Bridged pyrrolobenzodiazepine dimer (PBD) derivatives and their conjugates

Abstract

To provide antibody-drug conjugates (ADCs) that show superior antitumor antibiotic activity in clinical applications.SOLUTION: The present invention relates to novel cross-linked cytotoxic agents, pyrrolobenzo-diazepine dimer (PBD) derivatives, and their conjugates to cell-binding molecules, methods for preparation of the conjugates and the therapeutic use of the conjugates.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to novel cross-linked cytotoxic agents, pyrrolobenzodiazepine dimer (PBD) derivatives and their conjugates with cell-binding molecules, methods for preparing the conjugates, and therapeutic uses of the conjugates. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have shown great promise as anticancer agents, and four ADCs have been approved by the FDA: maytansine (Non-Patent Documents 1 and 2); auristatin and its parent compound dolastatin (Non-Patent Documents 3 and 4); and calicheamicin (Non-Patent Documents 5 and 6). Documents 5 and 6); duocarmycin and its analog CC-1065 (Non-Patent Documents 7 and 8); tubulysin (Non-Patent Documents 9-11 and Patent Document 1); eribulin (Patent Document 2); camptothecin and its analog SN-38 (Non-Patent Documents 12-14 and Patent Documents 3 and 4); amanitin (Non-Patent Document 15 and Patent Document 5); vinblastine (Non-Patent Document 1 6 and 17); doxorubicin (Non-patent Documents 18-20); cryptophycin (Non-patent Documents More than 100 ADCs using several different classes of cytotoxic payloads are in clinical trials, including eribulin (Non-Patent Document 21); eribulin (Non-Patent Document 22 and Patent Document 2); and pyrrolobenzodiazepine dimers (PBDs) (Non-Patent Documents 23 and 24). In recent years, the extreme potency of PBDs against most of the National Cancer Institute's (NCI) panel of NCI60 tumor cell lines and their unique mechanism of antitumor antibiotic activity (Non-Patent Document 23) have led to an increasing use of PBD-class cytotoxic payloads as ADC therapeutics (Non-Patent Documents 25-29 and Patent Document 6).

[0003] It is generally believed that ADCs are stable during circulation, limiting off-target toxicity, and that the drug will be released upon entry into target cancer cells. Therefore, all PBDs are usually conjugated to antibodies with conditionally stable linkers, as shown in the following structure (Non-Patent Document 30). Unfortunately, the most clinically advanced Vadastuxi-mab taliline (a CD33 antibody-PBD conjugate with a conditionally stable linker) (Non-Patent Document 31) was thwarted by the early deaths of four patients due to liver toxicity during clinical trials (Non-Patent Document 32).

[0004] [ka] TIFF0007811398000002.tif215150TIFF0007811398000003.tif82143 [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2014 / 009774 [Patent Document 2] US Patent Application Publication No. 2017 / 0252458 [Patent Document 3] International patent application PCT / JP2013 / 006069 [Patent Document 4] US Patent Application Publication No. 2016 / 0333112 [Patent Document 5] International patent application PCT / IB2016 / 052246 [Patent Document 6] International Publication No. WO2015 / 028850 [Non-patent literature]

[0006] [Non-Patent Document 1] Zhao, Robert Y, et al., 2011 J. Med. Chem. 54, 3606

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Summary of the Invention

発明の概要

Summary of the Invention

Summary of the Invention

非特許文献26

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[0007] Here, we disclose conjugates of PBD dimer derivatives via a dual cross-linker attached to both N10 positions of the PBD dimer. Thus, the DNA alkylation sites of the imine reactive groups regained their potency in the form of prodrugs, which required cleavage of the conjugate linker prior to conversion. Indeed, application of the dual cross-linker prodrug strategy to PBD conjugates demonstrated a much broader therapeutic window than single-linked PBD conjugates both in vitro and in vivo. Therefore, PBD conjugates may exhibit significantly superior antitumor antibiotic activity in clinical applications. [Means for solving the problem]

[0008] A first embodiment of the present invention discloses a double-bridged conjugate of a pyrrolo[2,1-c][1,4]benzodiazepine derivative via connections between both N10 positions of the PBD and a cell-binding molecule, as shown in formula (I), or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure thereof, or an optical isomer, racemate, diastereomer or enantiomer of these compounds, for targeted therapy of cell proliferation.

[0009] [ka]

[0010] During the ceremony: [ka] optionally represents a single bond or may be absent.

[0011] [ka] optionally represents a single or double bond.

[0012] wherein V and V' are the same or different, H, OH, -NHOH; OR (ether); OCOR (ester); OCOOR (carbonate); NR5R5', NR5COR5', or NR5NR5'NR5'' (amine); OCONR5R5' (carbamate); NR5(C=NH)NR5'R5'' (guanidinium); NR5CONR5'R5'' (urea); OCSNHR5 (thiocarbamate); -SH (thiol); -SR5 (sulfide); SOR5 sulfoxide (sulfoxide); SOOR5 (sulfone); SO3, HSO3, HSO2, or HSO 3- , SO3 2- , or -HSO2 - salts (sulfites); OSO3 (bisulfites); NR5SOOR5' (sulfonamides); H2S2O5 or S2O5 2- Salts of (metabisulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2, or PO3S 3- , PO2S2 3- ,POS3 3- , PS4 3- Salts of (mono-, di-, tri-, and tetra-thiophosphates); (R5O)2POSR5' (thiophosphate esters); HS2O3 or S2O3 2- Salts of HS2O4 or S2O4 (thiosulfates) 2- Salts of (dithionous acid); P(=S)(OR5)(S)(OH) (phosphorodithioates) or salts thereof formed with cations; -NR5OR5' (hydroxylamine derivatives); R5C(=O)NOH (hydroxamic acid) or salts formed with cations; HOCH2SO2 - or its salts (formaldehyde sulfoxylate); NR5COR5' (amide); O-glycoside; N3 (azide); CN (cyano); X (halo: F, Cl, Br, or I); C(R5)(R5')(R5'') (trialkyl), OP(O)(OR5)(NHR5') or OP(O)(NHR5)(NHR5') (phosphoramidate (phosphoramidic acid), or P(R5)(R5')(R5'') triarylphosphonium; Aa (amino acid) or NR5CO(Aa) t(peptide), wherein Aa is an amino acid or a polypeptide comprising t=1 to 100 amino acid units; a group derived from an amino acid: an α-, β-, γ-, or ω-amino acid, or an unnatural amino acid; wherein R5, R5', and R5'' are as shown below.

[0013] l, m, q, l', m', and q' are independently 0, 1, 2, 3, 4, or 5; and n is 1 to 30.

[0014] In the formula, X, X', Y, and Y' independently represent the same or different N, O, S, or alkyl such as CH2 or CHR5, alkene such as =CH- or =CR5-, or ether such as -C(OR5)H.

[0015] In the formula, Z and Z' are independently the same or different N, CH, CR5, COH, CNH2 , CNHR5, or COR5, or Z and Z' are linked to form -COR5OC-, where R5 is independently selected from C1-C8 alkyl and aryl;

[0016] wherein R1, R2, R3, R4, R1', R2', R3', and R4' are independently the same or different and are -H, substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms -(OCH2CH2). t R5 (polyethylene glycol unit), halogen, NH(C=NH)NH2 (guanidinium), -OR5, -NR5R5', -NO2, -NCO, -NR5COR5', -SR5, -SOR5 (sulfur oxide), -SO2R5 (sulfone), -SO3 - M + or -SO3H (sulfonate), -OSO3 - M +or OSO3H (sulfate), -SO2NR5R5' (sulfonamide), CN (cyano), N3 (azide), -COR5, -OCOR5, -OCONR5R5', CF3, OR5, aryl, heterocycle, P(O)R5R5'R5'' and a linking group (L'') having a reactive group or a cell binding agent that binds thereto when Q, Q', and T are not present.

[0017] wherein R5, R5', and R5'' are independently selected from H, C1-C8 alkyl, alkenyl, alkyl, heteroalkyl, aryl, arylalkyl, carbonyl, or a pharmaceutical salt.

[0018] Furthermore, R1 and R2 or R1' and R2' are bonded to each other to form a double bond containing a =O (ketone), =S, =NR, -C(=O)R, or =CR5R5' group; and R1 and R2, R1' and R2', R3 and R4, or R3' and R4' are bonded to form a C3-C 12 It forms an aromatic, heterocyclic, carbocyclic, or heteroaryl ring.

[0019] In the formula, G is -CH2-, O, -N(R5)-, S, -P(O)(OR5)-, -P(O)(NR5R5')-, [ka] where Z and Z' are as defined above.

[0020] In the formula, U and U' are independently C(O), C(O)O, C(O)NH, C(O)N(R5), C(=NH), C(=NH)O, C(=NH), NH, C(=NH)N(R5), -C=N-, C(=S) , C(O)S, C(S)NH, C(S)N(R5), S(O), S(O)O, S(O)NH, S(O)(OR5), S(O)(N(R5)), S(O2), S(O2)O, P(O)(OR5), P(O )(OR5)O, P(O)(NH2), P(O)(NR5R5'), P(O)(OR5)NH-, P(O)(OR5)NR5'-, P(O)(N(R5R5')(N(R5), P(S)(OR5), P (S)(OR5)O, P(S)(NH2), P(S)(NR5R5'), P(S)(OR5)NH-, P(S)(OR5)NR5'-, P(S)(N(R5R5')N(R5), R5, R5O.

[0021] In the formula, E1 and E2 are independently S, R5S, C(O)S, C(O)NH, C(O)O, C(O)R5S, C(=NH)NH, C(=NH)N(R5), C(= NH)S, -C=N-, C(=S)S, C(O)S, C(=S)NH, C(=S)N(R5), Ar-S, NC(O)CH2S, ArC(O)CH2S, SS, [ka] where the chemical bond in the middle of the two atoms means that it can connect to either of the two adjacent atoms.

[0022] wherein L1 and L2 are independently a linker or a linker having a functional group capable of reacting with a cell-binding agent (CBA), Q. Preferably, L1, L2, and L' are independently releasable linkers having the formula -Ww-(Aa)r-Tt-, -Ww-(Aa)r-Tt-Q, or Q-Ww-(Aa)r-Tt. wherein -W- is an extender unit; w is 0 or 1; -Aa- is an independent amino acid unit; r is independently an integer between 0 and 100; -T- is a spacer unit and can be a linear or branched alkyl, or a polyethylene glycol spacer; and t is 0 or 1 to 100. The extender units W may independently comprise a self-immolative spacer, a peptide unit, a hydrazone bond, a disulfide bond, an ester bond, or a thioether bond; w is 1, 2, or 3; preferably, L1 and L2 are independently O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3'), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, a group of the formula (OCH2CH2) p OR3, (OCH2CH(CH3))pOR3, NH(CH2CH2O) p R3, NH(CH2CH(CH3)O) p R3, N[(CH2CH2O) p R3][(CH2CH2O) p’ R3'], (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR3 polyethyleneoxy units, where p and p' are independently from 0 to about 1000, or a combination thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or (Aa) r, r=1-12 (1 to 12 amino acid units), and are independently selected from dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units composed of natural or unnatural amino acids, or the same or different.

[0023] wherein Q is a functional group that allows reaction with a cell-binding molecule (CBA), or a cell-binding agent, or a functional group that can react with a linker attached to a cell-binding agent. The functional group is selected from thiol, amine, hydrazine, alkoxylamino, disulfide substituent, maleimide, haloacetyl group, N-hydroxysuccinimide ester, ketone, ester, aldehyde, alkynyl, alkenyl, or protected thiol or disulfide group, SAc, SSR1, or SSAr, where Ar is an aromatic or heteroaromatic group. The cell-binding agent / molecule can be an antibody, a single-chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a monoclonal antibody fragment that binds to a single-chain monoclonal antibody, or the like. The antibody is selected from a binding peptide, protein, or small molecule attached to a target cell, a target cell, a chimeric antibody, a chimeric antibody fragment that binds to a target cell, a domain antibody, a domain antibody fragment that binds to a target cell, an adnectin that mimics an antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule (transferrin), and an albumin, a polymer, a dendrite, a liposome, a nanoparticle, a vesicle, or a (viral) capsid.

[0024] In a second embodiment, the present invention discloses monoconjugates of PBD derivatives and cell-binding molecules for targeted treatment of cell proliferation, as shown in formulas (II), (III), and (IV). [ka]

[0025] During the ceremony, [ka] , X, X', Y, Y', Z, Z', l, l', m, m', n, q, q', R1, R1', R2, R2', R3, R3', R4, R4', V, V', U, U', L1, L2, G, Q, E1, and E2 have the same definitions as in formula (I).

[0026] In a third embodiment, the present invention provides: (1) a therapeutically effective amount of a compound having a conjugate structure represented by formula (I), pyrrolo[2, (1-c) [1,4] benzodiazepine derivatives; and (2) a pharmaceutically acceptable carrier. Therapeutic compositions comprising the compound, a diluent or excipient, a target cell or the application of formulas (I)-(IV) for killing a target cell are disclosed. [Brief explanation of the drawings]

[0027] [Figure 1] 1 shows the synthesis of a bridged benzodiazepine dimer. [Figure 2] 1 shows the synthesis of antibody conjugates of bridged benzodiazepine dimers. [Figure 3] 1 shows the synthesis of a bridged benzodiazepine dimer. [Figure 4] 1 shows the synthesis of bridged benzodiazepine dimers and conjugates. [Figure 5] 1 shows the synthesis of antibody conjugates of bridged benzodiazepine dimers. [Figure 6] 1 shows the synthesis of antibody conjugates of bridged benzodiazepine dimers. [Figure 7] The synthesis of benzodiazepine dimers is shown. [Figure 8] 1 shows the synthesis of bridged benzodiazepine dimers and conjugates. [Figure 9] The synthesis of benzodiazepine dimers is shown. [Figure 10] 1 shows the synthesis of antibody conjugates of benzodiazepine dimers. [Figure 11] The synthesis of benzodiazepine dimers is shown. [Figure 12] The synthesis of a benzodiazepine dimer and its conjugate with a linker is shown. [Figure 13] 1 shows the synthesis of a crosslinker for a benzodiazepine dimer conjugate. [Figure 14] 1 shows the synthesis of a crosslinker for a benzodiazepine dimer conjugate. [Figure 15] 1 shows the synthesis of a crosslinker for a benzodiazepine dimer conjugate. [Figure 16] 1 shows the synthesis of a bridged benzodiazepine dimer conjugate. [Figure 17] 1 shows the synthesis of an intermediate benzodiazepine dimer conjugate. [Figure 18] 1 shows the synthesis of a bridged benzodiazepine dimer conjugate. [Figure 19] 1 shows the synthesis of a bridged benzodiazepine dimer conjugate. [Figure 20] 1 shows the synthesis of a bridged benzodiazepine dimer conjugate. [Figure 21] 1 shows the synthesis of bridged benzodiazepine dimer conjugates. [Figure 22] 1 shows the synthesis of an intermediate benzodiazepine dimer conjugate. [Figure 23] 1 shows the synthesis of a bridged benzodiazepine dimer. [Figure 24] 1 shows the synthesis of a bridged benzodiazepine dimer conjugate. [Figure 25] 1 shows the synthesis of a bridged benzodiazepine dimer. [Figure 26] 1 shows the synthesis of a bridged benzodiazepine dimer. [Figure 27] 1 shows the synthesis of a bridged benzodiazepine dimer. [Figure 28] 1 shows the synthesis of an intermediate benzodiazepine dimer conjugate. [Figure 29] 1 shows the synthesis of a bridged benzodiazepine dimer conjugate. [Figure 30] 1 shows the synthesis of an intermediate benzodiazepine dimer conjugate. [Figure 31]1 shows the synthesis of an intermediate benzodiazepine dimer conjugate. [Figure 32] 1 shows the synthesis of benzodiazepine dimer conjugates. [Figure 33] Figure 1 shows the in vivo activity of bridged benzodiazepine dimer conjugates. [Figure 34] Hematoxylin and eosin staining of mouse liver tissues is shown on day 5 after administration of conjugates CC-4, CC-29, T-DM1, and PBS buffer to ICR mice. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition

[0029] "Alkyl" refers to an aliphatic hydrocarbon group or monovalent group derived from an alkane by removing one or two hydrogen atoms from a carbon atom. It may be straight-chain or branched, with C1-C8 (1 to 8 carbon atoms) in the chain. "Branched" refers to a straight-chain alkyl group to which one or more lower carbon number alkyl groups, such as methyl, ethyl, or propyl groups, are attached. Specific examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, and propyl. C1-C8 alkyl groups include butyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. The C1-C8 alkyl group can be unsubstituted or substituted with one or more substituents, including but not limited to the following: Examples of the substituent include -C1-C8 alkyl, -O-(C1-C8 alkyl), aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, wherein each R' is independently selected from C1-C8 alkyl and aryl.

[0030] "Halogen" refers to fluorine, chlorine, bromine, or iodine atoms, with fluorine and chlorine atoms being preferred.

[0031] "Heteroalkyl" refers to a C2-C8 alkyl in which one to four carbon atoms are independently replaced by a heteroatom selected from the group consisting of O, S, and N.

[0032] "Carbocycle" refers to a saturated or unsaturated monocyclic ring containing 3 to 8 carbon atoms or a bicyclic ring containing 7 to 13 carbon atoms. Monocyclic carbocycles have 3 to 6, more typically 5 or 6, ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms and are arranged as a [4,5], [5,5], [5,6], or [6,6] bicycle, or 9 to 10 ring atoms and are arranged as a [5,6] or [6,6] bicycle. Representative C3-C8 carbocycles include, 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.

[0033] A C3-C8 carbocycle refers to a saturated or unsaturated non-aromatic hydrocarbon carbocyclic compound having 3, 4, 5, 6, 7, or 8 carbon atoms. A C3-C8 carbocycle can be unsubstituted or substituted with one or more substituents, including, but not limited to, -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)R', -S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where each R' is independently selected from C1-C8 alkyl and aryl.

[0034] "Alkenyl" refers to an aliphatic hydrocarbon group that may be straight or branched and has 2 to 8 carbon atoms in the chain and contains a carbon-carbon double bond. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, and octenyl.

[0035] "Alkynyl" refers to an aliphatic hydrocarbon group that may be straight or branched and has 2 to 8 carbon atoms in the chain and contains a carbon-carbon triple bond. Alkynyl groups include, for example, ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n- Includes pentynyl, hexynyl, heptynyl, and octynyl.

[0036] "Alkylene" refers to a saturated, straight or branched chain or cyclic hydrocarbon group of 1 to 18 carbon atoms having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH-), 1,2-ethyl (-CHCH-), 1,3-propyl (-CHCHCH-), 1,4-butyl (-CHCHCHCH-), and the like.

[0037] "Alkenylene" refers to an unsaturated, straight or branched chain or cyclic hydrocarbon group of 2 to 18 carbon atoms 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 groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0038] "Alkynylene" refers to an unsaturated, straight or branched chain or cyclic hydrocarbon group of 2 to 18 carbon atoms 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 groups include, but are not limited to, acetylene, propargyl, and 4-pentynyl.

[0039] "Aryl" or Ar refers to an aromatic or heteroaromatic group consisting of one or several rings containing 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. The term "heteroaromatic group" refers to an aromatic group in which one or several carbons, preferably 1, 2, 3, or 4 carbon atoms, have been replaced by O, N, Si, Se, P, or S, preferably O, S, and N. The term aryl or Ar also refers to one or more H atoms independently replaced by -R', -halogen, -OR', or -SR', -NR'R'', -N=NR', -N=R', -NR'R'', -NO, -S(O)R', -S(O)R', -S(O)OR', -OS(O)OR', -PR'R'', -P(O)R'R'', -P(OR')(OR''), -P(O)(OR')(OR''), or -OP(O)(OR')(OR''), where R', R'' are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or a pharmaceutical salt.

[0040] "Heterocycle" refers to a ring system in which one to four ring carbon atoms are independently replaced with a heteroatom from the group O, N, S, Se, B, Si, and P. Preferred heteroatoms are O, N, and S. Heterocycles are described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, pp. 225-226, and the disclosure thereof. are incorporated herein by reference. Preferred non-aromatic heterocycles include, but are not limited to, epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as fused systems resulting from condensation with a phenyl group.

[0041] The term "heteroaryl" or aromatic heterocycle refers to a 5- to 14-membered, preferably 5- to 10-membered, aromatic heterocyclic, monocyclic, bicyclic, or polycyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl ... Included are pyrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, and fused systems resulting from condensation with a phenyl group.

[0042] "Alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," "heterocyclic," and the like, also refer to the corresponding "alkylene," "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," "heterocyclene," and the like, formed by removing two hydrogen atoms.

[0043] "Arylalkyl" refers to an alkyl group consisting of a carbon atom, typically a terminal or sp 3 It refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, etc.

[0044] "Heteroarylalkyl" refers to a heteroaryl group consisting of a carbon atom, typically a terminal or sp 3 It refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like.

[0045] Examples of "hydroxy protecting groups" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate ester, pivaloate, benzoate, methanesulfonate, and p-toluenesulfonate.

[0046] "Leaving group" refers to a functional group that can be replaced by another functional group. Such leaving groups are well known in the art and include, but are not limited to, halides (e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. The leaving group is preferably selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate; acid anhydrides formed with themselves or other acid anhydrides (e.g., acetic anhydride, formic anhydride); or intermediates generated by condensation reagents for peptide coupling reactions or Mitsunobu reactions.

[0047] The following abbreviations may be used herein and have the definitions given below: Boc, tert-butoxycarbonyl; BroP, bromotrispirolizinophosphonium hexafluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane; DEAD, diethyl azodicarboxylate; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethylphosphoroanidinium; DMA, N,N-dimethyl Acetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; DTT, dithioester; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; EtOAc, ethyl acetate; Fmoc, N-(9-fluorenylmethoxycarbonyl); HATU, O-(7-azabenzotriazol-1-yl)-N,N,N'-N'-tetramethyl Fluoruronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high-pressure liquid chromatography; NHS, N-hydroxysuccinimide; MeCN, acetonitrile; MeOH, methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate-buffered saline (pH 7.0–7.5); Ph, phenyl; phe, L-phenylalanine; PyBrop, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; PEG, polyethylene glycol; SEC , size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine; TLC, thin layer chromatography; UV, ultraviolet.

[0048] "Amino acids" can be natural and / or unnatural amino acids, preferably α-amino acids. Natural amino acids are those encoded by the genetic code: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. Unnatural amino acids are derived forms of protein-forming amino acids, including, for example, hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (a neurotransmitter), ornithine, citrulline, β-alanine (3-aminopropanoic acid), γ-carboxyglutamate, selenocysteine ​​(present in most eukaryotes but not directly encoded by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formylmethionine (often the first amino acid in 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 that have the same general HN(R)CHCOH structure of a natural amino acid, except that the R group is not one found in a natural amino acid. Examples of analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Preferably, an amino acid mimetic is a compound that has a structure different from the general chemical structure of an α-amino acid, but functions similarly to it. The term "unnatural amino acid" is intended to refer to the "D" stereochemical form, whereas natural amino acids are in the "L" form. When 1 to 8 amino acids are used herein, the amino acid sequence is preferably a cleavage recognition sequence for a protease.Many cleavage recognition sequences are known in the art, for example, Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 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). See, e.g., the disclosure of which is incorporated herein by reference. In particular, the sequence may be selected from the group consisting of Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu. be selected.

[0049] A "glycoside" is a molecule in which a sugar group is linked to another group through its anomeric carbon by a glycosidic bond. Glycosides include O-(O-glycosides), N-(glycosylamines), and N-(glycosylamines). ), S-(thioglycoside), or C-(C-glycoside) glycosidic bonds. Its core empirical formula is C m (H2O) n(where m is different from n and m and n are <36), where glycosides include glucose (dextrose), fructose (levulose), allose, altrose, mannose, gulose, iodose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucuronic acid (glucuronide), and stachyose. It may be in the D- or L-form, in the five-atom cyclic furanose form, the six-atom cyclic pyranose form, or in the acyclic form, in the α-isomer (-OH at the anomeric carbon below the plane of the carbon atoms in the Howarth projection), or in the β-isomer (-OH at the anomeric carbon above the plane of the carbon atoms in the Howarth projection). It is used herein to refer to a monosaccharide, disaccharide, polyol, or oligosaccharide containing 3 to 6 sugar units.

[0050] The term "antibody," as used herein, refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, including, but not limited to, cancer cells or cell populations that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. Preferably, however, the immunoglobulins are of human, murine, or rabbit origin. Antibodies useful in the present invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized, or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab') fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs, and epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen.

[0051] Just as left and right hands are identical except that they are opposites along one axis (simply changing their orientation does not make them appear identical), "enantiomers," also known as "optical isomers," are one of two stereoisomers that are non-superimposable (non-identical) mirror images of each other. A single chiral atom or similar structural feature within a compound causes that compound to have two non-superimposable structures, each a mirror image of the other. The presence of multiple chiral elements in a particular compound increases the number of possible geometric forms, but perfect mirror-image pairs may exist. A pure enantiomer is a sample that, within the limits of detection, has only one chirality. When present in a symmetric environment, enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light (+ / -) by equal amounts in opposite directions (although polarized light can be considered an asymmetric medium). For this reason, they are sometimes called optical isomers. A mixture of equal parts of an optically active isomer and its enantiomer is called a racemate; it has zero net rotation of plane-polarized light. This is because the positive rotation of each (+) form is exactly offset by the negative rotation of the (-) form. Enantiomeric members often have different chemical reactions than other enantiomeric substances. Because many biological molecules are enantiomers, there can be significant differences in the effects of the two enantiomers on an organism. For example, in drugs, often only one of the drug's enantiomers is responsible for the desired physiological effect, while the other enantiomer may be less active, inactive, or even have adverse effects. This discovery allows the development of drugs composed of only one enantiomer ("enantiopure") to enhance pharmacological efficacy and sometimes eliminate some side effects.

[0052] Isotopes are variations of a particular chemical element that vary in the number of neutrons. All isotopes of a particular element have the same number of protons in each atom. Each atomic number identifies a particular element, but not an isotope. Atoms of a given element can have a wide range of neutron numbers. The number of nucleons (both protons and neutrons) in the nucleus is the atom's mass number, and each isotope of a particular element has a different mass number. For example, carbon-12, carbon-13, and carbon-14 are three isotopes of carbon with mass numbers 12, 13, and 14, respectively. Carbon has an atomic number of 6. This is because every carbon atom has six protons, so the neutron numbers of these isotopes are 6, 7, and 8, respectively. Hydrogen atoms have protium ( 1 H), deuterium ( 2 H), and tritium ( 3 There are three isotopes of hydrogen (H): deuterium has twice the mass of protium, and tritium has three times the mass of protium. Isotope substitution can be used to determine the mechanism of a chemical reaction or through kinetic isotope effects. Isotope substitution can be used to investigate how the body affects a particular xenobiotic / chemical through absorption and distribution mechanisms after administration, as well as the excretion pathways and effects of a drug's metabolites, as well as metabolic changes in the body (e.g., by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes). This study is called pharmacokinetics (PK). Isotope substitution can be used to study the biochemical and physiological effects of a drug. Effects can include those manifested in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). This study is called pharmacodynamics (PD). Effects can include those manifested in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). Together, both influence the administration, benefits, and adverse effects of a drug. Isotopes include elements that are stable (non-radioactive) or unstable. Isotopic substitutions of drugs may have different therapeutic effects than the original drug.

[0053] "Pharmaceutically" or "pharmaceutically acceptable" means that the corresponding compound or compound composition does not produce harmful, allergic or other adverse reactions when administered to animals or humans in a suitable manner.

[0054] A "pharmaceutically acceptable solvate" or "solvate" is disclosed as a compound containing one or more solvent molecules. Examples of solvents that form pharmacologically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0055] Pharmaceutically acceptable auxiliary materials include all carriers, diluents, auxiliaries, or excipients, such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coating agents, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, etc. In the pharmaceutical field, it is common practice to add these auxiliary materials to active drug components. It is considered reasonable to add auxiliary materials to drug components unless the auxiliary materials are incompatible with the active drug components. To achieve better results, active auxiliary materials may be added to drug components.

[0056] In the present invention, the term "pharmaceutical salt" refers to a salt derivative of the compound of the present invention. By appropriate modification, the compound of the present invention can be converted into a corresponding acid or alkali salt. Pharmaceutical salts include commonly used non-toxic salts or quaternary ammonium salts, which can be prepared with the compound of the present invention and a corresponding non-toxic inorganic or organic acid. For example, inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid. Organic acids include acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, and lactic acid, which can be used to form pharmaceutically acceptable salts. Other salts include ammonium salts of trometamol, meglumine, pyrroleethanol, etc., and salts of sodium, potassium, calcium, zinc, magnesium, etc. Contains metal salts of

[0057] In the present invention, pharmaceutical salts can be prepared from parent compounds containing acidic or basic residues by conventional chemical methods. Generally, these salts can be obtained by reacting the free acid or free base form of these compounds with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of the two. Generally, ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred as a non-aqueous reaction solvent. A list of suitable salts can be found in "Remington's Pharmaceutical Sciences," 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is incorporated by reference.

[0058] "Administering" or "Administration" refers to any manner of transferring, delivering, introducing, or carrying a pharmaceutical or other agent to a subject. Such manners include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, nasal, subcutaneous, or cavity administration. Also contemplated by the present invention is the use of a device or instrument in administering the agent. Such devices may utilize active or passive transport and may be slow-release or fast-release delivery devices.

[0059] "Therapeutically effective amount" means an amount of a compound / agent of the present invention effective to prevent or treat a pathological condition referred to herein.

[0060] The term "patient" or "patient in need thereof" is intended to refer to an animal or human suffering from or likely to suffer from a pathological condition referred to herein. Preferably, the patient is a human.

[0061] In the context of cancer, the term "treating" includes any or all of the following: inhibiting the growth of tumor or cancer cells, preventing tumor or cancer cell replication, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0062] In the context of autoimmune disease, the term "treating" includes any or all of the following: preventing the replication of cells associated with the pathology of the autoimmune disease, including but not limited to, cells capable of producing autoimmune antibodies, reducing the burden of autoimmune antibodies, and ameliorating one or more symptoms of the autoimmune disease.

[0063] In the context of infectious diseases, the term "treating" includes any or all of preventing the growth, proliferation, or replication of the pathogen causing the infectious disease and ameliorating one or more symptoms of the infectious disease.

[0064] Examples of "mammals" or "animals" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry.

[0065] The terms "compound," "cytotoxic agent," "cytotoxic compound," "cytotoxic dimer," and "cytotoxic dimeric compound" are used interchangeably. They include structures or formulas disclosed herein or any derivatives thereof, or structures or formulas incorporated by reference or any derivatives thereof. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts), and prodrugs, as well as prodrug salts, of compounds of all formulas disclosed herein. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The term "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," and the like in certain embodiments of the invention described herein are not intended to be limiting unless otherwise specified. The specific designation of "compound," "salt," "prodrug," "prodrug salt," "conjugate," "salt of conjugate," "solvate," "hydrate," or "polymorph" should not be construed as an intended omission of these other forms in other aspects of the invention where the term "compound" is used without recitation of these other forms.

