BR112017027445B1 - COMPOUNDS, PHARMACEUTICAL COMPOSITION AND USE THEREOF TO TREAT CANCER, A VIRAL DISEASE, AN AUTOIMMUNE DISEASE, AN ACUTE OR CHRONIC INFLAMMATORY DISEASE AND A DISEASE CAUSED BY BACTERIA, FUNGI, OR OTHER MICROORGANISMS - Google Patents

Abstract

RELEASE SYSTEMS FOR CONTROLLED DRUG RELEASE. The present invention provides a compound having the structure of Formula (I) or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, for controlled release and release agent.

Description

RELATED REQUEST

[001] This application claims the benefit and priority of US Provisional Patent Applications 62/182,219, filed June 19, 2015, 62/261,213, filed November 30, 2015, and 62/261,563, filed December 1 2015. Each of the foregoing orders is incorporated herein by reference in its entirety.

BASICS OF THE INVENTION

[002] Many drugs, particularly cancer therapies, have a narrow therapeutic window, in which their side effects limit their beneficial effects. Systemic administration of such drugs often results in a limited therapeutic effect, since the dose required to evoke a more robust effect results in unacceptable side effects to the patient. This is particularly critical in the case of those drugs that have a high cytotoxic potential, such as cytostatic agents, virostatic agents or immunosuppressive agents. Numerous research efforts have analyzed the release of a particular drug at a particular site of action. Often, this method results in a higher concentration of the drug at the site of action than would be achieved by systemic administration, while limiting side effects.

[003] Drug release in oncology is of particular interest due to the narrow therapeutic window of agents used in such an indication. Numerous research efforts have focused on conjugating anticancer drugs with a broad spectrum of low and high molecular weight carriers including sugars, growth factors, vitamins, peptides, antibodies, polysaccharides, lectins, serum proteins, and synthetic polymers. In most of these drug delivery systems, the drug is bound to the carrier through a Spacer that incorporates a predetermined breaking point that allows the bound drug to be released at the cellular target site. (Kratz et al., ChemMedChem, 3:20-53 (2008)).

[004] Conjugates are known in which cytostatic agents are bound to serum proteins, predominantly to specific carrier molecules such as human serum albumin and human serum transferin, and then administered. In some cases, the conjugates comprise a therapeutically effective substance and, upon administration, result in transport of the therapeutically effective substance to the target site where it is released (US 7,387,771). In other cases, conjugates comprising a therapeutically effective substance, a Spacer molecule and a protein binding molecule, covalently bind to circulating serum albumin after administration, which result in transport of the therapeutically effective substance to the target site where it is released ( US 7,387,771). In still other cases, antibody-drug conjugates (ADC) can transport the drug to the target site for local release (Kratz et al., ChemMedChem, 3:20-53 (2008); Panowski et al., mAbs, 6, 34 -45 (2014); Chari et al., Angewandte Chem. Int. Ed., 53, 3796-3827 (2014)).

[005] However, when designing drug delivery systems, the appropriate balance must be struck between preserving the target properties of the drug carrier while allowing controlled release of the drug. The drug release construct must have sufficient stability in the bloodstream, and yet allow for effective drug release at the tumor site by enzymatic cleavage, reduction, or in a pH-dependent manner (Kratz et al., ChemMedChem, 3:20-53 (2008)). For example, Gemcitabine (Gemzar®) is an anticancer nucleoside chemotherapy agent that is widely used to treat solid tumors. Unfortunately, at its recommended dose of ~1000 mg/m2 approximately 90% are deactivated by cytidine deaminase to the inactive uridine metabolite and excreted in the urine. Another disadvantage resulting in chemo-resistance is the low level of expression of human balanced nucleoside transporter 1 (hENT1) on the cell surface of cancer cells, thus preventing substantial absorption of gemcitabine.

[006] Therefore, there is still a need for efficient drug release and release systems in order to obtain more effective and controlled release and release of the drug.

SUMMARY OF THE INVENTION

[007] The present invention provides delivery systems for the effective and controlled release and release of therapeutic agents.

[008] The present invention relates to a drug delivery system comprising a hydrazone moiety that is cleaved in an acidic environment in a controlled manner, to provide controlled release of therapeutic agents. The present invention relates to a drug delivery system comprising an amide bond, a carbamate bond and/or an ester bond that is cleaved enzymatically, for example, by esterases or amidases and/or hydrolytically, to provide controlled release of therapeutic agents. In some embodiments, the present invention relates to a drug delivery system comprising an amide bond that is cleaved enzymatically, for example, by esterases or amidases and/or hydrolytically, to provide controlled release of therapeutic agents. In other embodiments, the present invention relates to a drug delivery system comprising a carbamate bond that is enzymatically and/or hydrolytically cleaved. In still other embodiments, the present invention relates to a drug delivery system comprising an ester bond that is enzymatically and/or hydrolytically cleaved.

[009] The present invention relates to a drug delivery system comprising (i) a hydrazone moiety that is cleaved in an acidic environment in a controlled manner, and optionally (ii) an amide bond, a carbamate bond and /or an ester bond that is cleaved enzymatically, for example, by esterases or amidases and/or hydrolytically, to provide controlled release of therapeutic agents. In some embodiments, the present invention relates to a drug delivery system comprising a (i) hydrazone moiety that is cleaved in an acidic environment in a controlled manner, and optionally (ii) an amide bond that is enzymatically cleaved. , for example, by esterases or amidases and/or hydrolytically, to provide controlled release of therapeutic agents. In some embodiments, the present invention relates to a drug delivery system comprising a (i) hydrazone moiety that is cleaved in an acidic environment in a controlled manner, and optionally (ii) a carbamate bond that is enzymatically cleaved. and/or hydrolytically, to provide controlled release of therapeutic agents. In some embodiments, the present invention relates to a drug delivery system comprising a (i) hydrazone moiety that is cleaved in an acidic environment in a controlled manner, and optionally (ii) an ester bond that is enzymatically cleaved. and/or hydrolytically, to provide controlled release of therapeutic agents. In some embodiments, the present invention relates to a drug delivery system comprising (i) a hydrazone moiety that is cleaved in an acidic environment in a controlled manner, and optionally (ii) an amide bond that is selectively cleaved. by carboxylesterase 1 and/or 2, to provide controlled release of therapeutic agents.

[010] In some embodiments, the present invention relates to a drug delivery system comprising a (i) hydrazone moiety that is cleaved in an acidic environment in a controlled manner, and (ii) an amide bond that is selectively cleaved by carboxylesterase 2, to provide controlled release of therapeutic agents.

[011] The present invention provides a compound having the structure represented by Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein: Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an antirheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent , an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, an inhibitor of protease, an angiogenesis inhibitor, a hormone or hormone derivative, an antibody or a fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of the precedents; Spacer is missing, II or -X II ; n is 0 or 1; " optionally substituted C1-C18-NH-C(O)-R5-alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl and optionally substituted cycloalkyl; Y is absent or selected from the group consisting of optionally substituted C1-C6 alkyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O- and -OC(O) -; R1 is absent or selected from the group consisting of optionally substituted C1-C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-, or R1 is a naturally occurring amino acid or unnaturally, or R1 has the following formula: on what: is absent, or is selected from the group consisting of: R is: •~'OPO3M1where M-, = Mg2+,2 Na+, 2K+, 2H+, 2NH4 or ~'SO3M2 where M2 = Na+, K+, H+, NH4+ R2 is selected from the group consisting of -H, alkyl optionally substituted C1-C12, optionally substituted aryl and optionally substituted heteroaryl; Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, an electron withdrawing group and a water-soluble group; PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group , an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, an optionally substituted N-hydroxysuccinimide ester group, an antibody or fragment thereof, and an antibody derived from a fragment thereof; wherein when the Spacer is absent, the agent is linked to the nitrogen adjacent to the Spacer by a double bond; and wherein at least one of Z1, Z2, Z3 and Z4 is an electron withdrawing group.

[012] In some embodiments, PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group substituted, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, and an optionally substituted N-hydroxysuccinimide ester group.

[013] In some embodiments, the PBG is associated with an antibody or fragment thereof. In some embodiments, the PBG is covalently linked to an antibody or fragment thereof. In some embodiments, PBG is associated with albumin. In other embodiments, the PBG is covalently linked to endogenous or exogenous albumin. In other embodiments, the PGB is covalently linked to cysteine-34 of endogenous or exogenous albumin.

[014] The present invention provides a compound having the structure represented by Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an antirheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent, an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, a protease inhibitor , an angiogenesis inhibitor, a hormone or hormone derivative, an antibody or fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of the foregoing ; Spacer is missing, n is 0 or 1; " optionally substituted C1-C18-NH- C(O)-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18 alkyl-C(O)-NH-R5- wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl and optionally substituted cycloalkyl; Y is absent or selected from the group consisting of optionally substituted C1-C6 alkyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O- and -OC(O) -; R1 is absent or selected from the group consisting of optionally substituted C1C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5-alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl; Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H or an electron withdrawing group; PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group , an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, an optionally substituted N-hydroxysuccinimide ester group, and an antibody or fragment thereof; wherein when the Spacer is absent, the agent is linked to the nitrogen adjacent to the Spacer by a double bond; and wherein at least one of Z1, Z2, Z3 and Z4 is an electron withdrawing group.

[015] In some embodiments, PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group substituted, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, and an optionally substituted N-hydroxysuccinimide ester group.

[016] In some embodiments, the PBG is associated with an antibody or fragment thereof. In some embodiments, the PBG is covalently linked to an antibody or fragment thereof. In some embodiments, PBG is associated with albumin. In other embodiments, the PBG is covalently linked to endogenous or exogenous albumin. In other embodiments, the PGB is covalently linked to cysteine-34 of endogenous or exogenous albumin.

[017] In certain embodiments, the invention provides a compound represented by Formula (II): Formula (II) or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, PBG, Y, R1, Z1, Z2, Z3 and Z4 are as defined by a compound of Formula (I).

[018] In certain embodiments, the invention provides a compound having the structure represented by Formula (III): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, Spacer, PBG, Y, R1, Z2, Z3 and Z4 are as defined by a compound of Formula (I); and where Z1 is an electron withdrawing group.

[019] In certain embodiments, the invention provides a compound having the structure represented by Formula (IV): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, PBG, Y, R1, Z2, Z3 and Z4 are as defined by a compound of Formula (I); and where Z1 is an electron withdrawing group.

[020] In certain embodiments, the invention provides a compound having the structure represented by Formula (V): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, PBG, n, Y, R1, R2, Z1, Z2, Z3 and Z4 are as defined by a compound of Formula (I).

[021] In certain embodiments, the invention provides a compound having the structure represented by Formula (VI):: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein M is a pyrimidine or purine group that contains at least one primary or secondary amino group and optionally contains one or more halogen-selected substituents. X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3. X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3. R' is -R3 or -CH2R3; wherein each occurrence of R3 is independently selected from the group consisting of -OH, -CH3, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP (O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium, or an alkyl-substituted ammonium ion; and wherein X, n, Z1, Z2, Z3, Z4, Y, R1, R2 and PBG are as defined by a compound of Formula (V).

[022] In certain embodiments, the invention provides a compound having the structure represented by Formula (VI): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; where M is a pyrimidine or purine group that contains at least one primary or secondary amino group. X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3. X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3. R3 is selected from -OH, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, - OP(O)(OH)OP(O)(OH)OP (O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium or an alkyl-substituted ammonium ion; and wherein X, n, Z1, Z2, Z3, Z4, Y, R1, R2 and PBG are as defined by a compound of Formula (V).

[023] In certain embodiments, the invention provides a compound having the structure represented by Formula (VII): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein R' is -R3 or -CH2R3; and X, X1, X2, X3, X4, n, Y, R1, R2, R3, Z1, Z2, Z3, Z4 and PBG are as defined by a compound of Formula (VI).

[024] In certain embodiments, the invention provides a compound having the structure represented by Formula (VII): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein X, X1, X2, X3, X4, n, Y, R1, R2, R3, Z1, Z2, Z3, Z4 and PBG are as defined by a compound of Formula (V).

[025] In certain embodiments, the invention provides a compound having a structure represented by Formula (VIII): or pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; where Z1 is an electron withdrawing group; and wherein Agent, X, n, R2, PBG, Y, R1, Z2, Z3 and Z4 are as defined by a compound of Formula (V).

[026] In certain embodiments, the invention provides a compound having the structure represented by Formula (IX): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein: M is a pyrimidine or purine group that contains at least one primary or secondary amino group and optionally contains one or more selected halogen substituents; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; R' is -R3 or -CH2R3; wherein each occurrence of R3 is independently selected from the group consisting of -OH, -CH3, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP (O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium, or an alkyl-substituted ammonium ion; and wherein X, n, Y, Z1, Z2, Z3, Z4, R1, R2 and PBG are as defined by a compound of Formula (VIII).

[027] In certain embodiments, the invention provides a compound having the structure represented by Formula (IX): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein M is a pyrimidine or purine group containing at least one primary or secondary amino group; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; R3 is selected from -OH, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, - OP(O)(OH)OP(O)(OH)OP (O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium or an alkyl-substituted ammonium ion; and wherein X, n, Y, Z1, Z2, Z3, Z4, R1, R2 and PBG are as defined by a compound of Formula (VIII).

[028] In some embodiments, X1, X2, X3, and X4 are each independently selected from the group consisting of -H, -OH, -CH3, -F, -Cl, -Br, -I and - N3.

[029] In certain embodiments, the invention provides a compound having the structure represented by Formula (X): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein R' is -R3 or -CH2R3; and X1, X2, X3, X4, R3, X, n, Y, Z1, Z2, Z3, Z4, R1 and R2 are as defined by a compound of Formula (IX).

[030] In certain embodiments, the invention provides a compound having the structure represented by Formula (X): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein X1, X2, X3, X4, R3,

[031] In some embodiments, the Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 5'-deoxy-5-fluorocytidine, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1-(((2S,3R,4S,5R)- 3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 7-acetylcamptothecin, 9-formylcamptothecin, 9-acetylcamptothecin, 9-formyl-10-hydroxycamptothecin, 10-formylcamptothecin, 10-acetylcamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl-9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansine, maytansinol; auristatin (including but not limited to auristatin D, auristatin E, auristatin F, monomethyl auristatin D, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin F methyl ester, auristatin PYE, auristatin PHE, the related natural product dolastatin 10, and derivatives of these); amatoxins (including, but not limited to, α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid and derivatives thereof); duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C, duocarmycin SA, CC1065, adozelesin, bizelesin, carzelesin; eribulin; trabectedin; pyrrolobenzodiazepine, anthramycin, toamimycin, sibiromycin, DC-81, DSB-120; epothilones; bleomycin; dactinomycin; plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing.

[032] In some embodiments, the Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1- (((2S,3R,4S,5R)-3,4-dihydroxy-5 -methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 9-formylcamptothecin, 9-formyl-10-hydroxycamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl- 9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansinoids; auristatins; epothilones; bleomycin, dactinomycin, plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing.

[033] In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl , -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, - CF3 and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2 and -CF3. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2 , - OP(O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H and a pharmaceutically acceptable salt thereof. In some embodiments, at least one of Z1, Z2, Z3 and Z4 is not -H.

[034] In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl , -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, - CF3 and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2 and -CF3. In some embodiments, at least one of Z1, Z2, Z3 and Z4 is not -H.

[035] In some embodiments, Z1 is selected from the group consisting of halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-alkyl C1-C6 and -CN; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S (O)2-alkyl C1-C6 and -CN. In some embodiments, Z1 is selected from the group consisting of -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN. In some embodiments, Z1 is selected from the group consisting of -Cl, -F and -NO2; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2 and -CF3.

[036] In some embodiments, the portion of of a compound of the invention is selected from the group consisting of:

[037] In some embodiments, the portion of of a compound of the invention is selected from the group consisting of:

[038] In some embodiments, Y is -C(O)-NH-. In some embodiments, Y is -C(O)-O-. In some embodiments, Y is absent.

[039] In some embodiments, R1 is selected from the group consisting of optionally substituted C1-C18 alkyl in which, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)-R5-alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-.

[040] In some embodiments, R1 is selected from the group consisting of

[041] In some embodiments, R1 is absent.

[042] In some embodiments, PBG is an optionally substituted maleimide group. In some embodiments, PBG is

[043] In some embodiments, the portion of of a compound of the invention is selected from the group consisting of:

[044] In some embodiments, the Spacer is ; n is 0 or 1; and X is selected from the group consisting of optionally substituted C1-C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; and R2 is as defined by Formula I.

[045] In some embodiments, the Spacer is , wherein R2 is selected from the group consisting of: -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl; W1, W2, W3 and W4 are each independently selected from the group consisting of: -H, halogen, -C(O)OH, -C(O)O- C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and -CN. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: -H, -Cl, -F, -NO2 and -CF3. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: a phenoxy group, a primary, secondary or tertiary amine group, an ether group, a phenol group, an amide group, an ester group, an alkyl group, a substituted alkyl group, a phenyl group and a vinyl group. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: -OP(O)(OH)2, -P(O)(OH)OP(O)(OH) 2, - OP(O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H and a pharmaceutically acceptable salt thereof. In some embodiments, at least one of W1, W2, W3 and W4 is not -H.

[046] In some embodiments, W1 is selected from the group consisting of: halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2- C1-C6 alkyl and -CN; and W2, W3 and W4 are each independently selected from the group consisting of: -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, - S(O)2-C1-C6 alkyl and -CN. In some embodiments, W1 is selected from the group consisting of: -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN; and W2, W3 and W4 are each independently selected from the group consisting of: -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN . In some embodiments, W1 is selected from the group consisting of: -Cl, -F and -NO2; and Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, -Cl, -F, -NO2 and -CF3.

[047] In some embodiments, the Spacer is , wherein R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl.

[048] In some embodiments, the Spacer is

[049] In some embodiments, the Spacer is em is 1, 2, 3, 4, 5 or 6.

[050] In some embodiments, the Spacer is

[051] In some embodiments, the compound of the invention is selected from or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein: is absent, or is selected from the group consisting of:

[052] In some embodiments, the entire invention is selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[053] In some embodiments, the Agent is α-amanitin, and the compound of the present invention is selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[054] In some embodiments, the Agent is an auristatin or derivative thereof and the compound of the present invention is selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[055] In certain embodiments, the invention provides a pharmaceutical composition comprising a compound as disclosed herein, and a pharmaceutically acceptable carrier.

[056] In certain embodiments, the invention provides a method for treating a disease or condition selected from the group consisting of a cancer, a viral disease, autoimmune disease, acute or chronic inflammatory disease, and a disease caused by bacteria, fungi or other microorganisms, comprising administering to a patient in need thereof a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein.

[057] In some embodiments, the invention provides compounds and compositions for use as a medicine. In some embodiments, the invention provides compounds and compositions for use in treating a disease or condition selected from the group consisting of a cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi or other microorganisms.

[058] In some embodiments, the compound disclosed herein can be used in the manufacture or preparation of a medicament for the treatment of a disease or condition selected from the group consisting of a cancer, a viral disease, an autoimmune disease, an inflammatory disease acute or chronic, and a disease caused by bacteria, fungi or other microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[059] Figure 1 shows the effect of compound 15 and gemcitabine on tumor growth in the LXFE 397 NSLC xenograft model.

[060] Figure 2 shows the effect of compound 15 and gemcitabine on tumor growth in an LXFE 397 NSLC xenograft model.

[061] Figure 3 shows the effect of compound 15 and gemcitabine on body weight change in a human LXFE 937 non-small cell carcinoma xenograft model.

[062] Figure 4 shows the effect of compound 15 and gemcitabine on tumor growth in an OVXF 899 ovarian cancer xenograft model.

[063] Figure 5 shows the effect of compound 15 and gemcitabine on body weight change in an OVXF 899 human ovarian cancer xenograft model.

[064] Figures 6(A) and 6(B) show scatter plots for individual tumor volumes after treatment with compound 15 or gemcitabine in an OVXF 899 ovarian cancer xenograft model. Figure 6(A) shows the absolute tumor volume on day 0; Figure 6(B) shows absolute tumor volume at day 67.

[065] Figure 7 shows the effect of compound 15 and gemcitabine on tumor growth in a Panc11159 pancreatic cancer xenograft model.

[066] Figure 8 shows the effect of compound 15 and gemcitabine on body weight change in a Panc11159 pancreatic cancer xenograft model.

DETAILED DESCRIPTION OF THE INVENTION

[067] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. .

[068] All publications, patents and published patent applications referred to in this application are specifically incorporated by reference here. In the event of a conflict, this specification, including its specific definitions, will control. Unless otherwise specified, it is to be understood that each embodiment of the invention may be used alone or in combination with any one or more other embodiments of the invention.

[069] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of an established integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[070] Throughout the application, where a compound or composition is described as having, including or comprising specific components, it is considered that such compound or composition may also consist essentially of or consists of the recited components. Similarly, where methods or processes are described as having, including or comprising specific process steps, the processes may also consist essentially of or consist of the recited process steps. Furthermore, it should be understood that the order of steps or order to perform certain actions is irrelevant as long as the compounds, compositions and methods described herein remain operable. Furthermore, two or more steps or actions can be conducted simultaneously.

[071] The singular forms “a/a” and “the/a” include plurals unless the context clearly dictates otherwise.

[072] The term “including” is used to mean “including, but not limited to”. “Including” and “including, but not limited to” are used interchangeably.

[073] The term “or”, as used here, should be understood to mean “and/or”, unless the context clearly indicates otherwise.

