016, 5,545,806, ,436,149 and 5,569,825. In human therapy, murine variable regions and human constant regions can also be fused to construct called "chimeric antibodies" that are considerably less immunogenic in man than murine mAbs (Kipriyanov et al, Mol Biotechnol. 26: 39-60 (2004); Houdebine, Curr Opin Biotechnol. 13: 625-9 (2002) each incorporated herein by nce). In on, site-directed mutagenesis in the variable region of an antibody can result in an antibody with higher affinity and specificity for its antigen (Brannigan et al, Nat Rev Mol Cell Biol. 3: 964- 70, (2002)); Adams et al, J Immunol Methods. 231: 249-60 (1999)) and exchanging constant regions of a mAb can improve its ability to e effector functions of binding and cytotoxicity.
Antibodies immune specific for a malignant cell antigen can also be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunespecific for a malignant cell antigen can be obtained commercially, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
Apart from an antibody, a peptide or n that bind/block/target or in some other way interact with the epitopes or corresponding receptors on a targeted cell can be used as a binding molecule. These peptides or proteins could be any random peptide or proteins that have an affinity for the epitopes or corresponding receptors and they don't necessarily have to be of the globulin family. These peptides can be ed by similar techniques as for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003, 23(4): 307-49). The use of peptides from such random e libraries can be similar to antibodies and antibody fragments.
The binding molecules of es or proteins may be conjugated on or linked to a large molecules or materials, such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, a dendrimer, as long as such attachment permits the peptide or n to retain its antigen binding specificity. es of antibodies used for conjugation of drugs via the bridge linkers of this tion for treating cancer, autoimmune disease, and/or infectious disease include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), mab (anti CD41 rin alpha-lib), Adah mum ah (anti-TNF-a), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-a); Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2), ALD518 IL-6), Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), imumab (anti-Rhesus ), Bapineuzumab (anti-beta d), Basiliximab (Simulect, antiCD25 (a chain of IL-2 receptor), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab CD125), Bertilimumab (anti-CCLll (eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti­ fibrin II beta chain), Bivatuzumab CD44 v6), Blinatumomab (BiTE, anti-CD19), ximab (cACIO, anti-CD30 TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anAg), Capromab, Catumaxomab (Removab, anti- EpCAM, anti-CD3), CC49 TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimzia anti-TNF-a), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumab x (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzumab (anti-MUCl), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti- CD40), umab ax, anti-CD25 (a chain of IL-2 receptor)), mumab (anti-CD38 (cyclic ADP ribose ase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B- lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), omab (anti-endotoxin), Edrecolomab ex, MAM7-1A, anti- EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CDlla), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), El si 1i mom ah (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD 18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrin cwPs), Exbivirumab ( anti-hepatitis B e antigen), Fanolesomab (NeutroSpec, D15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folate receptor 1), Felvizumab (antirespiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-y), Foravirumab rabies virus glycoprotein), Fresolimumab (anti- TGF-P), Galiximab (anti-CD80), Gantenerumab (anti- beta amyloid), Gavilimomab (anti-CD 147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-a), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab ade, anti-TNF-a), Intetumumab (anti-CD51), Tnohmomah (anti-CD25 (a chain of IL-2 or)), Inotuzumab CD22), Ipilimumab (anti-CD 152), Iratumumab (anti- CD30 (TNFRSF8)), Keliximab (anti-CD4), zumab (CEA-Cide, anti-CEA), Lebrikizumab (anti- IL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), imumab TGF beta 2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab CD33), Lucatumumab (anti-CD40), Lumiliximab (anti- CD23 (IgE receptor), Mapatumumab (anti-TRAIL-Rl), Maslimomab (anti- T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti- Rhesus factor), Motavizumab (Numax, anti-respiratory syncytial vims), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab 5T4), Natalizumab ri, anti-integrin 0.4). Ncbacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-a), Nimotuzumab cim, oc, GFR), momab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CDlla)), Ofatumumab (Arzerra, D20), Olaratumab (anti-PDGF-R a), Omalizumab (Xolair, gE Fc region), Oportuzumab EpCAM), Oregovomab x, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF,anti-EGFR), Panobacumab (anti- Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUCl), Pertuzumab (Omnitarg, 2C4,anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab (anti­ adenocarcinoma antigen), imab (anti-CD4), Pritumumab (anti-vimentin), PRO 140 (anti- CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), Rafivirumab (anti-rabies vims glycoprotein), Ramucimmab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), mmab (anti-cytomegalovims glycoprotein B), Reslizumab IL-5), Rilotumumab (anti-HGF), Rituximab era, Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-a), Rovelizumab (LeukArrest, anti-CDll, CD 18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevimmab (anti-cytomegalovims), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-a), Siltuximab (anti-IL-6), Siplizumab (anti- CD2), (Smart) MI95 (anti-CD33), Solanezumab (anti-beta amyloid), izumab (anti- sphingosinephosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte antigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin aiibPs), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (antitenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti- IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor), Toralizumab (anti- CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, (anti-HER2/neu), Tremelimumab CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti­ hepatitis B virus), Urtoxazumab (anti- Escherichia coli), Ustekinumab (Stelara, E-12, IL- 23), Vapaliximab AOC3 (VAP-1)), Vedolizumab, (anti-integrin C4P7), Veltuzumab (anti- CD20), Vepalimomab AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), n (antivascular integrin avb3), Volociximab integrin asPO, Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD 147 (basigin)), Zolimomab (anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory n 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst, for melanoma and solid tumors), 1591 (anti-PSMA, Weill l Medical School for prostate cancers), 225.28S [anti-HMW-MAA (High molecular weight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L. (Milan, Italy) for ma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst.
USA for ctal and c cancers), CYT-356 (Oncoltad®, for prostate cancers), HNK20 (OraVax Inc. for atory syncytial virus), ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DRIO, Peregrine Pharm. for Cancers), 5F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis toxic shock], MEDI-500 [T10B9, anti-CD3, TRaP (T cell receptor alpha/beta), complex, from Medlmmune Inc for Graft-versushost disease], RING SCAN [ anti-TAG 72 r associated glycoprotein 72), from Neoprobe Corp. for , Colon and Rectal cancers], Avicidin (anti-EPCAM elial cell adhesion molecule), anti-TACSTDl (Tumor-associated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 elial glycoprotein 2); anti-KSA; KS1/4 n; M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian, Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart ID 10 (Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11 (ImClone, NJ) and Cetuximab (ImClone, NJ) .
Other antibodies as cell binding molecules/ligands include, but are not limited to, are antibodies against the following antigens: Aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various s), CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid atase ate), epidermal growth factor (carcinomas), CD2 (Hodgkin’s disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, c, ovarian cancers, mune diseases, malignant s), 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 mia), CD56 (small cell lung cancers, n cancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma), CD66e (cancers), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas), CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3 ectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast, colorectal and lung cancers), DLL4 (delta-like-4), EGER (Epidermal Growth Factor Receptor, various s), CTLA4 (melanoma), CXCR4 (CD 184, Heme-oncology, solid tumors), Endoglin (CD 105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor or 2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3 idiotype (cancers), Heat shock proteins (cancers), HER1 (lung, stomach cancers), HER2 (breast, lung and n cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R in-like growth factor 1 receptor, solid , blood cancers), IL-2 or (interleukin 2 receptor, T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman’s e, IL6 dependent tumors), Integrins (avP3, a5pi, a6p4, alip3, a5p5, avP5, for various cancers), MAGE- 1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti­ transferrin or (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, dgkin's B cell lymphoma, ia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA ectal), gplOO (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), Paratope of anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-like testicular alkaline phosphatase an, testicular cancers), PSMA (prostate tumors), PSA (prostate), R0B04, TAG 72 r associated glycoprotein 72, AML, c, colorectal, ovarian cancers), T cell transmembrane protein rs), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancers), TNFRSF13B (tumor necrosis factor receptor amily member 13B, le a, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cell carcinoma), TRAIL-R1 (Tumor necrosis sis Inducing ligand Receptor 1,lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD 106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor associated antigens recognized by antibodies have been reviewed (Gerber, et al, mAbs 1:3, 247-53 (2009); Novellino et al, Cancer Immunol Immunother. 54(3), 187-207 (2005). Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76).
The cell-binding agents, more preferred antibodies, can be any agents that are able to against tumor cells, virus infected cells, microorganism ed cells, parasite ed cells, autoimmune cells, activated cells, myeloid cells, activated T-cells, B cells, or melanocytes. More specifically the cell g agents can be any agent/molecule that is able to against any one of the following antigens or receptors: CD3, CD4, CDS, CD6, CD7, CD8, CD9, CD10, CDlla, CDllb, CDllc, CD12w, CD14, CD15, CD16, CDwl7, 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, CD 120, CD125, CD126, CD127, CD133, CD134, CD135, CD137, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152,CD154, CD156, CD158, CD163, CD166, .CD168, CD174, CD180, CD184, CDwl86, CD194, CD195, CD200, CD200a, CD200b, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326, 4-1BB, SAC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4, Wnt- Activated Inhibitory Factor 1 or , Adenocarcinomaantigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, Amino-peptidase N, Amyloid beta, Androgen receptor, Angiopoietin 2, Angiopoietin 3, Annexin Al, Anthrax rotective antigen, Anti-transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracisanthrax, BAFF (B-cell ting factor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic anhydrase IX, Cardiac myosin, CCL11(C-C motif chemokine 11), CCR4 (C-C ine receptor type 4, , CCR5, CD3E (epsilon), CEA noembryonic antigen), CEACAM3, 5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clumping factor A,CRIPTO, FCSF1R (Colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, Granulocyte-macrophage colony-stimulating factor(GM-CSF)), CTLA4 (cytotoxic T-lymphocyte associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184),C-X-C ine receptor type 4, cyclic ADP ribose hydrolase, Cyclin Bl, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL4 (delta-like-ligand 4), DPP4 (Dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shiga toxintype-1, E. coli shiga ype-2, ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD 105), Endothelin B receptor, xin, EpCAM (epithelial cell on le), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia TV6-AML, FAP (Fibroblast activation proteinalpha), FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectin extra domain-B, FOLR (folate receptor), Folate or alpha, Folate hydrolase, lated antigen 1, F protein of respiratory syncytial vims, Frizzled receptor, l GM1,GD2 ganglioside, G-28 (a cell surface n glyvolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor a-chain, Growth differentiation factor 8, GP100, GPNMB (Transmembrane glycoprotein NMB), GUCY2C late cyclase 2C, guanylyl cyclase C(GC-C), intestinal Guanylate cyclase, Guanylate cyclase-C receptor, Heatstable enterotoxin receptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor), HHGFR, HIV-1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB , HMWMAA, Human chorionic gonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype, IGF1R , insulin-like growth factor 1 receptor), IGHE, IFN-y, Influeza hemag-glutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R leukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (Insulin-like growth factor 2), Integrins (a4, aiibPs, avP3, cuP?, a5pi, a6p4, a7p7,alip3, a5p5, avP5), Interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, LCK, Le, in, Y antigen, LFA-1 (Lymphocyte function-associated antigen 1, CDlla), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, , MAGE-2, MAGE-3, MAGE Al, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (Macrophage migration inhibitory factor, or glycosylation-inhibiting factor (GIF)), MS4A1 (membrane-spanning 4-domains subfamily A member 1), MSLN (mesothelin ), MUCl(Mucin 1, cell surfaceassociated (MUC1) orpolymorphic epithelial mucin (PEM)), MUC1-KLH, MUG 16 (CA125), MCPl(monocyte chemotactic protein 1), MelanA/MART 1, MLIAP , MPG, MS4A1 (membrane-spanning 4-domains subfamily A), MYCN, -associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 locyte antigen), Nectin-4 (ASG-22ME), NGF, Neural sis-regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TESl,P21, p53 nonmutant, P97, Paged, PAP, Paratope of N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death n 1,CD279), PDGF-Ra (Alpha-type platelet-derived growth factor receptor ), PDGFR-P, PDL-1, PLAC1, PLAP-like ular alkaline phosphatase, Platelet-derived growth factor receptor beta, Phosphate-sodium co-transporter, PMEL 17, Polysialic acid, Proteinase3 (PR1), Prostatic carcinoma, PS (Phosphatidylserine ), Prostatic carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor,RANKL, RhoC, Ras mutant,RGS5, R0B04, Respiratory syncytial virus, RON, Sarcoma translocation breakpoints,SART3, Sclerostin, SLAMF7 (SLAM family member 7), Selectin P, SDC1 (Syndecan 1), sLe(a), medin C, SIP (Sphingosinephosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, T cell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-a, TGF-P (Transforming growth factor beta), TGF-P 1, TGF-P2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2, TIM-1 14), Tn, TNF, TNF-a, 8, TNFRSF10B (tumor necrosis factor or superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor specific glycosylation ofMUCl, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD 106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, or cells expressing any insulin growth factor receptors, or any epidermal growth factor receptors.
In r specific ment, the cell-binding ligand-drug conjugates via the bridge s of this invention are used for the targeted treatment of cancers. The targeted cancers include, but are not limited, Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, ood, Cerebellar ytoma, Cerebral Astrocytoma, Ependymoma, Medulloblastoma, Supratentorial Primitive Neuroectodermal and Pineal Tumors, Visual Pathway and Hypothalamic Glioma), Breast , Carcinoid Tumor, intestinal, oma of Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Gallbladder , Gastric Cancer (Stomach), Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell oma, Kidney Cancer (renal cell cancer), eal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Lymphoma (AIDS-Related, Central Nervous , Cutaneous T- Cell, n's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic me, roliferative Disorders, Nasopharyngeal Cancer, lastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, n Cancer (Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer myosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter (Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, Skin Cancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma, Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular , Thymoma (Malignant), Thyroid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), Unusual Cancer of ood, Vaginal Cancer, Vulvar Cancer, Wilms' Tumor.
In another specific embodiment, the cell-binding-drug conjugates via the bridge linkers of this ion are used in ance with the itions and methods for the treatment or prevention of an autoimmune disease. The autoimmune diseases e, but are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's e, globulinemia, Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopic y, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear e, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis, Balo disease/Balo tric sclerosis, Bechets Syndrome, Berger's disease, staff s encephalitis, Blau syndrome, Bullous Pemphigoid, Castleman's disease, Chagas disease, Chronic Fatigue Immune Dysfunction Syndrome, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis, Chronic lyme disease, Chronic ctive pulmonary disease, Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Cranial arteritis, CREST me, Crohns Disease (a type of idiopathic inflammatory bowel diseases), g's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous ic sclerosis, Dressler's me, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic tis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed obulinemia, Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's alitis, Hashimoto's thyroiditis, Haemolytic anaemia, Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis ativa, Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia , Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic ocytopenic a (See Autoimmune ocytopenic purpura), IgA nephropathy (Also Berger's disease), ion body myositis, Inflammatory demyelinating polyneuopathy, Interstitial cystitis, ble Bowel Syndrome , Juvenile idiopathic tis, Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou 's Disease (Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, e's disease, Microscopic polyangiitis, Miller-Fisher me, Mixed Connective Tissue Disease, Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, Multiple Myeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, epsy, yelitis optica (Devic's Disease), Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage- Tumer syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, sis, Psoriatic Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, d phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt me, Schnitzler syndrome, Scleritis, derma, Sjogren's syndrome, loarthropathy, Sticky blood syndrome, Still's Disease, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome, Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathic inflammatory bowel diseases), Undifferentiated connective tissue disease, erentiated spondyloarthropathy, Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome In another specific embodiment, a binding molecule used for the conjugate via the bridge linkers of this invention for the ent or prevention of an autoimmune disease can be, but are not limited to, anti-elastin antibody; Abys against epithelial cells antibody; Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody; Thyroglobulin dy, Anti SCL-70; Anti-Jo; Anti-U.sub.IRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; CANCA ; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody, anglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 dy; Anti-splOO antibody; Anti- Mitochondrial(M2) antibody; toid factor antibody; Anti-MCV antibody; Antitopoisomerase antibody; Anti-neutrophil cytoplasmic(cANCA) antibody.
In certain preferred embodiments, the binding molecule for the conjugate in the present ion, can bind to both a receptor and a or complex expressed on an activated lymphocyte which is associated with an autoimmune disease. The receptor or receptor complex can comprise an globulin gene superfamily member (e.g. CD2, CDS, CD4, CDS, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD137, CD138, CD147, CD152/CTLA-4, PD-1, or ICOS), a TNF receptor superfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), an integrin, a cytokine or, a chemokine receptor, a major histocompatibility protein, a lectin (C-type, S-type, or I-type), or a complement control protein.
In r specific embodiment, useful cell binding s that are immune ic for a viral or a microbial antigen are humanized or human monoclonal antibodies. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide n (e.g.
HIV gp!20, HIV nef, RSV F rotein, influenza virus neuramimi-dase, influenza virus hemagglutinin, HTLV tax, herpes simplex vims glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune se. As used herein, the term "microbial antigen" es, but is not d to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a ia, fungi, pathogenic protozoa, or yeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response. Examples of dies available 1 for the viral or microbial infection include, but are not limited to, Palivizumab which is a zed anti-respiratory syncytial virus monoclonal dy for the ent of RSV infection; PR0542 which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir which is a human antibody for the treatment of hepatitis B vims; PROTVIR which is a humanized IgG.sub.l antibody for the treatment of cytomegalovirus; and anti-LPS antibodies.
The cell binding molecules-drug conjugates via the bridge s of this invention can be used in the treatment of infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovims infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, oides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection irus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratch disease, Cellulitis, Chagas Disease can trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection, Cholera, blastomycosis, Clonorchiasis, idium ile infection, Coccidioidomycosis , Colorado tick fever, Common cold (Acute viral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic , Erythema infectiosum (Fifth e), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas ne (Clostridial myonecrosis), chosis, Gerstmann-Straussler-Scheinker me, Giardiasis, Glanders, Gnathosto-miasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus nzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, lasmosis, rm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumovirus ion, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza vims infection, Hymenolepiasis, Epstein-Barr Vims Infectious Mononucleosis (Mono), Influenza, riasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kum, Lassa fever, Legionellosis (Legionnaires’ disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, Measles, Melioidosis (Whitmore’s disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, cum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, al conjunctivitis (Ophthalmia neonatomm), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, rellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystis nia, Pneumonia, yelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsial infection, Rickettsial-pox, Rift Valley fever, Rocky mountain d fever, rus infection, Rubella, Salmonellosis, SARS (Severe Acute atory Syndrome), Scabies, Schistosomiasis, , Shigellosis (Bacillary dysentery), Shingles (Herpes ), Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, sis, Tetanus (Lockjaw), Tinea barbae (Barber’s itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete’s foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia, West Nile Lever, White piedra (Tinea blanca), Yersinia pseudotuber-culosis ion, iosis, Yellow fever, Zygomycosis.
The cell binding molecule, which is more preferred to be an antibody described in this patent that are against pathogenic strains include, but are not limit, Acinetobacter nii, Actinomyces ii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), eba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple ia, Bacteroides genus, Balantidium coli, ascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, myces dermatitides, Machupo virus, Borrelia genus, idium botulinum, Sabia, Brucella genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae , Campylobacter genus, usually Candida albicans and other Candida species, ella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma iense; multiple parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) - Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, irus, Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, irus B19, Human herpesvirus 6 and Human virus 7, Fasciolopsis buski, la hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium genus, Clostridium perfringens; other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli 7, Bunyaviridae family, tis A Virus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1, Herpes simplex virus 2, lasma capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human eumovirus, Ehrlichia chaffeensis, Human omavirus, Human parainfluenza viruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus, Orthomy-xoviridae , Isospora belli, Kingella kingae, Klebsiella pneumoniae, ella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, pira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia s, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous species of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, erca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus s, Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis, ella pertussis, Yersinia pestis, Streptococcus pneumoniae, cystis cii, Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformis and lum minus, Respiratory ial virus, Rhinosporidium i, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, phyton rubrum, Hortaea werneckii, Trichophyton genus, Malassezia genus, ra canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order ophthora-mycosis), Pseudomonas aeruginosa, obacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella , Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella s, Brucella suis, la melitensis, asma spp., tsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, a albicans, lasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Schistosoma japonicum, osoma mansoni, Schistosoma haematobium, and hookworms).
Other dies as cell binding ligands used in this invention for treatment of viral disease include, but are not limited to, antibodies against ns of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Adenoviridae, viridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial vims, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis vims, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovims [such as, HBV ocellular carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi's sarcoma-associated herpesvims (Kaposi's sarcoma), Epstein-Barr vims (Nasopharyngeal carcinoma, Burkitt's lymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cell cancer), SV40 (Simian vims 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell leukemia/lymphoma)], Immune disorders caused vims: [such as Human Immunodeficiency Vims (AIDS)]; Central nervous system vims: [such as, ICY (Progressive multifocal leukoencephalopathy), MeV (Subacute sing panencephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus alitis, yxoviridae (probable) (Encephalitis lethargica), RV (Rabies), Chandipura vims, Herpesviral meningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis, Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)]; Cytomegalovims (Cytomegalovims retinitis, HSV (Herpetic tis)); Cardiovascular vims [such as CBV (Pericarditis, Myocarditis)]; atory system/acute viral aryngitis/viral pneumonia: [Epstein-Barr vims (LBV ion/infectious mononucleosis), Cytomegalovims; SARS coronavims (Severe acute respiratory syndrome) Orthomyxoviridae: nzavims A/B/C (Influenza/Avian influenza), Paramyxovirus: Human parainfluenza vimses (Parainfluenza), RSV (Human respiratory syncytialvims), hMPV]; Digestive system vims [MuV (Mumps), Cytomegalovims (Cytomegalovims esophagitis); Adenovims (Adenovims infection); Rotavirus, Norovims, Astrovims, Coronavims; HBV (Hepatitis B vims), CBV, HAV (Hepatitis A vims), HCV (Hepatitis C vims), HDV (Hepatitis D vims), HEV (Hepatitis E vims), HGV (Hepatitis G vims)]; ital vims [such as, BK vims, MuV (Mumps)].
According to a further object, the present invention also concerns ceutical compositions comprising the conjugate via the bridge linkers of the invention together with a pharmaceutically acceptable carrier, diluent, or excipient for treatment of cancers, infections or autoimmune disorders. The method for treatment of cancers, infections and autoimmune disorders can be practiced in vitro, in vivo, or ex vivo. Examples of in vitro uses include ents of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill ts that express undesired antigen. Examples of ex vivo uses include ents of hematopoietic stem cells(HSC)prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells. For instance, clinical ex vivo treatment to remove tumour cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplant in order to prevent graft-versus- host disease, can be carried out as follows. Bone marrow is harvested from the patient or other individual and then ted in medium containing serum to which is added the conjugate of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37 °C. The exact conditions of tration and time of incubation (=dose) are readily determined by the skilled clinicians. After incubation, the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative herapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical ent.
For clinical in vivo use, the conjugate via the linkers of the invention will be supplied as solutions or as a lyophilized solid that can be redissolved in sterile water for ion. Examples of le protocols of conjugate administration are as follows. Conjugates are given weekly for 8-20 weeks as an i.v. bolus. Bolus doses are given in 50 to 500 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can be added. Dosages will be about 50 pg to 20 mg/kg of body weight per week, i.v. (range of 10 pg to 200 mg/kg per injection). 4-20 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by the d clinicians.
Examples of medical conditions that can be treated ing to the in vivo or ex vivo s of killing selected cell populations e malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite).
The amount of a conjugate which is required to achieve the desired biological effect, will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the s to which the patient s, the diseased state of the patient, the route of administration, all s which dictate the ed dose amounts, delivery and regimen to be administered.
In general terms, the conjugates via the linkers of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 10% w/v ates for parenteral administration. Typical dose ranges are from 1 pg/kg to 0.1 g/kg of body weight per day; a preferred dose range is from 0.01 mg/kg to 20 mg/kg of body weight per day, or per week, or an equivalent dose in a human child. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the route of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the conjugates by the chosen delivery route, and the speed (bolus or continuous infusion) and le of administrations (number of repetitions in a given period of time).
The ates via the linkers of the present ion are also capable of being administered in unit dose forms, wherein the term "unit dose" means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, remaining as a ally and chemically stable unit dose sing either the active conjugate , or as a pharmaceutically acceptable composition, as described after. As such, typical total daily/weekly/biweekly/monthly dose ranges are from 0.01 to 100 mg/kg of body weight. By way of general guidance, unit doses for humans range from 1 mg to 3000 mg per day, or per week, per two weeks (biweekly) or per month. Preferably the unit dose range is from 1 to 500 mg administered one to four times a week, and even more preferably from 1 mg to 100 mg, once a week. ates provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be prepared for use by oral administration, particularly in the form of tablets, simple capsules or soft gel capsules; or intranasal, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, , lotions, gels or sprays, or via transdermal patches.
CYTOTOXIC AGENTS Drugs that can be conjugated to a cell-binding molecule in the present invention are small molecule drugs including cytotoxic agents, which can be linked to or after they are modified for linkage to the cell-binding agent. A "small molecule drug" is broadly used herein to refer to an organic, inorganic, or organometallic compound that may have a molecular weight of, for e, 100 to 2500, more suitably from 120 to 1500. Small le drugs are well characterized in the art, such as in WO05058367A2, and in U.S. Patent No. 4,956,303, among others and are incorporated in their entirety by reference. The drugs include known drugs and those that may become known drugs.
Drugs that are known include, but not limited to, 1). Chemotherapeutic agents: a). Alkylating : such as Nitrogen mustards: chlorambucil, chlomaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, onitol, melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil d; CC-1065 ding its adozelesin, carzelesin and bizelesin synthetic ues); Duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); Benzodiazepine dimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines); Nitrosoureas: (carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine); Alkylsulphonates: (busulfan, treosulfan, improsulfan and piposulfan); Triazenes: (dacarbazine); Platinum containing compounds: (carboplatin, cisplatin, oxaliplatin); ines, such as opa, carboquone, meturedopa, and uredopa; nimines and methylamelamines ing altretamine, triethylenemel-amine, trietylenephosphoramide, triethylenethiophosphaoramide and hylolomel-amine]; b). Plant Alkaloids: such as Vinca alkaloids: istine, vinblastine, vindesine, vinorelbine, navelbin); Taxoids: taxel, xol) and their analogs, Maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocins) and their analogs, cryptophycins (particularly cryptophycin 1 and cryptophycin 8); epothilones, eleutherobin, discodermo-lide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c). DNA Topoisomerase Inhibitors: such as [Epipodophyllins: (9- aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (retinols), teniposide, topotecan, 9- nitrocamptothecin (RFS 2000)); cins: (mitomycin C)]; d). Anti-metabolites: such as {[Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or the other folic acid analogues); IMP dehydrogenase tors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotide ase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs: abine, idine, 6- azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil, floxuridine, ratitrexed (Tomudex)); ne analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acid isher, such as frolinic acid}; e). Hormonal therapies: such as {Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene, tamoxifen); FHRH agonists: (goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, lin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane and other androgens inhibitors)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A); Cytokines: feron-alpha, Interferon-gamma, tumor necrosis factor (TNFs), human proteins containing a TNF domain)]}; f). Kinase tors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), nib (SKI-606), ntinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, mab, Trastuzumab, Ranibizumab, Panitumumab, sib; g). antibiotics, such as the enediyne antibiotics (e.g. calicheamicins, especially calicheamicin yl, 81, al and pi, see, e.g., J. Med. Chem., 39 (11), 2103-2117 (1996), Angew Chem Inti. Ed. Engl. 33:183-186 ; dynemicin, including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, omycin, carzinophilin; chromomycins, omycin, daunorubicin, detorubicin, 6-diazooxo-L-norleucine, bicin, lino-doxombicin, orpholino-doxombicin, 2-pyrrolino-doxorubicin and deoxydoxombicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, ozocin, tubercidin, ubenimex, zinostatin, zorubicin; f). Others: such as Polyketides (acetogenins), especially bullatacin and bullatacinone; gemcitabine, epoxomicins (e. g. zomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, ge, Yervoy, Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins (such as l-methylphenylpyridinium ion), Cell cycle inhibitors (such as staurosporine), mycins (such as Actinomycin D, dactinomycin), Bleomycins (such as bleomycin A2, bleomycin B2, ycin), Anthracyclines (such as daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirambicin, zorubicin, mtoxantrone, MDR inhibitors (such as verapamil), Ca2+ATPase inhibitors (such as thapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A); Thapsigargin, Celecoxib, glitazones, locatechin e, Disulfiram, Salinosporamide A.; Anti-adrenals, such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine (DEMO), elfomithine; elliptinium acetate, etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan; mine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, n A and anguidine); urethane, siRNA, antisense drugs, and a nucleolytic . 2) . An utoimmune disease agent includes, but is not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g. nide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone nate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, sone, sirolimus, tacrolimus. 3) . An nfectious disease agent includes, but is not limited to, a). lycosides: amikacin, icin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin cin, cin, bekanamycin, cin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin; b). Amphenicols:azidamfenicol, chloramphenicol, florfenicol, henicol; c). Ansamycins: geldanamycin, herbimycin; d). enems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e). Cephems: carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, xin, cefaloglycin, cefamandole, rin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, xime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, ome, cefpodoxime, cefprozil, nome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f).
Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g. tigecycline; g). P-Lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).
Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide (telithromycin, mycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; 1).
Monobactams: aztreonam, tigemonam; m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, llin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam), illin, meticillin, lin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin, colistin, polymyxin B; p).
Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, xacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q). Streptogramins: pristinamycin, quinupristin/dalfopristin); r). Sulfonamides: mafenide, sil, sulfacetamide, sulfamethizole, ilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (cotrimoxazole ); s). Steroid antibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline, chlortetracycline, clomocycline, ocycline, lymecycline, meclocycline, cline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g. tigecycline); u). Other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, nem, c acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel, simycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin; 4). Anti-viral drugs: a). fusion inhibitors: aplaviroc, roc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturation inhibitors: bevirimat, n; d). Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e). Nucleosides &nucleotides: ir, aciclovir, adefovir, amdoxovir, tabine, brivudine, cidofovir, clevudine, dexelvucitabine, sine (ddl), elvucitabine, emtricitabine (FTC), entecavir, lovir, fluorouracil (5-FU), 3’-fluorosubstituted 2’, 3’-dideoxynucleoside analogues (e.g. 3’-fluoro-2’,3’-dideoxythymidine (FLT) and 3’-fluoro-2’,3’-dideoxyguanosine (FLG), fomivirsen, lovir, idoxuridine, lamivudine (3TC),1-nucleosides (e.g. //-l-thymidinc and /?-l-2’-deoxycytidine), penciclovir, racivir, rin, stampidine, stavudine (d4T), taribavirin (viramidine), udine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f). Non-nucleosides: dine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod ), tromantadine; g). Protease inhibitors: amprenavir, atazanavir,boceprevir, vir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h). Other types of anti-virus drugs: abzyme, arbidol, calanolide a, nin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet, thsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib.
). The drugs used for conjugates via a bridge linker of the present invention also include radioisotopes. Examples of radioisotopes (radionuclides) are 3H, UC, 14C, 18F, 32P, 35S, f>4Cu, 68Ga, 86Y, "Tc, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 213Bi. Radioisotope labeled antibodies are useful in receptor targeted imaging experiments or can be for targeted treatment such as with the dy-drug conjugates of the invention (Wu et al (2005) Nature Biotechnology 23(9): 1137- 46). The cell binding molecules, e.g. an antibody can be labeled with ligand reagents through the bridge linkers of the present patent that bind, chelate or otherwise complex a radioisotope metal, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, Pubs. (1991). ing ligands which may complex a metal ion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex. USA). 6). The pharmaceutically acceptable salts, acids or derivatives of any of the above drugs.
In another embodiment, the drug in the Formula (II) and/or (IV) can be a chromophore le, for which the conjugate can be used for ion, monitoring, or study the interaction of the cell binding molecule with a target cell. Chromophore molecules are a compound that have the y to absorb a kind of light, such as UV light, florescent light, IR light, near IR light, visual light; A chromatophore molecule includes a class or subclass of xanthophores, ophores, iridophores, leucophores, melanophores, and cyanophores; a class or subclass of fluorophore les which are fluorescent chemical compounds re-emitting light upon light; a class or subclass of visual phototransduction molecules; a class or subclass of photophore molecules; a class or subclass of luminescence molecules; and a class or subclass of luciferin compounds.
The chromophore molecule can be selected from, but not d, non-protein organic phores, such as: Xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, and Texas red); Cyanine derivatives: (cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine); Squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; Naphthalene derivatives (dansyl and prodan derivatives); Coumarin derivatives; Oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole); 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 (proflavin, acridine orange, acridine yellow etc). Arylmethine derivatives (auramine, crystal , malachite green). Tetrapyrrole tives (porphin, phthalocyanine, bilirubin).
Or a chromophore molecule can be ed from any analogs and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), or Dyes (Abberior), DY and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight Dyes (APC, RPEPerCP, Phycobilisomes)(Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech).
Examples of the widely used fluorophore compounds which are reactive or conjugatable with the linkers of the invention are: Allophycocyanin (APC), Aminocoumarin, APC-Cy7 ates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, IR-783,Lissamine Rhodamine B, r , Methoxycoumarin, NBD, Pacific Blue, c Orange, PE-Cy5 ates, PE-Cy7 conjugates, PerCP, R-Phycoerythrin (PE), Red 613, SetaAzide, SetaDBCO, SetaNHS, SetaNHS, SetaNHS, SetaNHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, SeTauNHS, SeTau Maleimide, SeTauNHS, SeTauNHS, SeTauNHS, Texas Red, TRITC, TruRed, X- Rhodamine.
The fluorophore compounds that can be linked to the linkers of the invention for study of nucleic acids or proteins are ed from the following nds or their derivatives: 7-A AD (7-aminoactinomycin D, CG-selective), Acridine Orange, Chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark), DAPI, DRAQ5, DRAQ7, um Bromide, Hoechst33258, Hoechst33342, LDS 751, Mithramycin, Propidiumlodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO: e Monomer, TOTO-1, TO-PRO-1, TOTO-3, -3, YOSeta-1, YOYO-1. The fluorophore compounds that can be linked to the linkers of the invention for study cells are selected from the following compounds or their derivatives: DCFH (2'7'Dichorodihydro-fluorescein, oxidized form), DHR (Dihydrorhodamine 123, oxidized form, light catalyzes ion), Fluo-3 (AM ester. pH > 6), Fluo-4 (AM ester. pH 7.2), Indo-1 (AM ester, low/high calcium (Ca2+)), and SNARF (pH 6/9). The preferred fluorophore compounds that can be linked to the s of the invention for study proteins/antibodies are selected from the following compounds or their tives: Ahophycocyanin (APC), AmCyanl (tetramer, Clontech), AsRed2 (tetramer, Clontech), Azami 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 on), GFP (S65C on), GFP (S65L mutation), GFP (S65T mutation), GFP (Y66F mutation), GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRedl, J-Red, Katusha, Kusabira Orange (monomer, MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL), mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFPl (monomer, Tsien lab), mStrawberry, mTFPl, oise2, P3 (phycobilisome x), Peridinin Chlorophyll (PerCP), R-phycoerythrin(RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP , n), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), FP (dimer, Evrogen), Venus, Wild Type GFP, YPet, ZsGreenl (tetramer, Clontech), ZsYehowl (tetramer, Clontech).
The examples of the structure of the conjugates of the antibody-chromophore molecules via the bridge linker are as following AcOl, Ac02, Ac03, Ac04 and Ac05: \ MxA-sO O m, / m2 AcOl so3- Xo o Xi s-n-x3 \ V % O m\b N (R2)-X2' m 2 n •X3' R1 o °3S o so3- X'X V V \ I mAb S_J°3 I / (Rhx*X' n Ac03 (FNIR-774 conjugate) ^ // O o X3.. x 3s Xf °\ // O' N Ri H mAb mi / // s/ O \ / m 2 O Ac04 &so3- ■o3s. o so3- » V .0 N N+' X,‘ s\ Rf I mi SI mAb i I •s' in 2 o n Ac05 (IR800CW conjugate) Wherein "==" represents either single bond or double bond; mAb is antibody, preferably monoclonal dy; n, mi, m2, Xi, X2, Ri, R2 and R3 are the same defined in Formula (I) and In another ment, the drug in the Formula (II) and (IV) can be polyalkylene s that are used for extending the ife of the cell-binding molecule when administered to a mammal. Polyalkylene glycols include, but are not limited to, poly(ethylene glycols) (PEGs), polypropylene glycol) and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are nctionally activated hydroxyPEGs (e.g., hydroxyl PEGs activated at a single terminus, including reactive esters of hydroxyPEG-monocarboxylic acids, hydroxyPEG-monoaldehydes, hydroxyPEG-monoamines, hydroxyPEG-monohydrazides, hydroxyPEG-monocarbazates, hydroxyl PEG- monoiodoacetamides, hydroxyl PEG-monomaleimides, hydroxyl PEG-monoorthopyridyl disulfides, hydroxyPEG-monooximes, hydroxyPEG-monophenyl carbonates, hydroxyl PEG- monophenyl glyoxals, hydroxyl PEG-monothiazolidinethiones, yl PEG- monothioesters, hydroxyl PEG-monothiols, hydroxyl PEG-monotriazines and hydroxyl PEG- monovinylsulfones).
In certain such embodiments, the polyalkylene glycol has a molecular weight of from about 10 Daltons to about 200 kDa, preferably about 88 Da to about 40 kDa; two es each with a molecular weight of about 88 Da to about 40 kDa; and more preferably two branches, each of about 88 Da to about 20 kDa. In one particular embodiment, the polyalkylene glycol is poly(ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa, or about 40 kDa.
In ic embodiments, the PEG is a PEG 10 kDa (linear or branched), a PEG 20 kDa (linear or branched), or a PEG 40 kDa (linear or branched). A number of US patents have disclosed the preparation of linear or ed "non-antigenic" PEG polymers and derivatives or conjugates thereof, e.g., U.S. Pat. Nos. 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; ,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,131; 5,900,402; 5,902,588; 455; ,951,974; 5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095. The structure of the conjugates of the antibodypolyalkylene glycols via the bridge linker is as following PgOl: R' O .o Xi r3 Rl m3 m, H, .H* inAb R s'' m2 O n Wherein mAb is an antibody; R’ is H or CH3; m3 is an integer from Ito 5000; R3 is OH, H, or Ri; "==" represents either single bond or double bond; mi, m2, n, "-----", Xi, X2, Ri, and R2 are the same defined in Formula (I) and (II). In on, Ri can be .
In yet another embodiment, the preferred xic agents that conjugated to a cell-binding molecule via a bridge linker of this patent are tubulysins, maytansinoids, ids (taxanes), CC- 1065 analogs, daunorubicin and doxorubicin compounds, amatoxins, iazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines), calicheamicins and the enediyne antibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, atins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, geldanamycins, methotrexates, thiotepa, vindesines, vincristines, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins, clerodermins, llamides, esperamicins, PNU-159682, and their analogues and derivatives above thereof.
Tubulysins that are preferred for conjugation in the present invention are well known in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods (e. g. Balasubramanian, R., et al. J. Med. Chem., 2009, 52, 238-40; Wipf, P., et al. Org. Lett., 2004, 6, 4057-60; Pando, O., et al. J. Am. Chem. Soc., 2011, 133, 7692-5; Reddy, J. A., et al. Mol. Pharmaceutics, 2009, 6, 1518-25; Raghavan, B., et al. J. Med. Chem., 2008, 51, 1530-33; son, A. W., et al. J. Org. Chem., 2008, 73, 4362-9; Pando, O., et al. Org. Lett., 2009, 11 (24), 5567-9; Wipf, P., et al. Org. Lett., 2007, 9 (8), 1605-7; Friestad, G. K., Org. Lett.,2004, 6, 3249-52; Peltier, H. M., et al. J. Am. Chem. Soc., 2006, 128, 16018-9; Chandrasekhar, S., et al J. Org. Chem., 2009, 74, ; Liu, Y., et al. Mol.
Pharmaceutics, 2012, 9, 168-75; Friestad, G. K., et al. Org. Lett., 2009, 11, ; Kubicek, K., et al., Angew Chem Int Ed Engl, 2010.49: 4809-12; Chai, Y., et ah, Chem Biol, 2010, 17: 296- 309; h, A., et ah, 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 ah, Angew Chem Int Ed Engl, 2006, 45, 7235-9; Patent applications: Zanda, M., et al, Can. Pat. Appl. CA 2710693 (2011); Chai, Y., et al. Eur.
Pat. Appl. 2174947 (2010), WO 4724; Leamon, C. et al, W02010033733, WO 2009002993; Ellman, J., et al, PCT WO2009134279; WO 2009012958, US appl. 20110263650, 20110021568; Matschiner, G., et al, W02009095447; Vlahov, I., et al, W02009055562, WO 2873; Low, P., et al, W02009026177; Richter, W., WO2008138561; Kjems, J., et al, WO 2008125116; Davis, M.; et al, W02008076333; Diener, J.; et al, U.S. pl. 41901, W02006096754; Matschiner, G., et al, W02006056464; Vaghefi, F., et al, W02006033913; ng, A., Ger. Offen. DEI02004030227, W02004005327, W02004005326, W02004005269; Stanton, M., et al, U.S. Pat. Appl. Publ. 20040249130; Hoefle, G., et al, Ger.
Offen. DE10254439, DE10241152, DE10008089; Leung, D., et al, W02002077036; Reichenbach, H., et al, Ger. Offen. DE19638870; ng, R., US20120129779; Chen, H., US appl. 20110027274. The preferred structures of tubulysins for conjugation of cell binding les are described in the patent application of .
Examples of the structures of the conjugates of the antibody-tubulysin analogs via the bridge linker are T01, T02, T03, T04, T05, T06 and T07 as following: OAc O N N DH / ! ^ I ° H /mi i iinAb O / m2 T01 WO 59622 os \ Y H O OAc N O I N. OH I \ m\l) I ° C/i O / ( R2)-X2 -/'I 2 NC) n m2 T02 r%, H H O OAc N o X,' SN N. OH S N' (-J 5 mAb ° V O , ' m I (riV-x2< s x /m2 . o n H O OAc \ o Xj N' Ri N o I ° V C/ s O m, mAb ^W^S n V y m2 o -1 T04 R1 o Z3 ov >Xl s H O o / <1 Js T OH mAb O mi \ 2) C/V O \ /m2 n T05 H O OAc I mAb >Vx i OH \ I Ri ° ^ I Cj\ O /m, V)rx2-fR ) _ O \ 2/ m2 n T06 Vx,/ \ I S. N. OH, mAb Ri Cifi' ‘6/m> Wherein mAb is an antibody; Z3 and Z’3 are independently H, 0P(0)(0Mi)(0M2), 0P(0)(0Mi)(0M2), 0S03Mi, Ri, or O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside, etc), NH-glycoside, S-glycoside or CH2-glycoside; Miand WO 59622 M2 are independently H, Na, K, Ca, Mg, NH4, NR|R2R-,;"====" represents either single bond or double bond; n, mi, m2, "---- ",Xi, X2, Ri, R2 and R3 are the same defined in Formula (I) and Calicheamicins and their related enediyne antibiotics that are preferred for cell-binding le-drug conjugates of this patent are described in: Nicolaou, K. C. et al, Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-8), U.S. Patent Nos. 4,970,198; ,053,394; 5,108,912; 5,264,586; 5,384,412; 5,606,040; 5,712,374; 5,714,586; 5,739,116; ,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562; 6,124,310; 8,153,768. An Example of the structure of the conjugate of the antibody-Calicheamicin analog via the bridge linker is C01 as the following: •R ■S, HQ 11£> V1' n-x mAb -A OCH3 C2h5 ^ I H >-xh3co A,, IS h3co mi ^fR2)m2 n Wherein mAb is an antibody; "==" represents either single bond or double bond; n, mi, m2, ---- ", Xi, X2, Ri, and R2 are the same defined in Formula (I) and (II).
Maytansinoids that are preferred to be used in the present invention ing maytansinol and its analogues are described in U.S. Patent Nos. 4,256,746, 4,361,650, 4,307,016, 4,294,757, 4,294,757, 4,371,533, 4,424,219, 4,331,598, 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, 063, 7,851,432, and 8,163,888. An example of the ure of the conjugate of the antibody- Maytansinoids via the bridge linker is as the ing MyOl: n n a Me' d: \ ■Xf 1 I mAb / (d 11 y h3c Wherein mAb is an antibody; n, mi, m2, "==" represents either single bond or double bond; n, mi, m2, "---- ", Xi, X2, Ri, and R2 are the same defined in Formula (I) and (II).
WO 59622 Taxanes, which includes Paclitaxel (Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic tive, and their analogs which are preferred for conjugation via the bridge linkers of the present patent are exampled in:. K C. Nicolaou et ah, J. Am. Chem. Soc. 117, 2409-20, ; Ojima et al, J. Med. Chem. 39:3889-3896 (1996); 40:267-78 (1997); 45, 5620-3 (2002); Ojima et ah, Proc. Natl. Acad. Sci., 96:4256-61 (1999); Kim et ah, Bull. Korean Chem. Soc., 20, 1389-90 (1999); Miller, et al. J. Med. Chem., 47, 4802-5(2004); U.S. Patent No. ,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; 290; and 7,667,054.
Examples of the structures of the conjugate of the antibody-taxanes via the bridge linker are as the ing TxOl, Tx02 and Tx03. () v Rf o, n >Cr-sHo jP mAb I i CrV«^-Oho rl o Hh * hOAc •X2 \ OH O " ° ^2) MeO m 1 ' 7 m 2 \^OMe ()v O mAb I S^\ OH 5 0 'TR2) '^OMe m 1 MeO n ' 7 m2 Tx02 o, -X Rf 0 ^2) in l ' 7 Ill2 -cT MeO n OMe Tx03 Wherein mAb is an dy; n, mi, m2, "==" represents either single bond or double bond; n, mi, m2, "---- ", Xi, X2, Ri, and R2 are the same defined in Formula (I) and (II).
CC-1065 analogues and doucarmycin analogs are also preferred to be used for a conjugate with the bridge linkers of the present patent. The examples of the CC-1065 analogues and doucarmycin analogs as well as their sis are described in: e.g. Warpehoski, et al, J. Med.
Chem. 31:590-603 (1988); D. Boger et ah, J. Org. Chem; 66; 6654-61, 2001; U. S. Patent Nos: 4169888, 4391904, 4671958, 4816567, 4912227, 4923990, 4952394, 8, 4978757, 8, 5037993, 5070092, 5084468, 5101038, 5117006, 5137877, 5138059, 5147786, 5187186, 5223409, 5225539, 5288514, 5324483, 0, 5332837, 8, 5403484, 5427908, 5475092, 5495009, 5530101, 5545806, 5547667, 5569825, 5571698, 5573922, 5580717, 5585089, 9, 5587161, 5595499, 5606017, 5622929, 5625126, 5629430, 5633425, 5641780, 5660829, 5661016, 5686237, 5693762, 0, 5712374, 5714586, 5739116, 5739350, 5770429, 5773001, 5773435, 5786377 5786486, 5789650, 5814318, 5846545, 9, 5877296, 5877397, 5885793, 5939598, 6, 5969108, 8, 8, 6066742, 6075181, 6103236, 6114598, 6130237, 2, 1, 6150584, 6162963, 6172197, 6180370, 6194612, 6214345, 6262271, 6281354, 6310209, 6329497, 6342480, 6486326, 6512101, 6521404, 6534660, 6544731, 6548530, 6555313, 3, 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, 026, 7,655,660, 7,655,661, 7,906,545, and 8,012,978. Examples of the structures of the conjugate of the antibody-CC-1065 analogs via the bridge linker are as the following CC01, CC02, and CC03.
Hv X3 \ x.^o N // Ri O N S I N H !mAb H m i (R2)rX2~*^s OZ4 ' 2'm2 O n .s. Q wmi■X, Cl o. mAb I I O N 'S' H o— O' x2 O •r2 ■X3 in 2 _ n CC02 I -("vO-’t xi^° 1 s f\J ° mAb z4o N. ">! O N (r2V-X2^^s H V /Ill2 O n CC03 Wherein mAb is an dy; Z4 and Z’4are independently H, PO(OMi)(OM2), SO3M1, CH2PO(OMi)(OM2), CH3N(CH2CH2)2NC(0)-, 0(CH2CH2)2NC(0)-, Ri, or glycoside; X3 is O, NH, NHC(O), OC(O), -C(0)0, Ri, or absent; "==" represents either single bond or double bond; n, mi, m2, "-----", Xi, X2, Ri, R2, Mi, and M2 are the same defined in Formula (I) and (II).
Daunorubicin/Doxorubicin Analogues are also red for ation via the bridge linkers of the present patent. The red structures and their sis are exampled in: Hurwitz, E., et ah, Cancer Res. 35, 1 (1975). Yang, H. M., and Reisfeld, R. A., Proc. Natl.
Acad. Sci. 85, 1189-93 (1988); Pietersz, C. A., E., etal., E., etal.," Cancer Res. 48, 926-311 (1988); Trouet, et ah, 79, 626-29 (1982); Z. Brich et ah, J. Controlled Release, 19, 245-58 (1992); Chen et ah, Syn. Comm., 33, 2377-90, 2003; King et ah, Bioconj. Chem., 10, 279-88, 1999; King et ah, J. Med. Chem., 45, 4336-43, 2002; Kratz et ah, J Med Chem. 45, 5523-33, 2002; Kratz et ah, Biol Pharm Bull. Jan. 21, 56-61, 1998; Lau et ah, Bioorg. Med. Chem. 3, 1305-12, 1995; Scott et ah, Bioorg. Med. Chem. Lett. 6, 1491-6, 1996; Watanabe et ah, Tokai J.
Experimental Clin. Med. 15, 327-34, 1990; Zhou et ah, J. Am. Chem. Soc. 126, 15656-7, 2004; WO 01/38318; U.S. Patent Nos. 5,106,951; 5,122,368; 5,146,064; 5,177,016; 5,208,323; ,824,805; 6,146,658; 6,214,345; 7569358; 7,803,903; 8,084,586; 8,053,205.Examples of the structures of the conjugate of the antibody-CC-1065 analogs via the bridge linker are as the following DaOl, Da02, Da03 and Da04.
O H O O H ■S. 'X2 mAb (^L 'S' h3co ° °" 'aX R OH o' X, Illlj n H DaOl H n^x3 S' ■XT | OH I mAb W/TpI O OH t) h3co OH m, n NH2 Da02 OH N- v X3 Oh Rt i I ^mAb H3CO VY OH /m. n MeO Da03 N X3 aJi,'OH OH O Rr H X, l in AI) OH O OMe WiTp Q’y,NK/' -Meo'fcT* mi n Wherein mAh is an antibody; X3 is O, NH, , NHC(0)NH, C(O), Ri, OC(O), or absent; "==" represents either single bond or double bond; n, mi, m2, "---- ", Xi, X2, Ri.and R2 are the same defined in Formula (I) and (II).
Auristatins and dolastatins are preferred in conjugation via the bridge linkers of this patent.
The auristatins (e. g. auristatin E (AE) atin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E , Monomethylauristatin (MMAF), Auristatin F ene e (AFP) and a phenylalanine variant of MMAE) which are synthetic analogs of dolastatins, are described in Int. J. Oncol. 15: 367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-32 (2004); U.S. ation Nos. 11134826, 20060074008, 2006022925. U.S. Patent Nos. 4414205, 4753894,4764368, 4816444, 4879278, 4943628, 4978744, 5122368, 5165923, 5169774, 5286637, 5410024, 4, 5530097, 5554725, 5585089, 5599902, 5629197, 5635483, 5654399, 5663149, 5665860, 6, 5714586, 5741892, 5767236, 5767237, 5780588, 5821337, 5840699, 5965537, 6004934, 6033876, 6034065, 6048720, 6054297, 6054561, 6124431, 6143721, 6162930, 6214345, 6239104, 6323315, 9, 6342221, 6407213, 6569834, 6620911, 6639055, 6884869, 6913748, 7090843, 7091186, 7097840, 7098305, 7098308, 7498298, 7375078, 2, 7553816, 7659241, 7662387, 7745394, 7754681, 7829531, 7837980, 7837995, 7902338, 6, 7, 7851437, 7994135. Examples of the structures of the conjugate of the antibody-auristatins via the bridge linker are as the following AuOl, Au02, Au03, Au04, and Au05.
Q H O H QH ■Xf Nv N ,N- N mAb c N 1 0 A () ^,() () ^X2-fR2) mi O ' /m2 n AuOl y 'WAV'* .N- N \ •zy/ml \ —O —o ° O (QH " Au02 [7 rWi H H O ,N- N V I -6 o Z3 ° OT^OH mi mAh (r2VX2^; s' \ / m2 'q n ,N- N ,() \1 ° ^N O J) o x4. ■xr fN ■RT mi I m A b \ z/m2 o n Au04 H H N Ri Xr^ A N ,N- 'N' / i ^6 o X3 J m, 0 O^OH mAh X2 / (R^ ■s' v 'm2 n Au05 Wherein mAh is an antibody; X3 is CH2, O, NH, NHC(O), NHC(0)NH, C(O), OC(O) Ri, or absent; X4 is CH2, C(O), C(0)NH, Ri), Ri, NHRi, NRi C(0)Ri or C(0)0; Z3is H, Ri, 0P(0)(0Mi)(0M2), NHRi, 0CH20P(0)(0Mi)(0M2), 0S03Mi, or O-glycoside (glucoside, galactoside, mannoside, glncuronoside, alloside, ide), NH-glycoside, S-glycoside, or CH2- glycoside; Mi and M2 are independently H, Na, K, Ca, Mg, NH4, NRiR2R3; "==" represents either single bond or double bond; n, mi, m2, "-----", Xi, X2, R| R2 and R3 are the same defined in a (I) and (II). In addition, Ri can be absent and R2 can be H.
The benzodiazepine dimers (e. g. dimmers of pyrrolobenzodiazepine (PBD) or (tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines ) which are preferred cytotoxic agents according to the present invention are exampled in the art: US Patent Nos . 8,163,736; 8,153,627; 8,034,808; 7,834,005; 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; 951; 7,326,700; 7,312,210; 7,265,105; 7,202,239; 7,189,710; 7,173,026; 193; 7,067,511; 7,064,120; 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; 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; US patent appl. 20100203007, 20100316656, 20030195196.Examples of the ures of the ate of the antibody- benzodiazepine dimers via the bridge linker are as the following PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB09, PB10 and PB11. 2016/059622 2016/050580 7 M103S H SO3M2 o HN- 1 n-x3 xi •N- -N- o mAb MeO' o mi 2tX2 m2 o n •N- Ri- X, rj ' N== •5 ■N- I •N- R3 r3 MeO' o OMe O o mi so3m2 M^Oj! ■N- xrVs'^ ■3 MeOxS3>^:'., O o / mAb ' H x4 O NC Rr -N- ■N- / mAb r3 MeO' OMe R3 J mi O O (R2 /^s' rm2 -O n x3' O MjOjS H Xi SN ■N I so3m2 HN-r^ \ •N- -N ^ Jj —y o Me MeO‘ mi O O (r2; X2 n m2 mi X4 .O °3Sv H R i-. Xi s> y X O O m.
(R n x3' O R, ■X,' m,o3s h I so3m2 s HN- r3 mAb I I s' 3 "Cy m.
Me MeO (R O O O n ^fe-X2 X3 -Ri o " m1o3si h so3m2 'y I hn-^e y mAh I I 4/ ' ■N- y. l>x x' Me MeO‘ m'jL o o (R^;X2^o X3 R1 o m1o3sv h X'-Vs Rr MeO^^Sr^ R3 m, O (r^Xz-^-o m2 n H HNwS°3M2 N % x3 ,o x^l I Xi -N- R1 s Me MeO‘ /mi mAb O O / ( R2)-X2 s' \ /m? . 2 O n PB10 0 0 (Rz&r^ O n PB11 Wherein mAb is an antibody; X3 is CH2, O, NH, , NHC(0)NH, C(O), OC(O), 0C(0)(NR3), Ri,NHRi, NRi C(0)Ri or absent; X4 is CH2, C(O), C(0)NH, C(0)N(Ri), Ri,NHRi, NRi C(0)Ri or C(0)0; Mi and M2 are independently H, Na, K, Ca, Mg, NH4, NRiR2R3; "=: represents either single bond or double bond; n, mi, m2, ", Xi, X2, R| R2 and R3 are the same defined in Formula (I) and (II). In addition, Ri can be absent.
Amatoxins which are a subgroup of at least ten toxic nds originally found in several genera of poisonous mushrooms, most notably Amanita phalloides and several other mushroom species, are also preferred for conjugation via the bridge linkers of the present patent. These ten amatoxins, named a-Amanitin, P-Amanitin, y-Amanitin, s-Amanitin, Amanullin, Amanullinic acid, Amaninamide, Amanin, Proamanullin, are rigid bicyclic peptides that are synthesized as 35-amino-acid proproteins, from which the final eight amino acids are d by a prolyl oligopeptidase (Litten, W. 1975 Scientific American232 (3): 90-101 ;H. E.
Hallen, et al 2007 Proc. Nat. Aca. Sci. USA 104, 101; K. Baumann, et al, 1993 Biochemistry 32 (15): 4043-50; n-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49; Horgen, P. A. et al. 1978 Arch. io. 118 (3): 317-9). Amatoxins kill cells by inhibiting RNA rase II (Pol II), shutting down gene transcription and protein biosynthesis (Brodner, O. G. and Wieland, T. 1976 Biochemistry, 15(16): 3480-4; Fiume, L., Curr Probl Clin Biochem, 1977, 7: 23-8; Karlson-Stiber C, Persson H. 2003, Toxicon 42(4): 339-49; Chafin, D. R. , Guo, H. & Price, D. H. 1995 J. Biol. Chem. 270 (32): 19114-19; Wieland (1983) Int. J.
Pept. Protein Res. 22(3): .). Amatoxins can be produced from collected Amanita ides mushrooms (Yocum, R. R. 1978 Biochemistry 17(18): 3786-9; Zhang, P. et al, 2005, FEMS Microbiol. Lett.252(2), 223-8), or from fermentation using a basidiomycete (Muraoka, S. and Shinozawa T., 2000 J. Biosci. Bioeng. 89(1): 73-6) or from fermentation using A. fissa (Guo, X. W., et al, 2006 Wei Sheng Wu Xue Bao 46(3): 373-8), or from culturing Galerina fasciculata or Galerina helvoliceps, a strain belonging to the genus (WG/1990/009799, JP11137291). However the yields from these isolation and fermentation were quite low (less than 5 mg/L culture). Several preparations of amatoxins and their analogs have been reported in the past three decades (W. E. Savige, A. Fontana, Chem. Commun. 1976, 600-1; Zanotti, G., et al, Int J Pept Protein Res, 1981. 18(2): 162-8; Wieland, T., et al, Fur. J. Biochem. 1981, 117, 161-4; P. A. Bartlett, et al, Tetrahedron Lett. 1982, 23, 619-22; Zanotti, G., et ah, Biochim Biophys Acta, 1986. 870(3): 454-62; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 323-9; Zanotti, G., et ah, Int. J. Peptide n Res. 1987, 30, 450-9; i, G., et ah, Int J Pept Protein Res, 1988. 32(1): 9-20; G. Zanotti, T. et al, Int. J. Peptide Protein Res. 1989, 34, 222-8; Zanotti, G., et ah, Int J Pept n Res, 1990. 35(3): 263-70; Mullersman, J. E. and J.