[0066] The term "imine-reactive reagent" refers to a reagent that can react with an imine group. Examples of imine-reactive reagents include, but are not limited to, sulfite (HSO, HSO, or HSO formed with a cation),- , SO3 2- , or HSO2 - salts), metabisulfite (H2S2O5 or S2O formed with cations 52- salts), mono-, di-, tri-, and tetrathiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3, or PO3S formed with cations 3- , PO2S2 3- ,POS3 3- , or PS4 3- salts), thiophosphates ((RO)PS(OR), RSH, RSOOH, RSOH, RSOH), various amines (hydroxylamine (NHOH), hydrazine (NHNH), NHOR, RNHHR, NHR), NH-CO-NH, NH-C(=S)-NH), thiosulfates (HSO or S2O3 formed with cations 2- salts), dithionite (H2S2O4 or S2O4 formed with cations 2- salts), phosphorodithioates (P(=S)(OR5)(SH)(OH) or salts thereof formed with cations), hydroxamic acids (RC(=O)NHOH or salts thereof formed with cations), hydrazine (R5CONHNH2), formaldehyde sulfoxylate (HOCH2SO2H or HOCH2SO2 - Na + HOCH2SO2 formed with cations such as - or a salt thereof), a glycosylated nucleotide (GDP-mannose, etc.), fludarabine, or a mixture thereof, wherein R5 and R5' are each independently a straight-chain or branched alkyl having 1 to 8 carbon atoms, and are substituted with at least one substituent selected from -N(R5)(R5'), -CO2H, -SO3H, and -PO3H. R5 and R5' can be optionally further substituted with alkyl substituents as described herein. Preferably, the cation is Na + or K +Preferably, the imine-reactive reagent is selected from sulfite, hydroxylamine, urea, and hydrazine. More preferably, the imine-reactive reagent is NaHSO or KHSO.

[0067] "Cell-binding agents" or "cell-binding molecules" can be of any type currently known or discovered, including peptides and non-peptides. Generally, they can include antibodies (especially monoclonal antibodies) or antibody fragments containing at least one binding site, lymphokines, hormones, growth factors, nutrient transport molecules (such as transferrin), or any other cell-binding molecule or substance (such as vitamins).

[0068] More specific examples of cell binding agents can include: monoclonal antibodies; single chain antibodies; Fab, Fab', F(ab'), F v , {Parham, 131 J. Immunol. 2895-2902 (1983); Spring et al, 113 J. Immunol. 470-478 (1974); Nisonoff et al, 89 Arch. Biochem. Biophys. 230-244 (1960)}. , fragments obtained by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; interferons; peptides; lymphokines such as IL-2, IL-3, IL-4, and IL-6; hormones, e.g., insulin, TRH (thyrotropin-releasing hormone), MSH (myosinogen-stimulating hormone), steroid hormones (e.g., androgens and estrogens); growth factors and colony-stimulating factors, e.g., EGF, TGFα, insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF, and GM-CSF {Burgess, 5 Immunology Today 155-158 (1984)}; vitamins such as folic acid and transferrin {O ' Keefe et al, 260 J. Biol. Chem. 932-937 (1985)}.

[0069] Monoclonal antibody technology provides highly selective cell binding in the form of specific monoclonal antibodies. This allows for the production of antibodies. Techniques for generating monoclonal antibodies by immunizing mice, rats, hamsters, or other mammals with an antigen of interest, such as intact target cells, antigens isolated from target cells, whole viruses, weakened whole viruses, and viral proteins such as viral coat proteins, are particularly well known in the art. The selection of an appropriate cell-binding agent is a matter of choice that depends on the particular cell population to be targeted, but generally, monoclonal antibodies are preferred when suitable ones are available.

[0070] Cross-linked PBDs and their conjugates

[0071] The bridged PBD dimeric derivatives conjugated or capable of being conjugated to cell-binding molecules for targeted treatment of cell proliferation have the structure of formula (I) shown below, or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure of these compounds, or an optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0072] During the ceremony, [ka] optionally represents a single bond or may be absent, [ka] optionally represents a single or double bond.

[0073] V and V' are the same or different, H, OH, -NHOH; OR (ether); OCOR (ester); OCOOR (carbonate); NR, R, NR, COR, or NR, NR', NR'' (amine); OCONR, R (carbamate); NR(C=NH)NR', ​​R'' (guanidium); NR, CONR, R'' (urea); OCSNHR (thiocarbamate); -SH (thiol); -SR (sulfide); SO, S, S, O, or HSO. 3- , SO3 2- , or -HSO2 - salts (sulfites); OSO3 (bisulfites); NR5SOOR5' (sulfonamides); H2S2O5 or S2O5 2- Salts of (metabisulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2, or PO3S 3- , PO2S2 3- ,POS3 3- , PS4 3- Salts of (mono-, di-, tri-, and tetra-thiophosphates); (R5O)2POSR5' (thiophosphate esters); HS2O3 or S2O3 2- Salts of HS2O4 or S2O4 (thiosulfates) 2- Salts of (dithionous acid); P(=S)(OR5)(S)(OH) (phosphorodithioates) or salts thereof formed with cations; -NR5OR5' (hydroxylamine derivatives); R5C(=O)NOH (hydroxamic acid) or salts formed with cations; HOCH2SO2 - or its salts (formaldehyde sulfoxylate); NR5COR5' (amide); O-glycoside; N3 (azide); CN (cyano); X (halo: F, Cl, Br, or I); C(R5)(R5')(R5'') (trialkyl), OP(O)(OR5)(NHR5') or OP(O)(NHR5)(NHR5') (phosphoramidate (phosphoramidic acid), or P(R5)(R5')(R5'') triarylphosphonium; Aa (amino acid) or NR5CO(Aa) t(peptide), wherein Aa is an amino acid or a polypeptide comprising t=1 to 100 amino acid units; a group derived from an amino acid: an α-, β-, γ-, or ω-amino acid, or an unnatural amino acid; wherein R5, R5', and R5'' are as shown below.

[0074] l, m, q, l', m', and q' are independently 0, 1, 2, 3, 4, or 5; and n is 1 to 30.

[0075] X, X', Y, and Y' independently represent the same or different N, O, S, or alkyl such as CH2 or CHR5, alkene such as =CH- or =CR5-, or ether such as -C(OR5)H.

[0076] Z and Z' independently represent the same or different N, CH, CR5, COH, CNH2, CNHR5, or COR5, or Z and Z' join to form -COR5OC-, where R5 is independently selected from C1-C8 alkyl and aryl.

[0077] G is -CH2-, O, -N(R5)-, S,-P(O)(OR5)-, -P(O)(NR5R5')-, [ka] where Z and Z' are as defined above.

[0078] U and U' are independently C(O), C(O)O, C(O)NH, C(O)N(R5), C(=NH), C(=NH)O, C(=NH), NH, C(=NH)N(R5), -C=N-, C(=S), C (O)S, C(S)NH, C(S)N(R5), S(O), S(O)O, S(O)NH, S(O)(OR5), S(O)(N(R5)), S(O2), S(O2)O, P(O)(OR5), P(O) (OR5)O, P(O)(NH2), P(O)(NR5R5'), P(O)(OR5)NH-, P(O)(OR5)NR5'-, P(O)(N(R5R5')(N(R5), P(S)(OR5), P (S)(OR5)O, P(S)(NH2), P(S)(NR5R5'), P(S)(OR5)NH-, P(S)(OR5)NR5'-, P(S)(N(R5R5')N(R5), R5, R5O.

[0079] E1 and E2 are independently S, R5S, C(O)S, C(O)NH, C(O)O, C(O)R5S, C(=NH)NH, C(=NH)N(R5), C(=NH)S, -C=N- , C(=S)S, C(O)S, C(=S)NH, C(=S)N(R5), Ar-S, NC(O)CH2S, ArC(O)CH2S, SS, [ka] where the chemical bond in the middle of the two atoms means that it can connect to either of the two adjacent atoms.

[0080] L1 and L2 are independently a releasable linker or a linker having a functional group capable of reacting with a cell-binding agent (CBA). L1 and L2 independently have the formula -Ww-(Aa)r-Tt-, -Ww-(Aa)r-Tt-Q, or Q-Ww-(Aa)r-Tt, where -W- is an extender unit; w is 0 or 1; -Aa- is an independent amino acid unit; r is independently an integer between 0 and 100; -T- is a spacer unit and can be a linear or branched alkyl, or a polyethylene glycol spacer; and t is 0 or 1 to 100; the extender units W may independently comprise a self-immolative spacer, a peptide unit, a hydrazone bond, a disulfide bond, an ester bond, or a thioether bond; and w is 1, 2, or 3. Preferably, L1 and L2 are independently O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3'), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, the formula (OCH2CH2) p OR3, (OCH2CH(CH3))pOR3, NH(CH2CH2O) p R3, NH(CH2CH(CH3)O) p R3, N[(CH2CH2O) p R3][(CH2CH2O) p’ R3'], (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR3 polyethyleneoxy units, where p and p' are independently from 0 to about 1000, or a combination thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or (Aa) r , r=1-12 (1 to 12 amino acid units), and are independently selected from dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units composed of natural or unnatural amino acids, or the same or different.

[0081] R1, R2, R3, R4, R1', R2', R3', and R4' are independently the same or different and are -H, substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms -(OCH2CH2). t R5 (polyethylene glycol unit), halogen, NH(C=NH)NH2 (guanidinium), -OR5, -NR5R5', -NO2, -NCO, -NR5COR5', -SR5, -SOR5 (sulfoxy) (sulfone), -SO2R5 (sulfone), -SO3 - M + or -SO3H (sulfonate), - OSO3 - M + or OSO3H (sulfate), -SO2NR5R5' (sulfonamide), CN (cyano), N3 (azide), -COR5, -OCOR5, -OCONR5R5', CF3, OR5, aryl, heterocycle, or P(O)R5R5'R5'' and a linking group (L'') having a reactive group or a cell binding agent that binds thereto when Q, Q', and T are not present.

[0082] R5, R5', and R5'' are independently selected from H, C1-C8 alkyl, alkenyl, alkyl, heteroalkyl, aryl, arylalkyl, carbonyl, or a pharmaceutical salt;

[0083] Additionally, R1 and R2 or R1' and R2' are joined together to form a double bond containing a =O (ketone), =S, =NR, -C(=O)R, or =CR5R5' group; and R1 and R2, R1' and R2', R3 and R4, or R3' and R4' are joined together to form a C3-C 12 It forms an aromatic, heterocyclic, carbocyclic, or heteroaryl ring.

[0084] Q is a functional group that allows reaction with a cell-binding molecule (CBA), or cell-binding agent, or a functional group that can react with a linker attached to a cell-binding agent, wherein the functional group is selected from thiol, amine, hydrazine, alkoxylamino, disulfide substituent, maleimide, haloacetyl group, N-hydroxysuccinimide ester, ketone, ester, aldehyde, alkynyl, alkenyl, or protected thiol or disulfide group, SAc, SSR1, or SSAr, where Ar is an aromatic or heteroaromatic group. Preferably, Q is a cell binding agent / molecule selected from antibodies, single chain antibodies, antibody fragments that bind to target cells, monoclonal antibodies, monoclonal antibody fragments that bind to single chain monoclonal antibodies, target cells, chimeric antibodies, chimeric antibody fragments that bind to target cells, domain antibodies, domain antibody fragments that bind to target cells, antibody mimicking adnectins, DARPins, lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules (transferrin), and binding peptides, proteins, or small molecules attached to albumin, polymers, dendrites, liposomes, nanoparticles, vesicles, or (viral) capsids.

[0085] The term releasable conjugate refers to a conjugate that includes at least one bond that can be cleaved under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzymatically labile bond. It is understood that such physiological conditions that result in bond disruption do not necessarily include biological or metabolic processes, but instead may include standard chemical reactions such as hydrolysis or substitution reactions, e.g., disulfide bond exchange reactions with intracellular thiols such as glutathione, which is abundant in the millimolar range in endosomes, which have a pH lower than the cytoplasmic pH, and / or malignant cells.

[0086] The extender unit (-W-), if present, may be attached to the target-binding molecule unit (CBA) with an amino acid unit, or with T if Aa is absent. The extender unit W may independently comprise a self-immolative spacer, a peptide unit, a hydrazone bond, a disulfide bond, an ester bond, or a thioether bond. In this regard, the cell-binding molecule (CBA) has a functional group capable of forming a bond with a functional group of the extender unit. Functional groups that may be present on the binding molecule naturally or through chemical manipulation include sulfhydryl (-SH), amino, hydroxy, carbonyl, oxyamino, alkynyl, heteroaromatic, anomeric hydroxy groups of carbohydrates, and carboxyl. Preferred functional groups are sulfhydryl, carboxy, and amino. Sulfhydryl groups can be generated by reduction of intramolecular disulfide bonds in a ligand (such as a protein or antibody). Alternatively, sulfhydryl groups can be generated by reacting amino groups of lysine moieties of cell-binding molecules using 2-iminothiolane (Traut's reagent), or by reacting thiolactones or other sulfhydryl groups. The cell-binding molecule can be modified with a disulfide or thiol-ester followed by reduction or hydrolysis, respectively.

[0087] Preferably, L1 and L2 are independently a chain of atoms selected from C, N, O, S, Si, and P, having 0 to 500 atoms. The atoms used to form L1 and L2 may be combined in any chemically relevant manner, and preferably are C1 to C2. 20Alkylene, alkenylene, and alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, alkoxylamine, urethane, amino acid, peptide, acyloxylamine, hydroxamic acid, or a combination thereof. More preferably, L1 and L2 are independently the same or different, O, NH, S, NHNH, N(R3), N(R3)N(R3'), C1-C8 alkyl, amide, amine, imine, hydrazine, hydrazone; C2-C8 heteroalkyl, alkylcycloalkyl, ether, ester, hydrazone, urea, semicarbazide, carbazide, alkoxyamine, alkoxylamine, urethane, amino acid, peptide, acyloxylamine, hydroxamic acid, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or a group of the formula (OCH2CH2) p OR3, or (OCH2CH(CH3)) p OR3, or NH(CH2CH2O) p R3, NH(CH2CH(CH3)O) p R3, N[(CH2CH2O) p R3]-[(CH2CH2O) p’ R3'], (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR polyethyleneoxy units, wherein p and p' are independently an integer selected from 0 to about 5000, or a combination thereof; wherein R and R' are independently H; C-C alkyl; C-C heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C-C aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or C-C ester, ether, or amide; or 1 to 8 amino acids; or a group of the formula (OCHCH) p or (OCH2CH(CH3)) pwhere p is selected from an integer from 0 to about 5000, or combinations thereof as described above.

[0088] Optionally, L1 and L2 are independently 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoate ("SPP"), 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ("MCC"), (4-acetyl)acetyl The linker moiety may be one or more of the following: amino-benzoate ("SIAB"), ethyleneoxy (-CHCHO-) ("EO" or "PEO") as one or more repeating units, 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), or one or more moieties that are natural or unnatural peptides having the same or different sequences of 1 to 8 natural or unnatural amino acid units. The natural amino acids are preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, and alanine.

[0089] L1 and L2 may also independently comprise a self-immolative or non-self-immolative moiety, a peptide unit, a hydrazone bond, a disulfide, an ester, an oxime, an amide, or a thioether bond, including, but not limited to, a para-aminobenzylcarbamoyl (PAB) group, a 2-aminoimidazole-5-methanol derivative, a heterocyclic PAB analog, a β-glucuronide, and an aromatic group electronically similar to o- or p-aminobenzyl acetal. Includes aromatic compounds.

[0090] Preferably, the self-immolative linker moiety has one of the following structures: [ka]

[0091] In the formula, ( * The atom labeled with X is an additional spacer or releasable linker unit, or a point of attachment to a cytotoxic agent and / or a binding molecule (CBA); X, Y, Z, and Z are independently NH, O, or S; Z 1 are independently H, OH, NHR1, OR1, SR1, COX1R1, where X1 and R1 are defined above; v is 0 or 1; U 1 are independently H, OH, C1-C6 alkyl, (OCH2CH2) n , F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NR5R5', NO2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5'), or OCH2PO(OR5(OR5')), where R5 and R5' are H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl, or amino acid; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or glycoside; or a medicinal cation salt.

[0092] The non-self-immolative linker component is one of the following structures: [ka] TIFF0007811398000018.tif89149

[0093] In the formula, ( * Atoms labeled with X are sites of attachment for additional spacers or releasable linkers, cytotoxic agents, and / or binding molecules; 1 , Y 1 , U 1 The definitions of R, R5, and R5' are as defined above. r is 0 to 100. m and n are each independently 0 to 6.

[0094] More preferably, L1 and L2 can independently be releasable conjugate moieties. The term releasable refers to a conjugate that includes at least one bond that can be cleaved under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzyme-labile bond. It is understood that such physiological conditions that result in bond rupture do not necessarily involve biological or metabolic processes, but instead may involve standard chemical reactions such as hydrolysis or substitution reactions, e.g., disulfide bond exchange reactions with intracellular thiols such as glutathione, which is abundant in the millimolar range in endosomes and / or malignant cells, which have a lower pH than the cytoplasmic pH.

[0095] Examples of releasable linkers L1 or L2 include, but are not limited to: -(CR5R6) m (Aa) r (CR7R8) n (OCH2CH2) t -, -(CR5R6) m (CR7R8) n (Aa) r (OCH2CH2) t -, -(Aa) r -(CR5R6) m (CR7R8) n (OCH2CH2) t -, -(CR5R6) m (CR7R8) n (OCH2CH2) r (Aa) t -, -(CR5R6) m -(CR7=R8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -, -(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -(CR5R6) m (Aa) t (NR 11CO)(CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (CO)(Aa) t -(CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5 R6) m -フェニル-(CO)(Aa) t (CR7R8) n -、-(CR5R6) m -フリル-(CO)(Aa) t (CR7R8) n -、-(CR5R6) m -オキサゾリル-(CO)(Aa) t (CR7R8) n-, -(CR5R6) m -thiazolyl-(CO)(Aa) t (CCR7R8) n -, -(CR5R6) t -Thienyl-(CO)(CR7R8) n -, -(CR5R6) t -Imidazolyl-(CO)(CR7R8) n -, -(CR5R6) t -morpholino-(CO)(Aa) t (CR7R8) n -, -(CR5R6) t -piperazino-(CO)(Aa) t (CR7R8) n -, -(CR5R6) t -N-methylpiperazine-(CO)(Aa) t (CR7R8) n -, -(CR5R) m -(Aa) t Phenyl-, -(CR5R6) m -(Aa) t Frill-,-(CR5R6) m -oxazolyl (Aa) t -, -(CR5R6) m -Thiazolyl (Aa) t -, -(CR5R6) m -Thienyl (Aa) t -, -(CR5R6) m -Imidazolyl (Aa) t -, -(CR5R6) m -Morpholino-(Aa) t -, -(CR5R6) m -Piperazino-(Aa) t -, -(CR5R6) m -N-methylpiperazino-(Aa) t -, -K(CR5R6) m (Aa) r (CR7R8) n (OCH2CH2) t -, -K(CR5R6) m (CR7R8) n (Aa) r (OCH2CH2) t -, -K(Aa)r (CR5R6) m (CR7R8) n (OCH2CH2) t -、-K(CR5R6) m (CR7R8) n (OCH2CH2) r (Aa) t -、-K(CR5R6) m (CR7=R8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -、-K(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (Aa) t (NR 11 CO)(CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n (OCH2CH2) r-, -K(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m -phenyl-(CO)(Aa) t (CR7R8) n -, -K(CR5R6) m -Frill-(CO)(Aa) t (CR7R8) n -, -K(CR5R6) m -oxazolyl-(CO)(Aa) t (CR7R8) n -, -K(CR5R6) m -thiazolyl-(CO)(Aa) t (CR7R8) n -, -K(CR5R6) t -Thienyl-(CO)(CR7R8) n -, -K(CR5R6) t -Imidazolyl-(CO)(CR7R8) n -, -K(CR5R6) t -morpholino-(CO)(Aa) t (CR7R8) n -, -K(CR5R6) t -piperazino-(CO)(Aa) t (CR7R8) n -, -K(CR5R6) t -N-methylpiperazine-(CO)(Aa) t (CR7R8) n -, -K(CR5R) m -(Aa) t Phenyl-, -K(CR5R6) m -(Aa) t Frill-, -K(CR5R6) m -oxazolyl (Aa) t -, -K(CR5R6) m -Thiazolyl (Aa) t-, -K(CR5R6) m -Thienyl (Aa) t -, -K(CR5R6) m -Imidazolyl (Aa) t -, -K(CR5R6) m -Morpholino-(Aa) t -, -K(CR5R6) m -Piperazino-(Aa) t G-, -K(CR5R6) m -N-methylpiperazino-(Aa) t wherein m, Aa, m, and n are as defined above; t and r are independently 0 to 100; R3, R4, R5, R6, R7, and R8 are independently selected from H; halide; C1-C8 alkyl; C2-C8 aryl, alkenyl, alkynyl, ether, ester, amine, or amide, optionally selected from one or more of halide, CN, NRR, CF, OR, aryl, heterocycle, S(O)R, SO2R, - may be substituted by CO2H, -SO3H, -OR1, -CO2R1, -CONR1, -PO2R1R2, -PO3H, or P(O)R1R2R3; K is NR1, -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=NN H-, -C(=O)NH-NH-, O, S, Se, B, Het (heterocyclic or heteroaromatic ring having C3-C8), or a peptide containing 1 to 20 amino acids;

[0096] Additionally, U, U', E, E', L1, and L2 can independently comprise one or more units of the following hydrophilic structures: [ka]

[0097] During the ceremony, [ka] is a linking moiety; X2, X3, X4, X5, or X6 are independently NH, NHNH; N(R3); N(R3)N(R3'); O; S; C1-C6 alkyl; C2-C6 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; selected from 1 to 8 of the same or different amino acids; wherein R3 and R3' are independently H; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or C2-C8 ester, ether, or amide; or a group of the formula (OCH2CH2) p or (OCH2CH(CH3)) p where p is an integer from 0 to about 5000, or a combination thereof.

[0098] More preferably, L1 or L2 are independently a linear alkyl having 1 to 6 carbon atoms, or a group of the formula (OCH2CH2) p (p=1 to 5000), or a peptide containing 1 to 4 amino acid units (L or D type), or a combination thereof.

[0099] Additionally, U, U', L1, L2, L', E1, or E2 may independently consist of one or more of the following moieties: [ka] TIFF0007811398000022.tif231143, and L- or D-, natural or non-natural peptides containing 1 to 20 identical or different amino acids.

[0100] In the formula, the connecting bond in the center of the atom can connect to any of the adjacent carbon atom bonds. The wavy line indicates a site where another bond can be attached.

[0101] Alternatively, U, U', E1, or E2 may independently be absent.

[0102] The compounds of formula (I) with their geometric and stereoisomers are also part of the present invention.

[0103] Preferred stereoisomers of formula (I) are represented by the following formulae (Ia), (Ib), (Ic), and (Ie). [ka] TIFF0007811398000024.tif55139

[0104] wherein Z1 is OH, NH2, OR1, NHR1, NR1R2, SR1, NHR1COX1R1, OR1COX1R1, or N(R2)R1COX1R1; [ka] , X, X', Y, Y', Z, Z', l, l', m, m', n, q, q', R1, R1', R2, R2', R3, R3', R4, R4', V, V', U, U', L1, L2, E1, E2, and Q are the same as in formula (I).

[0105] In more preferred embodiments of formula (I), the conjugates of the bridged PBD dimer derivatives of the present invention have the following formulae (I-01) to (I-18): [ka] TIFF0007811398000027.tif222152TIFF0007811398000028.tif212151TIFF0007811398000029.tif233153TIFF0007811398000030.tif233154

[0106] In the formula, V, V', n, and q are defined as above. mAb is a cell-binding molecule, preferably an antibody. r, r', and r'' independently represent 0 to 200.

[0107] In another embodiment, the present invention provides a compound represented by formula (II), (I As shown in (II) and (IV), monolinking of PBD derivatives to cell-binding molecules is disclosed. [ka]

[0108] During the ceremony, [ka] , X, X', Y, Y', Z, Z', l, l', m, m', n, q, q', R1, R1', R2, R2', R3, R3', R4, R4', V, V', U, U', L1, L2, G, Q, E1, and E2 are as defined in formula (I).

[0109] In preferred embodiments of formulas (II), (III), and (IV), the PBD derivative conjugates of the present invention have the following formulae (II-01) to (II-11), (III-01) to (III-06), and (IV-01) to (IV-11): [ka] TIFF0007811398000034.tif224157TIFF0007811398000035.tif211155TIFF0007811398000036.tif174150 TIFF0007811398000037.tif210158TIFF0007811398000038.tif233159TIFF0007811398000039.tif226161

[0110] During the ceremony, [ka] , m, m', n, q, and q' are the same as defined in formula (I); r, r', and r'' are independently 0 to 200, and m3 is 0 to 30.

[0111] In another preferred embodiment, the conjugates of formula (I), (II), (III), (IV), and (V) are prepared by coupling a PBD dimer derivative having the structure of formula (V), (VI), (VII), and (VIII) below with a cell-binding molecule: [ka]

[0112] During the ceremony, [ka] , X, X', Y, Y', Z, Z', l, l', m, m', n, q, q', R1, R1', R2, R2', R3, R3', R4, R4', V, V', U, U', L1, L2, E1, and E2 are as defined in formula (I).

[0113] wherein E3 and E'3 are independently selected from: [ka] TIFF0007811398000044.tif130144

[0114] wherein X1' and X3' are independently F, Cl, Br, I, or Lv3; X2' is O, NH, N(R1), or CH2; R3 and R5 are independently H, R1, aromatic, heteroaromatic, or an aromatic group in which one or more H atoms are independently replaced by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, or -COOR1; Lv3 is a leaving group selected from methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, phenylthio, pyridinylthio, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; Pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxy 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole- an intermediate molecule generated with oxadiazol-yl (ODA), oxadiazol-yl, or condensation reagent for Mitsunobu reaction, wherein R1 and R2 are defined as above;

[0115] More preferably, E3 and E'3 are independently selected from -SH, -S-SCH3, -S-SAc, -SS-pyridine, -SS-Ar(-NO2), -S-cell binding agent, or any of the following formulas: [ka]

[0116] where D is H, —NO2, SO3H, or F; where R 1 , R 2 , R 3 , R 4 , r, m, and n are defined as above, and w and w' are independently 0, 1, or 2.

[0117] In the formula, R 5 and R 5 ' is independently selected from C1-C6 alkyl, aryl, cyclic, cyclohetero, H, or M, where M is Na, K, Ca, ammonium, or other pharmaceutically acceptable salts.

[0118] In certain embodiments, the PBD derivatives of formulas (V), (VI), (VII), and (VIII) are represented by the following formulas (V-01) to (V-20), (VI-01) to (VI-05), (VII-01) to (VII-06), and (VIII-01) to (VIII-06): [ka] TIFF0007811398000047.tif233151TIFF0007811398000048.tif211146TIFF0007811398000049.t if216151TIFF0007811398000050.tif234157TIFF0007811398000051.tif200156TIFF0007811398 000052.tif225152TIFF0007811398000053.tif211142TIFF0007811398000054.tif181149TIFF00 07811398000055.tif196153TIFF0007811398000056.tif204156TIFF0007811398000057.tif89156

[0119] wherein U, U', V, V', n, n', X, X', and L are as defined in the claims; R and R are independently selected from C-C alkyl, aryl, cyclic, cyclohetero, halogen, haloalkyl, alkoxy, haloalkoxyalkylamino, -NO, -CN, or H; and X and X are independently H, F, Cl, Br, I, OTs (tosylates), OMs (mesylates), nitrophenol OAr(NO), OAr(NO) dinitrophenol, OAr(F) monofluorophenol, OAr(F) pentafluorophenol, OAr(F) difluorophenol, N-hydroxysuccinimide (NHS), phenol, tetrafluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, or 2-ethyl-5-phenylisoxazolium-3'-sulfonate.

[0120] Synthesis of Bridged PBD Dimer Derivatives of Formulae (V), (VI), (VII), and (VIII) as Cytotoxic Agents

[0121] The compounds and processes of the present invention can be prepared in many ways well known to those skilled in the art. The compounds can be synthesized, for example, by application or adaptation of the methods described in the Examples, or variations thereof as would be understood by one skilled in the art. Appropriate modifications and substitutions will be readily apparent to those skilled in the art and are well known or readily obtainable by those skilled in the art from the scientific literature. In particular, such methods are described in Richard C. Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, Two Volume Set, 2004, Vol. nd Edition, Wiley Publishers, 2010.

[0122] Because the cytotoxic agents of the present invention may contain one or more asymmetrically substituted carbon atoms and may be isolated in optically active or racemic forms, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of the structures are intended, unless the particular stereochemistry or isomer is specifically indicated. Methods for preparing and isolating such optically active forms are well known in the art. For example, but not limited to, mixtures of stereoisomers can be separated from racemic forms by standard techniques, including, but not limited to, reverse-phase and chiral chromatography, preferential salt formation, recrystallization, etc., or by chiral synthesis from chiral starting materials or by intentional synthesis of a targeted chiral center.

[0123] The cytotoxic agents of the present invention can be prepared by a variety of synthetic routes. The reagents and starting materials are commercially available or can be easily synthesized by one of ordinary skill in the art using well-known techniques. All substituents are as previously defined unless otherwise specified.

[0124] In the synthesis of the cytotoxic agents of the present invention, desired reactive functional groups in the final product, such as hydroxy, amino, imino, thio, or carboxy groups, may be protected as necessary to avoid undesired participation in the reaction. Conventional protecting groups may be used according to standard practice, for example, as described in Peter GM Wuts, Theodora W. Greene in Greene's Protective Groups in Organic Synthesis, 4 th edition, John Wileyand Sons, 2006; Ian T. Harrison, Shuyen Harrison in Compendium of Organic Synthetic Methods, Vol 1, 2 Vols. 1&2 By Ian T. Harrison&ShuyenHarrison, Vols 3-5 by Louis S. Hegedus, Leroy Wade Vols 6-Vol 12 by Michael B. Smith, John Wileyand Sons, 2006-2012. Light.

[0125] Typically, synthesis reactions are carried out in an appropriate solvent, at an appropriate temperature, and for an appropriate time. Various solvents that do not adversely affect the reaction or the reagents can be used in synthesis reactions of cytotoxic agents. Examples of suitable solvents include: hydrocarbons (which can be aromatic, aliphatic, or alicyclic hydrocarbons, such as hexane, cyclohexane, benzene, toluene, and xylene); halogenated hydrocarbons, such as chloroform, dichloromethane, and dichloroethane; amides, such as dimethylformamide or dimethylformamide; alcohols, such as ethanol or methanol; and ethers, such as diethyl ether and tetrahydrofuran. The reaction can be carried out over a wide temperature range, from -100°C to 300°C. Temperatures between 0°C and 150°C are more preferred. The time required for a synthesis reaction can also vary widely. Depending on many factors, particularly the reaction temperature and the nature of the reagents, the time can range from 5 seconds to 4 weeks, more preferably from 10 minutes to 24 hours. Furthermore, the prepared cytotoxic agent may be isolated or purified from the reaction mixture by conventional means, such as evaporating or distilling off the solvent from the reaction mixture, or by distilling off the solvent from the reaction mixture, followed by placing the residue in water, extracting with a water-immiscible organic solvent, and distilling off the solvent from the extract. Methods may also include recrystallization, reprecipitation, or various known techniques such as various chromatographic techniques, particularly column chromatography, preparative thin-layer chromatography, or high performance liquid chromatography.