[074] The terms “drug”, “agent”, “therapeutic agent” or “therapeutically effective substance” are used to mean any compound that brings about a pharmacological effect by itself or after its conversion in the organism in question, and thus also include derivatives from these conversions. The pharmacological effect of the drugs in the composition according to the present invention can be a single effect only, for example, a cytostatic effect, or a broad spectrum of pharmacological action, such as an immunosuppressive and antiphlogistic effect at the same time.

[075] The term “anthracycline” refers to a class of antineoplastic antibiotics having an anthracenedione structural unit (also called anthraquinone or dioxoanthracene). For example, the term “anthracycline” is specifically intended to individually include doxorubicin, daunorubicin, epirubicin, idarubicin, nemorrubicin, valrubicin, pyrarubicin, zorubicin, aclarrubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, PNU-159682, caminomycin, mitox anthrone and amethantrone.

[076] The terms “patient”, “subject”, or “individual” are used interchangeably and refer to a human or a non-human animal. These terms include mammals such as humans, primates, farm animals (e.g., cattle, porcines), companion animals (e.g., canines, felines), and rodents (e.g., mice and rats). In certain embodiments, the patient or subject is a human patient or subject, such as a human patient having a condition in need of treatment.

[077] The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in an animal in question, including humans and mammals, for example, combined with one or more pharmaceutically acceptable carriers, excipients or solvents. Such a composition may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art. In certain embodiments, a pharmaceutical composition encompasses a composition comprising the active ingredient(s), and the inert ingredient(s) comprising the excipient, carrier or diluent, as well as any product that results, directly or indirectly, from the combination, complexation or aggregation of any two or more of the ingredients, or from the dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the Ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by administering a compound of the disclosure and one or more pharmaceutically acceptable excipients, carriers and/or diluents.

[078] The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier that can be administered to a patient, together with a therapeutically effective substance of this invention, and that does not destroy the pharmacological activity of the agent. The term “excipient” refers to an additive in a formulation or composition that is not a pharmaceutically active ingredient. In certain embodiments, a “pharmaceutically acceptable” substance is suitable for use in contact with cells, tissues or organs of animals or humans without excessive toxicity, irritation, allergic response, immunogenicity or other adverse reactions, in the amount used in the dosage form. according to the dosing schedule, and proportionate with a reasonable risk/benefit ratio. In certain embodiments, a “pharmaceutically acceptable” substance that is a component of a pharmaceutical composition is, in addition, compatible with the other ingredient(s) of the composition. In certain embodiments, the terms “pharmaceutically acceptable excipient”, “pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent” encompass, without limitation, pharmaceutically acceptable inactive ingredients, materials, compositions and carriers, such as liquid fillers, solid fillers, diluents, excipients, , carriers, solvents and encapsulation materials. Carriers, diluents and excipients also include all pharmaceutically acceptable dispersion media, coatings, buffers, isotonic agents, stabilizers, absorption delaying agents, antimicrobial agents, antibacterial agents, antifungal agents, adjuvants, and so on. Except as any conventional excipient, carrier or diluent is incompatible with the active ingredient, the present disclosure covers the use of conventional excipients, carriers and diluents in pharmaceutical compositions. See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania, 2005); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); and Pharmaceutical Preformulation and Formulation, Gibson, Ed., CRC Press LLC (Boca Raton, Florida, 2004).

[079] The terms “pharmaceutically effective amount”, “therapeutically effective amount”, or “therapeutically effective dose” refer to an amount effective to treat a disease in a patient, for example, affecting a beneficial and/or desirable change in general health of a patient suffering from a disease (e.g. cancer), treatment, cure, inhibition or improvement of a physiological response or condition, etc. The full therapeutic effect does not necessarily occur after administering one dose, and may occur only after administering a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations. The precise effective amount required for a subject will depend on, for example, the size, health and age of the subject, the nature and extent of the disease, the therapeutics or combination of therapeutics selected for administration, and the mode of administration. The skilled worker can easily determine the effective amount for a given situation by routine experimentation. The skilled worker will recognize that treating cancer includes, but is not limited to, killing cancer cells, preventing the growth of new cancer cells, causing tumor regression (a decrease in tumor size), causing a decrease in metastasis, improving functional of a patient, improve the patient's well-being, decrease pain, improve appetite, improve the patient's weight, and any combination of these. The terms “pharmaceutically effective amount”, “therapeutically effective amount” or “therapeutically effective dose” also refer to the amount required to improve a patient's clinical symptoms. The therapeutic methods or methods of treating cancer described herein should not be construed or otherwise limited to “curing” cancer.

[080] As used herein, the term “treat” or “treatment” includes reversing, reducing or interrupting the symptoms, clinical signs, and underlying pathology of a condition in order to improve or stabilize a condition of the subject. As used herein, and as well understood in the art, “treatment” is a method of obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical outcomes may include, but are not limited to, relief, improvement, or slowing of the progression of one or more symptoms or conditions associated with a condition, e.g., cancer, decreased extent of the disease, stabilized state (i.e., not worsening) of the disease, delay or deceleration of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or not detectable. “Treatment” can also mean prolonging survival compared to the expected survival if not receiving treatment. Exemplary beneficial clinical results are described here.

[081] “Administering” or “administration of” a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or agent may be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, by example, through a skin duct). A compound or agent may also be suitably introduced by refillable or biodegradable polymeric devices or other devices, for example, patches and pumps, or formulations, that provide for extended, slow or controlled release of the compound or agent. Administration may also be carried out, for example, once, a plurality of times and/or for one or more extended times. In some respects, administration includes both direct administration, including self-administration, and indirect administration, including the age of prescription of a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or have the drug administered by another, and/or who provides a patient with a prescription for a drug is administering the drug to the patient. When a method is part of a therapeutic regimen involving more than one agent or treatment modality, the disclosure contemplates that the agents can be administered at the same or different times and through the same or different routes of administration. Appropriate methods of administering a substance, compound, or agent to a subject will also depend, for example, on the age of the subject, whether the subject is active or inactive at the time of administration, whether the subject is cognitively impaired at the time of administration, the extent of the deficiency, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity).

[082] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more backbone carbons of a chemical compound. It will be understood that “substitution” or “substituted with” includes the implicit condition that such substitution is in accordance with the permissible valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not undergo transformation. spontaneously such as by regrouping, cyclization, elimination, etc. As used herein, the term “substituted” is considered to include all permitted substituents of organic compounds. In a broad aspect, permitted substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Permitted substituents may be one or more and the same or different for appropriate organic compounds. For purposes of the invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permitted substituents of organic compounds described herein that satisfy the valences of the heteroatoms. Substituents may include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl or an acyl), a thiocarbonyl (such as a thioester, a thioacetate or a thioformate), an alkoxy, an alkylthio, an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, an amide, an amidine, an imine, a cyano, a nitro, an azide, a sulfhydryl, an alkylthio, a sulfate, a sulfonate , a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl or an aromatic or heteroaromatic moiety.

[083] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the application includes cases where the circumstance occurs and cases where it does not occur. For example, the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present on a given atom, and thus the application includes structures in which a non-hydrogen substituent is present and structures in which a substituent that no hydrogen is not present.

[084] Unless specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include substituted variants. For example, reference to an “alkyl” group or moiety implicitly includes both substituted and unsubstituted variants. Examples of substituents on chemical moieties include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxyl, alkylthio, acyloxy, phosphoryl, phosphate , phosphonate, amino, starch, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aryl or heteroaryl moiety.

[085] The term “acyl” is recognized in the art and refers to a group represented by the general formula hydrocarbyl-C(O)-, preferably alkyl-C(O)-.

[086] The term “alkyl” refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups and branched-chain alkyl groups. In preferred embodiments, a straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its structure (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or less. In certain embodiments, the alkyl groups are lower alkyl groups, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl and n-pentyl. Furthermore, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen. on one or more carbons of the hydrocarbon structure. In certain embodiments, a straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its structure (e.g., C1-C30 for straight chains, C3-C30 for branched chains). In preferred embodiments, the chain has ten or fewer carbon atoms (C1-C10) in its structure. In other embodiments, the chain has six or fewer carbon atoms (C1-C6) in its structure.

[087] Such substituents may include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl or an acyl), a thiocarbonyl (such as a thioester, a thioacetate or a thioformate), an alkoxy, an alkylthio, an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, an amide, an amidine, an imine, a cyano, a nitro, an azide, a sulfhydryl, an alkylthio, a sulfate, a sulfonate , a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aryl or heteroaryl moiety.

[088] The term “amide”, as used here, refers to a group represented wherein RX and RY independently represent a hydrogen or hydrocarbyl group, or RX and RY taken together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[089] In some embodiments, the amide is -NH-C(O)- or -C(O)-NH-.

[090] The terms “amine” and “amino” are recognized in the art and refer to both unsubstituted and substituted amines and salts thereof, for example, a portion that can be represented by wherein RX, RY and RZ independently represent a hydrogen or a hydrocarbyl group, or RX and RY taken together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[091] The term “aryl”, as used herein, includes substituted or unsubstituted single ring aromatic groups in which each ring atom is carbon. Preferably, the ring is a 5 to 7 membered ring, more preferably a 6 membered ring. Aryl groups include phenyl, phenol, aniline, and the like. The terms "aryl" also include "polycyclyl", "polycycle" and "polycyclic" ring systems having two or more rings in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings", wherein at least one of the rings is aromatic, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls. In some preferred embodiments, polycycles have 2 to 3 rings. In certain preferred embodiments, polycyclic ring systems have two cyclic rings in which both rings are aromatic. Each of the polycycle rings can be replaced or not replaced. In certain embodiments, each ring of the polycycle contains from 3 to 10 ring atoms, preferably from 5 to 7. For example, aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as such as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl, and the like.

[092] In some embodiments, aryl is a single-ring aromatic group. In some embodiments, the aryl is a two-ring aromatic group. In some embodiments, the aryl is a three-ring aromatic group.

[093] The term “cycloalkyl”, as used here, refers to the radical of a saturated aliphatic ring. In preferred embodiments, cycloalkyls have from 3 to 10 carbon atoms in their ring structure, and more preferably from 5 to 7 carbon atoms in the ring structure. In some embodiments, the two cyclic rings may have two or more atoms in common, for example, the rings are “fused rings”. Suitable cycloalkyls include cycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl and cyclopropyl.

[094] In some embodiments, the cycloalkyl is a monocyclic group. In some embodiments, the cycloalkyl is a bicyclic group. In some embodiments, the cycloalkyl is a tricyclic group.

[095] The term “haloalkyl”, as used here, means an alkyl group substituted with one or more halogens. When more than one halogen is present, the halogens may be the same or different. For examples, haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, and the like.

[096] The terms “halo” and “halogen”, as used here, mean halogen and include chlorine, fluoro, bromine and iodine.

[097] The term "heteroaryl" includes substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5 to 6-membered rings, which ring structures include at least one heteroatom (e.g., O , N or S), preferably one to four or one to 3 heteroatoms, more preferably one or two heteroatoms. When two or more heteroatoms are present in a heteroaryl ring, they can be the same or different. The term "heteroaryl" also includes "polycyclyl", "polycyclo" and "polycyclic" ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, e.g., the rings are "fused rings". , wherein at least one of the rings is heteroaromatic, for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and/or heterocyclyl. In some preferred embodiments, preferred polycycles have 2 to 3 rings. In certain embodiments, preferred polycyclic ring systems have two cyclic rings wherein both rings are aromatic. In certain embodiments, each ring of the polycycle contains 3 to 10 ring atoms, preferably 5 to 7. For examples, heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine , pyrazine, pyridazine, quinoline, pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazole, benzofuran, benzothiophene, cinoline, phthalazine, quinazoline, carbazole, phenoxazine, quinoline, purine and the like.

[098] In some embodiments, the heteroaryl is a single-ring aromatic group. In some embodiments, the heteroaryl is a two-ring aromatic group. In some embodiments, the heteroaryl is a three-ring aromatic group.

[099] The terms “heterocyclyl”, “heterocycle” and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3 to 10 membered rings, more preferably 3 to 7 membered rings, whose structures of the ring include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. In certain embodiments, the ring structure may have two cyclic rings. In some embodiments, the two cyclic rings may have two or more atoms in common, for example, the rings are “fused rings”. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0100] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon-hydrogen bond and a carbon structure primarily, but may optionally include heteroatoms. Thus, methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl groups are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =O substituent on the carbon bond) and ethoxy (which is bonded through oxygen, not carbon ) they are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0101] The terms “hydrazone moiety” or “hydrazone” refer to The stereochemistry of the hydrazone moiety can be E or Z. The term hydrazone as used herein includes both the E and Z isomers.

[0102] In several places in the present specification, the compound substituents of the disclosure are disclosed in groups or bands. It is specifically intended that the disclosure includes each and every individual subcombination of the members of such groups and bands. For example, the term “C1-C6 alkyl” is specifically intended to individually disclose methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, etc.

[0103] A “pharmaceutically acceptable salt” is a salt of a compound that is suitable for pharmaceutical use, including, but not limited to, metallic salts (e.g., sodium, potassium, magnesium, calcium, etc.), metal addition salts acid (e.g. mineral acids, carboxylic acids, etc.), and base addition salts (e.g. ammonia, organic amines, etc.). The acid addition salt form of a compound occurring in its free form as a base can be obtained by treating said free base form with an appropriate acid such as an inorganic acid, for example, a hydrohalic acid such as such as hydrochloric or hydrobromic, sulfuric, nitric, phosphoric and the like; or an organic acid, such as, for example, acetic, hydroxyacetic, propanoic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclic, salicylic, p -aminosalicylic, pamoic and similar. See, for example, WO 01/062726. Some pharmaceutically acceptable salts listed by Berge et al., Journal of Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference in their entirety. Compounds containing acidic protons can be converted to their therapeutically active non-toxic base addition salt form, e.g., metal or amine salts, by treatment with appropriate organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts or ions, for example, lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. of N-methyl-D-glucamine, hydrabamine, and salts with amino acids such as, for example, arginine, lysine and the like. Conversely, said salt forms can be converted into free forms by treatment with an appropriate base or acid. Compounds and their salts can be in the form of a solvate, which is included within the scope of the present disclosure. Such solvates include, for example, hydrates, alcoholates and the like. See, for example, WO 01/062726.

[0104] The disclosure further provides pharmaceutical compositions comprising one or more compounds of the disclosure together with a pharmaceutically acceptable carrier or excipient. Compounds or pharmaceutical compositions of the disclosure can be used in vitro or in vivo.

[0105] The term “isomer”, as used herein, includes, but is not limited to tautomers, cis and trans isomers (E (entgegen), Z (zusammen)), R and S enantiomers (said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30), diastereomers, (D)-isomers, (L)-isomers, stereoisomers, racemic mixtures thereof, and other mixtures thereof. All such isomers, as well as mixtures thereof, are intended to be included in this invention. Tautomers, while not explicitly indicated in the formulas described herein, are intended to be included within the scope of the present invention. Compounds of the Invention

[0106] The present invention provides a compound having the structure represented by Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein: Agent is selected from the group consisting of: a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an anti-rheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent inflammatory agent, an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, an protease, an angiogenesis inhibitor, a hormone or hormone derivative, an antibody or a fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of precedents; Spacer is missing, or is selected from the group consisting of 0 or n is 0 or 1 optionally substituted C1-C18-NH- C(O)-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with - OCH2CH2- C1-C18-C(O) alkyl optionally substituted -NH-R5- wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl and optionally substituted cycloalkyl; Y is absent or selected from the group consisting of optionally substituted C1-C6 alkyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O- and -OC(O) -; R1 is absent or selected from the group consisting of optionally substituted C1-C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-, or R1 is a naturally occurring amino acid or unnaturally, or R1 has the following formula: on what: is absent, or is selected from the group consisting of: R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl; Z1, Z2, Z3 and Z4 are each independently selected from -H, an electron withdrawing group and/or a water-soluble group; PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group , an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, an optionally substituted N-hydroxysuccinimide ester group, an antibody or fragment thereof, and an antibody derived from a fragment thereof; wherein when the Spacer is absent, the agent is linked to the nitrogen adjacent to the Spacer by a double bond; and wherein at least one of Z1, Z2, Z3 and Z4 is an electron withdrawing group.

[0107] In some embodiments of the compounds described herein, PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, and an optionally substituted N-hydroxysuccinimide ester group.

[0108] In some embodiments, the PBG is associated with an antibody or fragment thereof. In some embodiments, the PBG is covalently linked to an antibody or fragment thereof. In some embodiments, PBG is associated with albumin. In other embodiments, the PBG is covalently linked to endogenous or exogenous albumin. In other embodiments, the PGB is covalently linked to cysteine-34 of endogenous or exogenous albumin.

[0109] In certain embodiments, the present invention provides a compound having the structure represented by Formula (I): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an antirheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent, an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, a protease inhibitor , an angiogenesis inhibitor, a hormone or hormone derivative, an antibody or fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of the foregoing ; 'θ'n [] HN JXAAT I x/WV' Spacer is missing, n is 0 or 1; " optionally substituted C1-C18-NH- C(O)-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl and optionally substituted cycloalkyl; Y is absent or selected from the group consisting of optionally substituted C1-C6 alkyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O- and -OC(O) -; R1 is absent or selected from the group consisting of optionally substituted C1-C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5-alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl; Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H and an electron withdrawing group; PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group , an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, an optionally substituted N-hydroxysuccinimide ester group and an antibody or fragment thereof; wherein when the Spacer is absent, the agent is linked to the nitrogen adjacent to the Spacer by a double bond; and at least one of Z1, Z2, Z3 and Z4 is an electron withdrawing group.

[0110] In some embodiments of the compounds described herein, PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, and an optionally substituted N-hydroxysuccinimide ester group.

[0111] In some embodiments, the PBG is associated with an antibody or fragment thereof. In some embodiments, the PBG is covalently linked to an antibody or fragment thereof. In some embodiments, PBG is associated with albumin. In other embodiments, the PBG is covalently linked to endogenous or exogenous albumin. In other embodiments, the PGB is covalently linked to cysteine-34 of endogenous or exogenous albumin.

[0112] In certain embodiments, the invention provides a compound having the structure represented by Formula (II): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, PBG, Y, R1, Z1, Z2, Z3 and Z4 are as defined by a compound of Formula (I).

[0113] In certain embodiments, the invention provides a compound having the or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, Spacer, PBG, Y, R1, Z2, Z3 and Z4 are as defined by a compound of Formula (I); and where Z1 is an electron withdrawing group.

[0114] In certain embodiments, the invention provides a compound having the structure represented by Formula (IV): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, PBG, Y, R1, Z2, Z3 and Z4 are as defined by a compound of Formula (I); and where Z1 is an electron withdrawing group.

[0115] In certain embodiments, the invention provides a compound having the structure represented by Formula (V): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein Agent, PBG, n, Y, R1, R2, Z1, Z2, Z3 and Z4 are as defined by a compound of Formula (I).

[0116] In certain embodiments, the invention provides a compound having the structure represented by Formula (VI): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein: M is a pyrimidine or purine group that contains at least one primary or secondary amino group and optionally contains one or more selected halogen substituents;

[0117] X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; R’ is -R3 or -CH2R3; wherein each occurrence of R3 is independently selected from the group consisting of -OH, -CH3, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP (O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium, or an alkyl-substituted ammonium ion; and wherein X, n, Z1, Z2, Z3, Z4, Y, R1, R2 and PBG are as defined by a compound of Formula (V).

[0118] In certain embodiments, the invention provides a compound having the structure represented by Formula (VI): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; where M is a pyrimidine or purine group that contains at least one primary or secondary amino group. X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3. X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3. R3 is selected from the group consisting of -OH, -OP(O)(OH)2, - P(O)(OH)OP(O)(OH)2, -OP(O)(OH)OP(O )(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium or an alkyl-substituted ammonium ion; and wherein X, n, Z1, Z2, Z3, Z4, Y, R1, R2 and PBG are as defined by a compound of Formula (V).

[0119] In certain embodiments, the invention provides a compound having the structure represented by Formula (VII): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein R' is -R3 or -CH2R3; and X, X1, X2, X3, X4, n, Y, R1, R2, R3, Z1, Z2, Z3, Z4 and PBG are as defined by a compound of Formula (VI).

[0120] In certain embodiments, the invention provides a compound having a structure represented by Formula (VII): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein X, X1, X2, X3, X4, n, Y, R1, R2, R3, Z1, Z2, Z3, Z4 and PBG are as defined by a compound of Formula (V).

[0121] In certain embodiments, the present invention provides a compound or pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; where Z1 is an electron withdrawing group; and wherein Agent, X, n, R2, PBG, Y, R1, Z2, Z3 and Z4 are as defined by a compound of Formula (V).

[0122] In certain embodiments, the invention provides or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein: M is a pyrimidine or purine group that contains at least one primary or secondary amino group and optionally contains one or more selected halogen substituents; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; R' is -R3 or -CH2R3; wherein each occurrence of R3 is independently selected from the group consisting of -OH, -CH3, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP (O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium, or an alkyl-substituted ammonium ion; and wherein X, n, Y, Z1, Z2, Z3, Z4, R1, R2 and PBG are as defined by a compound of Formula (VIII).

[0123] In certain embodiments, the present invention provides a compound having the structure represented by Formula (IX): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein M is a pyrimidine or purine group containing at least one primary or secondary amino group; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen and -N3; R3 is selected from -OH, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, - OP(O)(OH)OP(O)(OH)OP (O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium or an alkyl-substituted ammonium ion; and wherein X, n, Y, Z1, Z2, Z3, Z4, R1, R2 and PBG are as defined by a compound of Formula (VIII).