F. Preston, 3rd, Int J Pept Protein Res, 1991. 37(6): ; Mullersman, J.E., et al, Int J Pept Protein Res, 1991. 38(5): 409-16; Zanotti, G., et al, Int J Pept Protein Res, 1992. 40(6): 551-8; Schmitt, W. et al, J. Am. Chem. Soc. 1996, 118, 4380-7; Anderson, M.O., et al, J. Org. Chem., 2005, : 4578-84; J. P. May, et al, J. Org. Chem. 2005, 70, 8424-30; F. Brueckner, P.
Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; J. P. May, D. M. Perrin, Chem. Fur. J. 2008, 14, 3404-9; J. P. May, et al, Chem. Fur. J. 2008, 14, 7; Q. Wang, et al, Fur. J. Org.
Chem. 2002, 834-9; May, J. P. and D. M. Perrin, Biopolymers, 2007. 88(5): ; May, J.P., et al., Chemistry, 2008. 14(11): 3410-7; S. De Lamo Marin, et al, Fur. J. Org. Chem. 2010, 3985-9; Pousse, G., et ah, Org Lett, 2010. 12(16): 3582-5; Luo, H., et ah, Chem Biol, 2014. 21(12): 1610-7; Zhao, L., et ah, Chembiochem, 2015. 16(10): 1420-5) and most of these ations were by l sis. Because of their extreme potency and unique mechanism of cytotoxicity, amatoxins have been used as payloads for conjugations (Fiume, L., Lancet, 1969. 2 (7625): 853-4; Barbanti-Brodano, G. and L. Fiume, Nat New Biol, 1973. 243(130): 281-3; Bonetti, E., M. et al, Arch l, 1976. 35(1): p. 69-73; Davis, M. T., n, J. F.
Science 1981, 213, 1385-1388; Preston, J.F., et al, Arch Biochem Biophys, 1981. 209(1): 63- 71; H. Faulstich, et al, Biochemistry 1981, 20, 6498-504; Barak, F.S., et ah, Proc Natl Acad Sci USA, 1981. 78(5): 3034-8; Faulstich, H. and F. Fiume, Methods Enzymol, 1985. 112: 225-37; Zhelev, Z., A. et al, Toxicon, 1987. 25(9): 981-7; Khalacheva, K., et al, Eksp Med Morfol, 1990. 29(3): 26-30; U. Bermbach, H. Faulstich, Biochemistry 1990, 29, 6839-45; sman, J. E. and J. F. Preston, Int. J. Peptide Protein Res. 1991, 37, 544-51; Mullersman, J.E. and J.F.
Preston, Biochem Cell Biol, 1991. 69(7): 418-27; J. Anderl, H. Echner, H. Faulstich, Beilstein J. Org. Chem. 2012, 8, 2072-84; Moldenhauer, G., et al, J. Natl. Cancer Inst. 2012, 104, 622- 34; A. Moshnikova, et al; mistry 2013, 52, 1171-8; Zhao, F., et ah, ochem, 2015. 16(10): 1420-5; Zhou, B., et ah, Biosens Bioelectron, 2015. 68: 189-96; W02014/043403, US20150218220, EP 1661584). We have been working on the ation of amatoxins for a while. Examples of the structures of the conjugate of the antibody- amatoxins via the bridge linker are preferred as the following structures of AmOl, Am02, Am03, and Am04.
V r8 r9 Q rr HN ■N' R74* \ H o ii\ ° / ^fmVX2 II2 / Ril mi O n AmOl Q •Xf o O yRf R7\ \ H O ° / N* ^---- -- (rCV In yet another embodiment, two or more different xic agents are preferred conjugated to a cell-binding molecule via a bridge linker of this patent. The two or more different cytotoxic agents can be selected from any combinations of tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs, daunombicin and doxorubicin compounds, iazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines), calicheamicins and the enediyne antibiotics, actinomycins, amanitins, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, mides, microginins, umins, alterobactins, microsclerodermins, theonellamides, esperamicins, PNU-159682, and their analogues and derivatives above thereof.
Examples of the structures of the conjugates containing two or more different cytotoxic agents via the bridge linker are as the following Z01, Z02, Z02, Z04, Z05, Z06, Z07, Z08, Z09, Z10, Z12, Z13, Z14, Z15, Z16, Z17 and Z18: m A li mi X2 r2 AcO 1 Rj I I ■N- ■N- r3 3 —"O' 'Y o o OH m,K H O OAc mAh N O a N N, X/ R2 X 2 l I o n n I LT n OH O s ’ H O Ill 2 n mAh Vv O ^2 X2''b n I m2 .rCOXAW^^ o*° -1 mi mAh N O OAc O Xa’ R N. 2 X2 aT o N ° x»'' N I ^ /y n OH s—y h n I O Z05 O N ci \ ,9 % ts O \ X3 Ri MeO. N x, /9 I s o \ ^ i^N^O mAh ^ HO H / H3CO o / ■X4 s OA*. H ^R2 OAc O X3’ O N. X2 N O I V // N - I S—^ H 1*12 n O Z06 O o n O' / R1 Cl \ ' O N^x3‘ MeO N' XiAs I X ^ H3CON HO H / X2 WX,m2 _l n O O Z07 O ° / o Cl \ u o N ,Ri MeO. N' > X3 I SX O mAb ml a ! H3C() HO H X O Z08 /"V* X s xi Ri < ! O \ cCCo^xO^ O _____ O m.
X sV' X* o R2 1 n = 1 ^() () _() o 1 m2 n m1o3 so3m2 A A H HN- mAh Vi i o OMe Me° o ■N- r3 O I O —‘ "O O —o ° O^OH J -ln m1o3, ^so3m2 O D NH HN X3 V-N Me' ■N- mAb Me ni| XX’3^ O r2 1 ° 1 -o ° -() O o^nw J -J11 zn MiOjS. so3m2 x’4. D-WNi f \ N n ^ uivie Mevy - n /2)-x=/ iii| mAb _\ I o I ,o s -O o (Aoh x'/ /b>2 n Z12 Mj03i wHN-^S°3M2 X NH WV° rv-x^'W^ ■N. MeO / m, US n Z13 yO "I 1'VSN I \ N O OAc o JUiJr^rx'* N. xy^Rj N N I O ^ N ~xMs / S H m2 O -In O Z14 m1o3s H so3m2 N. HN /Ri U x>t< N-V-A. X, \ OMe Me< mixM mAb O / (9M Zi OH OAc O x3'-r2 V >y N OH n S-y H O Z15 jso3m2 Ml°3SV-N o HN Rt I X ,X4' X3 \ ■N- MeO‘ ■N- Me m. mAb O O X'4 / N O OAc O X N 'N 'X3' n O m2 O Z16 'X3 — X! a /? P () MeO N Tr^^b I \ mAb v* « N^O H3CO HO H *yX2 X’ o r2 1 O 1 -o O _J O o-k^U, Rl- X3----- Xi MeO 'V \ z8 I mAb # 4 N rrij H3CO HO H H O X-4 s-\x2. NA **&*» Zi R2 ss N’ PH o \ X'3 O \\N'' \ "o ' m2 -1 n Z18 Wherein mAb is an antibody; X3 and X’3 are independently CH2, O, NH, NHC(O), NH, C(O), OC(O), 0C(0)(NR3), Ri,NHRi, NRi,C(0)Ri or absent; X4and X’4are independently H, CH2, OH, O, C(O), C(0)NH, C(0)N(Ri), Ri,NHRi, NRi C(0)Ri or C(0)0; Mi and M2 are independently H, Na, K, Ca, Mg, NH4, NRiR2R3; n, mi, m2, "-----", Xi, X2, R^ R2and R3 are the same defined in a (I) and (II). In addition, Ri and/or R2 can be absent independently.
In yet another embodiment, cell-binding ligands or cell receptor agonists can be conjugated to a cell-binding molecule via a bridge linker of this patent. These conjugated cell-binding ligands or cell receptor ts, in particular, antibody-receptor conjugates, can be not only to work as a targeting tor/director to r the conjugate to malignant cells, but also be used to modulate or co-stimulate a desired immune response or altering signaling pathways.
In the immunotherapy, the cell-binding ligands or receptor agonists are preferred to conjugate to an dy of TCR (T cell receptors) T cell, or of CARs (chimeric antigen receptors) T cells, or of B cell or (BCR), or the cytotoxic cells. The cell-binding ligands or receptor agonists are selected, but not limited, from: Folate derivatives (binding to the folate receptor, a protein over-expressed in ovarian cancer and in other ancies) (Low, P. S. et al 2008, Acc. Chem. Res. 41, 120-9); Glutamic acid urea derivatives (binding to the prostate specific membrane n, a surface marker of prostate cancer cells) (Hillier, S. M.et al, 2009, Cancer Res. 69, 6932-40); Somatostatin (also known as growth hormone-inhibiting hormone (GHIH) or somatotropin release-inhibiting factor (SRIF)) or somatotropin release- inhibiting hormone) and its analogues such as octreotide (Sandostatin) and tide uline) (particularly for neuroendocrine tumors, GH-producing pituitary adenoma, paraganglioma, nonfunctioning pituitary a, pheochromocytomas) (Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. U.S.A. 103, 16436-41). In general, Somatostatinand its receptor subtypes (sstl, sst2, sst3, sst4, and sst5) have been found in many types of tumors, such as neuroendocrine tumors, in particular in GH-secreting pituitaryadenomas (Reubi J. C., t, A. M. 1984 J. Clin.
Endocrinol Metab 59: 1148-51; Reubi J. C., Landolt A. M. 1987 J Clin Endocrinol Metab 65: 65-73; Moyse E, et al, J Clin Endocrinol Metab 61: 98-103) and gastroenteropancreatic tumors (Reubi J. C., et al, 1987 J Clin Endocrinol Metab 65: 1127-34; Reubi, J. C, et al, 1990 Cancer Res 50: 5969-77), pheochromocytomas (Epel-baum J, et al 1995 J Clin Endocrinol Metab 80:1837-44; Reubi J. C., et al, 1992 J Clin Endocrinol Metab 74: 1082-9), neuroblastomas (Prevost G, 1996 Neuroendocrinology 63:188-197; Moertel, C. L, et al 1994 Am J Clin Path 102:752-756), medullary thyroid cancers (Reubi, J. C, et al 1991 Lab Invest 64:567-573) small cell lung cancers n U, et al, 1990 Cancer 66:2129-2133), nonneuroendocrine tumors including brain tumors such as meningiomas, medulloblastomas, or gliomas (Reubi J. C., et al 1986 J Clin Endocrinol Metab 63: 433-8; Reubi J. C., et al 1987 Cancer Res 47: 5758-64; Fmhwald, M. C, et al 1999 Pediatr Res 45: 697-708), breast omas (Reubi J. C., et al 1990 Int J Cancer 46: 416-20; Srkalovic G, et al 1990 J Clin Endocrinol Metab 70: 661-669), lymphomas (Reubi J. C., et al 1992, Int J 50: 0), renal cell cancers (Reubi J. C., et al 1992, Cancer Res 52: 6074-6078), mesenchymal tumors (Reubi J. C., et al 1996 Cancer Res 56: 1922-31), tic (Reubi J. C., et al 1995, J. Clin. Endocrinol Metab 80: 2806-14; et al 1989, Prostate 14:191-208; Halmos G, et al J. Clin. Endo-crinol Metab 85: 2564-71), ovarian (Halmos, G, et al, 2000 J Clin Endocrinol Metab 85: 3509-12; Reubi J. C., et al 1991 Am J Pathol 138:1267-72), gastric (Reubi J. C., et al 1999, Int J Cancer 81: ; Miller, G. V, 1992 Br J Cancer 66: 391-95), hepatocellular (Kouroumalis E, et al 1998 Gut 42: 442-7; Reubi J. C., et al 1999 Gut 45: ) and nasopharyngeal carcinomas (Loh K. S, et al, 2002 Virchows Arch 441: 444-8); certain Aromatic sulfonamides, specific to carbonic anhydrase IX (a marker of hypoxia and of renal cell carcinoma) (Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-7); Pituitary adenylate cyclase activating peptides (PACAP) (PAC1) for pheochromocytomas and paragangliomas; Vasoactive intestinal es (VlP)and their receptor subtypes (VPAC1, VPAC2) for cancers of lung, stomach, colon, rectum, breast, prostate, pancreatic ducts, liver, urinary bladder and epithelial tumors; a-Melanocyte-stimulating hormone (a-MSH) receptors for s tumors; Cholecystokinin (CCK)/gastrin receptors and their receptor subtypes (CCK1 (formerly CCK-A) and CCK2 for small cell lung cancers, ary thyroid carcinomas, astrocytomas, insulinomas and ovarian cancers;Bombesin(Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp- Ala-Val-Gly-His-Leu-Met-NH2)/gastrin-releasing e (GRP) and their receptor subtypes (BB1, GRP receptor subtype (BB2), the BBS and BB4) for renal cell, breast, lung, gastric and prostate carcinomas, and neuroblastoma (and neuroblastoma (Qhlsson, B., et al, 1999, Scand. J.
Gastroenterology 34 (12): 1224-9; Weber, H. €., 2009, Cur. Qpin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008, Cur. Opin. Endocri. Diab. Obesity 15(1), 58-64 ); Neurotensin ors and its receptor subtypes(NTRl, NTR2, NTR3) for small cell lung cancer, neuroblastoma, pancreatic, colonic cancer and Ewing sarcoma; Substance P receptors and their receptor subtypes(such as NK1 receptor for Glial tumors, Hennig I. M., et al 1995 Int. J. Cancer 61, 786-792); eptide Y (NPY) receptors and its receptor subtypes )for breast carcinomas; Homing Peptides include RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), the dimeric and multimeric cyclic RGD peptides (e.g. cRGDfV) that recognize receptors (integrins) on tumor surfaces (Laakkonen P, Vuorinen K. 2010, Integr Biol (Camb). 2(7-8): 326-337; Chen K, Chen X. 2011, ostics. 1:189-200; er E, et al, Anti-Cancer Agents Med Chem. 7 (5): 552- 558; Kerr, J. S. et al, Anticancer Research, 19(2A), 8; Thumshim, G, et al, 2003 Chem.
Eur. J. 9, 2717- 2725), and TAASGVRSMH or GLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptides (31 amino acid peptide that binds to cell surfaceexpressed nucleolin receptor) (Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143, Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrative Biol, 2(7-8), 326-337; M. A. Burg, 1999 Cancer Res., 59(12), 2869-2874; K.
Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11), ); Cell Penetrating Peptides (CPPs) (Nakase I, et al, 2012, J. Control Release. ,181-188); e es, such as luteinizing hormone-releasing hormone (LHRH) agonists and antagonists, and gonadotropinreleasing hormone (GnRH) agonist, acts by targeting follicle stimulating hormone (FSH) and luteinising hormone (LH), as well as terone production, e.g. buserelin (Pyr-His-Trp-Ser- Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), Gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro- Gly-NHi), Goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), Histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu-Arg-Pro-NHEt), leuprolide (Pyr-His-Trp-Ser-Tyr- D-Leu-Leu-Arg-Pro-NHEt), Nafarelin (Pyr-His-Trp-Ser-Tyr-lNal-Leu-Arg-Pro-Gly-NHi), Triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHi), Nafarelin, elirs, Abarelix 2Nal-DchloroPhe-D(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro- DAla-NHi), Cetrorelix (Ac-D-2Nal-Dchloro-Phe-D(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg- Ala-NHi), Degarelix (Ac-D-2Nal-DchloroPhe-D(3-pyridyl)Ala-SeraminoPhe(L- rotyl)-DaminoPhe(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH2), and Ganirelix (Ac- D-2Nal-DchloroPhe-D(3-pyridyl)Ala-Ser-Tyr-D-(N9, N10-diethyl)-homoArg-Leu-(N9, N10-diethyl)-homoArg-Pro-D-Ala-NH2) (Thundimadathil, J., J. Amino Acids, 2012, 967347, doi: 10.1155/2012/967347; Boccon-Gibod, L.; et ai, 2011, Therapeutic es in Urology 3(3): 127 -140; Debmyne, F., 2006, Future Oncology, 2(6), 677-696; Schally A. V; Nagy, A. 1999 Fur J Endocrinol 141:1-14; Koppan M, et al 1999 Prostate 38:151-158); and n Recognition Receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins and Nodlike Receptors (NLRs) (Fukata, M., et al, 2009, Semin. Immunol. 21, 242-253; Maisonneuve, C., et al, 2014, Proc. Natl. Acad. Sci. U. S. A. Ill, 1-6; Botos, I., et al, 2011, Structure 19, 447-459; Means, T.
K. , et al, 2000, Life Sci. 68, 241-258) that range in size from small molecules (imiquimod, guanisine and adenosine analogs) tolarge and complex biomacromolecules such as lipopolysaccharide (EPS), nucleic acids (CpG DNA, polyLC) and lipopeptides (Pam3CSK4) ri, S. P., et al, 2011, Nature 470, 543-547; Lane, T., 2001, J. R. Soc. Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228; Dudek, A. Z., et al, 2007, Clin.
Cancer Res. 13, 7119-25); onin receptors which is a 32-amino-acid neuropeptide involved in the tion of calcium levels largely through its effects on osteoclasts and on the kidney (Zaidi M, et al, 1990 Crit Rev Clin Lab Sci 28, 109-174; Gom, A. H., et al 1995 J Clin Invest 95:2680-91); And integrin receptors and their receptor subtypes (such as avPi, civPs, avPs, a.vPe.
(X6P4,0.7P1 (XlPi, a.iibPv etc) which generally play important roles in angiogenesis are sed on the surfaces of a variety of cells, in particular, of osteoclasts, endothelial cells and tumor cells (Ruoslahti, E. et al, 1994 Cell 77, 477-8; Albelda, S. M. et al, 1990 Cancer Res., 50, 6757-64).
Short peptides, GRGDSPK and Cyclic RGD pentapeptides, such as cyclo(RGDfV) (El) and its derives [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo-(RG-N(Me)D-fV), cyclo(RGDN (Me)f-V), cyclo(RGDf-N(Me)V-)(Cilengitide)] have shown high binding affinities of the intergrin ors (Dechantsreiter, M. A. et al, 1999 J. Med. Chem. 42, 3033-40, n, S.
L. , et al, 2002 J. Med. Chem. 45, 1045-51).
The cell-binding ligands or cell receptor agonists can be Ig-based and non-Ig-based protein scaffold molecules. The Ig-Based lds can be selected, but not limited, from Nanobody (a derivative of VHH id Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-97); Domain antibodies (dAb, a derivative of VH or VL domain) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-90); ific T cell Engager (BiTE, a ific y) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); Dual Affinity ReTargeting (DART, a bispecific diabody) (Moore P. A. P, et al. 2011, Blood 117(17), 4542-51); Tetravalent tandem antibodies (TandAb, a dimerized bispecific y) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336- 4341). The Non-Ig lds can be ed, but not d, from Anticalin (a derivative of Lipocalins) (Skerra A. 2008, FEES J., 275(11): 3; Beste G, et al, 1999 Proc. Nat. Acad.
USA. 96(5): 1898-903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2): 337-50; , A. 2007, Curr Opin Biotechnol. 18(4): 295-304; Skerra, A. 2008, FEES J. 275(11):2677-83); ins (10th FN3 (Fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4):1141-51; Batori V, 2002, Protein Eng. 15(12): 1015-20; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2): 363-71; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4): 211-19); Designed Ankyrin Repeat Proteins (DARPins) (a derivative of ankrin repeat (AR) proteins) (Boersma, Y.L, et al, 2011 Curr Opin Biotechnol. 22(6): 849-57), e.g. DARPin C9, DARPin Ec4 and DARPin E69_LZ3_E01 (Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 3; Patricia M-K. M., et al, Clin Cancer Res. 2011; 17(1): 100-10; Boersma Y. L, et al, 2011 J. Biol. Chem. 286(48), 41273-85); Avimers (a domain A/low-density lipoprotein (LDL) receptor) (Boersma Y. L, 2011 J. Biol. Chem. 286(48): 41273- 41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12): 1556-61).
Examples of the structures of the conjugate of the antibody-cell-binding ligands or cell receptor agonists via the bridge linker are as the following: LB01 (PMSA ligand conjugate), LB02 (Folate conjugate), LB03 (Somatostatin conjugate), LB04 (Octreotide, a Somatostatin analog conjugate), LB05 (Lanreotide, a Somatostatin analog conjugate), LB06 (Vapreotide (Sanvar), a Somatostatin analog conjugate), LB07 (CAIX ligand conjugate), LB08 (CAIX ligand ate), LB09 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB 10 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate), LB 11 (GnRH antagonist, Abarelix conjugate), LB 12 (cobalamin, VB12 analog conjugate), LB 13 (Gastrin releasing peptide receptor (GRPr), MBA conjugate), LB 14 (for avP3 integrin receptor, cyclic RGD pentapeptide conjugate), LB 15 (hetero-bivalent peptide ligand ate for VEGF or), LB 16 (Neuromedin B conjugate), LB 17 (bombesin conjugate for a G-protein coupled receptor), LB 18 (TLR2 conjugate for a Toll-like receptor,), LB 19 (for an androgen or), LB20 (Cilengitide/cyclo(-RGDfV-) conjugate for an av intergrin receptor) and LB21-LB29 (Glucocorticoids’ conjugates for glucocorticoid receptor (GR, or GCR) also known as NR3C1 (nuclear receptor subfamily 3, group C, member 1). y """s o ; m,,t. ■ r mAb H H i* y (R2^X2^S v /m2 o n LB01 o o °^OH O "I X. N = v 1 SN 3 Xi H o mi mAb h2n N ( r2)—X2-^ ■s' in2 o n LB 02 Zi OH OL X, /^"XV s R1 HH o H]y S\ H mAb S VN, C TVT H N nh2 S 7T'X2 N O mi o 't'R!)m2 O HO O n LB 03 ox; H X4 R| ,o ' rYv k V'Xj, 2 \ I ° N R6^n xr O-P s I R1 \ o OH ^Co3+ , N / v n H mAh N iN f h om,(42 OH nh2 O n o^nh2 h2n LB 12 ° V'/3 O NH j2n sn/'Xi HN-A H 9 V-N. A Xj^Vy^V1' mAb i N N "1 H W O i H o o o ml xxH2N'\) X VvS‘/ISstr"X2'-f Ri) m2 o n LB 13, o O o H \ xr\ls HN Ri \ JjO^NH O mAb NH S o NH H X2 HN \ o o o NH2 /mi ( rL, n LB 14 S S H o P 1 Ac-A-G-P-T-W-C-E-D-D-W-Y-Y-C-W-L-F-G-T-G-G-G NH2 R x,- 4xaU 1 mi mAb (Ri^' .. m2 O n LB 15 o Q H Ri h2n g-n-l-w-a-t-g-h-f-m-nh2 /s \ z Xi x3nh X^RJn,2 O n LB 16 / HR -V-x f n-Arg-Lcu-Gly-Asn-GIn-Trp-Ala-Val-Gly-His-Lcu-IVlct~N^.^/ 1 J i SL (uAXiTC-ssr ' 'm2 O n LB 17 o « n1 R’ H I O O i LB27 (Prednisolone conjugate) O O HO Ri xi-VN ^OH \ Me H mAh = i / H H m i i n Me «‘r8. LB28 (Methylprednisolone Ov jo Me \ HO Rl L"ilOH XiA/\ Me H Me mAh | = / F H mi o ( In yet another embodiment, IgG antibody conjugates conjugated with one, or two, or more differently function molecules or drugs are preferred to be conjugated specifically to a pair of thiols (through reduction of the disulfide bonds) between the light chain and heavy chain, the upper disulfide bonds between the two heavy chains, and the lower disulfide bonds between the two heavy chains as shown in the following structure, ST1, ST2, STS, ST4, STS, ST6, ST7 or |-X'fR.-Drugl)mi lx>'K)m2 o 2 o Xi’ ~Drug2)in3 (rWX2^P V /m2 N0 ST2 ^X' f R,"-Drug3) O m5 x^R,-Drugl)m o Xi’ (Rfexl >X^R^)m4 o M o (R2"fc6 o>Xl" T ^X‘" tR,"-Drug3) (R2'"fcx^ R1'" —Drug4)m7 o x'iK (Rfe 2 ............ . XY- jT ' fR^'-Drugj) (R2'"t8X^ X1",^Ri"' —Drug4) (r2"m4-x2^ ° mio ^ 1 !°X,'"4Ri""'Drug5) II \ J m9 O o ^R.-Urug.) o ,~l^ n x2'-(R2')m4 V z'm2 Q (R2"^x> rX|"tR i"-Drug3) (r2".)_x2.. x1-(Rr"-Dr,,84) (r2"m4-"X2'^'° mio 7T ICT ' l^-X,""(R'""'Dru84 /m9 (r2...4-x2’^J° £x,...(R1.....DruS5) 1,112 77 o 111 ii o ST8 Wherein Ri, Ri , Ri , Ri , Ri , and Ri are independently defined as Ri which is bed the same in Formula (I); Wherein R2 , Ri , Ri , Ri and R2 are independently defined as R2 which is described the same in Formula (I); Xi , Xi , Xi , Xi , and Xi are independently defined as Xi which is described the same in claim Formula (I); X2 , X2 , X2 , X2 , and X2 are independently d as X2 which is described the same in claim Formula (I); "==" represents either single bond or double bond; mi, m2, m3, 1114, ms, me, m? and mg are independently an integer from 1 to 30, preferably from 1 to 10; Dmgi, Dmg2, Drugs, Drugs.
Drugs and Drugs is independently selected from tubulysins, maytansinoids, taxanoids es), analogs, daunombicin and doxorubicin compounds, iazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, mycin, nobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines), calicheamicins and the ne otics, actinomycin, amanitin (amatoxins), azaserines, bleomycins, icin, tamoxifen, idambicin, dolastatins, auristatins (e.g. monomethyl auristatin E, MMAE , MMAF, auristatin PYE, auristatin TP, atins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins, microsclerodermins, theonellamides, esperamicins, PNU-159682, and their analogues and derivatives, as well as the function molecules bed throughout this patent application. In addition, Ri, Ri, Ri , Ri , Ri and/or Ri can be absent.
In yet another embodiment, a pharmaceutical composition comprising a therapeuticcally effective amount of the conjugate of Formula (II) or any ates described through the present patent can be administered concurrently with the other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates for istically effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease. The synergistic agents are preferably selected from one or several of the following drugs: Abatacept (Orencia), Abiraterone acetate (Zytiga®), Acetaminophen/hydrocodone, Adalimumab, afatinib dimaleate (Gilotrif®), alemtuzumab (Campath®), Alitretinoin (Panretin®), ado-trastuzumab emtansine (Kadcyla™), Amphetamine mixed salts (Amphetamine/dextroamphetamine, or Adderall XR), anastrozole (Arimidex®), Aripiprazole, Atazanavir, Atezolizumab (MPDL3280A), statin, axitinib (Inlyta®), AZD9291, stat (Beleodaq™), Bevacizumab (Avastin®), Cabazitaxel (Jevtana®), Cabozantinib (Cometriq™), bexarotene (Targrtin®), blinatumomab (Blincyto™), Bortezomib (Velcade®), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), Budesonide, Budesonide/formoterol, Buprenorphine, Capecitabine, carfilzomib (Kyprolis®), Celecoxib, ceritinib 8/Zykadia), Cetuximab (Erbitux®), Ciclosporin, Cinacalcet, crizotinib (Xalkori®), Dabigatran, dabrafenib lar®), Darbepoetin alfa, Damnavir, imatinib mesylate (Gleevec®), dasatinib (Sprycel®), denileukin diftitox (Ontak®), Denosumab (Xgeva®), te, Dexlansoprazole, Dexmethylphenidate, Dinutuximab (Unituxin™), Doxycycline, Duloxetine, Durvalumab 736), Emtricitabine/Rilpivirine/Tenofovir disoproxil fumarate, itbine/tenofovir/efavirenz, Enoxaparin, Enzalutamide (Xtandi®), Epoetin alfa, erlotinib (Tarceva®), Esomeprazole, Eszopiclone, Etanercept, Everolimus (Afinitor®), exemestane (Aromasin®), imus (Afinitor®), Ezetimibe, Ezetimibe/simvastatin, brate, Filgrastim, imod, Fluticasone propionate, Fluticasone/salmeterol, fulvestrant (Faslodex®), gefitinib (Iressa®), Glatiramer, Goserelin acetate (Zoladex), Icotinib, ib (Gleevec), Ibritumomab tiuxetan (Zevalin®), ibmtinib (Imbmvica™), idelalisib (Zydelig®), Infliximab, n aspart, Insulin detemir, Insulin glargine, Insulin lispro, Interferon beta la, eron beta lb, lapatinib (Tykerb®), Ipilimumab (Yervoy®), Ipratropium bromide/salbutamol, tide acetate (Somatuline® Depot), lenaliomide (Revlimid®), lenvatinib mesylate (Lenvima™), ole (Femara®), Levothyroxine, Levothyroxine, Lidocaine, Linezolid, Liraglutide, Lisdexamfetamine, MEDI4736 (AstraZeneca, e), Memantine, Methylphenidate, Metoprolol, Modafinil, Mometasone, Nilotinib (Tasigna®), Nivolumab (Opdivo®), ofatumumab (Arzerra®), obinutuzumab (Gazyva™), olaparib rza™), Olmesartan, Olmesartan/hydrochlorothiazide, Omalizumab, Omega-3 fatty acid ethyl esters, Oseltamivir, Oxycodone, palbociclib (Ibrance®), Palivizumab, panitumumab (Vectibix®), panobinostat (Farydak®), pazopanib (Votrient®), pembrolizumab uda®), Pemetrexed (Alimta), pertuzumab (Perjeta™), Pneumococcal conjugate vaccine, pomalidomide (Pomalyst®), Pregabalin, Quetiapine, Rabeprazole, radium 223 chloride (Xofigo®), Raloxifene, Raltegravir, rumab (Cyramza®), Ranibizumab, regorafenib (Stivarga®), Rituximab (Rituxan®), Rivaroxaban, romidepsin (Istodax®), Rosuvastatin, tinib ate i™) , Salbutamol, Sevelamer, Sildenafil, siltuximab (Sylvant™), Sitagliptin, Sitagliptin/ metformin, nacin, Sorafenib (Nexavar®), Sunitinib (Sutent®),Tadalafil, tamoxifen, Telaprevir, temsirolimus (Torisel®), Tenofovir/emtricitabine, Testosterone gel, Thalidomide (Immunoprin, Talidex), Tiotropium bromide, toremifene (Fareston®), trametinib (Mekinist®), Trastuzumab, Tretinoin (Vesanoid®), Ustekinumab, Valsartan, vandetanib (Caprelsa®), vemurafenib (Zelboraf®), vorinostat (Zolinza®), ziv-aflibercept (Zaltrap®), Zostavax., and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients thereof, or a combination above thereof.