[0126] Some of the synthetic reactions for cytotoxic agents and their conjugates with cell-binding agents are further exemplified, without limitation, in Figures 1-32 and the following description.

[0127] Conjugates of cross-linked PBD dimers with cell-binding molecules

[0128] The present invention provides a conjugate molecule comprising at least one PBD derivative covalently bonded to a cell-binding agent (Q) via a linking group of a conjugate linker. Preferably, the conjugate comprises 1 to 20 molecules of a bridged PBD dimer derivative of the present invention covalently bonded to the cell-binding agent via a linking group of a linker on the bridged PBD dimer derivative.

[0129] As noted above, the cell surface binding molecule-crosslinked PBD dimer derivative conjugates are shown in formulas (I), (II), (III), and (IV) above.

[0130] Drug loading can range from 1 to 30 drug moieties (D) per antibody, preferably an average of 2 to 8 drug residues per antibody in molecules of Formula (I)-(IV). The average number of drug residues per antibody from the conjugation reaction can be determined by conventional means, such as mass spectrometry (HPLC-MS, UPLC-QTOF, HPLC-MS / MS), ELISA assays, and HPLC (SEC-HPLC, HIC-HPLC). From the perspective of drug loading, The quantitative distribution of the conjugates can also be determined. In some instances, separation, purification, and characterization of homogeneous conjugates with a particular drug loading from conjugates with loaded drug can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0131] Cell-binding agents (CBAs) can be of any type, including peptides and non-peptides. In general, cell-binding agents include, but are not limited to, large molecular weight proteins, such as full-length antibodies (polyclonal or monoclonal), dimers, multimers, and multispecific antibodies (e.g., bispecific antibodies); single-chain antibodies; antibody fragments, such as Fab, Fab', F(ab')2, and Fv [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments obtained by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs, bispecific antibodies, trispecific antibodies, epitope-binding fragments of any of the above that immunospecifically bind to a protein generated by the immune system that can recognize, bind to, or exhibit a desired biological activity against a specific antigen; interferons (e.g., I , II, III types); peptides; lymphokines, such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, or interferon gamma (IFN-γ); hormones, such as insulin, TRH (thyrotropin-releasing hormone), MSH (myocyte-stimulating hormone), or steroid hormones such as androgen, estrogen, or melanocyte-stimulating hormone (MSH); growth factors and colony-stimulating factors, such as epidermal growth factor (EFG), granulocyte-macrophage colony-stimulating factor (GM-CSF); transforming growth factors (TGF), such as TGFα, TGFβ; insulin and insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF, and GM-CSF [Burgess, Immunology Today, 5, 155-158 (1984)]; vaccine growth factors ( VGF); fibroblast growth factor (FGF); small molecular weight proteins, polypeptides, peptides, and peptide hormones, e.g., bombesin, gastrin, and gastrin-releasing peptide; platelet-derived growth factor; interleukins and cytokines, e.g., interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins such as folic acid; apoproteins and glycoproteins, e.g., transferrin [O'Keefe et al., J. Bio. Chem. 260, 932-927 (1985)]; carbohydrate-binding proteins or lipoproteins, such as lectins; cellular nutrient transport molecules; and small molecule inhibitors, e.g., inhibitors of prostate-specific membrane antigen (PSMA), small molecule tyrosine kinase inhibitors (TKIs), non-peptides, or other cell-binding molecules or substances, e.g., bioactive polymers (Dhar, et al., J. Bio. Chem. 260, 932-927 (1985)). al,Proc.Natl.Acad.Sci.2008,105,17356-61), bioactive dendrimers (Lee,et al,Nat.Biotechnol.2005,23,1517-26;Almutairi,et al;Proc.Natl.Acad.Sci.2009,106,685-90), nanoparticles (Liong,et al,ACS Nano, 2008, 19, 1309-12; Medarova, et al., Nat. Med. 2007, 13, 372-7; Javier, et al., Bioconjugate Chem. 2008, 19, 1309-12), liposomes (Medinai, et al., Curr. Phar. Des. 2004, 10, 2981-9), and viral capsids (Flenniken, et al., Viruses Nanotechnol. 2009, 327, 71-93). Generally, monoclonal antibodies are preferred as cell surface binding molecules, when suitable ones are available.

[0132] Linkers for use in the conjugates of the invention include, but are not limited to, disulfide linkages, thioether linkages, amide bond linkages, peptidase labile linkages, photolabile linkages, acid labile linkages (such as hydrazone linkers), esterase labile linkages, oxidation labile linkages, metabolic labile linkages, and biochemically labile linkages.

[0133] Preferably, the linker is connected to the cell-binding molecule via a reaction between a thiol group and an amino group of the cell-binding molecule derived from a reduced disulfide bond and a lysine residue, respectively. More specifically, the derivative is connected to the cell-binding molecule by forming an amide bond with the amino group of the lysine residue of the cell-binding molecule through the —CO— group.

[0134] Furthermore, the linker may be composed of one or more linker moieties. Exemplary linker moieties include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), glycine-glycine, p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), and one or more repeating units containing an ethyleneoxy (-CHCHO-) unit (EO or PEO). The linker may also be a "cleavable linker" that facilitates intracellular drug release. Additional linker moieties are known in the art, some of which are described herein.

[0135] [ka] TIFF0007811398000059.tif219160TIFF0007811398000060.tif86159

[0136] In the formula, R7, R8, and R9 each independently represent -C1-C8 alkylene-, -C1-C7 carbocycle-, -O-(C1-C8 alkyl)-, -arylene-, -C1-C8 alkylene-arylene-, -arylene, -C1-C8 alkylene-, -C1-C8 alkylene-(C1-C8 carbocycle)-, -(C3-C7 carbocycle)-C1-C8 alkylene-, -C3-C8 heterocyclo-, -C1-C8 alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C9 alkylene-, -(CH2CHO) k -, -(CH(CH3)CH2O) k - and -(CH2CH2O) k -CH2-; k is an integer ranging from 1 to 30; X''', Y''', and Z''' are independently selected from NH, O, or S. Q, R1, and R2 are as defined above.

[0137] In preferred embodiments, the conjugates of the invention are antibody / cytotoxic agent, antibody fragment / cytotoxic agent, diabody / cytotoxic agent, tri(a)body / cytotoxic agent, epidermal growth factor (EGF) / cytotoxic agent, prostate-specific membrane antigen (PSMA) inhibitor / cytotoxic agent, melanocyte-stimulating hormone (MSH) / cytotoxic agent, thyroid-stimulating hormone (TSH) / cytotoxic agent, polyclonal antibody / cytotoxic agent, somatostatin / cytotoxic agent, folic acid / cytotoxic agent, matriptase inhibitor / cytotoxic agent, estrogen / cytotoxic agent, estrogen analog / cytotoxic agent, designed ankyrin repeat proteins (DARPins) / cytotoxic agent, androgen / cytotoxic agent, and androgen analog / cytotoxic agent.

[0138] In a more preferred embodiment, the cell-binding molecule of the present invention is a monoclonal antibody. Examples of antibodies conjugated with cytotoxic drugs for this prevention include, but are not limited to, 3F8 (anti-GD2 antibody), abagovomab (anti-CA-125 antibody), abciximab (anti-CD41 antibody (integrin α-IIb)), adalimumab (anti-TNF-α antibody), adalimumab (anti-EpCAM antibody, CD326), afelimomab (anti-TNF-α); afutuzumab (anti-CD20 antibody), alacizumab pegol (Alacizumab pegol) (anti-VEGFR2 antibody), ALD518 (anti-IL-6 antibody), alemtuzumab (also known as Campus, MabCampus, anti-CD52 antibody), altumomab (anti-CEA antibody), anatumomab (anti-tag-72 antibody), anrukinzumab (IMA-638, anti-IL-13 antibody), apolizumab (anti-HLA-DR antibody), arcitumomab (anti-CEA antibody), acelizumab (anti-L-selectin (CD62L) antibody), atlizumab ( Other names: Tocilizumab, Actemra, RoActemra, anti-IL-6 receptor antibody), Atroli Atorolimumab (anti-rhesus factor antibody), Bapineuzumab (anti-β-amyloid antibody), Basiliximab (Simulect, anti-CD25 (IL-2 receptor α chain) antibody), Bavituximab (anti-phosphatidylserine antibody), Bectumomab (also known as LymphoScan, anti-CD22 antibody), Belimumab (also known as BENLYSTA, LymphoStat-B, anti-BAFF antibody), Benralizumab (anti-CD125 antibody), Belchi Limumab (anti-CCL11 (eotaxin-1) antibody), besilesomab (also known as Scintimun , anti-CEA-related antigen antibody), bevacizumab (also known as Avastin, anti-VEGF antibody), biciromab (also known as FibriScint, anti-fibrin II beta chain antibody), bivatuzumab (anti-CD44v6 antibody), blinatumomab (also known as BiTE, anti-CD19 antibody), brentuximab (cAC10, anti-CD30 TNFRSF8 antibody), Briakinumab (anti-IL-12, IL-23 antibody), canakinumab (also known as Ilaris, anti-IL-1 antibody), cantuzumab (also known as C242, anti-CanAg antibody), capromab, catumaxomab (also known as removab, anti-EpCAM, anti-CD3 antibody), CC49 (anti-TAG-72 antibody), Cedelizumab (anti-CD4 antibody), CC49 (anti-TAG-72 antibody), Cedelizumab (anti-CD4 antibody), body), certolizumab pegol (also known as CIMZIA, anti-TNF-α antibody), cetuximab (also known as Erbitux, IMC-C225, anti-EGFR antibody), sitatuzumab (anti-EpCAM antibody), cixutumumab (anti-IGF-1 antibody), clenoliximab (anti-C D4 antibody), clivatuzumab (anti-MUC1 antibody), conatumumab (anti-TRAIL-R2 antibody), CR6261 (anti-influenza A red blood cell agglutinin antibody) Antibody), Dacetuzumab (anti-CD40 antibody), Daclizumab (also known as Zenapax, anti-CD25C (IL-2 receptor alpha chain) antibody), Daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase) antibody), Denosumab (also known as Prolia, anti-RANKL antibody), Detumomab (anti-B-lymphoma cell antibody), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglionic acid antibody) Osido antibody), eculizumab (also known as Soliris, anti-C5 antibody), edovacomab (anti-endo toxin antibody), edrecolomab (also known as Panorex, MAb17-A1, anti-EpCAM antibody body), efalizumab (also known as Raptiva, anti-LFA-1 (CD11a) antibody), Efan Efungumab (also known as Mycograb, anti-Hsp90 antibody), Elotuzumab (anti-SLAMF7 antibody), Elsilimomab (anti-IL-6 antibody), Enlimomab pegol (anti-ICAM-1 (CD54) antibody), Epitumomab (anti-episialin antibody), Epratuzumab (anti-CD22 antibody), Erlizumab (anti-ITGB2 (CD18) antibody), ertumaxomab (also known as Rexomun, anti-HER2 / neu, CD3 antibody), etaracizumab (also known as Abegrin, anti-integrin αvβ3), Exibirumab (anti-hepatitis B surface antigen antibody (HBs antibody)), Fanolesomab (also known as NeutroSpec, anti-CD15 antibody), Faralimomab (anti-interferon receptor antibody), Farletuzumab (anti-folate receptor 1 antibody), Felvizumab (anti-RS virus antibody), Fezakinumab (anti-IL-22 antibody), Figitumumab (anti-IGF-1 receptor antibody), Fontolizumab (anti-IFN-γ antibody), Foravirumab (anti-rabies virus glycoprotein antibody), Fresolim Fresolimumab (anti-TGF-β antibody), Galiximab (anti-CD80 antibody), Gantenerumab (anti-β-amyloid antibody), Gavilimomab (anti-CD147 (basigin) antibody), Gemtuzumab (anti-CD33 antibody), Girentuximab (anti-carbonic anhydrase 9 antibody), Glembatumumab (also known as CR011, anti-GPNMB antibody), Golimumab (also known as Simponi, anti-TNF-α antibody) antibody), Gomiliximab (anti-CD23C (IgE receptor) antibody), Ibalizumab (anti-CD4 antibody), Ibritumomab (anti-CD 20 antibody), Igovomab (also known as Indimacis-125, anti-CA-125 antibody), Imciromab (also known as Myoscint, an anti-cardiac myosin antibody), infliximab (also known as Remicade, anti-TNF-α antibody), intetumumab (anti-CD5 1 antibody), Inolimomab (anti-CD25 (IL-2 receptor α chain) antibody), Inotuzumab (anti-CD22 antibody), Ipilimumab (anti-CD152 antibody), Iratumumab (anti-CD30 (TNFRSF8) antibody), Keliximab (anti-CD4 antibody), Labetuzumab (also known as CEA-Cide, anti-CEA antibody), Lebriki Lebrikizumab (anti-IL-13 antibody), Lemalesomab (anti-NC A-90 (granulocyte antigen) antibody), Lerdelimumab (anti-TGFβ-2 antibody), Lexatumumab (anti-TRAIL-R2 antibody), Ribivirumab (anti-hepatitis B surface antigen antibody), Lintuzumab (anti-CD33 antibody), Lucatumumab (anti-CD40 antibody), Lumiliximab (anti-CD23 (IgE receptor) antibody), Mapatumumab (anti-TRAIL-R1 antibody), Maslimomab (anti-T cell receptor antibody), Matuzumab (anti-EG FR antibody), Mepolizumab (also known as Bosatria, anti-IL-5 antibody), Metelimumab (anti-TGFβ-1 antibody), Milatuzumab (anti-CD74 antibody), Minretumomab (anti-TAG-72 antibody), Mitumomab (also known as BEC-2, anti-ganglioside antibody-GD3), Morolimumab (anti Rhesus factor antibody), Motavizumab (also known as Numax, anti-RS virus antibody), Muromonab-CD3 (also known as Orthoclone OKT3, anti-CD3 antibody), Nacolomab (anti-C242 antibody), Naptumomab (anti-5T4 antibody) body), natalizumab (also known as Tysabri, anti-integrin α4 antibody), nebacumab (anti-endotoxin antibody), necitumumab (anti-EGFR antibody), Nerelimomab (anti-TNF-α antibody), Nimotuzumab (also known as Theracim, Theraloc, anti-EGFR antibody), Nofetumomab, Ocrelizumab (anti-CD20 antibody), Odulimomab (also known as Afolimomab, anti-LFA-1 (CD11a) antibody), Ofatumumab (also known as Arzerra, anti-CD20 antibody), Olaratumab (anti-P DGF-Rα antibody), Omalizumab (also known as Xolair, anti-IgE Fc domain antibody), Oportuzumab (anti-EpCAM antibody), Oregovomab (also known as OvaRex, anti-CA-125 antibody), Otelixizumab (anti-CD3 antibody), Pagibaximab (anti-LTA antibody), Palivizumab (also known as Synagis, Abbosynagis, anti-RS virus antibody), Panimumab (also known as Vectibix, ABX-E GF, anti-EGFR antibody), panobacumab (anti-Pseudomonas aeruginosa antibody), Pascoli Pascolizumab (anti-IL-4 antibody), Pemtumomab (also known as Theragyn, anti-MUC1 antibody), Pertuzumab (also known as Omnitarg, 2C4, anti-HER2 / neu antibody), Pexelizumab (anti-C5 antibody), Pintumomab ( Anti-adenocarcinoma antigen antibody), Priliximab (anti-CD4 antibody), Pritumumab (anti-vimentin antibody), PRO140 (anti-CCR5 antibody), Racotumomab (also known as 1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGN) A)-Ganglioside (GM3) antibody), Rafivirumab (anti-rabies antibody) anti-VEGFR2 antibody), ramucirumab (anti-VEGFR2 antibody), Visumab (also known as Lucentis, anti-VEGF-A antibody), Raxibacumab ( anti-anthrax toxin, protective antigen antibody), regavirumab (anti-CMV glycoprotein) B antibody), Reslizumab (anti-IL-5 antibody), Rilotumumab (anti-HGF antibody), Rituximab (also known as MabThera, Rituxanmab, anti-CD20 antibody) , Robatumumab (anti-IGF-1 receptor antibody), Rontalizumab (anti-IFN-α antibody), Rovelizumab (also known as LeukArrest, anti-CD11, CD18 antibody), Ruplizumab (also known as Antova, anti-CD154 (CD40L) antibody), Satumomab (anti-TAG-72 antibody), Sevirumab (anti-CMV antibody), Sibrotuzumab (anti-FAP antibody), Sifalimumab (anti-IFN-α antibody), Siltuximab (anti-IL-6 antibody), Siplizumab (anti-CD2 antibody), (Smart) MI95 (anti-CD33 antibody), solanezumab (anti-β-amyloid antibody), sonepcizumab (anti-s Fingosine-1-phosphate antibody), sontuzumab (anti-episialin antibody), stamulumab (anti-myostatin antibody), sulesomab (another Name: LeukoScan (anti-NCA-90 (granulocyte antigen) antibody), tacatuzumab (anti-alpha-fetoprotein antibody), tadocizumab (anti-integrin antibody) αIIbβ3 antibody), talizumab (anti-IgE antibody), tanezumab (anti-NG F antibody), taplitumomab (anti-CD19 antibody), tefibazumab (also known as Aurexis, anti-clumping factor A antibody), telimomab, Tenatumomab (anti-tenascin-C antibody), teneliximab (anti-CD40 antibody), teplizumab (anti-CD3 antibody), TGN1412 (anti-CD28 antibody), ticilimumab (also known as tremelimumab, anti-CTLA-4 antibody), tigatuzumab (anti-TRAIL-R2 antibody), TNX-650 (anti-IL-13 antibody) antibody), Tocilizumab (also known as Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor antibody ), Toralizumab (anti-CD154 (CD40L) antibody), Tositumomab (anti-CD20 antibody), trastuzumab (also known as Herceptin, anti-HER2 / neu antibody), Tremelimumab (anti-CTLA-4 antibody), Tucotuzumab celmoleukin (anti-EpCAM antibody), Tuvirumab (anti-hepatitis B antibody), Urtoxazumab (anti-colon Anti-IL-12 antibody), Ustekinumab (also known as Stelara, anti-IL-12, IL-23 antibody), Vapaliximab (anti-AOC3 (VAP-1) antibody), Vedriz Vedolizumab (anti-integrin α4β7 antibody), veltuzumab (anti-CD20 antibody) body), Vepalimomab (anti-AOC3 (VAP-1) antibody), Visilizumab (also known as Nuvion, anti-CD3 antibody), Vitaxin (anti-angiogenic integrin avb3 antibody), Volociximab (anti-integrin α5β1), Votumumab (also known as HumaSPECT, anti-tumor antigen CTAA16.88 antibody), Zalutumumab (also known as HuMax-EGFr, (anti-EGFR antibody), Zanolimumab (also known as HuMax-CD4, anti-CD4 antibody), Ziralimumab (anti-CD147 (basic immunoglobulin) antibody), Zolimomab (zolimomab) (anti-CD5 antibody), etanercept (registered trademark "Enbrel"), alefase Alefacept (registered trademark "Amevive"), abatacept (registered trademark "Orencia"), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron regulatory protein 2) antibody], 14G2a (anti-cancer drug for melanoma and solid tumors from Nat. Cancer Institute) rioside GD2 antibody), J591 (anti-PSMA antibody for treating prostate cancer from Weill Cornell Medical School), 225.28S [anti-HMW-MAA (high molecular weight melanoma-associated antigen) antibody for melanoma, Sorin Radiofarmaci SRL (Milan, Italy)], CO L-1 (anti-CEACAM3 antibody, CGM1, from Nat. Cancer Inst. for colon and gastric cancer), CYT-356 (Oncoltad®, prostate cancer), HNK20 (for respiratory syncytial virus, from Ora Vax Inc.), ImmuRAIT (for non-Hodgkin's lymphoma, from IMMUNOMEDICS), Lym-1 (anti-HLA-DR10 antibody, for oncology, from Peregrine Pharm) , MAK-195F [anti-TNF (tumor necrosis factor; TNFA, TNF-α; TNFSF2) antibody for sepsis and toxic shock from Abbott / Knoll], MEDI-500 [also known as T10B9, anti-CD3 antibody, TRαβ (T-cell receptor α / β) for graft-versus-host disease from MediImmune Inc.], RING SCAN [anti-TAG72 (tumor-associated glycoprotein 72) antibody for breast cancer, colon cancer, and colorectal cancer from Neoprobe Corp.], Avicidin (anti-EPCAM (epithelial cell adhesion molecule) antibody), anti-TACSTD1 (tumor-associated calcium signal transducer 1) antibody, anti-GA733-2 (gastrointestinal tumor-associated protein 2) antibody, anti-EGP-2 (epithelial glycoprotein 2) antibody; anti-KSA antibody; KS1 / 4 antigen; M4S; tumor antigen 17-1A; NeoRx Corp. for colon cancer, ovarian cancer, prostate cancer, and non-Hodgkin's lymphoma CD326 for VEGF; LYMPHOCIDE, Smart ID10, Oncolym, Allomune, anti-VEGF antibody (Genentech, CA); CEAcide, IMC-1C1 1, and cetuximab.

[0139] Other antibodies that bind to the ligand include, but are not limited to, antibodies against the following antigens: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA242 (colorectal), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma). , CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3ε (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma and liquid tumors, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (carcinoma), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer, and lung cancer), DLL4 (Delta-like-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hemoglobin, solid tumors), endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL, prostate, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2; lung, breast, and prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28G (cell surface glycolipid antigen, melanoma) , GD3 idiotype (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor-1 receptor, solid tumors, blood cancer), IL-2 receptor (interleukin-2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin-6 receptor, multiple myeloma, RA, Castleman's disease, IL-6-dependent tumors), integrin (αVβ3, α5β1, α6β4, αIIβ3, α5β5, αVβ5 for various cancers), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MS4A1 (transmembrane 4-domain family A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colon), gp100 (melanoma) , MART1 (melanoma), MPG (melanoma), MS4A1 (transmembrane 4-domain family A member 1, small cell lung cancer, NHL), nucleolin, neuronal oncogene product (carcinoma), P21 (carcinoma), anti-(paratope of N-glycolylneuraminic acid) (breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate cancer), PSA (prostate), ROBO4, TAG72 (tumor-associated glycoprotein 72, leukemia (AML), gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer) , Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related apoptosis ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers).Other tumor-associated antigens recognized by antibodies have already been reported (Gerber, et al., mAbs 1:3, 247-253 (2009); Novellino et al., Cancer Immunol Immunother. 54 (3), ). 187-207 (2005)Franke et al, cancer biother radiopharm. 2000,15,459-76). anti Examples of these antigens for the body are: CD2, CD2R, CD3, CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD12w, CD13, CD14, CD15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CD17w, 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, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, 4, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47 R, CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, C D62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD74, CD75 , CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD93, C D94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, C D112, CD113, CDw113, CD114, CD115, CD116, CD117, CD118, CD119, CDw119, CD120a, CD120b, CD121a, CD121b, CDw121b, CD122, CD123, CDw123, CD124,CD125、CDw125、CD126、CD127、CD128、CDw128、CD129、CD130、CD131、CDw131、CD132、CD133、CD134、CD135、CD136、CDw136、CD137、CDw137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CDw145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CDw156c、CD157、CD158a、CD158b、CD159a、CD159b、CD159c、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175、CD176、CD177、CD178、CD179、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CDw186、CD187、CD188、CD189、CD190、Cd191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CDw198、CD199、CDw199、CD200、CD200a、CD200b、CD201、CD202、CD202b、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210、CD212、CD213a1、CD213a2、CDw217、CDw218a、CDw218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235ab、CD235b、CD236、CD236R、CD238、CD239、CD240、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD2、 53, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265, CD266, CD267, CD268, CD269, CD271 , CD273, CD274, CD275, CD276(B7-H3), CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338, CD339), Annexin A1, Nucleolin, Endoglin (CD105), ROBO4, Aminopeptidase N, Δ-like-3 (DLL3), Δ-like-3 (DLL4), VEGFR-2 (CD309), CXCR49 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPVE6E7, EGFRvIII, HER-2 / neu, Idiotype, MAGEA3, Non-mutant p53, NY-ESO-1, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, Proteinase 3 (PR1), bcr -abl, tyrosinase, survivin, hTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, PSCA, MAGEA1, sLe(a), CYP1B1, PLA C1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, Fos-related antigen 1.

[0140] The production of antibodies used in the present invention can involve in vivo or in vitro production processes, or a combination thereof. Methods for preparing anti-receptor peptide polyclonal antibodies are well known, as shown, for example, in U.S. Patent No. 4,493,795 (Nestor et al.). A typical method for preparing monoclonal antibodies involves fusing mouse spleen cells isolated from mice immunized with a specific antigen with myeloma cells (Kohler, G; Milstein, C. 1975. Nature 256:495-497). Detailed procedures are described in "Antibodies—A Laboratory Manual," Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988), the contents of which are incorporated herein by reference. In particular, monoclonal antibodies can be obtained by immunizing mice, rats, hamsters, or other mammals with the antigen of interest, which can be, for example, intact target cells, antigens isolated from target cells, whole viruses, weakened whole viruses, or viral proteins. Spleen cells are fused with myeloma cells using PEG6000. The resulting hybridomas are screened for their sensitivity to HAT. Hybridomas producing monoclonal antibodies useful in the practice of the present invention are identified by immunoreaction with or inhibition of receptor activity of specific target cell receptors.

[0141] The monoclonal antibodies used in the present invention can be obtained by initiating the culture of monoclonal hybridoma cells in a nutrient medium containing hybridoma cells secreting antibodies with appropriate antigen specificity. The culture must be maintained for a time and under conditions sufficient for the hybridoma cells to secrete the antibodies into the culture medium. After collecting the antibody-containing culture supernatant, Antibodies can be isolated by well-known techniques, such as protein A affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, and molecular sieve chromatography (particularly affinity chromatography and molecular sieve chromatography using antigen-crosslinked protein A), centrifugation, precipitation, or other standard methods for purifying proteins.

[0142] Media useful for preparing these compositions are well known in the art and commercially available, including synthetic media. An exemplary synthetic medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm / L glucose, 20 mm glutamine, 20% fetal bovine serum, and an antifoaming agent (e.g., polyoxyethylene-polyoxypropylene block copolymer).

[0143] Furthermore, besides cell fusion techniques, cell lines for producing antibodies can also be constructed by, for example, direct transformation of B lymphocytes with oncogenic DNA or by transfection with tumor viruses, such as Epstein-Barr virus (EBV, also known as human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated virus (KSHV) (see U.S. Patent 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 for details). It can be prepared by using anti-receptor peptides or peptides containing terminal carboxyl groups (for details, see Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Lei et al., Biochemistry 34(20):6675-6688 (1995)). Usually, anti-receptor polypeptides are used. The peptide or polypeptide analog can be used alone or in combination with a cross-linked immunogenic carrier as an immunogen to prepare monoclonal antibodies against the receptor polypeptide.

[0144] There are also many other well-known techniques for producing monoclonal antibodies as binding molecules in the present invention. Particularly useful are methods for producing fully human antibodies. One method is phage display technology, which can be used to select a series of human antibodies that specifically bind to an antigen using affinity enrichment methods. Phage display has been thoroughly described in the literature, and the construction and screening of phage display libraries are well known in the art, see, for example, Dente et al., Gene. 148(1): 7-13 (1994); Little et al, Biotechnol Adv. 12(3): 539-55 (1994); Clackson et al. Nature 352: 264-628 (1991); Huse et al. Science 246: 1275-1281 (1989), Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (2001) (O' Brienet al. ed. Human Press, Totowa, NJ) and Lee et al. J. Mol. Biol. 340: 1073-1093 (2004).

[0145] Monoclonal antibodies obtained from non-human species (e.g., mice) using hybridoma technology can be humanized to avoid human anti-mouse antibodies when administered to humans. Among these, well-known methods for antibody humanization are complementarity-determining region (CDR) grafting and remodeling. These methods have been extensively described, see, for example, U.S. Patent Nos. 5,859,205 and 6,797,492; Liu et al., Immunol Rev. 222:9-27 (2008); Almagro et al., Front Biosci. 1;13:1619-33 (2008); Lazar et al., Mol Immunol. 44(8):1986-98 (2007); Li et al., Proc. Natl. Acad. Sci. USA. 103(10):3557-62 (2006), the disclosures of which are incorporated by reference. Fully human antibodies retain most of the human immunoglobulin light and heavy chains. Transgenic mice can be prepared by immunizing transgenic mice, rabbits, monkeys, and other mammals with an antigen. Examples of such mice include the Xenomouse (Abgenix, Inc.), HuMab-Mouse (Medarex / BMS), and VelociMouse (Regeneron). For details, see U.S. Patent Nos. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806, 5,436,149, and 5,569,825. For human therapy, mouse antibody variable region genes and human antibody constant region genes can be fused to obtain constructs called "chimeric antibodies," which are much less immunogenic in humans than mouse antibodies (Kipriyanov et al., Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002)), the disclosures of which are incorporated by reference. Furthermore, site-directed mutagenesis of antibody variable regions can improve antibody affinity and specificity (Brannigan et al., Nat Rev Mol Cell Biol. 3:964-70 (2002); Adams et al., J. Immunol. Methods. 23 1:249-60(1999)). Cytotoxic effects can be enhanced by partially replacing the constant region of an antibody, effectively enhancing its affinity for immune effector cells.

[0146] Immunospecific antibodies against malignant cell antigens can be obtained commercially or by several conventional techniques, such as chemical synthesis or recombinant expression techniques. Similarly, nucleotide sequences encoding immunospecific antibodies against malignant cell antigens can be obtained commercially from the GenBank database or other similar databases, from the literature, or by routine cloning and sequencing.

[0147] DNA encoding monoclonal antibodies derived from hybridoma cells or phage-displayed Fv clones of the antibodies can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide primers designed to specifically amplify the regions encoding the desired heavy and light chains from a hybridoma or phage DNA template). Once isolated, the DNA can be inserted into an expression vector and then transfected into host cells such as Escherichia coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulin proteins, to produce the desired monoclonal antibody in a recombinant host cell (Skerra, et al., Curr. Opinion in Immunol., 5, 256 (1993); Pluckthun, Immunol. Revs., 130, 151 (1992)). Antibodies can be prepared by cleaving the expressed polypeptides to improve antibody production yields and ensure proper assembly of the antibody. Antibody antibodies can also be produced using expression systems that control the quantitatively measured ratios of the polypeptide components. Such control is achieved, at least in part, by simultaneously controlling the strength of translation of the polypeptide components. After fermentation as known, the resulting antibody protein is further purified to obtain a substantially homogeneous product for further analysis and use. Known standard protein purification methods can be used. Exemplary purification methods include immunoaffinity fractionation (e.g., protein A column) or ion exchange column fractionation, ethanol precipitation, reverse-phase HPLC, silica gel column chromatography or cation exchange column chromatography (e.g., DAEA), chromatographic separation chromatography, SDS-PAGE, ammonium sulfate precipitation, and gel filtration (e.g., Sephadex G-75).