[0124] In certain embodiments, X1, X2, X3, and X4 are each independently selected from the group consisting of -H, -OH, -CH3, -F, -Cl, -Br, -I and - N3.

[0125] In certain embodiments, the invention provides or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein R' is -R3 or -CH2R3; and X1, X2, X3, X4, R3, X, n, Y, Z1, Z2, Z3, Z4, R1 and R2 are as defined by a compound of Formula (IX).

[0126] In certain embodiments, the present invention provides a compound having the structure represented by Formula (X): or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein X1, X2, X3, X4, R3,

[0127] In some embodiments, the Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 5'-deoxy-5-fluorocytidine, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1-(((2S,3R,4S,5R)- 3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 7-acetylcamptothecin, 9-formylcamptothecin, 9-acetylcamptothecin, 9-formyl-10-hydroxycamptothecin, 10-formylcamptothecin, 10-acetylcamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl-9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansin, maytansinol; auristatin (including but not limited to auristatin D, auristatin E, auristatin F, monomethyl auristatin D, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin F methyl ester, auristatin PYE, auristatin PHE, the related natural product dolastatin 10, and derivatives of these); amatoxins (including, but not limited to, α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid and derivatives thereof); duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C, duocarmycin SA, CC1065, adozelesin, bizelesin, carzelesin; eribulin; trabectedin; pyrrolobenzodiazepine, anthramycin, toamimycin, sibiromycin, DC-81, DSB-120; epothilones; bleomycin; dactinomycin; plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing.

[0128] In some embodiments, the Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolinoanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansinoids; auristatins; epothilones; bleomycin, dactinomycin, plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing.

[0129] In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, halogen, -C(O)OH, -C(O)O-C1-alkyl- C6, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, -Cl, -F, -NO2 and -CF3. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -OP(O)(OH)2, -P(O)(OH)OP(O)(OH) 2, - OP(O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H and a pharmaceutically acceptable salt thereof. In some embodiments, at least one of Z1, Z2, Z3 and Z4 is not -H.

[0130] In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl , -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, - CF3 and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2 and -CF3. In some embodiments, at least one of Z1, Z2, Z3 and Z4 is not -H.

[0131] In some embodiments, Z1 is selected from halogen, -C(O)OH, - C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and - CN; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S (O)2-alkyl C1-C6 e - CN. In some embodiments, Z1 is selected from the group consisting of -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN. In some embodiments, Z1 is selected from the group consisting of -Cl, -F and -NO2; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2 and -CF3.

[0132] In some embodiments, R2 is methyl.

[0133] In some embodiments, X1 is -H, X2 is -F, X3 is -F and X4 is -OH.

The Hydrazone Portion

[0134] The drug delivery system contains an acid-labile, cleavable hydrazone moiety. The cleavage of the hydrazone moiety and the half-life of drug release vary depending on the removal of substituent electrons and their position on the phenyl ring to which the hydrazone is attached. The phenyl ring can be replaced as follows , where PBG, Ri, Y, Zi, Z2, Z3 and Z4 are as defined herein.

[0135] In some embodiments, the phenyl ring comprises at least one electron-withdrawing group. In some preferred embodiments, the phenyl ring comprises an electron-withdrawing group attached to the i-position. In some embodiments, the phenyl ring comprises an electron-withdrawing group attached to position 2. In some embodiments, the phenyl ring comprises an electron-withdrawing group attached to position 3. In some preferred embodiments, the phenyl ring comprises two groups of In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions i and 3. In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions i and 4. In In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions i and 5. In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions 2 and 3. In some embodiments, the phenyl ring comprises two groups electron-withdrawing groups attached to positions 2 and 4. In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions 2 and 5. In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions 3 and 4. In some embodiments, the phenyl ring comprises two electron-withdrawing groups attached to positions 3 and 5. In some embodiments, the phenyl ring comprises three electron-withdrawing groups attached to positions 1, 3, and 5.

[0136] In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, halogen, -C(O)OH, -C(O)O-C1-alkyl- C6, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, -Cl, -F, -NO2 and -CF3. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of: -OP(O)(OH)2, -P(O)(OH)OP(O)(OH) 2, - OP(O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H and a pharmaceutically acceptable salt thereof. In some embodiments, at least one of Z1, Z2, Z3 and Z4 is not -H.

[0137] In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, - NO2, -CF3 and -CN, wherein at least one of Z1, Z2, Z3 and Z4 is not -H. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2 and -CF3 wherein at least one of Z1, Z2, Z3 and Z4 is not -H. In some embodiments, Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -F, -NO2 and -CF3 wherein at least one of Z1, Z2, Z3 and Z4 is not -H.

[0138] In some embodiments, the half-life of drug release is about 1.5 hours, about 2.0 hours, about 2.5 hours, about 3.0 hours, about 3.5 hours , about 4.0 hours, about 4.5 hours, about 5.0 hours, about 5.5 hours, about 6.0 hours, about 6.5 hours, about 7.0 hours, about 7.5 hours, about 8.0 hours, about 8.5 hours, about 9.0 hours, about 9.5 hours, about 10.0 hours, about 10.5 hours, about 11.0 hours, about 11.5 hours, about 12.0 hours, about 12.5 hours, about 13.0 hours, about 13.5 hours, about 14.0 hours, about 14.5 hours, about 15.0 hours, about 15.5 hours, about 16.0 hours, about 16.5 hours, about 17.0 hours, about 17.5 hours, about 18 .0 hours, about 18.5 hours, about 19.0 hours, about 19.5 hours or about 20.0 hours.

[0139] Without being bound by theory, a phenyl ring comprising an electron-withdrawing group attached to position 1 stabilizes the hydrazone moiety, resulting in a prolonged release of Agent in acidic conditions. In some embodiments, the position of the electron withdrawing group on the phenyl ring with respect to the position of the hydrazone moiety on the phenyl ring provides a method for controlling drug release.

[0140] In some embodiments, the invention provides a method of manufacturing a compound to control the release of a drug by modifying the drug to add a portion of described herein by Formulas I, II, III, IV, V, VI, VII.VIII, IX and X.

[0141] Some embodiments of the invention include conjugates with the substitution patterns and corresponding half-lives of drug release shown in Table A (HSA is for human serum albumin). The structure of nemorubicin The nemorubicin conjugates represented in Table A have the following structure:

[0142] In some embodiments, the compound of the invention is selected from: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein: is absent, or is selected from the group consisting of:

[0143] In some embodiments, the compound is selected from the group consisting of:

[0144] In some embodiments, the compound of the present invention is selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[0145] In some embodiments, the compound of the present invention is selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[0146] In some embodiments, the compound is compound 15, having the structure:

[0147] In some embodiments, compound 15 prevents the deactivation of gemcitabine, resulting in sustained tumor exposure.

The agent

[0148] In some embodiments, the Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressant, an anti-rheumatic, an antiphlogistic, an antibiotic, an analgesic, an anti-inflammatory agent , a virostatic, an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, an angiogenesis inhibitor, a hormone or hormone derivative, an antibody or fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, a derivative of any of the foregoing, a pharmaceutically acceptable salt, hydrate, solvate or isomer of any of the preceding.

[0149] In some embodiments, the Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 5'-deoxy-5-fluorocytidine, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1-(((2S,3R,4S,5R)- 3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 7-acetylcamptothecin, 9-formylcamptothecin, 9-acetylcamptothecin, 9-formyl-10-hydroxycamptothecin, 10-formylcamptothecin, 10-acetylcamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl-9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansine, maytansinol; auristatin (including but not limited to auristatin D, auristatin E, auristatin F, monomethyl auristatin D, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin F methyl ester, auristatin PYE, auristatin PHE, the related natural product dolastatin 10, and derivatives of these); amatoxins (including, but not limited to, α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid and derivatives thereof); duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C, duocarmycin SA, CC1065, adozelesin, bizelesin, carzelesin; eribulin; trabectedin; pyrrolobenzodiazepine, anthramycin, toamimycin, sibiromycin, DC-81, DSB-120; epothilones; bleomycin; dactinomycin; plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing.

[0150] In some embodiments, the Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1- (((2S,3R,4S,5R)-3,4-dihydroxy-5 -methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 9-formylcamptothecin, 9-formyl-10-hydroxycamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl- 9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansinoids; auristatins; epothilones; bleomycin, dactinomycin, plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing.

[0151] In some embodiments, the Agent is an N-nitrosourea.

[0152] In some embodiments, the Agent is an anthracycline, such as but not limited to doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone or amethanthrone.

[0153] In some embodiments, the Agent is an alkylating agent, such as but not limited to chlorambucil, bendamustine, melphalan or oxazaphosphorines.

[0154] In some embodiments, the Agent is an antimetabolite, such as but not limited to 5-fluorouracil, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1-(( (2S,3R,4S,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one or thioguanine. In some embodiments, the antimetabolite is a pyrimidine, or purine analog containing at least one primary or secondary amino group.

[0155] In some embodiments, the Agent is a folic acid antagonist, such as but not limited to methotrexate, raltitrexed, pemetrexed, or plevitrexed.

[0156] In some embodiments, the Agent is a taxane, such as but not limited to paclitaxel or docetaxel.

[0157] In some embodiments, the Agent is a camptothecin, such as but not limited to topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-amino camptothecin, camptothecin, -formylcamptothecin, 9-formylcamptothecin, 9 -formyl-10-hydroxycamptothecin, 7-butyl-10-aminocamptothecin or 7-butyl-9-amino-10,11,-methylenedioxocamptothecin.

[0158] In some embodiments, the Agent is a Vinca alkaloid, such as but not limited to vinblastine, vincristine, vindesine, vinorelbine or calicheamicins.

[0159] In some embodiments, the Agent is a maytansinoid, an auristatin, epothilones, a bleomycin, dactinomycin, plicamycin, myromycin C, a cis-configured platinum(II) complex, or a derivative of any of the foregoing.

The Spacer The Amide Bond

[0160] In some embodiments, the Spacer is <t<DRAW-CODE>>, where n, X and R2 are as defined here.

[0161] In some embodiments, the Spacer where n, X and R2 are as defined here.

[0162] In some embodiments, n is 0 or 1.

[0163] In some embodiments, optionally substituted C1-C18-NH-C(O)-R5-alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl.

[0164] In some embodiments, X is optionally substituted C1-C18 alkyl. In some embodiments, X is optionally substituted C1-C18 alkyl wherein a carbon atom in said C1-C18 alkyl is substituted with -OCH2CH2-. In some embodiments, X 1 is optionally substituted C1-C18 alkyl wherein two carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, X is optionally substituted C1-C18 alkyl wherein three carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, X is optionally substituted C1-C18 alkyl wherein four carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, X is optionally substituted C1-C18 alkyl wherein five carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, X is optionally substituted C1-C18 alkyl wherein six carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-.

[0165] In some embodiments, X is optionally substituted C1-C18-NH-C(O)R5- alkyl. In some embodiments, In some embodiments, In some embodiments, In some embodiments, In some embodiments, In some embodiments,

[0166] In some embodiments, X is optionally substituted C1-C18-C(O)-NH-R5- alkyl. In some embodiments, In some embodiments, In some embodiments, In some embodiments, In some embodiments, In some embodiments,

[0167] In some embodiments, X is optionally substituted aryl. In some embodiments, X is , wherein Z5, Z6, Z7 and Z8 are each independently selected from the group consisting of -H, halogen, -OH, -NO2, -CN, -O-C1-C6 alkyl, optionally substituted C1-C6 alkyl , optionally substituted C1-C6 haloalkyl and optionally substituted C1-C6 haloalkoxy.

[0168] In some embodiments, X is , wherein Z5, Z6, Z7 and Z8 are each independently selected from the group consisting of -H, halogen, -OH, -NO2, -CN, -O-optionally substituted C1-C6 alkyl, optionally C1C6 alkyl substituted, optionally substituted C1-C6 haloalkyl and optionally substituted C1-C6 haloalkoxy. In some embodiments, Z5, Z6, Z7 and Z8 are each -H.

[0169] In some embodiments, X is optionally substituted heteroaryl.

[0170] In some embodiments, X is optionally substituted cycloalkyl.

[0171] In some embodiments, R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl and optionally substituted cycloalkyl. In some embodiments, R5 is an optionally substituted aryl.

[0172] In some embodiments, R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl.

[0173] In some embodiments, the Spacer is absent.

[0174] In some embodiments, the Spacer comprises a moiety or bond that is enzyme cleavable. In some embodiments, the bond being cleaved is a peptide bond, an imide bond, or an amide bond. In some embodiments, the amide bond can be designed to be specifically cleavable by an esterase and/or amidases. In some embodiments, the Spacer comprises an amide bond that is selectively cleaved by carboxylesterases. In preferred embodiments, the Spacer comprises an amide bond that is selectively cleaved by carboxylesterase 2. In preferred embodiments, the Spacer comprises an amide bond that is attached to an aryl ring as H follows: In some embodiments, the Spacer comprises an amide bond that is linked to an aryl ring as follows: wherein Z5, Z6, Z7 and Z8 are each independently selected from the group consisting of -H, halogen, -OH, -NO2, -CN, -O-C1-C6 alkyl, optionally substituted C1-C6 alkyl , optionally substituted C1-C6 haloalkyl and optionally substituted C1-C6 haloalkoxy. In some embodiments, the Spacer comprises an amide bond that is linked to an aryl ring as follows: , wherein Z5, Z6, Z7 and Z8 are each independently selected from the group consisting of -H, halogen, -OH, -NO2, -CN, -O- C1-C6 alkyl, C1-C6 alkyl optionally substituted, optionally substituted C1-C6 haloalkyl and optionally substituted C1-C6 haloalkoxy. In some embodiments, Z5, Z6, Z7 and Z8 are each -H.

[0175] In some embodiments, the Spacer is that R2 is selected from the group consisting of: -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl; and W1, W2, W3 and W4 are each independently selected from the group consisting of: -H, halogen, -C(O)OH, -C(O)O- C1-C6 alkyl, -NO2, haloalkyl , -S(O)2-C1-C6 alkyl and -CN. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: -H, -Cl, -F, -NO2 and -CF3. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: a phenoxy group, a primary, secondary or tertiary amine group, an ether group, a phenol group, an amide group, an ester group, an alkyl group, a substituted alkyl group, a phenyl group and a vinyl group. In some embodiments, W1, W2, W3 and W4 are each independently selected from the group consisting of: -OP(O)(OH)2, -P(O)(OH)OP(O)(OH) 2, - OP(O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H and a pharmaceutically acceptable salt thereof. In some embodiments, at least one of W1, W2, W3 and W4 is not -H.

[0176] In some of the above embodiments, W1 is selected from the group consisting of: halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, - S(O) 2-C1-C6 alkyl and -CN; and W2, W3 and W4 are each independently selected from the group consisting of: -H, halogen, -C(O)OH, -C(O)O- C1-C6 alkyl, -NO2, haloalkyl, - S(O)2-C1-C6 alkyl and -CN. In some embodiments, W1 is selected from the group consisting of: -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN; and W2, W3 and W4 are each independently selected from the group consisting of: -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3 and -CN . In some embodiments, W1 is selected from the group consisting of: -Cl, -F and -NO2; and Z2, Z3 and Z4 are each independently selected from the group consisting of: -H, -Cl, -F, -NO2 and -CF3.

[0177] In some embodiments, the Spacer is, where R2 is as defined here. In some embodiments, the Spacer is

[0178] In some embodiments, the Spacer is in is 1,2, 3, 4, 5 or 6. o

[0179] In some embodiments, the Spacer is .

[0180] In some embodiments, R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl and optionally substituted heteroaryl.

[0181] In one aspect, the Spacer can operate as a protective group, preventing metabolic degradation of Agent, thereby considering a targeted use of higher doses of Agent. In some embodiments, the Agent is gemcitabine.

The Ligand

[0182] In some embodiments, the linker comprises Y and R1. In some embodiments, Y is absent. In some embodiments, R1 is absent. In some embodiments, both Y and R1 are absent.

[0183] In some embodiments, Y is selected from the group consisting of methyl, ethyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O- and -O-C( O)-.

[0184] In some embodiments, R1 is optionally substituted C1-C18 alkyl. In some embodiments, R1 is optionally substituted C1-C18 alkyl wherein a carbon atom in said C1-C18 alkyl is substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18 alkyl wherein two carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18 alkyl wherein three carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18 alkyl wherein four carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18 alkyl wherein five carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18 alkyl wherein six carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-.

[0185] In some embodiments, R1 is optionally substituted C1-C18-NH-C(O)R5- alkyl. In some embodiments, R1 is optionally substituted C1-C18-NH-C(O)R5- alkyl wherein a carbon atom in said C1-C18 alkyl is substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-NH- C(O)R5- alkyl wherein two carbon atoms in said C1C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-NH-C(O)R5-alkyl wherein three carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-NH-C(O)R5- alkyl wherein four carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-NH-C(O)R5- alkyl wherein five carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-NH-C(O)R5- alkyl wherein six carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-.

[0186] In some embodiments, R1 is optionally substituted C1-C18-C(O)-NH-R5- alkyl. In some embodiments, R1 is optionally substituted C1-C18-C(O)-NH- R5- alkyl wherein a carbon atom in said C1-C18 alkyl is substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-C(O)-NH- R5- alkyl wherein two carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein three carbon atoms in said C1C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18- C(O)-NH-R5- alkyl wherein four carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein five carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-. In some embodiments, R1 is optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein six carbon atoms in said C1-C18 alkyl are substituted with -OCH2CH2-.

[0187] In some embodiments, R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl and optionally substituted cycloalkyl.

[0188] In some embodiments, the portion of a compound of the invention is selected from the group consisting of:

The Protein Liaction Groups

[0189] Protein binding groups (“PBG”) include, but are not limited to, a substituted or unsubstituted maleimide group, a substituted or unsubstituted haloacetamide group, a substituted or unsubstituted haloacetate group, a substituted pyridylthio group or unsubstituted, a substituted or unsubstituted isothiocyanate group, a substituted or unsubstituted vinylcarbonyl group, a substituted or unsubstituted aziridine group, a substituted or unsubstituted disulfide group, a substituted or unsubstituted acetylene group, an N-hydroxysuccinimide ester group substituted or unsubstituted, an antibody or fragment thereof. In some embodiments, protein binding groups (“PBG”) include, but are not limited to, a substituted or unsubstituted maleimide group, a substituted or unsubstituted haloacetamide group, a substituted or unsubstituted haloacetate group, a substituted pyridylthio group, or unsubstituted, a substituted or unsubstituted isothiocyanate group, a substituted or unsubstituted vinylcarbonyl group, a substituted or unsubstituted aziridine group, a substituted or unsubstituted disulfide group, a substituted or unsubstituted acetylene group, or an N ester group. -substituted or unsubstituted hydroxysuccinimide. In some embodiments, the protein binding group is a substituted or substituted maleimide group. In some embodiments, the protein binding group is

[0190] In some embodiments, PBG is substituted with C1-C6 alkyl or halogen. In some embodiments, PBG is substituted with methyl, -Cl, or -Br. In some embodiments, PBG includes an antibody or a fragment thereof. In some embodiments, PBG includes a ligand with specificity for the receptor, for example, a low or high molecular weight compound such as folic acid, vitamins, peptides, sugars, native or modified proteins.

[0191] A disulfide group can be activated through a thionitrobenzoic acid (e.g., 5'-thio-2-nitrobenzoic acid) as the exchangeable group. A maleimide, pyridyldithio, or N-hydroxysuccinimide ester group may, when appropriate, be substituted by an alkyl group or the above water-soluble groups. In general, a protein binding group has protein binding properties, that is, it binds covalently (“a covalent protein binding group”) or noncovalently (“a noncovalent protein binding group”). ), in a physiological environment, to particular amino acids on the surface of the protein. The maleimide group, the haloacetamide group, the haloacetate group, the pyridyldithio group, the disulfide group, the vinylcarbonyl group, the aziridine group, and/or the acetylene group preferentially react with cysteine thiol groups (-SH), while the ester group of N-hydroxysuccinimide and/or the isothiocyanate group preferentially react with the amino group of lysines (-NH) on the surface of a protein. For example, the protein binding group, such as a maleimide group, can bind to the cysteine-34 of albumin. In some embodiments, the albumin is not modified (e.g., it is not modified to be charged, either positively or negatively). In some embodiments, a PBG as described herein can bind to an antibody or fragment thereof, such as those described herein.

[0192] The compound used in the invention includes any and all combinations of one or more agents, separators, ligands, protein binding groups, cleavable fragments, i.e., hydrazone, amide bond. The compounds may comprise an anthracycline, and an acid-cleavable moiety that can be cleaved so as to control the release of the anthracycline. In certain embodiments, the therapeutically effective substance comprises an anthracycline, a hydrazone as an acid-cleavable moiety whose cleavage and drug release half-life vary according to the removal of electron substituents and their position on the phenyl ring to which the hydrazone attaches. is attached, and a maleimide group as the covalent protein-binding group. In some other embodiments, the compounds may comprise gemcitabine, and an acid-cleavable moiety that may be cleaved so as to control the release of gemcitabine. In some other embodiments, the compounds may comprise a vinca alkaloid, and an acid-cleavable moiety that can be cleaved so as to control the release of vinca alkaloid.