The drugs/ cytotoxic agents used for conjugation via a bridge linker of the present patent can be any analogues and/or derivatives of drugs/molecules described in the present patent. One skilled in the art of dmgs/cytotoxic agents will readily tand that each of the dmgs/cytotoxic agents described herein can be modified in such a manner that the resulting compound still retains the specificity and/or activity of the ng compound. The skilled artisan will also understand that many of these compounds can be used in place of the drugs/cytotoxic agents described herein. Thus, the drugs/cytotoxic agents of the present invention include analogues and derivatives of the compounds described .
All references cited herein and in the examples that follow are expressly incorporated by reference in their entireties.
EXAMPLES The invention is further described in the following examples, which are not intended to limit the scope of the ion. Cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, chweig, Germany , or The Shanghai Cell Culture Institute of Chinese Acadmy of Science, unless otherwise specified. Cell culture reagents were obtained from Invitrogen Corp., unless otherwise specified.
All anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All other reagents and ts were purchased as the highest grade available and used without r purification. The preparative HPLC separations were performed with Varain PreStar HPLC. NMR spectra were recorded on Varian Mercury 400 MHz Instrument. Chemical shifts (.delta.) are ed in parts per million (ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J) are reported in Hz. The mass spectral data were ed on a Waters Xevo QTOF mass um equipped with Waters Acquity UPLC separations module and Acquity TUV detector.
Example 1. Synthesis of 2,3-dibromosuccinic anhydride (7) Biv__ Br To a solution of 2,3-dibromosuccinic acid (10.00 g, 36.51 mmol) in dry CH2CI2 (100 ml) at 0 °C was added phosphorus pentoxide (12.21 g, 85.84 mmol). The mixture was stirred at 0 °C for 2 h and then r.t. for 5 h, ed h short Si02 column, and rinsed the column with EtOAc/CH2Cl2 (1:6). The filtrate was concentrated and triturated with EtOAc/hexane to afford the title compound (6.63 g, 71% yield). ESI MS m/z Czd^B^Os [M+H] +, cacld. , found 256.70.
Example 2. Synthesis of (S, E)methyl-N-(3-methylbutanylidene)propane sulfonamide (122) II \N—( O / \ WO 59622 To a solution of (S)methylpropanesulfinamide (100 g, 0.825 mol, 1.0 eq.) in 1 L THF was added )4 (345 mL, 1.82 mol, 2.2 eq.) and 3-methylbutanone (81 mL, 0.825 mol, 1.0 eq.) under Ni at r.t. The reaction mixture was ed for 16 h, then cooled to r.t. and poured onto iced water. The mixture was filtered and the filter cake was washed with EtOAc. The organic layer was separated, dried over anhydrous NaiSC^ and concentrated to give a residue which was purified by vacuum distillation (15-20 torr, 95 °C) to afforded product 122 (141 g, 90% yield) as a yellow oil. 1H NMR (500 MHz, CDCI3) 5 2.54 - 2.44 (m, 1H), 2.25 (s, 3H), 1.17 (s, 9H), 1.06 (dd, / = 6.9, 5.1 Hz, 6H). MS ESIm/z calcd for NOS [M+Na]+ 212.12; found 212.11.
Example 3. Synthesis of (2S,3S)azidomethylpentanoic acid (124) \ ^N3 To a solution of NaNs (20.0 g, 308 mmol) in a e of water (50 mL) and dichloromethane (80 mL), cooled at 0 °C, TLO (10 mL, 59.2 mmol, 2.0 eq.) was added slowly.
After addition, the reaction was stirred at 0 °C for 2 h, then the organic phase was separated and the aqueous phase was extracted with dichloromethane (2 x 40 mL). The combined organic phases were washed with saturated NaHCCL, on and used as is. The dichloromethane on of l azide was added to a mixture of (L)-isoleucine (4.04 g, 30.8 mmol, 1.0 eq.), K2CO3 (6.39 g, 46.2 mmol, 1.5 eq.), CuSCL'SHiO (77.4 mg, 0.31mmol, 0.01 eq.) in water (100 ml) and methanol (200 ml). The mixture was stirred at r.t. for 16 h. The organic solvents were removed under reduced pressure and the aqueous phase was diluted with water (250 mL) and acidified to pH 6 with concentrated HC1 and diluted with phosphate buffer (0.25 M, pH 6.2, 250 mL). The aqueous layer was washed with EtOAc (5 x 100 mL) to remove the sulfonamide byproduct , and then acidified to pH 2 with concentrated HC1, extracted with EtOAc (3x150 mL).
The combined organic layers were dried over anhydrous NaiSOzt, filtered and concentrated to give product 124 (4.90 g, 99% yield) as a colorless oil. ^ NMR (500 MHz, CDCI3) 8 12.01 (s, 1H), 3.82 (d, / = 5.9 Hz, 1H), 2.00 (ddd, / = 10.6, 8.6, 5.5 Hz, 1H), 1.54 (dqd, / = 14.8, 7.5, 4.4 Hz, 1H), 1.36 - 1.24 (m, 1H), 1.08 - 0.99 (m, 3H), 0.97 - 0.87 (m, 3H).
Example 4. Synthesis of D-A-methyl pipecolinic acid (126) ,,,/co2h To a solution of D-pipecolinic acid (10.0 g, 77.4 mmol, 1.0 eq.) in methanol (100 mL) was added formaldehyde (37% aqueous solution, 30.8 mL, 154.8 mmol, 2.0 eq.), followed by Pd/C (10 wt%, 1.0 g). The reaction mixture was d under H2 (1 atm) overnight, and then filtered WO 59622 through Celite, with washing of the filter pad with methanol. The filtrate was concentrated under reduced pressure to afford nd 126 (10.0 g, 90% yield) as a white solid.
Example 5. sis of (R)-perfluorophenyl l-methylpiperidinecarboxylate (127) 2c6f5 To a solution of D-/V-methyl pipecolinic acid (2.65 g, 18.5 mmol) in EtOAc (50 mL) were added pentafluorophenol (3.75 g, 20.4 mmol) and DCC (4.21 g, 20.4 mmol). The reaction mixture was stirred at r.t. for 16 h, and then filtered over Celite. The filter pad was washed with mL of EtOAc. The filtrate was used immediately without further purification or concentration.
Example 6. Synthesis of 2,2-diethoxyethanethioamide (129) EtO AOEtNH2 10 2,2-diethoxyacetonitrile (100 g, 0.774 mol, 1.0 eq.) was mixed with (NtLOiS aqueous solution (48%, 143 mL, 1.05 mol, 1.36 eq.) in methanol (1.5 L) at room temperature. After stirring for 16 h, the reaction mixture was concentrated and the residue was taken up in dichloromethane, washed with saturated NaHCOs on and brine, dried over anhydrous Na2S04 and concentrated. The residue was triturated with a solvent mixture of petroleum ether and dichloromethane. After filtration, the desired t 2 as a white solid was ted (100 g, 79% yield). NMR (500 MHz, CDC13) 5 7.81 (d, / = 71.1 Hz, 2H), 5.03 (s, 1H), 3.73 (dq, / = 9.4, 7.1 Hz, 2H), 3.64 (dq, / = 9.4, 7.0 Hz, 2H), 1.25 (t, / = 7.1 Hz, 6H). e 7. Synthesis of ethyl 2-(diethoxymethyl)thiazolecarboxylate (130) EtO">j>-co2e, 90 g of molecular sieves (3A) was added to a e of thioamide 129 (100 g, 0.61 mol, 1.0 eq.) and ethyl bromopyruvate (142 mL, 1.1 mol, 1.8 eq.) in 1 L EtOH. The mixture was refluxed (internal temperature about 60 °C) for Ih, then ethanol was removed on rotovap and the residue was taken up in dichloromethane. The solid was filtered off and the filtrate was concentrated and purified by column chromatography (PE/EtOAc 5:1-3:1) to give thiazole carboxylate 130 (130 g, 82% yield) as a yellow oil.
Example 8. Synthesis of ethyl 2-formylthiazolecarboxylate (131) To a solution of compound 130 (130 g, 0.50 mol) in acetone (1.3 L) was added 2 N HC1 (85 mL, 0.165 mol, 0.33 eq.). The reaction mixture was ed (internal temperature about 60 °C), monitored by TLC is until starting material was completely consumed (about 1-2 h).
Acetone was removed under reduced re and the residue was taken up in romethane (1.3 L), washed with saturated NaHCO, solution, water and brine, and then dried over anhydrous NaiSOzj. The solution was filtered and concentrated under reduced pressure. The crude product was purified by recrystallization from petreolum ether and diethyl ether to afford a white solid 131 (40 g, 43% yield). NMR (500 MHz, CDCI3) 5 10.08 - 10.06 (m, 1H), 8.53 - 8.50 (m, 1H), 4.49 (q, / = 7.1 Hz, 2H), 1.44 (t, / = 7.1 Hz, 3H). MS ESI m/z calcd for C7H8N03S [M+H]+ 186.01; found 186.01.
Example 9. Synthesis of ethyl 2-((R,E)(((S)-tert-butylsulfinyl)imino)-l-hydroxy methylpentyl)thiazolecarboxylate (132) L/'COlEt 'Bu*S*0 To a solution of diisopropylamine (121 mL, 0.86 mol, 4.0 eq.) in dry THE (300 mL) was added n-butyllithium (2.5 M, 302 mL, 0.76 mol 3.5 eq.) at -78 °C under N2. The reaction mixture was warmed to 0 °C over 30 min and then cooled back to -78 °. Compound 122 (57 g, 0.3 mol, 1.4 eq.) in THE (200 mL) was added. The reaction mixture was stirred for 1 h before ClTi(0IPr)3 (168.5 g, 0.645 mol, 3.0 eq.) in THE (350 mL) was added dropwise. After stirring for 1 h, compound 131 (40 g, 0.215 mol, 1.0 eq.) dissolved in THE (175 mL) was added dropwise and the resulting reaction mixture was stirred for 2 h. The completion of the reaction was ted by TLC analysis. The reaction was quenched by a mixture of acetic acid and THE (v/v 1:4, 200 mL), then poured onto iced water, extracted with EtOAc (4 x 500 mL). The organic phase was washed with water and brine, dried over anhydrous NaiSCL, filtered and concentrated. The residue was purified by column chromatography (DCM/EtOAc/PE 2:1:2) to afforded the compound 132 (60 g, 74% yield) as a ess oil. NMR (500 MHz, CDC13) 5 8.13 (s, 1H), 6.63 (d, /= 8.2 Hz, 1H), 5.20-5.11 (m, 1H), 4.43 (q, 7 = 7.0 Hz, 2H), 3.42-3.28 (m, 2H), 2.89 (dt, / = 13.1, 6.5 Hz, 1H), 1.42 (t, /= 7.1 Hz, 3H), 1.33 (s, 9H), 1.25 - 1.22 (m, 6H). MS ESI m/z calcd for Ci6H26NaN2O4S2 [M+Na]+ 397.13, found 397.11.
Example 10. Synthesis of ethyl 2-((lR,3R)((S)-l,l-dimethylethylsulfinamido)-lhydroxymethylpentyl )thiazolecarboxylate (133) HN LrC02E*N A solution of compound 132 (23.5 g, 62.7 mmol) dissolved in THE (200 mL) was cooled to - 45 °C. Ti(OEt)4 (42.9 mL, 188 mmol, 3.0 eq.) was added slowly. After the completion of addition, the mixture was stirred for 1 h, before NaBH4 (4.75 g, 126 mmol, 2.0 eq.) was added in portions. The reaction mixture was d at -45 °C for 3 h. TEC analysis showed some starting material still remained. The reaction was quenched with HF (v/v 1:4, 25 mL), followed by EtOH (25 mL). The reaction mixture was poured onto ice (100 g) and warmed to r.t. After filtration over Celite, the organic phase was separated and washed with water and brine, dried over anhydrous NaiSC^, filtered, and trated. The residue was purified by column chromatography (EtOAc/PE 1:1) to deliver product 133 (16.7 g, 71% yield) as a white solid. ^ NMR (500 MHz, CDC13) 5 8.10 (s, 1H), 5.51 (d, 7 = 5.8 Hz, 1H), 5.23 - 5.15 (m, 1H), 4.41 (q, / = 7.0 Hz, 2H), 3.48-3.40 (m, 1H), 3.37 (d, 7=8.3 Hz, 1H), 2.29 (t, 7=13.0 Hz, 1H), 1.95- 1.87 (m, 1H), 1.73 - 1.67 (m, 1H), 1.40 (t, 7 = 7.1 Hz, 3H), 1.29 (s, 9H), 0.93 (d, 7 = 7.3 Hz, 3H), 0.90 (d, 7= 7.2 Hz, 3H). MS ESI m/z calcd for Cie^gNaNiCESi [M+Na]+ 399.15, found 399.14.
Example 11. Synthesis of ethyl 2-((lR,3R)amino-l-hydroxymethylpentyl)thiazole carboxylate hydrochloride (134) HC1H2N tN To a solution of nd 133 (6.00 g, 16.0 mmol, 1.0 eq.) in l (40 mL) was added 4 N HC1 in dioxane (40 mL) slowly at 0 °C. The reaction was allowed to warm to r.t. and stirred for 2.5 h then concentrated and triturated with petreolum ether. A white solid (4.54 g, 92% yield) was collected and used in the next step.
Example 12. Synthesis of ethyl 2-((lR,3R)((2S,3S)azidomethylpentanamido)-l- hydroxymethylpentyl)thiazolecarboxylate (135) O OH N3v N N H L^co2Et Azido-Ile-OH 124 (5.03g, 28.8 mmol, 2.0 eq.) was dissolved in THF (120 mL) and cooled to 0 °C, to which NMM (6.2 mL, 56.0 mmol, 4.0 eq.) and isobutylchloroformate (3.7 mL, 28.8 mmol, 2.0 eq.) were added in ce. The reaction was stirred at 0 °C for 30 min and r.t. 1.0 h, and then cooled back to 0 °C. nd 134 (4.54 g, 14.7 mmol, 1.0 eq.) was added in portions.
After stirring at 0 °C for 30 min, the reaction was warmed to r.t. and stirred for 2 h. Water was added at 0 °C to quenched the reaction and the resulting e was ted with ethyl acetate for three times. The combined organic layers were washed with IN HC1, saturated NaHCO, and brine, dried over anhydrous NaiSCL, filtered and concentrated. The residue was purified by column chromatography (0-30% EtOAc/PE) to give a white solid 135 (4.55 g, 74% yield).
Example 13. Synthesis of ethyl 2-((lR,3R)((2S,3S)azidomethylpentanamido) methyl- iethylsilyl)oxy)pentyl)thiazolecarboxylate (136) o OTES N !P^C02EtN To a solution of compound 135 (5.30 g, 12.8 mmol, 1.0 eq.) in CH2CI2 (50 mL) was added imidazole (1.75 g, 25.6 mmol, 2.0 eq.), followed by chlorotriethylsilane (4.3 mL, 25.6 mmol, 2.0 eq.) at 0 °C. The reaction mixture was allowed to warm to r.t. over 1 hour and stirred for an additional hour. Brine was added to the reaction e, the organic layer was separated and the aqueous layer was extracted with EtOAc. The combined organic phases were dried, filtered, concentrated under reduced re, and purified by column chromatography with a gradient of -35% EtOAc in petreolum ether to afford product 136 (6.70 g , 99% yield) as a white solid, 'fi NMR (500 MHz, CDCI3) 5 8.12 (s, 1H), 6.75 (d, /= 8.0 Hz, 1H), 5.20 - 5.12 (m, 1H), 4.44 (q, / = 7.0 Hz, 2H), 4.06 - 3.97 (m, 1H), 3.87 (d, / = 3.8 Hz, 1H), 2.14 (d, /= 3.8 Hz, 1H), 2.01 1.91 (m, 3H), 1.42 (t, 7 = 7.1 Hz, 3H), 1.34- 1.25 (m, 2H), 1.06 (d, 7 = 6.8 Hz, 3H), 1.00-0.93 (m, 18H), 0.88 (dd, 7 = 19.1, 6.8 Hz, 6H). MS ESI m/z calcd for €2^44^04881 [M+H]+ 526.28, found 526.28.
Example 14. Synthesis of ethyl 2-((lR,3R)((2S,3S)azido-N,3-dimethyl pentanamido)- 4-methyl-1 -((triethylsilyl)oxy)pentyl)thiazolecarboxylate (137) O OTES N3V N N rj-co2Et A solution of 136 (5.20 g, 9.9 mmol, 1.0 eq.) in THF (50 mL) was cooled to -45 °C and KHMDS (1M in toluene, 23.8 mL, 23.8 mmol, 2.4 eq.) was added. The resulting mixture was stirred at -45°C for 20 min. Methyl iodide (1.85 mL, 29.7 mmol, 3.0 eq.) was then added, and the WO 59622 reaction mixture was allowed to warm to r.t. over 4.5 h, at which time the reaction was quenched with EtOH (10 mL). The crude product was diluted with EtOAc (250 mL) and washed with brine (100 mL). The aqueous layer was extracted with EtOAc (3 x 50 ml). The organic layers were dried, filtered, concentrated and purified by column chromatography with a nt of 15-35% EtOAc in petreolum ether to afford product 137 (3.33 g, 63% yield) as a light yellow oil. 1H NMR (500 MHz, CDC13) 5 8.09 (s, 1H), 4.95 (d, / = 6.6 Hz, 1H),4.41 (q, / = 7.1 Hz, 2H), 3.56 (d, /= 9.5 Hz, 1H), 2.98 (s, 3H), 2.27 - 2.06 (m, 4H), 1.83 - 1.70 (m, 2H), 1.41 (t, 7 = 7.2 Hz, 3H), 1.29 (ddd, 7=8.9, 6.8, 1.6 Hz, 3H), 1.01 (d, 7=6.6 Hz, 3H), 0.96 (dt,7=8.0, 2.9 Hz, 15H), 0.92 (d, 7 = 6.6 Hz, 3H), 0.90 (d, 7 = 6.7 Hz,3H). MS ESI m/z calcd for C25H46N504SSi [M+H]+ 540.30, found 540.30.
Example 15. Synthesis of ethyl 2-((3S,6R,8R)((S)-sec-butyl)-10,10-diethyl isopropyl- -methyl-1 1 -methylpiperidinyl)-1,4-dioxooxa-2,5-diaza siladodecan-8 - yl)thiazolecarboxylate (138) 0 H O OTES If N ljrco2FAN 1 O I Dry Pd/C (10 wt%, 300 mg) and azide compound 137 (3.33 g, 6.61 mmol) were added to the above pentafluorophenyl ester 127 in EtOAc. The on mixture was d under hydrogen atmosphere for 27 h, and then filtered through a plug of Celite, with washing of the filter pad with EtOAc. The combined organic portions were concentrated and purified by column chromatography with a gradient of 0-5% methanol in EtOAc to deliver product 138 (3.90 g, 86% . MS ESI m/z calcd for CsiHsg^OsSSi [M+H]+ 639.39, found 639.39. e 16. Synthesis of ethyl 2-((lR,3R)((2S,3S)-N,3-dimethyl((R)-l-methyl piperidinecarboxamido)pentanamido)-1 -hydroxymethylpentyl)thiazolecarboxylate (139) U nH O OH N N T'J^C02Et 1 O I The ng product 138 (3.90 g, 6.1 mmol) was dissolved in deoxygenated AcOH/water/THF (v/v/v 3:1:1, 100 mL), and stirred at r.t. for 48 h. The reaction was then concentrated and purified by column chromatography (2:98 to 15:85 MeOH/EtOAc) to afford compound 139 (2.50 g, 72% yield over 2 steps). MS ESI m/z calcd for C26H45N4O5S [M+H]+ 525.30, found 525.33.
Example 17. Synthesis of 2-((lR,3R)((2S,3S)-N,3-dimethyl((R)-l-methylpiperidine carboxamido)pentanamido)- l-hydroxymethylpentyl)thiazolecarboxylic acid (140) O nH O OH if N ^co2HN I o I An aqueous solution of LiOH (0.4 N, 47.7 mL, 19.1 mmol, 4.0 eq.) was added to a solution of compound 139 (2.50 g, 4.76 mmol, 1.0 eq.) in dioxane (47.7 mL) at 0 °C. The reaction mixture was stirred at r.t. for 2 h and then concentrated. Column chromatography (100% CH2CI2 then CH2Cl2/Me0H/NH40H 80:20:1) ed compound 140 (2.36 g, 99% yield) as an ous solid. MS ESI m/z calcd for C24H41N4O5S [M+H]+ 497.27, found 497.28.
Example 18. sis of 2-((lR,3R)-l-acetoxy((2S,3S)-N,3-dimethyl((R)-lmethylpiperidinecarboxamido )pentanamido)methylpentyl)thiazolecarboxylic acid (141)o H Q OAc N' yN N N t^co2h 1 O I To a solution of compound 140 (2.36 g, 4.75 mmol) in pyridine (50 mL) at 0 °C, acetic anhydride (2.25 mL, 24 mmol) was added slowly. The reaction mixture was allowed to warm to r.t. over 2 h and stirred at r.t. for 24 h. The reaction was concentrated and the e was purified by reverse phase HPLC (Cig column, 10-90% acetonitrile/water) to afford compound 141 (2.25 g, 88% yield) as an amorphous white solid. MS ESI m/z calcd for C26H43N4O6S [M+H]+ 539.28, found 539.28.
Example 19. Synthesis of (lR,3R)((2S,3S)-N,3-dimethyl((R)-l-methylpiperidine carboxamido)pentanamido)methyl- l-(4-(perfluorobenzoyl)thiazolyl)pentyl acetate (142) O. NH O OAcrM Sc'Y N 1 O I XX"' oc6F5 To a solution of compound 141 (86 mg, 0.16 mmol, 1.0 eq.) in dichloromethane (2 mL) was added pentafluorophenol (44 mg, 0.24 mmol, 1.5 eq.) and /V,/V’-diisopropylcarbodiimide (22 mg, 0.175 mmol, 1.1 eq.) at 0 °C. The reaction mixture was warmed to room temperature and stirred over night. After the solvent was removed under reduced pressure, the on mixture was diluted with EtOAc (2 mL) then filtered over Celite. The filtrate was concentrated to afford crude pentafluorophenyl ester 142, which was used directly without further purification.
Example 20. sis of te/t-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoate (145). x'\^C02,Bu To a solution of 2,2’-(ethane-l,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then te/t-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise.
The mixture was d overnight and then quenched by HC1 solution (20.0 mL, IN) at 0 °C.
THF was removed by rotary evaporation, brine (300 mL) was added and the resulting mixture was extracted with EtOAc (3 x 100 mL). The organic layers were washed with brine (3 x 300 mL), dried over ous NaiSCL, filtered and concentrated to afford a colourless oil (30.20 g, 79.0% yield), which was used without further cation. MS ESI m/z calcd for C13H27O6 [M + H]+ 278.1729, found 278.1730. e 21. Synthesis of te/t-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate (146) T (VBu To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol, 2.0 eq.) in anhydrous DCM (220 mL) at 0 °C was added TEA (30.0 mL, 217.0 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight, and then washed with water (3 x 300 mL) and brine (300 mL), dried over anhydrous NaiSCL, filtered, trated and purified by Si CL column chromatography (3:1 hexanes/ EtOAc) to give a colorless oil (39.4 g, 84.0% yield). MS ESI m/z calcd for C20H33O8S [M + H]+ 433.1818, found 433.2838.
Example 22. sis of tert-butyl 2-(2-azidoethoxy)ethoxy)ethoxy) propanoate (147) To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF(100 mL) was added NaNs (20.67 g, 316.6 mmol, 3.5 eq.).
The mixture was stirred at room ature overnight. Water (500 mL) was added and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with water (3 x 900 mL) and brine (900 mL), dried over anhydrous NaiSOzt, filtered, concentrated and purified by Si CL column chromatography (5:1 hexanes/ EtOAc) to give a light yellow oil (23.8 g, 85.53% yield).
MS ESI m/z calcd for CoHisOsNsNa [M + Na]+ 326.2, found 326.2.
Example 23. Synthesis of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) propanoate (148) O O Ni (7.5 g, suspended in water) was washed with water (three times) and isopropyl alcohol (three times) and mixed with compound 147 (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under a Hi balloon at r.t. for 16 h and then filtered over a Celite pad, with washing of the pad with isopropyl alcohol. The filtrate was concentrated and purified by column chromatography (5-25% MeOH/DCM) to give a light yellow oil (2.60 g, 57% yield). MS ESI m/z calcd for CnHigNOs [M+H]+ 279.19; found 279.19.
Example 24. Synthesis of di-/er/-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa- 13,18- diazatriacont- 15-yne-1,30-dioate (149) jC02'Bu Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved in NMP (10 mL) and cooled to 0 °C, to which compound 148 (2.06 g, 7.43 mmol, 2.4 eq.) was added, followed by DMTMM (2.39 g, 8.65 mmol, 2.8 eq.) in portions. The on was stirred at 0 °C for 6 h and then diluted with ethyl acetate and washed with water and brine. The c solution was trated and triturated with a mixture solvent of ethyl acetate and eum ether. The solid was filtered off and the te was concentrated and ed by column chromatography (80- 90% EA/PE) to give a light yellow oil (2.26 g, >100% yield), which was used without further purification. MS ESI m/z calcd for C30H53N2O12 [M+H]+ 633.35; found 633.30.
Example 25. Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18 -diazatriacont yne-1,30-dioic acid (150) H O HO2C'K'o^ny^An^4o^L o co2h H Compound 149 (2.26 g) was dissolved in romethane (15 mL) and cooled to 0 °C then treated with TEA (15 mL). The reaction was warmed to r.t. and stirred for 45 min, and then the solvent and residual TEA was removed on rotovap. The crude product was purified by column chromatography (0-15% MeOH/DCM) to give a light yellow oil (1.39 g, 86% yield for two . MS ESI m/z calcd for C22H37N2O12 [M+H]+ 521.23; found 521.24.
Example 26. Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol (152). 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and K2C03(83.00 g, 600 mmol, 3.0 eq.) in acetonitrile (350 mL) was added BnBr(57.0 mL, 480 mmol, 2.4 eq.). The e was refluxed overnight. Water (1 L) was added and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrous Na2S04, filtered, concentrated and purified by SiCb column chromatography (4:1 hexanes/ EtOAc) to give a ess oil (50.97 g, 89.2% yield). MS ESI m/z calcd for CigffeNCENa [M + Na]+ 309.1729, found 309.1967.
Example 27. Synthesis of te/t-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate (153).
To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 eq.), tertbutyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and n-Bu4NI (6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL) was added sodium hydroxide solution (300 mL, 50%). The mixture was stirred overnight. The organic layer was separated and the water layer was extracted with EtOAc (3 x 100 mL). The organic layers were washed with water(3 x 300 mL) and brine (300 mL), dried over anhydrous NaiSOzt, filtered, concentrated and purified by SiOi column tography (7:1 hexanes/ EtOAc) to give a colorless oil (61.08 g, 89.4% . MS ESI m/z calcd for C25H36NO4 [M + H]+ 414.2566, found 84.
Example 28. Synthesis of te/t-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (154) To a on of tert-butyl 2-(dibenzylamino)ethoxy)ethoxy) propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THE (30 mL) and MeOH (60 mL) was added Pd/C (2.00 g, 10 wt%, 50% wet) in a hydrogenation bottle. The mixture was shaken overnight, filtered through Celite (filter aid), and the filtrate was concentrated to afford a ess oil (10.58 g, 93.8% yield). MS ESI m/z calcd for C11H24NO4 [M + H]+ 234.1627, found 234.1810.
Example 29. Synthesis of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (156) HO "O To a on of 2,2'-oxydiethanol (19.7 mL, 206.7 mmol, 3.0 eq.) in anhydrous THE (100 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then tert-butyl te (10.0 mL, 68.9 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight, and brine (200 mL) was added and extracted with EtOAc (3 x 100 mL). The organic layers were washed with brine (3 x 300 mL), dried over anhydrous Na2S04, filtered, concentrated and purified by Si02 column chromatography (1:1 hexanes/ EtOAc) to give to a colorless oil (8.10 g, 49.4% yield). MS ESI m/z calcd for C11H23O5 [M +H]+ 235.1467, found 235.1667.