[0148] In addition to antibodies, polypeptides or proteins that bind / block / attack or otherwise interact with the corresponding receptor or epitope on the surface of target cells can be used as binding molecules. As long as these peptides or proteins can specifically bind to the epitope or its corresponding receptor, they do not have to belong to the immunoglobulin family. These polypeptides can also be isolated by techniques similar to phage display antibodies. (Szardenings, J Recept Signal Transduct Res. 2003;23(4):307-49). Peptide fragments obtained from random peptide libraries are similar in application to antibodies and antibody fragments. Peptide or protein binding molecules may be conjugated or bound to macromolecules or vehicles, such as, but not limited to, albumin, polymers, liposomes, or nanoparticles, as long as the antigen-binding specificity of the peptide or protein is maintained.

[0149] The cell-binding molecule / ligand or cell receptor agonist can be an Ig-based or non-Ig-based protein scaffold molecule. Ig-based scaffolds include, but are not limited to, nanobodies (VHH (camelid Ig) derivatives) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-797); domain antibodies (dAbs, VH or VL domain derivatives) (Holt, L. J, et al., 2003, Trends Biotechnol. 21, 484-490); bispecific T cell endonucleases (BECs) ... Gaeger (BiTE, bispecific dimer) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); Dual affinity retargeting (DART, bispecific dimer) (Moore PA P, et al. 2011, Blood 117(17), 4542-4551);Tetravalent tandem antibody (TandAb , bispecific dimers) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341). Non-Ig scaffolds can be selected from, but are not limited to, anticalins (derivatives of lipocalins) (Skerra A. 2008, FEBS J., 275(11): 2677-2683; Beste G, et al. al, 1999 Proc. Nat. Acad. USA. 96(5):1898-1903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2):337-350; Skerra, A. 2007, Curr Opin Biotechnol. 18(4):295-304; Skerra, A. 2008, FEBS J. 275(11):2677-2683); Adnectins (10FN3 (fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4):1141-1151; Batori V, 2002, Protein Eng. 15(12): 1015-1020; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2):363-371; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4):211-219); Developed ankyrin repeat proteins (DARPins) (derivatives of ankyrin repeat (AR) proteins) (Boersma, Y. L., et al., 2011 Curr Opin Biotechnol. 22(6): 849-857), such as DARPinC9, DARPinEc4, and DARPinE69_LZ3_E01 (Winkler J., et al., 2009 Mol Cancer Ther. 8(9), 2674-2683; Patricia M. K. M., et al., Clin Cancer Res. 2011;17(1):100-110; Boersma Y. L., et al., 2011 J. Biol. Chem. 286(48),41273-41285; Avimer (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):1556-1561) You can choose.

[0150] Examples of small molecule structures of cell binding molecules / ligands or cell receptor agonists of the present patent application are as follows, the structures of which are shown below: LB01 (folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, somatostatin analog), LB08 (ramareotide, somatostatin analog), LB09 (vapreotide (sambar), somatostatin analog). , LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (gastrin-releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, abarelix), LB16 (cobalamin, vitamin B12 analog), LB17 (cobalamin, vitamin B12 analog), LB18 (α v β3 integrin receptor), LB19 (heterobivalent peptide ligand for VEGF receptor), LB20 (neuromedin B), LB21 (bombesin for G protein-coupled receptor), LB22 (TLR2 for toll-like receptor), LB23 (for androgen receptor), LB24 (α v Cilengitide / cyclo(-RGDfV-), LB23 (Fluid) for integrin receptors LB25 (rifabutin analogs), LB26 (rifabutin analogs), LB27 (rifabutin analogs), LB28 (fludrocortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (beclomethasone dipropionate), LB32 (triamcinolone acetonide), LB33 (prednisone), LB34 (prednisolone), LB35 (methylprednisolone), LB36 (betamethasone), LB37 (irinotecan analogs), LB38 (crizotinib analogs), LB39 (bortezomib analogs), carfilzo mib analog), LB40 (carfilzomib analog), LB41 (carfilzomib analog), LB42 (leuprolide analog), LB43 (triptorelin analog), LB44 (clindamycin), LB45 (liraglutide analog), LB46 (semaglutide analog), LB47 (retapamulin analog), LB48 (indibulin analog), LB49 (vinblastine analog), LB50 (lixisenatide analog), LB51 (osimertinib analog), LB52 (nucleoside analog), LB53 (erlotinib analog), or LB54 (lapatinib analog);

[0151] [ka] TIFF0007811398000062.tif232153TIFF0007811398000063.tif204138TIFF0007811398000064.tif223151TIFF0007811398000065.tif207151 TIFF0007811398000066.tif210152TIFF0007811398000067.tif233161TIFF0007811398000068.tif226149TIFF0007811398000069.tif110149

[0152] During the ceremony, [ka] is the moiety for attaching the side chain linker of the present invention; X4 and Y1 are independently O, NH, NHNH, NR1, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH2, C(O)NHNHC(O), and C(O)NR1; X1 is H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1, or C(O)O; X5 is H, CH3, F, or Cl; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, N(R1R2R3R4); R1, R2, R3, and R4 are defined in formula (I).

[0153] Any one of several different reactive groups on a cell-binding molecule, preferably an antibody, may be the conjugation site, such as the ε-amino group of a lysine residue, the side chain of a carbohydrate residue, a carboxylic acid group, a disulfide group, or a thiol group. For reviews of antibody reactive groups suitable for conjugation, see GT Hermanson, Bioconjugate Techniques, Academic Press, 2008; Garnett, Adv. Drug Delivery Rev., 2001, 53, 171-216; Dubowchik, Walker, Pharmacology & Therapeutics, 1999, 83, 67-123, etc., the disclosures of which are incorporated herein by reference.

[0154] The cytotoxic agents of the present invention can be conjugated to cell-binding molecules directly or via bifunctional linkers or cross-linking reagents. The bifunctional linkers contain two reactive groups: one that reacts with the cell-binding molecule, while the other can react with one or more of the cytotoxic molecules of the present invention. Bifunctional linkers are widely known in the art (e.g., U.S. Patent 5,208,020; Isalm and Dent in "Bioconjugation" chapter 5, pp. 218-363, Groves Dictionaries Inc. New York, 1999). Examples of bifunctional linkers include N-succinimidyl- 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)valerate (SPP), N-succinimidyl-3-(2-pyridyldithio)butyrate (SDPB), 2-iminothiolane, N-succinimidyl-4-(5-nitro-2-pyridyldithio)butyrate (SNPB), N-succinimidyl- 4-(5-nitro-2-pyridyldithio)valerate (SNPP), N-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio)butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)valerate (SMNP), N-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio)valerate (SSNPP), 4-succinimidyl-oxycarbonyl-α-methyl-α -(2-pyridyldithio)-toluene (SMPT), N-sulfosuccinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)valerate (SSMNP), N-succinimidyl-4-methyl-4-(2-pyridyldithio)valerate (SMPDP), N-succinimidyl-4-(5-N,N-dimethyl-carboxamido-2-pyridyldithio)butyrate (SCPB), N-sulfosuccinimidyl-4-(5-N,N-dimethyl N-carboxamido-2-pyridyldithio)butyric acid ester (SSCPB), N-succinimidyl-4,4-dimethyl-4-(2-pyridyldithio)valeric acid ester (SDMPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(iodoacetyl)-aminobenzoic acid ester (SIAB), bismaleimide polyethylene glycol (BMPEG), BM(PEG) 1-20 , N-(β-maleimidopropyloxy)-succinimide ester (BMPS), iminothiolane (IT), dimethyl adipimidate hydrochloride or derivatives of imidate esters, active esters (disuccinimidyl suberate) 1614666467490_0 , aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-( p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), bis-activated fluorine-based compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), γ-maleimidobutyric acid N-succinimide ester (GMBS), E-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-aminocaproic acid ester) (long-chain analog of SMCC (LC-SMCC)), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4 -(4-N-maleimidophenyl)-butyric acid hydrazide or hydrochloride (MPBH), N-succinimidyl-3-(bromoacetamido)propionate (SBAP), N-succinimidyl-iodoacetate (SIA), κ-maleimidoundecanoic acid succinimide ester (KMUA), N-succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl-6-(β-maleimidopropionylamido)-hexa Phosphonic acid ester (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate ester (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(γ-maleimidobutyloxy)sulfosuccinimide ester (sulfo-GMBS), N-(ε-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), N-(κ-maleimido or commercially available linkers (e.g., imidoester conjugates available from Thermo Scientific's Pierce: DMA (dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate dihydrochloride), DMS (dimethyl suberimidate dihydrochloride), DTBP (dimethyl 3,3-dithiobispropionimidate dihydrochloride); NHS-ester crosslinking amines: BS(PEG)5 (bis(succinimidyl)penta(ethylene glycol), BS(PEG)9 (bis(succinimidyl)nona(ethylene glycol), BS 3 (Bis[sulfosuccinimidyl]suberate), BSOCOES (Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfonate), DSG (Disuccinimidyl glutarate), DSP (Dithiobis[succinimidyl]propionate), DSS (Disuccinimidyl suberate), DST (Dithiobis[succinimidyl]tartrate) succinimidyl), DTSSP (3,3'-dithiobis[sulfosuccinimidyl propionate]), EGS (ethylene glycol bis[succinimidyl succinate]), Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)), TSAT (tris-succinimidyl aminotriacetate), DFDNB (1,5-difluoro-2,4-dinitrobenzene); amine-sulfhydryl crosslinker: Sulfo-SI AB (Sulfosuccinimidyl (4-iodoacetyl)aminobenzoate), SIAB (Succinimidyl (4-iodoacetyl)aminobenzoate), SBAP (Succinimidyl-3-(bromoacetamido)propionate), SIA (Succinimidyl N-iodoacetate), Sulfo-SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), SM (PEG) n(NHS-PEG-maleimide crosslinker: succinimidyl-([N-maleimidopropionamido]-ethylene glycol) ester, #=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24), LC-SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproic acid ester)), Sulfo-EMCS (N-ε-maleimidocaproyloxy sulfosuccinimide ester), EMCS (N-ε-maleimidocaproyloxy sulfosuccinimide ester), Sulfo-GMBS (N-γ-maleimidobutyloxysulfosuccinimide ester), Sulfo-GMBS (N-γ-maleimidobutyloxysulfosuccinimide ester), GMBS (N-γ-maleimidobutyloxysuccinimide ester), Sulfo-KMUS (N-κ-maleimidoundecanoyloxysulfosuccinimide ester), Sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-S MPB ((Sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate ester), SMPB ((Succinimidyl-4-(p-maleimidylphenyl)butyrate ester), AMAS (N-(α-maleimidoacetoxy)succinimide ester), BMPS (N-β-maleimidylpropyloxysuccinimide ester), SMPH (Succinimidyl-6-(β-maleimidopropionamido)hexanoate ester), PEG12-SPDP (2-pyridyldithiol-tetraoxaoctatriacontane-N-hydroxybenzoate), hydroxysuccinimide ester), PEG4-SPDP (2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide ester), Sulfo-LC-SPDP (sulfosuccinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoic acid ester), LC-SPDP (succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoic acid ester), SMPT (4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene);Carbonyl-amine crosslinkers: DCC (dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide); photoactivated crosslinkers: ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide ester); NHS-diazirine (SDA) crosslinkers: SDA (NHS-diazirine) (succinimidyl-4,4'; -azipentanoate), LC-SDA (NHS-LC-diazirine) (succinimidyl-6-(4,4'-azipentanamido)hexanoate), SDAD (NHS-SS-diazirine) (succinimidyl-2-([4,4'-azipentanamido]ethyl)-1,3'-dithiopropionic acid ester), Sulfo-SDA (Sulfo-NHS-diazirine) (sulfosuccinimidyl-4,4'-azipentanoate), Sulfo-LC-SDA (Sulfo-NHS-LC-diazirine) (sulfosuccinimidyl-6-(4,4'-azipentanamido)hexanoate), Sulfo-SD AD (Sulfo-NHS-SS-diazirine) (sulfosuccinimidyl-2-([4,4'-azipentanamido]ethyl)-1,3'-dithiopropionic acid ester), Sulfo-SANPAH (sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoic acid ester), SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyric acid ester); sulfhydryl-hydrocarbon crosslinkers: BMPH (N-β-maleimidylpropionic acid hydrazide-TFA), EMCH (N-ε-maleimididocaproic acid hydrazide-TFA), KMUH (N-κ-maleimidoundecanoic acid hydrazide-TFA), MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride), PDPH ( 3-(2-pyridyldithio)propionylhydrazide; sulfhydryl-hydroxy crosslinker: PMPI (p-maleimidophenyl isocyanate); sulfhydryl-sulfhydryl crosslinker: BM(PEG)2 (1,8-bismaleimido-diethylene glycol), BM(PEG)3 (1,11-bismaleimido-triethylene glycol), BMB (1,4-bismaleimidobutane), BMDB (1,4-bismaleimido-2,3-dihydroxybutane), BMH (bismaleimidohexane), BMOE (bismaleimidoethane), DTME (dithiobismaleimidoethane), TMEA (tris(2-maleimidoethyl)amine), and SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0155] Bismaleimide or bis-2-pyridyldithiol reagents can be used to sequentially or concurrently connect the thiol groups of thiol-containing cell-binding molecules (e.g., antibodies) with thiol-containing drug residues, markers, or linker intermediates. Other functional groups besides bismaleimides and pyridyldithiols that can react with thiol-containing cell adhesion molecules, drug residues, markers, or linker intermediates include, for example, iodoacetamide, bromideacetylamide, vinylpyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

[0156] In additional embodiments, the conjugate may be composed of one or more conjugate moieties. Exemplary conjugate moieties are as follows:

[0157] 1. Examples of self-immolative linker components: [ka]

[0158] In the formula, ( * Atoms labeled with a ) may be additional spacers or releasable linker units, or is the point of attachment to the cytotoxic agent and / or binding molecule (CBA); X1, Y1, Z2, and Z3 are independently NH, O, or S; Z 1 are independently H, NH, O, or S. v is 0 or 1; Q 1 are independently H, OH, C1-C6 alkyl, (OCH2CH2) n , F, Cl, Br, I, OR1, SR1, NR1R2, N=NR1, N=R1, NR1R2, NO2, SOR1R2, SO2R1, SO3R1, OSOR1, PR1R2, POR1R2, PO2R1R2, OPO(OR1)(OR2), or OCH2PO(OR1(OR2)), where R1 and R2 are as defined above and are preferably H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or glycoside; or a medicinal cation salt.

[0159] 2. Examples of non-self-immolative linker components: [ka] TIFF0007811398000073.tif129157

[0160] In the formula, ( * Atoms labeled with X are sites of attachment for additional spacers or releasable linkers, cytotoxic agents, and / or binding molecules; 1, Y 1 , Q 1 The definitions of R1, R', and R'' are as described above. r is 1 to 20. m and n are 1 to 6.

[0161] 3. Exemplary linker moieties may include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ("SMCC"), N-succinimidyl(4-acetyl)amino-benzoate ("SIAB"), ethyleneoxy (-CHCHO-) ("EO" or "PEO") as one or more repeating units. Additional linker moieties are known in the art, and some are described throughout this patent application.

[0162] In additional embodiments, the conjugate may comprise an amino acid residue. Exemplary amino acid conjugate moieties include a dipeptide, tripeptide, tetrapeptide, or pentapeptide. Exemplary dipeptides include valine-citrulline ("VC" or "val-cit"), alanine-phenylalanine ("af" or "ala-phe"). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues that comprise an amino acid conjugate moiety include naturally occurring as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. An amino acid conjugate moiety may be a specific These can be designed and optimized for selectivity of enzymatic cleavage by enzymes such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0163] In the cell-binding agent-drug conjugates of the invention, the cell-binding agent (Q) is conjugated via a bifunctional linker (L) to one or more drug moieties (drugs or PBD derivatives), e.g., about 1 to about 20 drug moieties per cell-binding agent. Conjugates of formulas (I), (II), (III), and (IV) can be prepared by several routes using organic chemistry reactions, conditions, and reagents known in the art, including: (1) reaction of a nucleophilic group on the cell-binding agent with a bivalent linker reagent to form QL via a covalent bond, followed by reaction with a drug moiety; or (2) reaction of a nucleophilic group on the drug moiety with a bivalent linker reagent to form drug-L via a covalent bond, followed by reaction with a nucleophilic group on the cell-binding agent.

[0164] To synthesize conjugates of general formulas (I), (II), (III), and / or (IV), functional groups E3 and / or E3' of general formulas (V), (VI), (VII), and (VIII) are reacted with one, two, or more residues of a cell-binding molecule in an aqueous medium at 0-60°C and pH 5-9.5, with or without 0-30% water-miscible organic solvents (DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene glycol), followed by dialysis or chromatographic purification to form conjugate compounds of formulas (I), (II), (III), and / or (IV). Some residues of the cell-binding molecule (reactive groups for conjugation) can be obtained by protein engineering.

[0165] Thiol or amino groups on cell-binding agents (such as antibodies) are nucleophilic and can form covalent bonds with electrophilic groups on linker reagents and drug linker intermediates, including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehyde, ketone, carboxyl, and maleimide groups; and (iv) disulfides such as pyridyl disulfides via sulfide exchange. Nucleophilic groups on drug moieties include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylic acid, and arylhydrazide groups, which can react with electrophilic groups on linker residues and linker reagents to form covalent bonds.

[0166] Nucleophilic groups on antibodies or proteins can react with a cytotoxic agent and then with an electrophilic group on a functional linker, or they can react directly with the linker-cytotoxic agent moiety to form a covalent cell-binding agent-cytotoxic agent conjugate. Nucleophilic groups on antibodies or proteins include, but are not limited to, (i) the N-terminal amino group, (ii) side chain amino groups such as lysine, (iii) side chain thiol groups such as cysteine, and (iv) sugar hydroxy or sugar amino groups on glycosylated antibodies. Amine, thiol, and hydroxy groups are nucleophilic and can react with electrophilic groups on the linker moiety and the linker-cytotoxic agent moiety to form covalent bonds, including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Some antibodies have reducible interchain disulfide bonds, i.e., cysteine ​​bridges, that can be rendered reactive by treatment with reducing agents such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) (Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.), dithioerythritol (DTE), L-glutathione (GSH), 2-mercaptoethylamine (β-MEA), or / and β-mercaptoethanol (β-ME, 2-ME). Thus, each cysteine ​​bridge would theoretically form two reactive thiol nucleophiles. Alternatively, sulfhydryl groups can be converted to thiols by, for example, reacting lysine residues with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. Reactive thiol groups can be introduced into antibodies via modifying lysine residues by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more non-naturally occurring cysteine ​​amino acid residues). Thus, free thiols on a cell-binding agent can be conjugated to thiol-reactive groups, such as maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive groups on a cytotoxic agent or conjugate-cytotoxic agent intermediate of the invention. Some unconjugated free thiols on an antibody can be reoxidized to reform inter- and intrachain disulfide bonds.

[0167] The antibody-drug conjugates of the present invention can also be produced by the reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, and a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one embodiment, an antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. In another embodiment, the sugars of a glycosylated antibody may be oxidized, such as with a periodate oxidation reagent, to form aldehyde or ketone groups that can react with amino groups on a linker reagent or drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, such as with a borohydride reagent, to form a stable amine bond. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on a drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to generate an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992), Bioconjugate Chem. 3:1). 38-146; U.S. Patent No. 5,362,852). Such aldehydes can be reacted with nucleophiles on the drug moiety or linker.

[0168] Examples of these types of two-step bonds are shown below: [ka]

[0169] where E includes, but is not limited to, hydroxysuccinimidyl esters (such as NHS and sulfo-NHS), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl (including sulfo-tetrafluorophenyl) esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. R' and R" are independently H, CH, or C2H5; J is F, Cl, Br, I, tosylate (TsO), mesylate (MsO), nitrophenol, dinitrophenol, or pentafluorophenol.

[0170] When two or more nucleophilic groups are present on the cell-binding agent, a reagent such as an antibody can be reacted with a drug-conjugate intermediate or conjugate reagent, followed by a drug residue reagent. The resulting product is a mixture of cell-binding agent-cytotoxic agent conjugates with a distribution of one or more drug residues attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by antibody-specific, drug-specific double ELISA antibody assay. Individual conjugate molecules are identified in the mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography. In certain embodiments, homogeneous conjugates with a single loading value can be isolated from the conjugate mixture by electrophoresis or chromatography.

[0171] In conjugates, the loading of the ADC (drug / antibody ratio) can be controlled in various ways, such as: (i) limiting the molar excess of the drug-conjugate intermediate or conjugate reagent compared to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limited reduction conditions of cysteine ​​thiol modifications; and (iv) recombinantly engineering the amino acid sequence of the antibody so that the number and position of lysine or cysteine ​​residues are altered to control the number or position of the conjugate-drug linkages (e.g., For example, it can be controlled by thioMab or thioFab).

[0172] The synthesized conjugates can be purified by standard biochemical methods, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, ion exchange, or dialysis. In some cases, when small molecules (e.g., folic acid, melanocyte-stimulating hormone, EGF, etc.) are conjugated to small molecule drugs as cell-binding molecules, they can be purified by chromatography, such as HPLC, medium-pressure column chromatography, or ion-exchange chromatography.

[0173] Aqueous solutions for modifying cell-binding agents are buffered to a pH between 4 and 9, preferably between 6.0 and 7.5, and may contain non-nucleophilic buffer salts useful in these pH ranges. Representative buffers include phosphate, acetate, triethanolamine HCl, HEPES, and MOPS buffers, and may further contain additional components such as cyclodextrin, hydroxypropyl-β-cyclodextrin, polyethylene glycol, sucrose, and salts (e.g., NaCl, KCl). After adding the drug conjugate of Formula (V), (VI), (VII), or (VIII) to the solution containing the reduced cell-binding molecule, the reaction mixture is incubated at ambient temperature between 4°C and 55°C, preferably 15°C. The progress of the reaction can be monitored by measuring the decrease in absorbance at 252 nm, the increase in absorbance at 280 nm, or other suitable wavelengths. After the reaction is complete, isolation of the modified cell-binding agent can be carried out by conventional methods, such as gel filtration chromatography, ion exchange chromatography, adsorption chromatography, or column chromatography on silica gel or alumina, crystallization, preparative thin layer chromatography, ion exchange chromatography, or HPLC.

[0174] The degree of modification can be assessed by measuring the absorbance of the emitted nitropyridinethione, dinitropyridinedithione, pyridinethione, carboxyamidopyridinedithione, and dicarboxyamidopyridinedithione groups via UV spectroscopy. For conjugates without chromophore groups, the modification or conjugation reaction can be monitored by LC-MS, preferably UPLC-QTOF mass spectrometry, or capillary electrophoresis (CEMS). The side-chain crosslinkers described herein have a variety of functional groups that can react with any drug, preferably a cytotoxic drug, bearing the appropriate substituent. For example, modified cell-binding molecules bearing amino or hydroxy substituents can react with drugs bearing N-hydroxysuccinimide (NHS) esters, and modified cell-binding molecules bearing thiol substituents can react with drugs bearing maleimide or haloacetyl groups. Furthermore, modified cell-binding molecules bearing carbonyl substituents (ketones or aldehydes) can react with drugs bearing hydrazides or alkyloxyamines. One of skill in the art can readily determine which linker to use based on the known reactivity of the available functional groups on the linker.

[0175] Other additional exemplary methods for preparing ADCs are described in Figures 1-32 and in the examples within the patent specification.

[0176] Application of cell-binding agent and cross-linking PBD-reducing agent conjugates.

[0177] The cell-binding agent-crosslinked PBD dimer conjugates of the present invention, preferably antibody-crosslinked PBD dimer conjugates (PBD dimer ADCs), can be used to treat a variety of diseases or disorders, such as those characterized by overexpression of tumor antigens. Exemplary diseases or hyperproliferative disorders include benign or malignant tumors, leukemias, and lymphoid malignancies. Others include neuronal, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastoid, inflammatory, angiogenic, and immune disorders, including autoimmune disorders.

[0178] In another specific embodiment, the conjugates of the invention are used in the treatment of cancer, in accordance with the compositions and methods of the invention, including, but not limited to, adrenocortical carcinoma, anal carcinoma, bladder cancer, brain tumors (adult, brainstem glioma, childhood, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumor, visual pathway and hypothalamic glioma), breast cancer, carcinoid tumor, gastrointestinal, carcinoma of unknown primary, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal carcinoma, extrahepatic bile duct carcinoma, Ewing family Tumors (PNET), extracranial malignant germ cell tumors, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumors, extragonadal, gestational trophoblastic tumors, head and neck cancer, hypopharyngeal cancer, pancreatic islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal carcinoma, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, small cell, lymphoma (AIDS-related, central nervous system, skin T-cell) alveolar cysts, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, metastatic squamous neck cancer of unknown primary site, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative syndrome, nasopharyngeal carcinoma, neuroblastoma, oral cancer, pharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low malignant potential tumor), pancreatic cancer (exocrine gland, islet cell carcinoma), paranasal sinus and nasal cavity cancer, parathyroid carcinoma, These include penile cancer, pheochromocytoma cancer, pituitary cancer, plasmacytoma, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma (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 cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), abnormal cancers of children, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0179] In another specific embodiment, the cell-binding molecule-drug conjugate via a bridge linker of the present invention can be used to treat or prevent autoimmune diseases, depending on its components and methods, including, but not limited to, autoimmune gastric achlorhydria, chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GMB / TBM nephritis, antiphospholipid syndrome, antisynthetase syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral nervous system disease, autoimmune pancreatitis, multiple autoimmune endocrinopathy types I, II, and III, autoimmune progesterone-induced dermatitis, autoimmune thrombocytopenic purpura, and autoimmune steroid ... Immune uveitis, Barlow's disease / Barlow's concentric sclerosis, Behçet's disease, Berger's disease, Bickerstaff's encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, allergic granulomatous vasculitis, cicatricial pemphigoid, celiac disease, Cogan's syndrome, cold agglutinin disease, complement component C2 deficiency, arteritis capitis, CREST syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic vasculitis, malignant atrophic papulosis, solitary steatoderma, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse cutaneous scleroderma, myocardial infarction, Discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, idiopathic mixed cryoglobulinemia, Evans syndrome, fibrodysplasia ossificans progressiva, fibromyalgia, fibromyositis, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes gestationis, hidradenitis suppurativa, Hughes' syndrome (antiphospholipid antibody syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic hematopoietic syndrome Thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, mucocutaneous lymph node syndrome, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), Lou Gehrig's disease (amyotrophic lateral sclerosis), lupus-like hepatitis, lupus erythematosus, Blau syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, scleroderma, Mucha-Jakob disease, Muckle-Wells syndrome syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neuromuscular, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatism, Panda syndrome (childhood autoimmune neuropsychiatric disorder complicated by streptococcal infection), tumor-associated cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, Parsonage-Georgia syndrome, pars planus inflammation, pemphigus, pemphigus vulgaris, pernicious anemia , perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathies, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplastic anemia, Rasmussen's encephalitis, Raynaud's disease, relapsing polychondritis, Reiter's syndrome, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, These include Sjögren's syndrome, spondyloarthropathy, sticky blood syndrome, Still's disease, stiff-man syndrome, subacute bacterial endocarditis, Susac's syndrome, acute febrile neutrophilic dermatosis, Sydenham's chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, and Westcott-Aldrich syndrome.

[0180] In another specific embodiment, binding molecules used to conjugate via the bridge linkers of the present invention for the treatment or prevention of autoimmune diseases include, but are not limited to, anti-elastin antibodies; Abys anti-epithelial cell antibodies; anti-basement membrane type IV collagen protein antibodies; anti-nuclear antibodies; anti-double-stranded DNA antibodies, anti-single-stranded DNA antibodies, anti-cardiolipin antibodies IgM, IgG; anti-celiac antibodies; anti-phospholipid antibodies IgK, IgG; anti-SM antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies, T-cell antibodies; thyroglobulin antibodies, anti-scleroderma-70 antibodies (AntiSCL-70); These include anti-Jo antibodies (Anti-Jo), anti-U1RNP antibodies (Anti-U1RNP); anti-La / SSB antibodies; anti-SSA antibodies; anti-SSB antibodies; anti-parietal cell antibodies; anti-histone antibodies; anti-RNP antibodies; C-ANCA; P-ANCA; anti-centromere antibodies; anti-fibrin antibodies, anti-GBM antibodies, anti-ganglioside antibodies; anti-desmosomal glycoprotein 3 core antibodies (anti-Desmogein3); anti-p62 antibodies; anti-sp100 antibodies; anti-mitochondrial (M2) antibodies; rheumatoid factor antibodies; anti-MCV antibodies; anti-topoisomerase antibodies; and anti-neutrophil cytoplasmic (cANCA) antibodies.

[0181] In certain preferred embodiments, the binding molecule for the conjugate of the invention is capable of binding to a receptor or receptor complex expressed on activated lymphocytes associated with autoimmune disease. The receptor or receptor complex can be, for example, a member of the immunoglobulin gene superfamily (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD90, CD125, CD147, CD152 / CTLA-4, PD-1, or ICOS), a member of the TNF receptor superfamily (e.g., CD27, CD40, CD95), or a member of the TNF receptor superfamily (e.g., CD27, CD40, CD95). / Fas, CD134 / OX40, CD137 / 4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (C-type, S-type, or I-type), or complement regulatory proteins.

[0182] In another specific embodiment, useful binders immunospecific for viral or bacterial antigens are humanized or human monoclonal antibodies. As used herein, the term "viral antigen" includes any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD, and gE), ... and Hepatitis B surface antigen). As used herein, the term "bacterial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule capable of eliciting an immune response (e.g., bacterial, fungal, pathogenic protozoan, yeast polypeptides (e.g., LPS and 5 / 8)). Type I antibodies useful in treating viral or bacterial infections include palivizumab (used to treat RVS infections). These include, but are not limited to, HIV-1 (humanized anti-respiratory syncytial virus monoclonal antibody), PRO542 (CD4 fusion antibody used to treat HIV infection), Ostavir (human antibody used to treat hepatitis B virus), PROTVIR (humanized IgG1 antibody used to treat cytomegalovirus), and anti-LPS antibodies.