Antibodies as Transporters

[0193] Cancer cells have specific markers, known as antigens, that play a role in tumor growth and progression. Antigens are proteins, often located on the surface of the tumor. An important characteristic of antibodies is their ability to bind target antigens with high specificity. However, despite specific binding to antigens, antibodies often lack therapeutic activity (Panowski et al., mAbs, 6, 34-45 (2014); Chari et al., Angewandte Chem. Int. Ed., 53, 3796- 3827 (2014)). Because of their high specificity for antigens, antibodies can be used as carriers for drug release. They can be conjugated with a therapeutically effective substance—forming an antibody-drug conjugate (ADC)—to deliver the therapeutically effective substance to a target site such as a tumor. After binding of the ACD to the antigen, the antigen-ADC complex is internalized through endocytosis. Once inside the tumor, the ADC releases the therapeutically effective substance. For an ADC to be successful, the choice of the therapeutically effective ligand and antibody substance is important. The selection of the antigen, and consequently a corresponding antibody, is important since the efficiency of internalization of the antigen-antibody complex influences the amount of therapeutically effective substance provided. Furthermore, for an ADC to be effective, the number of drug molecules needed to kill a cell must be below the amount that the drug can deliver. Therefore, a potent drug, with potency in the picomolar range, is preferred. Finally, once the ADC is internalized, the linker must be cleaved to release the therapeutically effective substance. Preferred ligands provide efficient and controlled release of the therapeutically effective substance from the acidic tumor environment while preventing release of the therapeutically effective substance into the plasma, which can lead to off-target toxicity and a narrower therapeutic window for use of the therapeutically effective substance. effective. The ligands disclosed herein can be conjugated to antibodies that are directed to any antigens or receptors expressed on a malignant cell. The antibody may be chimeric, humanized, human, or a genetically engineered or chemically modified antibody, such as a thio antibody (THIOMAB), and allow binding of the linker without significantly reducing the target binding capacity of the antibody.

[0194] In some embodiments, the protein binding groups (PBG) of the compounds described here are associated with an antibody or fragment thereof as described, thereby forming an ADC. In some embodiments, the PBG is covalently linked to an antibody or fragment thereof. In other embodiments, the PBG is not covalently linked to an antibody or fragment thereof.

[0195] In certain aspects, the PBG of the present application can bind to a carrier. In some embodiments, the carrier is a polypeptide binding agent. The disclosure covers polypeptide binding agents, such as antibodies, antigen-binding portions of antibodies, and non-immunoglobulin antigen-binding scaffolds in part, which can effectively target target antigens.

[0196] Monoclonal antibodies that bind to antigens present on the surface of the tumor can be used in the disclosed methods and compositions.

[0197] In some embodiments, the antibodies may be modified antibodies or antigen-binding fragments thereof that bind to the extracellular domain of the antigen.

[0198] In some embodiments, the deimmunized antibody or antigen-binding fragment that binds to the extracellular domain of the antigen, including a heavy chain variable region and a light chain variable region, wherein each variable region is between 2 to 20 amino acid substitutions in the structure of the region compared to a non-human antibody or precursor that binds to the extracellular domain of the antigen.

[0199] In some embodiments, the deimmunized antibody or antigen-binding fragment has one or more complementarity-determining regions (CDRs) of a non-human antibody or precursor that binds to the extracellular domain of the antigen. In one embodiment, between 1 and 5 substitutions are present in the complementarity determining regions (CDRs).

[0200] In some embodiments, single-chain antibodies and chimeric antibodies, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-extracted single-chain antibodies, comprising portions derived from different species, can be used as portions of binding to the antigen of an antibody. The various portions of these antibodies can be chemically brought together using conventional techniques, or they can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, for example, Cabilly et al., Pat. US No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., Pat. US No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, Pat. US No. 5,225,539; and Winter, European Patent No 0,239,400 B1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See, for example, Ladner et al., Pat. US No. 4,946,778; and Bird, R. E. et al., Science, 242: 423-426 (1988)), in relation to single chain antibodies.

[0201] In addition, functional antibody fragments can also be used, including chimeric, humanized, primatized antibody fragments or single-chain antibodies. The functional fragments of the antibodies in question retain at least one binding function and/or modulating function of the full-length antibody from which they are derived. In certain embodiments, the functional fragments retain an antigen-binding function corresponding to a full-length antibody (e.g., tumor specificity).

[0202] For example, antibody fragments capable of binding to an antigen receptor or a portion, including, but not limited to, Fv, Fab, Fab' and F(ab')2 fragments are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, cleavage of papain or pepsin can generate Fab or F(ab')2 fragments, respectively. Antibodies produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site are encompassed by the invention. For example, a chimeric gene encoding an F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and the heavy chain hinge region.

[0203] The term “humanized immunoglobulin”, as used herein, refers to an immunoglobulin comprising portions of immunoglobulins of different origin, at least one portion of which is of human origin. Accordingly, the present invention encompasses a humanized immunoglobulin having binding specificity for an antigen (e.g., human antigen), said immunoglobulin comprising an antigen-binding region of non-human (e.g., rodent) origin and at least a portion of a immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof). For example, the humanized antibody may comprise portions derived from an immunoglobulin of non-human origin with the requisite specificity, such as mouse, and from immunoglobulin sequences of human origin (e.g., a chimeric immunoglobulin), chemically brought together by conventional techniques ( e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide).

[0204] Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of non-human origin (e.g., one or more CDRs derived from an antibody of non-human origin) and a framework region derived from a light and/or heavy chain of human origin (e.g. CDR-grafted antibodies with or without structural changes).

[0205] In some embodiments, the antibody is an antagonistic antibody. As described herein, the term “antagonist antibody” refers to an antibody that can inhibit one or more functions of the target, such as a binding activity (e.g., ligand binding) and a signaling activity (e.g., transport amino acid). For example, an antagonist antibody may inhibit (reduce or prevent) the transport of glutamine across the antigen.

[0206] In some embodiments, anti-idiotypic antibodies are also useful. Anti-idiotypic antibodies recognize antigenic determinants associated with the antigen-binding site of another antibody. Anti-idiotypic antibodies can be prepared against a second antibody by immunizing an animal of the same species, and can be from the same strain as the animal used to produce the second antibody. See, for example, Pat. US No. 4,699,880. In one embodiment, antibodies are raised against the receptor or portion thereof, and these antibodies are in turn used to produce an anti-idiotypic antibody. Such an anti-idiotypic antibody may also be an inhibitor of an antigen transducer function, although it does not bind to the antigen itself. An anti-idotypic antibody can also be called an antagonist antibody.

[0207] In some embodiments, suitable antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for all antibodies. For example, fragments of F(ab)2 can be generated by treating antibodies with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bonds to produce Fab fragments.

[0208] In some embodiments, suitable antibodies are further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an antigen polypeptide conferred by at least one CDR region of the antibody. Techniques for producing single-chain antibodies (US Patent No. 4,946,778) can also be adapted to produce single-chain antibodies. Also, transgenic mice or other organisms, including other mammals, can be used to express humanized antibodies. Methods for generating these antibodies are known in the art. See, for example, Cabilly et al., Pat. US No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Queen et al., European Patent No. 0,451,216 B1; Boss et al., Pat. US No. 4,816,397; Boss et al., European Patent No. 0,120,694 E1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, Pat. US No. 5,225,539; winter, European Patent No 0,239,400 B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1. See also, Ladner et al., Pat. US No. 4,946,778; Houston, Pat. US No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)).

[0209] Such humanized immunoglobulins can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) that encode the desired humanized chain. For example, nucleic acid sequences (e.g., DNA) encoding humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized strand, such as a DNA template from a previously humanized variable region (see, for example, Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 24712476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be easily produced. In one embodiment, the cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see, e.g., Krebber et al., Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published April 1, 1993).

[0210] In some embodiments, an antibody is a monoclonal antibody. For example, a method for generating a monoclonal antibody that specifically binds to an antigen polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response by obtaining antibody-producing cells ( e.g., mouse spleen cells) and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monoclonal antibody that specifically binds to the myeloma polypeptide. antigen. Once obtained, a hybridoma can be propagated in a cell culture, optionally under culture conditions where the hybridoma-derived cells produce the monoclonal antibody that specifically binds to an antigen polypeptide. The monoclonal antibody can be purified from cell culture.

[0211] Furthermore, the techniques used to screen for antibodies in order to identify a desirable antibody can influence the properties of the antibody obtained. For example, an antibody to be used for therapeutic purposes may be capable of reaching a particular target cell type. Consequently, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells expressing the antigen of interest (e.g., through fluorescence-activated cell sorting). Likewise, if an antibody is to be used to bind an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing antibodies: antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., Biacore binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system from IGEN International, Inc., Gaithersburg, Mariland), Western blots, immunoprecipitation assays and immunohistochemistry.

Pharmaceutical Compositions

[0212] In some embodiments, the invention provides a pharmaceutical composition comprising a compound described herein.

[0213] The total amount of a compound in a composition to be administered to a patient is one that is suitable for the patient. One of skill in the art would appreciate that different individuals may require different total amounts of the therapeutically effective substance. In some embodiments, the amount of the compound is a pharmaceutically effective amount. The skilled worker would be able to determine the amount of the compound in a composition needed to treat a patient based on factors such as, for example, the patient's age, weight and physical condition. The concentration of the compound depends on its solubility in the intravenous administration solution and the volume of fluid that can be administered. For example, the concentration of the compound may be from about 0.1 mg/ml to about 50 mg/ml in the injectable composition. In some embodiments, the concentration of the compound may be from about 0.1 mg/ml to about 25 mg/ml, from about 7 mg/ml to about 17 mg/ml, from about 0.1 mg/ml. ml to about 5 mg/ml or from about 0.25 mg/ml to about 4.5 mg/ml. In particular embodiments, the concentration of the compound may be about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0. 5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2.0 mg/ml, about 2.1 mg/ml, about of 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7 mg /ml, about 2.8 mg/ml, about 2.9 mg/ml, about 3.0 mg/ml, about 3.1 mg/ml, about 3.2 mg/ml, about 3.3 mg/ml, about 3.4 mg/ml, about 3.5 mg/ml, about 3.6 mg/ml, about 3.7 mg/ml, about 3.8 mg/ml ml, about 3.9 mg/ml, about 4.0 mg/ml, about 4.1 mg/ml, about 4.2 mg/ml, about 4.3 mg/ml, about 4 .4 mg/ml, about 4.5 mg/ml, about 4.6 mg/ml, about 4.7 mg/ml, about 4.8 mg/ml, about 4.9 mg/ml , about 5.0 mg/ml, about 5.1 mg/ml, about 5.2 mg/ml, about 5.3 mg/ml, about 5.4 mg/ml, about 5, 5 mg/ml, about 5.6 mg/ml, about 5.7 mg/ml, about 5.8 mg/ml, about 5.9 mg/ml or about 6.0 mg/ml. In some embodiments, the concentration of the compound may be about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml. ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml , about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml, about 26 mg/ml, about 27 mg/ml, about 28 mg/ml, about 29 mg/ml or about 30 mg/ml.

[0214] The pharmaceutical compositions and kits of the present invention may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.

[0215] The compositions can be administered in a variety of conventional ways. Exemplary routes of administration that may be used include oral, parenteral, intravenous, intra-arterial, cutaneous, subcutaneous, intramuscular, topical, intracranial, intraorbital, ophthalmic, intravitreal, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, administration. central nervous system (CNS) or suppository administration. In some embodiments, the compositions are suitable for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously or intrathecally. In some embodiments, the compositions are injected intravenously. In some embodiments, a reconstituted formulation can be prepared by reconstituting a lyophilized anthracycline compound composition in a reconstitution liquid comprising ethanol and water. Such reconstitution may comprise adding the reconstitution liquid and mixing, for example, by swirling or vortexing the mixture. The reconstituted formulation can then be made suitable for injection by mixing, for example, Lactated Ringer's Solution with the formulation to create an injectable composition. A person of skill in the art would appreciate that a method of administering a therapeutically effective substance formulation or composition would depend on factors such as the age, weight and physical condition of the patient being treated, and the disease or condition being treated. The skilled worker would thus be able to select an optimal administration method for a patient on a case-by-case basis.

[0216] In some embodiments, the invention provides compounds and compositions for use as a medicine. In some embodiments, the invention provides compounds and compositions for use in treating a cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi or other microorganisms.

[0217] In some embodiments, the compound disclosed herein can be used in the manufacture or preparation of a medicament to treat a disease or condition selected from a group consisting of a cancer, a viral disease, autoimmune disease, acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, and other microorganisms.

[0218] In some embodiments, the cancer is a blood cancer or a solid tumor cancer. In some embodiments, the cancer is selected from carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, or melanoma.

[0219] In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, anal carcinoma, astrocytomas, basal cell carcinoma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, bronchial carcinoma of non-small cell, breast cancer, Burkitt's lymphoma, corpus carcinoma, CUP syndrome, colon cancer, small bowel cancer, ovarian cancer, endometrial carcinoma, gallbladder cancer, gallbladder carcinomas, uterine cancer, cancer cervical, neck, nose and ear tumors, hematological neoplasms, hairy cell leukemia, urethral cancer, skin cancer, gliomas, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal carcinoma, head/neck tumors , colon carcinoma, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, stomach cancer, colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer, renal cell carcinomas, oligodendroglioma, esophageal carcinoma, osteolytic carcinomas and osteoplastic carcinomas, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, penile cancer, prostate cancer, tongue cancer, ovarian carcinoma or lymph gland cancer.

[0220] In some embodiments, the present invention provides a kit comprising a compound as described herein and a pharmaceutically acceptable excipient, carrier and/or diluent.

[0221] In some embodiments, one or more excipients may be included in the composition. One of skill in the art would appreciate that the choice of any one excipient can influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients since the combination of excipients would produce undesirable effects. A person of skill in the art would be able to empirically determine which excipients, if any, would be included in the compositions. Excipients may include, but are not limited to, co-solvents, solubilizing agents, buffers, pH-adjusting agents, bulking agents, surfactants, encapsulating agents, tonicity-adjusting agents, stabilizing agents, protectants, and viscosity modifiers. In some embodiments, it may be beneficial to include a pharmaceutically acceptable carrier in the compositions.

[0222] In some embodiments, a solubilizing agent may be included in the compositions. Solubilizing agents may be useful for increasing the solubility of any of the components of the composition, including a compound or an excipient. The solubilizing agents described here are not intended to constitute an exhaustive list, but are provided merely as exemplary solubilizing agents that can be used in the compositions. In certain embodiments, solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, and any pharmaceutically acceptable salts and/or combinations thereof.

[0223] The pH of the compositions can be any pH that provides desirable properties for the formulation or composition. Desirable properties may include, for example, compound stability, increased compound retention compared to compositions at other pHs, and improved filtration efficiency. In some embodiments, the pH of the compositions may be from about 3.0 to about 9.0, for example, from about 5.0 to about 7.0. In particular embodiments, the pH of the compositions may be 5.5 ± 0.1, 5.6 ± 0.1, 5.7 ± 0.1, 5.8 ± 0.1, 5.9 ± 0.1, 6.0 ± 0.1, 6.1 ± 0.1, 6.2 ± 0.1, 6.3 ± 0.1, 6.4 ± 0.1, or 6.5 ± 0.1.

[0224] In some embodiments, it may be beneficial to buffer the pH by including one or more buffers in the compositions. In certain embodiments, a buffer may have a pKa of, for example, about 5.5, about 6.0, or about 6.5. One of skill in the art would appreciate that an appropriate buffer can be chosen for inclusion in compositions based on pKa and other properties. Tampons are well known in the art. Accordingly, the buffers described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary buffers that may be used in the formulations or compositions of the invention. In certain embodiments, a buffer includes, but is not limited to, Tris, Tris HCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium and potassium phosphate, Tris/Tris HCl, sodium bicarbonate , arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof .

[0225] In some embodiments, a pH adjusting agent may be included in the compositions. Modifying the pH of a composition may have beneficial effects on, for example, stability or solubility of a compound, or may be useful in manufacturing a composition suitable for parenteral administration. pH adjusting agents are well known in the art. Accordingly, the pH-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary pH-adjusting agents that may be used in the compositions. pH adjusting agents may include, for example, acids and bases. In some embodiments, a pH adjusting agent includes, but is not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

[0226] In some embodiments, a bulking agent may be included in the compositions. Bulking agents are commonly used in lyophilized compositions to provide added volume to the composition and aid in visualization of the composition, especially in cases where the lyophilized pellet would otherwise be difficult to observe. Bulking agents can also help prevent a breakdown of the active component(s) of a pharmaceutical composition and/or aid in cryoprotection of the composition. Bulking agents are well known in the art. Accordingly, the bulking agents described here are not intended to constitute an exhaustive list, but are provided merely as exemplary bulking agents that can be used in the compositions.

[0227] Exemplary bulking agents may include carbohydrates, monosaccharides, disaccharides, polysaccharides, sugar alcohols, amino acids and sugar acids, and combinations thereof. Carbohydrate bulking agents include, but are not limited to mono-, di- or poly-carbohydrates, starches, aldoses, ketoses, amino sugars, glyceraldehyde, arabinose, lyxose, pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose, thalose, heptose, glucose, fructose, methyl a-D- glucopyranoside, maltose, lactone, sorbose, erythrose, threose, arabinose, allose, altrose, gulose, idose, thalose, erytrulose, ribulose, xylulose, psychosis, tagatose, glucosamine , galactosamine, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galactocarolose, pectins, amylose, pullulan, glycogen, amylopectin, cellulose, pustulan, chitin, agarose, keratin, chondroitin , dermatan, hyaluronic acid, xanthine gum, sucrose, trehalose, dextran and lactose. Sugar alcohol bulking agents include, but are not limited to, alditols, inositols, sorbitol, and mannitol. Amino acid bulking agents include, but are not limited to, glycine, histidine, and proline. Sugar acid bulking agents include, but are not limited to, aldonic acids, uronic acids, aldaric acids, gluconic acid, isoascorbic acid, ascorbic acid, glucaric acid, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, neuraminic acid, pectic acids and alginic acid.

[0228] In some embodiments, a surfactant may be included in the compositions. Surfactants, in general, reduce the surface tension of a liquid composition. This may provide beneficial properties such as improved ease of filtration. Surfactants can also act as emulsifying agents and/or solubilizing agents. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary surfactants that may be used in the formulations or compositions of the invention. Surfactants that may be included include, but are not limited to, sorbitan esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (i.e., pluronics) , ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

[0229] In some embodiments, an encapsulating agent may be included in the compositions. Encapsulating agents can sequester molecules and help stabilize or solubilize them. Encapsulating agents are well known in the art. Accordingly, the encapsulating agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary encapsulating agents that can be used in the compositions. Encapsulating agents that may be included in compositions include, but are not limited to, dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and trimethyl-β-cyclodextrin, and combinations thereof.

[0230] In some embodiments, a tonicity adjusting agent may be included in the compositions. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration. Tonicity adjusting agents, therefore, can be used to assist in making a composition suitable for administration. Tonicity adjusting agents are well known in the art. Accordingly, the tonicity-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary tonicity-adjusting agents that may be used in the compositions. Tonicity adjusting agents can be ionic or non-ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose and mannitol.

[0231] In some embodiments, a stabilizing agent may be included in the compositions. Stabilizing agents help increase the stability of a compound in compositions. This can occur, for example, by reducing degradation or preventing aggregation of a compound. Without wishing to be bound by theory, mechanisms for enhancing stability may include sequestration of the compound from a solvent or inhibition of free radical oxidation of the therapeutically effective substance. Stabilizing agents are well known in the art. Accordingly, the stabilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary stabilizing agents that may be used in the compositions. Stabilizing agents may include, but are not limited to, emulsifiers and surfactants.

[0232] In some embodiments, a protectant may be included in the compositions. Protectants are agents that protect a pharmaceutically active ingredient (e.g., a therapeutically effective substance or compound) from an undesirable condition (e.g., instability caused by freezing or freeze-drying, or oxidation). Protectants may include, for example, cryoprotectants, lyoprotectants and antioxidants. Cryoprotectants are useful in preventing the loss of potency of an active pharmaceutical ingredient (e.g., an anthracycline compound) when a composition is exposed to a temperature below its freezing point. For example, a cryoprotectant can be included in a reconstituted lyophilized formulation so that the formulation can be frozen prior to dilution for intravenous (IV) administration. Cryoprotectants are well known in the art. Accordingly, the cryoprotectants described here are not intended to constitute an exhaustive list, but are provided merely as exemplary cryoprotectants that can be used in the compositions. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Cryoprotectants may include, for example, disaccharides (e.g. sucrose, lactose, maltose and trehalose), polyols (e.g. glycerol, mannitol, sorbitol and dulcitol), glycols (e.g. ethylene glycol, polyethylene glycol and propylene glycol).

[0233] Lyoprotectants are useful in stabilizing the components of a composition. For example, a therapeutically effective substance may be lyophilized with a lyoprotectant before reconstitution. Lyoprotectants are well known in the art. Accordingly, the lyoprotectants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary lyoprotectants that can be used in the compositions. Lyoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Exemplary lyoprotectants can be, for example, sugars and polyols. Trehalose, sucrose, dextran and hydroxypropyl-beta-cyclodextrin are not limiting examples of lyoprotectants.

[0234] Antioxidants are useful in preventing the oxidation of the components of a composition. Oxidation may result in aggregation of a drug product or other effects detrimental to the purity of the drug product or its potency. Antioxidants are well known in the art. Accordingly, the antioxidants described here are not intended to constitute an exhaustive list, but are provided merely as exemplary antioxidants that can be used in the compositions. Antioxidants can be, for example, sodium ascorbate, citrate, thiols, metabisulfite, and combinations thereof.