Example 30. Synthesis of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate (157) —A O2 Bu To a solution of te/t-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (6.24 g, 26.63 mmol, 1.0 eq.) and TsCl (10.15 g, 53.27 mmol, 2.0 eq.) in anhydrous DCM(50 mL) at 0 °C was added ne (4.3 mL, 53.27 mmol, 2.0 eq.). The mixture was stirred at room ature overnight, and then washed with water (100 mL) and the water layer was extracted with DCM (3 x 50 mL).
The combined c layers were washed with brine (300 mL), dried over anhydrous NaiSCL, filtered, concentrated and purified by Si CL column chromatography (5:1 hexanes/ EtOAc) to give a colorless oil (6.33 g, 61.3% . MS ESI m/z calcd for C18H27O7S [M + H]+ 389.1556, found 389.2809.
Example 31. Synthesis of te/t-butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (158) 02*Bu To a solution of te/t-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate (5.80 g, 14.93 mmol, 1.0 eq.) in anhydrous DMF (20 mL) was added NaNs (5.02 g, 77.22 mmol, 5.0 eq.). The mixture was stirred at room temperature overnight. Water (120 mL) was added and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water (3 x 150 mL) and brine (150 mL), dried over anhydrous NaiSOzj, filtered, concentrated and purified by Si CL column chromatography (5:1 hexanes/ EtOAc) to give a colorless oil (3.73 g, 69.6% yield). MS ESI m/z calcd for CntLiOs^NaCM + H]+ 260.1532, found 260.2259. e 32. Synthesis of te/t-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (159) n2\^^()——co2*Bu Compound 158 (0.18 g, 0.69 mmol) was dissolved in MeOH (3.0 mL, with 60 pL trated HC1) and hydrogenated with Pd/C (10 wt%, 20 mg) under a H2 balloon for 30 min.
The catalyst was filtered through a Celite pad, with washing of the pad with MeOH. The te was concentrated to give a colorless oil (0.15 g, 93% yield). MS ESI m/z calcd for C11H24NO4 [M+H]+ 234.16; found 234.14.
Example 33. Synthesis of di-/er/-butyl ll,14-dioxo-4,7,18,21-tetraoxa-10,15 -diazatetracos- -l,24-dioate (160) CBu02C4^ol^NYr=~JJ-N'''40—-LcO^Bu O H *2 2 Acetylenedicarboxylic acid (26.5 mg, 0.232 mmol, 1.0 eq.) was dissolved in NMP (1.0 mL) and cooled to 0 °C, to which compound 159 (0.15 g, 0.557 mmol, 2.4 eq.) was added, followed by DMTMM (0.18 g, 0.65 mmol, 2.8 eq.) in portions. The reaction was stirred at 0 °C for 5 h and then diluted with ethyl acetate and washed with water and brine. The organic solution was concentrated and ed by column chromatography (80-90% EA/PE). The appropriate fractions were concentrated and kept in refrigerator overnight. A solid formed and was filtered off. The filtrate was trated to give a light yellow oil (0.37 g, >100% , which was used without further purification. MS ESI m/z calcd for C26H45N2O10 [M+H]+ 545.30; found 545.30.
Example 34. Synthesis of ll,14-dioxo-4,7,18,21-tetraoxa-10,15-diaza tetracosyne-l,24- dioic acid (161) H O O H Compound 160 (0.21 g) was dissolved in dichloromethane (2.5 mL) and cooled to 0 °C then treated with TEA (2.5 mL). The reaction was warmed to r.t. and stirred for 45 min, and then the solvent and residual TEA was removed on p. The crude product was purified by column tography (0-15% MeOH/DCM) to give a corlorless oil (58.7 mg, 99% yield for two steps). MS ESI m/z calcd for C18H29N2O10 [M+H]+ 433.17; found .
Example 35. 3-(2-(2-azidoethoxy)ethoxy)propanoic acid (165) N3^^0v^^q^^C02H The azide compound 158 (2.51 g, 9.68 mmol) dissolved in 1,4-dioxane (30 mL) was treated with 10 ml of HC1 (cone.) at r.t. The mixture was stirred for 35 min, diluted with EtOH (30 ml) and toluene (30 ml) and trated under vacuum. The crude mixture was ed on silica gel using a mixture of methanol (from 5% to 10%) and 1% formic acid in methylene chloride as the eluant to give title compound 165 (1.63 g, 83% yield), ESI MS m/z C7H12N3O4 , cacld. 202.06, found 202.30.
Example 36. 2,5-dioxopyrrolidin-l-yl 2-azidoethoxy)ethoxy)propanoate (166) To compound 165 (1.60 g, 7.87 mmol) in 30 mL of dichloromethane was added NHS (1.08 g, 9.39 mmol) and EDC (3.60 g, 18.75 mmol) with stirring. After 8 h TEC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 10%) in methylene chloride as the eluant to give title compound 166 (1.93 g, 82% yield). ESI MS m/z C11H17N4O6 [M+H]+, cacld.301.11, found 301.20.
Example 37. Synthesis of (E)bromo-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa azaheptadecenoic acid (167) HOOC H —°—^C02?Bu To a solution of 3-bromofuran-2,5-dione (89 mg, 0.5 mmol) in THF (5 mL), / Example 38. Synthesis of (E)-l-/er/-butyl 18-methyl 13-bromo-ll,14-dioxo-4,7- dioxa- ,15-diazaoctadecene-1,18-dioate (169) H Br 'Bu02CX^°^^0X^NY=S-N/VC02Me o cr h Compound 167 (205 mg, 0.5 mmol) and methyl 3-aminopropanoate hydrochloride (70 mg, 0.5 mmol) were dissolved in DCM (20 mL), to which DIPEA (0.26 mL, 1.5 mmol) and EDC (144 mg, 0.75 mmol) were added. The resulting solution was stirred at r.t. overnight, and then washed with brine (50 mL), dried over anhydrous Na2S04. Concentration and purification by column chromatography (0 to 10% MeOH/DCM) yielded compound 169 (88 mg, 36% .
MS ESI m/z calcd for CiglLiBrNiOg [M+H]+ 495.13, found 495.25.
Example 39. Synthesis of bromo-3,7,10-trioxo-2,14,17-trioxa-6,ll-diaza icosen- -oic acid (170) °o^jy—s^C02Me nd 169 (88 mg, 0.18 mmol) in DCM (3 mL) was treated with formic acid (6 ml) at 38 °C overnight. All volatiles were removed under vacuum to yield compound 170 (about 78 mg, -90% yield).
Example 40. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid (172) CbzHN^^^C02H A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in ELO (40 mL) was cooled to 0 °C and treated with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reaction was allowed to warm to r.t. and d for 3 h. THF was removed under vacuum, the pH of the aqueous on was adjusted to 1.5 by addition of 6 N HC1. Extracted with ethyl e, and the organic layer was washed with brine, dried and concentrated to give compound 172 (16.4 g, 92% yield). MS ESI m/z calcd for C12H16NO5 [M+H]+238.10, found 238.08.
Example 4E Synthesis of te/t-butyl enzyloxy)carbonyl)amino)butanoate (173) ^^COi'Bu DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to a solution of 4- (((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and /-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring at r.t. ght, the reaction was filtered and filtrate concentrated. The residue was dissolved in ethyl acetate and the washed with IN HC1, brine and dried over NaiSCE. Concentration and purification by column chromatography (10 to 50% EtOAc/hexanes) yielded compound 173 (7.5 g, 37% yield). MS ESI m/z calcd for CielfeNCENa [M+Na]+ 316.16, found 316.13.
Example 42. Synthesis of te/t-butyl 4-aminobutanoate (174) te/t-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was ved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt%, 100 mg) then hydrogenated (1 atm) at room temperature for 3 h. The catalyst was ed off and all volatiles were removed under vacuum to afford compound 174 (272 mg, 90% yield). MS ESI m/z calcd for CgHigNOi [M+H]+ , found 160.13.
Example 43. Synthesis of (E)bromo((4-(tert-butoxy)oxobutyl)amino) oxobut enoic acid (175) HOOC H ,N. .CCVBu 3-Bromofuran-2,5-dione (300 mg, 1.71 mmol) was dissolved in THE (20 mL), to which tertbutyl 4-aminobutanoate(272 mg, 1.71 mmol) was added and the resulting solution was stirred at r.t. for 3 h. The solvent was removed under vacuum to afford compound 175 (572 mg, theoretical yield). MS ESI m/z calcd for CiiHigBrNOs [M+H]+338.03, found 338.04.
Example 44. sis of (E)-/er/-butyl romo((2-methoxyethyl) amino)oxobut- 2-enamido)butanoate (177) ?Bu02Cv^v^NT CN^^OMe O H 2-Bromo((4-(tert-butoxy)oxobutyl)amino)oxobutenoic acid(286mg, 0.85mmol) and 2-methoxyethanamine (128mg, 1.7mmol) were dissolved in DCM (40 mL), to which DIPEA (329 mg, 2.55 mmol) and EDC (490 mg, 2.55 mmol) were added. The resulting solution was stirred at r.t. for 24 h and then washed with brine, dried over NaiSCE. Concentration and cation by column chromatography (0 to 10% MeOH/DCM) yielded nd 177 (102 mg , 31% yield). MS ESI m/z calcd for CisfteBrNiOs [M+H]+ 393.09, found 393.11.
Example 45. Synthesis of (E)(3-bromo((2-methoxyethyl)amino)oxobut enamido)butanoic acid (178) H xP /^OMe J[ Br inr O H Compound 177 (52 mg, 0.132 mmol) was dissolved in DCM (3 mL), to which formic acid (6 ml) was added. The resulting solution was stirred at 38 °C overnight then concentrated to afford compound 178 (45 mg, theoretical yield). MS ESI m/z calcd for CnHigBrNiOs [M+H]+339.03, found 339.05.
Example 46. Synthesis of (E)-2,5-dioxopyrrolidin-l-yl 4-(3-bromo((2- methoxyethyl)amino)oxobutenamido)butanoate (179) O H Br To a solution of compound 178 (45 mg, 0.132 mmol) in DCM (10 ml) NHS (23 mg, 0.199 mmol) and EDC (38 mg, 0.199 mmol) were added. After stirring at r.t. for 3 h, the reaction was trated and purified by column chromatography (10 to 50% EtOAc/hexanes) to yield compound 179 (57 mg, 99% yield). MS ESI m/z calcd for CisHiiBrNsO? [M+H]+436.05, found Example 47. Synthesis of (Z)-di-/er/-butyl 4,4'-((2-bromofumaroyl)bis(azane diyl))dibutanoate (180) iBu02CX^XNN N^v^C02'Bu te/t-Butyl 4-aminobutanoate (174) (477 mg, 3 mmol) and 2,3-dibromosuccinic acid (414 mg, 1.5 mmol) was ved in DCM (35 mL), to which DIPEA (1.16 g, 9 mmol) and EDC (0.86 g, 4.5 mmol) were added. The ing solution was stirred at r.t. overnight and then washed with brine, dried over Na2S04. Filtration, concentration and purification by column chromatography (0 to 10% MeOH/DCM) yielded compound 180 (160 mg, 22% yield). MS ESI m/z calcd for Cio^BrNiOe 477.15, found 477.16.
Example 48. Synthesis of (Z)-4,4'-((2-bromofumaroyl)bis(azanediyl))dibutanoic acid (181) o Br H ho2c^^ ( ( ) 211 H O Compound 180 (80 mg, 0.168 mmol) was dissolved in DCM (5 mL) and treated with formic acid (8 mL) at 38 C overnight. All volatiles were removed under vacuum to afford compound o 181 (61 mg, 99% yield). MS ESI m/z calcd for CiiHigBrNiOe [M+H]+365.03, found 365.05. e 49. Synthesis of (Z)-bis(2,5-dioxopyrrolidin-l-yl) 4,4'-((2-bromo fumaroyl)bis(azanediyl))dibutanoate (182) 2 ¥r h O SuO N N OSu O O NHS (60 mg, 0.504 mmol) and EDC (97 mg, 0.504 mmol) were added to a solution of compound 181 (61 mg, 0.168 mmol) in DCM (10 mL). After stirring at r.t. ght, the on mixture was concentrated and purified by column tography (0 to 10% MeOH/DCM) to afford compound 182 (72 mg, 77% yield). MS ESI m/z calcd for C2oH24BrN4Oio [M+H]+559.06, found 559.78.
Example 50. Synthesis of te/t-butyl 2-(triphenylphosphoranylidene)propanoate (184) C02Bu A e of / The dichloromethane layers were combined and washed with brine (50 mL) once, then dried over Na2S04, filtered and concentrated, giving the ylide as a yellow solid (16.8 g, 58%).
Example 51. Synthesis of (S)-methyl 3-(4-(benzyloxy)phenyl)((/er/-butoxy carbonyl)amino)propanoate (186) BocHN Me02C To a mixture of Boc-L-Tyr-OMe (20.0 g, 67.7 mmol, 1.0 eq.), K2CO3 (14.0 g, 101.6 mmol, 1.5 eq.) and KI (1.12 g, 6.77 mmol, 0.1 eq.) in acetone (100 mL) was added BnBr (10.5 mL, 81.3 mmol, 1.2 eq.) slowly. The mixture was then refluxed overnight. Water (250 mL) was added and the reaction mixture was extracted with EtOAc (3x100 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous NaiSOzj, filtered, concentrated and purified by SiOi column chromatography (4:1 hexanes/EtOAc) to give a white solid 186 (26.12 g, 99% yield). NMR (500 MHz, CDC13) 5 7.44 - 7.41 (m, 2H), 7.41 - 7.36 (m, 2H), 7.35 - 7.30 (m, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.93 - 6.89 (m, 2H), 5.04 (s, 2H), 4.97 (d, J = 7.7 Hz, 1H), 4.55 (d, 7 = 6.9 Hz, 1H), 3.71 (s, 3H), 3.03 (dd,/= 14.4, 5.7 Hz, 2H), 1.44 (d,/= 18.6 Hz, 10H). MS ESI m/z calcd for CiiHiTNOsNa [M+Na]+408.18, found 408.11. e 52. Synthesis of (S)-tert-butyl (l-(4-(benzyloxy)phenyl)oxopropan yl)carbamate (187) BocHNTOOLOBn To a solution of ester 186 (26.1 g, 67.8 mmol, 1.0 eq.) in ous dichloromethane (450 mL) at -78 °C was added DIBAL (1.0 M in s, 163 mL, 2.2 eq.) in 1 h. The mixture was stirred at -78 °C for 3 h and then quenched with 50 mL of ethanol. IN HC1 was added dropwise until pH 4 was reached. The resulting mixture was allowed to warm to 0 °C. Layers were separated and the aqueous layer was further extracted with EtOAc (3 x 100 mL). The combined organic solution was washed with brine, dried over anhydrous NaiSOzj, and concentrated.
Trituration with PE/EtOAc and filtration gave a white solid 187 (18.3 g, 76% yield). MS ESI m/z calcd for Cii^NOsNa + 378.11, found 378.11.
Example 53. Synthesis of (S,Z)-/er/-butyl benzyloxy)phenyl)((/er/-but bonyl)amino)methylpentenoate (188) 'BuO,C^ VJL, Aldehyde 187 (0.84 g, 2 mmol, 1.0 eq.) was dissolved in dry dichloromethane (50 mL), to which te/t-butyl ester ylide 184 (1.6 g, 4 mmol, 2.0 eq.) was added and the solution was stirred at r.t. for 1.5 h as determined te by TLC. Purification by column chromatography (10-50% EtOAc/hexanes) afforded compound 188 (1.16g, 98% yield).
Example 54. Synthesis of (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino)(4- hydroxyphenyl)methylpentanoate (189) BocHN^s^^ •buo^^t yi Compound 188 (467 mg, 1 mmol) was dissolved in methanol (30 mL) and hydrogenated (1 atm) with Pd/C catalyst (10 wt%, 250 mg) at r.t. overnight. The catalyst was filtered off and the filtrate were concentrated under reduced pressure to afford compound 189 (379mg, 99% yield).
Example 55. Synthesis of (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino) (4-hydroxy nitrophenyl)methylpentanoate (190) ^uOjC no2 Compound 189 (379mg, 1 mmol, 1.0 eq.) was dissolved in THE (20 mL), to which a solution of te/t-butyl nitrite (315 mg, 3 mmol, 3.0 eq.) in THE (2 mL) was added. The reaction was stirred at r.t. for 3 h and then poured onto water, extracted with EtOAc (2 x 50 mL) and the combined organic phases were washed with brine (50 mL), dried over anhydrous NaiSOzj, filtered and trated. cation by column chromatography (10-50% EtOAc/hexanes) afforded compound 190 (300 mg, 71% yield).
Example 56. Synthesis of (4R)-/er/-butyl 4-((/er/-butoxycarbonyl)amino)(4- ((lerlbutyldimethylsilyl )oxy)nitrophenyl)methylpentanoate (191) BocHN^^^_OTBS tBu02C no2 To a solution of compound 190 (424 mg, 1 mmol) in DCM (20 mL), imidazole (408 mg, 6 mmol) and / Example 58. Synthesis of (E)-methyl 13-bromo-l-((5-((2R)(tert-butoxy) ((tertbutoxycarbonyl )amino)methyloxopentyl)((tert-butyldimethylsilyl)oxy)phenyl)-amino)- 1,11,14-trioxo-4,7-dioxa-10,15-diazaoctadec- 12-enoate (193) ^>OTBS -o2c BocHN'" NH O Y ‘BuOjC Compound 192 (90 mg, 0.178 mmol) and compound 170 (78 mg, 0.178 mmol) were dissolved in DCM (5 mL) and DMA (5 mL). To which TEA (36 mg, 0.356 mmol) and HATU (102 mg, 0.267 mmol) were added. The resulting mixture was stirred at r.t. for 4 h, and then diluted with EA and washed with brine, dried over anhydrous NaiSCE. tion and cation by column chromatography (0 to 10% MeOH/DCM) d compound 193 (87 mg, 53% yield).
Example 59. Synthesis of (E)-methyl 13-bromo-l-((5-((2R)(tert-butoxy) ((tertbutoxycarbonyl )amino)methyloxopentyl)hydroxyphenyl)amino)-1,11,14-trioxo-4,7- dioxa-10,15-diazaoctadecenoate (194) 7 V-OH Me02C‘ "| BocHN^ tBu02C H Br Compound 193 (87 mg, 0.0936 mmol) was dissolved in DCM (10 mL) and treated with TBAF (1M in THE, 0.3 mL). The resulting mixture was stirred at r.t. for 0.5 h and washed with brine, dried over ous Na2S04. Filteration and purification by column chromatography (0 to % MeOH/DCM) yielded nd 194 (53 mg, 69% yield). MS ESI m/z calcd for CseHseBrN^ii [M+H]+ 817.30, found 817.51.
Example 60. Synthesis of (4R)amino(3-((E)bromo-3,7,10-trioxo-2,14, 17-trioxa- 6,1 l-diazaicosenamido)hydroxyphenyl)methylpentanoic acid (195) Me02C' \ H2N =Nh nd 194 (18 mg, 0.022 mmol) was dissolved in DCM (2 mL) and treated with TEA (2 mL) at r.t. for 3 h. All volatiles were removed under vacuum to afford compound 195 (14.5 mg, theoretical yield). MS ESI m/z calcd for CiTHztoBrNztOlo [M+H]+ 659.18, found 659.18. e 61. Synthesis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3- dimethyl((R)-lmethylpiperidinecarboxamido )pentanamido)methylpentyl)thiazolecarboxamido)(3- ((E)bromo-3,7,10-trio xo-2,14,17-trioxa-6,ll-diazaicosenamido)hydroxyphenyl) methylpentanoic acid (196) O nkN^ H O OAc O If Me02C^ | N \ ' HN^O 1 o I v''° ^ S' To a solution of compound 195 (14.5 mg, 0.022 mmol) and perfluorophenyl ester 142 (18.5 mg, 0.026 mmol) in DMA (2.5 mL) was DIPEA (15 mg, 0.116 mmol) added. The ing mixture was stirred at r.t. for 1.5 h and then the solvent was removed under vacuum. The residue was purified on reverse phase ative HPLC (Cig column, 10-90% MeCN/H20) to afford compound 196 (12.6 mg, 49% yield). MS ESI m/z calcd for CssHgoBrNgOis [M+H]+l 181.45, found 1181.45.
Example 62. Synthesis of (4R)-/er/-butyl 5-(3-aminohydroxyphenyl) {{tertbutoxycarbonyl )amino)methylpentanoate (197) BocHN // \ tBu02C OH -- nh2 Compound 190 (56 mg, 0.132 mmol) was dissolved in EtOAc (20 mL) and mixed with Pd/C catalyst (10 wt%, 50 mg) and hydrogenated (1 atm) at r.t. for 3 h. The catalyst was filtered off and all les were removed under vacuum to afford compound 197 (52 mg, 99% yield).
MS ESI m/z calcd for C21H35N2O5 [M+H]+ 395.25, found 395.26.
Example 63. Synthesis of (4R)-/er/-butyl 5-(3-(4-((E)bromo((2-methoxy ethyl)amino)oxobutenamido)butanamido)hydroxyphenyl)((/e/tbutoxycarbonyl )amino)methylpentanoate (198) OH H BocHN N. Br 'BuO.C, N1 O ^.'N^OMc H O H NaH2P04 (0.1M in water, 2 ml) was added to a solution of compound 179 (57 mg, 0.1323 mmol) and compound 197 (52 mg, 0.1323 mmol) in EtOH (10 mL). The ing solution was stirred at r.t. for 72 h, concentrated and purified by column chromatography (30 to 100% hexanes) to yield compound 198 (50 mg, 53% yield). MS ESI m/z calcd for C32H5oBrN409 [M+H]+715.27, found 715.27.
Example 64. Synthesis of (4R)amino(3-(4-((E)bromo((2-methoxy ethyl) amino)oxobutenamido)butanamido)hydroxyphenyl)methylpentanoic acid (199) :!tf0H0 H Jr h2n 2 ho2c H O rVN nd 198 (25 mg, 0.035 mmol) in DCM (2 mL) was treated with TEA (2mL) at r.t. for 1 h. All volatiles were removed under vacuum to afford compound 199 (19 mg, 99% yield). MS ESI m/z calcd for Cist^Br^O? [M+H]+557.15, found 557.27.
Example 65. Synthesis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3- dimethyl((R)-lmethylpiperidinecarboxamido )pentanamido)methylpentyl)thiazolecarboxamido)(3- (4-((E)bromo((2-methoxyethyl)amino)oxobutenamido)butanamido) hydroxyphenyl)methylpentanoic acid (200) OAc 7 xy-oii ,N, H 1 oNX, ■0JBr ^OMe I ur h NH N ho2c O H Compound 199 (19 mg, 0.035 mmol) and perfluorophenyl ester 142 (33 mg, 0.046 mmol) was dissolved in DMA (5 mL), to which DIPEA (lOmg, 0.077 mmol) was added. The reaction mixture was stirred at r.t. for 2 h then concentrated and purified on ative HPLC (Cig column, 10-90% McCN/fEO) to afford the compound 200 (9 mg, 24% . MS ESI m/z calcd for C49H74BrN8Oi2S [M+H]+ 1079.43, found 1079.66.
Example 66. Synthesis of (4R)-tert-butyl 5-(3-((Z)bromo-l-hydroxy-73,78, oxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxa-72,77,82- hexaoctacontenamido)hydroxyphenyl)((tert-butoxycarbonyl)amino) methylpentanoate (203) n.OH O Br jj O BocHN o H o ^uOjC 23 203 NaHiPCXt (0.1M in water, 1 mL) was added to a solution of compound 182 (36 mg, 0.065 mmol) and compound 197 (25 mg, 0.063 mmol) in EtOH (5 mL).The resulting solution was stirred at r.t. overnight and then HO-(PEG)24-NH2 (95 mg ) was added to the mixture and stirred at r.t. overnight. All volatiles were removed under vacuum and the residue was purified by column chromatography (0 to 10% MeOH/DCM) to yield compound 203 (28 mg, 24% yield).
MS ESIm/z 1798.93 +).
Example 67. Synthesis of (4R)amino(3-((Z)bromo-l-hydroxy-73,78, 81-trioxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxa-72,77,82- triazahexaoctacontenamido)hydroxyphenyl)methylpentanoic acid (204) ft H O H2N NH OH Compound 203 (28 mg, 0.0156 mmol) was dissolved in DCM (2 mL) and treated with TEA (2 mL) at r.t.for 2 h. All volatiles were d under vacuum to afford compound 204 (25 mg, 98% . MS ESI m/z 1642.82 ([M+H]+).
Example 68. Synthesis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3 -dimethyl((R)- l-methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazolecarboxamido)(3- ((Z)bromo-1 -hydroxy-73,78,81 -trioxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxa-72,77,82- triazahexaoctacontenamido)hydroxyphenyl)methylpentanoic acid (205) H o OAc Br HN fj^OH ^ // O O I o L I c.£H N NH w" H N' H O ho2c HO nd 204 (25 mg, 0.0152 mmol) and perfluorophenyl ester 142 (15 mg, 0.0213 mmol) were dissolved in DMA (5 mL). To the mixture, DIPEA (10 mg, 0.077 mmol) was added.
The resulting e was stirred at r.t. overnight, concentrated and purified by preparative HPLC (Ci§ column, 10-90% MeCN/H20) to afford compound 205 (13 mg, 40% yield).MS ESI m/z 2163.82 ([M+H]+).
Example 69. Synthesis of (S)-/ Example 70. Synthesis of /7-butyl 2-formylpyrrolidine-l-carboxylate (209)or° To a on of alcohol 208 (13.0 g, 64.6 mmol) in dimethyl sulfoxide (90 mL) was added ylamine (40 mL) and the stirring was continued for 15 min. The mixture was cooled over ice bath and sulfur trioxide-pyridine complex (35.98 g, 226 mmol) was added in portions over a 40 min . The reaction was warmed to r.t. and stirred for 2.5 h. After addition of ice (250 g), the mixture was extracted with dichloromethane (150 mL x 3). The organic phase was washed with 50% citric acid solution (150 mL), water (150 mL), ted sodium bicarbonate solution (150 mL), and brine (150 mL), dried over anhydrous NaiSOzj. Removal of solvent in vacuo yielded aldehyde 209 (10.4 g, 81% yield) as a dense oil which was used without further purification. NMR (500 MHz, CDC13) 5 9.45 (s, 1H), 4.04 (s, 1H), 3.53 (dd, /= 14.4, 8.0 Hz, 2H), 2.00 - 1.82 (m, 4H), 1.44 (d, / = 22.6 Hz, 9H).
Example 71. Synthesis of (4R,5S)methylphenylpropionyloxazolidinone (211) n-Butyllithium in hexane (21.6 mL, 2.2 M, 47.43 mmol) was added dropwise at -78 °C to a stirred solution of 4-methylphenyloxazolidinone (8.0 g, 45.17 mmol) in THE (100 mL) under Ni. The solution was maintained at -78 °C for 1 h then propionyl chloride (4.4 mL, 50.59 mmol) was added slowly. The reaction mixture was warmed to -50 °C, stirred for 2 h then quenched by on of a saturated solution of ammonium chloride (100 mL). The organic solvent was removed in vacuo and the resultant solution was extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with saturated sodium bicarbonate on (100 mL) and brine (100 mL), dried over NaiSOzj, filtered and concentrated in vacuo. The residue was purified by column chromatography (20% ethyl acetate/hexanes) to afford the nd 211 as a dense oil (10.5 g, 98% yield). 1H NMR (500 MHz, CDC13) 5 7.45 - 7.34 (m, 3H), 7.30 (d, / = 7.0 Hz, 2H), 5.67 (d, 7 = 7.3 Hz, 1H), 4.82 - 4.70 (m, 1H), 2.97 (dd,/= 19.0,7.4 Hz, 2H), 1.19 (t,/ = 7.4 Hz, 3H), 0.90 (d, /= 6.6 Hz, 3H).
Example 72. Synthesis of (S)-/er/-butyl 2-((lR,2R)-l-hydroxymethyl-3 -((4R,5S) methyloxophenyloxazolidinyl)oxopropyl)pyrrolidine-l-carboxylate (212) N Ph Boc OH O To a solution of 209 (9.40 g, 40.4 mmol) in dichloromethane (60 mL) was added Et-,N (6.45 mL, 46.64 mmol) at 0 °C, followed by 1M dibutylboron triflate in romethane (42 mL, 42 mmol). The mixture was stirred at 0 °C for 45 min, cooled to -70 °C, aldehyde 211 (4.58 g, 22.97 mmol) in dichloromethane (40 mL) was then added slowly over a 30 min period. The reaction was stirred at -70 °C for 2 h, 0 °C 1 h, and r.t. 15 min, and then quenched with phosphate buffer solution (pH 7, 38 mL). After the addition of MeOH-30% H2O2 (2:1, 100 mL) at below 10 °C and stirring for 20 min, water (100 mL) was added and the mixture was concentrated in vacuo.
More water (200 mL) was added to the residue and the mixture was extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with IN KHSO4 (100 mL), sodium bicarbonate solution (100 mL) and brine (100 mL), dried over ous Na2S04 and concentrated in vacuo.
The residue was purified by flash column chromatography (10% - 50% ethyl acetate/hexanes) to afford the nd 212 as a white solid (7.10 g, 71% . ^ NMR (500 MHz, CDCI3) 8 7.39 (dt, / = 23.4, 7.1 Hz, 3H), 7.30 (d, / = 7.5 Hz, 2H), 5.67 (d, 7 = 7.1 Hz, 1H), 4.84 - 4.67 (m, 1H), 4.08 - 3.93 (m, 3H), 3.92 - 3.84 (m, 1H), 3.50 (d, J = 9.0 Hz, 1H), 3.24 (d, J = 6.7 Hz, 1H), 2.15 (s, 1H), 1.89 (dd, 7 = 22.4, 14.8 Hz, 3H), 1.48 (d, 7= 21.5 Hz, 9H), 1.33 (d,7=6.9 Hz, 3H), 0.88 (d, 7= 6.4 Hz, 3H).