[0183] The cell-binding molecule-cytotoxic agent conjugates of the present invention can be used to treat infectious diseases, including Acinetobacter infections, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immune deficiency syndrome), amebiasis, Anaplasmosis, Bacillus anthracis, bacterial tuberculosis infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, Balantidiosis, Strongyloides lumbricoides infection, BK virus infection, black sand mites, Blastocys hominis infection, Blastomyces, Bolivian hemorrhagic fever, Borrelia infection, and botulism. Poisoning (and infant botulism), Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (norovirus, sapovirus), Campylobacter infection, Candida infection (candidiasis, thrush), cat scratch disease, cellulitis, Chagas disease (American trypanosomiasis), chancroid, chickenpox, Clostridium difficile, Clostridium pneumoniae infection, cholera, chromomycosis, liver fluke disease, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever, cold (acute viral nasopharyngitis, acute rhinitis), Creutzfeldt-Jakob disease, Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcus, Cryptosporidium, cutaneous larval migration, Cyclospora infection, cysticercosis, cytomegalovirus infection, dengue fever, dienteramebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, hydatid disease, ehrlichiosis, pinworms (pinworm infection), enterococcal infection, enterovirus infection, typhus, erythema infectiosum (fifth disease), acute exanthema in children, fascioliasis, fluke thrombocytopenia, fatal familial insomnia, filariasis, food poisoning caused by Clostridium perfringens, non-parasitic amoebic infection, fuzobium infection, gas gangrene (Clostridial myonecrosis), diotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, glanders, gnathostomiasis, gonorrhea, inguinal granuloma (donovanosis), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand, foot and mouth disease (HFMD), hantavirus pulmonary syndrome, Helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human Balkan virus infection,Human ehrlichiosis Evans, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, microtaeniasis, influenza, isosporosis, Kawasaki disease, mononucleosis, Mycobacterium kumamotoi infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, malaria Burg hemorrhagic fever, measles, melioidosis (Whitmore's disease), meningitis, meningococcal disease, metagonism, microsporidiosis, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis (ophthalmia neonatorum), Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (blinding filariasis), paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasteurellosis, head lice (head lice), body lice (body lice), pubic lice (Crab lice, Crabb lice) ice), pelvic inflammatory disease, whooping cough, plague, pneumococcal infection, Pneumocystis carinii pneumonia, pneumonia, polio, Prevotella infection, PAME, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, Infection, rhinovirus infection, rickettsial infection, rickettsia, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, schistosomiasis, sepsis, diarrhea (dysentery), shingles (Herpes zoster), smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, nematodes, syphilis, taeniasis, tetanus (openis), tinea barbae (Barber's disease) itch), tinea manubriata, pityriasis nigricans, tinea pedis, tinea unguium, tinea versicolor, toxocariasis (ocular larva migrans), toxocariasis (visceral larva migrans), toxoplasmosis, Trichinella spiralis, trichomoniasis, cryptobiosis (whipworm infection), pulmonary tuberculosis, tularemia, urea-degrading Mycoplasma infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, rhizobacterium tuberculosis, Mycobacterium pseudotuberculosis infection, yersiniosis, yellow fever, and zygomycosis.

[0184] The cell binding molecules, more preferably antibodies against pathogenic strains described in this patent include Acinetobacter baumannii, Actiomyces israelii, Actinomyces gerenseriae, Propionibacterium propionicus, Trypanosoma brucei, HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma spp., Bacillus anthracis, and Arcanobacterium methylschaemolyticum. haemolyticum), Junin virus, Roundworms, Aspergillus, Astroviridae, Babesia, Bacillus cereus, Multiplex bacteria, Bacteroides, Colonic pouch ciliates, Roundworm of Bayley, Strongyloides, BK virus, Piedraia hortae, Blastocystis hominis, Dermatitis ulcerans, Macupo virus, Borrelia, Clostridium botulinum, Sabia, Brucella, typically Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter, typically Candida albicans and other Candida species, Bartonella henselaehenselae), Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella-zoster virus (VZV), Chlamydia trachomatis, Chlamydia pneumoniae, Vibrio cholerae, Fonseca pedrosoi, Clonorchiasis, Clostridium difficile, Coccidioides immitis, Coccidioides posadasi, Colorado tick fever virus, rhinovirus, coronavirus, Creutzfeldt-Jakob disease prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium spp., Ancylostoma cati, Co-parasites, Cyclospora, Taenia solium, Cytomegalovirus, Dengue virus (DE N-1, DEN-2, DEN-3 and DEN-4) - Flavivirus, Dentameba, Corynebacterium diphtheriae, Diphyllobothrix spp., Dracunculus medinensis, Ebola virus, Echinococcus spp., Ehrlichia spp., Pinworms, Enterococcus spp., Enterovirus spp., Typhi Rickettsia, Parvovirus B19, Human Herpesvirus 6, Human Herpesvirus 7, Fluke, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea, Clostridium perfringens, Fusobacterium, Clostridium perfringens, other Clostridia, Geotrichum candidum, GSS prion, Giardia lamblia lamblia), Burkholderia mallei, Gnathostoma nematode, Gnathostoma nigricans, Neisseria gonorrhoeae, Bordetella granulomatosis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, enteric viruses, most Coxsackievirus A, enteric virus type 71, Sin Nombre virus, Helicobacter pylori, Lori, Escherichia coli O158:H7, Bunyaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus type 1, Herpes simplex virus type 2, Histoplasma capsulatum, Oncocytoplasm of the duodenum, American hookworm, Haemophilus influenzae, Boca human virus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza virus, Tapeworm dwarf, Tapeworm diminuta, Epstein-Barr virus, Orthomyxoviridae, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella otsena, Klebsiella Klebsiellarhinoscleromotis, Coulour Provence, Lassa Fever Virus, Legionella pneumophila, Leishmania, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira spp., Listeria monocytogenes, Borreliosis and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic Choriomeningitis Virus (LCMV), Plasmodium spp., Marburg virus, Measles virus, Burkholderiapseudomallei), Neisseria meningitidis, Fluke yokogawai, Microsporidia, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, various bacteria (actinomycetoma) and fungi (mycosis fungi), Diptera parasitic fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Blastomyces brasiliensis, Paragonimus and other Paragonimus species, Pasteurella spp., Head lice, Body lice M. spp., Phthirus pubis, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis cysticercosis, Poliovirus, Prevotella spp., Naegleria ameba, JC virus, Chlamydia psittacosis, Coxiella burnetii, Rabies virus, Bead-chain Escherichia coli and Rat Bite Fever spirochetes, Respiratory Syncytial Virus, Rhinosporidium sayberi, Rhinovirus, Rickettsia spp., Rickettsia mites, Rift Valley fever virus, Rocky Mountain spotted fever rickettsia, Rotavirus , rubella virus, Salmonella spp., atypical pneumonia coronavirus, scabies mites, Schistosoma spp., Shigella spp., Varicella zoster virus, Variola major or minor, Sporothrix schenckii, Staphylococcus spp., Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, syphilis spirochetes, Cestodes spp., Clostridium tetani, ringworm, Trichophyton tonsurans, ringworm, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, Trichophyton rubrum, Holtea werneckii, ringworm, Malassezia spp., roundworm, dog roundworm and cat roundworm, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Tula hot Francis bacteria, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio cholerae, Guanarito virus, West Nile virus, Trichosporonium leuconostoma, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales (mucormycosis) and Entomophthora spp. (entomophthorosis), Pseudomonas aeruginosa, Campylobacter fetus (Vibrio), Aeromonas bacteria, Edwardsiella spp. tarda, Yersinia pestis, Shigella dysenteriae, Shigella sonnei, Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Fensenburgdorferi, Borrelia burgdorferi, Leptospira hemorrhagic jaundice, Pneumocystis carinii, Brucella abortus, Brucella abortus, Malta fever, Mycoplasma spp., Rickettsia typhi, Rickettsia tsutsugamushi, Chlamydia spp., Pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum) ); protozoa (Entamoeba histolytica, Trichomonas vaginalis, Trichomonas mansoni, Trypanosoma gambiens, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropicalis, Leishmania braziliensis, Pneumocystis carinii pneumoniae, Plasmodium vivax, Plasmodium falciparum, malignant malaria); or helminths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, hookworms).

[0185] Other antibodies as cell-binding ligands used in the present invention for the treatment of viral diseases include, but are not limited to, antibodies against pathogenic virus antigens, and examples of such pathogenic viruses include, but are not limited to, Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, arbovirus, rhabdovirus, Arenaviridae, non-A / non-B hepatitis virus, rhinovirus, coronavirus, rotavirus, tumor viruses [e.g., HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma)], EB virus (nasopharyngeal carcinoma, Burkitt's lymphoma, primary central nervous system lymphoma, etc.], and the like. tumor), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatocellular carcinoma), HTLV-I (adult T-cell leukemia / lymphoma)]; immune diseases caused by viruses: [e.g., human immunodeficiency virus (AIDS)], CNS viruses: [e.g., JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), arboviral encephalitis, orthomyxovirus Viral family (presumed) (encephalitis lethargica), RV (rabies), vesicular stomatitis, herpesvirus meningitis, Ramsay-Hunt syndrome type II; poliovirus (acute poliomyelitis, post-polio syndrome), HTLV-I (tropical spastic paralysis)]; cytomegalovirus (CMV retinitis, HSV (herpetic keratitis)); cardiovascular disease viruses [e.g., CBV (pericarditis, myocarditis)]; respiratory system / acute nasopharyngitis / viral pneumonia: [Epstein-Barr virus (EBV)] Infectious diseases / infectious mononucleosis, cytomegalovirus, SARS coronavirus (severe acute respiratory syndrome), orthomyxoviridae: influenza virus A / B / C (influenza / avian influenza), paramyxovirus: human parainfluenza virus (parainfluenza), RSV (human respiratory syncytial virus), hMPV); digestive system viruses [MuV (mumps), cytomegalovirus (CMV esophagitis); adenovirus (adenovirus infection); rotavirus, norovirus, astrovirus, coronavirus, HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus)]; urogenital system viruses [e.g., BK virus, MuV (mumps)].

[0186] Conjugate formulation and applications

[0187] The conjugates of the present application are suitable for formulation into liquids or for lyophilization and subsequent reconstitution into liquid formulations. Liquid formulations containing the conjugate active ingredient at a concentration of 0.1 g / L to 300 g / L as the active ingredient for delivery to patients without high levels of antibody aggregation may contain one or more polyols (e.g., sugars), a buffer having a pH of 4.5 to 7.5, a surfactant (e.g., polysorbate 20 or 80), an antioxidant (e.g., ascorbic acid and / or methionine), an isotonicity agent (e.g., mannitol, sorbitol, or NaCl), a chelating agent such as EDTA, a metal complex (e.g., Zn-protein complex), a biodegradable polymer such as a polyester, a preservative (e.g., benzyl alcohol), and / or free amino acids.

[0188] In a preferred embodiment, the conjugates of the present invention for in vivo clinical use will be supplied as a lyophilized solid (e.g., powder) that can be redissolved in a solution or sterile water for injection. The conjugate in a liquid formulation or formulated lyophilized powder may comprise 0.01% to 99% by weight of the major component of the formulation. The remainder of the formulation is an excipient consisting of one or more of the following compounds: 0.5% to 25% buffer, 0% to 20% polyol, 0% to 2.0% surfactant, 0% to 5% preservative, 0% to 30% amino acid or bulky compound, 0% to 5% antioxidant, and 0% to 0.3% chelating agent.

[0189] Buffers suitable for use in the formulation include, but are not limited to, organic acid salts such as sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, sulfate, or phosphate buffers. Additionally, amino acid cationic components can also be used as buffers. Examples of such amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, and the like. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, and histidine succinate. Examples of suitable organic acid salts include arginine diphosphate succinate, etc. The pH of the buffer solution is 4.5 to 7.5, preferably about 4.5 to about 6.5, and more preferably about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer solution is about 10 mM to about 500 mM.

[0190] "Polyols," optionally included in formulations, are substances with multiple hydroxyl groups. Polyols can be used as stabilizing excipients and / or isotonicity agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from physical and chemical degradation pathways. Preferentially excluded cosolvents increase the effective surface tension of the solvent at the protein interface, thereby ensuring that the most energetically favorable structural conformation is the one with the smallest surface area. Polyols include sugars (reducing and nonreducing sugars), sugar alcohols, and sugar acids. "Reducing sugars" contain a hemiacetal group that can reduce metal ions or covalently react with lysine and other amino groups in proteins, while "nonreducing sugars" lack these properties of reducing sugars. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Nonreducing sugars include sucrose, trehalose, sorbose, melezitose, and raffinose. The sugar alcohol is selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, and glycerol. Sugar acids include L-gluconate and its metal salts. Preferably, a non-reducing sugar, sucrose or trehalose, at a concentration of about 0.01% to 15% is selected in the formulation, with trehalose being preferred over sucrose due to the solution stability of trehalose.

[0191] Surfactants optionally included in the formulation include polysorbates (such as polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85), poloxamers (such as poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407, and polyethylene-polypropylene glycol); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl betaine; The surfactant may be selected from the group consisting of camidopropyl, linoleamidopropyl, myristamidopropyl, palmimidopropyl, or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl, palmidopropyl, or isostearamidopropyl dimethylamine; sodium methyl cocoyl or sodium methyl oleyl taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine, and cocoamphoglycinate, the "MONAQUAT" (trademark) series (e.g., isostearyl ethylimidonium ethosulfate); polyethyl glycol, polypropylene glycol, and ethylene and propylene glycol copolymers (e.g., Pluronics, PF68, etc.). Suitable surfactants are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). The concentration of surfactant in the formulation ranges from 0.0001% to about 1.0%. In certain embodiments, the concentration of surfactant is from about 0.01% to about 0.1%. In one embodiment, the concentration of surfactant is about 0.02%.

[0192] "Preservatives" that are optionally included in the formulation are compounds that essentially reduce bacterial activity. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexyl paraben, and cyclohexyl paraben. Preservatives include benzoxanol, 3-pentanol, and m-cresol. The preservative content in the formulation is less than 5%, preferably 0.01% to 1%. In one embodiment, the preservative herein is benzyl alcohol.

[0193] Suitable free amino acids for optional use in the formulation include, but are not limited to, arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycerin, glutamic acid, or aspartic acid. Basic amino acids, i.e., those containing arginine, lysine, and / or histidine, are preferred. When a composition contains histidine, it can function as both a buffer and a free amino acid; however, when a histidine buffer is used, a non-histidine free amino acid, e.g., a histidine buffer and lysine, are typically included. Amino acids may be in either the D- or / and L-form, but are typically in the L-form. Amino acids may be present as any stable salt, e.g., hydrochloride salts such as arginine-HCl. The concentration of the amino acid ranges from 0.0001% to about 15%, preferably from 0.01% to 5%.

[0194] The formulation may optionally contain methionine or ascorbic acid as an antioxidant at a concentration of 0.01 mg / ml to 5 mg / ml.The formulation may optionally contain a metal chelating agent, such as EDTA, EGTA, etc., at a concentration of about 0.01 mM to about 2 mM.

[0195] The final formulation can be adjusted to the desired pH with a buffer adjuster (e.g., acids such as HCl, H2SO4, acetic acid, H3PO4, citric acid, etc., or bases such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine, or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, or tromethamine). The formulation must be "isotonic," meaning that the formulation has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of approximately 250-350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice-type osmometer.

[0196] Other excipients that may be useful in either the liquid or lyophilized formulations of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluoronic F-127, cellulose, cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, dextran (10, 40, or 70 kD), polydextrose, maltodextrin, ficoll, gelatin, hydroxypropyl methacrylate, sodium phosphate, potassium phosphate, ZnCl2, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohols, or hydrogenated forms of carbohydrates having a carbonyl group reduced to a primary or secondary hydroxyl group.

[0197] Other contemplated excipients that may be utilized in the aqueous pharmaceutical compositions of the present application include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such as sodium, etc. These and additional known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are well known in the art, as listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003)" and "Remington: The Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005)."

[0198] To reduce patient pain during injection, topical analgesics can be used before or in conjunction with the injection. Commonly used analgesics include benzyl alcohol (0.01% to 1%), procaine hydrochloride (0.2% to 2.0%), lidocaine hydrochloride (0.2% to 2.0%), 2-trichloromethyl-2-propanol (0.3% to 0.5%), tramadol, morphine, morphine sulfate, hydromorphone, oxycodone hydrochloride, dobutamine, gabapentin, cyclobenzaprine, trazodone, clonidine, and codeine.

[0199] The conjugate formulations of the present application can be prepared as pre-filled syringe solutions, lyophilized powders, or high-efficiency spray-dried powders. A pharmaceutical container or vessel is used to hold the pharmaceutical formulation of the conjugate. The vessel can be a vial, bottle, pre-filled syringe, or pre-filled autoinjector syringe.

[0200] In a further embodiment, the present invention provides a method for preparing a formulation, comprising the steps of: (a) lyophilizing a formulation comprising a conjugate, excipients, and a buffer system; and (b) reconstituting the lyophilized mixture of step (a) in a reconstitution medium such that the reconstituted formulation is stable. The formulation of step (a) may further comprise a stabilizer and one or more excipients selected from the group including bulking agents, salts, surfactants, and preservatives described above. The reconstitution medium may be any of several diluted organic acids or water, i.e., sterile water or bacteriostatic water for injection (BWFI). The reconstitution medium may be selected from the group consisting of water, i.e., sterile water, bacteriostatic water for injection (BWFI), or acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, an acidic solution of sodium chloride, an acidic solution of magnesium chloride, and an acidic solution of arginine, in an amount of about 10 to about 250 mM.

[0201] Liquid pharmaceutical formulations of the present conjugates must exhibit various predefined properties. One of the main concerns for liquid pharmaceuticals is stability, as proteins / antibodies tend to form soluble and insoluble aggregates during manufacturing and storage. Furthermore, various chemical reactions (e.g., deamination, oxidation, clipping, isomerization) can occur in solution, leading to increased levels of degradation products and loss of biological activity. Preferably, the conjugates in liquid or lyophilized formulations should exhibit a shelf life of greater than 6 months at 25°C. More preferably, the conjugates, either in liquid or lyophilized formulations, should exhibit a shelf life of greater than 12 months at 25°C. Most preferably, liquid formulations should exhibit a shelf life of approximately 24-36 months at 2-8°C, and lyophilized formulations should exhibit a shelf life of up to approximately 60 months at 2-8°C. Both liquid and lyophilized formulations should exhibit a shelf life of at least 2 years at -20°C or -70°C.

[0202] In some embodiments, the formulation is stable after freezing (e.g., at -20°C or -70°C) and thawing, e.g., after one, two, or three freeze-thaw cycles. Stability can be qualitatively and / or quantitatively assessed by a variety of methods, including assessment of drug / antibody (protein) ratio and aggregate formation (e.g., using ultraviolet light, size-exclusion chromatography, turbidity measurements, and visual inspection); assessment of charge heterogeneity using cation-exchange chromatography, electric focusing, or capillary zone electrophoresis (e.g., image capillary); amino- or carboxyl-terminal sequence analysis; mass spectrometry, or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS), or HPLC-MS / MS; SDS-PAGE analysis to compare antibody reduction or integrity; peptide map (e.g., trypsin or lysine-carbon) analysis; and assessment of antibody biological activity or antigen-binding function. The instability can include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shearing / hydrolysis / cleavage (e.g., hinge rupture), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, and catabolic glycosylation.

[0203] A stable conjugate should also "retain its biological activity" in a pharmaceutical formulation, e.g., the biological activity of the conjugate at a particular time, e.g., 12 months, should be within about 20%, preferably within about 10% (within the error of the assay) of the biological activity at the time the pharmaceutical formulation was prepared, as determined by, e.g., antigen binding assays and / or in vitro cytotoxicity assays.

[0204] Examples of suitable conjugate administration methods are as follows: The conjugate is administered intravenously as a bolus injection daily, weekly, every two weeks, every three weeks, every four weeks, or once a month for 8 to 54 weeks. The bolus dose is dissolved in 50 to 1000 mL of saline, to which human serum albumin can be optionally added (e.g., 0.5 to 1 mL of concentrated human serum albumin solution at 100 mg / mL). The drug dosage is approximately 50 μg to 20 mg / kg body weight per week, administered intravenously (injections ranging from 10 μg to 200 mg / kg). After 4 to 54 weeks of treatment, patients can receive a second course of treatment. The exact treatment method, including the route of administration, excipients, diluents, dosage, and duration of treatment, can be determined by an experienced surgeon.

[0205] Examples of in vitro therapies include cell culture treatments to kill all cells except for desired variants that do not express the target antigen, or to kill variants that express undesired antigens. Examples of ex vivo therapies include treatment of hematopoietic stem cells (HSCs) prior to transplantation (HSCT) and subsequent return to the same patient to kill diseased or malignant cells. For example, clinical ex vivo treatments to remove tumor or lymphoid cells from bone marrow prior to autologous transplantation in the treatment of cancer and autoimmune diseases, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplantation to prevent graft-versus-host disease, can be performed as follows: After obtaining bone marrow cells from a patient or other individual, they are cultured at 37°C for 30 minutes to approximately 48 hours in serum-containing medium containing the conjugate of the present invention at a concentration ranging from 1 pM to 0.1 mM. The appropriate concentration conditions and culture time (= dose) can be easily determined by an experienced clinician. After the culture is complete, the bone marrow cells are washed with serum-containing medium and returned to the body by known methods, such as intravenous injection. If the patient is undergoing other treatments (e.g., ablative chemotherapy or total body irradiation) between the bone marrow cell harvest and reinfusion treatment, the processed bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0206] Examples of diseases that can be treated by selectively killing cell populations by in vivo or ex vivo methods include any type of cancer, autoimmune diseases, transplant rejection, and infectious diseases (including viral, bacterial, or parasitic).

[0207] Examples of diseases treated by selectively killing cell populations by in vivo or ex vivo methods include malignant tumors of all types, such as cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphoid organs; melanoma; autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis; transplant rejection, such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; graft-versus-host disease; viral infections, such as cytomegalovirus infection, HIV infection, and AIDS; bacterial infections; and parasitic infections identified by one of skill in the art, such as giardiasis, amebiasis, schistosomiasis, and others.

[0208] Identification of subjects in need of treatment for the diseases and conditions described herein is within the ability and knowledge of one of ordinary skill in the art. A veterinarian or physician of ordinary skill in the art can readily identify subjects in need of such treatment through the use of clinical tests, physical examinations, medical / family history, or biological and diagnostic tests.

[0209] As known to those skilled in the art, a therapeutically effective amount can be readily determined by the attending physician using routine skills and by observing results obtained under analogous circumstances. In determining the dosage regimen, the attending physician will take into account many factors, including but not limited to, the subject's race; size, age, and general health; the specific disease involved; the severity of the disease; the individual's responsiveness; the particular compound being administered; the mode of administration; the bioavailability characteristics of the dosage formulation; the selected dosage regimen; and other relevant circumstances surrounding the use of the drug.

[0210] The amount of conjugate required for the desired biological effect will vary depending on several factors, including the nature of the compound, the potency and bioavailability of the conjugate, the type of disease, the patient's race, the patient's pathological condition, the route of administration, and all factors that determine the required dosage, method of administration, and dosing regimen.

[0211] In general terms, the conjugates of the present invention may be formulated for parenteral administration in a physiological buffer solution at a concentration of 0.1 to 30% w / v. Typical dosage ranges are 1 μg / kg body weight to 0.1 g / kg body weight daily, weekly, biweekly, triweekly, or monthly, with preferred dosage ranges being 0.01 mg / kg body weight to 20 mg / kg body weight weekly, biweekly, triweekly, or monthly. The preferred drug dosage may depend appropriately on variables such as the type and extent of progression of the disease or disorder, the overall health of the individual patient, the relative biological activity of the selected drug, the compound dosage form, the mode of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the drug in the selected mode of administration, and the administration rate (single injection or continuous infusion) and schedule (frequency of administration over a given period of time).

[0212] The cell-binding agent-cytotoxic agent conjugates of the present invention can be administered in unit doses, where "unit dose" refers to a single dose administered to a single patient. The unit dose can be easily and conveniently packaged and maintains a physically and chemically stable unit dose, either as the active conjugate itself or as a pharmaceutically acceptable composition (described below). Typical daily doses range from 0.01 to 100 mg / kg. Generally, unit doses range from 1 to 3,000 mg per day. A preferred unit dose is 1 mg to 500 mg administered once or twice a week, every two weeks, three weeks, or month, and more preferably 10 mg to 500 mg administered every two or three weeks. The conjugates provided herein can be prepared by adding one or more pharmaceutically acceptable excipients to a pharmaceutical composition. The unit dosage of the drug can be administered orally as a tablet, simple capsule, or soft capsule; intranasally as a powder, nasal drops, or aerosol; or dermal as, for example, an ointment, cream, lotion, gel, or spray or skin patch. The composition can be conveniently administered in unit dosage form and can be prepared by any method known in the pharmaceutical arts, for example, as described in "Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005." A preferred dosage form of the pharmaceutical composition is one in which the compound of the present invention is formulated for oral or parenteral administration. For example, oral dosage forms such as tablets, pills, powders, capsules, troches, etc. may contain one or more of the following ingredients or other compounds of similar properties: binders such as microcrystalline cellulose or gum tragacanth; diluents such as starch or lactose; dispersing agents such as starch and cellulose derivatives; lubricants such as magnesium stearate; glidants such as colloidal silica; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint or methyl salicylate.Capsules are available in hard or soft form, blended with gelatin, optionally blended with a plasticizer, as are starch capsules. Furthermore, dosage units can contain a variety of different materials to modify their physical form, such as sugar coatings, shellac, or enteric coatings. Other oral dosage forms, such as syrups or elixirs, can contain sweeteners, preservatives, pigments, coloring agents, and flavorings. Furthermore, active compounds can be processed and formulated differently to produce fast-dissolving, sustained-release, or extended-release dosage forms, with such extended-release formulations preferably being dual-mode. A preferred tablet formulation contains a mixture of lactose, corn starch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, or talc, or a combination thereof. Liquid formulations for parenteral administration. These formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Liquid formulations may contain binders, buffers, preservatives, chelating agents, sweeteners, flavors, and colorants. Non-aqueous solvents include alcohol, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic acid esters such as ethyl oleate. Aqueous carriers include mixtures of water and ethanol, buffers, and saline. In particular, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers can be used as materials to control the release of active compounds. Vehicles for intravenous administration include fluid and nutrient replenishers and Ringer's dextrose-based electrolyte replenishers. Other possible parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

[0213] Other alternative administration methods include inhalants, including dry powders, aerosols, and drops. Inhalants may be, for example, aqueous solutions containing polyoxyethylene-9-lauryl ether, glycocholate, deoxycholate, or oily solutions for administration in the form of nasal drops or as gels for intranasal application. Formulations for buccal administration, such as tablets or lozenges, may contain flavorings such as sucrose or gum arabic and other additives such as glycocholate. Formulations suitable for rectal administration are preferably given as unit-dose suppositories, for example, containing a solid base such as cocoa butter and may contain salicylic acid. Topical formulations for the skin are preferably in the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Bases include petrolatum, lanolin, polyethylene glycols, and alcohols, as well as mixtures thereof. Formulations suitable for transdermal administration can be given as discrete patches and may be lipophilic emulsions or buffered aqueous solutions, dissolved or dispersed in a polymer or adhesive.

[0214] In certain embodiments, the cell-binding molecule-cytotoxic agent conjugates of the invention are administered simultaneously with other known or future drugs, such as chemotherapeutic agents, radiation therapy, immunotherapy agents, autoimmune disease treatment agents, anti-infective agents, or other antibody-drug conjugates, to achieve a synergistic effect. In another specific embodiment, the synergistic drug or radiation therapy can be administered prior to or following administration of the conjugates of the invention, and in some aspects, is administered prior to or following the conjugates of the invention by at least 1 hour, 12 hours, 1 day, 1 week, 2 weeks, 3 weeks, 1 month, or even several months.

[0215] The synergist is preferably selected from one or more of the following agents:

[0216] (1) Chemotherapeutic agents: a) alkylating agents: for example, [nitrogen mustards (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, trofosfamide); nitrosourea compounds (carmustine, lomustine); alkylsulfonates (busulfan, treosulfan); triazenes: (dacarbazine); platinum-containing compounds: (carboplatin, cisplatin, oxaliplatin)]; b) plant alkaloids: for example, [vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine); taxoids: (paclitaxel, docetaxel)]; c) DNA topoisomerase inhibitors: for example, [epipodophyllins: (9 - Aminocamptothecin, camptothecin, crisnatol, etoposide, etoposide phosphate, irinotecan, teniposide, topotecan; mitomycins (mitomycin C); d) antimetabolites: for example, {[antifolates: DHFR inhibitors (methotrexate, trimetrexate); IMP dehydrogenase inhibitors (mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); [pyrimidine analogs: uracil analogs (5-fluorouracil, doxifluridine, floxuridine, raltitrexed (Tomudex)); cytosine analogs (cytarabine, cytosine arabinoside) e) Hormonal therapy agents: for example, {receptor antagonists: [antiestrogens: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goserelin, leuprolide acetate); antiandrogens: (bicalutamide, flutamide)]; retinoids / deltoids: [vitamin D3 analogues: (CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents: (verteporfin, phthalocyanines, photosensitizer Pc4, demethoxy-hypocrelin A); cytokines: (interferon alpha, interferon gamma, tumor necrosis factor (TNF), including TNF domain-containing human proteins); f) kinase inhibitors: for example, BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VE GFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesib;g) Others: for example, gemcitabine, epoxomicins (carfilzomib, etc.), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Zygeba, Provenge, Elvoy, isoprenylation inhibitors (lovastatin, etc.), dopaminergic neurotoxins (1-methyl-4-phenylpyridinone), zinc ion, etc.), cell cycle inhibitors (staurosporine, etc.), actinomycins (actinomycin D, dactinomycin, etc.), bleomycins (bleomycin A2, bleomycin B2, peplomycin, etc.), anthracyclines (daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, etc.), mitoxantrone, MDR inhibitors (verapamil, etc.), Ca; 2+ ATP inhibitors (thapsigargin, etc.), vismodegib, histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD0103), belinostat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A, etc.); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A. Known and future known anti-cancer drugs that can be used as combination therapy (synergy) with the compounds and conjugates of the present invention are listed on the American Cancer Society (US) website (www.cancer.gov / cancertopics / druginfo / alphalist), the American Cancer Society (www.cancer.org / treatment / index), the Royal Cancer Research Foundation (www. 1614666467490_1 ) website.

[0217] (2) Anti-autoimmune disease drugs: including, but not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticoids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, and beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenolate, prednisone, sirolimus, and tacrolimus.