[0235] In some embodiments, a viscosity modifying agent may be included in the composition. Viscosity modifiers change the viscosity of liquid compositions. This can be beneficial since viscosity plays an important role in how easily a liquid composition is filtered. A composition may be filtered before lyophilization and reconstitution, or after reconstitution. Viscosity modifiers are well known in the art. Accordingly, the viscosity modifiers described here are not intended to constitute an exhaustive list, but are provided merely as exemplary viscosity modifiers that can be used in the compositions. Viscosity modifiers include solvents, solubilizing agents, surfactants and encapsulating agents. Exemplary viscosity modifiers that may be included in compositions include, but are not limited to, N-acetyl-DL-tryptophan and N-acetyl-cysteine.

Treatment Methods

[0236] The compounds and compositions described here are useful for a variety of clinical applications.

[0237] The compounds and compositions of this invention are capable of inducing prolonged or long-term inhibition of tumor growth. In certain embodiments, prolonged or long-term inhibition of tumor growth is without any loss in body weight or bone marrow toxicity.

[0238] The disclosure also provides a method of treating a condition or disease in a patient, said condition or disease selected from a cancer, a viral disease, autoimmune disease, acute or chronic inflammatory disease, and a disease caused by bacteria, fungi or other microorganisms, comprising administering to the patient a compound or pharmaceutical composition as described herein.

[0239] In some embodiments, the cancer is a blood cancer or a solid tumor cancer. In some embodiments, the cancer is selected from carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, or melanoma.

[0240] In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, anal carcinoma, astrocytomas, basal cell carcinoma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, bronchial carcinoma of non-small cell, breast cancer, Burkitt's lymphoma, corpus carcinoma, CUP syndrome, colon cancer, small bowel cancer, ovarian cancer, endometrial carcinoma, gallbladder cancer, gallbladder carcinomas, uterine cancer, cancer cervical, neck, nose and ear tumors, hematological neoplasms, hairy cell leukemia, urethral cancer, skin cancer, gliomas, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal carcinoma, head/neck tumors , colon carcinoma, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, stomach cancer, colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer, renal cell carcinomas, oligodendroglioma, esophageal carcinoma, osteolytic carcinomas and osteoplastic carcinomas, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, penile cancer, prostate cancer, tongue cancer, ovarian carcinoma or lymph gland cancer.

Variations and Modifications

[0241] Variations, modifications and other implementations of which are described here will occur to those of ordinary skill without departing from the spirit and scope of the invention. Accordingly, the invention should not be limited to the preceding description or the following examples.

Exemplification

[0242] With aspects of the invention now being generally described, these will be more readily understood by reference to the following examples, which are included merely for purposes of illustrating certain features and embodiments of the invention and are not intended to be limiting. Equivalents

[0243] Those skilled in the art will recognize, or be able to verify using no more than routine experimentation, numerous equivalents to the compounds, compositions and methods of use described herein. Such equivalents are considered to be within the scope of the invention.

Examples Example 1 Evaluation of gemcitabine and the albumin-binding gemcitabine phosphate compound 15 in a LXFE 397 human xenograft non-small cell lung cancer model.

[0244]Female NMRI nude mice received unilateral tumor implants subcutaneously into the left flank while under isoflurane anesthesia with human LXFE 397 (squamous cell carcinoma, differentiation: deficient) until the tumors were palpable and reached a volume of 50 to 150 mm3 . The animals were kept in cages, the temperature inside the cages maintained at 25 + 1 °C with a relative humidity of 45 to 65% and an air change rate in the cage of 60 times per hour. Animals were fed autoclaved Teklad Global 19% Protein Extruded Diet (T.2019S.12) from Harlan Laboratories and had access to sterilized acidified filtered tap water (pH 2.5) that was changed twice a week. Food and water are provided ad libitum. Before therapy, animals are randomized (8 mice per group) considering a comparable median and group mean tumor volume. Animals are routinely monitored twice a day on weekdays and once a day on Saturdays and Sundays. Starting on day 0, animals are weighed twice a week. Relative body weights (RBW) of individual animals are calculated by dividing the individual absolute body weight on day X (BWx) by the individual body weight on the day of randomization. Tumor volume is determined by a two-dimensional measurement with calipers on the day of randomization (Day 0) and twice weekly thereafter. Tumor volumes are calculated according to the following equation: Tumor Vol[mm3] = a [mm] x b2 [mm2] x 0.5, where “a” is the largest diameter and “b” is the perpendicular diameter of the tumor representing an idealized ellipsoid. The relative volume of an individual tumor on day X (RTVx) is calculated by dividing the absolute volume [mm3] of the respective tumor on day X (Tx) by the absolute volume of the same tumor on the day of randomization. Schedules are applied to the extent that animal welfare policies allow. Termination of individual mice at tumor volume > 2000 mm3. Stock solutions of compound 15 were prepared as follows for the two treatment groups: 1) 8 mice, 40 g average weight: 24 mg/kg Gemcitabine equivalents = 78.48 mg/kg = 3.14 mg/ mouse. Sample preparation: 154 mg weighed in a 20 mL vial dissolved in 14.7 mL of 20 mM sodium phosphate buffer pH 7; 4 aliquots of 3.6 mL each in 10 mL vials. Frozen in liquid nitrogen, freeze-dried (> 48 h) and covered. 2) 8 mice, 40 g average weight: 36 mg/kg Gemcitabine equivalents = 117.7 mg/kg = 4.71 mg/mouse. Sample preparation: 231 mg weighed in a 20 mL vial dissolved in 14.7 mL of 20 mM sodium phosphate buffer pH 7; 4 aliquots of 3.6 mL each in 10 mL vials. Frozen in liquid nitrogen, freeze-dried (> 48 h) and covered.

[0245] On the day of treatment, the lyophilized samples were dissolved in 20 mM sodium phosphate buffer pH 7, containing 5% glucose and injected intravenously. Intravenous administration with vehicle (20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0), gemcitabine (dissolved in 5% D-glucose; dose 240 mg/kg) and compound 15 (dissolved in 20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0; 24 mg/kg gemcitabine equivalents) was performed on days 1, 8, 15 and 22.

[0246] On day 23, 3 mice from the control group and 2 mice from the gemcitabine-treated group had to be sacrificed due to tumor volume exceeding 2000 mm3. Body weight change (BWC) versus control: Gemcitabine (day 23) approximately -6%; compound 15 (day 27) ~0.5%. The development of tumor growth in the LXFE 397 xenograft model of NSLC shows superior antitumor efficacy of compound 15 versus gemcitabine at one-tenth the gemcitabine dose (p < 0.05) at equitoxic toxicity as indicated by comparable and low body weight loss. See, Figure 1.

[0247] Figure 1 shows the effect of compound 15 and gemcitabine on tumor growth in the LXFE 397 xenograft model of NSLC. Example 2 Evaluation of gemcitabine and albumin-binding gemcitabine phosphate compound 15 in a human LXFE 937 non-small cell carcinoma xenograft model.

[0248]Female NMRI nude mice received unilateral tumor implants subcutaneously in the left flank while under isoflurane anesthesia with LXFE 937 human tumor pieces until the tumors were palpable and reached a volume of ~100 mm3. The animals were kept in cages, the temperature inside the cages maintained at 25 + 1 °C with a relative humidity of 45 to 65% and an air change rate in the cage of 60 times per hour. Animals were fed autoclaved Teklad Global 19% Protein Extruded Diet (T.2019S.12) from Harlan Laboratories and had access to sterilized acidified filtered tap water (pH 2.5) that was changed twice a week. Food and water were provided ad libitum. Before therapy, the animals were randomized (8 mice per group) considering a comparable median and group mean tumor volume. The animals were routinely monitored twice a day on weekdays and once a day on Saturdays and Sundays. Starting on day 0, animals were weighed twice a week. Relative body weights (RBW) of individual animals are calculated by dividing the individual absolute body weight on day X (BWx) by the individual body weight on the day of randomization. Tumor volume is determined by a two-dimensional measurement with calipers on the day of randomization (Day 0) and twice weekly thereafter. Tumor volumes are calculated according to the following equation: Tumor Vol[mm3] = a [mm] x b2 [mm2] x 0.5, where “a” is the largest diameter and “b” is the perpendicular diameter of the tumor representing an idealized ellipsoid. The relative volume of an individual tumor on day X (RTVx) is calculated by dividing the absolute volume [mm3] of the respective tumor on day X (Tx) by the absolute volume of the same tumor on the day of randomization. Schedules are applied to the extent that animal welfare policies allow. Termination of individual mice at tumor volume > 2000 mm3. Stock solutions of compound 15 were prepared as follows for the two treatment groups: 1) 8 mice, 40 g average weight: 2 x 18 mg/kg gemcitabine equivalents (twice weekly dose; d 0.3 , 7, 10 etc. for 4 weeks) = 58.4 mg/kg = 2.35 mg/mouse. Sample preparation: 115.5 mg weighed in a 10 mL vial dissolved in 7.35 mL of 20 mM sodium phosphate buffer pH 7; 4 aliquots with 1.86 mL each in 10 mL bottles. Frozen in liquid nitrogen, freeze-dried (> 48 h) and covered.

[0249] On the day of treatment, the lyophilized samples were dissolved in 20 mM sodium phosphate buffer pH 7, containing 5% glucose and injected intravenously. Intravenous administration with vehicle (20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0), gemcitabine (dissolved in 5% D-glucose; dose of 240 mg/kg) and carried out in 1. 8, 15 and 22 and compound 15 (dissolved in 20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0; 18 mg/kg gemcitabine equivalents) was performed on days 0, 3, 7, 10 , 14, 17, 21 and 24.

[0250] Body weight change (BWC) versus control was comparable, approximately + 10% for the group treated with gemcitabine and approximately + 2% in the group treated with compound 15.

[0251] Tumors in the gemcitabine-treated group began regrowth ~20 days after therapy reaching an average tumor volume ~700 mm3 ~50 days after therapy with 2 mice from the gemcitabine-treated group had to be sacrificed between day 78 to 85 due to tumor volume exceeding 2000 mm3. In contrast, complete tumor remissions were observed ~60 days after the end of therapy in the 15-treated group. Thus, the development of tumor growth in the LXFE 937 human non-small cell carcinoma xenograft model shows superior antitumor efficacy of compound 15 versus gemcitabine at approximately one-seventh the gemcitabine dose (p < 0.05) in equitoxic toxicity as indicated by comparable body weight gain. See Figures 2 and 3.

[0252] Figure 2 shows tumor growth curves of the control group, the gemcitabine-treated group, and the compound 15-treated group in the LXFE 937 human non-small cell carcinoma xenograft model.

[0253] Figure 3 shows body weight change curves in the control group, the gemcitabine-treated group, and the compound 15-treated group in the LXFE 937 human non-small cell carcinoma xenograft model. Example 3 Evaluation of gemcitabine and of albumin-binding gemcitabine phosphate compound 15 in an OVXF 899 human ovarian carcinoma xenograft model.

[0254]Female NMRI nude mice received unilateral tumor implants subcutaneously in the left flank while under isoflurane anesthesia with OVXF 899 human tumor pieces until the tumors were palpable and reached a volume of ~200 mm3. The animals were kept in cages, the temperature inside the cages maintained at 25 + 1 °C with a relative humidity of 45 to 65% and an air change rate in the cage of 60 times per hour. Animals were fed autoclaved Teklad Global 19% Protein Extruded Diet (T.2019S.12) from Harlan Laboratories and had access to sterilized acidified filtered tap water (pH 2.5) that was changed twice a week. Food and water were provided ad libitum. Before therapy, the animals were randomized (8 mice per group) considering a comparable median and group mean tumor volume. The animals were routinely monitored twice a day on weekdays and once a day on Saturdays and Sundays. Starting on day 0, animals were weighed twice a week. Relative body weights (RBW) of individual animals are calculated by dividing the individual absolute body weight on day X (BWx) by the individual body weight on the day of randomization. Tumor volume is determined by a two-dimensional measurement with calipers on the day of randomization (Day 0) and twice weekly thereafter. Tumor volumes are calculated according to the following equation: Tumor Vol[mm3] = a [mm] x b2 [mm2] x 0.5, where “a” is the largest diameter and “b” is the perpendicular diameter of the tumor representing an idealized ellipsoid. The relative volume of an individual tumor on day X (RTVx) is calculated by dividing the absolute volume [mm3] of the respective tumor on day X (Tx) by the absolute volume of the same tumor on the day of randomization. Schedules are applied to the extent that animal welfare policies allow. Termination of individual mice at tumor volume > 2000 mm3. Stock solutions of compound 15 were prepared as follows for the two treatment groups: 8 mice, 40 g average weight: 2 x 18 mg/kg gemcitabine equivalents (dose twice weekly; d 1.4, 8 , etc. for 4 weeks) = 58.4 mg/kg = 2.35 mg/mouse. Sample preparation: 115.5 mg weighed in a 10 mL vial dissolved in 7.35 mL of 20 mM sodium phosphate buffer pH 7; 4 aliquots with 1.86 mL each in 10 mL bottles. Frozen in liquid nitrogen, freeze-dried (> 48 h) and covered.

[0255] On the day of treatment, the lyophilized samples were dissolved in 20 mM sodium phosphate buffer pH 7, containing 5% glucose and injected intravenously. Intravenous administration with vehicle (20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0) gemcitabine (dissolved in 5% D-glucose; dose 240 mg/kg) and carried out in 1.8 , 15 and 22 and compound 15 (dissolved in 20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0; 18 mg/kg gemcitabine equivalents) was performed on days 1, 4, 8, 11, 15, 18, 22 and 25.

[0256] Body weight change (BWC) versus control was comparable, approximately + 5% for the group treated with gemcitabine and approximately + 10% in the group treated with compound 15.

[0257] Tumors in the gemcitabine-treated group began regrowth ~10 days after therapy reaching an average tumor volume ~2000 mm3 50 days after therapy with 3 mice in the gemcitabine-treated group having to be sacrificed on day 74 due to volume tumor exceeding 2000 mm3. In contrast, complete tumor remissions were observed 50 days after the end of therapy in the 15-treated group. Thus, the development of tumor growth in the OVXF 899 ovarian carcinoma xenograft model shows superior antitumor efficacy of compound 15 versus gemcitabine in approximately one-seventh of the gemcitabine dose (p < 0.05) in equitoxic toxicity as indicated by comparable body weight gain. See Figures 4, 5 and 6.

[0258] Figure 4 shows tumor growth curves of the control group, the group treated with gemcitabine, and the group treated with compound 15 in the human ovarian OVXF carcinoma xenograft model.

[0259] Figure 5 shows body weight change curves in the control group, the gemcitabine-treated group, and the compound 15-treated group in the human ovarian OVXF carcinoma xenograft model.

[0260] Figure 6 shows scatterplots for individual tumor volumes after treatment with compound 15 or gemcitabine in the OVXF 899 xenograft model of ovarian cancer. Example 4 Evaluation of Gemcitabine and Albumin-Binding Gemcitabine Phosphate Compound 15 in a Human Pancreatic Carcinoma Xenograft Model Panc11159

[0261] Female NMRI nude mice (nu/nu) received unilateral Panc11159 tumor pieces subcutaneously implanted until the tumors were palpable and reached a volume of ~200 mm3. The animals were kept in cages (Type II Macrolon metal mesh), the temperature inside the cages was maintained at 22 + 1 °C with a relative humidity of 50 + 10%. Light period: artificial; 12 hours in the dark/12 hours light pace (light 06.00 to 18.00 hours). The health of the mice was examined at the beginning of the experiment and twice daily during the experiment; Identification: Ear tag and cage tags. The animals were fed with Ssniff NM, Soest, Germany and had access to autoclaved and acidified drink (pH 4.0). Food and water were provided ad libitum. Before therapy, animals are randomized (10 mice per group) considering a comparable median and group mean tumor volume.

[0262] Change in body weight was carried out two or three times a week. Tumor diameters (median and mean) were measured two or three times a week with a caliper. Tumor volumes were calculated according to V = (length x (width)2)/2. To calculate the relative tumor volume (RTV), tumor volumes on each day of measurement were related to the day of the first treatment.

[0263] Stock solutions of compound 15 were prepared as follows for the two treatment groups: 10 mice, 30 to 40 g average weight: 2 x 18 mg/kg gemcitabine equivalents (dose twice weekly for 4 weeks) = 58.4 mg/kg = 2.35 mg/mouse. Sample preparation: 115.5 mg weighed in a 10 mL vial dissolved in 7.35 mL of 20 mM sodium phosphate buffer pH 7; 4 aliquots with 1.86 mL each in 10 mL bottles. Frozen in liquid nitrogen, freeze-dried (> 48 h) and covered.

[0264] On the day of treatment, the lyophilized samples were dissolved in 20 mM sodium phosphate buffer pH 7, containing 5% glucose and injected intravenously. Intravenous administration with vehicle (20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0) gemcitabine (dissolved in 5% D-glucose; dose 240 mg/kg) and performed on d 36, 43, 50, 57 compound 15 (dissolved in 20 mM sodium phosphate buffer, 5% D-glucose - pH 7.0; 18 mg/kg gemcitabine equivalents) was performed on days d 36, 40, 43, 47 , 50, 54, 57 and 60.

[0265] Body weight change (BWC) versus control was comparable, approximately + 3% for both the gemcitabine-treated group and the compound 15-treated group.

[0266] Tumor volumes in the gemcitabine-treated group doubled by day 85 while tumor volumes in the 15-treated group demonstrated slight regression or stable disease. Thus, the development of tumor growth in the Panc11159 human pancreatic carcinoma xenograft model showed superior antitumor efficacy of compound 15 versus gemcitabine at approximately one-seventh of the gemcitabine dose (p < 0.05) in equitoxic toxicity as indicated by body weight gain. and comparable. See Figures 7 and 8.

[0267] Figure 7 shows tumor growth curves of the control group, the gemcitabine-treated group, and the compound 15-treated group in the Panc11159 xenograft model of pancreatic cancer.

[0268] Figure 8 shows body weight change curves in the control group, the gemcitabine-treated group, and the compound 15-treated group in the Panc11159 xenograft model of pancreatic cancer. Example 5 General procedure for the synthesis of substituted maleimidobenzoyl hydrazone derivatives of nemorrubicin

[0269] Typically, to a mixture of nemorubicin (1 equivalent) and substituted maleimidobenzoic acid hydrazide trifluoroacetate (2 equivalents) anhydrous methanol was added at room temperature (room temperature) and the reaction mixture was stirred at room temperature. After the reaction was completed, as shown by TLC, the substituted maleimidobenzoyl hydrazone derivatives of nemorubicin were isolated as a red solid by precipitation or crystallization with a mixture of isopropanol and diisopropylether, and the obtained product was dried under high vacuum. General procedure for release studies of albumin-bound substituted maleimidobenzoyl hydrazone derivatives of nemorrubicin

[0270] Typically, substituted maleimidobenzoyl hydrazone derivatives of nemorubicin were dissolved in EtOH/5% Glucose 20:80 and added to completely reduced human serum albumin (HSA) at cysteine position 34 in 4 mM phosphate buffer pH 7 .4 and incubated at 37 °C for 2 hours. The albumin drug conjugate was isolated using Sephadex G-25 and eluted with 4 mM phosphate buffer pH 7.4 containing 150 mM NaCl. The purity of the nemorubicin albumin conjugate was measured and analyzed by RP-HPLC and the anthracycline content in the sample was determined spectrophotometrically. For drug release studies at pH 5.0, a 200 μM solution of the respective nemorrubicin human serum albumin (HSA) conjugate, adjusted to pH 5.0 with 50 mM sodium acetate buffer, was monitored with the aid of RP-HPLC and the release of nemorubicin determined at 495 nm. The results are presented in Table A. Example 6 Method A: Preparation of Compound 15

[0271] Compound 15 can be prepared as described below and shown in Scheme 1. Synthesis of 4-acetyl-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1 ,2-dihydropyrimidin-4-yl)benzamide (B)

[0272] To a stirred solution of 4-acetylbenzoic acid (8.25 g, 50.30 mmol) in CH2Cl2 (165 mL) oxalyl chloride (8.31 mL, 95.57 mmol) and DMF (catalytic amount) were added. ) at 0°C. The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated to dryness under high vacuum to give 4-acetylbenzoyl chloride as a light yellow solid. The crude material was used as such in the next step without any further purification. Yield: 9.15 g, 99.67%. To a stirred solution of gemcitabine hydrochloride (15 g, 50 mmol) in pyridine (150 mL), chlorotrimethylsilane (31.6 mL, 250 mmol) was added dropwise [~1.0 mL/min] for 30 min at 0 °C. The resulting mixture was stirred at room temperature for 2 h. 4-Acetylbenzoyl chloride (9.12 g, 50 mmol) was added to the reaction mixture in portions (3 portions, ~3.04 g/5.0 min) over 15 min. The resulting mixture was stirred at 45 °C for 16 h. Then, ethanol (150 mL) was added to the above reaction mixture and stirred for 30 min. Subsequently, desalted water (75 mL) was added and stirred for another 5 h. Then, the reaction mixture was concentrated to dryness and the residue was quenched with ice water (300 mL). The resulting solid was filtered through Whatman filter paper (11 μm) and dried under high vacuum for 5 h. The crude product was purified by flash chromatography on silica gel. The bulk was dissolved in CH2Cl2 and adsorbed on silica gel (60 to 120 mesh, 60 g) and purified over a 60 x 12.5 cm flash column using 240 g of 60 to 120 mesh silica gel and ~20 L of 60% ethyl acetate in gasoline ether as an eluent. The resulting solid was washed with methanol (100 mL), filtered and dried under high vacuum for 5 h to give 4-acetyl-N-(1-((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (B) as a white solid. Yield: 5.2 g, 12.71 mmol, 25.42%. Synthesis of intermediate (C)