Example 73. Synthesis of (S)-/er/-butyl 2-((lR,2R)-l-methoxymethyl ((4R,5S) methyloxophenyloxazolidinyl)oxopropyl)pyrrolidine-l-carboxylate (213) N ■N. Ph Boc CL O To a mixture of 212 (5.1 g 11.9 mmol) and molecular sieves (4 A, 5 g) was added ous dichloroethane (30 mL) under N2. The mixture was stirred at room temperature for 20 min and cooled to 0 °C. Proton sponge (6.62 g, 30.9 mmol) was added, followed by trimethyloxonium tetrafluoroborate (4.40 g, 29.7 mmol). Stirring was continued for 2 h at 0 °C and 48 h at r.t. The reaction mixture was filtrated and the filtrate was concentrated and purified by column chromatography (20-70% ethyl e/hexanes) to afford compound 213 as a white solid (1.80 g, % yield). NMR (500 MHz, CDCI3) 5 7.46 - 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 = 22.3 Hz, 9H), 1.32 (t, J = 6.0 Hz, 3H), 0.91 - 0.84 (m, 3H).
Example 74. Synthesis of (2R,3R)((S)-l-(/er/-butoxycarbonyl)pyrrolidinyl) y hylpropanoic acid (214) BocrVvOH/() O To a on of 213 (1.80 g, 4.03 mmol) in THE (30 mL) and H2O (7.5 mL), 30% H2O2 (1.44 mL, 14.4 mmol) was added over a 5 min period at 0 °C , followed by a solution of LiOH (0.27 g, 6.45 mmol) in water (5 mL). After stirring at 0 °C for 3 h, 1 N sodium sulfite (15.7 mL) was added and the mixture was allowed to warm to r.t. and stirred overnight. THE was removed in vacuo and the aqueous phase was wash with dichloromethane (3 x 50 mL) to remove the oxazolidinone auxiliary. The aqueous phase was acidified to pH 3 with IN HC1 and extracted with ethyl acetate (3 x 50 mL). The organic layer was washed with brine (50 mL), dried over Na2S04, filtered and concentrated in vacuo to afford the compound 214 as a colorless oil (1.15 g, 98% yield). NMR (500 MHz, CDCI3) 5 3.99 - 3.74 (m, 2H), 3.44 (d, J = 2.6 Hz, 3H), 3.23 (s, 1H), .45 (m, 1H), 1.92 (tt, J = 56.0, 31.5 Hz, 3H), 1.79- 1.69 (m, 1H), 1.58- 1.39 (m, 9H), 1.30- 1.24 (m, 3H).
Example 75. Synthesis of (4S,5S)-ethyl 4-((/er/-butoxycarbonyl)amino)methyloxo heptanoate (217) Boc"SfOvOEt H o o To an ice-cooled solution of N-Boc-L-isoleucine (4.55 g, 19.67 mmol) in THE (20 mL) was added l,l’-carbonyldiimidazole (3.51 g, 21.63 mmol). After evolution of gas ceased, the ant mixture was stirred at r.t. for 3.5 h.
A solution of freshly prepared isopropylmagnesium e in THE (123 mmol, 30 mL) was added dropwise to a pre-cooled (0 °C) solution of ethyl hydrogen malonate (6.50 g, 49.2 mmol) at such a rate to keep the internal temperature below 5 °C. The mixture was stirred at r.t. for 1.5 h. This solution of the magnesium enolate was then cooled over an ice-water bath, followed by the gradual addition of the imidazolide solution over a 1 h period via a -ended needle at 0 °C. The resultant mixture was stirred at 0 °C for 30 min then r.t. 64 h. The reaction mixture was quenched by addition of 10% aqueous citric acid (5 mL), and acidified to pH 3 with an additional 10% aqueous citric acid (110 mL). The mixture was ted with ethyl acetate (3 x 150 mL). The organic extracts were washed with water (50 mL), saturated aqueous sodium hydrogen ate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over Na2S04, and concentrated in vacuo. The residue was ed by column chromatography on silica gel using ethyl acetate/hexane (1:4) as an eluent to give compound 217 (5.50 g, 93% yield). iH NMR (500 MHz, CDCI3) 5 5.04 (d, / = 7.8 Hz, 1H), 4.20 (p, / = 7.0 Hz, 3H), 3.52 (t, / = 10.7 Hz, 2H), 1.96 (d, 7=3.7 Hz, 1H), 1.69 (s, 2H), 1.44 (s, 9H), 1.28 (dd, 7 = 7.1, 2.9 Hz, 3H), 0.98 (t, J = 6.9 Hz, 3H), 0.92 - 0.86 (m, 3H).
Example 76. Synthesis of (3R,4S,5S)-ethyl 4-((/er/-butoxycarbonyl)amino) hydroxy methylheptanoate (218) Boc OEt H OH O To a solution of the compound 217 (5.90 g, 19.83 mmol) in ethanol (6 mL) at -60 °C was added sodium borohydride (3.77 g, 99.2 mmol) in one portion. The reaction mixture was stirred for 5.5 h below -55 °C then ed with 10% aqueous citric acid (100 mL). The resultant solution was acidified to pH 2 with an additional 10% aqueous citric acid, ed by extraction with ethyl acetate (3 x 100 mL). The organic ts were washed with saturated aqueous sodium chloride (100 mL), dried over NaiSOzt, and concentrated in vacuo. The residue was purified by column chromatography (10-50% ethyl acetate/hexane) to give pure diastereomer 218 (2.20 g, 37% yield) and a mixture of two diastereomers (2.0g, 34% yield, about 9:1 ratio). 1H NMR (500 MHz, CDCI3) 5 4.41 (d, /= 9.3 Hz, 1H), 4.17 (tt, / = 7.1, 3.6 Hz, 2H), 4.00 (t, / = 6.9 Hz, 1H), 3.55 (dd,/= 11.7, 9.3 Hz, 1H), 2.56-2.51 (m, 2H), 2.44 (dd,/= .0 Hz, 1H), 1.79 (d, 7=3.8 Hz, 1H), 1.60- 1.53 (m, 1H), 1.43 (s, 9H), 1.27 (dd, 7 = 9.3, 5.0 Hz, 3H), 1.03- 0.91 (m, 7H).
Example 77. Synthesis of (3R,4S,5S)((/er/-butoxycarbonyl)amino)hydroxy methyl heptanoic acid (219) Boc OH 'NtQy H OH O To a solution of compound 218 (2.20 g, 7.20 mmol) in ethanol (22 mL) was added 1 N s sodium hydroxide (7.57 mL, 7.57 mmol). The mixture was stirred at 0 °C for 30 min then r.t. 2 h. The resultant solution was acidified to pH 4 by addition of 1 N aqueous hydrochloric acid, which was then extracted with ethyl acetate (3 x 50 mL). The organic extracts were washed with 1 N aqueous ium hydrogen sulfate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over NaiSOzt, and concentrated in vacuo to give nd 219 (1.90 g, 95% yield). NMR (500 MHz, CDC13) 5 4.50 (d, 7= 8.7 Hz, 1H), 4.07 (d, 7= 5.5 Hz, 1H), 3.59 (d, 7 WO 59622 = 8.3 Hz, 1H), 2.56 - 2.45 (m, 2H), 1.76 - 1.65 (m, 1H), 1.56 (d, 7 = 7.1 Hz, 1H), 1.45 (s, 9H), 1.26 (t, / = 7.1 Hz, 3H), 0.93 (dd, /= 14.4, 7.1 Hz, 6H).
Example 78. Synthesis of (3R,4S,5S)((/er/-butoxycarbonyl)(methyl)amino)- 3-methoxy- -methylheptanoic acid (220) Boc* OH 1 O To a solution of compound 219 (1.90 g, 6.9 mmol) in THE (40 mL) was added sodium hydride (60% oil suspension, 1.93 g, 48.3 mmol) at 0 °C. After stirring for Ih, methyl iodide (6.6 mL, 103.5 mmol) was added. The stirring was continued at 0 °C for 40 h before saturated aqueous sodium hydrogen carbonate (50 mL) was added, followed by water (100 mL). The mixture was washed with diethyl ether (2 x 50 mL) and the aqueous layer was acidified to pH 3 by 1 N aqueous potassium en sulfate, then extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with 5% aqueous sodium lfate (50 mL) and saturated aqueous sodium chloride (50 mL), dried over NaiSOzj, and trated in vacuo to give compound 220 (1.00 g, 48% yield). NMR (500 MHz, CDC13) 5 3.95 (d, / = 75.4 Hz, 2H), 3.42 (d, / = 4.4 Hz, 3H), 2.71 (s, 3H), 2.62 (s, IH), 2.56 - 2.47 (m, 2H), 1.79 (s, IH), 1.47 (s, IH), 1.45 (d, / = 3.3 Hz, 9H), 1.13-1.05 (m, IH), 0.96 (d, / = 6.7 Hz, 3H), 0.89 (td, / = 7.2, 2.5 Hz, 3H).
Example 79. Synthesis of Boc-N-Me-L-Val-OH (222) BoCx OH To a solution of Boc-L-Val-QH (2.00 g, 9.2 mmol) and methyl iodide (5.74 mL, 92 mmol) in anhydrous THE (40 mL) was added sodium hydride (3.68 g, 92 mmol) at 0 °C. The reaction e was stirred at 0 °C for 1.5 h, then warmed to r.t. and stirred for 24 h. The reaction was quenched by ice water (50 mL). After addition of water (100 mL), the reaction mixture was washed with ethyl acetate (3 x 50 mL) and the aqueous solution was acidified to pH 3 then ted with ethyl acetate (3 x 50 mL). The combined organic phase was dried over NaaSCL and concentrated to afford Boc-N-Me-Val-OH (2.00 g, 94% yield) as a white solid. 'H NMR (500 MHz, CDCI3) 5 4.10 (d, / = 10.0 Hz, IH), 2.87 (s, 3H), 2.37 - 2.13 (m, IH), 1.44 (d, / = 26.7 Hz, 9H), 1.02 (d, / = 6.5 Hz, 3H), 0.90 (t, / = 8.6 Hz, 3H).
Example 80. Synthesis of (S)-/er/-butyl ,2R)-l-methoxy(((S)-l- methoxy-l-oxo phenylpropanyl)amino)methyloxopropyl)pyrrolidine-l-carboxylate (223) cVr"N^^Ph O C02Me To a solution of compound 214 (100 mg, 0.347 mmol) and L-phenylalanine methyl ester hydrochloride (107.8 mg, 0.500 mmol) in DMF (5 mL) at 0 °C was added diethyl cyanophosphonate (75.6 pL, 0.451 mmol), followed by EpN (131 pL, 0.94 mmol). The reaction mixture was stirred at 0 °C for 2 h, then warmed to r.t. and stirred ght. The reaction mixture was then diluted with ethyl acetate (80 mL), washed with 1 N s potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over NaiSCL, and concentrated in vacuo. The residue was purified by column chromatography (15-75% ethyl acetate/hexanes) to afford compound 223 (130 mg, 83% yield) as a white solid. ^ NMR (500 MHz, CDCI3) 8 7.28 (dd, / = 7.9, 6.5 Hz, 2H), 7.23 (t, /= 7.3 Hz, 1H), 7.16 (s, 2H), 4.81 (s, 1H), 3.98 - 3.56 (m, 5H), 3.50 (s, 1H), 3.37 (d, / = 2.9 Hz, 3H), 3.17 (dd, /= 13.9, 5.4 Hz, 2H), 3.04 (dd, /= 14.0, 7.7 Hz, 1H), 2.34 (s, 1H), 1.81-1.69 (m, 2H), 1.65 (s, 3H), 1.51-1.40 (m, 9H), 1.16 (d,/ = 7.0 Hz, Example 81. General ure for the removal of the Boc function with trifluoroacetic acid. To a on of the /V-Boc amino acid (1.0 mmol) in methylene chloride (2.5 mL) was added trifluoroacetic acid (1.0 mL). After being stirred at room temperature for 1-3 h, the reaction mixture was concentrated in vacuo. Co-evaporation with toluene gave the deprotected product, which was used without any further purification.
Example 82. Synthesis of thyl 2-((2R,3R)((S)-l-((3R,4S,5S) ((tertbutoxycarbonyl )(methyl)amino)methoxymethylheptanoyl)pyrrolidinyl)methoxy propanamido)phenylpropanoate (225). 1 '' '' OO C02Me To a solution of the Boc-deprotected product of compound 224 (0.29 mmol) and compound 220 (96.6 mg, 0.318 mmol) in DMF (5 mL) at 0 °C was added l cyanophosphonate (58 pi 0.347 mmol), ed by EpN (109 pL, 0.78 mmol). The reaction mixture was stirred at 0 °C for 2 h, then warmed to r.t. and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL), and saturated aqueous sodium de (40 mL), dried over Is^SCL and trated in vacuo. The residue was purified by column chromatography (15-75% ethyl acetate/hexanes) to afford compound 225 (150 mg, 81% yield) as a white solid. LC-MS (ESI) m/z calcd. for C34H55N3O8 [M+H]+: 634.40, found: 634.40.
Example 83. sis of (S)-methyl 2-((2R,3R)((S)-l-((3R,4S,5S) ((S)((/ertbutoxycarbonyl )amino)-/V,3-dimethylbutanamido)methoxymethylheptanoyl)pyrrolidin yl)methoxymethylpropanamido)phenylpropanoate (227).
BocHN N^^Ph 1 0.0 /() O C02Me To a solution of the Boc-deprotected product of compound 225 (0.118 mmol) and Boc-Val- OH (51.8 mg, 0.236 mmol) in DCM (5 mL) at 0 °C was added BroP(70.1 mg, 0.184 mmol), followed by diisopropylethylamine (70 pL, 0.425 mmol). The mixture was shielded from light and stirred at 0 °C for 30 min then at r.t. for 2 days. The reaction mixture was d with ethyl acetate (80 mL), washed with 1 N aqueous potassium hydrogen e (40 mL), water (40 mL), saturated aqueous sodium hydrogen ate (40 mL), and saturated aqueous sodium chloride (40 mL), dried over NaiSCL and concentrated in vacuo. The residue was purified by column chromatography 0% ethyl acetate/hexanes) to afford compound 227 (67 mg, 77% yield) as a white solid. LC-MS (ESI) m/z calcd. for [M+H]+: 733.47, found: 733.46.
Example 84. Synthesis of (S)-methyl 2-((2R,3R)((S)-l-((6S,9S,12S,13R) ((S)-secbutyl )-6,9-diisopropylmethoxy-2,2,5, ll-tetramethyl-4,7,10-trioxooxa-5,8,11- triazapentadecanoyl)pyrrolidinyl)methoxymethylpropanamido)phenylpropanoate (229). 1 O 1 O. O .OO C02Me To a solution of the Boc-deprotected product of compound 227 (0.091 mmol) and Boc-NMe-Val-OH (127 mg, 0.548 mmol) in DML (5 mL) at 0 °C was added diethyl cyanophosphonate (18.2 pL, 0.114 mmol), followed by iV-mcthy 1 morpholine (59 pL, 0.548 mmol). The on mixture was stirred at 0 °C for 2 h, then warmed to r.t. and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), washed with 1 N s ium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium en carbonate (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 e/hexanes) to afford compound 229 (30 mg, 39% yield) as a white solid. LC-MS (ESI) m/z calcd. for C45H75N5O10 [M+H]+: 846.55, found: 846.56. e 85. Synthesis of (S)-methyl 2-((2R,3R)((S)-l-((3R,4S,5S) ((S)-N,3-dimethyl- 2-((S)methyl(methylamino)butanamido)butanamido)methoxymethylheptanoyl )pyrrolidinyl)methoxymethylpropanamido)phenylpropanoate (230). 1 O ^ 1 CL O .O O C02Me To a solution of compound 229 (75.0 mg, 0.0886 mmol) in methylene chloride (5 mL) was added trifluoroacetic acid (2 mL) at room temperature. After being stirred at room temperature for 1 h, the reaction mixture was concentrated in vacuo. Co-evaporation with toluene gave the deprotected product 230, which was used without further purification.
Example 86. Synthesis of (2S,4R)-methyl 4-hydroxypyrrolidinecarboxylate hydrochloric (232).
Me HO"* To a on of transhydroxy-L-proline (15.0 g, 114.3 mmol) in dry methanol (250 mL) was added thionyl chloride (17 mL, 231 mmol) se at 0 to 4 °C. The resulting mixture was stirred for at r.t. ght, concentrated, llized with EtOH/hexane to provide the title compound (18.0 g, 87% yield). ESI MS m/z 168.2 ([M+Na]+).
Example 87. Synthesis of (2S,4R)-l-/er/-butyl 2-methyl 4-hydroxypyrrolidine- 1,2- dicarboxyl ate (233).
ESI MS m/z 268.2 ([M+Na]+).
Example 88. Synthesis of (S)-1 -/ C02Me The title compound ed through Dess-Martin oxidation was described in: Franco Manfre et al. J. Org. Chem. 1992, 57, 2060-2065. Alternatively Swern oxidation procedure is as following: To a solution of (COCl)2 (13.0 ml, 74.38 mmol) in CH2CI2 (350 ml) cooled to -78 °C was added dry DMSO (26.0 mL). The solution was stirred at -78 °C for 15 min and then compound 233 (8.0 g, 32.63 mmol) in CH2CI2 (100 ml) was added. After stirring at -78 °C for 2 h, triethylamine (50 ml, 180.3 mmol) was added dropwise, and the reaction solution was warmed to room temperature. The mixture was diluted with aq. NaFDPC^ solution (1.0 M, 400 ml) and phases separated. The aqueous layer was extracted with CH2CI2 (2 x 60 ml). The organic layers were combined, dried over MgSOzt, filtered, concentrated and purified by Si02 column chromatography (7:3 hexanes/EtOAc) to give the title compound (6.73 g, 85% yield). ESI MS m/z 266.2([M+Na]+). e 89. Synthesis of (S)-l-tert-butyl yl 4-methylenepyrrolidine-l,2- dicarboxylate (235). _ A()2Me To a suspension of methyltriphenylphosphonium bromide (19.62 g, 55.11 mmol) in THF (150 mL) at 0 °C was added potassium-t-butoxide (6.20 g, 55.30 mmol) in anhydrous THF (80 mL). After stirring at 0 °C for 2 h, the resulting yellow ylide was added to a solution of compound 234 (6.70 g, 27.55 mmol) in THF (40 mL). After ng at r.t. for 1 h, the reaction mixture was concentrated, diluted with EtOAc (200 mL), washed with H2O (150 mL), brine (150 mL), dried over MgSCL, concentrated and purified on SiCL column chromatography (9:1 hexanes/EtOAc) to yield the title compound (5.77 g, 87% yield). El MS m/z 264 ]+).
Example 90. Synthesis of (S)-methyl 4-methylenepyrrolidinecarboxylate hydrochloride (236) ^ X02Me VNH HCl To a solution of nd 235 (5.70 g, 23.63 mmol) in EtOAc (40 ml) at 4 °C was added HCl (12 M, 10 ml). The mixture was stirred for 1 h, diluted with toluene (50 ml), concentrated, and crystallized with EtOH/hexane to yield the title compound as HCl salt (3.85 g, 92% yield). El MS m/z 142.2 ([M+H]+).
Example 91. sis of 4-(benzyloxy)methoxybenzoic acid (238).
MeO co2h To a mixture of 4-hydroxymethoxybenzoic acid (50.0 g, 297.5 mmol) in ethanol (350 ml) and aq. NaOH solution (2.0 M, 350 ml) was added BnBr (140.0 g, 823.5 mmol). The mixture was stirred at 65 °C for 8 h, concentrated, co-evaporated with water (2 x 400 ml) and concentrated to -400 ml, acidified to pH 3.0 with 6 N HC1. The solid was collected by filtration, crystallized with EtOH, dried at 45 °C under vacuum to afford the title compound (63.6 g, 83% yield). ESI MS m/z 281.2 ([M+Na]+). e 92. Synthesis of 4-(benzyloxy)methoxynitrobenzoic acid (239).
BnOUno2 MeO co2h To a solution of compound 238 (63.5 g, 246.0 mmol) in CH2CI2 (400 ml) and HO Ac (100 ml) was added HNO3 (fuming, 25.0 ml, 528.5 mmol). The mixture was stirred for 6 h, concentrated, crystallized with EtOH, dried at 40 °C under vacuum to afford the title compound (63.3 g, 85% yield). ESI MS m/z 326.1 ([M+Na]+).
Example 93. Synthesis of (S)-methyl l-(4-(benzyloxy)methoxynitro benzoyl) methylenepyrrolidinecarboxylate (240).
BnO. N02 C02Me MeO ■N O A catalytic amount of DMF (30 pi) was added to a solution of compound 239 (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CH2CI2 (70 mL) and the ing mixture was stirred at room ature for 2 h. Excess CH2CI2 and oxalyl de was removed with rotavap. The acetyl chloride was re-suspended in fresh CH2CI2 (70 mL) and was added slowly to a pre-mixed solution of 236 (1.58 g, 8.91 mmol) and Et^N (6 mL) in CH2CI2 at 0 °C under N2 atmosphere. The on mixture was allowed to warm to r.t. and stirring was continued for 8 h. After removal of CH2CI2 and Et^N, the residue was partitioned between H20 and EtOAc (70/70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried (MgSO^O and concentrated.
Purification of the residue with flash chromatography (silica gel, 2:8 hexanes/EtOAc) d compound 240 (2.88 g, 76% yield). El MS m/z 449.1 ]+).
Example 94. Synthesis of (S)-l-(4-(benzyloxy)methoxynitrobenzoyl) methylenepyrro-lidinecarbaldehyde (241).
BnO. no2 cho r MeO ■N To a vigorously stirred solution of compound 240 (2.80 g, 6.57 mmol) in anhydrous CH2CI2 (60 mL) was added H (IN in CH2CI2, 10 mL) dropwise at -78 °C under N2 atmosphere.
After the mixture was d for an additional 90 min, excess reagent was decomposed by addition of 2 ml of methanol, followed by 5% HC1 (10 mL). The resulting e was allowed to warm to 0 °C. Layers were separated and the aqueous layer was further extracted with CH2CI2 (3 x 50 mL). ed organic layers were washed with brine, dried (MgSCL) and trated.
Purification of the residue with flash tography (silica gel, 95:5 CHCL/McOH) yielded compound 241 (2.19 g, 84% yield). EIMS m/z 419.1 ([M+Na]+).
Example 95. Synthesis of (S)(benzyloxy)methoxymethylene-2,3 -dihydro-lH- benzo[e]-pyrrolo[l,2-a]azepin-5(llaH)-one (242).
MeO ■N.
A mixture of compound 241 (2.18 g, 5.50 mmol) and ^2826)4 (8.0 g, 45.97 mmol) in THE (60 ml) and H2O (40 ml) was stirred at r.t. for 20 h. Solvents were removed under high vacuum.
The residue was re-suspended in MeOH (60 mL), and HC1 (6M) was added dropwise until pH ~ 2 was reached. The ing mixture was stirred at r.t. for 1 h. The reaction was -up by removing most of MeOH, then diluted with EtOAc (100 mL). The EtOAc solution was washed with sat. NaHCOs, brine, dried (MgS04), and concentrated. Purification of the e with flash chromatography (silica gel, 97:3 CHCL/McOH) yielded compound 242 (1.52 g, 80%). EIMS m/z 372.1 ([M+Na]+).
Example 96. Synthesis of (S)hydroxymethoxymethylene-2,3 -dihydro-lH-benzo[e]- pyrrolo[l,2-a]azepin-5(l laH)-one (243) MeO ■N.
To a solution of compound 242 (1.50 g, 4.32 mmol) in 70 ml of CH2CI2 was added 25 ml of CH3SO3H at 0 °C. The mixture was stirred at 0 °C for 10 min then r.t. for 2 h, diluted with CH2CI2, pH adjusted with cold 1.0 N NaHCO , to 4 and filtered. The aqueous layer was extracted with CH2CI2 (3 x 60 ml). The organic layers were combined, dried over Na2S04, filtered, ated and purified on Si02 column chromatography (CH3OH/CH2CI2 1:15) to afford 811 mg (73% yield) of the title product. EIMS m/z 281.1 ([M+Na]+).
Example 97. Synthesis of (llaS,lla'S)-8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(llaH)-one) (244). r^N. N^, N- OMe MeO •N.
O O To a stirred suspended solution of CS2CO3 (0.761 g, 2.33 mmol)in butanone (8 ml) were added compound Cll (401 mg, 1.55 mmol) and 1,5-diiodopentane (240 mg, 0.740 mmol). The mixture was stirred at r.t. overnight, trated, and purified on SiCT chromatography (EtOAc/CFbCh 1:10) to afford 337 mg (78% yield) of the title product. EIMS m/z 607.2 ([M+Na]+).
Example 98. Synthesis of (S)methoxy((5-(((S)methoxymethyleneoxo- 2,3,5,10,11,11 a-hexahydro-1 H-benzo [e]pyrrolo [ 1,2-a] [ 1,4]diazepinyl)oxy)pentyl)oxy) ene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(llaH)-one (245).
O«CKN OMe MeO O O To a solution of compound 244 (150 mg, 0.256 mmol) in anhydrous dichloromethane (1 mL) and absolute ethanol (1.5 mL) was added sodium borohydride in methoxyethyl ether (85pl, 0.5 M, 0.042mmol) at 0 °C. The ice bath was removed after 5 minutes and the mixture was stirred at room temperature for 3 hours, then cooled to 0 °C, quenched with saturated ammonium chloride, diluted with dichloromethane, and phases separated. The c layer was washed with brine, dried over anhydrous NaiSOzj, filtered through Celite and trated. The residue was purified by reverse phase HPLC (Cig column, acetonitrile/water). The corresponding ons were extracted with dichloromethane and concentrated to afford the title compound 245 (64.7 mg, 43%), MS m/z 609.2 ([M+Na]+), 625.3 +) and 627.2 ([M+Na+H20]+); the fully reduced compound was obtained (16.5 mg, 11%), MS m/z 611.2 ([M+Na]+), 627.2 ([M+K]+), 629.2 ([M+Na+H20]+); and the unreacted ng material was also recovered (10.2 mg, 7%), MS m/z 607.2 ([M+Na]+), 625.2 ([M+Na+H20]+).
Example 99. Synthesis of ((5-(((S)(3-(2-(2-azidoethoxy)ethoxy) propanoyl) methoxymethylene-5 -oxo-2,3,5,10,11,11 a-hexahydro-1 H-benzo [e]pyrrolo [1,2- a] [ 1,4] diazepin- 8-yl)oxy)pentyl)oxy)methoxymethylene-2,3 ro-1H- benzo [e]pyrrolo [ 1,2-a] [ 1,4] diazepin-5( 11 aH)-one (246). jdCcc o OMe MeO •N.
O O To the mixture of compound 245 (60.0 mg, 0.102 mmol) and compound 166 (40.5 mg, 0.134 mmol) in dichloromethane (5 ml) was added EDC (100.5 mg, 0.520 mmol). The mixture was stirred at r.t. overnight, concentrated and purified on SiCT column chromatography (EtOAc/CfECh, 1:6) to afford 63.1 mg (81% yield) of the title product 246. ESI MS m/z C40H50N7O9 [M+H] +, cacld.772.36, found 772.30.
Example 100. Synthesis of (S)((5-(((S)(3-(2-(2-aminoethoxy)ethoxy) propanoyl) methoxymethylene-5 -oxo-2,3,5,10,11,11 a-hexahydro-1 H-benzo [e]pyrrolo [1,2- a] [ 1,4] diazepin- 8-yl)oxy)pentyl)oxy)methoxymethylene-2,3 -dihydro-1H- benzo [e]pyrrolo [ 1,2-a] [ 1,4] diazepin-5( 11 aH)-one (247).
(H'v'V'Mb OMe MeO ■N.
O O To a solution of compound 246 (60 mg, 0.078 mmol) in the mixture of THE (5 ml) and NaH2P04 buffer solution (pH 7.5, TO M, 0.7 ml) was added PPI13 (70 mg, 0.267 mmol). The mixture was d at r.t. ght, concentrated and purified on Cig preparative HPLC, eluted with water/CHsCN (from 90% water to 35% water in 35 min) to afford 45.1 mg (79% yield) of the title product 247 after drying under high . ESI MS m/z C40H52N5O9 [M+H]+, cacld.746.37, found 746.50.
Example 101. Synthesis of(2S)((2R,3R)((2S)-l-((HS,14S,17S)-l-azido((R)-sec- butyl)-l 1,14-diisopropylmethoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazai- 20-oyl)pyrrolidinyl)methoxymethylpropanamido)phenylpropanoic acid (257) N3v^fo N^ph 1 O 1 O^O /() o co2h To the crude compound 331 (22 mg, 0.030 mmol) in a mixture of DMA (0.8 ml) and NaH2P04 buffer on (pH 7.5, 1.0 M, 0.7 ml) was added compound 166 (18.0 mg, 0.060 mmol) in four portions in 2 h. The mixture was stirred overnight, concentrated and ed on Si02 column chromatography (CH3OH/CH2CI2/HOAC 1:8:0.01) to afford the title compound (22.5 mg, 82% yield). LC-MS (ESI) m/z for C46H77N80n [M+H]+: 917.56, found: 917.60.