[0218] (3) Anti-infectives include, but are not limited to: a) Aminoglycosides: amikacin, astromycin, gentamicins (netimicin, sisomicin, a) Amphenicols: Azidamphenicol, Chloramphenicol, Florfenicol, Thiamphenicol; c) Ansamycins: Geldanamycin, Herbimycin; d) Carbapenems: Biapenem, Doripenem, Ertapenem, Imipenem / Cilastatin, Meropenem, Panipenem; e) Cephems: Carbacephem (Loracarbef), Cephacetrile, Cefaclor, Cephradine , cefadroxil, cephalonium, cephaloridine, cephalothin or cephalosporin, cephalexin, cephaloglycin, cefamandole, cephapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cephalosporin, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycins (cefoxitin, cefotetan, cefmetazole), oxacephem ( Flomoxef, latamoxef; f) glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g) glycylcyclines: tigecycline; h) β-lactamase inhibitors: penams (sulbactam, tazobactam), clavams (clavulanic acid); i) lincosamides: clindamycin, lincomycin; j) lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDAs);k) Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolides (telithromycin, cethromycin), midecamycin, miokamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin l) Monobactams: aztreonam, tigemonam; m) Oxazolidinones: linezolid; n) Penicillins: amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin fluticasone, mecillinam (pivmecillinam), mezlocillin, methicillin, nafcillin, oxacillin, penamecillin, penicillin, phenethicillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o) polypeptides: bacitracin, colistin, polymyxin B; p) quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxacin, galactosamine, galactosamine; Noxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotrovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin;q) streptogramins: pristinamycin, quinupristin / dalfopristin;r) Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sul; Famethoxazole (cotrimoxazole);s) steroidal antibacterial agents: selected from fusidic acid;t) tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (including tigecycline);u) other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors ( Bacitracin), DADAL / AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampin, tazobactam, uvaricin;

[0219] (4) Antiviral drugs: a) entry / fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO140, CD4 (ibalizumab); b) integrase inhibitors: raltegravir, elvitegravir, globoidan A; c) maturation inhibitors: bevirimat, vivicon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, Cidofovir, clevudine, dexeruvucitabine, didanosine (DDI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'-fluoro-substituted 2',3'-deoxynucleoside analogues (e.g., 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG)), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), L-nucleosides (e.g., β -L-thymidine and β-L-2'-deoxycytidine), penciclovir, razivir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine, valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleosides: amantadine, ateviridine, capravirine, diallylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscal Net (phosphoryl formate), imiquimod, interferon alpha, loviride, rhodenosine, methisazone, nevirapine, NOV-205, pegylated interferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g) protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir;h) Other antiviral drugs: Abzyme, arbidol, calanolide A, ceragenin, cyanovirin-N, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib;

[0220] 5) Other immunotherapeutic drugs: for example, imiquimod, interferons (e.g., α, β), granulocyte colony-stimulating factors, cytokines, interleukins (IL-1 to IL-35), antibodies (e.g., trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab), protein-binding drugs (e.g., Abraxane), as well as calicheamicin derivatives, maytansine derivatives (DM1 and DM4), CC-1065 and duocarmycin minor groove binders, effective paclitaxel derivatives, doxorubicin, and auristatin-based mitotic inhibitors (e.g., trastuzumab-DM1, inotuzumab ozogamicin, brentuximab). buvedotin, glenbatumumab vedotin, lorvotuzumab mertansine, AN-152LMB2, TP-38, VB4-845, cantuzumab mertansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, milatuzumab-doxorubicin, SGN-75 (anti-CD70), anti-CD22-MCC-DM1, IMGN853, anti-CD22-MMAE, anti-CD22-MMAF, and anti-CD22 calicheamicin.

[0221] In further embodiments, the synergist is selected from one or several of the following drugs: abatacept, abemaciclib, abiraterone acetate, abraxane, acetaminophen / hydrocodone, acalabrutinib, adacanumab, adalimumab, ADXS31-142, ADXS-HER2, afatinib dimaleate, aldesleukin, alectinib, alemtuzumab, alitretinoin, adretinoin, anthracyclines, aripiprazole Zolazol, atazanavir, atezolizumab, atorvastatin, avelumab, axicarbagene ciloleucel, axitinib, belinstat, BCG Live, bevacizumab, bexarotene, cabzonib, blinatumomab, bortezomib, bosutinib, brentuximab capmatinib, capecitabine, carfilzomib, chimeric antigen receptor-engineered T (CAR-T) cells, celecoxib, ceritinib, cetuximab, chidamide, cyclosporine, cina Calcet, crizotinib, cobimetinib, Cosentyx, crizotinib, CTL019, dabigatran, dabrafenac dacarb, daptomycin, daratumumab, darbepoetin alfa, darunavir, dasatinib, denileukin diftitox, denosumab, Depakote, dexlansoprazole, dexmethylphenidate, dexamethasone, DigniCap cooling system, dinutuximab, doxycycline, duloxetine, Duvelisib, durvalumab, elotuzumab, emtricitine / rilpivirine / tenofovir, disoproxil fumarate, emtricitine / tenofovir, etanercept, everolimus, exemestane, everolimus, exenatide ER, ezetimibe, ezetimibe / simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone / salmeterol ... Bestrant, Gadiva, gefitinib ibrutinib, idelalisib, ifosfamide, infliximab, imiquimod, ImmuCyst, immune BCG, iniparib, insulin aspart, insulin detemir, insulin glargine, insulin lispro, interferon alpha, interferon alpha-1b, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma-1a, lapatinib, ipilimumab, ipratropium bromide / sa Rubutamol, Ixazomib, Kanuma, Lanreotide acetate, Lenalidomide, Lenaliomide, Lenvatinib mesylate, Letrozole, Levothyroxine, Lidocaine, Linezolid, Liraglutide, Lisdexamfetamine, LN-144, Loratinib, Memantine, Methylphenidate, Metoprolol, Mekinist, Memantine, Methylphenidate, Metoprolol, Mekinist C, Necitumumab, Neratinib, Nilotinib, Niraparib, Nivolumab, Ofatumumab, Obinutuzumab, Olaparib, Olmesartan, Olmesartan / Hydroxyprogesterone Lochlorothiazide, omalizumab, omega-3 fatty acid ethyl ester, oncoline, oseltamivir, osimertinib, oxicodomab, osimertinib, oxycodon pazopanib, pembrolizumab, PD-1 antibody, PD-L1 antibody, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, proscavacx, propranolol, quetiapine, rabeprazole, radium-223 chloride, raloxifene, raltegravir, lasilituximab, rivaroxaban romidepsin, rosuvastatin, ruxolitinib phosphate, salbutamol, savolitinib, semaglutide, sevelamer, sildenafil, siltuximab, sipuleucel-T, sitagliptin, sitagliptin / metformin, solifenacin, solanezumab, sonidegib, sorafenib, sunitinib, tacrolimus, taclimus, tadalafil, tamoxifen, Tafinlar, talimogene laherparepvec, tenofovir disoproxil fumarate, testosterone gel, thalidomide Id, TICE BCG, tiotropium bromide, tisagenlecleucel, toremifene, trametinib, trastuzumab, trabectedin (ecteinascidin 743), trabectedin (ecteinascidin 743), trametinib, tremelimumab, trifluridine / tipiravandetanib, vemurafenib, venetoclax, vorinostat, difluaflibercept, zostavax, and analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients thereof, or combinations thereof.

[0222] The present invention is further illustrated, but not limited, by the following examples. [Example]

[0223] The following examples further illustrate the present invention but are not intended to limit the scope of the present application. Unless otherwise specified, the cell lines described in the following examples were maintained in culture media based on conditions specified by the American Type Culture Collection (ATCC), the German Collection of Microbial Cell Cultures (DMSZ), or the Shanghai Institute of Cell Culture, Chinese Academy of Sciences. Unless otherwise specified, all cell culture reagents were provided by Invitrogen. All anhydrous solvents were commercially available and stored in nitrogen-sealed bottles. All other reagents and solvents were purchased according to the highest standards and used without further purification. Preparative HPLC separations were performed on a Varian PreStar HPLC. NMR spectra were detected on a Varian Mercury 400 MHz instrument. Chemical shifts (Δ) are in ppm and referenced to tetramethylsilane at 0.00. Coupling constants (J) are in Hz. Mass spectrometry data were acquired on a Waters Xevo QTOF mass spectrometer equipped with a Waters Acquity UPLC separator and an Acquity TUV detector.

[0224] Example 1: Synthesis of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate [ka]

[0225] To di-tert-butylhydrazine-1,2-dicarboxylate (8.01 g, 34.4 mmol) in DMF (150 ml) was added NaH (60% in petroleum, 2.76 g, 68.8 mmol). After stirring at room temperature for 30 minutes, tert-butyl 2-bromoacetate (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, quenched by the addition of methanol (3 ml), concentrated, diluted with ethyl acetate (100 ml) and water (100 ml), separated, and the aqueous layer extracted with ethyl acetate (2 x 50 ml). The organic layers were combined, dried over magnesium sulfate, filtered, evaporated, and purified by silica column chromatography (ethyl acetate / hexane 1:5 to 1:3) to give the title compound as a colorless oil (12.98 g, 82% yield). MS ESI m / z C 22 H 41 N2O8[M+H] + Calculated value: 461.28, measured value: 461.40.

[0226] Example 2: Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid [ka]

[0227] To di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) in 1,4-dioxane (40 ml) was added HCl (12 M, 10 ml). The mixture was stirred for 30 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated, and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to give the crude title product (2.15 g, 103% yield, ∼93% purity), which was used in the next step without further purification. MS ESI m / z C4H9N2O4 [M+H] + Calculated value: 149.05, actual value: 149.40.

[0228] Example 3: Synthesis of 2,2'-(1,2-bis((benzyloxy)carbonyl)hydrazine-1,2-diyl)diacetic acid [ka]

[0229] To 2,2'-(hydrazine-1,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in a mixture of THF (200 mL) and disodium phosphate (0.1 M, 250 mL, pH 8.0) was added benzyl carbonochloridate (5.01 g, 29.47 mmol) in four portions over 2 h. The mixture was stirred for an additional 6 h, concentrated, and purified on a silica column eluted with water / acetonitrile (1:9) containing 1% formic acid to give the title compound (2.26 g, 73% yield, approximately 95% purity). MS ESI m / z C 20 H 21 N2O8[M+H] + Calculated value 417.12, actual Measured value: 417.40.

[0230] Example 4: Synthesis of dibenzyl 1,2-bis(2-chloro-2-oxoethyl)hydrazine-1,2-dicarboxylate [ka]

[0231] To a solution of 2,2'-(1,2-bis((benzyloxy)carbonyl)hydrazine-1,2-diyl)diacetic acid (350 mg, 0.841 mmol) in dichloroethane (30 mL) was added (COCl) (905 mg, 7.13 mmol), followed by 0.030 mL of DMF. After stirring at room temperature for 2 h, the mixture was diluted with toluene, concentrated, and coevaporated with dichloroethane (2 × 20 mL) and toluene (2 × 15 mL) to dryness to give the title crude product (unstable) (365 mg, 96% yield), which was used in the next step without purification. MS ESI m / z C 20 H 19 Cl2N2O6[M+H] + Calculated value: 453.05, measured value: 453.50.

[0232] Example 5: Synthesis of 1,2-bis(2-(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylic acid di-tert-butyl ester [ka]

[0233] Di-tert-butylhydrazine-1,2-dicarboxylate (0.50 g, 2.16 mmol, 1.0 equiv) and anhydrous DMF (8 mL) were added to a suspension of sodium cyanide (0.259 g, 6.48 mmol, 3.0 equiv) in anhydrous DMF (2 mL) at room temperature under a nitrogen atmosphere within 10 minutes. The mixture was stirred at room temperature for 10 minutes and then cooled to 0 °C. To this was added tert-butyl 2-bromoacetate (1.4 mL, 8.61 mmol, 4.0 equiv). The resulting mixture was allowed to warm to room temperature and stirred overnight. Saturated ammonium chloride solution (100 mL) was added. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 50 mL). The combined organic solution was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica column chromatography (10:1 hexane / ethyl acetate) to give the title compound as a colorless oil (0.94 g, 99.6% yield). ESI MS m / z [M+Na] + 483.4.

[0234] Example 6: Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid [ka]

[0235] To a solution of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (0.94 g, 2.04 mmol) in DCM (4 mL) was added TFA (4 mL) at 0° C. The reaction was stirred for 30 minutes, then warmed to room temperature and stirred overnight. The mixture was concentrated, diluted with DCM, and concentrated. This procedure was repeated three times to give a white solid. The white solid was triturated with DCM and collected by filtration (0.232 g, 76.8% yield). ESI MS m / z [M+H] + 149.2.

[0236] Example 7: Synthesis of 2,2'-(1,2-bis(2-chloroacetyl)hydrazine-1,2-diyl)diacetic acid [ka]

[0237] A solution of 2,2'-(hydrazine-1,2-diyl)diacetic acid (0.232 g, 1.57 mmol, 1.0 equiv) in anhydrous THF (10 mL) was added to a solution of 2-chloroacetyl chloride (0.38 mL, 4.70 mmol, 3.00 equiv) at 0 °C. The reaction was allowed to warm to room temperature. The mixture was heated, stirred overnight, and concentrated. The residue was co-evaporated three times with THF to give a white solid (0.472 g, theoretical yield). ESI MS m / z [M+H] + 301.1.

[0238] Example 8: Synthesis of tert-butyl 2,8-dioxo-1,5-oxazolidine-5-carboxylate [ka]

[0239] A solution of 3,3'-azanediyldipropionic acid (10.00 g, 62.08 mmol) in 1.0 M NaOH (300 mL) was added to a solution of di-tert-butyl dicarbonate (22.10 g, 101.3 mmol) in THF (200 mL) at 4 °C for 1 hour. After the addition, the mixture was stirred at 4 °C for 2 hours. The mixture was carefully acidified with 0.2 M H3PO4 to pH ~4, concentrated in vacuo, extracted with C2Cl2, dried over Na2SO4, evaporated, and purified by high-performance silica chromatography eluting with acetic acid / methanol / dichloromethane (0.01:1:5) to give 3,3'-((tert-butoxycarbonyl)azanediyl)dipropionic acid (13.62 g, 84% yield). ESI MS m / z C 11 H 19 NO6[M+H] + Calculated value: 262.27, measured value: 262.40.

[0240] To a solution of 3,3'-((tert-butoxycarbonyl)azanediyl)dipropionic acid (8.0 g, 30.6 mmol) in CHCl (500 mL) at 0°C was added phosphorus pentoxide (8.70 g, 61.30 mmol). The mixture was stirred at 0°C for 2 hours and then at room temperature for 1 hour. It was filtered through a short silica column and the column was rinsed with ethyl acetate / dichloromethane (1:6). The filtrate was concentrated and triturated with ethyl acetate / hexane to give the title compound (5.64 g, 74% yield). ESI MS m / z C 11 H 17 NO5[M+H] + Calculated value 244.11, measured value 244.30 .

[0241] Example 9: Synthesis of tert-butyl 3-((benzyloxy)amino)propionate [ka]

[0242] To a solution of O-benzylhydroxylamine hydrochloride (10.0 g, 62.7 mmol) in THF (100 mL) was added EtN (15 mL) and tert-butyl acrylate (12.1 g, 94.5 mmol). The mixture was refluxed overnight, concentrated, and purified on a silica column eluted with ethyl acetate / hexane (1:4) to give the title compound 3 (13.08 g, 83% yield). 1 ESI MS m / z+ C 14 H 21 NNaO3(M+Na) calculation Value: 274.15, actual value: 274.20.

[0243] Example 10: Synthesis of tert-butyl 3-(hydroxyamino)propionate [ka]

[0244] To tert-butyl 3-((benzyloxy)amino)propanoate (13.0 g, 51.76 mmol) in methanol (100 ml) was added Pd / C (0.85 g, 10% Pd, 50% wet) in a hydrogenation vessel. The reaction was evacuated under vacuum and placed under 2 atmospheres of hydrogen gas, after which the reaction mixture was stirred at room temperature overnight. The crude reaction was passed through a short pad of Celite, rinsed with ethanol, concentrated, and purified on a silica column eluting with methanol / DCM (1:10 to 1:5) to give the title compound (7.25 g, 87% yield). 1 H NMR (CDCl3) 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI MS m / z+ C7H 15 NNaO3 (M+Na) calculated value 184.10, found value 184.30.

[0245] Example 11: Synthesis of tert-butyl 3-((toluenesulfonyloxy)amino)propionate [ka]

[0246] To tert-butyl 3-(hydroxyamino)propionate (5.10 g, 31.65 mmol) in a mixture of DCM (50 mL) and pyridine (20 mL) was added the tosylate chloride (12.05 g, 63.42 mmol) at 4° C. After the addition, the mixture was stirred at room temperature overnight, concentrated, and purified on a SiO column eluted with ethyl acetate / DCM (1:10 to 1:6) to give the title compound (8.58 g, 86% yield). 1 H NMR(CDCl3)7.81(s, 2H), 7.46(s, 2H), 3.22(t, J=6.4Hz, 2H), 2.55(t, J=6.4Hz, 2H), 2.41(s, 3H), 1.49 (s, 9H); ESI MS m / z + C 14 H 21 NNaO5S (M+Na), calculated 338.11, found 338.30.

[0247] Example 12: Synthesis of di-tert-butyl 3,3'-(hydrazine-1,2-diyl)dipropionate [ka]

[0248] To a solution of tert-butyl 3-aminopropanoate (3.05 g, 21.01 mmol) in THF (80 mL) was added tert-butyl 3-((tosyloxy)amino)propanoate (5.10 g, 16.18 mmol). The mixture was stirred at room temperature for 1 hour and then at 45° C. for 6 hours. The mixture was concentrated and purified on a silica column eluted with methanol / DCM / triethylamine (1:12:0.01 to 1:8:0.01) to give the title compound (2.89 g, 62% yield). ESI MS m / z + C 14 H 28 N2NaO4 (M+Na) calculated value 311.20, actual value 311.40.

[0249] Example 13: Synthesis of di-tert-butyl 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionyl)propanoyl)hydrazine-1,2-diyl)dipropionate [ka]

[0250] To a solution of 3-maleidopropanoic acid (1.00 g, 5.91 mmol) in DCM (50 mL) was added oxalyl chloride (2.70 g, 21.25 mmol) and DMF (50 μL). The mixture was stirred at room temperature for 2 hours, evaporated, and co-evaporated with DCM / toluene to give crude 3-maleidopropanoic acid chloride. To a mixture of 3,3'-(hydrazine-1,2-dipropyl)di-tert-butyldipropionate (0.51 g, 1.76 mmol) in DCM (35 mL) was added crude 3-maleidopropanoic acid chloride. The mixture was stirred overnight, evaporated, concentrated, and purified on a silica column eluted with ethyl acetate / DCM (1:15 to 1:8) to give the title compound (738 mg, 71% yield). ESI MS m / z+ C 28 H 38 N4NaO 10 (M+Na), calculated 613.26, found 613.40.

[0251] Example 14: Synthesis of 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-hydrazine-1,2-diyl)dipropanoic acid [ka]

[0252] To a solution of compound 14 (700 mg, 1.18 mmol) in dioxane (4 ml) was added concentrated HCl (1 ml). The mixture was stirred for 30 minutes, diluted with ethanol (10 ml) and toluene (10 ml), evaporated, and co-evaporated with ethanol (10 ml) and toluene (10 ml) to give the crude title product (560 mg) for the next step, which was used in the next step without further purification. ESI MS m / z- C 20 H 21 N4O 10 (MH) Calculated value 477.13, measured value 477.20.

[0253] Example 15: Bis(2,5-dioxopyrrolidin-1-yl)-3,3'-(1,2- Synthesis of bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1)-yl)propanoyl)hydrazine-1,2-diyl)dipropanoate [ka]

[0254] To a solution of crude 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-hydrazine-1,2-diyl)dipropanoic acid (~560 mg, ~1.17 mmol) in DMA (8 ml) was added NHS (400 mg, 3.47 mmol) and EDC (1.01 g, 5.26 mmol). The mixture was stirred overnight, evaporated, concentrated, and purified on a silica column eluting with ethyl acetate / DCM (1:12 to 1:7) to give the title compound (520 mg, 65% yield over two steps). ESI MS m / z+ C 28 H 28 N6NaO 14 (M+Na) calculated 695.17, found 695.40.

[0255] Example 16: Synthesis of tert-butyl 3-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionate. [ka]

[0256] To 350 mL of anhydrous THF, 80 mg (0.0025 mol) of sodium metal and triethylene glycol (150.1 g, 1.00 mol) were added with stirring. After the sodium was completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol) was added. The solution was stirred at room temperature for 20 hours and neutralized with 8 mL of 1.0 M HCl. The solvent was removed in vacuo, and the residue was suspended in brine (250 mL) and extracted with ethyl acetate (3 × 125 mL). The combined organic layers were washed with brine (100 mL), then water (100 mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried under vacuum to give 69.78 g (76% yield) of the title product. 1 H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J = 6.4 Hz), 3.59-3.72 (m, 14H). ESI MS m / z-C 13 H 25 O6(MH) calculated value 277.17, observed value 277.20.

[0257] Example 17: Synthesis of tert-butyl 3-(2-(2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)ethoxy)propionate. [ka]

[0258] tert-Butyl 3-(2-(2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)ethoxy)propionate (10.0 g, 35.95 mmol) in acetonitrile The tolyl solution (50.0 mL) was treated with pyridine (20.0 mL). A solution of tosyl chloride (7.12 g, 37.3 mmol) in 50 mL of acetonitrile was added dropwise via an addition funnel over 30 minutes. After 5 hours, TLC analysis revealed the reaction to be complete. The formed pyridine hydrochloride salt was filtered off and the solvent was removed. The residue was purified on silica gel by eluting with 20% ethyl acetate in hexane to neat ethyl acetate to give 11.2 g (76% yield) of the title compound. 1 H NMR: 1.40 (s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4 Hz), 3.52-3.68 (m, 14H), 4.11 (t, 2H, J=4.8 Hz), 7.30 (d, 2H, J=8.0 Hz), 7.75 (d, 2H, J=8.0 Hz); ESI MS m / z+ C 20 H 33 Calculated for O8S (M+H) 433.18, observed Value 433.30.

[0259] Example 18: Synthesis of tert-butyl 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate [ka]

[0260] To 50 mL of DMF was added tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)-propanoate (4.0 g, 9.25 mmol) and sodium azide (0.737 g, 11.3 mmol) and stirred. The reaction was heated to 80 °C. After 4 h, TLC analysis confirmed the reaction was complete. The reaction was cooled to room temperature and quenched with water (25 mL). The aqueous layer was separated and extracted with ethyl acetate (3 × 35 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo. The crude azide product (2.24 g, 98% yield, approximately 93% purity by HPLC) was used in the next step without further purification. 1H NMR (CDCl3): 1.40 (s, 9H), 2.45 (t, 2H, J=6.4 Hz), 3.33 (t, 2H, J=5.2 Hz), 3.53-3.66 (m, 12H). ESI MS m / z+ C 13 H 26 N3O8 (M+H) calculated 304.18, found 304.20.

[0261] Example 19: Synthesis of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid [ka]

[0262] To a solution of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate (2.20 g, 7.25 mmol) in 1,4-dioxane (40 ml) was added HCl (12 M, 10 ml). The mixture was stirred for 40 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated, and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to give the crude title product (1.88 g, 105% yield, approximately 92% purity by HPLC), which was used in the next step without further purification. MS ESI m / z C9H 18 N3O5[M+H] + Calculated value 248.12, measured value 248.40.

[0263] Example 20: Synthesis of tert-butyl 13-amino-4,7,10-trioxadodecanoate and tert-butyl 13-amino-bis(tert-butyl 4,7,10-trioxadodecanoate) [ka]

[0264] The crude azide material, 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (5.0 g, ∼14.84 mmol), was dissolved in ethanol (80 mL) and 300 mg of 10% Pd / C was added. The reaction was evacuated under vacuum and placed under 2 atmospheres of hydrogen gas via a hydrogenation reactor with vigorous stirring. The reaction was then stirred at room temperature overnight and TLC confirmed the disappearance of the starting material. The crude reaction was passed through a short pad of Celite and rinsed with ethanol. The solvent was removed and the amine was purified on silica gel using a mixture of methanol (5% to 15%) and 1% triethylamine in methylene chloride as the eluent to give tert-butyl 13-amino-4,7,10-trioxadodecanoate (1.83 g, 44% yield, ESI MS m / z + C 13 H 27 NO5 (M+H) calculated 278.19, found 278.30) and 13-amino-bis(tert-butyl 4,7,10-trioxadodecanoate) (2.58 g, 32% yield, ESI MS m / z+ C 26 H 52 NO 10 (M+H) calculated value: 538.35, found value: 538.40).

[0265] Example 21: Synthesis of 3-(2-(2-(2-(aminoaminoethoxy)ethoxy)ethoxy)propanoic acid hydrochloride [ka]

[0266] To a solution of tert-butyl 13-amino-4,7,10-trioxadodecanoate (0.80 g, 2.89 mmol) in dioxane (30 mL) was added 10 mL of HCl (36%) with stirring, and after 0.5 h, TLC analysis confirmed the reaction was complete. Upon completion, the reaction mixture was evaporated and co-evaporated with ethanol and ethanol / toluene to form the title product as the HCl salt (>90% purity, 0.640 g, 86% yield) without further purification. ESI MS m / z + C9H 20 NO5 (M+H) calculated value 222.12, found value 222.20.

[0267] Example 22: 13-aminobis(4,7,10-trioxododecanoic acid hydrochloride) [ka]

[0268] To a solution of 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester) (1.00 g, 1.85 mmol) in dioxane (30 mL) was added 10 mL of HCl (36%) with stirring. After 0.5 h, TLC analysis indicated the reaction was complete. The reaction mixture was evaporated and co-evaporated with ethanol and ethanol / toluene to form the hydrochloride salt of the title product without further purification (purity >90%, 0.71 g, 91% yield). ESI MS m / z+ C 18 H 36 NO 10 (M+H) calculated 426.22, found 426.20.

[0269] Example 23: 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)pro Synthesis of tert-butyl pantoate [ka]

[0270] To a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 equiv.) in anhydrous THF (200 mL) was added sodium (0.1 g). After stirring until the sodium disappeared, tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 equiv.) was added dropwise. After stirring overnight, the mixture was quenched with HCl (20.0 mL, 1N) at 0 °C. After removal by rotary evaporation, brine (300 mL) was added, and the resulting mixture was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a colorless oil (30.20 g, 79.0% yield), which was used in the next step without further purification. MS ESI m / z C 13 H 27 O6 [M+H] + Calculated value: 278.1729, measured value: 278.1730.

[0271] Example 24: Synthesis of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate [ka]

[0272] To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 equiv.) and TsCl (41.37 g, 217.0 mmol, 2.0 equiv.) in anhydrous DCM (220 mL) was added TEA (30.0 mL, 217.0 mmol, 2.0 equiv.) at 0° C. The mixture was stirred overnight at room temperature and then washed with water (3×300 mL) and brine (300 mL). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica column chromatography (3:1 hexane / ethyl acetate) to give a colorless oil (39.4 g, 84.0% yield). MS ESI m / z C 20 H 33 O8S [M+H] + Calculated value 433.1818, measured value 433.2838.

[0273] Example 25: Synthesis of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate [ka]

[0274] 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoic acid To a solution of tert-butyl ether (39.4 g, 91.1 mmol, 1.0 equiv.) in anhydrous DMF (100 mL) was added NaN3 (20.67 g, 316.6 mmol, 3.5 equiv.). The mixture was stirred overnight at room temperature, water (500 mL) was added, and the mixture was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were washed with water (3 x 900 mL) and brine (900 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica column chromatography (5:1 hexane / ethyl acetate) to give a pale yellow oil (23.8 g, 85.53% yield). MS ESI m / z C 13 H 25 O3N5Na [M+Na] + Calculated value: 326.2, measured value: 326.2.

[0275] Example 26: Synthesis of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate [ka]

[0276] Raney Ni (7.5 g, suspended in water) was washed with water (3 times) and isopropyl alcohol (3 times) and mixed with tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under a hydrogen balloon at room temperature for 16 hours and then filtered through a pad of Celite, washing the pad with isopropyl alcohol. The filtrate was concentrated and purified by column chromatography (5-25% methanol / DCM) to give a pale yellow oil (2.60 g, 57% yield). MS ESI m / z C13 H 28 NO5[M+H] + Calculated value 279.19, measured value 279.19.

[0277] Example 27: Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol [ka]

[0278] To a solution of 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 equiv.) and potassium carbonate (83.00 g, 600 mmol, 3.0 equiv.) in acetonitrile (350 mL) was added BnBr (57.0 mL, 480 mmol, 2.4 equiv.). The mixture was refluxed overnight. Water (1 L) was added and extracted with ethyl acetate (3 x 300 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica column chromatography (4:1 hexane / ethyl acetate) to give a colorless oil (50.97 g, 89.2% yield). MS ESI m / z C 18 H 23 NO2Na [M+Na] + Calculated value: 309.1729, measured value: 309.1967.

[0279] Example 28: Synthesis of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate [ka]

[0280] To a solution of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 equiv.), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0 equiv.), and n-Bu4NI (6.10 g, 16.53 mmol, 0.1 equiv.) in DCM (560 mL) was added sodium hydroxide solution (300 mL, 50%). The mixture was stirred overnight. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with water (3 × 300 mL) and brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica column chromatography (7:1 hexane / ethyl acetate) to give a colorless oil (61.08 g, 89.4% yield). MS ESI m / z C 25 H 36 NO4[M+H] + Calculated value: 414.2566, measured value: 414.2384.

[0281] Example 29: Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate [ka]

[0282] 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid tert-butyl ester To a solution of methyl methyl ether (20.00 g, 48.36 mmol, 1.0 equiv) in THF (30 mL) and methanol (60 mL) was added Pd / C (2.00 g, 10 wt %, 50% wet). The mixture was shaken overnight under 1 atmosphere of hydrogen gas, filtered through Celite (filter aid), and the filtrate was concentrated to give a colorless oil (10.58 g, 93.8% yield). MS ESI m / z C 11 H 24 NO4[M+H] + Calculated value: 234.1627, measured value: 234.1810.

[0283] Example 30: Synthesis of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate [ka]

[0284] To a solution of 2,2'-oxydiethanol (19.7 mL, 206.7 mmol, 3.0 equiv.) in anhydrous THF (100 mL) was added sodium (0.1 g). The mixture was stirred until the sodium disappeared, and then tert-butyl acrylate (10.0 mL, 68.9 mmol, 1.0 equiv.) was added dropwise. The mixture was stirred overnight, brine (200 mL) was added, and the mixture was extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with brine (3 x 300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica column chromatography (1:1 hexane / ethyl acetate) to give a colorless oil (8.10 g, 49.4% yield). MS ESI m / z C 11 H 23 O5 [M+H] + The calculated value was 235.1467, and the measured value was 235.1667.

[0285] Example 31: Synthesis of tert-butyl 3-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)propanoate. [ka]

[0286] To a solution of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (6.24 g, 26.63 mmol, 1.0 equiv.) and TsCl (10.15 g, 53.27 mmol, 2.0 equiv.) in anhydrous DCM (50 mL) was added pyridine (4.3 mL, 53.27 mmol, 2.0 equiv.) at 0° C. The mixture was stirred overnight at room temperature, then washed with water (100 mL), and the aqueous layer was extracted with DCM (3×50 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica column chromatography (5:1 hexane / ethyl acetate) to give a colorless oil (6.33 g, 61.3% yield). MS ESI m / z C 18 H 27 O7S [M+H]+ Calculated value: 389.1556, measured value: 389.2809.

[0287] Example 32: Synthesis of tert-butyl 3-(2-(2-azidoethoxy)ethoxy)propionate [ka]

[0288] To a solution of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate (5.80 g, 14.93 mmol, 1.0 equiv.) in anhydrous DMF (20 mL) was added NaN (5.02 g, 77.22 mmol, 5.0 equiv.). The mixture was stirred at room temperature overnight. Water (120 mL) was added and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water (3 × 150 mL) and brine (150 mL), dried over anhydrous NaSO, filtered, concentrated, and purified by silica column chromatography (5:1 hexane / ethyl acetate) to give a colorless oil (3.73 g, 69.6% yield). MS ESI m / z C 11 H 22 O3N4Na [M+H] + Calculated value 260.1532, measured value 260.2259.