[0273] In a 50 ml flask, 1.816 ml (19.54 mmol) phosphoryl chloride was added to 12.2 ml of trimethyl phosphate. The clear, colorless solution was cooled in an ice bath, followed by the addition of 2 g (4.89 mmol) of (B). The suspension was cleared after ∼15 min and the resulting pale yellow solution was stirred in an ice bath. After 2 h, 5 μL of the reaction mixture was carefully added to 1 ml of a 1:1 mixture of diethyl ether and saturated aqueous sodium bicarbonate. The milky suspension in the organic phase dissipates after thorough mixing. 50 μL of the aqueous phase was removed, diluted with 150 μL of Millipore water and analyzed by LC-MS. After ~2.5 h, the reaction mixture was added dropwise with intermittent stirring to a mixture of 140 mL of Millipore water, 140 mL of saturated NaHCO3, and 600 mL of diethyl ether in a 1000 mL Erlenmeyer flask cooled in a ice bath. The mixture was transferred to a 1000 mL separatory funnel and stirred well. The aqueous phase was washed with another 400 ml diethyl ether. The aqueous phase (pH 7 - 8) was isolated, filtered through a fritted funnel (pore size 3) into a 500 mL flask and acidified to pH 4 with conc. HCl. The mixture was transferred into eight 50 mL Falcon tubes (~40 mL each), then stored at 4 °C in the refrigerator overnight. The suspension was centrifuged (3220 rpm/30 min), the supernatant removed by pipetting and the residue lyophilized. Yield: (4.195 g/175%, theoretical: 2.393 g). The solid was dissolved in 275 mL of methanol, then 75 mL of tetrahydrofuran was added and the mixture was stored at 5 °C for 3 days. The solvent was removed in vacuo to ~150 mL, then 60 mL of tetrahydrofuran was added forming a white precipitate which was filtered and washed with 20 mL of tetrahydrofuran/methanol (1:1). (C) was isolated as a white solid from the filtrate. Yield: 3 g. Ligand synthesis (A)

[0274] 4-Amino-2-nitrobenzoic acid (4.62 g, 25.36 mmol) was stirred with tert-butyl carbazate (5.04 g, 38.14 mmol), HOBt hydrate (5.297 g, 34.6 mmol) and EDC (4.84 g, 25.25 mmol) in 120 mL of a 1:2 dimethylformamide/dichloromethane mixture in an ice bath for 15 min, then at room temperature overnight. TLC (CHCl3/methanol, 9:1) shows completion of the reaction after 26 h. The crude product was purified by column chromatography on silica yielding the pure compound as a yellow foam (6.2 g, 82%). In a 250 ml flask, 4-amino-2-nitro carbazate (2.827 g, 9.54 mmol) was dissolved in 80 ml of tetrahydrofuran. To the stirring solution, 6-maleimidocaproic acid chloride (2.41 g, 10.5 mmol), dissolved in 30 mL of tetrahydrofuran, was added. Triethylamine (1.455 mL, 10.5 mmol), mixed with 40 mL of THF, was added dropwise to the mixture over 1 h using a dropping funnel. The resulting light brown suspension was stirred at room temperature for 15 h. TLC (CHCl3/methanol, 9:1) showed completion of the reaction. The crude product was purified by flash chromatography on silica gel (CHCl3/methanol, 9:1), yielding the pure product as a yellow solid (3 g, 64%). The product (3 g, 6.13 mmol) was suspended in 10 mL of dichloromethane and cooled in an ice bath. 10 mL of trifluoroacetic acid was added dropwise over 30 min using a dropping funnel to the stirred, cooled mixture. TLC (CHCl3/methanol, 9:1) of the yellow solution after 3 h shows complete cleavage of the BOC group. All solvents were removed under reduced pressure and the residue dried under high vacuum overnight. The residue was dissolved in a minimal amount of tetrahydrofuran and precipitated in a 1:1 mixture of diisopropyl ether and n-hexane, then stored at 5 °C overnight. The cream-colored precipitate was isolated by centrifugation, washed with diethyl ether and dried under high vacuum to provide 2.5 g (81%) of ligand A as a beige solid. Synthesis of compound 15

[0275](C) was dissolved (2 x 375mg (1.226 mmol) in 30 mL of anhydrous methanol each in two 50 mL Falcon tubes), centrifuged (3220 rpm, 10 min), the supernatants added to the ligand (A) ( 1.235 g, 2.452 mmol), suspended in 20 mL of anhydrous methanol in a 250 mL flask. An additional 40 mL of methanol and 2 mL of acetonitrile were added and the cloudy, light yellow solution was stirred at room temperature. TLC (RP-18, acetonitrile/NaH2PO4, 30:70) of the reaction mixture after 15 h shows complete consumption of (C). The mixture was evaporated to ∼30 mL, transferred to a 50 mL Falcon tube, and stored at 5 °C (1 h). The precipitate was collected by centrifugation (3220 rpm, 10 min), the supernatant (3 x 10 mL) was added to a cold 3:1 mixture of diisopropyl ether/isopropanol (3 x 30 mL) in three 50 mL Falcon tubes. and stored at 5°C overnight. The precipitate was collected by centrifugation (3220 rpm, 15 min), washed with 5 mL of diethyl ether, dried in air and under high vacuum to provide 903 mg (85.6% of theory 1.055 g) of compound 15 as a yellow solid. . The crude product was combined in a 50 mL Falcon tube, washed with tetrahydrofuran (6 x 10 to 15 mL) and centrifuged after each washing step (3220 rpm, 10 min). Finally, the product was washed 3 times with 5 to 10 mL of diethyl ether and dried in air and under high vacuum to provide 694.4 mg (66%) of compound 15 as a yellowish solid. pH-dependent stability of HSA compound 15 at pH 7.0 and 5.0

[0276] Stability at pH 7.0 (4 mM phosphate buffer, 150 mM NaCl): compound 15 was added to a completely reduced HSA solution (human serum albumin) and complete binding to cysteine position 34 verified by HPLC. A 200 μM solution of the HSA conjugate obtained from compound 15 was incubated at 37 °C and analyzed by HPLC every hour. Approximately 0.18% of (C) was released per hour. Stability at pH 5.0 (4 mM phosphate buffer, 150 mM NaCl, adjusted with 1 M HCl): The HSA conjugate of compound 15 was prepared at pH 7.0 (see, above) and adjusted to pH 5.0 by addition of 1 M HCl. A 200 μM solution of the HSA conjugate obtained from compound 15 was incubated at 37 °C and analyzed by HPLC every hour. The half-life (t1/2) for the release of (C) is approximately 13 h. Method B: Preparation of Compound 15.

[0277] Compound 15 can be prepared by the following alternative method. Synthesis of 4-acetyl-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1 ,2-dihydropyrimidin-4-yl)benzamide (B)

[0278] Synthesis of 2,5-dioxopyrrolidin-1-yl 4-acetylbenzoate: Under a nitrogen atmosphere a 4-liter three-necked round-bottom flask was charged with 4-acetylbenzoic acid (1.0 equiv., 487 .8 mmol, 80.00 g), N-hydroxysuccinimide (NHS) (1.1 equiv., 536.6 mmol, 61.75 g), and anhydrous THF (1.80 L). A Eurostar IKA digital overhead stirrer (power control at 190 rpm) was used to stir the contents of the reaction solution at constant speed. The solution was cooled using an ice bath (60 min). A solution of N,N'-dicyclohexylcarbodiimide (DCC) (1.1 equiv, 536.6 mmol, 110.71 g) in anhydrous THF (720 mL) was then added dropwise (over the course of 90 min), under nitrogen while stirring (the addition funnel was washed with anhydrous THF, 2 x 5 mL). After about 30 minutes of adding DCC the reaction mixture became cloudy due to the formation of N,N'-dicyclohexylurea (DCU) as a white precipitate. The reaction mixture was stirred at 0 °C for an additional 2 hours although ice was continually added to the ice bath. The reaction mixture was then allowed to slowly warm to room temperature without removing the ice bath to react until HPLC analysis indicated complete consumption of the carboxylic acid after 25 h 30 min. Solid material was removed by suction filtration. The solid was washed with dry THF (4 x 100 mL), and the combined fractions were evaporated to dryness in vacuo at 40°C. The residue was dissolved in dry dichloromethane (400 mL), and the solution was stored at 4 °C for 15 h and then filtered again. The solid was rinsed with pre-cooled dry dichloromethane (2 x 20 mL). The solvent was removed in vacuo at 30°C, the residue was redissolved in dry DCM (750 mL), and the organic layer was washed with cold 5% NaOH from aq. (2 x 400 mL). The organic layer was washed with distilled H 2 O (400 mL), then dried over sodium sulfate (~20 g), suction filtered, and concentrated in vacuo at 30 °C to provide the title compound. The compound was dried in high vacuum for 24 h to yield (100.2 g, 79%) as a yellowish-white solid (HPLC250nm > 95.9%).

[0279] Synthesis of (B): Gemcitabine (1.0 equiv., 76.02 mmol, 20.00 g) was added to THF (532 mL) and distilled water (45.6 mL) was added in one portion to form a fine suspension that became a clear solution after refluxing (67 °C) for 5 min. Then, a solution of 2,5-dioxopyrrolidin-1-yl 4-acetylbenzoate (1.05 equiv., 79.82 mmol, 20.84 g) in THF (229 mL) was added to the drops via cannula. during the 6 min period. After the addition was complete, the reaction mixture was stirred while refluxing (67 °C) for 16 h. After 16 h, HPLC (PDA 266 nm) and LC-MS analyzes showed approximately 20% gemcitabine of unreacted starting material. According to HPLC analysis (PDA 266 nm), the reaction solution contained 53.8% of the product, 33.6% of total polar impurities, and 12.6% of total nonpolar impurities. Purification of the product from this batch utilized successive and selective washing steps of the crude product to remove any unwanted polar and non-polar impurities. The process comprised mud washing steps with different solvents, which selectively dissolved polar and non-polar impurities, in which the product is ideally insoluble or only slightly soluble. Therefore, after 16 h the light yellow reaction solution was allowed to reach room temperature, and the solvents were evaporated under vacuum at 40 °C to provide a sticky residue.

[0280] The first purification step was applied to remove polar impurities by washing with water followed by filtration and additional washing with water. Therefore, water (300 mL) was added to the above crude product, and the contents of the vial were ground at room temperature (30 min) until a fine white suspension was obtained. The resulting suspension was then stirred at 50°C for 15 min, separated by filtration through a filter funnel (Por. 4), and washed with water (2 x 25 mL) to provide an amorphous solid (solid 1) which was dried under vacuum for 15 h yielding 26.34 g. HPLC analysis (266 nm) of this solid (solid 1) showed a purity of 91.9%, with a total of 3.7% polar impurities and 4.4% non-polar impurities.

[0281] The second purification step was applied to remove the remaining non-polar and polar impurities of solid 1 by slurry washing with a mixture of water and chloroform (2:1, v/v). Thus, water (200 mL) was poured into the flask containing solid 1 and the suspension obtained was stirred at 50 °C for 5 min, allowed to cool to room temperature (0.5 h), slurried with chloroform (100 mL) in the room temperature and stirred for 10 minutes until homogeneous. The mixture was then allowed to settle (approximately 5 minutes) and the heterogeneous mixture was separated by filtration through a filter funnel (Por. 4), and washed with cold water (2 x 15 mL) to provide an amorphous solid (solid 2 ) which was then dried under high vacuum for 24 h to provide the title compound (23.60 g, 76%) as a white solid (HPLC (266 nm) > 94.21%). HPLC analysis (266 nm) of the filtered solid 2 showed a purity of 94.2%, with a total of 1.7% polar impurities and 4.1% non-polar impurities.

[0282]Chemical Formula: C18H17 F2N3O6, [M+H]+ calculated: 410.12, [M+H]+ found: 410.15 Synthesis of intermediate (C)

[0283] Phosphorus oxychloride (4.2 equiv., 102.6 mmol, 9.59 mL) was added to cold (0° C, ice water bath) trimethyl phosphate (60 mL) and the clear solution was maintained at 0°C for 20 min. Then, solid (B) (1.0 equiv., 24.42 mmol, 10.00 g) was added in three portions (3 g, 3 g, and 4 g). After about 20 minutes, the reaction mixture became homogeneous and turned light yellow, and was kept at 0°C for 3.5 h. HPLC and LC-MS analyzes after 3 h showed approx. 99% conversion. Once phosphorylation was complete (3.5 h), the mixture was filtered through a deep-fried funnel, and then poured dropwise for 30 min into a freshly prepared cold (0 °C) vigorously stirred mixture of sodium hydrogen carbonate. saturated aqueous solution (NaHCO3) and diethyl ether (450 mL, 300 mL). Stirring was continued for 10 min at 0 °C and for 60 min at room temperature until a clear aqueous phase was obtained (internal temperature 15 °C).

[0284] The organic layer was removed and the aqueous layer was washed with diethyl ether (1 x 300 mL). The aqueous phase remained partially emulsified and subsequently more saturated NaHCO3 was added to achieve demulsification conditions. The addition of 330 mL of saturated NaHCO3 helped to break the emulsified droplets and phases were separated. The combined aqueous extracts (780 mL) were stirred at room temperature and adjusted to pH 4.0 with concentrated HCl (approximately 19 mL) and the solution was divided evenly into 50 mL Falcon tubes (16 in total) and stored at 4 °C for 2.5 days. After this period, a precipitated white solid was formed and the following steps were performed before analysis:

[0285] The samples were centrifuged for 20 minutes at 10 °C and 4,000 rpm.

[0286] The supernatants (aqueous phase) were removed by decantation, combined and dried under vacuum at 50 °C and the solid obtained was dried under high vacuum for 17 h.

[0287] The amorphous precipitates were redissolved in methanol (400 ml). The solution was transferred to a 1 liter round bottom flask and the solvents were evaporated under vacuum at 40 °C to give a white solid (solid 1, 10.96 g) which was further dried under high vacuum for 20 h.

[0288] HPLC analysis of solid 95.4% (220 nm).

[0289]Chemical formula: C18H18F2N3O9P, [MH]+ calculated: 488.07, [MH]+ found: 488.55 Ligand synthesis (A)

[0290] Synthesis of tert-butyl 2-(4-amino-2-nitrobenzoyl)hydrazine-1-carboxylate: 4-Amino-2-nitrobenzoic acid (1.0 eq, 164.7 mmol, 30.00 g ) was dissolved in a mixture of acetonitrile and tetrahydrofuran (1:1; 720 mL) and cooled to 6 °C with an ice-salt bath, then tert-butyl carbazate (1.5 eq, 247.1 mmol , 32.65 g), HOBt (1.1 eq, 181.2 mmol, 27.93 g) and EDC-HCl (1.0 eq, 164.7 mmol, 31.64 g) were added, and the mixture The reaction mixture was stirred for 90 min on ice, after which the ice bath was removed and the solution was stirred at room temperature for 16 h. The solid initially remaining in the reaction mixture dissolved under stirring within the first two hours. After stirring the reaction solution for 16 h, the solvents were removed under reduced pressure (water bath: 40 °C). The crude (a viscous dark brown oil) was dissolved in 2% n-butanol in dichloromethane (600 mL), washed with saturated NH4Cl (2 x 600 mL), saturated NaHCO3 (1 x 600 mL), and distilled water (1 x 600 mL). The organic layer was then dried over sodium sulfate, Na2SO4, (200 g). The solvents were removed under reduced pressure (water bath: 40°C) yielding the title compound as a brown foam (purity: 98.4%, according to HPLC (220 nm); yield: 30.42 g (62 %).Chemical Formula: C12H16N4O5, [M+Na]+ calculated: 319.10, [M+Na]+ found: 319.08.

[0291] Synthesis of 6-maleimidocaproic acid chloride: Dry dichloromethane (250 mL) was added at room temperature under stirring in one portion to a one-liter one-neck round-bottom flask containing 6-maleimidocaproic acid (1. 0 equiv., 236.6 mmol, 49.97 g) resulting in a yellow solution. A small amount of insoluble impurities was removed by filtration through a filter paper (MN 617%, 0185 mm) followed by rinsing with dry DCM (25 mL). To the solution at room temperature, oxalic acid chloride (1.1 equiv., 259.6 mmol, 22.50 mL) was added dropwise via a dropping funnel (over the course of 2h) while stirring the solution. reaction. Caution: Gas evolution was observed during the addition process. The reaction mixture was stirred at room temperature and left to react until HPLC analysis indicated complete consumption of 6-maleimidocaproic acid after 7 h 30 min. The color of the reaction solution changed to dark yellow during the reaction time. 5 h 30 min after complete addition of oxalic acid chloride, the solvent was removed in vacuum at 30 °C. The resulting dark yellow oil was then dried in high vacuum for 20 hours. The obtained light brownish solid was crushed with a spatula and dried for another 20 h under high vacuum to give (53.35 g, 98 %) as a light brownish solid (HPLC (220 nm) > 97.6 % as the methyl ester ).

[0292] Synthesis of Boc-protected ligand A: A solution of 6-maleimidocaproic acid chloride (1.1 eq, 216.6 mmol, 49.70 g) in dry THF, (382 mL) was added in one portion at room temperature to a solution of tert-butyl 2-(4-amino-2-nitrobenzoyl)hydrazine-1-carboxylate (1.0 eq, 196.9 mmol, 58.34 g) in dry THF (580 mL) . The clear reaction mixture was stirred at room temperature for 10 min. A solution of DIPEA (1.1 eq, 216.6 mmol, 37.7 mL) in dry THF (120 mL) was then added to the drops via a dropping funnel (over the course of one hour) while stirring. moderately at room temperature. After completion of the reaction as indicated by HPLC, the reaction mixture was stored at 4 °C for 14 h. The solvent was then removed under reduced pressure to give a dark brown viscous oil, which was then dissolved in DCM (450 mL) while stirring using a KL 2 stirrer (approx. 150 rpm; Edmund Bühler GmbH, Hechingen, Germany) for 10 min at room temperature. This solution was transferred to a 2 L separatory funnel, and the round bottom flask was rinsed with additional DCM (450 mL) which was then added to the separatory funnel. The organic phase was successively washed with 5% HCl (900 mL), aq. Sat. (900 mL) and 1 M NaH2PO4 (900 mL). After aqueous work-up, the organic phase was dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was dried for 16 h under high vacuum to provide the title compound (79.25 g) as a light brown foam. The crude product was further purified by flash chromatography to give 31.21 g with 99.6% HPLC purity (220 nm). Chemical Formula: C22H27N5O8, [M+Na]+ calculated: 512.18, [M+Na]+ found: 512.04.