Example 102. sis of (2S)((2R,3R)((2S)-l-((llS,14S,17S)-l- amino((R)-secbutyl )-11,14-diisopropylmethoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16- triazaicosanoyl)pyrrolidinyl)methoxymethylpropanamido)phenylpropanoic acid (258).
■N^rb 0^0 o co2h To compound 257 (22.0 mg, 0.024 mmol) in methanol (5 ml) in a hydrogenation bottle was added Pd/C (5 mg, 10% Pd, 50% wet). After air was vacuumed out and 25 psi H2 was conducted in, the mixture was shaken for 4 h, filtered through celite. The filtrate was trated to afford the crude title product (-20 mg, 92% yield), which was used in the next step without further purification. ESI MS mlz+ N6O11 (M+H), cacld.891.57, found 891.60.
Example 103. Synthesis of (S)((2R,3R)((S)-l-((6S,9S,12S,13R)((S)-sec- butyl)- 6,9-diisopropylmethoxy-2,2,5,11-tetramethyl-4,7,10-trioxooxa-5,8,11-triazapenta-decan- -oyl)pyrrolidinyl)methoxymethylpropanamido)phenylpropanoic acid (330). 1 o ^ 1 0^0 /() o co2h To a solution of compound 229 (30 mg, 0.035 mmol) in THE (1.0 ml) was added LiOH in water (1.0M, 0.8 ml). The mixture was stirred at r.t. for 35 min, neutralized with 0.5 M H3PO4 to pH 6, concentrated and purified on Si02 column chromatography (CH3OH/CH2CI2/HOAC 1:10:0.01) to afford the title compound 330 (25.0 mg, 85% yield). LC-MS (ESI) m/z calcd.for C44H74N5O10 [M+H]+: 832.54, found: .
Example 104. Synthesis of (S)((2R,3R)((S)-l-((3R,4S,5S)((S)-N,3-dimethyl((S)- 3-methyl(methylamino)butanamido)butanamido)methoxymethylheptanoyl)-pyrrolidin- 2-yl)methoxymethylpropanamido)phenylpropanoic acid (331) ■^VrrrVirHY'phco2h Compound 330 (25 mg, 0.030 mmol) in the mixture of cone. HC1 (0.3 ml) and 1,4-dioxane (0.9 ml) was d at r.t. for 35 min. The mixture was diluted with EtOH (1.0 ml) and toluene (1.0 ml), concentrated and co-evaporated with EtOH/toluene (2:1) to afford the title compound 331 as a white solid (22 mg, -100% yield), which was used in the next step without r purification. LC-MS (ESI) m/z calcd.for CsgHeeNsOg [M+H]+: 732.48, found: 732.60.
WO 59622 e 105. Synthesis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3- dimethyl((R)- l-methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazolecarboxamido)(3- (3-(2-(2-azidoethoxy)ethoxy)propanamido)hydroxyphenyl)methylpentanoic acid (335) O HkN^"" O OAc I U ' u H ho2c O To a solution of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3-dimethyl((R) methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazolecarboxamido)(3- aminohydroxyphenyl)methylpentanoic acid 285 (Huang Y. et al, Med Chem. #44, 249th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; W02014009774) (100 mg, 0.131 mmol) in the mixture of DMA (10 ml) and NaH2P04 buffer solution (pH 7.5, 1.0 M, 0.7 ml) was added compound 166 (80.0 mg, 0.266 mmol) in four portions in 2 h. The mixture was stirred overnight, concentrated and purified on Cig ative HPLC (3.0 x 25 cm, 25 ml/min), eluted with from 80% water/methanol to 10% water/methanol in 45 min to afford the title compound (101.5 mg, 82% yield). LC-MS (ESI) m/z calcd.for C45H70N9O11S [M+H]+: 944.48, found: 944.70.
Example 106. Synthesis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3- dimethyl((R)- l-methyl-piperidinecarboxamido)pentanamido)methylpentyl)thiazolecarboxamido) (3-(3-(2-(2-aminoethoxy)ethoxy)propanamido)hydroxyphenyl)methylpentanoic acid (336)9Yy; OAc O if vS—OH I HN NH2 ho2c O 2 To a solution of compound 332 (100.0 mg, 0.106 mmol) in methanol (25 ml) ning 0.1% HC1 in a hydrogenation bottle was added Pd/C (25 mg, 10% Pd, 50% wet). After air was vacuumed out in the vessel and 35 psi H2 was conducted in, the mixture was shaken for 4 h, filtered h celite. The filtrate was concentrated and purified on Cig preparative HPLC (3.0 x cm, 25 ml/min), eluted with from 85% water/methanol to 15% water/methanol in 45 min to afford the title compound (77.5 mg, 79% yield). LC-MS (ESI) m/z calcd.for C45H72N7O11S [M+H]+: 918.49, found: 918.60.
Example 107. Synthesis of 2,3-dibromo((2-(2-(3-((S)methoxy((5-(((S)-7 xy- 2-methyleneoxo-2,3,5,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin yl)oxy)pentyl)oxy)methyleneoxo-2,3,11,11 a-tetrahydro-1 H-benzo rolo [1,2- a][l,4]diazepin-10(5H)-yl)oxopropoxy)ethoxy)ethyl)amino)oxobutanoic acid (340).
WO 59622 o NH r^N. N-. o Br N- OMe MeO •N. Br C02H O O To a solution of compound 247 (40.0 mg, 0.068 mmol) in the mixture of DCM (4 ml) and DIPEA (12 uL, 0.069 mmol) was added 7 (38.0 mg, 0.148 mmol) at 0 °C. The mixture was stirred at 0 °C for 2 h and then r.t. 5h. The mixture was concentrated and purified on SiCT column eluted with CH3OH/CH2CI2/HOAC (1:6:0.01) to afford the title compound 340 (56.5 mg, 83% yield). ESI MS m/z calcd. for €4411536^5012 [M+H]+: 1002.21, found: 1002.40, 1004.40 [M+2+H]+.
Example 108. Synthesis of 2,5-dioxopyrrolidin-l-yl 2,3-dibromo((2-(2-(3-((S) methoxy((5-(((S)methoxymethyleneoxo-2,3,5,lla-tetrahydro-lH- e]pyrrolo[ 1,2-a] [ 1,4] -diazepinyl)oxy)pentyl)oxy)methyleneoxo-2,3,11,11atetrahydro-1 H-benzo [e]pyr-rolo [ 1,2-a] [ 1,4] diazepin- 10(5H)-yl)-3 - oxopropoxy)ethoxy)ethyl)amino)oxobutanoate (341) o-rKVNH r^N. N-. o Br N- OMe MeO ■N. Br C02Su O O To a solution of 2,3-dibromo((2-(2-(3-((S)methoxy((5-(((S)-7 -methoxy methyleneoxo-2,3,5,11 a-tetrahydro-1 H-benzo [e]pyrrolo [ 1,2-a] [ 1,4]diazepin yl)oxy)pentyl)oxy)methyleneoxo-2,3,11,11 a-tetrahydro-1 o [e]pyrrolo [1,2- ]diazepin-10(5H)-yl)oxopropoxy)ethoxy)ethyl)amino)oxobutanoic acid (55.0 mg, 0.054 mmol) in CH2CI2 (3 ml) was added NHS (10.0 mg, 0.086 mmol) and EDC (30.5 mg, 0.158 mmol). The mixture was stirred at r.t. overnight, concentrated and purified on Si02 column eluted with EtOAc/CH2Cl2 (1:5) to afford the title compound 341 (50.5 mg, 85% yield). ESI MS m/z calcd. for C48H56Br2N6Oi4 [M+H]+: 1099.22, found: 1099.40, 1101.40 ]+, 1119.50 [M+2+H+H20]+.
Example 109. Synthesis of (2S)((2R,3R)((2S)-l-((13S,26S,29S,32S)-12,13 -dibromo- )-sec-butyl)-26,29-diisopropylmethoxy-l-((S)methoxy((5-(((S)methoxy methyleneoxo-2,3,5,11 a-tetrahydro-1 H-benzo rolo [ 1,2-a] [ 1,4]diazepin yl)oxy)pentyl)oxy)methyleneoxo-2,3,11,11 a-tetrahydro-1 H-benzo [e]pyrrolo [1,2- a][l,4]diazepin-10(5H)-yl)-25,31-dimethyl-l,ll,14,24,27,30-hexaoxo-4,7,18,21-tetraoxa- ,15,25,28,31 -pentaazapentatriacontan-3 5-oyl)pyrrolidinyl)-3 -methoxy methylpropanamido)phenylpropanoic acid (344).
OMe MeO ■N.
O O T5r H Br HN—H-p N'^Ph 1 O I (L O O O CP2H To a solution of compound 258(~20 mg, 0.022 mmol) in the mixture of THF (1.5 mL) and NaHiPCXj buffer solution (0.9 mL, 0.15 M, pH 7.5) was added compound 2,5-dioxopyrrolidin-l- yl 2,3-dibromo((2-(2-(3-((S) methoxy((5-(((S)methoxymethyleneoxo-2,3,5,l la- tetrahydro-1 H-benzo [e]pyrrolo [ 1,2-a] [ 1,4] -diazepinyl)oxy)pentyl)oxy)methyleneoxo- 2,3,11,11 a-tetrahydro-1 H-benzo -rolo [ 1,2-a] [ 1,4] diazepin-10(5H)-yl)-3 - oxopropoxy)ethoxy)ethyl)amino)oxobutanoate 341 (30.0 mg, 0.027 mmol). The mixture was stirred at r.t. for 7 h, concentrated, and purified on Cig preparative HPLC ( 2.0 cm x 25 cm) eluted with watcr/CH^CN (from 90% water to 15% water in 50 min at 10 ml/min) to afford the title product 344 (26.1 mg, 63% yield) after drying under high vacuum. ESI MS m/z CgoHBoBriNiiOii [M+H]+, cacld. 1874.77, found 1874.50.
Example 110. Conjugated compound 344 to an antibody for 345.
N- OMe MeO mAb O s'/T° v "> P-v\ T^ph /n 345 JO o co2h To a mixture of 2.0 mL of 10 mg/ml tin in pH 6.0-8.0, were added 0.70 - 2.0 mL PBS buffer of 100 mM NaHiPOzt, pH 6.5-7.5 buffers, TCEP (28 pL, 20 mM in water) and the compound 344 (14 pL, 20 mM in DMA). The mixture was incubated at RT for 4-16 h, then DHAA (135 pL, 50 mM) was added. After uous incubation at RT overnight, the e was purified on G-25 column eluted with 100 mM NaHiPCL, 50 mM NaCl pH 6.0-7.5 buffer to afford 16.5-17.7 mg of conjugate compound 345 8% yield) in 13.1-15.0 ml . The drug/antibody ratio (DAR) (the combination of PBD dimer and MMAF per antibody) was 3.85, determined by UPLC-QTof mass um. The product was 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel.
Example 111. sis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3- dimethyl((R)- l-methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazolecarboxamido)(3- (12,13-dibromo((S)methoxy((5-(((S)methoxymethyleneoxo-2,3,5,llatetrahydro-1 H-benzo [e]pyrrolo [ 1,2-a] [ 1,4]diazepinyl)oxy)pentyl)oxy)methyleneoxo- 2,3,11,1 rahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-10(5H)-yl)-l 1,14,24-trioxo- 4,7,18,21-tetraoxa-10,15-diazatetracosanamido)hydroxyphenyl)methylpentanoic acid (346) OMe MeOH-a Q Br O O OAc o 7 'VOH HN. Br ■N. Pn/^O^ N N I S~J H 346 HO,C o To a solution of ccompound 336 (20 mg, 0.021 mmol) in the mixture of THE (1 ml) and NaH2P04 buffer solution (0.6 ml, 0.15 M, pH 7.5) was added nd 341 (30.0 mg, 0.027 mmol). The e was stirred at r.t. for 8 h, concentrated, and purified on Cig preparative HPLC ( 2.0 cm x 25 cm) eluted with watcr/CH^CN (from 90% water to 20% water in 50 min at 10 ml/min) to afford the title product 346 (26.6 mg, 64% yield) after drying under high vacuum. ESI MS m/z iNuOnS [M+H]+, cacld. 1901.69, found 1901.90.
Example 112. Conjugated compound 346 to an antibody for 347.
AC®OMe MeO H OAc q /f^VOH N N iwvO n I k-J H HO,C o To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, were added 0.70 ~ 2.0 mL PBS buffer of 100 mM NaHiPOzt, pH 6.5-7.5 buffers, TCEP (28 pL, 20 mM in water) and the compound 346 (14 pL, 20 mM in DMA). The mixture was incubated at RT for 4-16 h, then DHAA (135 pL, 50 mM) was added. After continuous incubation at RT overnight, the mixture was purified on G-25 column eluted with 100 mM NaHiPOzt, 50 mM NaCl pH 6.0-7.5 buffer to afford 16.4-17.6 mg of the conjugate compound 347(82%~88% yield) in 13.2-15.1 ml buffer.
The drug/antibody ratio (DAR) (the combination of PBD dimer and tubulysin analog per antibody) was 3.9, determined by UPLC-QTof mass spectrum. The t was 96-99% r analyzed by SEC HPLC (Tosoh Bioscience, Tskgel W, 7.8 mm ID x 30 cm, 0.5 , 100 min) and a single band measured by SDS-PAGE gel.
Example 113. Synthesis of (4R)-/er/-butyl 5-(4-acetoxynitrophenyl)((/er/- butoxycarbonyl)amino)methylpentanoate (349) BocHN ^ // OAc u02C no2 To a solution of compound 190 (107.1 mg, 0.252 mmol) in dichloromethane (4.0 mL) at 0 °C was added acetic anhydride (0.11 mL, 1.17 mmol) and triethylamine (0.16 mL) in sequence.
The reaction was then warmed to r.t. and stirred for 1 h, diluted with dichloromethane and washed with water and brine, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by column chromatography (0-15% EA/PE) to give a ess oil (120.3 mg, theoretical yield). MS ESI m/z calcd for C23H35N2O8 [M+H]+ 467.23, found 467.23. e 114. Synthesis of (4R)-/er/-butyl 5-(4-acetoxyaminophenyl) (Uerl- butoxycarbonyl)amino)methylpentanoate (349) BocHN^^"^_OAc u02C nh2 Phenyl nitrile 348 (120.3 mg, 0.258 mmol) was dissolved in ethyl acetate (5 mL) and acetic acid (0.5 mL). To which Pd/C (10 wt%, 10 mg) was added and the mixture was stirred under H2 balloon at r.t. for 30 min before filtration through a celite pad with washing of the pad with ethyl acetate. The filtrate was concentrated and purified by column chromatography (0-25% EA/PE) to give a yellow oil (120.9 mg, theoretical yield). MS ESI m/z calcd for C23H37N2O6 [M+H]+ 437.26, found 437.28.
Example 115. Synthesis of30-((2-acetoxy((2R)(tert-butoxy)((tertbutoxycarbonyl )amino)methyloxopentyl)phenyl)amino)-14,17,30-trioxo-4,7,10,21,24,27- hexaoxa-13,18-diazatriacontyn-l-oic acid (350) OAc O I O H N' O 'N^K^c°2H BocHN H 3 H To a solution of aniline 349 (106.9 mg, 0.245 mmol) and diacid 150 (140.2 mg, 0.269 mmol) in DMF (4.5 mL) at 0 °C was added HATU (93.2 mg, 0.245 mmol) and DIPEA (0.13 mL) in sequence. The reaction was then warmed to r.t. and stirred for 18 h, diluted with ethyl acetate and washed with water for three times and brine, dried over anhydrous NaiSOzj, filtered and trated. The residue was purified by column tography (0-15% McOH/CfTCh) to give a light yellow oil (70.9 mg, 31% yield). MS ESI m/z calcd for C45H71N4O17 [M+H]+ 939.47, found 939.47.
Example 116. Synthesis of 2,5-dioxopyrrolidin-l-yl 30-((2-acetoxy((2R) (tertbutoxy )((tert-butoxycarbonyl)amino)methyloxopentyl)phenyl)amino)-14,17,30-trioxo- 4,7,10,21,24,27-hexaoxa-13,18-diazatriacontyn-1 -oate (351) BocHN I O H O ,N. .O. tBu02C N' •°T3 N r'co2su H H A mixture of acid 350 (22.3 mg, 0.0237 mmol), N-hydroxysuccinimide (13.7 mg, 0.119 mmol) and DCC (15.0 mg, 0.0728 mmol) were stirred at r.t. for 16 h. The solid was filtered off and the filtrate trated and purified by column chromatography (0-10% McOH/CfECE) to give colorless oil (14.7 mg, 60% yield). MS ESI m/z calcd for C49H74N5O19 [M+H]+ 1036.49, found 1036.49.
Example 117. Synthesis of utyl 5-(4-acetoxy(l-hydroxy-73,86,89- trioxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,76,79,82,93,96,99- nonacosaoxa-72,85,90-triazadohectynamido)phenyl)((tert-butoxycarbonyl)amino) methylpentanoate (352) OAc o I NH BocHN H O *1 tBu02C ho^42VVVV-' To a solution of H2N-PEG24-OH (15.2 mg, 0.0142 mmol) in a mixture of Et0H/CH2Cl2/H20 (v/v/v 5:3:1, 0.5 mL), triethylamine (25 pL) was added, followed by a on of NHS ester 351 (14.7 mg, 0.0142 mmol) in H2Cl2/H20 (v/v/v 5:3:1, 1.0 mL).
After stirring at r.t. for 1 h, the reaction was concentrated and purified by column chromatography (0-15% MeOH/CH2Cl2) to give colorless oil. MS ESI m/z calcd for C93H168N5O40 [M+H]+ 2, found 1995.12.
Example 118. Synthesis of (4R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3-di methyl ((R)-l-methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazolecarboxamido)- -(4-acetoxy(l-hydroxy-73,86,89-trioxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,76,79,82,93,96,99- nonacosaoxa-72,85,90-triazadohectynamido)phenyl)methylpentanoic acid (353)cwI ° OAcrM, O O H N 'N- ,N. NH I H o'i3 O / ho2c HO Compound 352 (0.005 mmol) was dissolved in CH2CI2 (1.0 mL) and treated with TFA (1.0 mL) at r.t. for 1.5 h then concentrated and re-dissolved in DMF (0.5 mL), to which luorophenyl ester 142 (0.02 mmol) in DMF (0.5 mL) and DIPEA (3.5 pL) were added. The reaction was stirred at r.t. for 2 h and then concentrated. The residue was purified by preparative reverse phase HPLC (Cig column, acetonitrile/water) to give a colorless oil (1.13 mg, 10% .
MS ESI m/z calcd for C110H192N9O43S [M+H]+ 2359.28, found 2359.28.
Example 119. sis of (3R,4S,7S,10S)((S)-sec-butyl)-7,10-diisopropyl (2-((S)- 2-((lR,2R)-l-methoxy(((S)-l-methoxy-l-oxophenylpropanyl)amino)methyl oxopropyl)pyrrolidin-l-yl)oxoethyl)-5,11-dimethyl-6,9,12,25,28-pentaoxo- 2,15,18,21,32,35,38-heptaoxa-5,8,ll,24,29-pentaazahentetracontynoic acid (355) L oJ ° ()\ O /O O C02Me To a solution of 230 (26.1 mg, 0.035 mmol) and diacid 150 (55 mg, 0.106mmol) in dichloromethane (2 mL) at 0 °C was added Brop (16 mg, 0.042 mmol), followed by diisopropylethylamine (22 uL, 0.12 mmol). The reaction mixture was d at 0 °C for 10 min, then warmed to r.t. and stirred overnight. The reaction mixture was diluted with ethyl acetate (50 mL), washed with water (10 mL), saturated aqueous sodium de (10 mL), dried over sodium sulfate, and concentrated in vacuo. The residue was ed by reverse phase HPLC with a gradient of MeCN/H20 to afforded compound 355 (30 mg, 68% yield) as a white solid. MS ESI m/z calcd. for C62H101N7O19 [M+H]+: 1248.72, found: 1248.72.
Example 120. Synthesis of (3R,4S,7S,10S)-perfluorophenyl4-((S)-sec- butyl)-7,10- diisopropyl(2-((S)((lR,2R)-l-methoxy(((S)-l-methoxy-l-oxophenylpropan no)methyloxopropyl)pyrrolidin-l-yl)oxoethyl)-5,1 l-dimethyl-6,9,12,25,28- pentaoxo-2,15,18,21,32,35,38-heptaoxa-5,8,ll,24,29-pentaazahentetracontynoate (356). q h H l^N'l^'° O = I (V O O C02Me O^'-N't"—°4^CO2C6F5 U H 3 To a on of compound 355 (15 mg, 0.012 mmol) dissolved in ethyl acetate (1 mL) was added pentafluorophenol (3.3 mg, 0.018 mmol) and DCC (3.7 mg, 0.018 mmol). The reaction mixture was stirred at room temperature overnight and then filtered, washing the filter cake with 2 mL of EtOAc. The filtrate was used immediately without further purification or concentration.
Example 121. Synthesis of (S)-methyl 2-((2R,3R)((S)-l-((104S,107S, 110S,111R)- 110-((S)-sec-butyl)-l-hydroxy-104,107-diisopropyl-l 1 l-methoxy-103,109-dimethyl- 73,86,89,102,105,108-hexaoxo- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,76,79,82,93,96,99- saoxa-72,85,90,103,106,109-hexaazatridecahectynoyl)pyrrolidinyl) methoxymethylpropanamido)phenylpropanoate (357) 0VNVvo N^ph C02Me o OH H O To a solution of compound 356 (0.012 mmol) dissolved in DMA (1 mL) was added HO- PEG24-NH2 (19 mg, 0.018 mmol) and ropylethylamine (4.2 pL, 0.024 mmol). The reaction mixture was d overnight and concentrated in vacuo. The residue was purified by reverse phase HPLC with a nt of MeCN/H20 to afforded compound 357 (5 mg, 18% yield) as a colorless oil. MS ESI m/z calcd. for CnoHiggNgCLK [M+H]+ 2304.36, found 2304.36.
Example 122. Synthesis of compound 361 OWovA/VV0 O \ A I A C VV / Vo n'1—Ns 1 °,.-k 1 S- o H HN Et02C 0 H 361 To a solution of nd 161 (4.7 mg, 0.011 mmol) dissolved in ethyl acetate (1 mL) was added pentafluorophenol (10.1 mg, 0.055 mmol) and DCC (11.3 mg, 0.055 mmol). The reaction mixture was d at room temperature overnight and then filtered through celite, washing the filter cake with 2 mL of EtOAc. The filtrate was concentrated and re-dissolved in DMA (2 mL), to which thyl 4-(2-((lR,3R)-l-acetoxy((2S,3S)-N,3-dimethyl((R)-lmethylpiperidinecarboxamido )pentanamido)methylpentyl)thiazolecarboxamido)(3- aminohydroxyphenyl)methylpentanoate 360 (8 mg, 0.0102 mmol) and DIPEA (3.8 pL, 0.022 mmol) were added. The reaction mixture was stirred at r.t. overnight, and then the solvent was removed under reduced pressure and the residue was purified by reverse phase HPLC with a gradient of McCN/fEO to give the title t 361 (4.5mg, 38% yield). ESI MS m/z: calcd for C58H87N8Oi6S [M+H]+ 1183.59, found 1183.58.
Example 123. sis of compound 364 O H BocHN ho2c HN'VVWs H O 364 To a on of compound 161 (0.43 g, 0.997 mmol) dissolved in ethyl acetate (20 mL) was added pentafluorophenol (0.92 g, 4.98 mmol) and DCC (1.02 g, 4.98 mmol). The reaction e was stirred at room temperature overnight and then ed through celite, washing the filter cake EtOAc. The filtrate was trated and re-dissolved in DMA (10 mL), to which (4R)(3-aminohydroxyphenyl) ((tert-butoxycarbonyl)amino)methylpentanoic acid 363 (0.34 g, 0.997 mmol) and DIPEA (0.35 mL, 2 mmol) were added. The reaction mixture was stirred at r.t. overnight and concentrated under reduced pressure. The residue was d with H2O and extracted with EtOAc. The combined the organic layers were dried over NaiSOzj, concentrated and purified by SiOi column chromatography to give the title product 364 (0.3 g, 42% yield). ESI MS m/z: calcd for C35H51N4O13 [M+H]+:735.34, found 735.34.
Example 124. Synthesis of compound 365 Qjyws.H O OAcr>% H fYo^O 0A/W’ 1 1 O H HN Compound 364 (0.1 g, 0.14 mmol) was dissolved in DCM (10 mL), treated with TEA (5 mL) and stirred at r.t. for 2 h, then concentrated. The residue was dissolved in DMA (8 ml), to which pentafluorophenyl ester 142 (0.15 g, 0.21 mmol) and DIPEA (37 pi, 0.21mmol) were added and the reaction was stirred overnight. The reaction solution was concentrated and purified by reverse phase HPLC with a gradient of 20 to give the product 365 (47.2 mg, 30% yield). ESI MS m/z: calcd for CseHssNsOieS [M+H]+: 1155.56, found 1155.56.
Example 125. Synthesis of 24-((5-((2R)(/er/-butoxy)((/er/-butoxycarbonyl) - 4-methyloxopentyl)((/er/-butyldimethylsilyl)oxy)phenyl)amino)-l 4-trioxo-4,7,l 8,21- tetraoxa-10,15-diazatetracosyn-l-oic acid (367) I O H BocHN tBu02C H02Cf^0^N-i0 367 Compound 192 (0.085 g, 0.197 mmol), compound 161 (0.1 g, 0.197 mmol) and HATU (0.112 mg, 0.30 mmol) were mixed in DMF (6 ml), to which TEA (55 pL, 0.40 mmol) was added. The reaction mixture was stirred at r.t. overnight. The solvent was removed under reduced pressure and the residue purified by SiCT column chromatography to give the title product 367 (0.065 mg, 36% yield). ESI MS m/z: calcd for €451175^01481 [M+H]+ 923.50, found 923.50. e 126. Synthesis of 24-((5-((2R)(tert-butoxy)((tert-butoxycarbonyl) - 4-methyloxopentyl)hydroxyphenyl)amino)-ll,14,24-trioxo-4,7,18,21-tetraoxa-10,15- diazatetracosyn-l-oic acid (368) O H BocHN t ^Op^N^0 Bu02C 368 Compound 367 (64.8 mg, 0.070 mmol) was dissolved in DCM (3 mL), TBAF(1 M, 0.1 mL) was added, and the reaction was stirred at r.t. for 1 h, then trated and purified by Si02 column chromatography to give the title product 368 (34.5 mg, 61% yield). ESI MS m/z: calcd for C39H61N4O14 [M+H]+ 809.41, found 809.41.
Example 127. Synthesis of 24-((5-((2R)(2-((lR,3R)-l-acetoxy((2S,3S)-N,3 - dimethyl((R)-l-methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazole carboxamido)carboxypentyl)hydroxyphenyl)amino)-ll,14,24-trioxo-4,7,18,21-tetraoxa- ,15-diazatetracosyn-l-oic acid (369) W OAcrVV OH o Ij H I °A| I H02cfv°'h^N^o Compound 368 (34.5 mg, 0.043 mmol) was dissolved in DCM (1 mL), and then TEA (0.5 mL) was added. The reaction was stirred at r.t. for 2 h, then concentrated and re-dissolved in DMA( 4 mL), to which pentafluorophenyl ester 142 (45 mg, 0.065 mmol) and DIPEA (15 pL, 0.09mmol) were added and the reaction was stirred ght and then concentrated and purified by reverse phase HPLC with a gradient of McCN/FEO to give the title product 369 (22.8 mg, 46% yield). ESI MS m/z: calcd for CseHgsNgOnS [M+H]+ 1173.57, found 1173.58.
Example 128. Synthesis of (4R)-ethyl 4-(((benzyloxy)carbonyl)amino) butanamido)((tert-butyldimethylsilyl)oxy)phenyl)((tert-butoxycarbonyl)amino) methylpentanoate (372) ft \ OTBS BocHN O HN NHCbz Et02C 2,5-dioxopyrrolidin-l-yl 4-(((benzyloxy)carbonyl)amino)butanoate (0.396 g, 1.2 mmol) and (4R)-ethyl 5-(3-aminohydroxyphenyl)((tert-butoxycarbonyl) amino) methylpentanoate (0.44 g, 1.2 mmol) were dissolved in EtOH (10 mL), and phosphate buffer solution (pH=7.5, 0.1M, 2ml) was added. The reaction mixture was stirred at r.t. overnight and then the solvent was removed under reduced pressure and the residue purified by SiCE column chromatography to give the title product 372 (0.485g, 70%). ESI: m/z: calcd for C31H44N3O8 [M+H]+:586.31, found .
Example 129. Synthesis of (4R)-ethyl 4-aminobutanamido)((tert-butyl dimethylsilyl)oxy)phenyl)((tert-butoxycarbonyl)amino)methylpentanoate (373) '/ \ OTBS BocHN- Et02C 373 Compound 372 (0.35 g, 0.5 mmol) was dissolved in MeOH (5 ml), and Pd/C (10 wt%, 35 mg) was then added. The reaction mixture was stirred at r.t. under H2 balloon overnight, then filtered through celite and the te was concentrated under reduced pressure to give the title product 373 (0.22 g, 79% yield). ESI MS m/z: calcd for CigHsiNsOeSi [M+H]+:566.35, found Example 130. Synthesis of 4-((4-((5-((2R)((tert-butoxycarbonyl)amino) ethoxy oxopentyl)((tert-butyldimethylsilyl)oxy)phenyl)amino)oxobutyl)amino) oxobutynoic acid (374) OTBS Q BocHN ^ // O co2h Et02C- HN^t NH Compound 373 (0.22 g, 0.39 mmol) was dissolved in NMP (2 mL), and actylenedicarboxylic acid (0.089g, 0.78mmol) was added at 0 °C. After stirring for 10 min, DMTMM (0.1 g, 0.39 mmol) was added. The mixture was stirred at 0 °C ght. The reaction was washed with H2O (100 mL) and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over , filtered, concentrated under reduced re and the residue was purified by SiCT column chromatography with a gradient of DCM/ MeOH to give the title product 374 (0.05 g, 20% yield). ESI MS m/z: calcd for CsstfeNsOgSi :662.34, found 662.34.
Example 131. Synthesis of 4-((4-((5-((2R)((tert-butoxycarbonyl)amino)ethoxy methyloxopentyl)hydroxyphenyl)amino)oxobutyl)amino)oxobutynoic acid (375) ^ // OH O BocHN O > = co2h Et02C 11N —\ f^MI Compound 374 (50 mg, 0.076 mmol) was dissolved in DCM (2 mL), and then TBAF (1 M, 0.05 mL) was added. The reaction was stirred at r.t. for 1 h, and then concentrated and purified by SiOi column chromatography to give the title product 375 (10.5 mg, 25% . ESI MS m/z: calcd for CiTHggNsOg [M+H]+ 548.25, found 548.25.
Example 132. Synthesis of 4-((4-((5-((2R)(2-((lR,3R)-l-acetoxy((2S,3S)- N,3- dimethyl((R)-l-methylpiperidinecarboxamido)pentanamido)methylpentyl)thiazole carboxamido)ethoxymethyloxopentyl)hydroxyphenyl)amino)oxobutyl)amino) oxobutynoic acid (376) H O OAc O co2h N. O ■N H N\ 'N- .M T 8 s H C02Et O 376 Compound 375 (10.5 mg, 0.02 mmol) was dissolved in DCM (1 mL), and TEA (0.5 mL) was added. The reaction was stirred at r.t. for 2 h, then concentrated and re-dissolved in DMA (2 mL), to which pentafluorophenyl ester 142 (21 mg, 0.03 mmol) and DIPEA(7 pi, 0.04 mmol) were added. The reaction was stirred overnight and then concentrated and the residue was purified by reverse phase HPLC with a gradient of MeCN/H20 to give the title product 376 (9 mg, 47% yield). ESI MS m/z: calcd for C48H70N7O12S [M+H]+ 968.47, found 968.47.
Example 133. Synthesis of 2-((6S,9S,12R,14R)((S)-sec-butyl)hydroxy -6,12- diisopropyl-2,2,5,1 amethyl-4,7,10-trioxooxa-5,8,11-triazatetradecan- Id-y^thiazole-dcarboxylic acid (381) V" H O Y OHX 'N XvIf™*1 To a solution of Boc-N-Me-L-Val-OH (33 mg, 0.14 mmol) in EtOAc was added pentafluorophenol (39 mg, 0.21 mmol) and DCC (32 mg, 0.154 mmol). The on mixture was stirred at r.t. for 16 h and then ed over a celite pad, with washing of the pad with EtOAc. The filtrate was concentrated and re-dissolved in DMA (2 mL), and then ,3R)((2S,3S) amino-N,3-dimethylpentanamido)-l-hydroxy methylpentyl)thiazolecarboxylic acid 380 (52 mg, 0.14 mmol) and DIPEA (48.5 pL, 0.28mmol) were added. The reaction mixture was stirred at r.t. for 24 h and then concentrated and purified by reverse phase HPLC (Cig column, % acetonitrile/water) to afford compound 381 (40.2 mg, 49% yield). ESI MS m/z: calcd for C28H49N4O7S [M+H]+: 585.32, found 585.32.
Example 134. Synthesis of 2-((6S,9S,12R,14R)((S)-sec-butyl)-6,12-di- isopropyl- 2,2,5,1 l-tetramethyl-4,7,10,16-tetraoxo-3,15-dioxa-5,8,11-triazaheptadecanyl)thiazole carboxylic acid (382) H o OAc Boc N 'N N nr-co2HN 1 o l o'" 382 Compound 381 (40 mg, 0.069 mmol) was dissolved in pyridine (8 mL), to which acetic anhydride (20.4 mg, 0.2 mmol) was added at 0 °C and the reaction was allowed to warm to r.t. and stirred overnight. The mixture was concentrated and the e purified by SiCT column chromatography with a gradient of OH to give the title product 382 (48.1 mg, -100% yield). ESI MS m/z: calcd for C30H51N4O8S [M+H]+ , found 627.33.
Example 135. Synthesis of (4R)(2-((6S,9S,12R,14R)((S)-sec-butyl)-6,12- diisopropyl-2,2,5,1 l-tetramethyl-4,7,10,16-tetraoxo-3,15-dioxa-5,8,11-triazaheptadecan yl)thiazolecarboxamido)methylphenylpentanoic acid (383) H O OAc Boc* r>% ll N N 1 O I H COOH To a solution of compound 382 (48.1 mg, 0.077 mmol) in EtOAc was added pentafluorophenol (21.2 mg, 0.115 mmol) and DCC (17.4 mg, 0.085 mmol). The reaction mixture was stirred at r.t. for 16 h and then filtered over a celite pad, with washing of the pad with EtOAc. The filtrate was concentrated and solved in DMA (4 mL), and then (4R) aminomethylphenylpentanoic acid (20.7 mg, 0.1 mmol) and DIPEA (26.8 pL, 0.154 mmol) were added. The on mixture was stirred at r.t. for 24 h and then concentrated and purified by reverse phase HPLC (Cig , % acetonitrile/water) to afford compound 383 (63 mg, -100% yield). ESI MS m/z: calcd for C42H66N509S [M+H]+ 816.45, found 816.45.
Example 136. Synthesis of ll,14,24-trioxo(perfluorophenoxy)-4,7,18,21- tetraoxa- ,15-diazatetracosyn-l-oic acid (378) H O co2c6f5 378 o H To a solution of compound 161 (45.4 mg, 0.11 mmol) in EtOAc was added pentafluorophenol (21.3 mg, 0.12 mmol) and DCC (24.7 mg, 0.12 mmol). The reaction mixture was stirred at r.t. for 16 h and then ed over a Celite pad, with washing of the pad with EtOAc. The filtrate was trated and used for the next step without further purification.
Example 137. Synthesis of (4R,6R,9S,12S)((S)-sec-butyl)(4-(((2R) carboxy phenylpentanyl)carbamoyl)thiazolyl)-6,12-diisopropyl-7,13-dimethyl-2,8,11,14,24,27- hexaoxo-3,17,20,31,34-pentaoxa-7,10,13,23,28-pentaazaheptatriacontynoic acid (384) H O H O OAc N LHN O 2 V MN N 1 0 ' O^N^°^trC02H Compound 383 (63 mg, 0.077 mmol) was dissolved in DCM (5 mL), and TEA (2.5 mL) was then added. The on was stirred at r.t. for 2 h, then concentrated and re-dissolved in DMA (4 mL), to which compound 378 (65.8mg, 0.11 mmol) and DIPEA (27 pL, 0.154 mmol) were added. The reaction mixture was stirred at r.t. overnight and then concentrated and the residue purified by reverse phase HPLC (Cig , 10-100% acetonitrile/water) to afford compound 384 (14 mg, 16% yield). ESI MS m/z: calcd for C55H84N7O16S [M+H]+: 1130.56, found 1130.57.
Example 138. General method of conjugation of compound 196, 200, 205, 259, 267, 269, 271, 273, 274, 276, 288, 292, 299, 301, 307, 310, 313, 316, 318, 320, 325, 327, 328, 333, 337, 339, 342, 344, 346, 353, 355, 357, 361, 365, 369, 376, 384, 388, 393, 394, 397, 400, 401, 404, 406, 409, 411, or 413 independently to an antibody (Herceptin).
To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, were added of 0.70 - 2.0 mL PBS buffer of 100 mM NaH2P04, pH 6.5-8.5 buffers, TCEP (14-35 pL, 20 mM in water) and the compound 196, 200, 205, 259, 260, 267, 269, 271, 273, 274, 276, 288, 292, 299, 301, 307, 310, 313, 316, 318, 320, 325, 327, 328, 333, 337, 339, 342, 344, 346, 353, 355, 357, 361, 365, 369, 376, 384, 388, 393, 394, 397, 400, 401, 404, 406, 409, 411, or 413 (14-28 pL, 20 mM in DMA).
The mixture was incubated at RT for 4-16 h, then DHAA (135 pL, 50 mM) was added in. After continuous incubation at RT overnight, the mixture was purified on G-25 column eluted with 100 mM NaHiPCXj, 50 mM NaCl pH 6.0-7.5 buffer to afford 12.0-18.4 mg of the ate compound 196a, 201, 206, 261, 267, 270, 272, 275, 277, 289, 293, 300, 302, 308, 311, 314, 317, 319, 321, 326, 329, 334, 338, 339a, 343, 345, 347, 354, 355a, 358, 362, 366, 370, 377, 385, 386, 389, 395, 396, 398, 402, 405, 407, 410, 412, or 414 (60%~91% yield) accordingly in 13.0-15.8 ml buffer. The drug/antibody ratio (DAR) was 1.9-4.0, which was ined via UPLC-QTOF mass spectrum. It was 94-99% monomer ed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE Example 139. l method of conjugation of compound 248, 250, 252, 254, and 255, ndently to Herceptin (an antibody).
To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, 0.70 - 2.0 mL of 100 mM NaHiPOzt, 1 mM NaiSOs, pH 6.5-8.5 buffer, and the compound 248, 250, 252, 254 and 255 independently (21-28 pL, 20 mM in DMA) incubated for 1 h, was added TCEP (14-35 pL, 20 mM in water). After the mixture was continued to incubate at RT for 8-24 h, DHAA (135 pL, 50 mM) was added in. And the solution was continuously incubated for another 12 h. then purified on G-25 column eluted with 100 mM NaHiPOzt, 50 mM NaCl pH 6.0-7.5 buffer to afford 13.8-17.6 mg of the conjugate compound 249, 251, 253, and 256 respectively (69%~88% yield) in 13.6-15.2 ml buffer. The drug/antibody ratio (DAR) was 1.9-4.0, which was determined via UPLC-QTOF mass um. It was 96-99% r analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGE gel or two bands when a reduce reagent DTT was in the SDS Page.
Example 140. In vitro cytotoxicity evaluation of ates 196a, 201, 206, 354, 358, 362, 366, 370, 377, 385 and 407 in comparison with T-DM1: The cell line used in the cytotoxicity assays was NCI-N87, a human gastric carcinoma cell line; The cells were grown in RPMI-1640 with 10% FBS. To run the assay, the cells (180 pi, 6000 cells) were added to each well in a 96-well plate and incubated for 24 hours at 37°C with % COi- Next, the cells were treated with test compounds (20 pi) at various concentrations in appropriate cell e medium (total volume, 0.2 mL). The control wells contain cells and the medium but lack the test compounds. The plates were incubated for 120 hours at 37°C with 5% CO2. MTT (5mg/ml) was then added to the wells (20 jal) and the plates were incubated for 1.5hr at 37°C. The medium was carefully removed and DMSO (180 pi) was added afterward. After it was shaken for 15min, the absorbance was measured at 490nm and 570nm with a reference filter of 620nm. The inhibition% was calculated according to the following equation: inhibition% = [1- (assay-blank)/(control-blank)] x 100.
The cytotoxicity results: DAR (drug ratio ) N87 cell (Ag+) IC50 (nM) Conjugate 196a 2.8 3.9 nM Conjugate 201 2.7 1.3 nM ate 206 2.9 1.7 nM Conjugate 354 2.1 28.4 nM Conjugate 358 2.2 643.7 nM Conjugate 362 2.8 3.1 nM Conjugate 366 2.9 2.3 nM Conjugate 370 3.2 1.3 nM ate 377 2.5 62.7 nM Conjugate 385 2.8 561.6 nM Conjugate 407 3.1 0.51 nM T-DM1 3.5 0.07 nM The conjugates with the bridge linkers were less potent than T-DM1 in vitro. e 141. Antitumor Activity In vivo.
The in vivo efficacy of conjugates 196a, 201, 206, 354, 358, 370, 377, 385, and 407 along with T-DM1 were evaluated in a human gastric carcinoma N-87 cell line tumorxenograft models.
Five-week-old female BALB/c Nude mice (30 animals) were ated subcutaneously in the area under the right shoulder with N-87 carcinoma cells (5 x 106 cells/mouse) in O.lmL of serum- free medium. The tumors were grown for 8 days to an average size of 130 mm3. The animals were then randomly divided into 10 groups (6 animals per group). The first group of mice served as the control group and was treated with the phosphate-buffered saline vehicle. The remaining 9 groups were treated with conjugates 196a, 201, 206, 354, 358, 370, 377, 385, 407 and T-DM1 respectively at dose of 6 mg/Kg administered enously. Three dimensions of the tumor were ed every 4 days and the tumor volumes were calculated using the a tumor volume =1/2 (length x width x height). The weight of the animals was also ed at the same time. A mouse was sacrificed when any one of the following criteria was met: (1) loss of body weight of more than 20% from pretreatment weight, (2) tumor volume larger than 1500 mm , (3) too sick to a reach food and water, or (4) skin necrosis. A mouse was considered to be tumor-free if no tumor was palpable.
The results were plotted in Figures 36. All the 9 conjugates did not cause the animal body weight loss. And the animals at control group were sacrificed at day 30 due to the tumor volume larger than 1200 mm and they were too sick. All 6/6 animals at the groups of compounds 201 and a 407 had completely no tumor measurable at day 18 till day 68 (the end of ment). In contrast T-DM1 at dose of 6 mg/Kg was not able to eliminate the tumors and it only inhibited the tumor growth for 38 days. Conjugate compounds 196a, 206, 277, and 354 did not ate the tumor at dose of 6 mg/Kg, but had better antitumor activity than T-DM1. The conjugates 358 and 385 had worse antitumor ty than T-DM1. More importantly, all animals treated with conjugate compounds 196a, 201, 206, 277, 354, 358, 385 and 407 had no or less liver toxicities than the animals treated with T-DM1 when measured levels of alanine aminotransferase ( ALT) and aspartame aminotransferase ( AST) in serum at the end of experiment (data not shown). This trates that the conjugates with the bridge linkers of this patent application would have broader therapeutical applications than the traditional conjugates.
MAR KED-UP

Claims (7)

1. A compound of formula (II): wherein: “ ” represents a triple bond; Drug1 represents a cytotoxic agent or a drug, R1 is absent, or selected from the group consisting of C1-C8 of alkyl; C2-C8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 of aryl, Ar-alkyl, heterocyclic, yclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; C1-C8 of esters, ether, or amide; polyethyleneoxy unit of formula (OCH2CH2)p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or combination thereof; or R1 is a chain of atoms selected from C, N, O, S, Si, and P; X1 and X2 are independently absent or independently ed from NH; NHNH; N(R3); (R3’); O; S; C1-C8 of alkyl; C2-C8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, arbonyl, heteroaryl; or 1~8 amino acids; wherein R3 and R3’ are independently H; C1-C8 of alkyl; C2-C8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, aryl; C1-C8 esters, ether, or amide; or polyethyleneoxy unit of formula H2)p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or a combination f; R2 is selected from OH, H, NH2; SH; NHNH2; N(R3)(R3’); N(R3)NH(R3’); polyethyleneoxy unit of formula (OCH2CH2)pOR3, or (OCH2CH(CH3))pOR3, or NH(CH2CH2O)pR3, or NH(CH2CH(CH3)O)pR3, or N[(CH2CH2O)pR3][ (CH2CH2O)p’R3’], or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; C1-C8 of alkyl; C2-C8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; wherein R3 and R3’ are independently H; C1-C8 of alkyl; C2-C8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 of aryl, Ar-alkyl, cyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or C1-C8 esters, ether, or amide; or 1~8 amino M AR KED-UP acids; or polyethyleneoxy unit of formula (OCH2CH 2)p or (OCH2CH(CH 3)) p, wherein p is an integer from 0 to about 1000, or combination f; and m1 and m2 are ndently an integer from 1 to 30.
2. The compound of formula (II) of claim 1, wherein the Drug1 is independently selected from the group consisting of: (1) a chemotherapeutic agent: a) an alkylating agent: including nitrogen mustards: chlorambucil, chlornaphazine, cyclophosphamide , dacarbazine, estramustine, ifosfamide, rethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, novembichin , phenesterine, prednimustine, thiotepa, famide, uracil mustard; CC-1065, or its adozelesin, esin and bizelesin synthetic analogues; duocarmycins, or its synthetic analogues , including 9 and CBI-TMI; benzodiazepine dimers, including dimers of pyrrolobenzodiazepine (PBD); or tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines , or oxazolidinobenzodiazepines; nitrosoureas, including carmustine, lomustine, chlorozotocin , fotemustine, nimustine, stine; alkylsulphonates, inclduingbusulfan, treosulfan, improsulfan and piposulfan; triazenes, including dacarbazine; Platinum containing compounds, including latin, cisplatin, oxaliplatin; aziridines, including carboquone; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaor-amide and trimethylolomelamine; b) a plant alkaloid: including vinca alkaloids including vincristine, vinblastine, vindesine, vinorelbine, in; taxoids including paclitaxel, docetaxol, and their analogs; Maytansinoids including DM1, DM2, DM3, DM4, DM5, DM6, DM7, sine and ansamitocins and their analogs; cryptophycins, particularly cryptophycin 1 and cryptophycin 8; epothilones, eleutherobin, discodermolide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c) a DNA omerase inhibitor: including epipodophyllins, 9-aminocamptothecin, camptothecin, tol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000); mitomycins, including mitomycin C; d) an antimetabolite: including {[anti-folate: DHFR tors: (methotrexate, trimetrexate, erin, pteropterin, aminopterin (4-aminopteroic acid) or the other folic acid ues); IMP dehydrogenase tors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors: yurea, deferoxamine; pyrimidine analogs: uracil M AR KED-UP analogs: ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil, floxuridine, exed (Tomudex; cytosine s: cytarabine, cytosine arabinoside, fludarabine; purine analogs:(azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine; folic acid replenisher, including frolinic acid; e) a hormonal therapies: receptor antagonists: anti-estrogen: megestrol, raloxifene, tamoxifen; LHRH ts: goscrclin, lide acetate; anti-androgens: tamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane and other androgens inhibitors; retinoids/deltoids: Vitamin D3 analogs: CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol; ynamic therapies: verteporfin, ocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; cytokines: interferon-alpha, interferon-gamma, tumor necrosis factor , human ns containing a TNF ; f) a kinase inhibitor, including BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, anib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), nib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, ib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab, ispinesib; g) antibiotic, enediyne: calicheamicins, especially calicheamicin .γ1, δ1, α1 and β1; dynemicin, including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, zinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores, aclacinomysins, actinomycin, authramycin, ine, cins, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazooxo-L-norleucine, doxorubicin, lino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, icin, idarubicin, lomycin, nitomycins, mycophe-nolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; h) polyketides acetogenins, bullatacin and bullatacinone; gemcitabine, epoxomicins, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA- 9090, stimuvax, allovectin-7, xegeva, provenge, yervoy, isoprenylation inhibitors,dopaminergic neurotoxins, cell cycle inhibitors, staurosporine, actinomycins, actinomycin D, dactinomycin, amanitins, bleomycins, bleomycin A2, bleomycin B2, peplomycin, anthracyclines, M AR KED-UP daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDR inhibitors, verapamil, Ca2+ ATPase inhibitors, thapsigargin, histone deacetylase inhibitors stat, romidepsin, panobinostat, ic acid, mocetinostat (MGCD0103), belinostat, PCI-24781, entinostat, SB939, resminostat, givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A) ; thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A.; anti-adrenals aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; uone; eflornithine (DFMO), elfomithine; elliptinium acetate, etoglucid; gallium nitrate; gacytosine, yurea; ibandronate, lentinan; lonidamine; mitoguazone; mitoxantrone; mol; nitracrine; pentostatin; et; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; quone; 2, 2',2''-trichlorotriethylamine; trichothecenes, especially T-2 toxin, verrucarin A, roridin A and anguidine; urethane, siRNA, antisense drugs; (2) anti-autoimmune disease agent: cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, riptine, chlorambucil, chloroquine, hosphamide, corticosteroids (including amcinonide, betamethasone, budesonide, hydrocortisone, olide, asone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, sone, sirolimus, tacrolimus; (3) anti-infectious disease agent, a) aminoglycosides: amikacin, astromicin, gentamicin, netilmicin, sisomicin, isepamicin, hygromycin B, kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, ycin, neomycin, framycetin, paromomycin, amycin, netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin; b) amphenicols: azidamfenicol, chloramphenicol, florfenicol, thiamphenicol; c) snsamycins: geldanamycin, herbimycin; d) enems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephems: carbacephem (loracarbef), cefacetrile, or, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, flur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, razone, ceforanide, M AR KED-UP cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f) glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), anin; g) glycylcyclines: tigecycline; h) β-lactamase inhibitors: penam, sulbactam, tazobactam, clavam, clavulanic acid; i) lincosamides: clindamycin, lincomycin; j) lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k) macrolides: azithromycin, cethromycin, hromycin, romycin, erythromycin, flurithromycin, josamycin, ketolide, telithromycin, cethromycin, midecamycin, miocamycin, oleandomycin, cins, rifampicin, rifampin, rifabutin, rifapentine, mycin, roxithromycin, spectinomycin, ycin, tacrolimus (FK506), troleandomycin, telithromycin; l) monobac-tams: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) llins: illin, ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin, carindacillin, cloxacillin, dicloxacillin, epicillin, flucloxacillin, inam (pivmecillinam), mezlocillin, meticillin, nafcillin, lin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o) polypeptides: bacitracin, colistin, polymyxin B; p) quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, loxacin, danofloxacin, acin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, oxacin, nadifloxacin, norfloxacin, oxacin, ofloxacin, acin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) streptogramins: namycin, quinupristin/dalfopristin); r) sulfonamides: mafenide, prontosil, sulfacetamide, ethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole); s) steroid antibacterials: fusidic acid; M AR KED-UP t) tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, cline, ycline, cline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines, tigecycline; u) antibiotics: annonacin, arsphenamine, bactoprenol inhibitors, Bacitracin, DADAL/AR tors, cycloserine, dictyostatin, discodermolide, erobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, idazole, mupirocin, mycolactone, NAM synthesis inhibitors, fosfomycin, nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin; (4) an anti-viral drug: a) entry/fusion inhibitors: aplaviroc, maraviroc, iroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b) integrase inhibitors: ravir, elvitegravir, globoidnan A; c) maturation inhibitors: bevirimat, vivecon; d) inidase inhibitors: oseltamivir, vir, peramivir; e) nucleosides & nucleotides: abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3’-fluoro-substituted 2’, 3’- dideoxynucleoside analogues (including 3’-fluoro-2’,3’-dideoxythymidine (FLT) and 3’-fluoro- 2’,3’-dideoxyguanosine (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), lnucleosides (including β-l-thymidine and β-l-2’-deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), udine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, enz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g) protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, vir, nelfinavir, pleconaril, vir, saquinavir, telaprevir (VX-950), tipranavir; h) other types of anti-virus drugs: abzyme, arbidol, calanolide a, nin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet, thsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib; M AR KED-UP (5) a radioisotope that can be selected from uclides including 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Ga, 86Y, 99Tc, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 213Bi; (6) a chromophore le, which can be one that has the ability to absorb a kind of light, including UV light, florescent light, IR light, near IR light, visual light; A class or subclass of xanthophores, erythrophores, iridophores, leucophores, melanophores, cyanophores, fluorophore molecules which are fluorescent chemical compounds re-emitting light upon light, visual phototransduction molecules, photophore molecules, luminescence molecules, luciferin compounds; Non-protein organic fluorophores, including: Xanthene tives: fluorescein, rhodamine, eosin; cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine; ine derivatives and ring-substituted ines: Seta, SeTau, and Square dyes; naphthalene derivatives, dansyl and prodan derivatives; coumarin derivatives; oxadiazole derivatives: loxazole, enzoxadiazole and benzoxadiazole; anthracene derivatives, quinones; pyrene derivatives: cascade blue; oxazine derivatives, acridine derivatives: proflavin, acridine orange, acridine yellow, arylmethine derivatives: auramine, crystal violet, ite green, tetrapyrrole derivatives: n, phthalocyanine, bilirubin; (7) a cell-binding ligand or receptor agonist, which can be selected from: folate derivatives; glutamic acid urea derivatives; somatostatin and its analogs: octreotide, Sandostatin and lanreotide. Somatuline; aromatic sulfonamides; pituitary adenylate e activating peptides; vasoactive intestinal peptides VIP/PACAP and VPAC1, VPAC2; melanocyte-stimulating hormones (α-MSH); cholecystokinins (CCK) /gastrin receptor agonists; bombesins /gastrinreleasing peptide (GRP); neurotensin receptor ligands (NTR1, NTR2, NTR3); substance P (NK1 receptor) ligands; neuropeptide Y (Y1–Y6); homing es include RGD: Arg-Gly- Asp, NGR: Asn-Gly-Arg, the dimeric and multimeric cyclic RGD es, TAASGVRSMH and LTLRWVGLMS and F3 peptides; cell penetrating pe ptides; peptide hormones: luteinizing hormone-releasing hormone (LHRH) ts and antagonists, and gonadotropin-releasing hormone (GnRH) agonist, acts by targeting follicle ating hormone (FSH) and luteinising hormone (LH), testosterone production: buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg- Pro-NHEt), gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), goserelin (Pyr-His- Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N- benzyl)-Leu-Arg-Pro-NHEt), lide (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), triptorelin (Pyr-His-Trp-Ser-Tyr- D-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, deslorelin, abarelix (Ac-D-2Nal-DchloroPhe-D (3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-NH2), cetrorelix (Ac-D- M AR KED-UP 2Nal-DchloroPhe-D(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix 2Nal-DchloroPhe-D(3-pyridyl)Ala-SeraminoPhe(L-hydroorotyl)-D aminoPhe(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH2), and ganirelix (Ac-D-2Nal-D chloroPhe-D(3-pyridyl)Ala-Ser-Tyr-D-(N9, N10-diethyl)-homoArg-Leu-(N9, N10-diethyl)- homoArg-Pro-D-Ala-NH2); pattern recognition receptor (PRRs): Toll-like receptors’ (TLRs) ligands, C-type lectins and nodlike receptors’ (NLRs) ligands; calcitonin receptor agonists; integrin receptors’ and their receptor subtypes’ (including αVβ1, αVβ3, αVβ5, αVβ6, α6β4, α7β1, αLβ2, αIIbβ3) agonists: GRGDSPK, cyclo(RGDfV) (L1) and its tives [cyclo(-N(Me)R-GDfV), R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)f-V), RGDf-N(Me)V- )(Cilengitide); nanobody (a derivative of VHH (camelid Ig)); Domain antibodies (dAb, a derivative of VH or VL domain); ific T cell Engager (BiTE, a bispecific diabody); Dual Affinity ReTargeting (DART, a bispecific diabody); Tetravalent tandem antibodies (TandAb, a dimerized bispecific diabody); Anticalin (a derivative of Lipocalins); Adnectins (10th FN3 (Fibronectin)); Designed Ankyrin Repeat ns (DARPins); Avimers; EGF receptors and VEGF receptors’ agonists; or (8) a pharmaceutically acceptable salt, acid or derivative of any of the above drugs.
3. The compound of formula (II) of claim 1, wherein the Drug1 is a chromophore molecule.
4. The compound of Formula (II) of claim 1, wherein the Drug1 is a polyalkylene glycol or a polyalkylene glycol analog; wherein the polyalkylene glycol is a poly(ethylene s) (PEGs), poly(propylene ), or a copolymer of ne oxides or ene oxides that has a molecular weight of from about 44 Daltons to about 300 kDa.
5. The nd of Formula (II) of claim 1, wherein the Drug1 is a cell-binding ligand or a cell receptor agonist, or a cell receptor binding m olecule.
6. The compound of Formula (II) of claim 1, wherein the Drug1 is selected from tubulysins, calicheamicins, auristatins, maytansinoids, CC-1065 analogs, daunorubicin and doxorubicin compounds, taxanoids (taxanes), cryptophycins, epothilones, benzodiazepine dimers, including dimers of pyrrolobenzodiazepine (PBD), ycins, anthramycins, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiaze-pines), calicheamicins and the enediyne antibiotics, actinomycins, amanitins, azaserines, bleomycins, epirubicins, tamoxifen, idarubicin, dolastatins/auristatins (including thyl auristatin E, MMAE , MMAF, auristatin PYE, M AR KED-UP atin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, geldanamycins, methotrexates, thiotepa, vindesines, vincristines, hemiasterlins, nazumamides, microginins, radiosumins, alterobactins, microsclerodermins, theonellamides, esperamicins, siRNA, nucleolytic enzymes, and/or pharmaceutically acceptable salts, acids, or/and their analogues tives of any of the above molecules.
7. The compound according to claim 1 having the Formula 40, 73, 79, 87, 252, 267, 353, 355, 357, 369, 376, 384, 388, 406, 409, 411, 413, or 418 as shown in below: M AR KED-UP R 9 OAc .OAc O O n O N N NH I H I '-A H ° o^SK y^pii C02Me °Wh ^1 ^ ^ ' o ^ I 0^0 ^O O C02Me '1^0't?>'0H H O 23 357 H O OAc OH /? H O i \'v‘ ho2c H02C*h^01^g '0 H O OAc q N,. U .N n 0 co2h N N
NZ744940A 2016-02-04 Conjugation linkers, antibody-drug conjugates thereof, and methods of synthesis and use of such conjugates NZ744940B2 (en)

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