[0289] Example 33: Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propionate [ka]

[0290] Tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (0.18 g, 0.69 mmol) was dissolved in a methanol solution (3.0 mL, containing 60 μL of concentrated HCl) and hydrogenated over Pd / C (10 wt%, 20 mg). The mixture was left under a hydrogen gas balloon for 30 minutes. The catalyst was filtered through a Celite pad, washing the pad with methanol. The filtrate was concentrated to give a colorless oil (0.15 g, 93% yield). MS ESI m / z C11 H 24 No. 4 [M+H] + Calculated value 234.16, measured value 234.14.

[0291] Example 34: Synthesis of 3-(2-(2-azidoethoxy)ethoxy)propanoic acid [ka]

[0292] tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (2.51 g, 9.68 mmol) dissolved in 1,4-dioxane (30 mL) was treated with 10 mL of concentrated HCl at room temperature. The mixture was stirred for 35 minutes, diluted with ethanol (30 mL) and toluene (30 mL), and concentrated in vacuo. The crude mixture was purified on silica gel using a mixture of methanol (5% to 10%) and 1% formic acid in methylene chloride as eluent to give the title compound (1.63 g, 83% yield). ESI MS m / z C7H 12 N3O4[MH] - calculation Value 202.06, actual value 202.30.

[0293] Example 35: Synthesis of 2,5-dioxapyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate [ka]

[0294] To 3-(2-(2-azidoethoxy)ethoxy)propanoic acid (1.60 g, 7.87 mmol) in 30 mL of CHCl was added NHS (1.08 g, 9.39 mmol) and EDC (3.60 g, 18.75 mmol). After 8 hours, when TLC analysis showed the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (5% to 10%) in methylene chloride as the eluent to give the title compound (1.93 g, 82% yield). ESI MS m / z C11 H 17 N4O6[M+H] + Calculated value: 301.11, measured value: 301.20.

[0295] Example 36: Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate [ka]

[0296] To 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propionic acid (4.50 g, 18.21 mmol) in 80 mL of dichloromethane was added NHS (3.0 g, 26.08 mmol) and EDC (7.60 g, 39.58 mmol) and stirred. After 8 h, TLC analysis revealed the reaction was complete, and the reaction mixture was concentrated and purified by silica gel column chromatography using a mixture of ethyl acetate (5% to 10%) in methylene chloride as the eluent. The title compound was obtained by purification using a HPLC column (5.38 g, 86% yield). ESI MS m / z C 13 H 20 N4O7[M+H] + , Calculated value 345.13, measured value 345.30.

[0297] Example 37: Synthesis of (14S,17S)-1-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)-amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanoic-18-oic acid [ka]

[0298] To a solution of (S)-2-((S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)-4-(tert-butoxy)-4-oxobutanoic acid (2.81 g, 6.73 mmol) in a mixture of DMA (70 mL) and 0.1 M NaH2PO4 (50 mL, pH 7.5) was added 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy)-ethoxy)propanoate (3.50 g, 10.17 mL). The mixture was stirred for 4 hours, evaporated in vacuo, and purified on silica gel using a mixture of methanol (5% to 15%) in methylene chloride containing 0.5% acetic acid as the eluent to give the title compound (3.35 g, 77% yield). ESI MS m / z C 28 H 51 NO 11 [M+H] + Calculated value: 647.35, measured value: 647.80.

[0299] Example 38: (14S,17S)-tert-butyl 1-azido-14-(4-((tert-butoxycarbonyl)-amino)butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,1 Synthesis of 6-diazanonadecane-19-oate [ka]

[0300] To (14S,17S)-1-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)-amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanoic-18-oic acid (3.30 g, 5.10 mmol) and (4-aminophenyl)methanol (0.75 g, 6.09 mmol) in DMA (25 ml) was added EDC (2.30 g, 11.97 mmol). The mixture was stirred overnight, evaporated in vacuo, and purified on silica gel using a mixture of methanol (5% to 8%) in methylene chloride as the eluent to give the title compound (3.18 g, 83% yield). ESI MS m / z C 35 H 58 N7O 11 [M+H] + The calculated value was 752.41 and the measured value was 752.85.

[0301] Example 39: Synthesis of (14S,17S)-tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)-butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate [ka]

[0302] To a solution of (14S,17S)-tert-butyl 1-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate (1.50 g, 1.99 mmol) in THF (35 mL) was added Pd / C (200 mg, 10% Pd, 50% wet) in a hydrogenation bottle. The mixture was shaken overnight under a hydrogen atmosphere at 1 atmosphere pressure, filtered through Celite (filter aid), and the filtrate was concentrated to give the title compound (1.43 g, 99% yield), which was used immediately in the next step without further purification. ESI MS m / z C 35 H60 N5O 11 [M+H] + Calculated value: 726.42, measured value: 726.70.

[0303] Example 40: Synthesis of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecane-1-carboxylic acid [ka]

[0304] To a solution of (S)-2-(2-amino-3-methylbutanamido)acetic acid (Val-Gly) (1.01 g, 5.80 mmol) in DMA (50 mL) and 0.1 M sodium dihydrogen phosphate (50 mL, pH 7.5) was added 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (1.90 g, 6.33 mL). The mixture was stirred for 4 hours, evaporated in vacuo, and purified on silica gel using a mixture of methanol (5% to 15%) in methylene chloride containing 0.5% acetic acid as eluent to give the title compound (1.52 g, 73% yield). ESI MS m / z C 14 H 26 N5O6[M+H] + , total Calculated value: 360.18, actual value: 360.40.

[0305] Example 41: Synthesis of (S)-2,5-dioxapyrrolidin-1-yl 15-azido-5-isopropyl-4,7-dioxa-10,13-dioxa-3,6-diazapentadecan-1-oate [ka]

[0306] To a solution of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oic acid (1.50 g, 4.17 mmol) in 40 mL of dichloromethane, NHS (0.88 g, 7.65 mmol) and EDC (2.60 g, 13.54 mmol) were added with stirring. After 8 hours, TLC analysis revealed the reaction to be complete, and the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (5% to 20%) in methylene chloride as the eluent to give the title compound (1.48 g, 78% yield). ESI MS m / z C 18 H 29 N6O8[M+H] + , Calculated value 457.20, Actual measurement Value 457.50.

[0307] Example 42: Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid [ka]

[0308] A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in water (40 mL) was cooled to 0° C. and treated dropwise with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 mL). After 1 h, the reaction was allowed to warm to room temperature and stirred for 3 h. The THF was removed in vacuo and the pH of the aqueous solution was adjusted to 1.5 by the addition of 6 N HCl. The ethyl acetate and organic layer were washed with brine, dried, and concentrated to give the title compound (16.4 g, 92% yield). MS ESI m / z C 12 H 16 NO5[M+H] + Calculated value: 238.10, measured value: 238.08.

[0309] Example 43: 4-(((benzyloxy)carbonyl)amino)butanoate tert Synthesis of -butyl [ka]

[0310] 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 t-BuOH (15.4 g, 208 mmol) dissolved in DCM (100 mL). After stirring overnight at room temperature, the reaction was filtered and the filtrate was concentrated. The residue was dissolved in ethyl acetate, washed with 1N hydrogen chloride, brine, and dried over Na2SO4. Concentration and purification by column chromatography (10-50% ethyl acetate / hexane) gave the title compound (7.5 g, 37% yield). MS ESI m / z C 16 H 23 NO4Na [M+Na] + Calculated value 316.16, measured value 316.13.

[0311] Example 44: Synthesis of tert-butyl 4-aminobutanoate [ka]

[0312] tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in methanol (50 mL), mixed with Pd / C catalyst (10 wt%, 100 mg), and hydrogenated (1 atm) at room temperature for 3 hours. The catalyst was filtered off and all volatiles were removed under vacuum to give the title compound (272 mg, 90% yield). MS ESI m / z C8H 18 NO2[M+H] + Calculated value 160.13, actual value 160.13.

[0313] Example 45: Synthesis of 3,3'-(benzylazadiyl)di-tert-butyl dipropionate [ka]

[0314] A mixture of phenylmethanamine (2.0 mL, 18.29 mmol, 1.0 equiv) and tert-butyl acrylate (13.3 mL, 91.46 mmol, 5.0 equiv) was refluxed at 80° C. overnight and concentrated. The crude product was purified by silica column chromatography (20:1 hexane / ethyl acetate) to give the title compound as a colorless oil (5.10 g, 77% yield). ESI MS m / z: C 21 H 34 NO4[M+H] + Calculated value: 364.2, measured value: 364.2. 1 H NMR (400 MHz, CDCl3) δ 7.38-7.21 (m, 5H), 3.58 (s, 2H), 2.76 (t, J=7.0 Hz, 4H), 2.38 (t, J=7.0 Hz, 4H), 1.43 (s, 17H).

[0315] Example 46: Synthesis of di-tert-butyl 3,3'-azadimalonate [ka]

[0316] To a solution of di-tert-butyl 3,3'-(benzylazanediyl)dipropanoate (1.37 g, 3.77 mmol, 1.0 equiv.) in methanol (10 mL) was added Pd / C (0.20 g, 10% Pd / C, 50% wet). The mixture was shaken overnight under an atmosphere of hydrogen gas in a hydrogenation bottle and then filtered through a pad of Celite. The filtrate was concentrated to give the title compound as a colorless oil (1.22 g, 89% yield). ESI MS m / z: C 14 H 28 NO4[M+H] + Calculated value: 274.19, measured value: 274.20.

[0317] Example 47: Synthesis of tert-butyl 4-(2-((((benzyloxy)carbonyl)amino)propanamido)butanoate [ka]

[0318] To a solution of tert-butyl 4-aminobutanoate (1.00 g, 6.28 mmol, 1.0 equiv) and ZL-alanine (2.10 g, 9.42 mmol, 1.5 equiv) in anhydrous DCM (50 mL) at 0 °C, HATU (3.10 g, 8.164 mmol, 1.3 equiv) and TEA (2.6 mL, 18.8 mmol, 3.0 equiv) were added. The reaction was stirred at 0 °C for 10 min, warmed to room temperature, and stirred overnight. The mixture was diluted with DCM, washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica column chromatography (10:3 petroleum ether / ethyl acetate) to give the title compound as a colorless oil (1.39 g, 61% yield). ESI MS m / z C 19 H 29 N2O5Na [M+H] + Calculated value 387.2, measured value 387.2.

[0319] Example 48: Synthesis of tert-butyl 4-(2-aminopropanamido)butanoate [ka]

[0320] To a solution of tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)propanamido)butanoate (1.39 g, 3.808 mmol, 1.0 equiv) in methanol (12 mL) in a hydrogenation bottle was added Pd / C (0.20 g, 10 wt%, 10% wet). The mixture was shaken for 2 h, then filtered through Celite (filter aid) and concentrated to give the title compound as a pale yellow oil (0.838 g, 95% yield). ESI MS m / z C 11 H 23 N2O3[M+H] + Calculated value 231.16, measured value 231.15.

[0321] Example 49: Synthesis of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propionic acid [ka]

[0322] To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propionate (2.3 g, 5.59 mmol, 1.0 equiv.) in DCM (10 mL) was added TFA (5 mL) at room temperature. After stirring for 90 min, the reaction mixture was diluted with anhydrous toluene and concentrated; this procedure was repeated three times to give the title compound as a pale yellow oil (2.0 g, theoretical yield), which was used directly in the next step. ESI MS m / z C 21 H 28 NO4[M+H] + Calculated value: 358.19, measured value: 358.19.

[0323] Example 50: Perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) Synthesis of (thoxy)-propanoate [ka]

[0324] To a solution of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propionic acid (2.00 g, 5.59 mmol, 1.0 equiv) in anhydrous DCM (30 mL) at 0° C., DIPEA was added until the pH was neutral. PFP (1.54 g, 8.38 mmol, 1.5 equiv) and DIC (1.04 mL, 6.70 mmol, 1.2 equiv) were then added. After 10 min, the reaction was allowed to warm to room temperature and stirred overnight. The mixture was filtered, concentrated, and purified by silica column chromatography (15:1 petroleum ether / ethyl acetate) to give the title compound as a colorless oil (2.10 g, 72% yield). ESI MS m / z: C 27 H 27 F5NO4[M+H] + Calculated value 524.2, measured value 524.2.

[0325] Example 51: Synthesis of tert-butyl 2-benzyl-13-methyl-11,14-dioxa-1-phenyl-5,8-dioxa-2,12,15-triazanonadecan-19-oate [ka]

[0326] To a solution of tert-butyl 4-(2-aminopropanamido)butanoate (0.736 g, 3.2 mmol, 1.0 equiv) and perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propionic acid (2.01 g, 3.84 mmol, 0.2 equiv) in anhydrous DMA (20 mL) was added DIPEA (1.7 mL, 9.6 mmol, 3.0 equiv) at 0 °C. After stirring at 0 °C for 10 min, the reaction was warmed to room temperature and stirred overnight. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with water (3 × 200 mL) and brine (200 mL), dried over Na SO , filtered, concentrated, and purified by silica column chromatography (25:2 DCM / methanol) to give the title compound as a colorless oil (1.46 g, 80% yield). ESI MS m / z C 32 H 48 N3O6[M+H] + Calculated value: 570.34, measured value: 570.33.

[0327] Example 52: Synthesis of 2-benzyl-13-methyl-11,14-dioxo-1-phenyl-5,8-dioxa-2,12,15-triazanonadecan-19-oic acid [ka]

[0328] To a solution of tert-butyl 2-benzyl-13-methyl-11,14-dioxo-1-phenyl-5,8-dioxa-2,12,15-triazanonadecan-19-oate (0.057 g, 0.101 mmol, 1.0 equiv.) in DCM (3 mL) was added TFA (1 mL) and stirred for 40 minutes at room temperature. The reaction mixture was diluted with anhydrous toluene and concentrated. This procedure was repeated three times to give the title compound as a colorless oil (0.052 g, theoretical yield). This was used directly in the next step. ESI MS m / z C 28H 40 N3O6[M+H] + Calculated value 514.28, measured value 514.28.

[0329] Example 53: Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid [ka]

[0330] A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and sodium hydroxide (6 g, 150 mmol) in water (40 mL) was cooled to 0° C. and treated dropwise with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 mL). After 1 h, the reaction was warmed to room temperature and stirred for 3 h. The THF was removed in vacuo and the pH of the aqueous solution was adjusted to 1.5 by the addition of 6N HCl. After extraction with ethyl acetate, the organic layer was washed with brine, dried, and concentrated to give the title compound (16.4 g, 92% yield). MS ESI m / z C 12 H 16 NO5[M+H] + Calculated value 238.10, Actual measured value: 238.08.

[0331] Example 54: Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate [ka]

[0332] 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4g, 69.2 To a solution of (15.4 g, 208 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL) was added DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol). After stirring overnight at room temperature, the reaction was filtered and the filtrate was concentrated. The residue was dissolved in ethyl acetate, washed with 1N HCl, brine, and dried over sodium sulfate. Concentration and purification by column chromatography (10-50% ethyl acetate / hexanes) gave the title compound (7.5 g, 37% yield). MS ESI m / z C 16 H 23 NO4Na [M+Na] + Calculated value: 316.16, measured value: 316.13.

[0333] Example 55: Synthesis of tert-butyl 4-aminobutanoate [ka]

[0334] tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in methanol (50 mL), mixed with Pd / C catalyst (10 wt%, 100 mg), and hydrogenated (1 atm) at room temperature for 3 hours. The catalyst was filtered off and all volatiles were removed under vacuum to give the title compound (272 mg, 90% yield). MS ESI m / z C8H 18 NO2[M+H] + Calculated value 160.13, actual value 160.13.

[0335] Example 56: 2-(2-(((benzyloxy)carbonyl)amino)propanamide ) Synthesis of tert-butyl acetate [ka]

[0336] 2-(((benzyloxy)carbonyl)amino)propanoic acid (0.84 g, 5 mmol), tert-butyl 2-aminoacetate (0.66 g, 5 mmol), HOBt (0.68 g, 5 mmol), and EDC (1.44 g, 7.5 mmol) were dissolved in DCM (20 ml), and then DIPEA (1.7 ml, 10 mmol) was added. The reaction mixture was stirred overnight at room temperature, washed with water (100 ml), and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over MgSO4, filtered, evaporated under reduced pressure, and the residue was purified on a silica column to give the title product 1 (0.87 g, 52%). ESI: m / z: C 17 H 25 N2O5[M+H] + Calculated value 337.17, measured value 337.17.

[0337] Example 57: 2-(2-(((benzyloxy)carbonyl)amino)propanamide ) Synthesis of acetic acid [ka]

[0338] tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamide)acetate (0.25 g, 0.74 mmol) was dissolved in DCM (30 ml), followed by the addition of TFA (10 ml). The mixture was stirred at room temperature overnight and concentrated to give the title compound, which was used in the next step without further purification. ESI: m / z C 17 H 25 N2O5[M+H] + Calculated value 337.17, measured value 337.17.

[0339] Example 58: Synthesis of di-tert-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacontan-15-yne-1,30-dioate [ka]

[0340] Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 equiv.) was dissolved in NMP (10 mL), cooled to 0 °C, and the compound tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-propanoate (2.06 g, 7.43 mmol, 2.4 equiv.) was added, followed by the portionwise addition of DMTMM (2.39 g, 8.65 mmol, 2.8 equiv.). The reaction was stirred at 0 °C for 6 h, then diluted with ethyl acetate and washed with water and brine. The organic solution was concentrated and triturated with a mixture of ethyl acetate and petroleum ether. The solid was filtered off, and the filtrate was concentrated and purified by column chromatography (80-90% EA / PE) to give a pale yellow oil (2.26 g, >100% yield), which was used without further purification. MS ESI m / z [M+H] + 633.30.

[0341] Example 59: Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioic acid [ka]

[0342] The compound 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-di-tert-butyl ester (2.26 g) was dissolved in dichloromethane (15 mL). The mixture was cooled to 0 °C and treated with TFA (15 mL). The reaction was warmed to room temperature and stirred for 45 min, then the solvent and residual TFA were removed by rotary evaporation. The crude product was purified by column chromatography (0–15% methanol / DCM) to give a pale yellow oil (1.39 g, 86% yield over two steps). MS ESI m / z [M+H] + 521.24.

[0343] Example 60: 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazatetradodecan-21-yne-1,42-diester [ka]

[0344] 14,17-dioxo-4,7,10,21,24,27-hexyl ether in DMA (40 ml) To a mixture of oxaoxa-13,18-diazatriacontan-15-yne-1,30-diester (1.38 g, 2.65 mmol) and tert-butyl 2-(2-aminopropanamido)propanoate (0.75 g, 3.47 mmol) was added EDC (2.05 g, 10.67 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / dichloromethane (1:5 to 1:1) to give the title compound (2.01 g, 82% yield, approximately 95% purity by HPLC. MS ESI m / z C 42 H 73 NO 16 [M+H] + total Calculated value: 917.50, actual value: 917.90.

[0345] Example 61: Synthesis of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-1,42-dioic acid [ka]

[0346] Di-di-tert-butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-1,42-dioate (1.50 g, 1.63 mmol) was dissolved in a mixture of dichloromethane (10 mL) and TFA (10 mL). The mixture was stirred overnight, diluted with toluene (20 mL), and concentrated to give the title compound (1.33 g, 101% yield, approximately 92% purity by HPLC), which was used in the next step without further purification. MS ESI m / z C 34 H 56 NO 16 [M+H] + Calculated value: 805.37, measured value: 805.85.

[0347] Example 62: Bis(2,5-dioxopyridin-1-yl)2,5,38,41-tet Synthesis of tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaaza-1,42-dicarboxylate [ka]

[0348] 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-1,42-dioic acid (1.30 g, 1.61 m To a mixture of 10 ml of DMA (1.0 mol) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / dichloromethane (1:4 to 2:1) to give the title compound (1.33 g, 83% yield, approximately 95% purity by HPLC). MS ESI m / z C 42 H 63N8O 20 [M+H] + Calculated value 999.40, actual value 999.95.

[0349] Example 63: Synthesis of 2,3-bis(2-bromoacetamido)succinyl dichloride [ka]

[0350] To a solution of 2,3-diaminosuccinic acid (5.00 g, 33.77 mmol) in a mixture of THF / water / DIPEA (125 ml / 125 ml / 8 ml) was added 2-bromoacetyl bromide (25.0 g, 125.09 mmol). The mixture was stirred overnight, evaporated, and purified by silica column chromatography (water / acetonitrile 5:95) to give 2,3-bis(2-bromoacetamido)succinic acid as a pale yellow oil (9.95 g, 76% yield). MS ESI m / z C8H 11 Br2N2O6[M+H] + Calculated value: 388.89, measured value: 388.68.

[0351] To a solution of 2,3-bis(2-bromoacetamido)succinic acid (3.50 g, 9.02 mmol) in dichloromethane (80 ml) was added oxalyl chloride (5.80 g, 46.05 mmol) and DMF (0.01 ml). The mixture was stirred for 2.5 hours and diluted with toluene. The mixture was dried by coevaporation with dichloroethane (2 × 20 mL) and toluene (2 × 15 mL) to give 2,3-bis(2-bromoacetamido)succinyl dichloride as a crude (not stable) product (3.90 g, 102% yield), which was used in the next step without further purification. MS ESI m / z calculated for C8H9Br2Cl2N2O4 [M+H]+ 424.82, found 424.90.

[0352] Example 64: Synthesis of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid [ka]

[0353] To a mixture of 2,3-diaminosuccinic acid (4.05 g, 27.35 mmol) in THF (250 mL) and NaH2PO4 (0.1 M, 250 mL, pH 8.0), benzyl carboxylochloridate (15.0 g, 88.23 mmol) was added in four portions over 2 hours. The mixture was stirred for an additional 6 hours, concentrated, and purified on a silica column eluted with water / acetonitrile (1:9) containing 1% formic acid to give the title compound (8.65 g, 76% yield, approximately 95% purity). MS ESI m / z C 20 H 21 N2O8[M+H] + Calculated value 417.12, actual value 417.60.

[0354] Example 65: Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(((benzyloxy)carbonyl)-amino)succinate [ka]

[0355] To a mixture of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (4.25 g, 10.21 mmol) in DMA (70 ml) was added NHS (3.60 g, 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / dichloromethane (1:6) to give the title compound (5.42 g, 87% yield, approximately 95% purity). MS ESI m / z C 28 H 27 N4O 12 [M+H] + Calculated value: 611.15, measured value: 611.60.

[0356] Example 66: Synthesis of 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinic acid [ka]

[0357] To a mixture of 2,3-diaminosuccinic acid (5.00 g, 33.77 mmol) in THF / HO / DIPEA (125 mL / 125 mL / 2 mL) was added maleic anhydride (6.68 g, 68.21 mmol). The mixture was stirred overnight and evaporated to give 2,3-bis((Z)-3-carboxyacrylamide)succinic acid as a white solid (11.05 g, 99% yield). MS ESI m / z C 12 H9N2O8[M+H] + Calculated value: 309.03, measured value: 309.30.

[0358] To a solution of 2,3-bis((Z)-3-carboxycalilamide)succinic acid (11.05 g, 33.43 mmol) in a mixture of HOAc (70 mL), DMF (10 mL), and toluene (50 mL) was added acetic anhydride (30 mL). The mixture was stirred for 2 hours, refluxed at 100°C for 6 hours using a Dean-Stark trap, concentrated, co-evaporated with ethanol (2 x 40 mL) and toluene (2 x 40 mL), and purified on a silica column eluted with water / acetonitrile (1:10) to give the title compound (7.90 g, 76% yield, approximately 95% purity). MS ESI m / z C 12 H9N2O8[M+H] + Calculated value: 309.03, measured value: 309.30.

[0359] Example 67: Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinate [ka]

[0360] To a mixture of 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinic acid (4.00 g, 12.98 mmol) in DMF (70 mL) was added NHS (3.60 g, 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / dichloromethane (1:6) to give the title compound (5.73 g, 88% yield, approximately 96% purity by HPLC). MS ESI m / z C 20 H 15 N4O 12 [M+H] + Calculated value 503.06, measured value 503.45.

[0361] Example 68: (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tert-butoxycarbonyl)amino)butyl)-22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,42-bis((4-( Synthesis of (hydroxymethyl)phenyl)carbamoyl)-5,8,21,24,37,40-hexaoxo-11,14,17,28,31,34-hexaoxa-4,7,20,25,38,41-hexaazatetratetradecane-1,44-diester [ka]

[0362] A solution of (14S,17S)-tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate (1.43 g, 1.97 mmol) and 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinic acid (0.30 g, 0.97 mmol) in DMA (25 ml) was added to EDC (1.30 g, 6.77 mmol). The mixture was stirred overnight, evaporated in vacuo, and purified on silica gel using a mixture of methanol (5% to 8%) in methylene chloride as the eluent to give the title compound (1.33 g, 80% yield). ESI MS m / z C 82 H 123 N 12 O 28 [M+H] + Calculated value 1722.85, actual value 1722.98.

[0363] Example 69: Synthesis of tert-butyl 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate [ka]

[0364] To a mixture of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (1.55 g, 6.27 mmol), tert-butyl 2-(2-aminopropanamido)propanoate (1.35 g, 6.27 mmol) in DMA (60 ml) was added EDC (3.05 g, 15.88 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / dichloromethane (1:3) to give the title compound (2.42 g, 86% yield, approx. 95% purity by HPLC). MS ESI m / z C 19 H 36 N5O7[M+H] + Calculated value: 446.25, actual value: 446.60.

[0365] Example 70: Synthesis of 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanoic-18-oic acid [ka]

[0366] To a solution of 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate (2.20 g, 4.94 mmol) in 1,4-dioxane (40 mL) was added HCl (12 M, 10 mL). The mixture was stirred for 40 minutes, diluted with dioxane (20 mL) and toluene (40 mL), evaporated, co-evaporated with dioxane (20 mL) and toluene (40 mL), and dried to give the crude title compound (1.92 g, 100% yield, approximately 94% purity by HPLC), which was used in the next step without further purification. MS ESI m / z C 15 H 28 N5O7[M+H] + Calculated value: 390.19, measured value: 390.45.

[0367] Example 71: Synthesis of 21,22-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazatetradodecane-1,42-dioic acid [ka]

[0368] To a solution of 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanoic-18-oic acid (1.90 g, 4.88 mmol) in DMA (40 mL) was added Pd / C (0.20 g, 50% wet). The reaction was evacuated under vacuum and placed under 2 atmospheres of hydrogen gas via a hydrogenation reactor with vigorous stirring. The reaction was then stirred at room temperature for 6 hours, at which point TLC confirmed the disappearance of the starting material. The crude reaction mixture was passed through a short pad of Celite and rinsed with ethanol. The solvent was concentrated under reduced pressure to give the crude product, 1-amino-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanoic-18-oic acid, which was used directly in the next step. ESI MS m / z + C 15 H 30 N3O7(M+H), calculated 364.20, found 364.30.

[0369] To a solution of the amino compound in DMA (~30 ml) was added 0.1 M disodium phosphate, pH 7.5 (20 ml), followed by bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) succinate (1.30 g, 2.59 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with 8% water in acetonitrile to give the title compound (1.97 g, 81% yield). ESI MS m / z + C 42 H 63 N8O 20 (M+H) calculated 999.41, found 999.95.

[0370] Example 72: Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 21,22-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazatetradodecane-1,42-dioate [ka]

[0371] To a solution of 21,22-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracontane-1,42-dioic acid (1.50 g, 1.50 mmol) in DMA (10 mL) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / CHCl (1:4 to 2:1) to give the title compound (1.50 g, 83% yield, approximately 95% purity by HPLC). MS ESI m / z C 50 H 69 N 10 O 24 [M+H] + Calculated value: 1193.44, actual value: 1193.95.

[0372] Example 73: (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate salt Synthesis of acid salts [ka]

[0373] To a solution of trans-4-hydroxy-L-proline (15.0 g, 114.3 mmol) in dry methanol (250 mL) was added thionyl chloride (17 mL, 231 mmol) dropwise at 0-4°C. The resulting mixture was stirred overnight at room temperature, concentrated, and crystallized from ethanol / hexane to give the title compound (18.0 g, 87% yield). ESI MS m / z 168.2 ([M+Na] + ).

[0374] Example 74: Synthesis of (2S,4R)-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate [ka]

[0375] To a solution of trans-4-hydroxy-L-proline methyl ester (18.0 g, 107.0 mmol) in methanol (150 mL) in a mixture of sodium bicarbonate solution (2.0 M, 350 mL) was added BocO (30.0 g, 137.6 mmol) in three portions over 4 hours. After stirring for an additional 4 hours, the reaction was concentrated to approximately 350 mL and extracted with ethyl acetate (4 x 80 mL). The combined organic layers were washed with brine (100 mL), dried (MgSO), filtered, concentrated, and purified by silica column chromatography (1:1 hexane / ethyl acetate) to give the title compound (22.54 g, 86% yield). ESI MS m / z 268.2 ([M+Na] + ).

[0376] Example 75: Synthesis of (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate [ka]

[0377] The title compound was prepared by Dess-Martin oxidation as described by Franco Manfre et al. J. Org. Chem. 1992, 57, 2060-2065. Alternatively, the procedure for Swern oxidation is as follows: To a solution of (COCl)2 (13.0 ml, 74.38 mmol) in dichloromethane (350 ml) cooled to -78 °C, dry DMSO (26.0 ml) was added. The solution was stirred at -78 °C for 15 minutes, and then (2S,4R)-1-tert-butyl-2-methyl-4-hydroxypyrrolidine-1,2-dicarboxylate in dichloromethane (100 ml) was added. To the resulting solution was added methyltriethylamine (8.0 g, 32.63 mmol). After stirring at -78 °C for 2 h, triethylamine (50 mL, 180.3 mmol) was added dropwise and the reaction was allowed to warm to room temperature. The mixture was diluted with aqueous NaH2PO4 (1.0 M, 400 mL) and the phases were separated. The aqueous layer was extracted with dichloromethane (2 x 60 mL). The organic layers were combined, dried over magnesium sulfate, filtered, concentrated, and purified by silica column chromatography (7:3 hexane / ethyl acetate) to give the title compound (6.73 g, 85% yield). ESI MS m / z 266.2 ([M+Na] + ).

[0378] Example 76: Synthesis of (S) tert-butyl-1,2-methyl-4-methylenepyrrolidine-1,2-dicarboxylate [ka]

[0379] To a suspension of methyltriphenylphosphonium bromide (19.62 g, 55.11 mmol) in THF (150 mL) was added potassium tert-butoxide (6.20 g, 55.30 mmol) in anhydrous THF (80 mL) at 0 °C. After stirring for 2 h at 0 °C, the resulting yellow ylide was added to a solution of (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate (6.70 g, 27.55 mmol) in THF (40 mL). After stirring for 1 h at room temperature, the reaction mixture was concentrated, diluted with ethyl acetate (200 mL), washed with water (150 mL), brine (150 mL), dried over magnesium sulfate, concentrated, and purified by silica column chromatography (9:1 hexane / ethyl acetate) to give the title compound (5.77 g, 87% yield). EI MS m / z 264 ([M+Na] + ).

[0380] Example 77: Synthesis of (S)-methyl 4-methylenepyrrolidine-2-carboxylate hydrochloride [ka]

[0381] To a solution of (S)-1-tert-butyl 2-methyl 4-methylenepyrrolidine-1,2-dicarboxylate (5.70 g, 23.63 mmol) in ethyl acetate (40 mL) was added HCl (12 M, 10 mL) at 4 °C. The mixture was stirred for 1 h, diluted with toluene (50 mL), concentrated, and crystallized from ethanol / hexane to give the title compound as the hydrochloride salt (3.85 g, 92% yield). EI MS m / z 142.2 ([M+H] + ).