[0293] Synthesis of ligand A: The protected ligand (1.0 equiv., 30.60 mmol, 15.06 g) was placed in a 250 mL one-neck round-bottom flask and pre-cooled in a water bath. ice for 10 minutes. Pre-cooled TFA (26 equiv., 784 mmol, 60 mL) was added in one portion while stirring with a magnetic stir bar (250 rpm). The mixture was further stirred under ice-bath cooling for 45 minutes until HPLC analysis indicated complete consumption of the protected binder. Because it was found that removal of TFA at room temperature or higher temperatures leads to dimerization of ligand A, TFA was removed at low temperature (ice bath) under high vacuum. To facilitate TFA removal, toluene was chosen as a carrier. Toluene (30 mL) was added to the reaction solution at 0 °C, and the TFA-toluene mixture was then removed at 0 °C (caution: 1) careful control of high vacuum, 2) no agitation of the solution to avoid its rapid transfer to the siphon) under high vacuum. An empty double-necked round-bottom flask (500 mL) cooled with liquid nitrogen was used as an additional cooling siphon for the TFA and condensed toluene. During TFA removal, the mixture bubbled from time to time and part of the solution spilled from the connecting tube. After 30 minutes most of the TFA-toluene mixture was removed, and an oily residue was obtained. For further removal of TFA, an additional 30 mL of toluene was added to the vial and TFA was removed as described above. After 80 minutes, this process was repeated after adding another 20 mL of toluene. After 110 minutes the additional cooling siphon was removed and the wax-like substance was directly connected to the high vacuum system. After 3 h 30 minutes the ice bath was removed and the oily residue was dried for a further 30 minutes at room temperature to provide a foam. The residue as foam was dissolved in N,N-dimethylformamide, DMF (30 ml) and the product was precipitated by dropwise addition of DMF solution at room temperature in a mixture of diisopropylether/methanol (45:5, 1 .5 L) under vigorous agitation. An additional 2 mL of DMF was used to rinse the reaction vial. The precipitate was filtered through a fritted filter (Por. 4) by suction, and the product (filter cake) was washed with diisopropylether (3 x 200 ml) without allowing the cake to become dry. After a final washing step with 200 ml of n-pentane the cake was dried by suction. The yellowish solid was collected in a flask and dried under high vacuum for 18 h. The product was stored at -20 °C. Yield: 10.94 g (91.8%). Purity (measurement average): 99.0% (220 nm), 98.7% (247 nm). [M+H]+ calculated: 390.14, [M+H]+ found: 390.12. Synthesis of compound 15

[0294] The gemcitabine derivative (C) (1.0 equiv., 9.29 mmol, 4.55 g) was suspended in anhydrous methanol (68.9 mL) followed by the addition of anhydrous DMSO (45.9 mL) and the clear transparent solution was stirred at room temperature for 5 minutes. After this period, ligand (A) (1.0 equiv., 9.29 mmol, 3.62 g) was added in three portions followed by 1.0 equiv. of TFA. The reaction mixture turned light yellow and was stirred at room temperature. Once the reaction was complete (4 h) as confirmed by HPLC (220 nm) and LC-MS analyses, the mixture was added dropwise for 2 - 3 minutes in a freshly cold 1:3 (0 °C) mixture. prepared from methyl tert-butyl ether and isopropanol (600 mL) under vigorous stirring. Stirring was continued for 10 min at 0 °C (internal temperature: 5 °C). The precipitate was filtered, washed four times with cold THF, transferred to a new fritted funnel and slurried with cold methyl tert-butyl ether (1 x 100 mL, 4 °C). This final amorphous precipitate was transferred to a 250 mL round bottom flask and dried under high vacuum for 20 h to provide the title compound 15 as a clear solid (4.52 g). HPLC analysis 96.2% (220 nm), Chemical Formula: C35H35F2N8O14P, [M-H]+ calculated: 859.19, [MH]+ found: 859.40. Example 7 Preparation of Compound 16

[0295] 80 mg of Doxorubicin-HCl (138 μmol) and 139 mg of Ligand A (276 μmol; 2 eq) were dissolved in 20 mL of anhydrous methanol under vigorous stirring at room temperature. The reaction was stirred for 4 h. Precipitation was performed with 105 mL of a 2:1 mixture of isopropanol and diisopropylether and the reaction stored at −20 °C overnight. The red precipitate was centrifuged (3200 x g, 10 min) and discarded, and the supernatant transferred into a fresh vessel. The supernatant was precipitated again with 150 mL of diisopropylether and stored overnight at −20 °C. The red precipitate was centrifuged and washed twice with 8 mL of acetonitrile and once with 40 mL of a 1:3 isopropanol/diisopropylether mixture. The product obtained was then dried under high vacuum to provide Compound 16 as a red solid. Yield: 53.9 mg (40%). Stability of HSA Conjugate of compound 16 at pH 7.0 and 5.0

[0296] Stability at pH 7 (Phosphate buffer 4 mM NaCl 150 mM):

[0297] Compound 16 was added to a completely reduced HSA solution (human serum albumin) and complete binding to cysteine position 34 verified by HPLC. A 200 μM solution of the HSA conjugate obtained from compound 16 was incubated at 37 °C and analyzed with HPLC every hour.

[0298] Only 0.36% of free doxorubicin was released per hour.

[0299] Stability at pH 5 (4 mM phosphate buffer, 150 mM sodium chloride buffer and 50 mM sodium acetate): the HSA conjugate of compound 16 was prepared at pH 7 (see, above) and a buffer of sodium acetate (50 mM) was added to adjust the pH value to 5.0. A 200 μM solution of the HSA conjugate obtained from compound 16 at pH 5.0 was incubated at 37 °C and analyzed with HPLC every hour. The half-life for doxorubicin release was approximately 9 h. Example 8 Synthesis of a vinblastine prodrug based on linker A

[0300]12'-Formylvinblastine was prepared from vinblastine and hexamethylenetetramine according to the procedure published in WO 2005/055939 A2,12'-Formylvinblastine (320 mg, 336 μmol) and ligand A (172.3 mg, 442.5 μmol ; 1.31 eq.) were dissolved in dry MeOH (32 mL) under vigorous stirring in a 50 mL reaction tube. After 2 h, the reaction was completed by adding diisopropylether (34 mL), and the solution was stored at -20 °C for 2 h. A small amount of impurities was precipitated, which was centrifuged and discarded. A mixture of n-hexan/diisopropylether (20 mL, 50:50) was added to the supernatant and stored at -20 °C for 16 h. The precipitate was centrifuged, dissolved in 1 mL of CHCl3/MeOH (90:10) and reprecipitated from 20 mL of n-hexan/diisopropylether (50:50), centrifuged and washed with 8 mL of AcN/diethylether (1: 3) and additionally centrifuged. The product was dissolved in 5 mL of CHCl3/MeOH (99:1), reprecipitated from 20 mL of n-hexan/diisopropylether (50:50) and centrifuged. This washing step was repeated twice. Then, the product was dried under high vacuum to give a white solid.

[0301] Yield: 253 mg (57%), HPLC Purity: 90.1% (220 nm) 93.0% (310 nm)

[0302]Chemical Formula: C64H75N9O15, [M+H]+ calculated: 1210.55, [M+H]+ found: 1210.42 Example 9 Synthesis of vinblastine prodrug based on ligand B

[0303]12'-Formylvinblastine was prepared from vinblastine and hexamethylenetetramine according to the procedure published in WO 2005/055939 A2.

[0304]12'-Formylvinblastine (300 mg, 315 μmol) and ligand B (153 mg, 321 μmol; 1.02 eq.) were dissolved in dry MeOH (25 mL) and stirred at room temperature. After 1 h, the reaction was completed by adding n-hexan/diisopropylether (23 mL, 50:50), and the solution was stored at -20 °C for 4 h. A small amount of impurities was precipitated, which was centrifuged and discarded. Diisopropylether (20 mL) was then added to the supernatant and stored at -20 °C for 2 h.

[0305] The precipitate was centrifuged, dissolved in 1 mL of CHCl3/MeOH (99:1) and reprecipitated from 20 mL of n-hexan/diisopropylether (50:50), centrifuged and washed with 10 mL of AcN/diethylether (1:3) and additionally centrifuged.

[0306] The product was dissolved in 5 mL of CHCl3/MeOH (90:1), reprecipitated from 20 mL of diisopropylether, centrifuged and dried under high vacuum to give a white powder.

[0307] Yield: 251 mg (63%), HPLC Purity: 90.1% (220 nm) 94.0% (310 nm)

[0308]Chemical Formula: C64H75FN8O12, [M+H]+ calculated: 1183.55, [M+H]+ found: 1183.45 Preparation of ligand B

[0309] To a solution of 4-amino-2-fluorobenzoic acid (8.1 g, 52.22 mmol), tert-butyl carbazate (8.28 g, 62.66 mmol) and N-methylmorpholine (14 .35 ml, 130.54 mmol) in tetrahydrofuran (25 ml) and EtOAc 7 (5 ml), 1.67 M T3P (50% in ethyl acetate, 40.65 ml) were added dropwise via a funnel. The mixture was stirred at room temperature for 16 h. Water was added and the organic layer was washed with saturated KHCO3 and water, dried over MgSO4, filtered and concentrated in vacuo. The crude product was triturated in MTBE, filtered and dried in vacuo to give 9.7 g (69%) of tert-butyl 2-(4-amino-2-fluorobenzoyl)hydrazinecarboxylate as a white solid. Chemical Formula: C12H16FN3O3, [M-H]+ calculated: 268.11, [M- H]+ found: 268.00

[0310] To a solution of tert-butyl 2-(4-amino-2-fluorobenzoyl)hydrazinecarboxylate (9.68 g, 35.94 mmol), 6-maleimidohexanoic acid (6.6 g, 31. 25 mmol) and N-methylmorpholine (8.59 ml, 0.08 mol) in tetrahydrofuran (40 ml) and EtOAc (120 ml), 1.67M T3P (50% in ethyl acetate, 24.32 ml) were added and the mixture was stirred at 60 °C for 48 h. Water and EtOAc were added, and the organic layer was washed with saturated KHCO3, water and brine, dried over MgSO4, filtered and concentrated in vacuo. The crude was purified by column chromatography using 1:1 to 1:2 n-hexane:EtOAc as an eluent. The solid obtained was triturated in MTBE to provide 7.5 g (51.9%, purity > 98%) of N-Boc-O-fluoroligand as a white solid. Chemical Formula: C22H27FN4O6, [M-H]+ calculated: 461.18, [M-H]+ found: 461.00.

[0311] To a suspension of N-Boc-O-fluoroligand (7 g, 15.14 mmol) in dichloromethane (40 ml) at 0 °C, TFA (40.54 ml, 0.53 mol) was added. The mixture was stirred at 0 °C for 15 min and at room temperature for 15 min. The solvent was removed under vacuum at 0°C. The crude product was ground in MTBE/DCM 7:1. The solid was filtered and dried in vacuo to give 6.5 g (90.1%) of O-Fluor maleimide binder as a white solid. HPLC purity 96.8% (220 nm). Chemical Formula: C17H18FN4O4, [M-H]+ calculated: 361.13, [M-H]+ found: 361.10. Example 10 Preparation of linker C.

[0312] The ligands of this invention can be made by the methods represented in the reaction scheme shown below.

[0313] Synthesis of fmoc-L-proline acid chloride: Fmoc-L-proline (250 mg, 0.74 mmol) was dissolved in CH2Cl2 (4 mL). Thionyl chloride (800 μL, 10.36 mmol) was added and the solution was stirred at reflux for 3 h with monitoring by quenching with methanol as a diluent and after formation of the methanol adduct by TLC (CH2Cl2:MeOH; 9: 1). Subsequently, the reaction was dried under vacuum to give 250 mg of crude, which was then used in the subsequent reaction without any further purification.

[0314] Synthesis of tert-butyl 2-(2-nitro-4-(pyrrolidine-2-carboxamido)benzoyl)hydrazine-1-carboxylate: A solution of 2-(4-amino-2-nitrobenzoyl)hydrazine- tert-Butyl 1-carboxylate (104 mg, 0.35 mmol) in THF (2 mL) was added (9H-fluoren-9-yl)methyl (S)-2-(chlorocarbonyl)pyrrolidine-1-carboxylate ( 250 mg, 0.70 mmol) as a solution in THF (2 mL) followed by dropwise addition of triethyl amine (49 μL, 0.35 mmol) as a solution in THF (200 μL). After stirring for 12 h, TLC (CHCl3/acetone, 7:3) showed complete conversion of the starting material. The reaction mixture was filtered and dried under vacuum using rotoevaporation. The resulting oil was then purified via Biotage FCC with a gradient of methanol and chloroform to provide 279 mg of a clear solid.

[0315] To a solution of the previous solid in anhydrous CH2Cl2 (2 mL) was added DBU (10%, 200 μL) and the mixture was stirred at room temperature for 30 min. The reaction was monitored by LC/MS until starting material was consumed. The reaction mixture was dried in vacuum using rotoevaporation. The resulting oil was then purified via Biotage FCC with a gradient of methanol and chloroform to provide the title compound as a brown solid (63 mg, 46% yield, 2 steps). Chemical Formula: C17H23N5O6, [M+H]+ calculated: 394.17, [M+NH]+ found: 394.14.

[0316] Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)(sulfo)-D-alanine: L- Cysteic Acid (500 mg, 2.95 mmol) was suspended in a mixture of 10 % aqueous Na2CO3 (17.5 mL) and 1,4-dioxane (7.5 mL) and cooled in an ice bath. N-fluorenylmethoxycarbonyl succinimide (1.19 g, 3.54 mmol) was dissolved in 1,4-dioxane (12.5 mL) by gentle heating and the solution was added over 30 min via a dropping funnel with efficient stirring. . The reaction mixture was stirred overnight and the organic solvent was removed in vacuo. The suspension was diluted with H2O (10 mL), washed with tert-butyl methyl ether (2 x 10 mL) and the aqueous phase was acidified with conc. HCl. at pH 3.0. The solution was lyophilized to give 1.02 g of fmoc cysteic acid as a white hygroscopic solid. This solid was used without further purification. Chemical Formula: C18H17NO7S, [M-H]+ calculated: 390.06, [M-H]+ found: 390.07.

[0317] Synthesis of acid (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- ((S)-2-((4-(2-( tert - butoxycarbonyl)hydrazine-1-carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin- 1-yl)-3-oxopropane-1-sulfonic acid: In a flame-dried flask, L-Cysteic acid-fmoc (96 mg, 0.30 mmol) was dissolved in a mixture of DMSO:CH2Cl2:anhydrous DMF (1:1:1, 3 mL). HATU (114 mg, 0.30 mmol) was added, followed by HOAt (41 mg, 0.30 mmol). After 5 min, a solution of tert-butyl (S)-2-(2-nitro-4-(pyrrolidine-2-carboxamido)benzoyl)hydrazine-1-carboxylate (100 mg, 0.25 mmol) in DMF anhydrous (2 mL) was added followed by the addition of NMM drops (55 μL, 0.50 mmol) and the resulting solution was stirred at room temperature for 17 h. Normal phase flash chromatography (CHCl3/methanol gradient) yielded the title compound (152 mg, 78%). Chemical Formula: C35H38N6O12S, [M-CO2tBu+H]+ calculated: 667.18, [M-CO2tBu+H]+ found: 667.11.

[0318] Synthesis of acid (R)-2-amino-3-((S)-2-((4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin- 1-yl)-3- oxopropane-1-sulfonic acid: To a solution of (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-((S)- 2-((4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin-1-yl)-3-oxopropane-1-sulfonic acid (152 mg, 0.198 mmol) in CH2Cl2 anhydrous (2 mL) DBU (10%, 200 μL) was added at 0 °C and the mixture was stirred at room temperature for 30 min. The LC-MS chromatogram (sample in MeCN) showed complete conversion of the starting material. The organic solvent was evaporated under vacuum yielding an oily residue. Flash purification of this residue (CHCl3/methanol gradient; 2 to 95%) gave the title compound as a brown solid (80.4 mg, 75% yield). Chemical Formula: C20H28N6O10S, [M+Na]+ calculated: 567.15, [M+Na]+ found: 567.19.

[0319] Synthesis of acid (R)-3-((S)-2-((4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin-1-yl) -2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-oxopropane-1-sulfonic acid: A solution of (R)-2 acid -amino-3- ((S)-2-((4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin- 1-yl)-3-oxopropane-1- sulfonic acid (80.4 mg, 0.14 mmol) in THF (2 mL) was added 6-maleimidocaproic acid chloride (41.0 mg, 0.14 mmol) in one portion as a solution in THF (1 mL). After stirring for 5 min at room temperature, a solution of triethylamine (24 μL, 0.14 mmol) in THF (200 μL) was added to the drops for 10 min. The brown solution was stirred at room temperature for another 3 h. The reaction mixture was filtered and dried under vacuum yielding a brown oily residue. Purification of this residue by flash chromatography (CHCl3/MeOH gradient, 100:0 to 2:98) gave the title compound (71 mg, 65%). Chemical Formula: C30H39N7O13S, [M-CO2tBu+H]+ calculated: 638.18, [M-CO2tBu+H]+ found: 638.02.

[0320] (R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexamido)-3-((S)-2-(( 4-(hydrazinecarbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin-1-yl)-3-oxopropane-1-sulfonic acid: To an ice-cold stirred solution of (R)-3-((S)-2-((4) -(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin-1-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanamido)-3-oxopropane-1-sulfonic acid (71 mg, 0.09 mmol) in CH2Cl2 (1 mL) was added TFA (293 μL, 3.83 mmol). The reaction mixture was stirred for 2 h until LC-MS showed consumption of the starting material. The desired product was obtained from the reaction mixture by solvent evaporation and used without further purification. Brown solid (91 mg, traces of TFA). LRMS (ESI) by C25H31N7O11S, [M+H]+ calculated: 637.18, [M+H]+ found: 638.07 (M+H). Purity: 96% (HPLC, 220 nm). Example 11 Synthesis of gemcitabine hydrazine: acid (R)-3-((R)-2-((4-(2-((Z)-1-(4-((1- ((2R,4R,5R) -3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)phenyl)ethylidene)hydrazine-1 -carbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin-1-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- oxopropane-1-sulfonic acid.

[0321] For a stirred suspension of 4-acetyl-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)- 2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (3 mg, 0.071 mmol) and acid R)-2-(6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1- yl)hexanamido)-3-((S)-2-((4-(hydrazinecarbonyl)-3-nitrophenyl)carbamoyl)pyrrolidin-1-yl)-3- oxopropane-1-sulfonic acid (5 mg , 0.078 mmol) in methanol (250 μL) TFA (9 μL, 0.117 mmol) was added. The reaction mixture was stirred for 3 days monitoring by LC-MS. After this time, the clear solution was concentrated in vacuum. Normal phase flash chromatography (CHCl3/methanol gradient) yielded the title compound (5.6 mg, 74%). Chemical Formula: C43H46F2N10O16S, [M+H]+ calculated: 1028.27, [M+H]+ found: 1029.05. Example 12 Synthesis of nemorubicin hydrazone

[0322] Nemorrubicin (3 mg, 4.7 μmol, 1 eq) was dissolved in dry MeOH (750 μL) and added to Ligand C (10.5 mg, 14.0 μmol, 3 eq) in a reaction tube 2 mL. TFA (1.1 μL, 2 eq) was added and the reaction mixture was stirred overnight. During the reaction a precipitate may be observed.

[0323] After 16 h, MeOH (800 μL) was added and the reaction mixture was centrifuged (20000 x g, 2 min).

[0324] The supernatant was divided into 2 new 2 mL reaction tubes and further precipitated with 1 mL of diisopropylether each. The second precipitate was centrifuged, dried and analyzed.

[0325] Purity: 89% (220 nm) 80% (495 nm)

[0326]Chemical Formula: C57H66N8O23S, [MH]+ calculated: 1261.39, [MH]+ found: 1261.60 Example 13 Synthesis of N -(4-Acetylbenzoyl)-MMAE (compound 17)

[0327]54.9 mg of 4-acetylbenzoic acid (334 μmol, 2 eq), 127 mg of HATU (334 μmol, 2 eq) and 45.6 mg of HOAt (334 μmol, 2 eq) were dissolved in 5 mL of anhydrous DMF and 36.7 μL of NMM (2 eq) were added. The reaction mixture was stirred for 15 minutes at room temperature. Subsequently, a solution of 120 mg of MMAE (167 μmol, 1eq) and 36.7 μL of NMM (334 μmol, 2 eq) in 5 mL of anhydrous DMF were added and stirring was continued for 72 h at room temperature. The reaction mixture was diluted with 40 mL of chloroform and washed twice with 10 mL of 5% HCl solution and three times with 10 mL of saturated sodium bicarbonate solution. The organic layer was dried over sodium sulfate, filtered and the solvent was removed in vacuo. The residue was dissolved in 2 mL of CHCl3 and purified using flash chromatography (solvent A: CHCl3, solvent B: MeOH, 36 mL/min, column volume (CV) = 19 mL, gradient: 2 CV (0% B) , 4 CV (0 to 3% of B), 2.4 CV (3% of B), 2 CV (3 to 5% of B), 3 CV (5% of B), 1.5 CV (5 to 9% B), ZIP® Sphere 10 g, Biotage® Isolera™ One). After vacuum drying, compound 17 (115 mg, 80%) was obtained as a colorless foam. HPLC purity: 97.5% (220 nm). Synthesis of N-(4-Acetylbenzoyl)-MMAE Hydrazone with Maleimide Ligand A (compound 18) compound 18

[0328] To a solution of 20.6 mg of compound 17 (23.3 μmol, 1 eq) in 3 mL of methanol was added 23.5 mg of maleimide ligand A (46.6 μmol, 2 eq) in a 50 mL reaction tube. Subsequently, 3.7 μL of TFA (46.6 μmol, 2 eq) was added to the slightly cloudy reaction mixture under which the solution immediately cleared. After 4 h of stirring at room temperature, 4 mL of n-hexane/diisopropyl ether (1:1) followed by 16 mL of n-hexane were added. The precipitate formed was centrifuged (3.220 x g, 10 min), dissolved in 1 mL of chloroform/methanol (1:1) and purified using repeated flash chromatography (1. chromatography: solvent A: CHCl3, solvent B: MeOH, 32 mL/ min, column volume (CV) = 19 mL, gradient: 1 CV (1 to 5% B), 2 CV (5% B), 3 CV (5 to 8% B), 4 CV (8% of B), 1 CV (8 to 9% of B), 6 CV (9% of B), ZIP® Esfera 10 g, Biotage® Isolera™ One, 2. Chromatography: solvent A: water, solvent B: acetonitrile, 12 mL/min, column volume (CV) = 21 mL gradient: 1 CV (10% B), 4 CV (10 to 50% B), 3 CV (50% B), 6 CV (50 at 80% of B) SNAP® Ultra C18 12g, Biotage® Isolera™ One). After vacuum drying, compound 18 (17.0 mg, 58%) was obtained as a colorless powder. HPLC purity: 99.1% (220 nm). Synthesis of a conjugate of compound 18 with trastuzumab (compound 19)

[0329] Commercial trastuzumab (Herceptin®, Roche) was reconstituted with WFI and the pH was adjusted with 0.5 M Tris buffer, 25 mM EDTA (4% batch volume) to pH 8. The mAb solution was diluted to 10 mg/mL with 10 mM PBS pH 7.4. Then, 2.25 eq of TCEP (tris-(2-carboxyethyl)phosphine) was added and the mixture was incubated for 90 min at 20 °C. Subsequently, the NMA (N,N-dimethylacetamide) content of the mixture was adjusted to 5%, and 5.5 equivalents of compound 18 were added. The mixture was incubated for 60 min at 20 °C. Then, the reaction was quenched by adding 11 equivalents of NAC (N-acetyl cysteine). Removal of unbound drug and buffer exchange in 10 mM PBS pH 7.4 was achieved by Tangential Flow Filtration (10 day-volumes). The solution was concentrated and the protein concentration was determined. Then, the solution was diluted to 7.2 mg/mL with 10 mM PBS pH 7.4. The resulting trastuzumab conjugate (compound 19) had a DAR of 4.0 as determined by HIC (TOSOH Bioscience, Butyl-NPR 4.6 mm ID x 3.5 cm, 2.5 μm; mobile phase A: ammonium sulfate 3 M, 25 mM monobasic sodium phosphate monohydrate in purified water, pH 6.95; mobile phase B: 25% isopropanol and 75% 25 mM monobasic sodium phosphate monohydrate in purified water, pH 6.95 ; flow: 0.8 mL min-1 at 25 °C for 18 min using a systematic gradient) and 0.41% unbound compound 18 as determined by HPLC (Waters Xterra MS, C18, 3.5 μM, 2 .1 x 100 mm, gradient: mobile phase A: 20 mM ammonium acetate, pH 7.0 ± 0.1, mobile phase B: acetonitrile, gradient: 0 min: 70% A, 20 min: 30% A , 25 min: 10% A, 25.1 min: 70% A, 35 min: 70% A). Stability and release kinetics of compound 19 in buffer solutions at pH 4.0 and pH 7.4

[0330] To study stability and release kinetics, the conjugated compound 19 was incubated in buffer solutions at 37 °C. Thus, a 3 mL aliquot of a solution of compound 19 in phosphate buffer (7.2 mg/mL in 10 mM PBS) was acidified to pH 4.0 using 0.5 M acetic acid and placed together with an aliquot of 3 mL untreated (pH 7.4) in a heating block at 37 °C. After appropriate intervals, samples (50 μL aliquots) at both pHs were removed and stored at −20 °C until analysis. Before analysis, samples were removed from the freezer and 7 μL of 5 M NaCl as well as 93 μL of ice-cold methanol were added. Then, the samples were stored for 30 min at -20 °C, centrifuged at 4 °C and 200 rpm for 60 min. Subsequently, 75 μL of the supernatant was diluted with 75 μL of purified water and the mixture was vortexed and analyzed using HPLC (Waters Xterra MS, C18, 3.5 μM, 2.1 x 100 mm, gradient: mobile phase A: acetate 20 mM ammonium, pH 7.0 ± 0.1, mobile phase B: acetonitrile, gradient: 0 min: 70% A, 20 min: 30% A, 25 min: 10% A, 25.1 min: 70% A, 35 min: 70% A). A standard curve of compound 17 was prepared at 2.00, 1.00, 0.50, 0.30, 0.20, 0.10, 0.05, and 0.01 μM. A range of 2.00 to 0.05 μM was used for UV quantification at 214 nm. Compound 17 was found to be the only release product. After 24 h, 2.7% of compound 17 was released from ADC at pH 7.4 while at pH 4.0, 39.6% of free compound 17 was observed.