[0382] Example 78: Synthesis of (S)-tert-butyl 2-(hydroxymethyl)-4-methylenepyrrolidine-1-carboxylate [ka]

[0383] To a solution of (S)-1-tert-butyl 2-methyl 4-methylenepyrrolidine-1,2-dicarboxylate (5.20 g, 21.56 mmol) in anhydrous THF (100 mL) was added LiAlH (15 mL, 2 M in THF) at 0 °C. After stirring for 4 h at 0 °C, the reaction was quenched by the addition of methanol (5 mL) and water (20 mL). The reaction mixture was neutralized to pH 7 with 1 M HCl, diluted with ethyl acetate (80 mL), filtered through Celite, separated, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over NaSO, concentrated, and purified by silica column chromatography (1:5 ethyl acetate / DCM) to give the title compound (3.77 g, 82% yield). EI MS m / z 236.40 ([M+Na] + ).

[0384] Example 79: Synthesis of (S)-(4-methylenepyrrolidin-2-yl)methanol hydrochloride [ka]

[0385] To a solution of (S)-tert-butyl 2-(hydroxymethyl)-4-methylenepyrrolidine-1-carboxylate (3.70 g, 17.36 mmol) in ethyl acetate (30 mL) was added HCl (12 M, 10 mL) at 4°C. The mixture was stirred for 1 hour, diluted with toluene (50 mL), concentrated, and crystallized from ethanol / hexane to give the title compound as the hydrochloride salt (2.43 g, 94% yield). EI MS m / z 115.1 ([M+H] + ).

[0386] Example 80: Synthesis of 4-(benzyloxy)-3-methoxybenzoic acid [ka]

[0387] To 4-hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) in ethanol (350 ml) and aqueous NaOH (2.0 M, 350 ml) was added BnBr (140.0 g, 823.5 mmol). The mixture was stirred at 65° C. for 8 hours, concentrated, coevaporated with water (2×400 ml), concentrated to approximately 400 ml, and acidified to pH 3.0 with 6 N HCl. The solid was collected by filtration, crystallized from ethanol, and dried under vacuum at 45° C. to give the title compound (63.6 g, 83% yield). ESI MS m / z 281.2 ([M+Na] + ).

[0388] Example 81: Synthesis of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid [ka]

[0389] To a solution of 4-(benzyloxy)-3-methoxybenzoic acid (63.5 g, 246.0 mmol) in CHCl (400 mL) and HOAc (100 mL) was added nitric acid (fuming, 25.0 mL, 528.5 mmol). The mixture was stirred for 6 hours, concentrated, crystallized from ethanol, and dried under vacuum at 40°C to give the title compound (63.3 g, 85% yield). ESI MS m / z 326.1 ([M+Na] + ).

[0390] Example 82: Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone [ka]

[0391] A catalytic amount of DMF (30 μL) was added to a solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CHCl (70 mL), and the resulting mixture was stirred at room temperature for 2 h. Excess CHCl and oxalyl chloride were removed on a rotary evaporator. Acetyl chloride was resuspended in fresh dichloromethane (70 mL) and slowly added to a premixed solution of (S)-(4-methylenepyrrolidin-2-yl)methanol hydrochloride (1.32 g, 8.91 mmol) and EtN (6 mL) in CHCl at 0 °C under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and continued stirring for 8 h. After removing the CHCl and EtN, the residue was partitioned between water and ethyl acetate (70 / 70 mL). The aqueous layer was further extracted with ethyl acetate (2 x 60 mmol). The combined organic layers were washed with brine (40 mL), dried (MgSO4), and concentrated. The residue was purified by high-performance chromatography (silica gel, 2:8 hexane / ethyl acetate) to give the title compound (2.80 g, 79% yield). EI MS m / z 421.2 ([M+Na] + ).

[0392] Example 83: Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-1-yl)methanone [ka]

[0393] (S)-(4-(benzyl)-2-methyl-2-benzoyl)-1,2-dibenzofuran-2-one in a mixture of DCM (10 ml) and pyridine (10 ml) To (oxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone (2.78 g, 8.52 mmol) was added tert-butylchlorodimethylsilane (2.50 g, 16.66 mmol). The mixture was stirred overnight, concentrated, and purified on a silica column eluted with ethyl acetate / dichloromethane (1:6) to give the title compound (3.62 g, 83% yield, approximately 95% purity). MS ESI m / z C 27 H 37 N2O6Si [M+H] + Calculated value 513.23, measured value 513.65.

[0394] Example 84: Synthesis of (S)-(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone [ka]

[0395] To (S)-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone (2.80 g, 7.03 mmol) in a mixture of DCM (30 mL) and CHSOH (8 mL) was added PhSCH (2.00 g, 14.06 mmol). The mixture was stirred for 0.5 h, diluted with DCM (40 mL), and neutralized by careful addition of 0.1 M NaCO solution. The mixture was separated, and the aqueous solution was extracted with DCM (2 × 10 mL). The combined organic layers were dried over sodium sulfate, concentrated, and purified on a silica column eluted with methanol / dichloromethane (1:15 to 1:6) to give the title compound (1.84 g, 85% yield, ~95% purity). MS ESI m / z C 14 H 17 N2O6[M+H] + Calculated value: 309.10, measured value: 309.30.

[0396] Example 85: Synthesis of (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidon-1-yl)methanone) [ka]

[0397] To a solution of (S)-(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone (0.801 g, 2.60 mmol) in butanone (10 mL) was added CsCO (2.50 g, 7.67 mmol), followed by 1,5-diiodopentane (415 mmol, 1.28 mmol). The mixture was stirred for 26 h and then concentrated and purified on a silica column eluted with methanol / dichloromethane (1:15 to 1:5) to give the title compound (0.675 g, 77% yield). purity ~95%). MS ESI m / z C 27 H 37 N2O6Si [M+H] +Calculated value 513.23, measured value 513.65.

[0398] Example 86: Synthesis of (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone [ka]

[0399] To a solution (10 ml) of (S)-((pentane-1,5-diylbis(oxy)bis(5-methoxy-2-nitro-4,1-phenylene))bis((((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone (0.670 g, 0.98 mmol) in methanol was added a solution (8 ml) of NaSO (1.01 g, 5.80 mmol). The mixture was stirred at room temperature for 30 h. The reaction mixture was evaporated, co-evaporated with DMA (2 × 10 mL) and ethanol (2 × 10 mL), and dried under high vacuum to give the title compound (total weight 1.63 g) containing inorganic salts, which was used directly in the next step (without further separation). EIMS m / z 647.32 ([M+Na] + ).

[0400] Example 87: Synthesis of C-1 (PBD dimer analogue with bis-linker) [ka]

[0401] (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tert-butoxycarbonyl)amino)butyl)-22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,42-bis((4-(hydroxymethyl)phenyl)carbamoyl)) in pyridine (0.100 ml, 1.24 mmol) at 0°C. To a solution of 5,8,21,24,37,40-hexaoxo-11,14,17,28,31,34-hexaoxa-4,7,20,25,38,41-hexaazatetratetracontane-1,44-dioate (0.840 g, 0.488 mmol) in THF (8 mL) was added a solution of triphosgene (0.290 mg, 0.977 mmol) in THF (3.0 mL). The reaction mixture was stirred at 0 °C for 15 min and used directly in the next step.

[0402] At 0 °C, (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone) (0.842 mg, ∼0.49 mmol) containing inorganic salts was suspended in ethanol (10 ml), and the trichloride in THF prepared above was added. The mixture was warmed to room temperature for 1 hour, concentrated, and analyzed by reverse-phase HPLC (250 (L) mm × 10 (d )mm, C 18 The product was purified by column chromatography (10-80% acetonitrile / water in 40 min, v=8 ml / min) to give the title compound (561.1 mg, 48% yield over three steps). ESI MS m / z: C 117 H 163 N 16 O 38 [M+H] + Calculated value: 2400.12, actual value: 2400.90.

[0403] Example 88: Synthesis of C-2 (PBD dimer analogue with bis-linker) [ka]

[0404] To a solution of compound C-1 (132.0 mg, 0.055 mmol) in DCM (5.0 mL) was added Dess-Martin periodinane (138.0 mg, 0.329 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 2 h. A saturated solution of NaHCO3 / Na2SO3 (5.0 mL / 5.0 mL) was then added, and the mixture was extracted with DCM (3 × 25 mL). The combined organic layers were washed with NaHCO3 / Na2SO3 (5.0 mL / 5.0 mL), brine (10 mL), dried over Na2SO4, filtered, concentrated, and purified by reverse-phase HPLC (250 (L) mm × 10 (d) mm, C 18 Purification on a column (10-80% acetonitrile / water in 40 min, v=8 ml / min) gave the title compound as a foam (103.1 mg, 78% yield). ESI MS m / z: C 117 H 158 N 16 O 38 [M+H] + Calculated value 2396.09, actual value 2396.65.

[0405] Example 89: Synthesis of C-3 (PBD dimer analogue with bis-linker) [ka]

[0406] Compound C-2 (55.0 mg, 0.023 mmol) was dissolved in DCM (3 ml), followed by the addition of TFA (3 ml) at 4 °C. The reaction mixture was then stirred at room temperature for 1 h, then concentrated and co-evaporated with DCM / toluene to dryness to give crude product C-3 (48.0 mg, 100% yield, 92% purity by HPLC). Reverse-phase HPLC (250 (L) mm × 20 (d) mm, C 18 Purification on a column (5-60% acetonitrile / water in 40 min, v=8 ml / min) gave the pure product C-3 as a foam (42.1 mg, 88% yield, 96% purity). ESI MS m / z: C 99 H 126 N 16 O 34 [M+H] + Calculated value: 2083.86, actual value: 2084.35.

[0407] Example 90: Synthesis of C-4 (PBD dimer analogue with bis-linker) [ka]

[0408] Compound C-3 (35.0 mg, 0.017 mmol) was dissolved in a mixture of THF (3 mL) and 0.1 M disodium phosphate (3 mL), pH 7.5, followed by the addition of N-succinimidyl 2,5,8,11,14,17,20,23-octaoxahexacosane-26-olate (43.0 mg, 0.084 mmol) in four portions over 2 h. The reaction mixture was stirred at room temperature for 4 h and then coevaporated with DMF (10 mL) to dryness to give crude product C-4, which was purified by reverse-phase HPLC (250 (L) mm × 20 (d) mm, C 18 Further purification by column chromatography (20-60% acetonitrile / water in 40 min, v=8 ml / min) gave the pure product C-4 as a foam (39.4 mg, 81% yield, 96% purity). ESI MS m / z: C 135 H 195 N 16 O 52 [M+H] + Calculated value: 2872.30, actual value: 2871.65.

[0409] Example 91: Synthesis of C-5 (PBD dimer analogue with bis-linker) [ka]

[0410] To a solution of the C-4 compound (35.0 mg, 0.012 mmol) and 2,5,8,11,14,17,20,23-octaoxapentacosan-25-amine (15.1 mg, 0.0394 mmol) in dry DMA (2 mL) was added EDC (30.0 mg, 0.156 mmol). The reaction mixture was stirred at room temperature for 14 h, concentrated, and analyzed by reverse-phase HPLC (250 (L) mm × 20 (d) mm, C 18Purification by column chromatography (20-60% acetonitrile / water in 40 min, v=8 ml / min) gave the pure product C-5 as a foam (31.2 mg, 77% yield, 97% purity by HPLC). ESI MS m / z: C 161 H 249 N 18 O 62 [M+H] + Calculated value: 3426.68, measured value: 3427.21.

[0411] Example 92: Synthesis of (S)-methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylate [ka]

[0412] To a solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CHCl (70 mL) was added a catalytic amount of DMF (30 μL). The resulting mixture was stirred at room temperature for 2 h. Excess CHCl and oxalyl chloride were removed on a rotary evaporator. Acetyl chloride was resuspended in fresh CHCl (70 mL) and slowly added to a premixed solution of (S)-methyl 4-methylenepyrrolidine-2-carboxylate hydrochloride (1.58 g, 8.91 mmol) and EtN (6 mL) in CHCl at 0 °C under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and continued stirring for 8 h. After removing the CHCl and EtN, the residue was partitioned between water and ethyl acetate (70 / 70 mL). The aqueous layer was further extracted with ethyl acetate (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried (MgSO4), and concentrated. The residue was purified by flash chromatography (silica gel, 2:8 hexanes / ethyl acetate) to give the title compound (2.88 g, 76% yield). EI MS m / z 449.1 ([M+Na] + ).

[0413] Example 93: Synthesis of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carbaldehyde [ka]

[0414] At −78° C. under a nitrogen atmosphere, DIBAL-H (1N in dichloromethane, 10 mL) was added dropwise to a vigorously stirred solution of (S)-methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylate (2.80 g, 6.57 mmol) in anhydrous dichloromethane (60 mL). The mixture was stirred for an additional 90 minutes, after which the excess reagent was destroyed with 2 mL of methanol, followed by 5% HCl (10 mL). The resulting mixture was warmed to 0° C. The layers were separated, and the aqueous layer was further extracted with dichloromethane (3×50 mL). The combined organic layers were washed with brine, dried (magnesium sulfate), and concentrated. The residue was purified by flash chromatography (silica gel, 95:5 CHCl3 / methanol) to give the title compound (2.19 g, 84% yield). EIMS m / z 419.1 ([M + Na] + ).

[0415] Example 94: Synthesis of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]-pyrrolo[1,2-a]azepin-5(11aH)-one [ka]

[0416] A mixture of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carbaldehyde (2.18 g, 5.50 mmol) and sodium sulfate (8.0 g, 45.97 mmol) in THF (60 mL) and water (40 mL) was stirred at room temperature for 20 h. The solvent was removed under high vacuum. The residue was resuspended in methanol (60 mL) and HCl (6 M) was added until a pH of approximately 2 was reached. The resulting mixture was stirred at room temperature for 1 h. The reaction was worked up by removing most of the methanol and then diluted with ethyl acetate (100 mL). The ethyl acetate solution was washed with saturated NaHCO3, brine, dried (MgSO4), and concentrated. The residue was purified by flash chromatography (silica gel, 97:3 CHCl3 / methanol) to give the title compound (1.52 g, 80%). EIMS m / z 372.1 ([M + Na] + ).

[0417] Example 95: Synthesis of (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]-pyrrolo[1,2-a]azepin-5(11aH)-one [ka]

[0418] To a solution of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]-pyrrolo[1,2-a]azepin-5(11aH)-one (1.50 g, 4.32 mmol) in CHCl (70 mL) was added CHSOH (25 mL) at 0 °C. The mixture was stirred at 0 °C for 10 min, then at room temperature for 2 h, diluted with CHCl, the pH adjusted to 4 with cold 1.0 N NaHCO, and the aqueous layer extracted with CHCl (3 × 60 mL). The organic layers were combined, dried over NaSO, filtered, evaporated, and purified by SiO column chromatography (CHOH / dichloromethane 1:15) to give 811 mg of the title product (73% yield). EIMS m / z 281.1 ([M+Na] + ).

[0419] Example 96: Synthesis of (11aS,11a's)-8,8'-(pentane-1,5-di(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one [ka]

[0420] To a stirred suspension of CsCO (0.761 g, 2.33 mmol) in butanone (8 mL) was added (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]-pyrrolo[1,2-a]azepin-5(11aH)-one (401 mg, 1.55 mmol) and 1,5-diiodopentane (240 mg, 0.740 mmol). The mixture was stirred overnight at room temperature, concentrated, and purified by silica chromatography (ethyl acetate / dichloromethane 1:10) to give 337 mg of the title product (78% yield). EIMS m / z 607.2 ([M+Na] + ).

[0421] Example 97 Synthesis of (S)-7-methoxy-8-((5-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-5benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one and (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaz-5(10H)-one) [ka]

[0422] To (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one) (150 mg, 0.256 mmol) in anhydrous dichloromethane (1 mL) and absolute ethanol (1.5 mL) at 0 °C was added a solution of sodium borohydride in methoxyethyl ether (85 μL, 0.5 M, 0.042 mmol). After 5 min, the ice bath was removed and the mixture was stirred at room temperature for 3 h, then cooled to 0 °C, quenched with saturated ammonium chloride, diluted with dichloromethane, and the phases were separated. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered through Celite, and concentrated. The residue was purified by reverse-phase HPLC (C 18 The resulting mixture was purified by column chromatography (acetonitrile / water). The corresponding fractions were extracted with dichloromethane and concentrated to give the semi-reduced compound (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (64.7 mg, 43%), MS m / z 609.2 ([M+Na] + ), 625.3([M+K]+) and 627.2 ([M + Na + H2O] + ); the fully reduced compound (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(10H)-one) (16.5 mg, 11%), MS m / z 611.2 ([M+Na] + ) , 627.2([M+K] + ), 629.2 ([M + Na + H2O] + ) and the unreacted starting material is also recovered. (10.2mg, 7%), MS m / z 607.2([M+Na] +), 625.2 ([M + Na + H2O] + ).

[0423] Example 98: Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one [ka]

[0424] To a solution of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (60.0 mg, 0.102 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (40.5 mg, 0.134 mmol) in dichloromethane (5 mL) was added EDC (100.5 mg, 0.520 mmol). The mixture was stirred at room temperature. It was concentrated overnight and purified by silica column chromatography (ethyl acetate / dichloromethane 1:6) to give 63.1 mg (81% yield) of the title product. ESI MS m / z C 40 H 50 N7O9[M+H] + Calculated value: 0.772.36, measured value: 772.30.

[0425] Example 99 Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-aminoethoxy)ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one [ka]

[0426] To a solution of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (60 mg, 0.078 mmol) in THF (5 mL) and NaHPO buffer (pH 7.5, 1.0 M, 0.7 mL) was added PPh (70 mg, 0.267 mmol). The mixture was stirred overnight at room temperature, concentrated, and the resulting mixture was purified by HPLC. 18 After purification by preparative HPLC eluting with water / acetonitrile (90% water to 35% water in 35 min) and drying under high vacuum, 45.1 mg of the title product was obtained (79% yield). ESI MS m / z C 40 H 52 N5O9[M+H] + total Calculated value: 746.37, actual value: 746.50.

[0427] Example 100: (S)—N-(2-((S)-8-((5-(((11S,11aS)-10-((S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H- Benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy) Synthesis of propanamido-3-methylbutanamide [ka]

[0428] To (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (60.0 mg, 0.102 mmol) and (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazepinedecanoic-1-oic acid (90.2 mg, 0.25 mmol) in DMA (8 ml) was added BrOP (240.2 mg, 0.618 mmol). The mixture was stirred at 60° C. overnight, concentrated, and purified by silica column chromatography (methanol / dichloromethane 1:10 to 1:5) to give 97.1 mg of the title product (74% yield). ESI MS m / z C 61 H 87 N 14 O 17 [M+H] + , Calculated value 1287.63, measured value 1287.95.

[0429] Example 101: (S)—N-(2-((S)-8-((5-(((11S,11aS)-10-((S)-15-amino-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2- Synthesis of a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]-pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)-propanamido)-3-methylbutanamide (C-6) [ka]

[0430] (S)-N-(2-((S)-8-((5-(((11S,11aS)-10-((S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-o To a solution of (2-(2-azidoethoxy)ethoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-3-methylbutanamide (85 mg, 0.066 mmol) in THF (5 mL) was added PPh (100 mg, 0.381 mmol). The mixture was stirred for 2 h, then NaHPO buffer (pH 7.5, 1.0 M, 0.7 mL) was added, and the mixture was stirred for 10 min. After confirmation by LC-MS, bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) succinate (33 mg, 0.066 mmol) was added, and (S)-N-(2-((S)-8-((5-(((11S,11aS)-10-((S)-15-amino-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oyl)-11-hydroxy-7-methoxy-2-methylene -5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)-propanamido)-3-methylbutanamide (ESI MS m / z C 61 H 90 N 10 O 17 [M+Na] + Calculated value 1257.66, measured value 1257.90. The mixture was stirred for 4 hours and eluted with water / acetonitrile (90% water to 30% water in 35 minutes). 18 After concentration and purification by preparative HPLC and drying under high vacuum, the title product C-5 was obtained (40.1 mg, 40% yield). ESI MS m / z C 73 H 95 N 12 O 23 [M+H] + Calculated value: 1507.66, actual value: 1507.90.

[0431] Example 102: Synthesis of 4,4'-(pentane-1,5-diylbis(oxy))bis(3-methoxybenzyl) [ka]

[0432] To a vigorously stirred solution of vanillic acid (20.0 g, 119 mmol) in THF (150 mL) and aqueous NaOH (340 mL) at 65 °C in a light-free environment (foil-wrapped flask), a solution of diiodopropane (19.0 g, 58.6 mmol) in THF (75 mL) was added dropwise over 4 h. After heating to reflux in the dark for 48 h, the solution was cooled and the THF was removed by evaporation in vacuo. The residue was extracted with EA. The aqueous layer was separated and acidified to pH 2 with concentrated HCl. The resulting precipitate was collected by filtration, washed, dried, and recrystallized from glacial acetic acid to give the corresponding biscarboxylic acid as a white solid (14.0 g, 34.7 mmol). Yield (60%).

[0433] Example 103: 4,4'-(pentane-1,5-diylbis(oxy))bis(5-methyl) Synthesis of 2-nitrobenzoic acid [ka]

[0434] To a suspension of 4,4'-(pentane-1,5-diylbis(oxy))bis(3-methoxybenzoic acid) (18.0 g, 66.8 mmol) in HOAc (80 mL, 1800 mmol) was added dropwise HNO3 (80 mL, 1778 mmol) at room temperature. After stirring for 2 h, the mixture was poured onto 100 g of ice and extracted with EA (2 × 200 mL). The organic layer was separated and washed with water (2 × 100 mL), followed by the addition of 4 N sodium hydroxide (400 mL). After extraction with EA (2 × 100 mL), the basic aqueous layer was separated and acidified to pH 2 with concentrated HCl. The mixture was extracted with EA (2 × 250 mL). The combined organic extracts were washed with brine, dried, filtered, and concentrated. The residue was purified by flash chromatography (DCM / methanol=4 / 1) to give 4,4'-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid) (6.1 g, 12.3 mmol) as a pale yellow solid (18% yield). f 0.3 (DCM / methanol = 3 / 1)

[0435] Example 104: Synthesis of (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl))pyrrolidin-1-yl)methanone) [ka]

[0436] To a solution of L-(+)-prolinol (2.25 g, 22.3 mmol) in DMF (100 mL) was added 4,4'-(pentane-1,5-dialkylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid) (5.0 g, 10.0 mmol) at room temperature, TEA (4.0 g) was added. After stirring for 10 min, HATU (10.77 g, 28.3 mmol) was added. The mixture was stirred at room temperature overnight. After complete conversion, the mixture was diluted with water (100 mL) and extracted with EA (2 × 100 mL) and DCM (2 × 50 mL). The combined organic extracts were washed with brine, dried, filtered, and concentrated. The residue was purified by chromatography (DCM / methanol=15 / 1) to give (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone) as a white foam (6.025 g, 9.1 mmol, 91% yield).

[0437] Example 105: (S)-((pentane-1,5-diylbis(oxy))bis(2-amino) Synthesis of ((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone [ka]

[0438] To a solution of (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))-bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone) (6.0 g, 9.1 mmol) in MeOH (100 mL) was added 10% Pd / C (2.4 g), and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. After 14 h of stirring, the Pd / C was removed by filtration and washed with methanol. The filtrate was concentrated, and the residue was purified by chromatography (DCM / MeOH=10 / 1) to give (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone) as a white foam (3.54 g, 5.9 mmol, 65% yield).

[0439] Example 106: Synthesis of bis(4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxy-5,1-phenylene)) dicarbamate [ka]

[0440] To a solution of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (8.0 g, 21.3 mmol) in anhydrous THF (300 mL) was added a solution of DIPEA (5.5 g, 40.3 mmol) and triphosgene (3.2 g, 10.8 mmol) in dry THF (50 mL) at 5° C. After stirring for 15 minutes, the solution was recooled to 5° C., and a solution of (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-pyrrolidin-1-yl)methanone) (3.2 g, 5.3 mmol) and DIPEA (2.75 g, 21.6 mmol) in anhydrous THF (150 mL) was added. The resulting solution was allowed to warm to room temperature and stirred overnight. The THF was removed by evaporation in vacuo. The residue was purified by chromatography (DCM / methanol=20 / 1) to give bis(4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)-benzyl)((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxy-5,1-phenylene)) dicarbamate as a yellow foam (7.0 g, 4.97 mmol, 94% yield).

[0441] Example 107: Synthesis of (11S,11aS,11'S,11a'S)-bis(4-((S)-2-((S)-2-((allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl)8,8'-(pentane-1,5-diylbis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ka]

[0442] At room temperature under a nitrogen gas atmosphere, a dry DCM solution (15 mL) of bis(4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxy-5,1-phenylene))dicarbamate (300 mg, 0.21 mmol) was added to DMSO. P (280 mg, 0.66 mmol) was added. After the conversion was complete, aqueous NaSO was added to the reaction solution, followed by aqueous NaHCO, and the mixture was stirred for an additional 15 min and extracted with DCM (3 × 20 mL). The combined organic extracts were washed with brine, dried, filtered, and concentrated. The residue was purified by chromatography (DCM / methanol=20 / 1) to give (11S,11aS,11′S,11a′S)-bis(4-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)8,8′-(pentane-1,5-diylbis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) as a white foam (270 mg, 0.19 mmol, 92% yield).

[0443] Example 108: Synthesis of (11S,11aS,11'S,11a'S)-bis(4-((S)-2-((S)-2-((allyloxy)carbonyl)-amino)-3-methylbutamido)propanamido)benzyl)8,8'-(pentane-1,5-diylbis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ka]

[0444] To a dry solution of (11S,11aS,11'S,11a'S)-bis(4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)8,8'-(pentane-1,5-diylbis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-...

Claims

1. A bridged PBD dimer derivative having the structure of formula (V), (VI), or (VIII): 【Chemistry 14】 During the ceremony, 【Chemistry 2】 optionally represents a single bond or may be absent; 【Transformation 3】 optionally represents a single or double bond; V and V' are the same or different, H, OH; OR ５ (ether); wherein R 5 is as shown below; q and q′ are independently 0, 1, 2, 3, 4, or 5; l, m, l', and m' are 1; X, X', Y, and Y' are independently the same or different, CH, CR ５ or ═C—; wherein R 5 is as shown below; Z and Z' are CH, COH, or COR Ａ wherein R Ａ is C １ ~C ８ independently selected from alkyl and aryl; G is -CH ２ - is; U and U′ are independently C(O), C(O)O, C(O)NH, or C(O)N(R ５ ) wherein R 5 is as shown below: L １ and L ２ are independently a releasable linker having the formula -Ww-(Aa)r-Tt-, wherein: -W- is an expansion unit; w is 1; -Aa- is an independent amino acid unit; r is independently an integer between 0 and 100; -T- is a spacer unit, which is a linear or branched alkyl, or polyethylene glycol spacer; and t is 0 or 1 to 100; The expansion unit W independently includes the following self-collapsible spacers: 【Chemistry 28】 In the formula, ( ＊ The atom labeled with is the point of attachment to another moiety; X1 is NH; Y1 is O; Z １ are independently H, OH, and NHR １ , OR １ , S.R. １ , COX １ R １ where X １ and R １ is defined as above; v is 0 or 1; U １ are independently H, OH, or C １ ~C ６ It is alkyl. R １ , R ２ , R ３ , R ４ , R １ ', R ２ ', R ３ ', and R ４ ' are independently selected from the same or different: -H, substituted linear or branched alkyl, alkenyl, or aryl; R ５ is H or C １ ~C ８ selected from alkyl of the formula: In the formula, E ３ and E' ３ is independently selected from: 【Chemistry 16】 During the ceremony, X １ ' and X ３ ' are independently F, Cl, Br, I, or Lv ３ Is; Lv ３ is a leaving group selected from methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, phenylthio, pyridinylthio, N-succinimidyloxyl (NHS), phenoxyl, dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxyl, 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazol-yl (ODA), oxadiazol-yl.

2. The crosslinked PBD dimer derivative of claim 1, represented by the following formula: [Chemistry 18] 【change】 【change】 【change】 【change】 wherein V, V', q, m, m', X, and X' are as defined in claim 1; and r, r', and r'' are independently 0-200.

3. The crosslinked PBD dimer derivative of claim 1, represented by the following formula: 【Chemistry 20】 【change】 【change】 【change】 【change】 【change】 【change】 wherein r is independently 0 to 200.

4. A method for producing a conjugate of a crosslinked PBD dimer derivative and a cell-binding agent / molecule, or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, or an optical isomer, racemate, diastereomer, or enantiomer thereof, by reacting the crosslinked PBD dimer derivative according to any one of claims 1 to 3 with a cell-binding agent / molecule, comprising the steps of: The method, wherein the cell binding agent / molecule is selected from an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to a target cell, a chimeric antibody, a chimeric antibody fragment that binds to a target cell, a domain antibody, a domain antibody fragment that binds to a target cell, or an antibody-mimicking adnectin.

5. 5. The method of claim 4, wherein the conjugate has the structure of formula (I): 【Chemistry 1】 During the ceremony, 【Chemistry 15】 X, X', Y, Y', Z, Z', l, l', m, m', q, q', R １ , R １ ', R ２ , R ２ ', R ３ , R ３ ', R ４ , R ４ ', V, V', U, U', L １ , L ２ , E １ , and E ２ is the same as defined in claim 1; n is 1 to 30; Q is a cell binding agent / molecule.

6. 5. The method of claim 4, wherein the conjugate has a structure of formula (Ia), (Ib), or (Ic): 【Transformation 7】 During the ceremony, 【Transformation 8】 X, X', Y, Y', Z, Z', l, l', m, m', q, q', R １ , R １ ', R ２ , R ２ ', R ３ , R ３ ', R ４ , R ４ ', V, V', U, U', L １ , L ２ , E １ , and E ２ has the same definition as in claim 1, and n and Q have the same definition as in claim 5.

7. 5. The method of claim 4, wherein the conjugate has one of the following structures: 【Chemistry 9】 【change】 【change】 【change】 During the ceremony, V, V', and q have the same definition as in claim 1; n has the same definition as in claim 5; mAb is a cell binding agent / molecule; r and r' are independently 0-200.

8. The method of claim 4, wherein the conjugate has a structure of formula (II) or (IV): 【Chemistry 10】 During the ceremony, 【Chemistry 11】 , X, X', Y, Y', Z, Z', l, l', m, m', q, q', R １ , R １ ', R ２ , R ２ ', R ３ , R ３ ', R ４ , R ４ ', V, V', U, U', L １ , L ２ , G., E. １ , and E ２ has the same definition as in claim 1, and n and Q have the same definition as in claim 5.

9. 5. The method of claim 4, wherein the conjugate has one of the following structures: 【Chemistry 12】 【change】 【change】 During the ceremony, m, m', q, V, and V' are defined as in claim 1; n is defined as in claim 5; r, r １ , r ２ and r' are independently 0 to 200, m ３ is 0-30; mAb is a cell binding agent / molecule.

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