Claims (34)

1. Compound CHARACTERIZED by the fact that it has the structure of the Formula or a pharmaceutically acceptable salt thereof; wherein: a) Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an anti-rheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent, -inflammatory, an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, a angiogenesis inhibitor, a hormone or hormone derivative, an antibody or a fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of the foregoing; Spacer is missing, or is selected from the group consisting of n is 0 or 1 optionally substituted C1-C18-NH- C(O)-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl; Y is absent or selected from the group consisting of optionally substituted C1-C6 alkyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O-, and -OC( O)-; R1 is absent or selected from the group consisting of optionally substituted C1C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-, or R1 is a naturally occurring amino acid or unnaturally, or R1 has the following formula: on what: is absent, or is selected from the group consisting of: R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; Z1, Z2, Z3 and Z4 are each independently -H, an electron withdrawing group, and/or a water-soluble group; PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group , an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, an optionally substituted N-hydroxysuccinimide ester group, an antibody or fragment thereof, and an antibody derived from a fragment thereof; wherein when the Spacer is absent, the Agent is linked to the nitrogen adjacent to the Spacer by a double bond; and wherein at least one of Z1, Z2, Z3 and Z4 is an electron withdrawing group; or b) Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an anti-rheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent , an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, an inhibitor of angiogenesis, a hormone or hormone derivative, an antibody or a fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of the foregoing; Spacer is missing, n is 0 or 1; " optionally substituted C1-C18-NH- C(O)-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl; Y is absent or selected from the group consisting of optionally substituted C1-C6 alkyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O-, and -OC( O)-; R1 is absent or selected from the group consisting of optionally substituted C1-C18 alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5-alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H and an electron withdrawing group; PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group , an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, an optionally substituted N-hydroxysuccinimide ester group; and an antibody or fragment thereof; wherein, when the Spacer is absent, the Agent is linked to the nitrogen adjacent to the Spacer by a double bond; and at least one of Z1, Z2, Z3 and Z4 is an electron withdrawing group. 2. Compound, according to claim 1, CHARACTERIZED by the fact that it has a structure of Formula (II): or a pharmaceutically acceptable salt thereof; wherein Agent, PBG, Y, R1, Z1, Z2, Z3 and Z4 are as defined in claim 1. 3. Compound, according to claim 1, CHARACTERIZED by the fact that it has the structure of Formula (III): or a pharmaceutically acceptable salt thereof; wherein Agent, Spacer, Z2, Z3, Z4, Y, R1 and PBG are as defined in claim 1; and where Z1 is an electron withdrawing group. 4. Compound, according to claim 1, CHARACTERIZED by the fact that it has the structure of Formula (IV): or pharmaceutically acceptable salt thereof; wherein Agent, PBG, Y, R1, Z2, Z3 and Z4 are as defined in claim 1; and where Z1 is an electron withdrawing group. 5. Compound, according to claim 1, CHARACTERIZED by the fact that it has the structure of Formula (V): or a pharmaceutically acceptable salt thereof; wherein Agent, n, X, R1, R2, Z1, Z2, Z3, Z4, Y, R1, and PBG are as defined in claim 1. 6. Compound, according to claim 5, CHARACTERIZED by the fact that it has the structure of: a) Formula (VIa): or a pharmaceutically acceptable salt thereof; wherein: M is a pyrimidine or purine group that contains at least one primary or secondary amino group and optionally contains one or more selected halogen substituents; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; R' is -R3 or -CH2R3; wherein each occurrence of R3 is independently selected from the group consisting of -OH, -CH3, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP (O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group, and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium, or an alkyl-substituted ammonium ion; and wherein X, n, Z1, Z2, Z3, Z4, Y, R1, R2 and PBG are as defined in claim 5; or b) Formula (VIb): or a pharmaceutically acceptable salt thereof; wherein: M is a pyrimidine or purine group containing at least one primary or secondary amino group; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; R3 is selected from the group consisting of -H, -OH, -OP(O)(OH)2, - OP(O)(OH)OP(O)(OH)2, -OP(O)(OH) OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group, and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal, an alkali metal ion, an ammonium or an alkyl-substituted ammonium ion; and wherein X, n, Z1, Z2, Z3, Z4, Y, R1, R2 and PBG are as defined in claim 5. 7. Compound, according to claim 6, CHARACTERIZED by the fact that it has the structure of: a) Formula (VIIa): or a pharmaceutically acceptable salt thereof; wherein R' is -R3 or -CH2R3; and X, X1, X2, X3, X4, n, Y, R1, R2, R3, Z1, Z2, Z3, Z4 and PBG are as defined in claim 6; or b) Formula (VIIb) or a pharmaceutically acceptable salt thereof; wherein X, X1, X2, X3, X4, n, Y, R1, R2, R3, Z1, Z2, Z3, Z4 and PBG are as defined in claim 6. 8. Compound, according to claim 5, CHARACTERIZED by the fact that it has the structure of Formula (VIII): or a pharmaceutically acceptable salt thereof; wherein Agent, X, n, R2, PBG, Y, R1, Z2, Z3 and Z4 are as defined in claim 5; and where Z1 is an electron withdrawing group. 9. Compound, according to claim 8, CHARACTERIZED by the fact that it has the structure of: a) Formula (IXa): , or a pharmaceutically acceptable salt thereof; wherein: M is a pyrimidine or purine group that contains at least one primary or secondary amino group and optionally contains one or more selected halogen substituents; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; R' is -R3 or -CH2R3; wherein each occurrence of R3 is independently selected from the group consisting of -OH, -CH3, -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP (O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid, an acyl group, and a pharmaceutically acceptable salt thereof, wherein the salt contains an alkali metal ion, an alkali metal ion, an ammonium, or an alkyl-substituted ammonium ion; and wherein X, n, Y, Z1, Z2, Z3, Z4, R1, R2 and PBG are as defined in claim 8; or b) Formula (IXb): or a pharmaceutically acceptable salt thereof; wherein: M is a pyrimidine or purine group containing at least one primary or secondary amino group; X1 and X2 are each independently selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; X3 and X4 are each independently, as valence permits, absent or selected from the group consisting of -H, -OH, C1-C6 alkyl, halogen, and -N3; R3 is selected from the group consisting of -H, -OH, -OP(O)(OH)2, - OP(O)(OH)OP(O)(OH)2, -OP(O)(OH) OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), an amino acid or an acyl group, and a pharmaceutically acceptable salt thereof, wherein the salt contains an ion alkali metal, an alkali metal ion, an ammonium or an alkyl-substituted ammonium ion; and wherein X, n, Y, Z1, Z2, Z3, Z4, R1, R2 and PBG are as defined in claim 8. 10. Compound, according to claim 9, CHARACTERIZED by the fact that it has the structure of: a) Formula (Xa): or a pharmaceutically acceptable salt thereof; wherein R' is -R3 or -CH2R3; and X1, X2, X3, X4, R3, X, n, Y, Z1, Z2, Z3, Z4, PBG, R1 and R2 are as defined in claim 9; or b) Formula (Xb): or a pharmaceutically acceptable salt thereof; and wherein X1, X2, X3, X4, R3, X, n, Y, Z1, Z2, Z3, Z4, PBG, R1 and R2 are as defined in claim 9. 11. Compound, according to any one of claims 1 to 5 and 8, CHARACTERIZED by the fact that: a) Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 5'-deoxy-5-fluorocytidine, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1- (((2S,3R,4S,5R)- 3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 7-acetylcamptothecin, 9-formylcamptothecin, 9-acetylcamptothecin, 9-formyl-10-hydroxycamptothecin, 10-formylcamptothecin, 10-acetylcamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl-9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansine, maytansinol; auristatin (including but not limited to auristatin D, auristatin E, auristatin F, monomethyl auristatin D, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin F methyl ester, auristatin PYE, auristatin PHE, the related natural product dolastatin 10, and derivatives thereof); amatoxins (including, but not limited to, α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid and derivatives thereof); duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C, duocarmycin SA, CC1065, adozelesin, bizelesin, carzelesin; eribulin; trabectedin; pyrrolobenzodiazepine, anthramycin, toamimycin, sibiromycin, DC-81, DSB-120; epothilones; bleomycin; dactinomycin; plicamycin, myromycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing; b) Agent is selected from the group consisting of N-nitrosoureas; doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone; amethanthrone; chlorambucil, bendamustine, melphalan, oxazaphosphorines; 5-fluorouracil, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1-(((2S,3R,4S,5R)-3,4-dihydroxy-5 - methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one, thioguanine; methotrexate, raltitrexed, pemetrexed, plevitrexed; paclitaxel, docetaxel; topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, 7-formylcamptothecin, 9-formylcamptothecin, 9-formyl-10-hydroxycamptothecin, 7-butyl-10-aminocamptothecin, 7-butyl- 9-amino-10,11,-methylenedioxocamptothecin; vinblastine, vincristine, vindesine, vinorelbine; calicheamicins; maytansinoids; auristatins; epothilones; bleomycin, dactinomycin, plicamycin, mitomycin C, and cis-configured platinum(II) complexes; or a derivative of any of the foregoing; c) Agent is an N-nitrosourea or a derivative thereof; d) Agent is doxorubicin, 2-pyrrolpyrrolineanthracycline, morpholinoanthracycline, diacetatoxyalkylanthracycline, daunorubicin, epirubicin, idarubicin, nemorubicin, PNU-159682, mitoxantrone or ametanthrone, or a derivative of any of the foregoing; e) Agent is chlorambucil, bendamustine, melphalan or oxazaphosphorines or a derivative of any of the foregoing; f) Agent is 5-fluorouracil, 2'-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 4-amino-1-(((2S,3R,4S,5R)-3,4-dihydroxy -5-methyltetrahydrofuran-2-yl)methyl)-5-fluoropyrimidin-2(1H)-one or thioguanine; or a derivative of any of the foregoing; g) Agent is methotrexate, raltitrexed, pemetrexed or plevitrexed; or a derivative of any of the foregoing; h) Agent is paclitaxel or docetaxel; or a derivative of any of the foregoing; i) Agent is topotecan, irinotecan, SN-38, 10-hydroxycamptothecin, GG211, lurtotecan, 9-aminocamptothecin, camptothecin, -formylcamptothecin, 9-formylcamptothecin, 9-formyl-10-hydroxycamptothecin, 7-butyl-10-aminocamptothecin or 7 -butyl-9-amino-10,11,-methylenedioxocamptothecin; or a derivative of any of the foregoing; j) Agent is vinblastine, vincristine, vindesine, vinorelbine or calicheamicins; or a derivative of any of the foregoing; k) Agent is a maytansinoid, an auristatin, an epothilone, a bleomycin, dactinomycin, plicamycin, myromycin C, a cis-configured platinum(II) complex or a derivative of any of the foregoing. 12. Compound according to any one of claims 1 to 11, CHARACTERIZED by the fact that: a) Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, halogen, -C (O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl, and -CN, wherein at least one of Z1, Z2, Z3 and Z4 is different from -H; b) Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3, and -CN; c) Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2, and -CF3; d) Z1, Z2, Z3 and Z4 are each independently selected from the group consisting of -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, - OP(O)(OH)OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H, and a pharmaceutically acceptable salt thereof; e) Z1 is selected from the group consisting of halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl, and -CN; and Z2, Z3 and Z4 are each independently selected from -H, halogen, -C(O)OH, -C(O)O- C1-C6 alkyl, -NO2, haloalkyl, -S(O)2- C1-C6 alkyl, or -CN; f) Z1 is selected from the group consisting of -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3, and -CN; and Z2, Z3, and Z4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3, and -CN ; or g) Z1 is selected from the group consisting of -Cl, -F, and -NO2; and Z2, Z3 and Z4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2, and -CF3. 13. Compound, according to any of claims 1, 2, 5 to 7 and 12, CHARACTERIZED by the fact that is: a) selected from the group consisting of: b) selected from the group consisting of: wherein Y, Ri and PBG are as defined in claim 1. 14. Compound, according to any one of claims 1 to 13, CHARACTERIZED by the fact that Y is: a) -C(O)-NH-; b) -C(O)-O-; or c) absent. 15. Compound according to any one of claims 1 to 14, CHARACTERIZED by the fact that R1 is: a) selected from the group consisting of optionally substituted C1-C18 alkyl in which, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH- C(O)-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each independently substituted with -OCH2CH2-; and optionally substituted C1-C18-C(O)-NH-R5- alkyl wherein, optionally, up to six carbon atoms in said C1-C18 alkyl are each c) absent; d) a naturally or non-naturally occurring amino acid; or e) on what: is absent, or is selected from the group consisting of: 16. Compound according to any one of claims 1 to 15, CHARACTERIZED by the fact that said PBG is a protein binding group selected from the group consisting of an optionally substituted maleimide group, an optionally substituted haloacetamide group, a haloacetate group optionally substituted, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group, and an optionally substituted N-hydroxysuccinimide ester group. 17. Compound, according to claim 16, CHARACTERIZED by the fact that the PBG is associated with an antibody or fragment thereof. 18. Compound, according to claim 16, CHARACTERIZED by the fact that the PBG is covalently linked to an antibody or fragment thereof. 19. Compound, according to claim 16, CHARACTERIZED by the fact that PBG is associated with albumin. 20. Compound, according to claim 16, CHARACTERIZED by the fact that PBG is covalently linked to endogenous or exogenous albumin. 21. Compound according to claim 16, CHARACTERIZED by the fact that PGB is covalently linked to endogenous or exogenous albumin cysteine-34. 22. Compound according to any one of claims 1 to 16, CHARACTERIZED by the fact that PBG is an optionally substituted maleimide group. 23. Compound, according to any one of claims 1 to 16, CHARACTERIZED by the fact that PBG is 24. Compound CHARACTERIZED by the fact that it has the structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein: Agent is selected from the group consisting of a cytostatic agent, a cytotoxic agent, a cytokine, an immunosuppressive agent, an antirheumatic, an antiphlogistic, an antibiotic, an analgesic, a virostatic agent, an anti-inflammatory agent , an antimycotic agent, a transcription factor inhibitor, a cell cycle modulator, an MDR modulator, a proteasome or protease inhibitor, an apoptosis modulator, an enzyme inhibitor, a signal transduction inhibitor, an inhibitor of angiogenesis, a hormone or hormone derivative, an antibody or a fragment thereof, a therapeutically or diagnostically active peptide, a radioactive substance, a light-emitting substance, a light-absorbing substance, and a derivative of any of the foregoing; Spacer is missing, or is selected from the group consisting of oo n is 0 or 1; X is selected from the group consisting of optionally substituted C1-C18 alkylene wherein, optionally, up to six carbon atoms in said C1-C18 alkylene are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-NH-C(O)-R5- alkylene wherein, optionally, up to six carbon atoms in said C1-C18 alkylene are each independently substituted with -OCH2CH2-; optionally substituted C1-C18-C(O)-NH-R5- alkylene wherein, optionally, up to six carbon atoms in said C1-C18 alkylene are each independently substituted with -OCH2CH2-; optionally substituted aryl; optionally substituted heteroaryl; and optionally substituted cycloalkyl; R5 is selected from the group consisting of an optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl; R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; on what is selected from the group consisting of: 25. Compound, according to any one of claims 1, 3 and 11 to 24, CHARACTERIZED by the fact that the Spacer is: n is 0 or 1; " optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl; and R2 is as defined in claim 1; B) wherein R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and W1, W2, W3 and W4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl, and -CN; w) wherein R2 is selected from the group consisting of -H, optionally substituted C1 -C12 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and W1, W2, W3 and W4 are each independently selected from a phenoxy group, a primary, secondary or tertiary amine group, an ether group, a phenol group, an amide group, an ester group, an alkyl group, an substituted alkyl group, a phenyl group, and a vinyl group; w, w2 O. ) < R2 d) , wherein R2 is selected from the group consisting of -H, optionally substituted C1 -C12 alkyl, optionally substituted aryl or optionally substituted heteroaryl; and W1, W2, W3 and W4 are each independently selected from -OP(O)(OH)2, -P(O)(OH)OP(O)(OH)2, - OP(O)(OH) )OP(O)(OH)OP(O)(OH)2, -OP(O)(OH)(NH2), -P(O)(OH)2, -SO3H, and a pharmaceutically acceptable salt thereof; It is) , wherein R2 is selected from the group consisting of -H, optionally substituted C1 -C12 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; W1 is selected from the group consisting of halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S(O)2-C1-C6 alkyl, and -CN ; and W2, W3 and W4 are each independently selected from the group consisting of -H, halogen, -C(O)OH, -C(O)O-C1-C6 alkyl, -NO2, haloalkyl, -S (O)2-C1-C6 alkyl, e - CN; f) wherein R2 is selected from the group consisting of -H, optionally substituted C1-C12 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; g) H) wherein m is 1, 2, 3, 4, 5, or 6, and R2 is as defined in claim 1; or 26. Compound according to claim 25, CHARACTERIZED by the fact that: a) W1, W2, W3 and W4 are each independently selected from the group consisting of -H, -Cl, -Br, -I , -F, -C(O)OH, -NO2, -CF3, and -CN; b) W1, W2, W3 and W4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2, and -CF3; c) W1 is selected from the group consisting of -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3, and -CN; and W2, W3, and W4 are each independently selected from the group consisting of -H, -Cl, -Br, -I, -F, -C(O)OH, -NO2, -CF3, and -CN ; or d) W1 is selected from the group consisting of -Cl, -F, and -NO2; and W2, W3 and W4 are each independently selected from the group consisting of -H, -Cl, -F, -NO2, and -CF3. 27. Compound according to any one of claims 6, 7, 9 and 10 to 26, CHARACTERIZED by the fact that X1, X2, X3, and X4 are each independently selected from the group consisting of -H, -OH, -CH3, -F, -Cl, -Br, -I, and -N3. 28. Compound CHARACTERIZED by the fact that it is selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein: is absent, or is selected from the group consisting of: 29. Compound CHARACTERIZED by the fact that it is selected from the group consisting of: and a pharmaceutically acceptable salt thereof. 30. Compound CHARACTERIZED by the fact that it is selected from the and a pharmaceutically acceptable salt thereof. 31. Compound CHARACTERIZED by the fact that it is selected from the group consisting of: and a pharmaceutically acceptable salt thereof. 32. Pharmaceutical composition CHARACTERIZED by the fact that it comprises a compound, as defined in any one of claims 1 to 31, and a pharmaceutically acceptable carrier. 33. Compound according to any one of claims 1 to 31, CHARACTERIZED by the fact that it is for use in the treatment of a disease or condition selected from the group consisting of a cancer, a viral disease, an autoimmune disease, a acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms. 34. Use of a compound, as defined in any one of claims 1 to 31, or a pharmaceutical composition, as defined in claim 32, CHARACTERIZED by the fact that it is for the manufacture of a medicament to treat a disease or condition selected from among a cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms.