Selective drug release from conjugates of internalized biologically active compounds.

Abstract

The present invention relates to conjugates of biologically active compounds, wherein such conjugates comprise a sequence of amino acids containing a tripeptide that confers selective cleavage by tumor tissue homogenates for release of free drug and / or improves biodistribution into tumor tissue compared to normal tissue homogenates from the same species, wherein the normal tissue is the site of an adverse event associated with administering to a human subject in need thereof a therapeutically effective amount of a comparison conjugate having an amino acid sequence that is a dipeptide known to be selectively cleavable by cathepsin B.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 902,888, filed September 19, 2019, the entire contents of which are incorporated herein by reference.

[0002] Background of the Invention The present invention relates to ligand drug conjugate (LDC) compounds, including antibody drug conjugates (ADC), and compositions thereof, that have improved selectivity for targeted cells compared to non-targeted cells. [Background technology]

[0003] Conventional ligand-drug conjugates exhibit biological activity against targeted cells, presenting the targeting moiety by binding to the targeting moiety recognized by the ligand unit of the conjugate, and then enter the cells through internalization of the conjugate.The selectivity of targeted cells over non-targeted cells in conventional ligand-drug conjugates is mainly achieved as a result of the targeting moiety being present at a higher abundance in targeted cells compared to non-targeted normal cells, which are cells that are not intended to be affected by the conjugate.After internalization of the conjugate, the peptide-based linker unit of the conjugate is enzymatically processed, where the conditional release of the conjugated compound, which has cytotoxicity in free form, is achieved by intracellular proteases.

[0004] The reduction of premature release of cytotoxic compounds from conventional dipeptide-based ligand-drug conjugates, which would otherwise cause undesirable side effects, is achieved by optimizing selectivity for specific lysosomal proteases that are thought to be upregulated in cancer cells. Because the proteases responsible for intracellular processing of conventional ligand-drug conjugates are common to all cells, selectivity for targeted cells is primarily due to the greater abundance of the targeted moiety in cells intended to be affected by the conjugate, despite differences in the intracellular activity levels of processing proteases between targeted cancer cells and non-targeted normal cells. However, this approach does not take into account possible differences in exposure to the released cytotoxic compounds between tumor tissues and normal tissues currently exploited by the ligand-drug conjugates of the present invention.

[0005] Therefore, the dipeptide sequence of a conventional ligand-drug conjugate, designed to be selectively affected by intracellular proteases upregulated in cancer cells of tumor tissues, can still be affected by proteases localized in normal tissues. Such action can occur either within the microenvironment of normal tissues or within normal tissue cells after immunologically specific or nonspecific uptake into these cells, resulting in on-target or off-target toxicity, respectively. These toxicities are urgent problems that need to be solved in the targeted delivery of highly cytotoxic compounds. Therefore, ligand-drug conjugates with improved peptide sequences that result in less exposure to normal tissues compared with conventional dipeptide-based ligand-drug conjugates, thus reducing exposure to cytotoxic compounds released from the ligand-drug conjugates while maintaining the efficacy provided by these conventional conjugates, are expected to improve the tolerability of treatment.

[0006] Ligand drug conjugates with improved peptide sequences that are more susceptible to proteolysis by tumor tissue than by normal tissue may also reduce exposure to released cytotoxic compounds compared to proteolysis of conventional dipeptide-based ligand drug conjugates by these tissues, which may contribute to improved tolerability of the treatment. Determining these differences in proteolysis using tissue homogenates should capture these differences that are driven by the microenvironment of these tissues and / or occur after cellular internalization.

[0007] To provide a solution to this problem in the art, ligand-drug conjugates having peptide-based linker units with sequences that provide more selective exposure of targeted cells in tumor tissue to the cytotoxic compound released from the conjugate compared to cells in normal tissue to the free cytotoxin, thus improving tolerability of the conjugate while retaining the efficacy of conventional dipeptide-based conjugates in treating cancer in mammalian subjects, are disclosed herein. The difference in exposure may be due to greater selectivity for proteolysis in tumor tissue of the ligand-drug conjugate with the selectivity-conferring peptide sequence compared to proteolysis in normal tissue, compared to proteolysis of conventional dipeptide-based conjugates. Alterations in the peptide sequence may also affect the physiochemical properties of the conjugate compound, resulting in greater exposure resulting from improved biodistribution into tumor tissue rather than normal tissue and / or improved disposal upon distribution within these tissues, resulting in preferential retention of the conjugate compound in tumor tissue and / or preferential elimination of the conjugate compound from normal tissue, respectively. These biodistribution effects may also be dominant factors for preferential proteolysis, which may be difficult to observe in vivo.

[0008] Thus, conjugate compounds having peptide sequences that result in enhanced exposure of tumor tissue to released free cytotoxic compounds compared to normal tissue should exhibit reduced undesired toxicity due to the peptide sequence being less susceptible to overall proteolysis in normal tissue or cells thereof compared to tumors, and / or due to improved pharmacokinetic properties of conjugate compounds incorporating peptide sequences that favor tumor tissue over normal tissue.

[0009] Thus, the Ligand Drug Conjugates of the present invention possess two levels of selectivity for targeted cells over non-targeted normal cells: (1) selective entry into targeted cells, and (2) reduced exposure of normal tissue to the conjugate compound compared to tumor tissue. This second level of selectivity is predicted to result in reduced normal tissue toxicity, resulting in fewer adverse events associated with conventional targeted therapies. Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention One main embodiment of the present invention is a compound represented by Formula 1: [ka] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, L is a ligand unit; LU is a linker unit; D' represents 1 to 4 drug units (D) within each drug linker moiety of formula -LU-D'; and the subscript p is a number from 1 to 12, 1 to 10, or 1 to 8, or is about 4 or about 8; wherein the Ligand unit is an antibody or an antigen-binding fragment of an antibody capable of selectively binding to an antigen in tumor tissue for subsequent release of the Drug unit as a cytotoxic compound; wherein the drug linker moiety of the formula -LU-D′ of each of the ligand drug conjugate compounds of the composition has the formula 1A: [ka] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, where the wavy line indicates a covalent bond to L; D is a drug unit of a cytotoxic compound; L B is the ligand covalent binding moiety; A is the first stretcher unit on demand; The subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional branching unit; The subscript b is 0 or 1, indicating the absence or presence of B, respectively; L O is a secondary linker moiety, wherein the secondary linker is [ka] where the wavy line adjacent to Y is L O indicates the site of covalent attachment to the Drug unit, and the wavy line adjacent to A' indicates the site of covalent attachment to the remainder of the Drug Linker moiety; A' is a second optional Stretcher unit that becomes a subunit of A in the absence of B; the subscript a' is 0 or 1, indicating the absence or presence of A', respectively; W is a peptide cleavable unit, wherein the peptide cleavable unit is a contiguous sequence of up to 12 (e.g., 3 to 12 or 3 to 10) amino acids, wherein the sequence includes a selectivity-conferring tripeptide that results in improved selectivity for exposure of tumor tissue over normal tissue to the free cytotoxic compound released from the ligand drug conjugate compound of the composition compared to the cytotoxic compound released from the ligand drug conjugate compound of a comparative ligand drug conjugate composition in which the peptide sequence of the peptide cleavable unit is the dipeptide-valine-citrulline- or -valine-alanine-; wherein the tumor tissue and normal tissue are of a rodent species, and wherein the composition of formula I the efficacy of the comparative ligand drug conjugate composition in the tumor xenograft model is maintained when administered at the same effective amount and dose schedule as previously determined for the comparative ligand drug conjugate composition; and When administered to non-tumor-bearing rodents at the same effective dose and dose schedule as in the tumor xenograft model, the composition demonstrates reduced plasma concentrations of cytotoxic compounds released from the Ligand Drug Conjugate compounds, and / or sparing of normal cells in tissues, compared to equivalent (e.g., identical) administration of a comparative Ligand Drug Conjugate composition in which the Ligand unit of both conjugate compositions is replaced by a non-binding antibody. resulting in the improved exposure selectivity demonstrated by wherein cytotoxicity to cells of a human tissue of the same tissue type as normal cells in the tissue of the non-tumor-bearing rodent is at least partially responsible for the adverse event in a human subject receiving a therapeutically effective amount of the comparative conjugate composition; Y is a self-immolative spacer unit; the subscript y is 0, 1, or 2, indicating the absence of Y or the presence of one or two Ys, respectively; and The subscript q is an integer ranging from 1 to 4, where subscript q is 1 if subscript b is 0, and subscript q is 2, 3, or 4 if subscript b is 1; and wherein the ligand drug conjugate compound of the composition has the structure of Formula 1 in which the subscript p is replaced by the subscript p', where the subscript p' is an integer from 1 to 12, 1 to 10, or 1 to 8, or is 4 or 8.

[0011] A related main embodiment is of formula I: [ka]

[0012] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, wherein in Formula I, LU' is capable of providing a covalent bond between L and LU of Formula 1 and is therefore sometimes referred to as a linker unit precursor; and D' represents 1 to 4 drug units, wherein the drug linker compound is of Formula IA: [ka]

[0013] is further defined by the structure of B ' is L in Equation 1A B whereby it is capable of forming a covalent bond to L of Formula 1 and is therefore sometimes referred to as a ligand covalent precursor moiety, and the remaining variables of Formula IA are as defined for Formula 1A.

[0014] In some embodiments, provided herein are compounds of Formula 1: [ka] or a pharmaceutically acceptable salt thereof, wherein in Formula 1: L is a ligand unit; LU is a linker unit; D' represents 1 to 4 drug units (D) within each drug linker moiety of formula -LU-D'; and the subscript p is a number from 1 to 12, 1 to 10, or 1 to 8, or is about 4 or about 8; wherein the Ligand unit is derived from an antibody or an antigen-binding fragment of an antibody that is capable of selectively binding to an antigen in tumor tissue for subsequent release of the Drug unit(s) as free drug; wherein the drug linker moiety of the formula -LU-D′ of each of the ligand drug conjugate compounds of the composition has the formula 1A: [ka] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, where the wavy line indicates a covalent bond to L; D is the drug unit; L B is the ligand covalent binding moiety; A is the first stretcher unit on demand; The subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional branching unit; The subscript b is 0 or 1, indicating the absence or presence of B, respectively; L O is a secondary linker moiety, wherein the secondary linker is [ka] and has the formula Here, the wavy line adjacent to Y is L O indicates the site of covalent attachment to the Drug unit, and the wavy line adjacent to A' indicates the site of covalent attachment to the remainder of the Drug Linker moiety; A' is a second optional Stretcher unit that becomes a subunit of A in the absence of B; The subscript a' is 0 or 1, indicating the absence or presence of A', respectively; W is a peptide cleavable unit, wherein the peptide cleavable unit comprises a tripeptide having the sequence -P3-P2-P1-, where P1, P2, and P3 are each an amino acid, wherein: the first amino acid of amino acids P1, P2, or P3 is negatively charged; a second amino acid among amino acids P1, P2, or P3 has an aliphatic side chain with a hydrophobicity not greater than that of leucine; and a third amino acid among amino acids P1, P2, or P3 has a hydrophobicity less than that of leucine; wherein the first amino acid among amino acids P1, P2, or P3 corresponds to any one of P1, P2, or P3, the second amino acid among amino acids P1, P2, or P3 corresponds to one of the remaining two amino acids P1, P2, or P3, and the third amino acid among amino acids P1, P2, or P3 corresponds to the last remaining amino acid P1, P2, or P3; However, -P3-P2-P1- is neither -Glu-Val-Cit- nor -Asp-Val-Cit-; Y is a self-immolative spacer unit; the subscript y is 0, 1, or 2, indicating the absence of Y or the presence of one or two Ys, respectively; and The subscript q is an integer ranging from 1 to 4; where subscript q is 1 if subscript b is 0, and subscript q is 2, 3, or 4 if subscript b is 1; and wherein the ligand drug conjugate compound of the composition has the structure of Formula 1 in which the subscript p is replaced by the subscript p', where the subscript p' is independently an integer from 1 to 12, 1 to 10, or 1 to 8, or is 4 or 8.

[0015] In some embodiments, provided herein is a ligand drug conjugate composition of Formula 1, wherein the ligand drug conjugate compound in the ligand drug conjugate composition is of Formula 1H: [ka] or a pharmaceutically acceptable salt thereof, and optionally having a minority of ligand drug conjugate compounds in which one or more of the drug linker moieties of each of the ligand drug conjugate compounds has its succinimide ring in hydrolyzed form, and HE is a hydrolysis enhancing unit; A', if present, is a subunit of the indicated first Stretcher unit (A); the subscript a' is 0 or 1, indicating the absence or presence of A'; and The wavy line indicates the site of covalent bonding to the sulfur atom of the Ligand unit.

[0016] In some embodiments that may be combined with any of the preceding embodiments, provided herein are Ligand Drug Conjugate compositions wherein HE is -C(=O).

[0017] In some embodiments that may be combined with any of the preceding embodiments, the present specification provides a group comprising -Y y -D is [ka] having the structure where -N(R y ) D' represents D, where D' is the remainder of D; The wavy line indicates the covalent binding site to P1; The dotted line is R y and optionally cyclization to D' of R y is optionally substituted C1-C6 alkyl in the absence of cyclization to D', or is optionally substituted C1-C6 alkylene when cyclized to D'; each Q is independently selected from the group consisting of -C1-C8 alkyl, -O-(C1-C8 alkyl), halogen, nitro, and cyano; and Ligand drug conjugate compositions are provided wherein the subscript m is 0, 1, or 2.

[0018] In some embodiments that may be combined with any of the preceding embodiments, provided herein is a compound having the formula D, where D is a cytotoxic drug, wherein the cytotoxic drug is a secondary amine-containing auristatin compound, where the nitrogen atom of the secondary amine is the site of covalent attachment to the drug linker moiety, and the secondary amine-containing auristatin compound has the formula D F / E-3 : [ka] and having the structure of formula D F / E-3where the dagger indicates the covalent bonding site of the nitrogen atom providing the carbamate functionality; R 10 and R 11 one is hydrogen and the other is methyl; R 13 is isopropyl or -CH2-CH(CH3)2; and R 19B is -CH(CH3)-CH(OH)-Ph, -CH(CO2H)-CH(OH)-CH3, -CH(CO2H)-CH2Ph, -CH(CH2Ph)-2-thiazolyl, -CH(CH2Ph)-2-pyridyl, -CH(CH2-p-Cl-Ph), -CH(CO2Me)-CH2Ph, -CH(CO2Me)-CH2CH2SCH3, -CH(CH2CH2SCH3)C(=O)NH-quinol-3-yl, -CH(CH2Ph)C(=O)NH-p-Cl-Ph, or R 19B teeth, [ka] and R 19B In the structure of Figure 1, the wavy line indicates the covalent bond to the remainder of the auristatin compound.

[0019] In some embodiments that may be combined with any of the preceding embodiments, provided herein are ligand drug conjugate compositions wherein the secondary amine-containing auristatin compound is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0020] In some embodiments that may be combined with any of the preceding embodiments, the subscript q is 1 and the ligand drug conjugate compound in the ligand drug conjugate composition is represented by the formula 1H-MMAE: [ka] or a pharmaceutically acceptable salt thereof, and optionally having a minority of ligand drug conjugate compounds wherein one or more of the drug linker moieties of each of the ligand drug conjugate compounds have its succinimide ring in hydrolyzed form; and The subscript a' is 0 and A' does not exist; and The wavy line indicates the site of covalent bonding to the sulfur atom of the ligand unit. Ligand drug conjugate compositions are provided.

[0021] In some embodiments that may be combined with any of the preceding embodiments, the peptide cleavable unit is a tripeptide having the sequence -P3-P2-P1-, where P1, P2, and P3 are each an amino acid, and wherein: The P3 amino acid of the tripeptide is in the D-amino acid configuration; one of the P2 amino acid and the P1 amino acid has an aliphatic side chain that has a hydrophobicity less than that of leucine; and The other of the P2 amino acid and the P1 amino acid is negatively charged, Ligand drug conjugate compositions are provided. In some embodiments, the P3 amino acid is D-Leu or D-Ala. In some embodiments, one of the P2 amino acid or the P1 amino acid has an aliphatic side chain with hydrophobicity not greater than that of valine, and the other of the P2 amino acid or the P1 amino acid is negatively charged at physiological pH of plasma. In some embodiments, the P2 amino acid has an aliphatic side chain with hydrophobicity not greater than that of valine, and the P1 amino acid is negatively charged at physiological pH of plasma. In some embodiments, -P2-P1- is -Ala-Glu- or -Ala-Asp-. In some embodiments, -P3-P2-P1- is -D-Leu-Ala-Asp-, -D-Leu-Ala-Glu-, -D-Ala-Ala-Asp-, or -D-Ala-Ala-Glu-. In some embodiments, the P3 amino acid is D-Leu or D-Ala, the P2 amino acid is Ala, Glu, or Asp, and the P1 amino acid is Ala, Glu, or Asp.

[0022] In some embodiments, the compound described herein is [ka] or a pharmaceutically acceptable salt thereof; Ligand drug conjugate compositions are provided wherein L is a Ligand unit and the subscript p' is an integer from 1 to 24.

[0023] In some embodiments that may be combined with any of the preceding embodiments, provided herein are ligand drug conjugate compositions wherein L is an antibody ligand unit of an intact antibody or antigen-binding fragment thereof. In some embodiments, the intact antibody or fragment thereof is capable of selectively binding to a cancer cell antigen. In some embodiments, the intact antibody is a chimeric, humanized, or human antibody, wherein the antibody is capable of selectively binding to a cancer cell antigen, or the antibody is a non-binding control antibody, thereby defining a non-binding control conjugate composition.

[0024] In some embodiments that may be combined with any of the preceding embodiments, provided herein are ligand drug conjugate compositions wherein the subscript p is in the range of about 2 to about 12, or about 2 to about 10, or about 2 to about 8; or the subscript p is about 2, about 4, or about 8.

[0025] In some embodiments that can be combined with any of the preceding embodiments, provided herein is a pharmaceutically acceptable formulation comprising an effective amount of a ligand drug conjugate composition described herein or an equivalent amount of a non-binding control conjugate and at least one pharmaceutically acceptable excipient. In some embodiments, the at least one pharmaceutically acceptable excipient is a liquid carrier that results in a liquid formulation, wherein the liquid formulation is suitable for lyophilization or administration to a subject in need thereof. In some embodiments, the formulation is a solid derived from lyophilization or a liquid formulation described herein, wherein at least one excipient in the solid formulation is a cryoprotectant.

[0026] In some embodiments, provided herein are compounds of formula IA: [ka] or a salt thereof, wherein: D is the drug unit; L B ' is a ligand covalent precursor moiety; A is the first stretcher unit on demand; The subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional branching unit; The subscript b is 0 or 1, indicating the absence or presence of B, respectively; L O is a secondary linker moiety, wherein the secondary linker is [ka] and has the formula Here, the wavy line adjacent to Y is L O indicates the site of covalent attachment to the Drug unit, and the wavy line adjacent to A' indicates the site of covalent attachment to the remainder of the Drug Linker compound; A' is a second optional Stretcher unit that becomes a subunit of A in the absence of B; The subscript a' is 0 or 1, indicating the absence or presence of A', respectively; W is a peptide cleavable unit, wherein the peptide cleavable unit comprises a tripeptide having the sequence -P3-P2-P1-, where P1, P2, and P3 are each an amino acid, wherein: the first amino acid of amino acids P1, P2, or P3 is negatively charged; a second amino acid among amino acids P1, P2, or P3 has an aliphatic side chain with a hydrophobicity not greater than that of leucine; and a third amino acid among amino acids P1, P2, or P3 has a hydrophobicity less than that of leucine; wherein the first amino acid among amino acids P1, P2, or P3 corresponds to any one of P1, P2, or P3, the second amino acid among amino acids P1, P2, or P3 corresponds to one of the remaining two amino acids P1, P2, or P3, and the third amino acid among amino acids P1, P2, or P3 corresponds to the last remaining amino acid P1, P2, or P3; However, -P3-P2-P1- is neither -Glu-Val-Cit- nor -Asp-Val-Cit-; Y is a self-immolative spacer unit; the subscript y is 0, 1, or 2, indicating the absence of Y or the presence of one or two Ys, respectively; and The subscript q is an integer ranging from 1 to 4; where subscript q is 1 if subscript b is 0, and subscript q is 2, 3, or 4 if subscript b is 1.

[0027] In some embodiments, provided herein is a drug linker compound having formula IH: [ka] or a salt thereof, wherein HE is a hydrolysis enhancing unit; A', if present, is a subunit of the first Stretcher unit (A) as indicated; the subscript a' is 0 or 1, indicating the absence or presence of A'; Drug-linker compounds of formula IA are provided:

[0028] In some embodiments that may be combined with any of the preceding embodiments, provided herein are drug linker compounds wherein HE is -C(=O).

[0029] In some embodiments that may be combined with any of the preceding embodiments, the present specification provides a group comprising -Y y -D is [ka] having the structure where -N(R y ) D' represents D, where D' is the remainder of D; The wavy line indicates the covalent binding site to P1; The dotted line is R yand optionally cyclization to D' of R y is optionally substituted C1-C6 alkyl in the absence of cyclization to D', or is optionally substituted C1-C6 alkylene when cyclized to D'; each Q is independently selected from the group consisting of -C1-C8 alkyl, -O-(C1-C8 alkyl), halogen, nitro, and cyano; and Drug-linker compounds are provided wherein the subscript m is 0, 1 or 2.

[0030] In some embodiments that may be combined with any of the preceding embodiments, provided herein is a compound having the formula D, where D is a cytotoxic drug, wherein the cytotoxic drug is a secondary amine-containing auristatin compound, where the nitrogen atom of the secondary amine is the site of covalent attachment to the drug linker moiety, and the secondary amine-containing auristatin compound has the formula D F / E-3 : [ka] and having the structure of formula D F / E-3 where the dagger indicates the covalent bonding site of the nitrogen atom providing the carbamate functionality; R 10 and R 11 one is hydrogen and the other is methyl; R 13 is isopropyl or -CH2-CH(CH3)2; and R 19B is -CH(CH3)-CH(OH)-Ph, -CH(CO2H)-CH(OH)-CH3, -CH(CO2H)-CH2Ph, -CH(CH2Ph)-2-thiazolyl, -CH(CH2Ph)-2-pyridyl, -CH(CH2-p-Cl-Ph), -CH(CO2Me)-CH2Ph, -CH(CO2Me)-CH2CH2SCH3, -CH(CH2CH2SCH3)C(=O)NH-quinol-3-yl, -CH(CH2Ph)C(=O)NH-p-Cl-Ph, or R 19B teeth, [ka] and R 19B In the structure of the drug linker compound, the wavy line indicates the covalent bond to the remainder of the auristatin compound.

[0031] In some embodiments that may be combined with any of the preceding embodiments, provided herein are drug linker compounds, wherein the secondary amine-containing auristatin compound is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0032] In some embodiments that may be combined with any of the preceding embodiments, the drug linker compound is described herein as having the formula IH-MMAE: [ka] or a salt thereof, wherein The subscript a' is 0 and A' does not exist. Drug linker compounds are provided.

[0033] In some embodiments that may be combined with any of the preceding embodiments, the peptide cleavable unit is a tripeptide having the sequence -P3-P2-P1-, where P1, P2, and P3 are each an amino acid, and wherein: The P3 amino acid of the tripeptide is in the D-amino acid configuration; one of the P2 amino acid and the P1 amino acid has an aliphatic side chain that has a hydrophobicity less than that of leucine; and The other of the P2 amino acid and the P1 amino acid is negatively charged, Drug linker compounds are provided. In some embodiments, the P3 amino acid is D-Leu or D-Ala. In some embodiments, one of the P2 amino acid or the P1 amino acid has an aliphatic side chain with hydrophobicity not greater than that of valine, and the other of the P2 amino acid or the P1 amino acid is negatively charged at physiological pH of plasma. In some embodiments, the P2 amino acid has an aliphatic side chain with hydrophobicity not greater than that of valine, and the P1 amino acid is negatively charged at physiological pH of plasma. In some embodiments, -P2-P1- is -Ala-Glu- or -Ala-Asp-. In some embodiments, -P3-P2-P1- is -D-Leu-Ala-Asp-, -D-Leu-Ala-Glu-, -D-Ala-Ala-Asp-, or -D-Ala-Ala-Glu-. In some embodiments, the P3 amino acid is D-Leu or D-Ala, the P2 amino acid is Ala, Glu, or Asp, and the P1 amino acid is Ala, Glu, or Asp.

[0034] In some embodiments, the drug linker compound is [ka] [ka] Drug linker compounds are provided having the structure:

[0035] In some embodiments, provided herein are compounds of formula IA-L: [ka] or a salt thereof, wherein the linker compound is of formula IA-L: RG is a reactive group; L B ' is a ligand covalent precursor moiety; A is the first stretcher unit on demand; The subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional branching unit; The subscript b is 0 or 1, indicating the absence or presence of B, respectively; L O is a secondary linker moiety, wherein the secondary linker is [ka] and has the formula Here, the wavy line adjacent to Y is L O indicates the site of covalent attachment to the Drug unit, and the wavy line adjacent to A' indicates the site of covalent attachment to the remainder of the Drug Linker compound; A' is a second optional Stretcher unit that becomes a subunit of A in the absence of B; The subscript a' is 0 or 1, indicating the absence or presence of A', respectively; W is a peptide cleavable unit, wherein the peptide cleavable unit comprises a tripeptide having the sequence -P3-P2-P1-, where P1, P2, and P3 are each an amino acid, wherein: the first amino acid of amino acids P1, P2, or P3 is negatively charged; a second amino acid among amino acids P1, P2, or P3 has an aliphatic side chain with a hydrophobicity not greater than that of leucine; and a third amino acid among amino acids P1, P2, or P3 has a hydrophobicity less than that of leucine; wherein the first amino acid among amino acids P1, P2, or P3 corresponds to any one of P1, P2, or P3, the second amino acid among amino acids P1, P2, or P3 corresponds to one of the remaining two amino acids P1, P2, or P3, and the third amino acid among amino acids P1, P2, or P3 corresponds to the last remaining amino acid P1, P2, or P3; However, -P3-P2-P1- is neither -Glu-Val-Cit- nor -Asp-Val-Cit-; Y is a self-immolative spacer unit; the subscript y is 0, 1, or 2, indicating the absence of Y or the presence of one or two Ys, respectively; and The subscript q is an integer ranging from 1 to 4; where subscript q is 1 if subscript b is 0, and subscript q is 2, 3, or 4 if subscript b is 1.

[0036] In some embodiments, the peptide cleavable unit described herein is a tripeptide having the sequence -P3-P2-P1-, where P1, P2, and P3 are each an amino acid, and wherein: The P3 amino acid of the tripeptide is in the D-amino acid configuration; one of the P2 amino acid and the P1 amino acid has an aliphatic side chain that has a hydrophobicity less than that of leucine; and The other of the P2 amino acid and the P1 amino acid is negatively charged, A linker compound is provided.

[0037] In some embodiments, the linker compound described herein has the formula IA-L-3: [ka] A linker compound is provided having the structure:

[0038] In some embodiments, the linker compound described herein is [ka] A linker compound is provided having the structure:

[0039] These and other embodiments of the present invention are described in further detail in the detailed description and claims below. [Brief explanation of the drawings]

[0040] [Figure 1A] Tumor volume versus days after implantation in a xenograft model treated with subcurative doses of a series of 4-loaded ADCs having various tripeptide sequences as peptide-cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE, compared with a subcurative dose of a 4-loaded ADC targeting the same cancer cell antigen and having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE. The compound in Figure 1A was tested at 4 mg / kg. The compounds in Figures 1B and 1D were tested at 3 mg / kg. The compound in Figure 1C was tested at 6 mg / kg. [Figure 1B] Tumor volume versus days after implantation in a xenograft model treated with subcurative doses of a series of 4-loaded ADCs having various tripeptide sequences as peptide-cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE, compared with a subcurative dose of a 4-loaded ADC targeting the same cancer cell antigen and having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE. The compound in Figure 1A was tested at 4 mg / kg. The compounds in Figures 1B and 1D were tested at 3 mg / kg. The compound in Figure 1C was tested at 6 mg / kg. [Figure 1C] Tumor volume versus days after implantation in a xenograft model treated with subcurative doses of a series of 4-loaded ADCs having various tripeptide sequences as peptide-cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE, compared with a subcurative dose of a 4-loaded ADC targeting the same cancer cell antigen and having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE. The compound in Figure 1A was tested at 4 mg / kg. The compounds in Figures 1B and 1D were tested at 3 mg / kg. The compound in Figure 1C was tested at 6 mg / kg. [Figure 1D]Tumor volume versus days after implantation in a xenograft model treated with subcurative doses of a series of 4-loaded ADCs having various tripeptide sequences as peptide-cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE, compared with a subcurative dose of a 4-loaded ADC targeting the same cancer cell antigen and having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE. The compound in Figure 1A was tested at 4 mg / kg. The compounds in Figures 1B and 1D were tested at 3 mg / kg. The compound in Figure 1C was tested at 6 mg / kg.

[0041] [Figure 2] Neutrophil counts after 4 days of administration at 10 mg / Kg of a series of 4-loaded non-binding control conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE compared to 4-loaded non-binding conjugates having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE or mp-val-cit-PABC-MMAE.

[0042] [Figure 3] Reticulocyte counts in rat plasma after 4 days of administration at 10 mg / kg to non-tumor-bearing animals of a series of 4-loaded non-binding conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE compared with 4-loaded non-binding conjugates having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE or mp-val-cit-PABC-MMAE.

[0043] [Figure 4]Histopathology of rat bone marrow after 4 days of administration to non-tumor-bearing animals of 10 mg / Kg of vehicle or 4-loaded non-binding conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE compared to 4-loaded non-binding conjugates having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE.

[0044] [Figure 5A] Free MMAE in rat plasma at various time points after administration of 10 mg / Kg of vehicle and four loaded non-binding conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE to non-tumor-bearing animals compared with four loaded non-binding conjugates having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE. [Figure 5B] Free MMAE in rat plasma at various time points after administration of 10 mg / Kg of vehicle and four loaded non-binding conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE to non-tumor-bearing animals compared with four loaded non-binding conjugates having a drug-linker moiety represented by the formula mc-val-cit-PABC-MMAE.

[0045] [Figure 6A] Percentage of drug cleaved in vitro by neutrophil elastase (Figure 6A) or cathepsin B (Figure 6B) from the heavy chain of tetra-loaded non-targeted conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE compared to tetra-loaded non-targeted conjugates having a drug-linker moiety represented by the formula mp-val-cit-PABC-MMAE. [Figure 6B]Percentage of drug cleaved in vitro by neutrophil elastase (Figure 6A) or cathepsin B (Figure 6B) from the heavy chain of tetra-loaded non-targeted conjugates having various tripeptide sequences as peptide cleavable units with a drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE compared to tetra-loaded non-targeted conjugates having a drug-linker moiety represented by the formula mp-val-cit-PABC-MMAE.

[0046] [Figure 7] Aggregation of a series of four-loaded non-targeted conjugates with various tripeptide sequences as peptide cleavable units with the drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE in rat plasma (Figure 7), cynomolgus monkey plasma (Figure 8), or human plasma (Figure 9) after 96 h of incubation. [Figure 8] Aggregation of a series of four-loaded non-targeted conjugates with various tripeptide sequences as peptide cleavable units with the drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE in rat plasma (Figure 7), cynomolgus monkey plasma (Figure 8), or human plasma (Figure 9) after 96 h of incubation. [Figure 9] Aggregation of a series of four-loaded non-targeted conjugates with various tripeptide sequences as peptide cleavable units with the drug-linker moiety represented by the formula mp-P3-P2-P1-PABC-MMAE in rat plasma (Figure 7), cynomolgus monkey plasma (Figure 8), or human plasma (Figure 9) after 96 h of incubation.

[0047] [Figure 10] Aggregation of non-targeted MMAF ADC in rat plasma at various time points.

[0048] [Figure 11] Correlation between reticulocyte depletion by non-targeted ADC in rats and ADC aggregation in rat plasma after 96 hours of incubation.

[0049] [Figure 12] Correlation between reticulocyte depletion by non-targeted ADC in rats and ADC aggregation in cynomolgus monkey plasma after 96 hours of incubation.

[0050] [Figure 13] Correlation between reticulocyte depletion by non-targeted ADC in rats and ADC aggregation in human plasma after 96 h of incubation.

[0051] [Figure 14] Concentration of antibody in the extracellular bone marrow compartment of rats administered non-targeted ADC.

[0052] [Figure 15] Amount of free MMAE in bone marrow cells from rats administered non-targeted ADC.

[0053] [Figure 16] Reticulocyte depletion at days 5 and 8 post-dose with a non-targeted tripeptide ADC after administration at 20 mg / kg in rats.

[0054] [Figure 17] Neutrophil depletion at days 5 and 8 post-dose with a non-targeted tripeptide ADC after administration at 20 mg / kg in rats.

[0055] [Figure 18] Bone histology at days 5 and 8 post-dosing with a non-targeted tripeptide ADC after administration at 20 mg / kg in rats.

[0056] [Figure 19] Correlation between the cLogP of the linker and the aggregation of the corresponding h00 conjugate in rat plasma after 96 h of incubation (expressed as %HMW = % high molecular weight species).

[0057] [Figure 20]Correlation between reticulocyte depletion caused by non-targeted ADC in rats and ADC aggregation in rat plasma after 96 hours of incubation (expressed as %HMW = % high molecular weight species).

[0058] [Figure 21] Correlation between reticulocyte depletion caused by non-targeted ADC in rats and ADC aggregation in human plasma after 96 hours of incubation (expressed as %HMW = % high molecular weight species).

[0059] [Figure 22] Correlation between reticulocyte depletion caused by non-targeted ADC in rats and ADC aggregation in cynomolgus monkey plasma after 96 hours of incubation (expressed as %HMW = % high molecular weight species). DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed Description of the Invention general

[0061] The present invention is based, in part, on the unexpected discovery that protease activity in tumor tissue and non-targeted normal tissue differs sufficiently to provide additional selectivity for cancer cells targeted by ligand drug conjugates bearing protease-activatable peptide sequences for conditional release of a conjugated cytotoxic compound. This difference is exploited by the protease-cleavable peptide sequences disclosed herein when these sequences are incorporated into the peptide-cleavable linker unit of the ligand drug conjugate compounds. In some cases, sequences with this property are believed to result in conjugate compounds with favorable susceptibility to proteolysis for biodistribution and / or release of free cytotoxic compound in tumor tissue compared to normal tissue.

[0062] 1.Definition

[0063] As used herein, unless otherwise stated or implied by context, the terms used herein have the meanings defined below. For example, in their definitions and throughout this specification, unless specifically contraindicated or implied by the inclusion of mutually exclusive elements or alternatives, the terms "a" and "an" mean one or more, and the term "or" means and / or, where the context allows. Thus, as provided in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context specifically dictates otherwise.

[0064] At various points in the present disclosure, for example, in any disclosed embodiment or in the claims, reference is made to compounds, compositions, or methods that "comprise" one or more specified components, elements, or steps. Inventive embodiments also specifically include compounds, compositions, compositions, or methods that are, consist of, or consist essentially of those specified components, elements, or steps. The term "consisting of" is used interchangeably with the term "comprising" and is stated as an equivalent term. For example, disclosed compositions, devices, products, or methods that "comprise" a component or step are open-ended; they include or indicate those compositions or methods plus additional component(s) or step(s). However, these terms do not encompass unrecited elements that would destroy the functionality of the disclosed composition, device, product, or method for its intended purpose. Similarly, disclosed compositions, devices, products, or methods that "consist of" a component or step are closed-ended; they do not include or indicate compositions or methods that have a significant amount of additional component(s) or additional step(s). Furthermore, the term "consisting essentially of" permits the inclusion of unrecited elements that do not materially affect the functionality of the disclosed composition, device, article, or method for its intended purpose as further defined herein. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed.

[0065] Unless the term is specifically stated or implied in the context, "about," when used herein in connection with a numerical value or range of values ​​provided to describe a particular property of a compound or composition, indicates that the value or range of values ​​may deviate to an extent considered reasonable by one of ordinary skill in the art while still describing the particular property. Reasonable deviations include those within the precision or accuracy of the device(s) used to measure, determine, or derive the particular property. Specifically, the term "about," when used in this context, indicates that the numerical value or range of values ​​may vary by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.01%, typically 10% to 0.5%, more typically 5% to 1%, of the stated value or range of values, while still describing the particular property.

[0066] With respect to the subscript p (which represents the average number of drug linker moieties in a ligand drug conjugate composition, as further defined herein), the term "about" reflects the uncertainty accepted in the art with respect to determining this value from the distribution of ligand drug conjugate compounds in the composition, as determined by standard methods of size exclusion or HIC chromatography or HPLC-MS.

[0067] The terms "essentially retain," "essentially retaining," and the like, as used herein, unless otherwise stated or implied by the context, refer to a property, characteristic, function, or activity of a compound or composition or portion thereof that does not detectably alter, or within experimental error of determination, the same activity, characteristic, or property of a compound or composition or portion of related structure.

[0068] The terms "substantially retain," "substantially retaining," and the like, as used herein, unless otherwise stated or implied by the context, refer to a measurement of a physical property or characteristic of a compound or composition or portion thereof that may differ statistically from the determination of the same physical property of another compound or composition or portion of related structure, but that such difference is not interpreted as a statistically significant or material difference in those activities or properties in a biological test system appropriate for assessing biological activity or pharmacological properties (i.e., the biological activity or property is retained or essentially retained). Thus, the phrase "substantially retain" is made with reference to the effect that a physical property or characteristic of a compound or composition has on a physicochemical or pharmacological property or biological activity that is clearly related to that physical property or characteristic.

[0069] Terms such as "negligible" or "negligible," as used herein, refer to an amount of impurity below the level of quantification by HPLC analysis, unless otherwise stated or implied in the context. Depending on the context, these terms may alternatively mean that no statistically significant difference is observed between measurements or results, or is within the experimental error of the equipment used to obtain these values. Negligible differences in the values ​​of experimentally determined parameters do not mean that the impurity characterized by that parameter is present in negligible amounts.

[0070] As used herein, terms such as "predominantly containing," "predominantly having," and the like refer to the major component of a mixture unless otherwise stated or implied by the context. When a mixture has two components, the major component accounts for more than 50% by weight of the mixture. In a mixture having three or more components, the major component is the one present in the greatest amount in the mixture and may or may not account for the majority of the mass of the mixture.

[0071] "Electron-withdrawing group," as that term is used herein, unless otherwise stated or implied in the context, refers to a functional group or electronegative atom that, via either inductive and / or resonance predominately (i.e., the functional group or atom may be electron-donating via resonance but overall inductively electron-withdrawing), tends to withdraw electron density from the atom to which it is bonded, stabilizing the anion or electron-rich moiety. The electron-withdrawing effect is transferred, even if in attenuated form, typically inductively, to other atoms bonded to the bonded atom made electron-deficient by the electron-withdrawing group (EWG), thus reducing the electron density of more distant reaction centers.

[0072] Electron withdrawing groups (EWG) are typically -C(=O)R', -CN, -NO2, -CX3, -X, -C(=O)OR', -C(=O)NH2, -C(=O)N(R')R op , -C(=O)R', -C(=O)X, -S(=O)2R op , -S(=O)2OR', -SO3H2, -S(=O)2NH2, -S(=O)2N(R')R op , -PO3H2, -P(=O)(OR')(OR op )2, -NO, -NH2, -N(R')(R op ), -N(R op )3 + and salts thereof, wherein X is -F, -Br, -Cl, or -I, and R op is independently selected at each occurrence from the groupings described above for optional substituents, and R′ is —H or R op , where R op is as defined above. In some aspects, each R op are independently C1 to C 12alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or independently selected from the group consisting of C1-C6 alkyl and optionally substituted phenyl, and R' is hydrogen. EWG can also be aryl (e.g., phenyl) or heteroaryl, depending on its substitution, and can be certain electron-deficient heteroaryl groups (e.g., pyridyl). Thus, in some aspects, an "electron-withdrawing group" is an electron-deficient C5-C 24 Heteroaryl and C6-C substituted with electron-donating substituents 24 Further encompassed is aryl. More typically, electron-withdrawing groups are independently selected from the group consisting of -C(=O)R', -CN, -NO2, -CX3, and -X, where X is a halogen, typically selected from the group consisting of -F and -Cl, and R' is H, C1-C6 alkyl, or C1-C4 alkyl. Depending on its substituents, an optionally substituted alkyl moiety can also be an electron-withdrawing group, and thus, in such cases, these aspects are encompassed by this term for the electron-withdrawing group.

[0073] "Electron-donating group," as the term is used herein, unless otherwise stated or implied by the context, refers to a functional group or electropositive atom that increases the electron density of the atom to which it is bonded, either through inductive and / or resonance (i.e., the functional group or atom may be inductively electron-withdrawing, but overall electron-donating through resonance), which tends to stabilize a cation or electron-deficient system. The electron-donating effect is transferred, typically by resonance, to other atoms bonded to the bonded atom made electron-rich by the electron-donating group (EDG), thus increasing the electron density of more distant reaction centers. Typically, the electron-donating group is selected from the group consisting of -OH, -OR', -NH2, -NHR', and N(R')2, where each R' is independently selected from C1 to C 12 alkyl, typically C1-C6 alkyl. Depending on the substituents, C6-C 24 Aryl, C5-C 24Heteroaryl or unsaturated C1-C 12 Alkyl moieties can also be electron donating groups, and in some aspects, such moieties are encompassed by this term for electron donating groups.

[0074] The term "compound," as used herein, refers to and includes the compound itself (whether named or represented by structure) and its salt form(s), unless the context clearly indicates otherwise or implies. Salts of the compound include zwitterionic salt forms with organic or inorganic counterions, as well as acid and base addition salt forms, and salt forms containing two or more counterions, which may be the same or different. In some aspects, the salt forms are pharmaceutically acceptable salt forms of the compound. The term "compound" further encompasses solvated forms of the compound, in which a solvent is noncovalently associated with the compound or reversibly covalently bonded to the compound, such as when a carbonyl group of the compound is hydrated to form a gem-diol. Solvate forms include solvate forms of the compound itself and its salt form(s), and encompass hemisolvates, monosolvates, disolvates (including hydrates), and when a compound may be associated with two or more solvent molecules, these two or more solvent molecules may be the same or different. In some instances, the compounds of the present invention include explicit reference to one or more of the above forms (e.g., salts and solvates), which does not imply any solid-state form of the compound. However, this reference is for emphasis only and should not be construed as excluding any other of the forms identified above. Furthermore, if an explicit reference to salts and / or solvate forms of a compound or ligand-drug conjugate composition is not made, this omission should not be construed as excluding salts and / or solvate forms of the compound or conjugate, unless the context makes it clear that such salts and / or solvate forms are to be excluded.

[0075] "Enantiomer," as the term is used herein, unless otherwise stated or implied in context, refers to a related compound that differs structurally by one or more chiral centers of opposite stereochemical configuration(s) compared to a reference compound, although both have the same atomic bonds.

[0076] "Moiety," as the term is used herein, means a specific segment, fragment, or functional group of a molecule or compound, unless otherwise stated or implied by context. A chemical moiety is sometimes referred to as a chemical component (i.e., a substituent or variable) embedded in or attached to a molecule, compound, or chemical formula.

[0077] Unless otherwise indicated or implied by context, for any substituent or moiety described herein with a given range of carbon atoms, the specified range means that any individual number of carbon atoms is described. Thus, for example, reference to "optionally substituted C1-C4 alkyl" or "optionally substituted alkenyl C2-C6 alkenyl" specifically means that there is a 1-, 2-, 3-, or 4-carbon optionally substituted alkyl moiety, as defined herein, or a 2-, 3-, 4-, 5-, or 6-carbon optionally substituted alkenyl, as defined herein, respectively. All such numerical designations are expressly intended to disclose all individual carbon atom groups; thus, "optionally substituted C1-C4 alkyl" includes methyl, ethyl, 3-carbon alkyl, and 4-carbon alkyl, whether substituted or unsubstituted, including all of these positional isomers. Thus, when an alkyl moiety is substituted, the numerical designation refers to the unsubstituted base moiety and is not intended to include carbon atoms not directly attached to the base moiety that may be present in a substituent of the base moiety. For esters, carbonates, carbamates, and ureas, as defined herein, specified with a given range of carbon atoms, the specified range includes the carbonyl carbon of the respective functional group. Thus, a C1 ester refers to a formate ester, and a C2 ester refers to an acetate ester.

[0078] The organic substituents, moieties, and groups described herein, as well as any other moieties described herein, are generally intended to exclude unstable moieties, except insofar as such unstable moieties are transient species that can be used to make compounds with sufficient chemical stability for one or more of the uses described herein. Specifically excluded are substituents, moieties, or groups that, by manipulating the definitions provided herein, result in those having a pentavalent carbon.

[0079] "Alkyl," as the term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, includes methyl, or an alkyl group in which one or more of the carbon atoms are saturated (i.e., one or more sp 3 "Alkyl" refers to a group of consecutive carbon atoms (one of which is monovalent) covalently linked together in a linear, secondary, tertiary, or cyclic arrangement (composed of carbon atoms), i.e., in a straight, branched, cyclic arrangement, or any combination thereof. When the consecutive saturated carbon atoms are in a cyclic arrangement, such an alkyl moiety is, in some aspects, referred to as a carbocyclyl, as further defined herein.

[0080] When an alkyl moiety or alkyl group is referred to as an alkyl substituent, the alkyl substituent on the Markush structure or another organic moiety to which it is associated provides the structure or moiety with the sp of the alkyl substituent. 3 and methyl or a chain of consecutive carbon atoms bonded via an alkyl group. Thus, an alkyl substituent, as used herein, contains at least one saturated moiety and can also be substituted with a cycloalkyl, aromatic, or heteroaromatic moiety or group, or substituted with an alkenyl or alkynyl moiety to provide an unsaturated alkyl. Thus, an optionally substituted alkyl substituent can further contain one, two, three, or more independently selected double and / or triple bonds, or can be substituted with an alkenyl or alkynyl moiety or any combination thereof to define an unsaturated alkyl substituent, and can be substituted with other moieties containing appropriate optional substituents as described herein. The number of carbon atoms in saturated alkyls can vary and is typically 1 to 50, 1 to 30, or 1 to 20, and more typically 1 to 8 or 1 to 6, and in unsaturated alkyl moieties or groups typically varies between 3 to 50, 3 to 30, or 3 to 20, and more typically between 3 to 8.

[0081] A saturated alkyl moiety is a group consisting of saturated, non-cyclic carbon atoms (i.e., non-cyclic sp 3 carbon) and sp 2 It contains neither sp carbon atoms nor sp carbon atoms, but may be substituted with optional substituents as described herein, provided that such substitution does not exceed the sp of such optional substituents. 3 Carbon atom, sp 2 It is not through a carbon atom or sp carbon atom, because this affects the identity of the base alkyl moiety so substituted in number of carbon atoms, except when the optional substituent is a basic unit as defined herein. Unless otherwise indicated or implied by context, the term "alkyl" refers to a saturated, acyclic hydrocarbon radical, wherein the hydrocarbon radical has the indicated number of covalently bonded, saturated carbon atoms, such that terms such as "C1-C6 alkyl" or "C1-C6 alkyl" refer to an alkyl moiety or group containing one saturated carbon atom (i.e., methyl), or two, three, four, five, or six consecutive acyclic, saturated carbon atoms, and "C1-C8 alkyl" refers to an alkyl moiety or group containing one saturated carbon atom, or two, three, four, five, six, seven, or eight consecutive acyclic, saturated carbon atoms. Typically, saturated alkyls do not contain sp carbon atoms in the continuous carbon chain. 2 A C1-C6 or C1-C4 alkyl moiety containing no carbon atoms or no sp carbon atoms, the latter sometimes referred to as lower alkyl, and in some aspects, when the number of carbon atoms is not specified, 1 to 8 consecutive acyclic sp 3 carbon atoms in the continuous carbon chain 2"C1-C8 alkyl" refers to a saturated C1-C8 alkyl moiety that contains no carbon atoms or no sp carbon atoms. In other aspects, when a range of consecutive carbon atoms defines the term "alkyl" but does not specify it as saturated or unsaturated, the term encompasses saturated alkyls in the specified range and unsaturated alkyls where the lower end of the range is increased by two carbon atoms. For example, the term "C1-C8 alkyl" includes saturated C1-C8 alkyls and C3-C8 unsaturated alkyls, without limitation to saturated alkyls.

[0082] Where saturated alkyl substituents, moieties, or groups are specified, the species includes those derived from removing a hydrogen atom from a parent alkane (i.e., the alkyl moiety is monovalent) and may include methyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl, -CH(CH3)2), 1-butyl (n-butyl), 2-methyl-1-propyl (iso-butyl, -CH2CH(CH3)2), 2-butyl (sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-butyl, -C(CH3)3), amyl, isoamyl, sec-amyl, and other straight- and branched-chain alkyl moieties.

[0083] "Alkylene," as that term is used herein, alone or as part of another term, means, unless otherwise indicated or implied by context, a saturated, branched or straight chain hydrocarbon diradical, substituted or unsubstituted, in which one or more of the carbon atoms are saturated (i.e., one or more sp 3 carbons), of the stated number of carbon atoms ranging from 1 to 50 or 1 to 30, typically 1 to 20 or 1 to 12 carbon atoms, more typically 1 to 8, 1 or 6, or 1 to 4 carbon atoms, and having the same or two different saturations (i.e., sp 3(-CH2CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and similar diradicals. Typically, alkylene is a divalent group having two radical centers (i.e., divalent) derived by removing two hydrogen atoms from a carbon atom. An alkylene moiety, in some aspects, is an alkyl radical as described herein in which a hydrogen atom has been removed from another of its saturated carbon atoms or from the radical carbon atom of an alkyl radical to form a diradical. In other aspects, the alkylene moiety is, or is further encompassed by, a divalent moiety derived by removing a hydrogen atom from a saturated carbon atom of a parent alkyl moiety, and is exemplified by, but not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and similar diradicals. Typically, alkylene is a divalent group having two radical centers (i.e., divalent) derived from a saturated carbon atom of a parent alkyl moiety. 3 It is a branched or straight chain hydrocarbon containing only carbon (i.e., fully saturated despite the radical carbon atom), and in some aspects is unsubstituted. In other aspects, the alkylene contains one or more internal unsaturated sites in the form of one or more double and / or triple bond functionalities (typically one or two such functionalities, more typically one), such that the terminal carbon of the unsaturated alkylene moiety is a monovalent sp 3 In yet another aspect, alkylene is substituted with 1 to 4, typically 1 to 3, or 1 or 2 substituents, as defined herein for optional substituents, on the saturated carbon atom(s) in a saturated alkylene moiety, or on the saturated and / or unsaturated carbon atom(s) in an unsaturated alkylene moiety, excluding alkyl, arylalkyl, alkenyl, alkynyl, and any other moiety where the resulting substituted alkylene differs in the number of consecutive non-aromatic carbon atoms compared to the unsubstituted alkylene (except when the optional substituents are basic units as defined herein).

[0084] "Carbocyclyl," as the term is used herein, alone or as part of another term, means, unless otherwise indicated or implied by context, a radical of a monocyclic, bicyclic, or tricyclic ring system in which each of the atoms forming the ring system (i.e., the skeletal atoms) is a carbon atom, and one or more of these carbon atoms in each ring of the cyclic ring system is saturated (i.e., one or more sp 3 Thus, a carbocyclyl is a cyclic arrangement of saturated carbons, but can also contain unsaturated carbon atom(s), and thus the carbocycle may be saturated or partially unsaturated or may be fused to an aromatic moiety, with the points of fusion between the cycloalkyl and the aromatic ring being adjacent unsaturated carbons in the carbocyclyl moiety and adjacent aromatic carbon atoms in the aromatic moiety.

[0085] Unless otherwise specified, a carbocyclyl may be substituted (i.e., optionally substituted) with moieties described for alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, etc., or may be substituted with another cycloalkyl moiety. Cycloalkyl moieties, groups, or substituents include cyclopropyl, cyclopentyl, cyclohexyl, adamantly, or other cyclic moieties having only carbon atoms in their cyclic ring system.

[0086] When carbocyclyl is used as a Markush group (i.e., a substituent), the carbocyclyl is attached to its attached Markush formula or another organic moiety through a carbon atom in the carbocyclic ring system of the carbocyclyl moiety, provided that the carbon is not an aromatic carbon. When an unsaturated carbon atom in the alkene moiety containing the carbocyclyl substituent is attached to its attached Markush formula, the carbocyclyl is sometimes referred to as a cycloalkenyl substituent. The number of carbon atoms in a carbocyclyl substituent is defined by the total number of skeletal atoms in the carbocyclic ring system. The number can vary and is typically in the range of 3 to 50, 1 to 30 or 1 to 20, and more typically in the range of 3 to 8 or 3 to 6 unless otherwise specified, for example, C3-C8 carbocyclyl means a carbocyclyl substituent, moiety or group containing 3, 4, 5, 6, 7 or 8 carbocyclic carbon atoms, and C3-C6 carbocyclyl means a carbocyclyl substituent, moiety or group containing 3, 4, 5 or 6 carbocyclic carbon atoms. A carbocyclyl can be derived by removing a hydrogen atom from a ring atom of a parent cycloalkane or cycloalkene. Representative C3-C8 carbocyclyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0087] Thus, a carbocyclyl substituent, moiety, or group typically has 3, 4, 5, 6, 7, or 8 carbon atoms in its carbocyclic ring system and may contain exo or endo ring double bonds or endo ring triple bonds, or a combination of both, which endo ring double or triple bonds, or a combination of both, do not form a 4n+2 electron conjugated ring system. Bicyclic ring systems can share two carbon atoms, and tricyclic ring systems can share a total of three or four carbon atoms. In some aspects, the carbocyclyl is a C3-C8 or C3-C6 carbocyclyl, which may be substituted (i.e., optionally substituted) with one or more, 1 to 4, typically 1 to 3, or 1 or 2 moieties described herein for alkyl, alkenyl, alkynyl, aryl, arylalkyl, and alkylaryl, and / or other moieties that include a substituent(s) as defined herein for optional substituents, and in some aspects, it is unsubstituted. In other aspects, the cycloalkyl moiety, group, or substituent is a C3-C6 cycloalkyl selected from the group consisting of cyclopropyl, cyclopentyl, and cyclohexyl, or a C3-C8 cycloalkyl that encompasses this group and other cyclic moieties having up to 8 carbon atoms in their cyclic ring system. If no number of carbon atoms is indicated, the carbocyclyl moiety, group, or substituent has 3 to 8 carbon atoms in its carbocyclic ring system.

[0088] "Carbocyclo," as that term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to an optionally substituted carbocyclyl, as defined above, in which another hydrogen atom of the cycloalkyl ring system has been removed (i.e., is divalent), and has a C3-C 50 or C3~C 30 Carbocyclo, typically C3-C 20 or C3~C 12carbocyclo, more typically C3-C8 or C3-C6 carbocyclo, and in some aspects unsubstituted or optionally substituted C3, C5 or C6 carbocyclo. If no number of carbon atoms is indicated, the carbocyclo moiety, group or substituent has from 3 to 8 carbon atoms in its carbocyclic ring system.

[0089] In some aspects, the other hydrogen atom is removed from the monovalent carbon atom of the cycloalkyl to provide a divalent carbon atom, which in some instances is a spiro carbon atom, interrupting the alkyl moiety with a carbocyclic carbon atom. In such instances, the spiro carbon atom results from the carbon atom index of the interrupting alkyl moiety and the carbocyclo ring system, and the carbocyclo is shown incorporated into the alkyl moiety. In these aspects, the carbocyclo moiety, group, or substituent is a C3-C6 carbocyclo in the form of a spiro ring system and is selected from the group consisting of cycloprop-1,1-diyl, cyclobutyl-1,1-diyl, cyclopenta-1,1-diyl, and cyclohexa-1,1-diyl, or a C3-C8 carbocyclo, which further includes other divalent cyclic moieties containing the group and having 8 or fewer carbon atoms in the cyclic ring system. The carbocyclo can be saturated or unsaturated and / or unsubstituted in the same manner as described for the carbocyclyl moiety. When unsaturated, one or both monovalent carbon atoms of the carbocyclo moiety may be bonded to sp 1 from the same or different double bond functional groups. 2 carbon atom, or both monovalent carbon atoms may be adjacent or non-adjacent sp 3 It may be a carbon atom.

[0090] "Alkenyl," as the term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to an organic moiety, substituent, or group that contains one or more double-bonded functional groups (e.g., a -CH=CH- moiety), or one, two, three, four, five, or six or more, typically one, two, or three, such functional groups, more typically one such functional group, and in some aspects may be substituted (i.e., optionally substituted) with an aryl moiety or group (e.g., a phenyl) or may include non-aromatically bonded normal, secondary, tertiary, or cyclic carbon atoms (i.e., straight-chain, branched-chain, cyclic, or any combination thereof) as part of the base moiety, unless the alkyl substituent, moiety, or group is a vinyl moiety (e.g., a -CH=CH2 moiety). Alkenyl moieties, groups, or substituents having multiple double bonds can have the double bonds positioned adjacently (i.e., 1,3 butadienyl moieties) or non-adjacently with one or more intervening saturated carbon atoms or combinations thereof, provided that the cyclic, adjacent positioning of the double bonds does not form a 4n+2 electron cyclic conjugated system (i.e., not aromatic).

[0091] The alkenyl moiety, group or substituent must have at least one sp 2 a carbon atom, wherein the carbon atom is divalent and is double-bonded to another organic moiety or Markush structure to which it is associated, or at least two sp 2 carbon atoms, where these sp 2 One of the carbon atoms is monovalent and is single-bonded to another organic moiety or Markush structure with which it is associated. Typically, when alkenyl is used as a Markush group (i.e., is a substituent), the alkenyl is bonded to the Markush formula or another organic moiety with which it is associated by the sp of the alkene functionality of the alkenyl moiety. 2In some aspects, when an alkenyl moiety is specified, the species includes those corresponding to any of the optionally substituted alkyl or carbocyclyl groups, moieties, or substituents described herein, which have one or more terminal double bonds, where the sp 2 The carbon atom is monovalent and the sp 2 A monovalent moiety derived by removing a hydrogen atom from carbon. Such monovalent moieties are exemplified by, but not limited to, vinyl (-CH=CH), allyl, 1-methylvinyl, butenyl, isobutenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, and cyclohexenyl. In some aspects, the term alkenyl refers to a group selected from the group consisting of sp 2 It includes these and / or other straight-chain, cyclic and branched-chain all-carbon moieties that contain at least one double bond functionality in which one of the carbon atoms is monovalent.

[0092] The number of carbon atoms in the alkenyl moiety is determined by the sp of the alkene functional group(s) that define it as an alkenyl substituent. 2 The number of carbon atoms, and the sp 2It is identified by the total number of consecutive non-aromatic carbon atoms attached to each carbon (not including carbon atoms from other moieties or Markush structures to which the alkenyl moiety is a variable, and any optional substituents on the alkenyl moiety), which ranges from 1 to 50 or 1 to 30, typically 1 to 20 or 1 to 12, more typically 1 to 8, 1 to 6, or 1 to 4 carbon atoms when the double-bonded functional group is double-bonded to the Markush structure (e.g., ═CH), or from 2 to 50, typically 2 to 30, 2 to 20, or 2 to 12, more typically 2 to 8, 2 to 6, or 2 to 4 carbon atoms when the double-bonded functional group is single-bonded to the Markush structure (e.g., —CH═CH). For example, C2-C8 alkenyl or C2-C8 alkenyl refers to a group containing 2, 3, 4, 5, 6, 7, or 8 carbon atoms, at least two of which are sp 2 carbon atoms, one of which is monovalent, and C2-C6 alkenyl or C2-C6 alkenyl means an alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms, at least two of which are conjugated with each other. 2 In some aspects, an alkenyl substituent or group has only two sp carbon atoms conjugated to each other. 2and in other aspects, the alkenyl moiety is unsubstituted or substituted with one to four or more, typically one to three, more typically one or two, independently selected moieties as disclosed herein, optionally including substituents as defined herein for substituents, and substituted alkenyl moieties exclude alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, and any other moiety when the number of consecutive non-aromatic carbon atoms is different compared to the unsubstituted alkenyl, where the substitution(s) is / are between consecutive sp 2 carbon and sp if present 3 It can occur at any of the carbon atoms. Typically, the alkenyl substituent has only two sp 2 An alkenyl moiety has carbons from C2 to C6 or C2 to C4. If the number of carbon atoms is not specified, the alkenyl moiety has from 2 to 8 carbon atoms.

[0093] "Alkenylene," as that term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to an alkyl group containing one or more double bond moieties as described above for alkenyl, and containing the same or two different sp's of an alkene functionality in a parent alkane. 2 An alkenylene moiety is an organic moiety, substituent, or group of the stated number of carbon atoms having two radical centers derived by the removal of two hydrogen atoms from a carbon atom or two hydrogen atoms from two separate alkene functional groups. In some aspects, an alkenylene moiety is a group consisting of the same or different sp 2 sp from carbon atoms or from different double bond moieties 2The alkenyl portion of an alkenyl radical as described herein, wherein a hydrogen atom has been removed from a carbon to provide a diradical. Typically, the alkenylene portion is -C=C- or -C=CX. 1 Diradicals containing the structure -C=C-, where X 1 is absent or is an optionally substituted saturated alkylene as defined herein, which is typically a C1-C6 alkylene, which is more typically unsubstituted. The number of carbon atoms in an alkenylene moiety is determined by the sp of its alkene functional group(s) that defines it as an alkenylene moiety. 2 The number of carbon atoms and their sp 2 The alkenyl moiety is identified by the total number of consecutive non-aromatic carbon atoms attached to each carbon (not including any carbon atoms in other moieties or Markush structures where the alkenyl moiety exists as a variable). This number, unless otherwise specified, ranges from 2 to 50 or 2 to 30, typically 2 to 20 or 2 to 12, more typically 2 to 8, 2 to 6, or 2 to 4 carbon atoms. For example, C2-C8 alkenylene or C2-C8 alkenylene refers to an alkylene group containing 2, 3, 4, 5, 6, 7, or 8 carbon atoms, at least two of which are conjugated to each other. 2 and C2-C6 alkenylene or C2-C6 alkenylene means an alkenylene moiety containing 2, 3, 4, 5, or 6 carbon atoms, at least two of which are conjugated to each other. 2 carbon, at least two of which are sp 2 In some aspects, the alkenylene moiety is a group of two sp carbons, one of which is divalent or both of which are monovalent. 2 carbon, and both sp 2C2-C6 or C2-C4 alkenylene, where the carbon atoms are monovalent, and in some aspects, unsubstituted. If the number of carbon atoms is not indicated, the alkenylene moiety has 2 to 8 carbon atoms and is unsubstituted or substituted in the same manner as described for alkenyl moieties.

[0094] "Alkynyl," as the term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to an organic moiety, substituent, or group that contains one or more triple bond functional groups (e.g., a -C≡C- moiety), or one, two, three, four, five, or six or more, typically one, two, or three, such functional groups, more typically one such functional group, and in some aspects may be substituted (i.e., optionally substituted) with an aryl moiety such as phenyl, or with an alkenyl moiety or attached normal, secondary, tertiary, or cyclic carbon atom (i.e., straight-chain, branched-chain, cyclic, or any combination thereof), unless the alkynyl substituent, moiety, or group is -C≡CH. Alkynyl moieties, groups, or substituents having multiple triple bonds can have the triple bonds positioned adjacently or non-adjacently with one or more intervening saturated or unsaturated carbon atoms or combinations thereof, provided that the cyclic, adjacent positioning of the triple bonds does not form a 4n+2 electron conjugated ring system (i.e., is not aromatic).

[0095] An alkynyl moiety, group, or substituent contains at least two sp carbon atoms, which are conjugated to each other, and one of the sp carbon atoms is single-bonded to another organic moiety or Markush structure with which it is associated. When alkynyl is used as a Markush group (i.e., a substituent), the alkynyl is single-bonded to the Markush formula or another organic moiety with which it is associated through the triple-bonded carbon (i.e., sp carbon) of the terminal alkyne functional group. In some aspects, when an alkynyl moiety, group, or substituent is specified, the species includes those corresponding to any of the optionally substituted alkyl or carbocyclyl groups, moieties, or substituents described herein, which have one or more endo-triple bonds and are monovalent moieties derived by removing a hydrogen atom from an sp carbon of a parent alkyne compound. Such monovalent moieties are exemplified by, but not limited to, -C≡CH, -C≡C-CH3, and -C≡C-Ph.

[0096] The number of carbon atoms in an alkynyl substituent is identified by the number of sp carbon atoms in the alkene functional group that defines it as an alkynyl substituent and the total number of consecutive non-aromatic carbon atoms bonded to each of these sp carbons (not including carbon atoms in other moieties or carbon atoms of Markush structures in which the alkenyl moiety is a variable). This number can range from 2 to 50, typically 2 to 30, 2 to 20, or 2 to 12, more typically 2 to 8, 2 to 6, or 2 to 4 carbon atoms (when a triple bond functionality is single-bonded to a Markush structure (e.g., -CH≡CH)). For example, C2-C8 alkynyl or C2-C8 alkynyl refers to an alkynyl moiety containing 2, 3, 4, 5, 6, 7, or 8 carbon atoms, at least two of which are sp carbon atoms conjugated to each other, and one of these carbon atoms is monovalent, and C2-C6 alkynyl or C2-C6 alkynyl refers to an alkynyl moiety containing 2, 3, 4, 5, or 6 carbon atoms, at least two of which are sp carbon atoms conjugated to each other, and one of these carbon atoms is monovalent. In some aspects, an alkynyl substituent or group is a C2-C6 or C2-C4 alkynyl moiety having two sp carbon atoms conjugated to each other, and one of these carbon atoms is monovalent, and in other aspects, the alkynyl moiety is unsubstituted. If the number of carbon atoms is not indicated, the alkynyl moiety, group, or substituent has from 2 to 8 carbon atoms. Alkynyl moieties are substituted or unsubstituted in the same manner as described for alkenyl moieties, except that substitution at monovalent sp carbons is not allowed.

[0097] "Aryl," as the term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to an organic moiety, substituent, or group having an aromatic or fused aromatic ring system with no ring heteroatoms, comprising or consisting of 1, 2, 3, or 4-6 aromatic rings, each of which is independently optionally substituted, typically consisting of 1-3 aromatic rings, more typically 1-2 aromatic rings, each of which is independently optionally substituted, and in which the rings consist only of carbon atoms participating in a cyclically conjugated system of 4n+2 electrons (Hückel's rule), typically 6, 10, or 14 electrons, some of which may further participate in exocyclic conjugation with heteroatoms (cross-conjugation, e.g., quinones). Aryl substituents, moieties or groups are typically formed by 6, 8, 10 or more consecutive aromatic carbon atoms up to 24 consecutive aromatic carbon atoms, C6-C 24 aryl, and in some aspects C-C 20 or C6 to C 12 Aryl substituents, moieties, or groups are optionally substituted, and in some aspects are unsubstituted or substituted with one, two, three, or more, typically one or two, independently selected substituents as defined herein for alkyl, alkenyl, alkynyl, or other moieties described herein (including another aryl or heteroaryl), to form biaryl and other optional substituents as defined herein. In other aspects, aryl is a C6-C 10Aryl, for example, phenyl, naphthalenyl, and phenanthryl. Because the aromaticity of a neutral aryl moiety requires an even number of electrons, it is understood that the ranges given for the moiety do not encompass species having an odd number of aromatic carbons. When aryl is used as a Markush group (i.e., a substituent), the aryl is attached to the Markush formula or another organic moiety to which it is attached via an aromatic carbon of the aryl group.

[0098] "Heterocyclyl," as this term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to a carbocyclyl in which one or more, but not all, skeletal carbon atoms, together with the hydrogen atoms attached thereto in its carbocyclic ring system, are replaced by independently selected heteroatoms or heteroatom moieties (optionally substituted where permissible, including but not limited to N / NH, O, S, Se, B, Si, and P), wherein two or more, typically two, heteroatoms or heteroatom moieties may be adjacent to each other or separated by one or more carbon atoms, typically one to three carbon atoms, in the same ring system. These heteroatoms or heteroatom moieties are typically N / NH, O, and S. A heterocyclyl typically contains a monovalent skeletal carbon atom or monovalent heteroatom or heteroatom moiety, and contains a total of 1 to 10 heteroatoms and / or heteroatom moieties, typically a total of 1 to 5, or more typically a total of 1 to 3, or 1 or 2 heteroatoms and / or heteroatom moieties, with the proviso that not all of these skeletal atoms are heteroatoms and / or heteroatom moieties in any one of the heterocyclic ring(s) in the heterocyclyl (i.e., at least one carbon atom is not replaced in each ring with at least one that is replaced in one of the rings), and wherein each optionally substituted heteroatom or heteroatom moiety, where permitted, in the ring(s) is independently selected from the group consisting of N / NH, O, and S, with the proviso that no one ring contains two adjacent O or S atoms.Exemplary heterocyclyls and heteroaryls, collectively referred to as heterocycles, are provided in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 1960, 82:5545-5473, especially 5566-5573).

[0099] When heterocyclyl is used as a Markush group (i.e., a substituent), the saturated or partially unsaturated heterocyclic ring of the heterocyclyl is attached to the associated Markush structure or other moiety through a carbon atom or heteroatom of the heterocyclic ring, and such attachment does not result in an unstable or disallowed formal oxidation state of the carbon atom or heteroatom. Heterocyclyl in that context is a monovalent moiety in which the heterocyclic ring of the heterocyclic ring system defining it as heterocyclyl is non-aromatic but may be fused to a carbocyclic ring, an aryl ring, or a heteroaryl ring, and includes phenyl (i.e., benzo)-fused heterocyclic moieties.

[0100] Heterocyclyl refers to a C3-C cycloalkyl ring system in which one, two, or three or more, but not all, carbons of the cycloalkyl ring system have been replaced, together with typically one, two, three, or four, more typically one or two, of the attached hydrogens, by a heteroatom or heteroatom moiety (optionally substituted where permitted) independently selected from the group consisting of N / NH, O, and S. 50 or C3~C 30 Carbocyclyl, typically C3-C 20 or C3~C 12carbocyclyl, more typically C3-C8 or C3-C6 carbocyclyl, and thus 50 or C3~C 30 Heterocyclyl, typically C3-C 20 or C3~C 12 Heterocyclyl is a heterocyclyl of the formula (I), more typically a C3-C6 or C5-C6 heterocyclyl, where the subscript indicates the total number of skeletal atoms (including the carbon atoms and heteroatoms) in the heterocyclic ring system(s) of the heterocyclyl. In some aspects, a heterocyclyl contains 0-2 N, 0-2 O, or 0-1 S skeletal heteroatoms, or any combination thereof, optionally substituted, provided that at least one of the heteroatoms is present in the heterocyclic ring system of the heterocyclyl. A heterocyclyl may be saturated or unsaturated and / or unsubstituted, substituted with oxo (=O) moieties at a skeletal carbon atom (as in pyrrolidin-2-one) and / or with one or two oxo moieties at a skeletal heteroatom, and may contain oxidized heteroatoms, as exemplified by, but not limited to, -N(=O), -S(=O)-, or -S(=O)2-. A fully saturated or partially unsaturated heterocyclyl may be substituted or further substituted with alkyl, (hetero)aryl, (hetero)arylalkyl, alkenyl, alkynyl, or other moiety described herein (including optional substituents as defined herein), or a combination of two, three, or more, typically one or two, such substituents. In certain aspects, the heterocyclyl is selected from the group consisting of pyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl.

[0101] "Heterocyclo," as this term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, refers to a heterocyclyl moiety, group, or substituent, as defined above, in which a hydrogen atom from its monovalent carbon atom, a hydrogen atom from a different skeletal atom (carbon atom or, if present, nitrogen atom), or an electron (if permitted) from a skeletal nitrogen atom has been removed, or an electron from a nitrogen ring atom that is no longer monovalent has been removed and replaced with a bond (i.e., it is divalent). In some aspects, the replaced second hydrogen is a hydrogen from a monovalent carbon atom of the parent heterocyclyl, thus forming a spiro carbon atom. This may, in some instances, disrupt an alkyl moiety at the carbocyclic carbon atom. In such instances, the spiro carbon atom contributes to the carbon atom count of the disrupted alkyl moiety, and the heterocyclo is shown to be incorporated into the alkyl moiety.

[0102] "Heteroaryl," as used herein by itself or as part of another term, refers, unless otherwise indicated or implied by context, to an aryl moiety, aryl group, or aryl substituent, as defined herein, in which one or more, but not all, aromatic carbons of the aryl's aromatic ring system are replaced by heteroatoms. A heteroaryl typically contains a total of 1 to 4 skeletal heteroatoms in the ring(s) of its heteroaryl ring system, provided that not all skeletal atoms of any one ring system in the heteroaryl are optionally substituted heteroatoms where permitted, with 0 to 3 N skeletal heteroatoms, 1 to 3 N skeletal heteroatoms, or 0 to 3 N skeletal heteroatoms, typically 0 to 1 O skeletal heteroatom and / or 0 to 1 S skeletal heteroatom, provided that at least one skeletal heteroatom is present. A heteroaryl can be monocyclic, bicyclic, or polycyclic. A polycyclic heteroaryl is typically a C5-C6 heterocyclic ring. 50 or C5~C 30 Heteroaryl, more typically C5-C 20 or C5~C12 Heteroaryl, bicyclic heteroaryl is typically C5-C 10 Heteroaryl, and monocyclic heteroaryl is typically C5-C6 heteroaryl, where the subscript indicates the total number of skeletal atoms (including the carbon atoms and heteroatoms) in the aromatic ring system of the heteroaryl. In some aspects, heteroaryl is a bicyclic aryl moiety in which 1, 2, 3, 4 or more, typically 1, 2 or 3, of one of the carbon atoms and their attached hydrogen atoms in the aromatic ring(s) of the parent bicyclic aryl moiety are replaced with independently selected heteroatoms or heteroatom moieties, or in which 1, 2, 3 or more, typically 1 or 2, of one of the carbon atoms and their attached hydrogen atoms in the aromatic ring(s) of the parent monocyclic aryl moiety are optionally replaced, where permitted. and a monocyclic aryl moiety replaced by an independently selected heteroatom or heteroatom moiety, where the heteroatom or heteroatom moiety is optionally substituted where permissible, and includes N / NH, O, and S, with the proviso that not all skeletal atoms of any one aromatic ring system in the parent aryl moiety are replaced by heteroatoms, more typically oxygen (—O—), sulfur (—S—), nitrogen (═N—), or —NR— (such that the nitrogen heteroatom is optionally substituted), where R is —H, a nitrogen protecting group, or an optionally substituted C-C heteroatom. 20 C6-C alkyl) or optionally substituted 24 Aryl or C5-C 24It is replaced by a heteroaryl to form a heterobiaryl. In another aspect, one, two, or three of the carbon atoms of the aromatic ring(s) of the parent aryl moiety and the hydrogen atoms bonded thereto are replaced by nitrogen substituted with another organic moiety in a manner that maintains the cyclic conjugation system. In yet another aspect, the aromatic carbon radical of the parent aryl moiety is replaced by an aromatic nitrogen radical. In any of these aspects, the nitrogen, sulfur, or oxygen heteroatom participates in the conjugation system either through a π bond with an adjacent atom in the ring system or a lone electron pair on the heteroatom. In yet another aspect, the heteroaryl has the structure of a heterocyclyl, as defined herein, whose ring system is aromatized.

[0103] Typically, heteroaryl is monocyclic with a 5- or 6-membered aromatic heterocyclic ring system in some aspects. Five-membered heteroaryl is a monocyclic C5 heteroaryl containing 1 to 4 aromatic carbon atoms and the required number of aromatic heteroatoms in its aromatic heterocyclic ring system. Six-membered heteroaryl is a monocyclic C6 heteroaryl containing 1 to 5 aromatic carbon atoms and the required number of aromatic heteroatoms in its aromatic heterocyclic ring system. Five-membered heteroaryls have 4, 3, 2, or 1 aromatic heteroatom, and six-membered heteroaryls include heteroaryls having 5, 4, 3, 2, or 1 aromatic heteroatom.

[0104] C5 heteroaryl, also referred to as 5-membered heteroaryl, is a monovalent moiety derived, where permitted, by removing a hydrogen atom from a skeletal aromatic carbon or an electron from a skeletal aromatic heteroatom of a parent aromatic heterocycle, which in some aspects is selected from the group consisting of pyrrole, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, triazole, and tetrazole. In other aspects, the parent heterocycle is selected from the group consisting of thiazole, imidazole, oxazole, and triazole, and is typically thiazole or oxazole, more typically thiazole.

[0105] A 6-membered C6 heteroaryl is a monovalent moiety obtained, where permitted, by removing a hydrogen atom from an aromatic carbon or an electron from an aromatic heteroatom of a parent aromatic heterocycle, which in certain aspects is selected from the group consisting of pyridine, pyridazine, pyrimidine, and triazine. The heteroaryl may be substituted or further substituted with alkyl, (hetero)arylalkyl, alkenyl, or alkynyl, or with an aryl or another heteroaryl to form a biaryl, or with other moieties as described herein (including optional substituents as defined herein), or with a combination of two, three, or more, typically one or two, such substituents.

[0106] "Arylalkyl" or "heteroarylalkyl," as the terms are used herein, alone or as part of another term, refers to an aryl or heteroaryl moiety bound to an alkyl moiety (i.e., (aryl)-alkyl-), where the alkyl and aryl groups are as described above. Typically, arylalkyl is an alkyl group selected from the group consisting of (C6-C 24 Aryl)-C1~C 12 Alkyl- is a moiety, group or substituent, and heteroarylalkyl is (C-C24 Heteroaryl)-C1-C 12 When a (hetero)arylalkyl is used as a Markush group (i.e., a substituent), the alkyl portion of the (hetero)arylalkyl is the sp 3 In some aspects, arylalkyl is a (C6-C 24 Aryl)-C1~C 12 Alkyl- or (C6-C 20 Aryl)-C1~C 20 Alkyl-, typically (C6-C 12 Aryl)-C1~C 12 Alkyl- or (C6-C 10 Aryl)-C1~C 12 Alkyl-, more typically (C6-C), exemplified by, but not limited to, C6H5-CH2-, C6H5-CH(CH3)CH2-, and C6H5-CH2-CH(CH2CH2CH3)- 10 (hetero)arylalkyl- may be unsubstituted or substituted in the same manner as described for the (hetero)aryl and / or alkyl moieties.

[0107] An "arylene" or "heteroarylene," as the term is used herein, alone or as part of another term, unless otherwise indicated or implied by context, is an aromatic or heteroaromatic diradical moiety that forms two covalent bonds (i.e., is divalent) within another organic moiety, and the bonds are in the ortho, meta, or para configuration. Arylene and some heteroarylenes comprise divalent species formed by the removal of hydrogen atoms from a parent aryl or heteroaryl moiety, group, or substituent, as defined herein. Other heteroarylenes are divalent species formed by the removal of hydrogen atoms from two different aromatic carbon atoms of a parent heteroaromatic ring to form the diradical species, or by the removal of a hydrogen atom from an aromatic carbon atom or heteroatom and another hydrogen atom or electron from a different aromatic heteroatom of a parent heteroaromatic ring to form the diradical species (where one aromatic carbon atom and one aromatic heteroatom are monovalent, or where two different aromatic heteroatoms are each monovalent). Heteroarylene further includes heteroatom(s) and / or heteroatom moiety(s) replacing one or more, but not all, of the aromatic carbon atoms of the parent arylene.

[0108] Non-limiting exemplary arylenes, optionally substituted at the remaining positions, have the following structure: [ka] These are phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene, as shown in

[0109] "Heteroalkyl," as used herein alone or in combination with another term, unless otherwise indicated or implied by context, refers to an optionally substituted, straight- or branched-chain hydrocarbon that is fully saturated or contains 1 to 3 degrees of unsaturation and has 1 to 12 carbon atoms and 1 to 6 heteroatoms, typically 1 to 5 heteroatoms, more typically 1 or 2 heteroatoms or heteroatom moieties (selected from the group consisting of O, N / NH, Si, and S, optionally substituted where permitted, and including each nitrogen and sulfur atom independently optionally oxidized to N-oxide, sulfoxide, or sulfone, or one or more of the nitrogen atoms optionally substituted or quaternized). The heteroatom(s) or heteroatom moieties O, N / NH, S, and / or Si can be located at any interior position of the heteroalkyl group or at the terminal position of the optionally substituted alkyl group of the heteroalkyl. In some aspects, heteroalkyl is fully saturated or contains one degree of unsaturation and contains 1 to 6 carbon atoms and 1 to 2 heteroatoms, and in other aspects, the heteroalkyl is unsubstituted. Non-limiting examples are -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, as exemplified by -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0110] Heteroalkyl is typically represented by the number of its consecutive heteroatom(s) and non-aromatic carbon atoms, including the consecutive carbon atom(s) bonded to those heteroatom(s), unless otherwise indicated or indicated by context (e.g., as described for aminoalkyl). Thus, -CH2-CH2-O-CH3 and -CH2-CH2-S(O)-CH3 are both C4-heteroalkyl, and -CH2-CH=NO-CH3 and -CH=CH-N(CH3)2 are both C5-heteroalkyl. A heteroalkyl can be unsubstituted or substituted (i.e., optionally substituted) at its heteroatom or heteroatom component with any one of the moieties described herein (including optional substituents as defined herein), and / or at its alkyl component with 1 to 4 or more, typically 1 to 3 or 1 or 2, independently selected moieties described herein (including optional substituent(s) as defined herein, and excluding an alkyl, (hetero)arylalkyl, alkenyl, alkynyl, another heteroalkyl, or any other moiety where a substituted alkenyl has a different number of consecutive non-aromatic carbon atoms than an unsubstituted aminoalkyl).

[0111] An aminoalkyl, as defined herein, is an exemplary heteroalkyl in which a terminal carbon atom of the alkyl moiety other than the monovalent carbon atom is replaced by an amino group. When shown as a substituent on a Markush structure or other related organic moiety, the monovalent carbon atom of the alkyl moiety is bonded to the other related organic moiety, typically a different carbon atom than that bonded to the amino group. An aminoalkyl differs from other heteroalkyls only by the numbering designation indicating the number of consecutive carbon atoms in the alkylene portion.

[0112] "Heteroalkylene," as this term is used herein alone or in combination with another term, unless otherwise indicated or implied by context, means a divalent group derived from a heteroalkyl (as discussed above) by removing a hydrogen atom or a heteroatom electron from a parent heteroalkyl to provide a divalent moiety exemplified, but not limited to, by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. In the case of heteroalkylene, the heteroatom(s) can be internal to the optionally substituted alkylene chain or can occupy one or both termini of the alkylene chain, such that one or both of the heteroatoms are monovalent. When heteroalkylene is a component of a linker unit, both orientations of the component within the linker unit are permitted unless indicated or implied by context. A heteroalkylene is typically represented by the number of its consecutive heteroatom(s) and non-aromatic carbon atoms, including the consecutive carbon atom(s) bonded to those heteroatom(s), unless otherwise indicated or indicated by context. Alkylene diamines are heteroalkylenes in which two monovalent carbon atoms of the alkylene are replaced by amino groups, and thus each of their nitrogen atoms is monovalent, and differ from other heteroalkylenes only by the numbering description indicating the number of consecutive carbon atoms of the alkylene portion.

[0113] "Aminoalkyl," as the term is used herein alone or in combination with another term, unless otherwise indicated or implied by context, refers to an optionally substituted C1-C amine, as described above, to provide a primary amine (not further substituted at the basic nitrogen), or a secondary or tertiary amine (where the basic amine is one or two independently selected C1-C amines, respectively). 12"Aminoalkyl" refers to a moiety, group, or substituent having a basic nitrogen attached to one radical terminus of an alkylene moiety, as defined above, so as to provide a group (further substituted with an alkyl moiety). In some aspects, the optionally substituted alkyl is a C1-C8 alkyl or a C1-C6 alkyl, and in other aspects, the alkyl is unsubstituted. In still other aspects, the basic nitrogen, together with the substituents, defines an optionally substituted C3-C8 heterocyclyl containing the basic nitrogen as a backbone atom, typically in the form of a nitrogen-containing C3-C6 or C5-C6 heterocyclyl (optionally substituted). When aminoalkyl is used as a variable to a Markush structure, the alkylene portion of the aminoalkyl is the sp of the moiety. 3 The alkylene group is bonded to the associated Markush carbon atom (which in some aspects is the other radical terminus of the alkylene group). Aminoalkyls are typically represented by the number of consecutive carbon atoms in the alkylene portion. Thus, C1 aminoalkyls are exemplified by, but not limited to, -CH2NH2, -CH2NHCH3, and -CH2N(CH3)2, and C2 aminoalkyls are exemplified by -CH2CH2NH2, -CH2CH2NHCH3, and -CH2CH2N(CH3)2.

[0114] As used herein, unless otherwise indicated or implied by context, terms such as "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted arylalkyl," "optionally substituted heterocycle," "optionally substituted aryl," "optionally substituted heteroaryl," "optionally substituted heteroarylalkyl," and the like refer to an alkyl, alkenyl, alkynyl, arylalkyl, heterocycle, aryl, heteroaryl, heteroarylalkyl, or other substituent, moiety, or group as defined or disclosed herein, in which a hydrogen atom(s) of the substituent, moiety, or group is / are optionally replaced with a different moiety(s) or group(s), or an alicyclic carbon chain comprising one of these substituents, moieties, or groups is / are interrupted by the replacement of a carbon atom(s) of the chain with a different moiety(s) or group(s). In some aspects, an alkene functional group is a group consisting of two consecutive sp 3 Carbon atoms are replaced except for the radical carbon of the alkyl portion, making the optionally substituted alkyl an unsaturated alkyl substituent.

[0115] The optional substituents replacing hydrogen in any one of the foregoing substituents, moieties or groups are independently C6 to C 24 Aryl, C5-C 24 Heteroaryl, Hydroxyl, C1-C 20 Alkoxy, C6-C 24 Aryloxy, cyano, halogen, nitro, C1-C 20 fluoroalkoxy and amino (including mono-, di-, and tri-substituted amino groups and protected derivatives thereof), or -X, -OR', -SR', -NH, -N(R')(R op ), -N(R op )3, =NR', -CX3, -CN, -NO2, -NR'C(=O)H, -NR'C(=O)Rop , -NR'C(=O)R op , -C(=O)R', -C(=O)NH2, -C(=O)N(R')R op , -S(=O)2R op , -S(=O)2NH2, -S(=O)2N(R')R op , -S(=O)2NH2, -S(=O)2N(R')R op , -S(=O)2OR', -S(=O)R op ,-OP(=O)(OR')(OR op ), -OP(OH)3, -P(=O)(OR')(OR op ), -PO3H2, -C(=O)R', -C(=S)R op , -CO2R', -C(=S)OR op , -C(=O)SR', -C(=S)SR', -C(=S)NH2, -C(=S)N(R')(R op )2, -C(=NR')NH2, -C(=NR')N(R')R op and salts thereof, wherein each X is independently selected from the group consisting of halogen: -F, -Cl, -Br, and -I; op are independently C1 to C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C6-C 24 Aryl, C3-C 24 Heterocyclyl, C5-C 24 a heteroaryl, a protecting group, and a prodrug moiety, or two R op These, together with the heteroatoms to which they are bonded, form C3 to C 24 R' is hydrogen or R op where R op is C1~C 20 Alkyl, C6-C 24 Aryl, C3-C 24 Heterocyclyl, C5-C 24 It is selected from the group consisting of heteroaryl and a protecting group.

[0116] Typically, the optional substituents present are -X, -OH, -ORop , -SH, -SR op , -NH2, -NH(R op ), -NR'(R op )2, -N(R op )3, =NH, =NR op , -CX3, -CN, -NO2, -NR'C(=O)H, NR'C(=O)R op , -CO2H, -C(=O)H, -C(=O)R op , -C(=O)NH2, -C(=O)NR'R op , -S(=O)2R op , -S(=O)2NH2, -S(=O)2N(R')R op , -S(=O)2NH2, -S(=O)2N(R')(R op ), -S(=O)2OR', -S(=O)R op , -C(=S)R op , -C(=S)NH2, -C(=S)N(R')R op , -C(=NR')N(R op )2 and salts thereof, wherein each X is independently selected from the group consisting of -F and -Cl, and wherein R op is typically C1-C6 alkyl, C6-C 10 Aryl, C3-C 10 Heterocyclyl, C5-C 10 R' is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C3-C 10 Heterocyclyl, C5-C 10 heteroaryl and protecting group (independently R op The compound is typically selected from the group consisting of:

[0117] More typically, the optional substituents present are -X, -R op , -OH, -OR op , -NH2, -NH(R op ), -N(R op )2, -N(R op )3, -CX3, -NO2, -NHC(=O)H, -NHC(=O)R op, -C(=O)NH2, -C(=O)NHR op , -C(=O)N(R op )2, -CO2H, -CO2R op , -C(=O)H, -C(=O)R op , -C(=O)NH2, -C(=O)NH(R op ), -C(=O)N(R op )2, -C(=NR')NH2, -C(=NR')NH(R op ), -C(=NR')N(R op ) 2, protecting groups and salts thereof, wherein each X is -F and R op are independently C1-C6 alkyl, C6-C 10 Aryl, C5-C 10 R' is selected from the group consisting of hydrogen, C1-C6 alkyl, and a protecting group (independently R op (selected from the group consisting of)

[0118] In some aspects, the optional alkyl substituents present are -NH, -NH(R op ), -N(R op )2, -N(R op )3, -C(=NR')NH2, -C(=NR')NH(R op ) and -C(=NR')N(R op )2, wherein R′ and R op is R' or R op In some of these aspects, R' and / or R op The substituents, together with the nitrogen atom to which they are attached, form R opare independently selected from the group consisting of hydrogen and C1-C6 alkyl, providing the basic functionality of the basic unit (BU). The alkylene, carbocyclyl, carbocyclo, aryl, arylene, heteroalkyl, heteroalkylene, heterocyclyl, heterocyclo, heteroaryl, and heteroarylene groups, as described above, are similarly substituted or unsubstituted, except as described in the definitions of these moieties.

[0119] Other optional substituents may replace carbon atoms in the acyclic carbon chain of an alkyl or alkylene moiety, group or substituent, and may be C3-C 12 Heteroalkyl or C3-C 12 Heteroalkylene is provided, and for this purpose is typically selected from the group consisting of optionally substituted -O-, -C(=O)-, -C(=O)O-, -S-, -S(=O)-, -S(=O)2-, -NH-, -NHC(=O)-, -C(=O)NH-, S(=O)2NH-, -NHS(=O)2-, -OC(=O)NH-, and -NHC(=O)O, where -NH- is an optionally substituted heteroatom moiety by replacing its hydrogen atom with a substituent independently selected from the group described above for the optional -NH- substituent.

[0120] The term "optionally substituted heteroatom," as used herein alone or in combination with other terms, unless otherwise indicated or implied by context, refers to a heteroatom or heteroatom moiety within a functional group or other organic moiety that is either not further substituted or is substituted with any one of the above moieties having a monovalent carbon atom (including, but not limited to, alkyl, cycloalkyl, alkenyl, aryl, heterocyclyl, heteroaryl, heteroalkyl, and (hetero)arylalkyl-) or is oxidized by substitution with one or two =0 substituents. In some aspects, "optionally substituted heteroatom" refers to an aromatic or non-aromatic -NH- moiety that is unsubstituted or has a hydrogen atom replaced with any one of the above substituents. In other aspects, "optionally substituted heteroatom" refers to the aromatic backbone nitrogen atom of a heteroaryl, where the heteroatom's electron has been replaced with any one of the above substituents. To encompass both of these aspects, the nitrogen heteroatom is sometimes referred to as optionally substituted N / NH.

[0121] Thus, in some aspects, the optional substituents on the nitrogen atom are C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C6-C 24 Aryl, C5-C 24 Heteroaryl, (C6-C 24 Aryl)-C1~C 20 Alkyl-, and (C5-C 24 Heteroaryl)-C1-C 20 In another aspect, the optional substituents present on the nitrogen atom are independently selected from the group consisting of C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C6-C24 Aryl, C5-C 24 Heteroaryl, (C6-C 24 Aryl)-C1~C 12 Alkyl-, and (C5-C 24 Heteroaryl)-C1-C 12 alkyl-(optionally substituted), or C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, (C6-C 10 aryl)-C1-C8 alkyl-, and (C5-C 10 heteroaryl)-C1-C8 alkyl, or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, (C6-C 10 aryl)-C1-C6 alkyl-, and (C5-C 10 heteroaryl)-C1-C6 alkyl-.

[0122] When an optionally substituted nitrogen atom is the point of covalent attachment of a peptide cleavable unit to the PAB or PAB-type portion of a self-immolative spacer unit, sometimes referred to as J, the optional substituents on that nitrogen atom, if present, must be monovalent sp , which does not adversely affect the electron-donating ability of the nitrogen atom compared to the unsubstituted nitrogen atom. 3 The carbon atom is limited to that attached, and upon cleavage of the cleavable unit, its electron-donating ability is restored, thereby allowing self-immolation to release the drug unit as free drug.

[0123] "O-linked moiety," as that term is used herein alone or in combination with other terms, refers to a moiety, group, or substituent that is attached to a Markush structure or another organic moiety that is related directly through the oxygen atom of the O-linked moiety, unless otherwise indicated or implied by context. A monovalent O-linked moiety has its attachment through a monovalent oxygen atom and is typically represented by -OH, -OC(=O)R b (acyloxy) (where R b -H, optionally substituted saturated C1-C 20 Alkyl, optionally substituted unsaturated C1-C 20 Alkyl, optionally substituted C3-C 20 Cycloalkyl (wherein the cycloalkyl moiety is saturated or partially unsaturated), optionally substituted C3-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl, optionally substituted C6-C 24 Aryl, optionally substituted C5-C 24 Heteroaryl or optionally substituted C3-C 24 heterocyclyl or R b is C1 to C, substituted as necessary 12 Alkyl, optionally substituted C3-C 12 Cycloalkyl, optionally substituted C3-C 12 Alkenyl or optionally substituted C2-C 12 alkynyl), and the monovalent O-linked moiety is an optionally substituted C1-C 12 Ether groups that are alkyloxy (i.e., C1-C 12 It further includes an aliphatic ether) moiety, where the alkyl portion is saturated or unsaturated.

[0124] In other aspects, the monovalent O-linked moiety is selected from the group consisting of optionally substituted phenoxy, optionally substituted C1-C8 alkyloxy (i.e., C1-C8 aliphatic ethers), and —OC(═O)R b where R is a monovalent moiety selected from the group consisting ofb is typically a saturated optionally substituted C1-C8 alkyl or an optionally substituted unsaturated C3-C8 alkyl.

[0125] In yet another aspect, the O-linked moiety is selected from the group consisting of -OH, saturated C1-C6 alkyl ethers, unsaturated C3-C6 alkyl ethers (optionally substituted), and -OC(=O)R b (where R b is typically a monovalent moiety selected from the group consisting of C1-C6 saturated alkyl, C3-C6 unsaturated alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, or phenyl (optionally substituted), or is selected from the group excluding —OH and / or phenyl, or R b is a monovalent moiety selected from the group consisting of optionally substituted C1-C6 saturated alkyl, C3-C6 unsaturated alkyl, and C2-C6 alkenyl, or the monovalent O-linked moiety is selected from the group consisting of saturated C1-C6 alkyl ether, unsaturated C3-C6 alkyl ether, and -OC(=O)R b (where R b is an unsubstituted O-linked substituent selected from the group consisting of unsubstituted saturated C1-C6 alkyl or unsubstituted unsaturated C3-C6 alkyl.

[0126] Other exemplary O-linked substituents are provided by the definitions for carbamates, ethers, and carbonates as disclosed herein, where the monovalent oxygen atom of the carbamate, ether, or carbonate functional group is bonded to the Markush structure or other organic moiety with which it is associated.

[0127] In other aspects, the O-linked moiety to the carbon is divalent and includes ═O and —X—(CH) n -Y-, where X and Y are independently S and O, and the subscript n is 2 or 3, forming a spiro ring system with the carbon to which both X and Y are attached.

[0128] "Halogen," as used herein by itself or in combination with other terms, refers to fluorine, chlorine, bromine, or iodine, unless otherwise indicated or implied by context, and is typically -F or -Cl.

[0129] "Protecting group," as the term is used herein alone or in combination with other terms, refers to a moiety that prevents or substantially reduces the ability of the atom or functional group to which it is attached to participate in undesired reactions, unless otherwise indicated or implied by context. Typical protecting groups for atoms or functional groups are provided in Greene (1999), "Protective groups in organic synthesis, 3rd ed.", Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur, and nitrogen are sometimes used to minimize or prevent these undesired reactions with electrophilic compounds. At other times, protecting groups are used to reduce or eliminate the nucleophilicity and / or basicity of the unprotected heteroatom. Non-limiting examples of protected oxygens include -OR, ... PR (In the formula, R PRis a protecting group for hydroxyl), which is typically protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for hydroxyl avoid interference with the nucleophilicity of organometallic or other highly basic reagents, and for this purpose, hydroxyl is typically protected as an ether, including, but not limited to, alkyl or heterocyclyl ethers (e.g., methyl or tetrahydropyranyl ethers), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ethers), optionally substituted aryl ethers, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include -NHR PR or -N(R PR )2(wherein, R PR at least one of R is a nitrogen atom protecting group, or both R PR together define a nitrogen atom protecting group), for primary or secondary amines.

[0130] A protecting group is suitable for a protecting group if it can prevent or substantially prevent undesired side reactions and / or premature loss of the protecting group under the reaction conditions required to effect the desired chemical transformation elsewhere in the molecule and, if desired, during purification of the newly formed molecule, and can be removed under conditions that do not adversely affect the structural or stereochemical integrity of the newly formed molecule. In some aspects, suitable protecting groups are those previously described for protecting functional groups. In other aspects, suitable protecting groups are those used in peptide coupling reactions. For example, a suitable protecting group for the basic nitrogen atom of an acyclic or cyclic basic unit is an acid-labile carbamate protecting group, such as t-butyloxycarbonyl (BOC).

[0131] "Ester," as that term is used herein alone or in combination with other terms, unless otherwise indicated or implied by context, refers to a substituent, moiety, or group having the structure -C(=O)-O-, which defines an ester functionality, wherein the carbonyl carbon atom of the structure is not directly connected to another heteroatom, but is directly connected to a hydrogen or another carbon atom of the organic moiety with which it is associated, and the monovalent oxygen atom is bonded to the same organic moiety at a different carbon atom to provide a lactone, or to a Markush structure or some other organic moiety. Typically, the ester comprises or consists of an organic moiety containing, in addition to the ester functional group, 1 to 50 carbon atoms, typically 1 to 20 carbon atoms, or more typically 1 to 8, 1 to 6, or 1 to 4 carbon atoms, and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si, but typically O, S, and N), typically 0 to 2 heteroatoms, where the organic moiety is attached to a -C(=O)-O- structure (i.e., via the ester functional group) to provide a structure having the formula organic moiety -C(=O)-O- or -C(=O)-O-organic moiety.

[0132] When an ester is a substituent or variable of a Markush structure or other organic moiety with which it is associated, the substituent is attached to that structure or other organic moiety through the monovalent oxygen atom of the ester functional group, and is thus a monovalent O-linked substituent, which is sometimes referred to as acyloxy. In such cases, the organic moiety attached to the carbonyl carbon of the ester functional group is typically a C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C6-C 24 Aryl, C5-C 24 Heteroaryl, C3-C 24 heterocyclyl or a substituted derivative of any one of these, for example having 1, 2, 3 or 4 substituents, more typically C1-C 12 Alkyl, C2-C12 Alkenyl, C2-C 12 Alkynyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C3-C 10 heterocyclyl, or a substituted derivative of any one of these, e.g., with 1, 2 or 3 substituents; or C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, or phenyl, or a substituted derivative of any one of these, e.g., with 1 or 2 substituents, wherein each independently selected substituent is as defined herein for an optional alkyl substituent, or is unsubstituted C1-C6 alkyl or unsubstituted C2-C6 alkenyl.

[0133] Exemplary esters include, but are not limited to, acetate, propionate, isopropionate, isobutyrate, butyrate, valerate, isovalerate, caproate, isocaproate, hexanoate, heptanoate, octanoate, phenylacetate, and benzoate, or -OC(=O)R b and structure (where R b is as defined for an acyloxy O-linked substituent and is typically selected from the group consisting of methyl, ethyl, propyl, iso-propyl, 2-methyl-prop-1-yl, 2,2-dimethyl-prop-1-yl, prop-2-en-1-yl, and vinyl.

[0134] "Ether," as used herein alone or in combination with other terms, unless otherwise indicated or implied by context, refers to an organic moiety, organic group, or organic substituent containing one, two, three, four, or more, typically one or two, -O- (i.e., oxy) moieties that are not bonded to a carbonyl moiety, where the two -O- moieties are not immediately adjacent to each other (i.e., not directly bonded). Typically, an ether comprises the formula -O-organic moiety, where the organic moiety is as described for an organic moiety bonded to an ester functional group or as described herein for an optionally substituted alkyl group. When an ether is described as a substituent or variable of an associated Markush structure or other organic moiety, the oxygen of the ether functional group is bonded to the associated Markush formula and is sometimes described as an "alkoxy" group, which is an exemplary O-linked substituent. In some aspects, an ether O-linked substituent is a C1-C 20 Alkoxy or C1-C 12 Alkoxy optionally substituted with 1, 2, 3, or 4, typically 1, 2, or 3, substituents, and in another aspect, C1-C8 alkoxy or C1-C6 alkoxy optionally substituted with 1 or 2 substituents, where each independently selected substituent is as defined herein for an optional alkyl substituent, and in yet another aspect, the ether O-linked substituent is an unsubstituted, saturated or unsaturated C1-C4 alkoxy (such as, by way of example and not limitation, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, and allyloxy (i.e., -OCH2CH=CH2)).

[0135] "Amide," as the term is used herein by itself or in combination with other terms, means, unless otherwise indicated or implied by context, RC(=O)N(R c )- or -C(=O)N(R c)2, in which no other heteroatoms are directly attached to the carbonyl carbon, and in which each R c are independently hydrogen, a protecting group, or an independently selected organic moiety, and R is hydrogen or an organic moiety, where R c The organic moieties independently selected from are as described herein for organic moieties attached to ester functional groups, or as described herein for optionally substituted alkyl groups. When an amide is listed as a substituent or variable for an associated Markush structure or other organic moiety, the amide nitrogen atom or carbonyl carbon atom of the amide functional group is attached to that structure or other organic moiety. Amides are typically prepared by condensing an acid halide, such as an acid chloride, with a molecule containing a primary or secondary amine. Alternatively, in some aspects, the amide coupling reaction, well known in the art of peptide synthesis, is used, which proceeds via an activated ester of a carboxylic acid-containing molecule. Exemplary preparations of amide bonds via peptide coupling methods are provided in Benoiton (2006) "Chemistry of peptide synthesis," CRC Press; Bodansky (1988) "Peptide synthesis: A practical textbook," Springer-Verlag; Frinkin, M. et al., "Peptide Synthesis," Ann. Rev. Biochem. (1974) 43:419-443. Reagents used for the preparation of activated carboxylic acids are provided in Han et al., "Recent development of peptide coupling agents in organic synthesis," Tet. (2004) 60:2447-2476.

[0136] Thus, in some aspects, amides are prepared by reacting a carboxylic acid with an amine in the presence of a coupling agent. As used herein, "in the presence of a coupling agent" includes contacting the carboxylic acid with a coupling agent, thereby converting the acid to its activated derivative (e.g., an activated ester or mixed anhydride), and subsequently or simultaneously contacting the resulting activated derivative with the amine, with or without isolating the resulting activated derivative of the acid. In some instances, the activated derivative is prepared in situ. In other instances, the activated derivative may be isolated to remove any undesired impurities.

[0137] "Carbonate," as used herein alone or in combination with other terms, means, unless otherwise indicated or implied by context, a substituent, moiety, or group containing a functional group having the structure -OC(=O)-O- (which defines a carbonate functional group). Typically, a carbonate group, as used herein, includes an organic moiety bonded to the -OC(=O)-O- structure, where the organic moiety is as described herein for an organic moiety bonded to an ester functional group (e.g., the organic moiety -OC(=O)-O-). When carbonate is described as a substituent or variable of an associated Markush structure or other organic moiety, one of the monovalent oxygen atoms of the carbonate functional group is bonded to the structure or other organic moiety with which it is associated, and the other is bonded to a carbon atom of another organic moiety as described above for an organic moiety bonded to an ester functional group, or as described herein for an optionally substituted alkyl group. In such cases, carbonate is an exemplary O-linked substituent.

[0138] "Carbamate," as the term is used herein by itself or in combination with other terms, means, unless otherwise indicated or implied by context, -OC(=O)N(R c )- or -OC(=O)N(R c)2, or —OC(═O)NH(optionally substituted alkyl)- or —OC(═O)N(optionally substituted alkyl)2, where the independently selected optionally substituted alkyl is an exemplary carbamate functional group substituent and typically includes an optionally substituted C1-C 12 alkyl or C1-C8 alkyl, more typically optionally substituted C1-C6 alkyl or C1-C4 alkyl, where each R c are independently selected and independently selected R c is hydrogen, a protecting group, or an organic moiety, which is as described herein for an organic moiety attached to an ester functional group, or as described herein for an optionally substituted alkyl group. Typically, a carbamate group is formed by bonding R c and further comprising an organic moiety independently selected from -OC(=O)-N(R c )-structure, the resulting structure is as described herein for the organic moiety attached to the ester functional group attached via the organic moiety -OC(=O)-N(R c )- or -OC(=O)-N(R c )-organic moiety. When a carbamate is described as a substituent or variable of an associated Markush structure or other organic moiety, the monovalent oxygen (O-linked) or nitrogen (N-linked) of the carbamate functional group is attached to the associated Markush formula. The bond of the carbamate substituent is either explicitly shown (N- or O-linked) or is implied in the context of the reference to the substituent. The O-linked carbamates described herein are exemplary monovalent O-linked substituents.

[0139] "Ligand Drug Conjugate," as that term is used herein, unless otherwise indicated or implied by context, refers to a construct comprising a Ligand unit (L) that incorporates or corresponds in structure to a targeting agent and a Drug unit (D) that incorporates or corresponds in structure to a free drug, where L and D are linked to each other via a Linker unit (LU), and where the Ligand Drug Conjugate is capable of selectively binding to a targeted portion of a targeted cell. In one aspect, the term Ligand Drug Conjugate (LDC) refers to a plurality of individual conjugate compounds (i.e., compositions) in which the number of auristatin Drug units conjugated to each Ligand unit and / or the position on the Ligand unit to which the Drug units are conjugated are the same or differ to some extent. In some aspects, this term refers to a collection (i.e., a population or plurality) of conjugate compounds having essentially the same Ligand unit, and the same Drug unit and Linker unit, which in some aspects have variable loading and / or distribution of auristatin drug linker moieties attached to each antibody residue (e.g., when any two Ligand Drug Conjugate compounds in a plurality of such compounds have the same number of Drug units but different locations of their attachment sites on the Ligand unit). In these examples, the Ligand Drug Conjugates are described by the average drug loading of the conjugate compounds.

[0140] The average number of Drug units per Ligand unit in a Ligand Drug Conjugate composition is the average number for a population of Ligand Drug Conjugate compounds, sometimes represented by the subscript p, which in some aspects reflects the distribution of these compounds that differ primarily in the number of Drug units conjugated to the Ligand unit and / or their position on the Ligand unit to which they are conjugated.

[0141] The ligand drug conjugate compounds of the present invention, alone or within a ligand drug conjugate composition, typically have the formula 1: [ka]

[0142] or a salt thereof, which in some aspects is a pharmaceutically acceptable salt, where L is a Ligand unit; LU is a Linker unit; the subscript p' is an integer ranging from 1 to 24; and D' represents 1 to 4 Drug units. In some aspects, the Ligand unit incorporates or corresponds in structure to an antibody or antigen-binding fragment thereof, thereby defining an antibody Ligand unit. In these aspects, the antibody Ligand unit is capable of selectively binding to an antigen of a cell to be targeted for subsequent release of free drug, where the targeted antigen in one aspect is a cancer cell antigen selectively recognized by the antibody Ligand unit, and upon said binding, is capable of internalization into the cancer cell along with the attached ADC compound to initiate intracellular release of free drug after internalization. In any of these aspects, each Drug Linker moiety in the Ligand Drug Conjugate compound is represented by Formula 1A: [ka]

[0143] or a salt thereof, which in some aspects is a pharmaceutically acceptable salt, wherein D of each Drug Linker moiety is a Drug unit; the wavy line indicates a covalent bond to L; L B is a Ligand covalent binding moiety; A is a first optional Stretcher unit; the subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional Branching unit; the subscript b is 0 or 1, indicating the absence or presence of B, respectively; L O is a secondary linker moiety; D is a Drug unit, where the Drug unit corresponds to the free drug in the structure; and the subscript q is an integer ranging from 1 to 4,

[0144] wherein a ligand drug conjugate composition comprising a distribution or collection of ligand drug conjugate compounds is represented by the structure of Formula 1, wherein the subscript p' is replaced by the subscript p, where the subscript p is a number ranging from about 2 to about 24.

[0145] "Ligand unit," as the term is used herein, unless otherwise indicated or implied by context, refers to a compound capable of selectively binding to the targeting moiety or its cognate targeted moiety of a ligand drug conjugate composition and incorporating or corresponding in structure to a targeting agent. Ligand units (L) include, but are not limited to, ligand units derived from receptor ligands, antibodies to cell surface antigens, and transporter substrates. In some aspects, the receptor, antigen, or transporter to which the conjugate compound of a ligand drug conjugate composition binds is present in greater abundance in abnormal cells as opposed to normal cells, thereby providing a desired improvement in tolerability or reducing the potential occurrence or severity of one or more adverse events associated with administration of the drug in its unconjugated form. In other aspects, the receptor, antigen, or transporter that binds to the Ligand unit of a ligand drug conjugate compound is present in greater abundance in normal cells near the abnormal cells as opposed to normal cells distal to the site of the abnormal cells, thereby selectively exposing nearby abnormal cells to the free drug. Various aspects of ligand units, including antibody ligand units, are further illustrated by embodiments of the present invention.

[0146] " Targeting agent ", as used herein, unless otherwise indicated or implied by context, refers to an agent that can selectively bind to the targeting moiety and substantially retains this ability when incorporated as a ligand unit in a ligand-drug conjugate.Therefore, the ligand unit of a ligand-drug conjugate corresponds structurally to a targeting agent, and the ligand unit is the targeting moiety of the conjugate.In some aspects, the targeting agent is an antibody or a fragment thereof that selectively binds to an accessible antigen that is characteristic of abnormal cells or exists on abnormal cells in a higher copy number than normal cells, or an accessible antigen that is specific to the surrounding environment in which these cells are found, to the extent that it achieves improved tolerability compared to the administration of free drugs.In other aspects, the targeting agent is a receptor ligand that specifically binds to an accessible receptor that is characteristic of or exists more abundantly on abnormal cells, or an accessible receptor on nominally normal cells that is specific to the surrounding environment of abnormal cells. Typically, the targeting agent is an antibody as defined herein that selectively binds to a targeted portion of abnormal mammalian cells, more typically, a targeted portion of abnormal human cells.

[0147] A "targeted moiety," as defined herein, is a moiety that is selectively recognized by a targeting agent or the targeting moiety of a ligand-drug conjugate (which is its ligand unit that incorporates or corresponds in structure to the targeting agent). In some aspects, the targeted moiety is present inside, on the surface of, or near abnormal cells, and typically is present in greater abundance or copy number in abnormal cells compared to normal cells or the environment of normal cells distant from the site of the abnormal cells, thereby providing improved tolerability compared to administration of the free drug or reducing the likelihood of one or more adverse events from its administration. In some aspects, the targeted moiety is an antigen accessible for selective binding by an antibody, which is an exemplary targeting agent incorporated into or corresponding in structure to the antibody-ligand unit of the antibody-drug conjugate composition or compound thereof. In other aspects, the targeted moiety is a ligand targeting moiety for an extracellularly accessible cell membrane receptor that, in some aspects, is internalized upon binding of the cognate targeting moiety by a ligand unit of a ligand drug conjugate compound that incorporates or structurally corresponds to the receptor ligand; in other aspects, the receptor is capable of passive or facilitated transport of the ligand drug conjugate compound after binding to the cell surface receptor. In some aspects, the targeted moiety is present on abnormal mammalian cells or on mammalian cells characteristic of the environment of such abnormal cells. In some of these aspects, the targeted moiety is an antigen of abnormal mammalian cells, more typically, a targeted moiety of abnormal human cells.

[0148] "Targeted cells," as the term is used herein, unless otherwise indicated or implied by context, are intended cells with which the Ligand Drug Conjugate is designed to interact to inhibit the proliferation or other undesired activity of abnormal cells. In some aspects, the targeted cells are hyperproliferating cells or hyperactivated immune cells, which are exemplary abnormal cells. Typically, these abnormal cells are mammalian cells, more typically human cells. In other aspects, the targeted cells are located in the vicinity of the abnormal cells, such that the action of the Ligand Drug Conjugate compound on these nearby cells has the intended effect on the abnormal cells. For example, the nearby cells may be epithelial cells characteristic of the abnormal vasculature of a tumor. Targeting these vascular cells with the Ligand Drug Conjugate is believed to result in the inhibition of nutrient delivery to abnormal cells near the tumor, indirectly exerting a cytotoxic or cytostatic effect on these cells. Such inhibition has an indirect cytotoxic or cytostatic effect on the abnormal cells, and also has a direct cytotoxic or cytostatic effect on nearby abnormal cells by releasing its drug payload in the vicinity of those cells.

[0149] "Antibody drug conjugate," as that term is used herein, unless otherwise indicated or implied by context, refers to a construct that is a subset of the Ligand Drug Conjugates of Formula 1 and thus is composed of an antibody ligand unit (L) that is incorporated into or corresponds to an antibody or antigen-binding fragment thereof, and a drug unit (D) that is incorporated into or corresponds in structure to a biologically active compound, often referred to as the free drug, where L and D are linked to each other via a linker unit (LU), and where the antibody drug conjugate is capable of selectively binding to a targeted antigen on a targeted cell via its targeting antibody ligand unit, where the targeted antigen is, in some aspects, an antigen of an abnormal cell such as a cancer cell.

[0150] The term antibody-drug conjugate (ADC) refers, in one aspect, to a plurality of individual conjugate compounds (i.e., compositions) that vary to some extent in the number of drug units conjugated to each antibody ligand unit and / or the positions on the antibody ligand unit to which the drug units are conjugated. In some aspects, the term refers to a distribution or collection (i.e., a population or plurality) of conjugate compounds having the same drug-linker moiety and antibody ligand unit, allowing for variant amino acid variations and varying glycosylation patterns as described herein to occur during antibody production from cell culture, and in some aspects, having variable loading and / or distribution of drug-linker moieties attached to each antibody residue (e.g., when any two antibody-drug conjugate compounds in a plurality of such compounds have the same number of drug units but different positions of these attachment sites of the drug-linker moieties to the targeting antibody ligand unit). In these examples, the antibody-drug conjugates are described by the average drug loading of the conjugate compounds.

[0151] The average number of drug units per antibody ligand unit or antigen-binding fragment thereof in an antibody drug conjugate composition having an intact drug linker moiety where the linker unit is unbranched is the average number for a population of antibody drug conjugate compounds, which in some aspects reflects a distribution of these compounds that differ primarily in the number and / or location of drug units conjugated to the antibody ligand unit. When the linker unit is branched, the average number reflects a distribution of drug linker moieties for the population of antibody drug conjugate compounds. In either situation, p is a number ranging from about 2 to about 24 or from about 2 to about 20, and is typically about 2, about 4, about 10, or about 8. In other situations, p represents the number of drug units covalently attached to one antibody ligand unit of an antibody drug conjugate within a population of antibody drug conjugate compounds, where the compounds in this population differ primarily in the number and / or location of drug units or drug linker moieties. In this context, p is designated p' and is an integer ranging from 1 to 24 or 1 to 20, typically from 1 to 12 or 1 to 10, and more typically from 1 to 8. In other aspects, essentially all of the available reactive functional groups of the antibody targeting agent form covalent bonds to drug linker moieties to provide the maximum number of antibody ligand units attached to drug linker moieties, such that the value of p of the antibody drug conjugate composition is the same or nearly the same as the value of each p' of each antibody drug conjugate compound of the composition, such that antibody drug conjugate compounds having smaller values ​​of p', if any, are present only in small amounts as detected using an appropriate chromatographic method such as electrophoresis, HIC, reverse-phase HPLC, or size exclusion chromatography.

[0152] The average number of drug units or drug linker moieties per antibody ligand unit in the preparation from the conjugation reaction is characterized in some aspects by combining conventional chromatographic means, as described above, with mass spectrometry detection. In other aspects, the quantitative distribution of conjugate compounds is determined in terms of the value of p'. In these examples, separation, purification, and characterization of homogeneous antibody drug conjugate compounds (where p' is a particular value) from the antibody drug conjugate composition from those with other drug unit or drug linker moiety loads can be achieved by means such as the chromatographic methods described above.

[0153] "Drug Linker Compound," as that term is used herein, unless otherwise indicated or implied by context, refers to a compound having a Drug Unit covalently attached to a Linker Unit Precursor (LU'), where LU' is L B ', which is sometimes composed of a ligand covalent precursor (L B The moiety is referred to as a "ligand covalent binding moiety" (L') because it contains a reactive or activatable functional group, which, after activation, reacts with a targeting agent to form a ligand covalent binding moiety (L B ) and a Ligand unit, thus providing a covalent bond to the Drug Linker moiety of Formula 1A for a Ligand Drug Conjugate compound of Formula 1, particularly an antibody Ligand unit that incorporates or corresponds in structure to an antibody.

[0154] The drug linker compounds of the present invention typically have the formula I: [ka]

[0155] or a salt thereof, which in some aspects is a pharmaceutically acceptable salt, where LU' is an LU precursor; and D' represents 1 to 4 Drug units, wherein the Drug Linker Compound has the general formula IA: [ka]

[0156] is further defined by the structure of B ' comprises a reactive or activatable functional group, and the remaining variables are as defined for Formula 1A.

[0157] A "cytotoxic agent," as that term is used herein, unless otherwise indicated or implied by context, is a compound capable of inducing cell death or inhibiting the growth or continued survival of cells, which are typically abnormal mammalian cells in vitro or in vivo. Included within this definition of cytotoxic agent are cytostatic agents that exert their therapeutic effect primarily by inhibiting the growth of abnormal cells rather than by directing cell death. In some aspects, the cytotoxic agent is the free drug resulting from the release of a drug unit from an antibody-drug conjugate.

[0158] "Drug Unit," as this phrase is used herein, unless otherwise indicated or implied by context, refers to a residue of a drug that is covalently attached to a Linker Unit (LU) within a Drug Linker Moiety of a Ligand Drug Conjugate (LDC) or to a Linker Unit Precursor (LU') of a Drug Linker Compound, and is releasable as a free drug from the Drug Linker Moiety or Drug Linker Compound. The free drug can be directly incorporated into the Drug Unit, or a component of the free drug can be covalently attached to an LU or LU' or an intermediate thereof, followed by further elaboration to complete the structure of the Drug Unit. The term "drug," when used herein alone or in conjunction with another term (e.g., "drug unit"), is not intended to imply that the compound has been approved, is approvable, or is intended to be approved by a governmental agency for medical or veterinary treatment.

[0159] In some aspects, the free drug incorporated into the Drug Unit is a cytotoxic compound, typically one having a secondary aliphatic amine as the conjugation handle, and including an auristatin compound as defined herein.

[0160] The terms "auristatin drug," "auristatin compound," and the like, as used herein, unless otherwise indicated or implied by context, refer to peptide-based tubulin-disrupting agents with cytotoxic, cytostatic, or anti-inflammatory activity that contain dolaproline and dolaisoleucine residues or amino acid residues related thereto.

[0161] Some exemplary auristatins are D E or D F : [ka]

[0162] where Z is —O—, —S—, or —N(R 19 )- and R 10 ~R 21 is as defined in embodiments for an Auristatin Drug Unit, and the indicated nitrogen atom (†) is the nitrogen atom of a secondary amine (e.g., R 10 , R 11 (One of which is hydrogen and the other is -CH3). In these aspects, the auristatin is incorporated into the Drug unit via a carbamate functional group containing its nitrogen atom. The carbamate functional group is an exemplary second spacer unit (Y'), capable of self-immolation, which in turn is attached to a PAB or PAB-type spacer unit (Y), such that the subscript y in any one of the Drug Linker moieties described herein is 2.

[0163] Other exemplary auristatins include but are not limited to AE, AFP, AEB, AEVB, MMAF and MMAE, and those further described in the embodiments of the present invention.The synthesis and structure of auristatins are described in US Patent Application Publication No. 2003-0083263, No. 2005-0238649, No. 2005-0009751, No. 2009-0111756 and No. 2011-0020343; International Patent Publication No. WO04 / 010957, International Patent Publication No. WO02 / 088172, and US Patent No. 7,659,241 and No. 8,343,928.Their structures and their synthetic methods disclosed therein are specifically incorporated herein by reference.

[0164] As used herein, the phrase "salt thereof" refers to a salt form of a compound (e.g., a drug, a drug linker compound, or an LDC compound), unless otherwise indicated or suggested by the context. A salt form of a compound is one or more intramolecular salt forms and / or involves the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion in the salt form of a compound is typically an organic or inorganic moiety that stabilizes the charge on the parent compound. A salt form of a compound has one or more charged atoms in its structure. When multiple charged atoms are part of the salt form, there are multiple counterions and / or multiple charged counterions. Thus, a salt form of a compound typically has one or more charged atoms and one or more counterions corresponding to the atoms in the non-salt form of the compound. In some embodiments, a non-salt form of a compound contains at least one amino group or other basic moiety, and therefore, in the presence of an acid, an acid addition salt with the basic moiety is obtained. In other embodiments, the non-salt form of the compound contains at least one carboxylic acid group or other acidic moiety, thus yielding a carboxylate or other anionic moiety in the presence of base.

[0165] Exemplary counteranions and countercations in salt forms of the compounds include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylenebis-(2-hydroxy-3-naphthoate)) salts.

[0166] The selection of a salt form of a compound depends on the properties that the pharmaceutical product must exhibit, such as sufficient aqueous solubility at various pH values ​​depending on the intended route(s) of administration, crystallinity with flow properties, and low hygroscopicity (i.e., water absorption versus relative humidity) suitable for handling and the required shelf life by determining chemical and solid-state stability under accelerated conditions (i.e., to determine degradation or solid-state changes when stored at 40°C and 75% relative humidity).

[0167] "Pharmaceutically acceptable salts" are salt forms of compounds suitable for administration to a subject as described herein, and in some embodiments, are those salts described in P.H. Stahl and C.G. Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wei Include countercations or counteranions as described by Wiley-VCH / VHCA, 2002.

[0168] As used herein, the term "antibody" is used in the broadest sense and, unless otherwise indicated or implied by context, specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity, provided that the antibody fragment has the necessary number of binding sites for the desired number of drug-linker moieties and is capable of specifically and selectively binding to the targeted cancer cell antigen. Antibodies in their natural form are tetramers and typically consist of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) together are primarily responsible for binding to the antigen. The light and heavy chain variable domains consist of framework regions separated by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." In some aspects, the constant region is recognized by and interacts with the immune system (see, e.g., Janeway et al., 2001, Immunol. Biology, 5th Ed., Garland Publishing, New York), thereby exerting effector function. Antibodies include any isotype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclasses thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Antibodies are derived from any suitable species. In some aspects, antibodies are of human or murine origin. Such antibodies include human antibodies, humanized antibodies, or chimeric antibodies.

[0169] In some aspects, the antibody is in a reduced form, where the antibody has undergone reduction of its hinge disulfide bonds. The antibody is then incorporated into an antibody-drug conjugate as an antibody ligand unit by reaction of one or more of the resulting cysteine ​​thiols with a suitable electrophile of a drug-linker compound, resulting in the covalent attachment of a drug-linker moiety to the antibody ligand unit or a linker intermediate that is further elaborated into its final form as a drug-linker moiety.

[0170] "Monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations and / or differences in glycosylation patterns, which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0171] "Selectively binds" and "selectively binds," as these terms are used herein, unless otherwise indicated or implied by context, refer to an antibody, fragment thereof, or antibody-ligand unit of an antibody-drug conjugate that is capable of binding in an immunologically selective and specific manner to its cognate cancer cell antigen, but not to numerous other antigens. Typically, the antibody or antigen-binding fragment thereof binds to the targeted cancer cell antigen at least about 1 x 10 -7 M, preferably about 1 x 10 -8 M~1×10 -9 M, 1 x 10 -10 M or 1×10 -11M and binds to its predetermined antigen with an affinity at least two-fold higher than its affinity for binding to a nonspecific antigen other than a closely related antigen (e.g., BSA, casein), wherein said affinity is substantially retained when the antibody or antigen-binding fragment thereof corresponds to an antibody drug conjugate or is incorporated into a ligand drug conjugate as an antibody-ligand unit.

[0172] "Antigen," as used herein, unless otherwise indicated or implied by context, refers to a moiety capable of specifically binding to an unconjugated antibody or its antigen-binding fragment, or to an antibody-drug conjugate compound comprising an antibody ligand unit incorporating or corresponding in structure to an unconjugated antibody. In some aspects, the antigen is an extracellularly accessible cell surface protein, glycoprotein, or carbohydrate, particularly a protein or glycoprotein, preferentially presented by abnormal cells compared to normal cells distal to the site of the abnormal cells. In these aspects, the cell surface antigen can be internalized upon selective binding by the conjugate compound of the antibody-drug conjugate composition. After internalization, intracellular processing of the linker unit of the antibody-drug conjugate compound of the composition releases the drug unit as a free drug. Examples of antigens associated with hyperproliferative cells that are accessible to antibody-drug conjugate compounds on the cell surface include, but are not limited to, the cancer-specific antigens described herein.

[0173] Typically, the antigen is associated with cancer. In some of these aspects, the antigen is preferentially presented by cancer cells compared to normal cells not localized in the abnormal cells, particularly when the cancer cells presenting the antigen are mammalian cancer cells. In other aspects, the cancer cell antigen is an extracellularly accessible antigen that is preferentially presented by nearby normal cells that are unique to the cancer cell's environment compared to normal cells distal to the cancer cell site. For example, the nearby cells may be epithelial cells characteristic of the abnormal vasculature of tumors. Targeting these vascular cells with antibody-drug conjugates is believed to have a cytotoxic or cytostatic effect on these cells, resulting in the inhibition of nutrient delivery to cancer cells near the tumor. Such inhibition may have an indirect cytotoxic or cytostatic effect on cancer cells, and may also have a direct cytotoxic or cytostatic effect on nearby cancer cells after the drug unit is released as a free drug following immunoselective binding by the antibody-drug conjugate (ADC) compound. In either of these aspects, the cell surface antigen is capable of being internalized by the targeted cell, allowing intracellular delivery of the free drug upon release from the conjugate.

[0174] Preferred internalizable antigens are those expressed on the surface of cancer cells at copy numbers of 10,000 or more per cell, 20,000 or more per cell, or 40,000 or more per cell. Cancer cell-associated antigens that are cell surface accessible to ADCs and internalizable include antigens expressed on Hodgkin's lymphoma cells, particularly Reed-Sternberg cells, exemplified by Karpas 299 cells, and certain cancer cells of high-grade lymphomas sometimes referred to as Ki-1 lymphomas. Other antigens include cancer cells of renal cell adenocarcinoma exemplified by 789-O cells, cancer cells of B-cell lymphomas or leukemias including non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) exemplified by CHO cells, cancer cells of acute myeloid leukemia (AML) exemplified by HL-60, and certain transporter receptors ubiquitously expressed on these and other cancer cells.

[0175] "Linker unit," as that term is used herein, unless otherwise indicated or implied by context, refers to the organic portion of a Ligand Drug Conjugate that is interposed between and covalently bonded to a Drug unit and a Ligand unit (L) (as these terms are defined herein), or is the organic portion of a Drug-Linker Compound that is covalently bonded to a Drug unit and has a reactive functional group or moiety that incorporates or corresponds in structure to a targeting agent, and a Linker unit (LU), for interacting with the targeting agent to form a covalent bond between L and L. Because the Linker unit of a Drug-Linker is capable of forming such a bond, it is considered a precursor to the Linker unit of a Ligand Drug Conjugate, and is sometimes denoted LU'. A Linker unit is a precursor to a primary linker (L R ) and L R and D in the drug linker portion of the ligand drug conjugate compound, or L R and a secondary linker (L O ) (in the latter case, the LR To explicitly indicate that it is a precursor of R ').

[0176] "Primary Linker," as that term is used herein, unless otherwise indicated or implied by context, refers to the required component of a Linker Unit (LU) in a Ligand Drug Conjugate that is covalently attached to the Ligand unit and the remainder of the LU. R ) is a covalent ligand bond (L B ) moiety, which in some aspects of the Ligand Drug Conjugates (LDCs) and Drug Linker Compounds described herein, is a self-stabilizing (L SS ) linker, whereby L SS In defining the primary linker and in other aspects of the LDC, SS The self-stabilizing (L S ) linker, whereby L S A primary linker is defined (as these terms are further described herein). The primary linker optionally contains a branching unit (B) and a first optional stretcher unit (A), depending on the values ​​of the subscripts a and b in Formula 1A, where A is L R L SS or L S Present if primary linker.

[0177] L in LDC or drug linker compound SS The primary linkers each have a succinimide (M 2 ) or maleimide (M 1 ) moiety, while the L in the LDC composition or compound thereof S The primary linker is a succinamide (M 3 The L of the present invention is characterized by the moiety SS or L S The primary linker is also present, M 1 Or M 2The imide nitrogen of the maleimide or succinimide ring system of 3 Optionally substituted C1-C bonded to the amide nitrogen 12 It is characterized by a first optional Stretcher unit (A) comprising an alkylene moiety, where the alkylene moiety in some aspects is substituted with an acyclic basic unit and may be further substituted with optional substituents, or in other aspects is optionally substituted and incorporates an optionally substituted cyclic basic unit.

[0178] L in drug linker compounds SS The primary linker (which is sometimes referred to as the L of a ligand-drug conjugate) SS To explicitly indicate that it is a precursor of SS The maleimide (M') covalently binds the ligand precursor 1 ) moiety reacts with the sulfur atom of the reactive thiol functional group of the targeting agent to form the L SS A thio-substituted succinimide moiety (M 2 ), where the thio substituent is a ligand unit that incorporates or corresponds in structure to the targeting agent. In aspects where the targeting agent is an antibody or antigen-binding fragment thereof, the antibody may be linked to M through the sulfur atom of a cysteine ​​residue resulting from disulfide bond reduction or introduced by genetic engineering. 2 As a result, the antibody or antigen-binding fragment thereof becomes bound to L SS Covalently attached to the primary linker. SS M of the primary linker 2 By hydrolysis of M 2 is the succinamide moiety (M 3 ) converted to L S This results in a primary linker, which exists in two regioisomers (M 3A and M 3B ) may exist as a mixture.

[0179] "Ligand covalent binding moiety," as that term is used herein, unless otherwise indicated or implied by context, refers to the portion of a Linker unit (LU) in a Ligand Drug Conjugate that interconnects that Ligand unit (L) with the remainder of the Linker unit, and is the corresponding Ligand covalent binding precursor (L) of a Linker unit precursor (LU') in a Drug-Linker Compound. B The L') moiety is derived from the reaction of the L') moiety with a targeting agent such as an antibody or antigen-binding fragment thereof. B ' is the maleimide moiety (M 1 ), the reaction of this moiety with a reactive thiol functional group of the targeting agent can be achieved by B ' to the ligand covalent bond (L B ) moiety, resulting in a thio-substituted succinimide moiety. When the targeting agent is an antibody or antigen-binding fragment thereof, the thio substituent comprises a sulfur atom of the antibody ligand unit, which in some aspects is provided by a cysteine ​​residue obtained by interchain disulfide bond reduction or genetic engineering.

[0180] In another example, L B When L' contains an activated carboxylic acid functional group, reaction of this functional group with a reactive amino group of a targeting agent, such as the epsilon amino group of a lysine residue of an antibody or antigen-binding fragment thereof, converts this functional group into an amide, wherein the amide functional group resulting from this reaction is B and the bound ligand unit, which in the case of an antibody or antigen-binding fragment is an antibody ligand unit. B Parts and their L B Conversions from '-containing moieties are described in embodiments of the present invention. In yet another example, a targeting agent having a reactive amino group is derivatized with a bifunctional molecule to provide an intermediate, which in some cases results in a reactive thiol functionality, which can be converted to a thiol group, such as L B As a result of this condensation, the L B The moiety is a bifunctional molecule and LB ' has atoms due to.

[0181] A "ligand covalent bond precursor moiety" is a portion of a linker unit of a drug-linker compound or intermediate thereof that contains a reactive or activatable functional group, where the reactive or activatable functional group can be activated and then covalently attached to a targeting agent, such as an antibody or antigen-binding fragment thereof, during the preparation of a ligand drug conjugate (LDC), including an antibody drug conjugate (ADC), whereby the ligand-binding moiety precursor (L B ') part is the ligand covalent bond (L B ) moiety. In some cases, L B ' part is L B ' possesses a functional group capable of reacting with a nucleophile or electrophile that is either native to the antibody or antigen-binding fragment thereof or introduced into the antibody or antigen-binding fragment by chemical transformation or genetic engineering (see above) for conversion of the moiety into an antibody-ligand unit. In some of these aspects, the nucleophile is the N-terminal amino group of the light or heavy chain of the antibody or antigen-binding fragment thereof, or the epsilon-amino group of a lysine residue of the light or heavy chain.

[0182] In other aspects, the nucleophile is a sulfhydryl group of a cysteine ​​residue introduced into the antibody light or heavy chain or antigen-binding fragment thereof by genetic engineering or by chemical reduction of the interchain disulfides of the antibody or antigen-binding fragment. In yet some aspects, the electrophile is an aldehyde introduced into the glycan component of the antibody or antigen-binding fragment thereof by selective oxidation of the carbohydrate moiety, or a ketone derived from an unnatural amino acid introduced into the antibody light or heavy chain or antigen-binding fragment thereof using a genetically engineered tRNA / tRNA synthetase pair. These and other methods for introducing reactive functional groups to provide conjugation sites in antibodies are reviewed by Behrens and Liu "Methods for site-specific drug conjugation to antibodies" mAB (2014) 6 (1): 46-53.

[0183] The terms "secondary linker," "secondary linker moiety," and the like, as used herein, unless otherwise indicated or implied by context, refer to an organic moiety in a linker unit (LU), where the secondary linker (L O ) is the linker between the drug unit and the primary linker (L R ) and the ligand covalent bond (L B ) moiety, a first optional stretcher unit and / or an optional branching unit (B), and in some aspects a self-stabilizing (L) drug linker compound useful in the preparation of or of a ligand drug conjugate (LDC), such as an antibody drug conjugate (ADC). SS ) provide a primary linker, or L SS The self-stabilization of LDC / ADC compounds during hydrolysis of S ) provides the primary linker. R L SS or L S In those instances, there is a first optional stretcher unit. Ris connected to L through a heteroatom or functional group from the first optional Stretcher unit (A) present O is combined with

[0184] The secondary linker of a Ligand Drug Conjugate Compound or a Drug Linker Compound is typically

[0185] [ka] having the structure

[0186] Here, if the subscript b is 0, the wavy line adjacent to A' is L O indicates the site of covalent attachment of Y to the primary linker; the wavy line adjacent to Y indicates the site of covalent attachment of L O represents the site of covalent attachment to the Drug unit; A' is a second optional Spacer unit, or in some aspects, a subunit of the first optional Stretcher unit, if present, where the subscript a' is 0 or 1, indicating the absence or presence of A', respectively; Y is a Spacer unit, and the subscript y is 0, 1, or 2, indicating the absence or presence of one or two Spacer units, respectively; and W is a peptide cleavable unit, where the peptide cleavable unit provides a recognition site with greater overall selectivity for proteases in tumor tissue homogenates compared to proteases in normal tissue homogenates, where tumor tissue is comprised of targeted cancer cells and normal tissue is comprised of non-targeted normal cells, to which off-target cytotoxicity by the Ligand Drug Conjugate is at least partially responsible for the adverse events often associated with administration of a therapeutically effective amount to a mammalian subject in need thereof. When the subscript b is 0, A', if present, becomes a subunit of A, in which case the secondary linker is -WY y In any of these aspects, W, Y and D have the structure -WY y-D, where W is a peptide cleavable unit and the subscript y is 0, 1, or 2. If the subscript y is 1 or 2, after protease cleavage, the self-immolative spacer unit attached to W self-immolates to release D or Y'-D, and if a second spacer unit (Y') is present, it decomposes to complete the release of D as the free drug.

[0187] The secondary linker (L O ) is typically represented by, as exemplified when only one Drug unit is attached to LU where W is a peptide cleavable unit:

[0188] If the subscript b is 1, then [ka] or, if subscript b is 0 and subscript a' is 1, then A' a’ is treated as a sub-unit of the first need-based stretcher unit,

[0189] [ka] is represented by the structure

[0190] where D is the Drug unit and the remaining variables are L O as defined herein;

[0191] And the drug linker moiety or drug linker compound including the secondary linker typically has the structure of Formula 1B and Formula 1B, respectively: [ka]

[0192] where L Bis the ligand covalent binding moiety as defined herein, which is the primary linker (L) of the linker unit (LU) of the drug linker portion of the ligand drug conjugate compound. R ) is a component of; and L B ' is a Ligand covalent binding moiety as defined herein, which is the primary linker (L R ') and sometimes, when a drug-linker compound is used in the preparation of a ligand-drug conjugate, the L R , L B and LU are referred to as Ligand Covalent Moiety Precursor, Primary Linker Precursor, and Linker Unit Precursor, respectively; A is a first optional Stretcher unit; subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional Branching unit, and subscript b is 0 or 1, indicating the absence or presence of B, respectively, where A' is a subunit of A, and when subscript b is 0, subscript a is 1 and subscript a' is 1; subscript q ranges from 1 to 4, where L B / L B ' and A and B, if present, L R / L R ', with the proviso that if subscript b is 1, then subscript q is in the range of 2 to 4, and if subscript b is 0, then subscript q is 1; and the remaining variables are L O is as defined herein.

[0193] "Maleimide moiety," as used herein, unless otherwise indicated or implied by context, refers to a component of the primary linker of a drug-linker compound, which in some aspects is a component of a self-stabilizing linker, where the primary linker is sometimes the L in a ligand drug conjugate. R / L SS To explicitly indicate that it is a precursor of R ' or L SSThe maleimide moiety (M 1 ) participates in a Michael addition (i.e., 1,4-conjugate addition) with the sulfur atom of a reactive thiol functional group of a targeting agent, such as an antibody or antigen-binding fragment thereof, to form a thio-substituted succinimide (M 2 ) moiety, where the thio substituent is a Ligand unit that incorporates or corresponds to the structure of a targeting agent as exemplified herein for the antibody Ligand unit of an antibody drug conjugate composition or compound thereof. 1 The moiety is attached to the remainder of the primary linker, typically L SS ' is a component of M 1 to a first optional stretcher unit (A) present as part of a second linker (L O ) via its imide nitrogen atom.

[0194] Other than the imide nitrogen atom, M 1 The moiety is usually unsubstituted, but may be asymmetrically substituted at the cyclic double bond of its maleimide ring system. Such substitution may result in regiochemically favorable conjugate addition of the sulfur atom of the reactive thiol functional group of the targeting agent to the less sterically hindered or more electron-deficient double-bonded carbon atom (depending on the more dominant contribution) of the maleimide ring system. The conjugate addition is carried out by a succinimide (M 2 ) moiety, which is thio-substituted by a Ligand unit via a sulfur atom from a thiol functional group provided by the targeting agent.

[0195] A "succinimide moiety," as used herein, unless otherwise indicated or implied by context, refers to one type of ligand covalent bond (L B ) moiety, which in turn is a component of the linker unit of a ligand drug conjugate, such as an antibody drug conjugate, and is one type of ligand covalent bond precursor (L) in a drug linker compound to the sulfur atom of a reactive thiol functional group of an antibody or antigen-binding fragment thereof. B The maleimide moiety (M1 ) or its M 1 It results from the Michael addition of a succinimide (M 2 The ) moiety comprises a thio-substituted succinimide ring system whose imide nitrogen atom is substituted with the remainder of a primary linker, typically the first optional Stretcher unit (A) present. In some aspects, the nitrogen atom is substituted with an optionally substituted C1-C aryl group that makes up the first optional Stretcher unit (A) present. 12 When the Primary Linker is a self-stabilizing linker, the alkylene moiety either incorporates a cyclic basic unit in the first optional Stretcher unit present, or is replaced by an acyclic basic unit, and is otherwise optionally substituted, as described elsewhere, and M 1 The substituent(s) on the succinimide ring system that may have been present in the precursor and the M 2 The parts are replaced as necessary.

[0196] Thus, the optionally substituted C1-C of A optionally combined with the optional hydrolysis enhancing unit [HE] 12 The alkylene moiety is the same as the optional secondary linker (L O ) or, when subscript b is 1, via —[HE]-B— in a Drug Linker moiety of Formula 1B or a Drug Linker compound of Formula IB O In these examples where subscript b is 0, subscript a is 1, and subscript a' is 1, A is represented by the formula -A1[HE]-A2-, where A1 is the first subunit of A and is an optionally substituted C1-C1 alkyl group optionally combined with HE. 12 contains an alkylene moiety, and L OA', shown above as a component of, becomes A2, which is now the second subunit of A. In these examples, when subscript b is 1, subscript a is 1, and subscript a' is 1, A' is a component of the secondary linker, and A is a single unit optionally combined with [HE], or optionally comprises two subunits, which is -A[HE]-A O - where A O is an optional subunit of A. A O When present, A is also represented by the formula -A1[HE]-A2-.

[0197] Self-stabilizing linkers (L) in ligand-drug conjugate compounds SS ), a thio-substituted succinimide (M 2 Hydrolysis of the succinimide ring system of the ) moiety, which is pH-controllable due to the presence of a basic functionality on a nearby acyclic or cyclic basic unit, can in some cases lead to the formation of self-stabilized linkers (L) due to asymmetric substitution by the thio substituent. S ) in succinamide (M 3 ) moiety. The relative amounts of these isomers are given by M 1 M may be at least partially attributed to any substituents present in the precursor. 2 This is due, at least in part, to the difference in reactivity of the two carbonyl carbons in L. R M does not contain a basic unit 2 While this is expected to occur to some extent when the moiety is present, it is highly variable compared to the controlled hydrolysis provided by the basic unit.

[0198] In some respects, M 2 and a first optional Stretcher unit is present, optionally bonded at a position distal to its attachment site to the imide nitrogen atom to an optional hydrolysis enhancing unit, [HE], an optionally substituted C1-C 12In that aspect, A is a single unit or further comprises A', which is an optional subunit of A present when subscript b is 0 and subscript a' is 1, and is linked to [HE], also present, so that A has the formula -A[HE]-A'-; or, when subscript b is 1 and subscript a' is 1, A' is an present component of a secondary linker, so that A is -A[HE]-A'-. O -It is expressed by the formula:

[0199] A "succinic acid amide moiety," as used herein, unless otherwise indicated or implied by context, refers to a self-stabilizing linker (L) of a linker unit in a ligand drug conjugate, such as an antibody drug conjugate. S ) and has the structure of succinic amide hemi-acid residue, L S wherein this moiety is typically the first optional Stretcher unit (A) present or a subunit thereof, and has a C1-C bond optionally bonded to [HE]. 12 When the subscript b is 0 and the subscript a is 0 or 1, the possible structures of A are -A[HE]-A' a’ -, where A' is present as a subunit of A when the A' previously associated with the secondary linker is not present, such that the subscript a' is 0, or when the subscript a' is 1. When this subunit is present, A is represented by the formula A1[HE]-A2-, where A1 is an optionally substituted C1-C1 bonded to [HE]. 12 A is the first subunit of A containing an alkylene moiety, and A2 is the second subunit of A, previously designated as A'. A possible structure of A when subscript b is 1 and subscript a is 1 is -A[HE]-A O - where A OIf present, is an optional subunit of A. If this subunit is absent, A is a single, separate unit, and A O When present, A is represented by the formula A1[HE]-A2-, where A1 is an optionally substituted C1-C 12 is a first subunit of A that includes an alkylene moiety, and A O A2, shown above as, is the second subunit of A.

[0200] In some aspects, the alkylene moiety incorporates a cyclic basic unit, and in other aspects is substituted by an acyclic basic unit, and in any aspect is otherwise optionally substituted, where succinamide (M 3 ) moiety has a further substitution by LS-, where L is a ligand unit, such as an antibody ligand unit, that incorporates or corresponds in structure to a targeting agent, such as an antibody or antigen-binding fragment thereof, and S is a sulfur atom from the targeting agent, antibody, or antigen-binding fragment. 3 The moiety is a self-stabilizing primary linker succinimide (M 2 ) moiety, the hydrolysis of which is assisted by the basic unit.

[0201] Therefore, M 3 The moiety has a free carboxylic acid functionality and an amide functionality, its nitrogen heteroatom is attached to the remainder of the primary linker, and its M 2 It is substituted by LS- at the carbon that is alpha to the carboxylic acid or amide functionality depending on the hydrolysis site of the precursor. 3 Such hydrolysis to yield a moiety is believed to provide a linker unit (LU) of a ligand drug conjugate that is less susceptible to premature loss by elimination of the thio substituent from its targeting ligand unit (L) conjugate.

[0202] "Self-stabilizing linker," as used herein, unless otherwise indicated or implied by context, refers to a linker that is 2 a primary linker of a linker unit (LU) in a ligand drug conjugate, such as an antibody drug conjugate, having a containing moiety, or M 1 refers to the primary linker of a Linker Unit Precursor (LU') in a Drug-Linker Compound having a containing moiety, where this moiety is L SS ', which is the L of LDC SS M 2 The self-stabilizing linker is then hydrolyzed under controlled hydrolysis conditions to form the corresponding self-stabilizing linker (L S ) which undergoes conversion to L SS This is facilitated by the basic unit component of L SS The LDC / ADC including S Due to its linker unit (LU) containing L, it is more resistant to premature loss of its ligand unit. SS The primary linker is the M 1 or M 2 In addition to the moiety, a first optional stretcher unit (A) must be present, where A is selected from the group consisting of C1-C in combination with [HE]. 12 and an alkylene moiety, where this combination is sometimes referred to as A1, and A is the optional subunit (A O ) or A further comprises A' when the subscript b is 0 and the subscript a' is 1, where either value of the subscript b further denotes a subunit, and is represented as A2. If A can exist as a single separate unit or in the form of two separate units, both possibilities are represented as -A[HE]-A when the subscript b is 1. O - or, if the subscript b is 0, A[HE]-A' a’and for any value of the subscript b, it becomes -A[HE]- or -A1[HE]-A2- depending on the absence or presence of the second subunit, respectively. SS In any variation of A within the formula (I), the alkylene portion incorporates a cyclic basic unit or is substituted by an acyclic basic unit, and is otherwise optionally substituted.

[0203] Therefore, the primary linker of a drug-linker compound is sometimes referred to as the L of a ligand-drug conjugate. SS L to indicate that it is a precursor of SS ' is displayed as L SS , the Primary Linker comprises a first optional Stretcher unit (A) that must be present, and a maleimide (M 1 ) moiety (through which the targeting agent is attached), which in the case of an antibody or antigen-binding fragment thereof provides an antibody ligand unit. SS A C1~C 12 The alkylene portion is M 1 and the remainder of the linker unit, the latter of which is optionally linked to the imide nitrogen of the maleimide ring system of A O Depending on the absence or presence of / A' and [HE], if the subscript b is 1, then [HE]-A O -B- or [HE]-A' if the subscript b is 0 a’ In some of these aspects, the hydrolysis enhancing moiety, [HE], consists of or includes an optionally substituted electron-withdrawing heteroatom or functional group, which in some aspects, in addition to BU, is linked to the corresponding L of the LDC / ADC compound. SS M in part 2 After the drug linker compound is incorporated into the LDC / ADC compound, the L SS where M is a thio-substituted succinimide (M 2 ) moiety (i.e., the attachment of the Ligand unit to its Drug Linker moiety is 1via Michael addition of the sulfur atom of the reactive thiol functionality of the targeting agent to the maleimide ring system of

[0204] In some aspects, the cyclized basic unit (cBU) corresponds in structure to the acyclic basic unit via formal cyclization to the basic nitrogen of that unit, and thus the cyclic basic unit structure is substituted with an optionally substituted spiro C4-C6 basic unit in the first optional Stretcher unit present. 12 In such a construction, the spiro carbon is incorporated as a heterocyclo. 1 The maleimide nitrogen of M 2 and further comprising a Drug Linker moiety of Formula 1B or a Drug Linker compound of Formula IB, wherein the Drug Linker moiety is -[HE]-A O - or [HE]-A a’ -L including the first optional stretcher unit (A) as described above optionally present through SS In these aspects, the cyclic BU is linked to the remainder of the primary linker. In these aspects, the cyclic BU is linked to the M in a manner qualitatively similar to the hydrolysis of the acyclic basic unit (which can also be enhanced by [HE]). 2 From the succinimide moiety of M 3 The hydrolysis of the ring-opened form of the formula:

[0205] In some aspects, L SS L for primary linker B '-AB b -(sometimes a self-stabilizing (L SS ) to explicitly indicate that it is a precursor of the primary linker. SS ') is M if the subscript b is 1. 1 -A(BU)-[HE]-A O -B-, or M if the subscript b is 0. 1 -A(BU)-[HE]-A' a’ -, where M 1is a maleimide moiety, A incorporates or is substituted by BU, and is otherwise optionally substituted, and C1-C optionally combined with an optional hydrolysis enhancing moiety, [HE]. 12 alkylene, where the formula is M when A is a single individual unit. 1 -A(BU)-[HE]-B- or M 1 -A(BU)[HE]- or M if A is two subunits 1 -A1(BU)-[HE]-A2-B- or M 1 -A1(BU)-[HE]-A2-, where A1 and A2 are subunits of A.

[0206] In another aspect, L of the drug linker moiety of Formula 1B of the ADC of Formula 1A SS The primary linker is -M if the subscript b is 1. 2 -A(BU)-[HE]-A O -B- or -M when the subscript b is 0 2 -A(BU)-[HE]-A a’ -, where M 2 is a succinimide moiety, A is the first optional Stretcher unit present and is C1-C2 optionally combined with [HE], an optional hydrolysis enhancing moiety, incorporating or substituted by BU and otherwise optionally substituted. 12 Contains alkylene, and A O / A' is an optional subunit of A. If A is a single, separate unit, L SS -M 2 -A(BU)-[HE]-B- or -M 2 -A(BU)-[HE]-, where A is two subunits, L SS is -M when the subscript b is 0 or 1, respectively. 2 -A1(BU)-[HE]-A2- or -M 2It is represented by the formula -A1(BU)-[HE]-A2-B-.

[0207] In yet another aspect, the L of the drug linker moiety of Formula 1B of the LDC / ADC of Formula 1A S The primary linker is -M if the subscript b is 1. 3 -A(BU)-[HE]-A O -B- or -M when the subscript b is 0 3 -A(BU)-[HE]-A a’ -, where M 3 is a succinimidic acid amide moiety, A is a C1-C6 group optionally combined with an optional hydrolysis enhancing moiety, [HE], incorporating or substituted by BU and optionally substituted otherwise. 12 alkylene, and A O / A' is an optional subunit of A, where -A(BU)-[HE]-A O -or-A(BU)-[HE]-A a’ - becomes -A(BU)-[HE]- when A is a single separate unit, or -A1(BU)-[HE]-A2- when A is or contains two subunits.

[0208] L in the Drug Linker Moiety of Formula 1B for Some Ligand Drug Conjugates of Formula 1 SS Exemplary, but non-limiting, primary linkers include -L B -A-structure is [ka]

[0209] where the wavy line indicates the site of covalent attachment to the Ligand unit and the number sign (#) indicates the site of covalent attachment to the Branching unit (B) in Formula 1B in the upper structure where subscript b is 1, or the optional secondary linker (L) present in the lower structure where subscript b is 0. O) to W, and the dashed curve indicates optional cyclization, which is present if BU is a cyclic basic unit or absent if BU is an acyclic basic unit, where [HE] is an optional hydrolysis-enhancing moiety, and A O / A' is an optional subunit of A, the subscript z is 0 or an integer ranging from 1 to 6; each R d1 are independently selected from the group consisting of hydrogen and optionally substituted C1-C6 alkyl, or two R d1 , the carbon atoms to which they are attached and any intervening carbon atoms define an optionally substituted C3-C8 carbocyclo, and the remaining R d1 is, if present, independently hydrogen or optionally substituted C1-C6; and R a2 is —H or optionally substituted C1-C8 alkyl when BU is an acyclic basic unit, and R is —H or optionally substituted C1-C8 alkyl when BU is a cyclic basic unit. a2 must be other than -H, and BU and R a2 Optionally substituted spiro C4-C having a secondary or tertiary skeletal basic nitrogen atom together with the carbon atom to which it is attached 12 The definition of heterocyclo is therefore acyclic or cyclic BU can be used in conjunction with the corresponding bond (R a2 is hydrogen and BU is replaced by hydrogen) compared to the succinimide (M 2 ) moiety to form succinamide (M 3 ) moiety, and the cyclic basic unit can be linked to the above conjugate (R a2 is hydrogen and BU is replaced by hydrogen) rather than the corresponding drug-linker moiety of the LDC / ADC (R a2 is hydrogen and BU is acyclic BU) substantially retains the increased hydrolysis rate.

[0210] Exemplary but non-limiting L SS L containing ' BThe '-A- structure, which is sometimes present in drug linker compounds of Formula I used as intermediates in the preparation of ligand drug conjugate compositions, is [ka]

[0211] where BU and other variables are represented by L SS LDC / ADC with primary linker B -A- as defined above for the structure. A self-stabilizing linker precursor (L) containing a maleimide moiety SS When a drug linker compound having a SS The L' moiety contains a succinimide moiety. SS Prior to condensation with a reactive thiol functional group from a targeting agent, such as an antibody or antigen-binding fragment thereof, the basic nitrogen atom of BU is typically protonated or protected by an acid-labile protecting group.

[0212] A "self-stabilizing linker" is a self-stabilizing linker (L S ) corresponding M 3 A self-stabilizing linker (L) in a ligand drug conjugate, such as an antibody drug conjugate, that undergoes hydrolysis under controlled conditions to provide a hydroxyl group moiety. SS )'s M 2 an organic moiety derived from a containing moiety, where the LU component is the original M 2 Contains L SS The targeting moiety and M 1 Low likelihood of reversing the condensation reaction with the containing moiety. 3 In addition to the moiety, a self-stabilizing linker (L S ) comprises a first Stretcher unit (A) present and incorporating a cyclic basic unit or replaced by an acyclic basic unit, where A is M 3 , and L Scovalently attached to the remainder of the primary linker (i.e., B) or to the secondary linker (Lo) if B is absent. 3 The moiety is L in the ligand drug conjugate. SS The succinimide moiety (M 2 ) transformation, where M 2 The moiety L in the drug linker compound SS 'M' part 1 and a thio-substituted succinimide ring system resulting from the Michael addition of the sulfur atom of the reactive thiol functional group of the targeting agent to the maleimide ring system of M 2 The part derived from M 2 The thio substituent is less reactive to elimination reactions than the corresponding substituent in 2 The part derived from M 2 The corresponding succinamide (M 3 ) moiety, where M 2 undergoes hydrolysis of one of the carbonyl-nitrogen bonds of the succinimide ring system, the hydrolysis being assisted by the basic functionality of BU due to the suitable proximity resulting from that bond. Thus, the product of the hydrolysis is a carboxylic acid functionality, and L S M to 2 Contains L SS The BU has an amide functionality substituted with the remainder of the primary linker (minimum including any optional Stretcher units present) at the amide nitrogen atom corresponding to the imide nitrogen atom in the precursor. In some aspects, the basic functionality is a primary, secondary, or tertiary amine of an acyclic basic unit, or a secondary or tertiary amine of a cyclic basic unit. In other aspects, the basic nitrogen of the BU is the heteroatom of an optionally substituted basic functionality, such as in a guanidino moiety. In either aspect, the reactivity of the basic functionality of the BU to base-catalyzed hydrolysis is controlled by pH through reduction of the protonated state of the basic nitrogen atom.

[0213] Therefore, the self-stabilizing linker (L S) typically represents M covalently bonded to the first optional Stretcher unit present. 3 The stretcher unit A then has the structure of L, and incorporates a cyclic basic unit or is replaced by an acyclic basic unit. In some aspects, A is a separate single unit, and in other aspects, it is two or more subunits, typically represented by A-A, where two subunits are present as A / A, optionally in combination with [HE]. The stretcher unit A is then L S B or L of the primary linker O covalently bonded to W and its M 3 , A, A' a’ / B and BU components are -M 3 -A(BU)-[HE]-A' a’ - or M 3 -A(BU)-[HE]-A O -B-, where each subscript b is 0 or 1. When A is a single, separate unit, L S If the subscript b is 1, then -M 3 -A(BU)-[HE]-B- or -M 3 -A(BU)-[HE]-, and when A is two subunits, L S is -M when the subscript b is 0 or 1, respectively. 3 -A1(BU)-A2- or -M 3 It is represented by -A1(BU)-A2-B-, where BU represents either kind of basic unit (cyclic or acyclic).

[0214] LDC / ADC SS and L S -L in the primary linker B -A-(Here L B is M 2 or M 3 and A(BU) / A1(BU), and [HE] within these structures are arranged in the manner shown above, where BU is an acyclic basic unit), an exemplary non-limiting structure of [ka]

[0215] where the —CH(CHNH)C(═O)— moiety is A, and if A is a single, separate unit, then A O Or A' does not exist, or A O When / A' is present as A2, A is A1-A2-, and A / A1 is substituted by BU, where BU is an acyclic basic unit, which is -CH2NH2, having an optionally protonated basic nitrogen atom, and -C(=O)- in that moiety is an optional hydrolysis enhancing moiety [HE] present, and the hash symbol in the top structure indicates a covalent bond to B, and the hash symbol in the bottom structure indicates L O to W. Their exemplary structures are succinimide (M 2 ) moiety or succinamide (M 3 ) moiety, the latter containing L SS L S M assisted by -CH2NH2 in the conversion to 2 It arises from hydrolysis of the succinimide ring of

[0216] LDC / ADC SS and L S -L in the primary linker B -A-(Here L B is M 2 or M 3 and A(BU) / A1(BU), A O An exemplary non-limiting structure of (wherein / A' and [HE] are arranged in the manner shown above and BU is a cyclic basic unit) is: [ka] [ka]

[0217] and wherein these -M 2 -A(BU)-[HE]-A O / A' a’ - and -M 3 -A(BU)-[HE]-A O / A' a’ -Structure is A O is absent or the subscript a' is 0, so that A exists as a single, distinct unit; 2 -A(BU)-[HE]- and -M 3 -A(BU)-[HE]- or A O -M when / A' is present as a subunit of A, denoted A2 2 -A1(BU)-[HE]-A2- and -M 3 -A 1 (BU)-[HE]-A2-, and in either structure, BU is a cyclic basic unit of the form of an optionally protonated azetidine-3,3-diyl, which structure is an exemplary heterocyclic basic unit incorporated into A / A1. The heterocyclic corresponds to the aminoalkyl of the acyclic basic unit in the -A1(BU)- or -A(BU)- moiety, where the basic nitrogen of the acyclic basic unit is bonded to the M 2 The carbon atom alpha to the succinimide nitrogen and R a2 is formally at least partially cyclized via

[0218] -L above B The wavy line in each of the -A- structures represents the structurally corresponding drug-linker compound or its M 1 M in the containing intermediate 1 The hash symbol (#) in the above structure indicates the site of covalent attachment of the sulfur atom of the Ligand unit, which is derived from the reactive thiol functionality of the targeting agent upon Michael addition of the sulfur atom of the targeting agent to the maleimide ring system of the L moiety. SS or L SIn the structure below, L indicates the site of covalent attachment to B, the remainder of the primary linker. O The covalent binding site of M to W is shown. 2 The succinimide ring system of is asymmetrically substituted due to its thio substituent, and therefore has a different position relative to the free carboxylic acid group, succinamide (M 3 ) moiety is a regiochemical isomer of M 2 In the above structure, A O The carbonyl functionality shown adjacent to exemplifies the hydrolysis enhancement factor [HE] as defined herein.

[0219] -M above 3 -A(BU)-[HE]-A O / A' a’ -, -M 3 -A(BU)- and -M 3 The -A1(BU)-[HE]-A2- moiety (where BU is an acyclic or cyclic basic unit) forms a self-stabilizing linker (L S ) Exemplary -L containing primary linkers B These structures represent the -A- structures from which they are derived. 2 -A(BU)-[HE]-A O / A' a’ -, -M 2 -A(BU)- or -M 2 -A1(BU)-[HE]-A2- SS It is so named because it is less likely to lose its targeting moiety due to the reduced likelihood of elimination of the thio substituent of the ligand unit compared to the M moiety. 2 Compared to M 3 This is believed to be due to the greater conformational flexibility in (which no longer constrains the thio substituent in a conformation favorable for the E2 elimination reaction).

[0220] "Basic unit," as used herein, unless otherwise indicated or implied by context, refers to L SS Contains M 2The succinimide ring system in the moiety is hydrolyzed by the base-catalyzed BU corresponding to the L S a self-stabilizing linker (L) as described herein that is carried over to the moiety (i.e., catalyzes the addition of a water molecule to one of the succinimide carbonyl-nitrogen bonds); SS ) refers to the organic moiety in the primary linker. In some aspects, base-catalyzed hydrolysis of L SS In another aspect, base-catalyzed hydrolysis is initiated under controlled conditions that are permissive for the targeting ligand unit attached to L. SS is initiated upon contacting a drug linker compound comprising: SS M 1 Without being bound by theory, the following aspects illustrate various considerations for the design of suitable basic units. In one such aspect, the basic functionality of the acyclic basic unit and L SS M in 2 Its relative position to the component is such that BU is M 2 In another such aspect, their selection is such that a water molecule (whose nucleophilicity is enhanced by hydrogen bonding with the basic functional group of BU) can hydrogen bond with the carbonyl group of M 2 In a third such aspect, the selection is made so that the basic nitrogen in the protonation does not increase the electrophilicity of the succinimide carbonyl by inductive electron withdrawal to such an extent that it promotes premature hydrolysis, which requires compensation by an undesired excess of the drug linker compound. In a further such aspect, some combination of these mechanistic effects may be present in the L SS From L S The catalyst serves as a catalyst for the controlled hydrolysis of hydroxybenzoates to hydroxybenzoates.

[0221] Typically, acyclic basic units that can act via any of the above mechanistic aspects contain one carbon atom or 2 to 6 consecutive carbon atoms, more typically one carbon atom or 2 or 3 consecutive carbon atoms, where the carbon atoms connect the basic amino functionality of the acyclic basic unit to the L SS Connect to the remainder of the primary linker. 2 ) moiety to the corresponding ring-opened succinamide (M 3 The amine-bearing carbon chain of the acyclic basic unit is typically a carbon atom such as M 2 The succinimide nitrogen of (and hence the corresponding M 1 -C1-C of that moiety alpha to the site of attachment of A to the maleimide nitrogen of the A structure 12 L at the carbon of alkylene SS -L B It is attached to A of the -A- moiety. Typically, the alpha carbon in the acyclic basic unit has the (S) stereochemical configuration, or a configuration corresponding to the configuration of the alpha carbon of an L-amino acid.

[0222] As previously described, the acyclic or cyclized BU is typically SS M 1 Or M 2 or L S M 3 otherwise, C1 to C are substituted as necessary. 12 connected via an alkylene moiety, which incorporates a cyclized basic unit or is replaced by an acyclic basic unit; M 1 Or M 2 the maleimide nitrogen or succinimide nitrogen of M 3 In some aspects, an otherwise optionally substituted C1-C cyclic basic unit incorporating a cyclic basic unit is bonded to the amide nitrogen atom of 12The alkylene moiety is covalently bonded to [HE], typically through the intermediation of an ether, ester, carbonate, urea, disulfide, amide carbamate or other functional group, more typically through an ether, amide or carbamate functional group. Similarly, acyclic forms of BU are typically bonded to M 2 M resulting from hydrolysis of the succinimide ring system of 1 Or M 2 the imino nitrogen atom of the maleimide or succinimide ring system of M 3 C1-C bonded to the amide nitrogen 12 Substitution of acyclic basic units at the same carbon of the alkylene moiety, L B '-A-(where L B ' is M1 or -L B -A-, where L B is M 2 or M 3 otherwise, optionally substituted C1 to C 12 L via the alkylene moiety SS M 1 Or M 2 or L S M 3 is connected to.

[0223] In some aspects, the cyclic basic unit is bonded to the same alpha carbon as the acyclic basic unit, independently selected from A / A, otherwise optionally substituted C-C 12 Alkylene (R a2 ) to form a spirocyclic ring system, the cyclic basic unit is incorporated into the structure of A / A1 rather than being a substituent of A / A1 as when BU is acyclic. In these aspects, the formal cyclization is to the acyclic basic unit to the basic amine nitrogen, thus forming an optionally substituted symmetrical or asymmetrical spiro C4-C system, depending on the relative carbon chain lengths of the two alpha carbon substituents. 12The cyclic basic unit is provided as a heterocyclo (where the basic nitrogen is a basic backbone heteroatom). In order for the cyclization to substantially retain the basic character of the acyclic basic unit in the cyclic basic unit, the basic nitrogen atom of the nitrogen of the acyclic basic unit must be a basic nitrogen atom of a primary or secondary amine, not a basic nitrogen atom of a tertiary amine, since this may result in a quaternized backbone nitrogen in the heterocyclo of the cyclic basic unit. In this aspect of the formal cyclization of the acyclic basic unit to the cyclic basic unit, the basic nitrogen is a basic nitrogen atom of L. SS From L S M in the conversion to 2 From M 3 To substantially retain the ability to support hydrolysis to C4-C6, the resulting cyclic basic unit structures in these primary linkers should have no more than three, typically one or two, intervening carbon atoms in a spiro C4-C6 12 The basic nitrogen atom of the heterocyclic component may be located between the basic nitrogen atom and the spiro carbon atom. SS and L S Primary linkers are further described by embodiments of the present invention.

[0224] "Hydrolysis enhancing moiety," as used herein, unless otherwise indicated or implied by context, refers to a hydrolysis enhancing moiety (L SS The primary linker and its hydrolysis product, L S L B '-A- or -L B The term "hydrolysis enhancing [HE] moiety" refers to an electron withdrawing group or moiety optionally present in the first optional Stretcher unit (A) in -A-. In some aspects, the term "hydrolysis enhancing [HE] moiety refers to an electron withdrawing group or moiety optionally present in the first optional Stretcher unit (A) in -A-. 2 L in the drug linker portion of LDC / ADC is attached to the imide nitrogen of the SS When present as a component of A / A1 in M 2 Due to the electron-withdrawing effect of [HE], depending on the proximity of the L S M of the primary linker 3In the case of A / A1 incorporating or substituted by a cyclic or acyclic basic unit, respectively, the derivatization of M 3 M to increase the rate of hydrolysis to 2 The potential effect of [HE] on the carbonyl group of M and the above-mentioned effect of either type of BU are 1 -A(BU)- and A / A1 combined with [HE] M 1 Formula M having two variants represented by the formula -A1(BU)-[HE]-A2- 1 -A(BU)-[HE]-A O / A' a’ -L B During the preparation of the ligand-drug conjugate from the drug-linker compound containing the '-A- structure, M 1 Instead, under controlled conditions (such as when the pH is deliberately increased to reduce the state of protonation of the basic units), hydrolysis (of the general formula -M of the ligand-drug conjugate compound) is achieved. 2 -A(BU)-[HE]-A O / A' a’ - or more specifically the formula -M 2 -A(BU)- or -M 2 -A1(BU)-A2-no-L B -A-structure to its corresponding -M 3 -A(BU)-[HE]-A O / A' a’ -, -M 3 -A(BU)- or M 3 The combined effect of BU and [HE] in promoting the conversion of BU to the -A1(BU)-[HE]-A2-formula is 1 An excessive molar excess of the drug linker compound is not required to compensate for hydrolysis of the moiety. 2 providing a targeting ligand unit attached to the succinimide ring system of 1 The Michael addition of the sulfur atom of a reactive thiol functional group of a targeting agent to the maleimide ring system of typically results in M 1Without being bound by theory, at low pH, such as when the basic amine of BU is in the form of a TFA salt, the hydrolysis of M in the drug-linker product occurs at a rate that effectively competes with the hydrolysis of M. 1 The premature hydrolysis of is believed to be much slower than when the pH is raised to a suitable pH for base catalysis using an appropriate buffer, and a tolerable molar excess of the drug linker compound can be used to increase the M 1 An early M Michael addition occurs during the time course for the sulfur atom of the reactive thiol functional group of the targeting agent to reach or near completion. 1 It is believed that this is sufficient to offset any loss due to hydrolysis.

[0225] As discussed previously, the enhancement of carbonyl hydrolysis by either type of basic unit depends on the basicity of the functional group and the M 1 / M 2 It depends on the distance of the basic functional group to the carbonyl group. Typically, [HE] is M 2 , or M derived from it 3 C1 to C of A / A1 are bonded to 12 A carbonyl moiety or other carbonyl-containing functional group located distal to the end of the alkylene, and which also provides a covalent bond to A2 or an optional secondary linker if B is absent and A is a single, separate unit. Carbonyl-containing functional groups other than ketones include esters, carbamates, carbonates, and ureas. L SS When [HE] is a carbonyl-containing functional group other than a ketone in the drug linker portion of an ADC having a primary linker, the functional carbonyl moiety shared with A / A1 is typically selected from the group consisting of M 2 Optionally substituted C1-C at other points in A / A1 distal to the bond site to the imide nitrogen atom of 12 In some aspects, the [HE] moiety may be sufficiently distal from the imide nitrogen to which A / A is covalently attached, such that M2 No discernible effect or only a small effect is observed on the hydrolytic susceptibility of the succinimide carbonyl-nitrogen bond of the containing moiety; instead, the effect is primarily driven by BU.

[0226] "Stretcher unit," as used herein, unless otherwise indicated or implied by context, refers to an optional organic moiety in a primary or secondary linker of a linker unit of a drug-linker compound or drug-linker moiety of a ligand drug conjugate, such as an antibody drug conjugate, that physically separates the targeting ligand unit (L) from the optional secondary linker, if present. SS or L S In the case of a Primary Linker, the first Optional Stretcher is present because it provides a basic unit for these types of Primary Linkers. In any type of Primary Linker, the L Optional Stretcher unit may be present if there is insufficient steric relief from the Ligand unit in the absence of that Optional Stretcher unit to allow efficient processing of the Secondary Linker to release the Drug unit as the free drug. R The presence of a first optional Stretcher unit (A) in may also be required. Alternatively, or in addition to steric relief, these optional components may be included for synthetic ease in preparing the Drug Linker Compound. In some aspects, when subscript b is 1, the first or second optional Stretcher unit (A or A', respectively) may be a single unit or contain multiple subunits (e.g., when A has two subunits, represented by -A1-[HE]-A2-). In other aspects, when subscript b is 0, typically when subscript b is 0 and subscript a' is 1, A is one separate unit or has two separate subunits. In still other aspects, B / A' has 2 to 4 independently selected separate subunits.

[0227] In some aspects, L R LSS / L S where M of the drug linker compound is 1 or M of the drug linker portion of the LDC / ADC compound 2 / M 3 In addition to the covalent bond to A, A[HE](A O / A' is absent) or A1-[HE]-A2 (A O / A' exists), A is used as needed. O / A' a’ via a branching unit (B) or an optional secondary linker (L O ) and is generally attached to W in A-[HE]-A O / A a’ -, where A, when present as A / A1 and A2, O / A a’ MoL SS / L S is a component of.

[0228] In some aspects, the subunits of A or A' or any of these Stretcher units are -L P (PEG)-, where L P is a parallel connector unit, and PEG is a PEG unit as defined elsewhere. Thus, in some of these aspects, the drug-linker portion of a ligand-drug conjugate or linker unit of a drug-linker compound where subscript b is 0 and subscript a' is 1 is -A1-[HE]-L P (PEG)-, where A' is L P (PEG)- and exists as A2. The subscript b is 1 and A O is present as A2, the drug linker portion of the ligand drug conjugate or the linker unit of the drug linker compound is -A1-[HE]-L P In yet another aspect, the subscript b is 1 and the subscript a' is 1, and the Ligand Drug Conjugate or Drug Linker Compound contains the formula -A-[HE]-A O -BLP (PEG), where A' is L P (PEG).

[0229] In some aspects, when the subscript a is 1, such that a first optional stretcher unit (A) is present, that unit typically has at least one carbon atom, where this atom is a group selected from the group consisting of L B / L B Connect ' to [HE]. L B ' is the L of the drug linker compound SS 'L for primary linker B In some of those aspects, the Stretcher unit is substituted by or incorporates a basic unit, and is otherwise optionally substituted, and one of its radical carbon atoms is bonded to a maleimide nitrogen atom and the other radical carbon atom is bonded to [HE] (where [HE] is an optional hydrolysis-enhancing moiety present), C1-C 12 In another aspect, L R ' is L SS ', but nevertheless containing a maleimide moiety or any other L B ' part, L B ' is attached to an optional first Stretcher unit (A), which in some aspects is an optionally substituted C1-C2 optionally combined with [HE]. 12 It is alkylene. Therefore, L R ' is L SS In some aspects, the first stretcher unit on demand exists, and C1 to C 12 The alkylene moiety, [HE], and the optional subunit (A when the subscript b is 1) O or A' if subscript b is 0 a’ ), including L R ' is L SS If all of these are L R ', where A is C1 to C 12Distal to the site of attachment of the alkylene moiety to the imide nitrogen atom, L R ' is a component of B or L O In another aspect, when the subscript a is 1 and A exists as a single separate unit or is of two subunits, A is -A-[HE]-A O / A a’ -having the general formula: O / A' a’ is an optional subunit of A, or more specifically, A O is present as the second subunit of A and subscript b is 1, or subscript a' is 1 and subscript b is 0, such that A' is present as the second subunit of A, then A has the formula -A1-[HE]-A2-. In such aspects, A O / A2 or A' / A2 is an α-amino acid, β-amino acid or other amine-containing acid residue.

[0230] "Branching unit," as used herein, unless otherwise indicated or implied by context, refers to a trifunctional or polyfunctional organic moiety that is an optional component of a linker unit (LU). A branching unit (B) is a unit that is formed by combining multiple -L O When the -D moiety is a single drug-linker moiety, it is present in the primary linker of the drug-linker moiety of Formula 1A of an LDC / ADC of Formula 1A. In an LDC / ADC having the above generalized formula, the absence or presence of a branching unit is determined by B b where each subscript b is 0 or 1. The branching units are at least trifunctional for incorporation into the primary linker. Multiple -L per drug linker moiety of formula -LU-D O Drug linkers or LDC / ADC compounds having branching units due to the -D moiety typically have the formula -A' a’ -WY y - each secondary linker (L O), where A' is a second optional Stretcher unit; the subscript a' is 0 or 1, indicating the absence or presence of A', respectively; W is a peptide cleavable unit; Y is a Spacer unit; and the subscript y is 0, 1, or 2, indicating the absence or presence of one or two Spacer units, respectively.

[0231] In some aspects, the residue of a natural or unnatural amino acid or another amine-containing acid compound having a functionalized side chain is selected from two -L O In some of these aspects, B is a lysine, glutamic acid, or aspartic acid residue in the L or D configuration, in which the epsilon-amino, gamma-carboxylic acid, or beta-carboxylic acid functional groups, respectively, together with their amino and carboxylic acid ends, interconnect B within the remainder of LU. O Branching units with greater functionality for attachment of -D moieties typically contain the required number of trifunctional subunits.

[0232] "Natural amino acid," as used herein, unless otherwise indicated or implied by context, refers to a naturally occurring amino acid, i.e., arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, or a residue thereof, in the L- or D-configuration, unless otherwise specified or implied by context.

[0233] "Unnatural amino acid," as used herein, unless otherwise indicated or implied by context, refers to an α-amino-containing acid or residue thereof that has the backbone structure of a natural amino acid but has a side chain group attached to the α-carbon that is not found in natural amino acids.

[0234] "Nonclassical amino acid," as used herein, unless otherwise indicated or implied by context, refers to an amine-containing acid compound whose amine substituent is not attached to a carbon that is alpha to a carboxylic acid and is therefore not an alpha-amino acid. Nonclassical amino acids include β-amino acids, in which a methylene is inserted between the carboxylic acid and amino functional groups of a natural or unnatural amino acid.

[0235] "Peptide," as used herein, unless otherwise indicated or implied by context, refers to a polymer of two or more amino acids in which the carboxylic acid group of one amino acid is linked to the alpha-amino group of the adjacent amino acid in a peptide sequence. Methods for preparing amide bonds in polypeptides are further provided in the definition of amide. Peptides can be composed of naturally occurring amino acids in the L- or D-configuration, and / or non-natural and / or non-classical amino acids.

[0236] "Protease," as defined herein, refers to a protein capable of enzymatic cleavage of carbonyl-nitrogen bonds, such as amide bonds typically found in peptides. Proteases are divided into six major classes: serine proteases, threonine proteases, cysteine ​​proteases, glutamic acid proteases, aspartic acid proteases, and metalloproteases, named according to the catalytic residue within the active site that is primarily responsible for cleaving the carbonyl-nitrogen bond of its substrate. Proteases are characterized by different specificities that depend on the identity of residues located on the N- and / or C-terminal sides of the carbonyl-nitrogen bond and their different distribution (intracellular and extracellular).

[0237] Regulatory proteases are typically intracellular proteases, and are required for the regulation of abnormal or otherwise undesirable cellular activities that are sometimes abnormal or dysregulated within cells. In some cases, when a peptide cleavage unit is directed to a protease that has a preferential distribution within cells, the protease is a regulatory protease, and is involved in the maintenance or proliferation of cells. These proteases include cathepsins. Cathepsins include serine proteases, cathepsin A, cathepsin G, aspartic acid proteases, cathepsin D, cathepsin E, and cysteine ​​proteases, cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin Z.

[0238] A "peptide cleavable unit," as used herein, unless otherwise indicated or implied by context, refers to an organic moiety within the Drug Linker portion of a Ligand Drug Conjugate Compound or a secondary linker of a Drug Linker Compound that provides a recognition site for a protease and, upon enzymatic action by that protease, is capable of enzymatically releasing the conjugated Drug unit (D) as the free drug.

[0239] The recognition site of protease cleavage is sometimes limited to that recognized by the protease found in the nominal normal cells targeted by the ligand-drug conjugate, which is specific to abnormal cells such as cancer cells or the environment of nearby abnormal cells, but it can also be found in normal cells.For this purpose, the peptide is typically resistant to circulating proteases to minimize the premature release of free drug or its precursor, which may otherwise cause off-target adverse events resulting from systemic exposure to free drug.In some aspects, the peptide has one or more D-amino acids or non-natural or non-classical amino acids to achieve its resistance.In some of these aspects, the sequence includes a dipeptide or tripeptide in which the P2' position contains a D-amino acid and the P1' position contains one of the 20 naturally occurring L-amino acids other than L-proline.

[0240] In these aspects, the reactive site is more likely to be activated enzymatically after immunological selective binding to the targeted antigen.In some of these aspects, the targeted antigen is present on abnormal cells, so that the recognition site is more likely to be activated enzymatically after the ligand-drug conjugate compound is internalized into the targeted abnormal cells.Therefore, these abnormal cells should present the targeted antigen in a higher copy number than normal cells, thereby alleviating on-target adverse events.In other of these aspects, the targeted antigen is present on normal cells that are in the environment of the abnormal cells and are unique to them, so that the recognition site is more likely to be activated enzymatically after the ligand-drug conjugate compound is internalized into these targeted normal cells.Therefore, these normal cells should present the targeted antigen in a higher copy number than normal cells that are distant from the site of cancer cells, thereby alleviating on-target adverse events.

[0241] In any one of the above aspects, the protease reactivity of the recognition site is higher in tumor tissue homogenate than in normal tissue homogenate.In some aspects, this higher reactivity is due to the fact that the amount of intracellular protease activity in the targeted cells of tumor tissue is higher than that in the normal cells of normal tissue, and / or the protease activity in the interstitial space of normal tissue is lower than that of the protease of the peptide cleavable unit of conventional ligand-drug conjugate.In these aspects, the intracellular protease is a regulatory protease, and in addition to being selectively cleaved by the protease in tumor tissue homogenate compared to that in normal tissue homogenate, the peptide bond of the peptide cleavable unit can be selectively cleaved by the intracellular regulatory protease compared to that of serum proteases.

[0242] Secondary linkers containing peptide cleavable units are typically -A' a’ -WY y -, where A' is a second optional spacer unit when subscript b is 1; subscript a' is 0 or 1, W is a peptide cleavable unit; Y is an optional spacer unit; and subscript y is 0, 1, or 2. When subscript b is 0 and subscript a' is 1, A' becomes a subunit of A, such that the secondary linker is -WY yFor a secondary linker of either formula, the action of a protease on the peptide sequence comprising the peptide cleavable unit results in the direct release of D when subscript y is 0, or results in a drug-linker fragment of formula Y-D as a precursor to the free drug when subscript y is 1 (where Y typically self-immolates to provide the free drug), or results in a first drug-linker fragment of formula Y-Y'-D when subscript y is 2 (where Y is a first spacer unit that self-immolates to provide a second drug-linker fragment of formula Y'-D, and where Y' is a second spacer unit that degrades to complete the release of D as the free drug).

[0243] In some aspects, the drug linker compound in which the secondary linker contains a peptide cleavable unit has the formula IC:

[0244] [ka]

[0245] and the corresponding drug linker portion of the ligand drug conjugate is represented by the structure of Formula 1D or Formula 1E:

[0246] [ka]

[0247] [ka]

[0248] where W is a peptide cleavable unit and M of formula IC 1 -A a -B b -, -M in Eq. 1D 2 -A a -B b - and -M in Formula 1E 3 -A a -B b- is a primary linker, where M 1 is the maleimide moiety; M 2 is the succinimide moiety; M 3 is a succinamide moiety; Y is an optional spacer unit, such that the subscript y is 0 or 1, or Y y is -Y-Y', such that the subscript y is 2, and Y and Y' are the first and second spacer units, respectively, and the remaining variables are as defined for the drug linker compounds of formula IA and the drug linker moieties of formula 1A. 1 L of a drug linker compound containing a moiety SS 'primary linker, and M 2 The L of the drug linker moiety in some LDCs / ADCs contains a SS Primary linkers are of the formula in which A or a subunit thereof is replaced by or incorporates a basic unit. Other primary linkers are of the formula 1C above, 2 Contains L SS from the primary linker by hydrolysis of the succinimide moiety to form M 3 L derived by providing a containing moiety S It is the primary linker.

[0249] In any one of the above aspects, the amide bond that is specifically cleaved by a protease produced by or present in the targeted cell is a bond to an amino group of the Spacer unit (Y) or Drug unit (if Y is absent). Thus, action of the protease on the peptide sequence within W results in the release of D as a free drug or its precursor Y. y -D, which spontaneously fragments to give the free drug.

[0250] A "spacer unit", as used herein, unless otherwise indicated or implied by context, is a group of the formula -A' a’ -WY y - the secondary linker (LO ), where the subscript y is 1 or 2 and indicates the presence of one or two spacer units within the linker unit of the drug-linker compound or the drug-linker portion of the ligand drug conjugate, where A' is a second optional spacer unit, as some aspects described herein, that becomes part of the primary linker to which the secondary linker is covalently attached as a subunit of the first optional spacer unit present, and the subscript a' is 0 or 1 and indicates the absence or presence of A'; Y is a spacer unit, and W is a group of the formula -P n··· [P3]-[P2]-[P1]- or -P n··· A peptide cleavable unit of the formula [P3]-[P2]-[P1]-[P-1]-, where subscript n ranges from 0 to 12 (e.g., 0 to 10, 3 to 12, or 3 to 10), and P1, P2, and P3 are amino acid residues that confer selectivity for protease cleavage by tumor tissue homogenate over normal tissue homogenate, as described herein. When subscript y is 1, the spacer unit is covalently attached to W and the drug unit (D), or when subscript y is 2, the spacer unit is covalently attached to another such moiety (Y') that is covalently attached to D. As further described by embodiments of the present invention, protease action on W initiates the release of D as a free drug.

[0251] A "self-immolative moiety," as used herein, refers to a bifunctional moiety within a self-immolative spacer unit (Y), where the self-immolative moiety is covalently bonded to a heteroatom of D or to a functional group shared between Y and D, and is optionally substituted where permitted, and is also covalently bonded to a peptide cleavable unit via another optionally substituted heteroatom (J), where J is -NH- or an appropriately substituted nitrogen atom within an amide functional group, such that the self-immolative moiety incorporates these drug linker components into a typically stable three-part molecule unless activated.

[0252] Upon cleavage of the peptide bond between P1 / P-1 and Y, the first drug linker fragment, D, or Y'-D, spontaneously separates from the tripartite molecule by self-destruction of the self-immolative portion of the self-immolative spacer unit. In some aspects, the component of the self-immolative spacer unit intervening between Y'-D or D and the optionally substituted heteroatom J of Y (where J is bonded to W) is -C6-C 24 Arylene-C(R 8 )(R 9 )-, -C5~C 24 Heteroarylene-C(R 8 )(R 9 )-, -C6~C 24 Arylene-C(R 8 )=C(R 9 )- or -C5~C 24 Heteroarylene-C(R 8 )=C(R 9 )-, optionally substituted, where R 8 and R 9 are as described in the embodiments of the present invention, and typically, C6 to C 10 Arylene-CH2- or C5-C 10Heteroarylene-CH2-, where the (hetero)arylene is optionally substituted, and where the moiety of the self-immolative spacer unit can undergo fragmentation to form an imino-quinone methide or related structure by a 1,4 or 1,6-elimination reaction, concomitantly releasing D or Y'-D upon cleavage of the protease-cleavable bond between J and W. In some aspects, self-immolative spacer units having the above moieties attached to J are exemplified by an optionally substituted p-aminobenzyl alcohol (PAB) moiety, ortho- or para-aminobenzyl acetal, or other aromatic compounds electronically similar to the PAB group (i.e., PAB-type), such as 2-aminoimidazole-5-methanol derivatives (see, e.g., Hay et al., 1999, Bioorg. Med. Chem. Lett. 9: 2237), or those in which the phenyl group of the p-aminobenzyl alcohol (PAB) moiety is replaced by heteroarylene.

[0253] Without being bound by theory, an aromatic carbon of the arylene or heteroarylene group of the PAB or PAB-type portion of a self-immolative spacer unit incorporated into a linker unit is substituted with J, where the electron-donating heteroatom of J is attached to the cleavage site of W and the electron-donating ability of that heteroatom is weakened (i.e., the EDG ability is masked by the incorporation of the self-immolative portion of the self-immolative spacer unit into the linker unit). The other substituent of the hetero(arylene) is an optionally substituted heteroatom of D, an optionally substituted functional group shared between Y and D, or a benzylic carbon attached to a second spacer unit (Y') attached to the drug unit (D), where the benzylic carbon is attached to another aromatic carbon atom of the central arylene or heteroarylene, where the aromatic carbon bearing the weakened electron-donating heteroatom is either adjacent (i.e., 1,2 related) or two positions further away from the benzylic carbon atom (i.e., 1,4 related). The functionalized EDG heteroatom is selected so that upon processing of the cleavage site of W, the electron-donating ability of the masked heteroatom is restored, thus causing a 1,4- or 1,6-elimination reaction to occur, resulting in either the expulsion of D from the benzyl substituent as the free drug, or the release of Y'-D, followed by self-immolation of Y' to provide the free drug and elicit a therapeutic effect. Exemplary self-immolative moieties and self-immolative spacer units having these self-immolative moieties are exemplified by embodiments of the present invention.

[0254] Other examples of self-immolative groups include, but are not limited to, aromatic compounds electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (see, e.g., Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization when amide bond is hydrolyzed can be used, such as substituted and unsubstituted 4-aminobutyric acid amide (see, for example, Rodrigues et al., 1995, Chemistry Biology 2: 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (see, for example, Storm et al., 1972, J. Amer. Chem. Soc. 94: 5815) and 2-aminophenylpropionic acid amide (see, for example, Amsberry et al., 1990, J. Org. Chem. 55: 5867). The removal of amine-containing drugs substituted at the a-position of glycine (see, for example, Kingsbury et al., 1984, J. Med. Chem. 27: 1447) is also an example of a self-immolative group. In one embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, which can be used for the incorporation and release of multiple drugs, as described in WO2007 / 011968. Additional self-immolative spacers are described in WO2005 / 082023.

[0255] A "methylene carbamate unit," as used herein, unless otherwise indicated or implied by context, refers to an organic moiety that is capable of self-immolation and is interposed between a first self-immolative spacer unit and a drug unit in a ligand drug conjugate or a linker unit of a drug linker compound, and is thus an exemplary second spacer unit.

[0256] The methylene carbamate (MAC) unit attached to the Drug unit has Formula III: [ka]

[0257] or a pharmaceutically acceptable salt thereof, wherein the wavy line indicates the covalent bond of the methylene carbamate unit to the first self-immolative spacer unit (Y); D is a drug unit having a functional group (e.g., a hydroxyl, thiol, amide, or amine functional group) incorporated into the methylene carbamate unit; and T* is a heteroatom from the functional group, including oxygen, sulfur, or optionally substituted nitrogen as -NH-. Upon cleavage of the linker unit containing the MAC unit, the first self-immolative spacer unit (Y) attached to the MAC unit as the second self-immolative spacer unit (Y') undergoes fragmentation to release -Y'-D of Formula III. The MAC unit then spontaneously decomposes, completing the release of D as the free drug, a presumed mechanism for which is illustrated by embodiments of the present invention.

[0258] "PEG unit," as used herein, means [ka] "PEG" refers to a group containing a polyethylene glycol moiety (PEG) having repeating ethylene glycol subunits having the formula:

[0259] PEGs include polydisperse PEGs, monodisperse PEGs, and discontinuous PEGs. Polydisperse PEGs are heterogeneous mixtures of sizes and molecular weights, while monodisperse PEGs are typically isolated from a discontinuous mixture and therefore provide a single chain length and molecular weight. Discontinuous PEGs are compounds synthesized in a stepwise manner without a polymerization process. Discontinuous PEGs provide a single molecule with a defined and specified chain length.

[0260] PEG units contain at least 2 subunits, at least 3 subunits, at least 4 subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits. Some PEG units contain up to 72 subunits.

[0261] A "PEG capping unit," as used herein, is a nominally non-reactive organic moiety or functional group that terminates the free or untethered end of a PEG unit, and in some aspects is other than hydrogen. In these aspects, the PEG capping unit is a methoxy, ethoxy, or C1-C6 ether, or -CH2-CO2H, or other suitable moiety. Thus, the ether, -CH2-CO2H, -CH2CH2CO2H, or other suitable organic moiety serves as a cap for the terminal PEG subunit of a PEG unit.

[0262] "Parallel connector unit," as used herein, unless otherwise indicated or implied by context, refers to the organic moiety of a Drug Linker Compound or the Drug Linker portion of a Ligand Drug Conjugate Compound, which is typically present in that Linker unit as a subunit of a first or second Stretcher unit, where the parallel connector unit (L P) can orient the PEG unit attached thereto in a parallel orientation with a hydrophobic drug unit, referred to herein as a hydrophobic drug unit, thereby at least partially reducing the hydrophobicity of that drug unit. P and related PEG unit and PEG capping unit structures are described in WO2015 / 5057699, which is specifically incorporated herein by reference, and in some aspects, L P is a trifunctional α-amino acid, β-amino acid or other trifunctional amine-containing acid residue.

[0263] As used herein, terms such as "cleaved intracellularly" and "intracellular cleavage" refer to a metabolic process or reaction within a target cell that occurs in a Ligand Drug Conjugate or the like, whereby the covalent bond between the Drug unit and the Ligand unit of the conjugate through its Linker unit is broken, resulting in the release of a D + As described herein, in some embodiments, D is initially released as an adduct of the Drug Unit with one or more self-immolative spacers, which subsequently spontaneously separate from the Drug Unit to release D as the free drug.

[0264] "Hematologic malignancies," as used herein, unless otherwise indicated or implied by context, refer to blood cell tumors originating from cells of lymphoid or myeloid origin and are synonymous with the term "liquid tumors." Hematologic malignancies can be classified as indolent, moderately aggressive, or highly aggressive.

[0265] "Lymphoma," as used herein, unless otherwise indicated or implied by context, refers to a hematological malignancy that usually arises from hyperproliferative cells derived from the lymphatic system. Lymphomas are sometimes classified into two major types: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Lymphomas can also be classified according to the normal cell type that most closely resembles the cancer cells, according to phenotypic, molecular, or cytogenetic markers. Lymphoma subtypes under this classification include, but are not limited to, mature B-cell neoplasms, mature T-cell and natural killer (NK) cell neoplasms, Hodgkin's lymphoma, and lymphoproliferative disorders associated with immune deficiency. Lymphoma subtypes include precursor T-cell lymphoblastic lymphoma (sometimes called lymphoblastic leukemia because the T-cell lymphoblasts are generated in the bone marrow), follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic lymphoma (sometimes called leukemia because of peripheral blood involvement), MALT lymphoma, Burkitt lymphoma, mycosis fungoides and its more aggressive variant, Sézary syndrome, peripheral T-cell lymphoma not otherwise specified, nodular sclerosis of Hodgkin lymphoma, and mixed cellularity subtype Hodgkin lymphoma.

[0266] "Leukemia," as used herein, unless otherwise indicated or implied by context, refers to a hematological malignancy that typically arises from hyperproliferative cells of myeloid origin, including, but not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL). Other leukemias include hairy cell leukemia (HCL), T-cell lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia.

[0267] "Hyperproliferative cells," as used herein, unless otherwise indicated or implied by context, refer to abnormal cells characterized by unwanted cell growth or an abnormally high or sustained rate of cell division or other cellular activity unrelated to or uncoordinated with that of surrounding normal tissue. In some aspects, the hyperproliferative cells are hyperproliferative mammalian cells. In other aspects, the hyperproliferative cells are overstimulated immune cells, as defined herein, where these states of cell division or activation persist after cessation of stimuli that may have initially triggered these changes in cell division. In other aspects, the hyperproliferative cells are transformed normal cells or cancer cells, where these states of uncontrolled and progressive cell proliferation can result in benign, potentially malignant (premalignant), or actually malignant tumors. Hyperproliferative states resulting from transformed normal cells or cancer cells include, but are not limited to, those characterized by precancer, hyperplasia, dysplasia, adenoma, sarcoma, blastoma, carcinoma, lymphoma, leukemia, or papilloma. Precancer is usually defined as a lesion that exhibits histological changes and is associated with an increased risk of cancer development, and sometimes possesses some, if not all, of the molecular and phenotypic characteristics that characterize cancer. Hormone-associated or hormone-responsive precancers include prostatic intraepithelial neoplasia (PIN), particularly high-grade PIN (HGPIN), atypical small acinar proliferation (ASAP), cervical dysplasia, and ductal carcinoma in situ. Hyperplasia generally refers to cell proliferation within an organ or tissue beyond that normally observed, which may lead to the enlargement of the entire organ or the formation or growth of a benign tumor. Hyperplasia includes, but is not limited to, endometrial dysplasia (endometriosis), benign prostatic hyperplasia, and ductal hyperplasia.

[0268] "Normal cells," as used herein, unless otherwise indicated or implied by context, refer to cells that undergo coordinated cell division associated with maintaining the cellular integrity of normal tissues, or with the replenishment of circulating lymphoid or blood cells required by regulated cell turnover, or with tissue repair necessitated by injury, or with a regulated immune or inflammatory response resulting from exposure to pathogens or invasion by other cells, with the induced cell division or immune response terminating upon completion of the necessary maintenance, replenishment, or pathogen clearance. Normal cells include cells that proliferate normally, normal quiescent cells, and normally activated immune cells. Normal cells include normal quiescent cells, which are capable of regulating these quiescent G o These are non-cancerous cells in the immune system that have not been stimulated by stress or mitogens, or that are normally inactive or have not been activated by exposure to pro-inflammatory cytokines.

[0269] "Abnormal cells," as used herein, unless otherwise indicated or implied by context, refer to normal cells that have become dysfunctional due to an inability to properly regulate their spontaneous intracellular activity, either in a disproportionate response to external stimuli or, in some cases, due to mutations. Abnormal cells include hyperproliferative cells and overstimulated immune cells, as these terms are defined elsewhere. When present in an organism, these cells typically interfere with the function of otherwise normal cells, causing harm to the organism and increasing their destructive potential over time. Abnormal cells include cancer cells, overactivated immune cells, and other unwanted cells of an organism. Abnormal cells are also referred to as nominally normal cells that are apparently in the environment of abnormal cells but nevertheless support the growth and / or survival of these other abnormal cells (e.g., tumor cells); as a result, targeting these normally normal cells indirectly inhibits the growth and / or survival of tumor cells.

[0270] "Overstimulated immune cells," as used herein, unless otherwise indicated or implied by context, refer to cells involved in innate or adaptive immunity characterized by a state of abnormal persistent proliferation or inappropriate stimulation that occurs after the cessation of a stimulus that may have initially induced a change in proliferation or stimulation, or that occurs in the absence of any external insult. In many cases, a state of persistent proliferation or inappropriate stimulation leads to a chronic state of inflammation characteristic of a disease state or condition. In some cases, the stimulus that may have initially induced a change in proliferation or stimulation is not due to an external insult but is internally derived, as in the case of autoimmune diseases. In some aspects, overstimulated immune cells are proinflammatory immune cells that have been overactivated through chronic exposure to proinflammatory cytokines.

[0271] In some aspects of the present invention, the ligand drug conjugate compounds of the ligand drug conjugate compositions bind to antigens preferentially presented by aberrantly proliferating or inappropriately or persistently activated proinflammatory immune cells, including classically activated macrophages or type 1 T helper (Th1) cells, which are activated by interferon-gamma (INF-γ), interleukin-2 (IL-2), interleukin-10 (IL-10), and tumor necrosis factor-beta (TNF-β), i.e., macrophages and CD8 + Produces cytokines involved in T cell activation.

[0272] "Bioavailability," unless otherwise indicated or implied by context, refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term indicating measurement of both the time (rate) and total amount (extent) of drug that reaches the systemic circulation from an administered dosage form.

[0273] "Subject," unless otherwise indicated or implied by context, refers to a human, non-human primate, or mammal having or prone to a hyperproliferative disorder, inflammatory disorder, or immune disorder, or other disorder caused by abnormal cells, and which may benefit from the administration of an effective amount of a Ligand Drug Conjugate. Non-limiting examples of subjects include humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. Typically, the subject is a human, non-human primate, rat, mouse, or dog.

[0274] "Carrier," unless otherwise specified or implied by context, refers to a diluent, adjuvant, or excipient with which a compound is administered. Such pharmaceutical carriers can be liquids such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea. In addition, auxiliary substances, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, when administered to a patient, the compound or composition and pharmaceutically acceptable carrier are sterile. When a compound is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions of the present invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0275] "Salt form," as used herein, unless otherwise indicated by context, refers to a charged compound ionically associated with a countercation(s) and / or a counteranion to form an overall neutral species. In some aspects, a salt form of a compound arises through the interaction of a basic or acidic functional group of the parent compound with an external acid or base, respectively. In other aspects, the charged atom of the compound associated with the counteranion is permanent in the sense that spontaneous dissociation to a neutral species cannot occur without changing the structural integrity of the parent compound, such as when a nitrogen atom is quaternized. Thus, a salt form of a compound may include a quaternized nitrogen atom within the compound, and / or a protonated form of a basic functional group, and / or an ionized carboxylic acid of the compound, each of which is ionically associated with a counteranion.

[0276] In some aspects, salt forms may result from the interaction of a basic functional group and an ionizable acid functional group within the same compound, or may involve the inclusion of a negatively charged molecule (e.g., acetate, succinate, or other counter anion). Thus, a compound in salt form may have more than one charged atom in its structure. In instances where multiple charged atoms of a parent compound are part of the salt form, the salt form may have multiple counter ions, such that the salt form of the compound may have one or more charged atoms and / or one or more counter ions. The counter ion may be any charged organic or inorganic moiety that stabilizes the opposite charge of the parent compound.

[0277] Protonated salt forms of a compound typically occur when a basic functional group of the compound (e.g., a primary, secondary, or tertiary amine or other basic amine functional group) interacts with an organic or inorganic acid that has a suitable pKa for protonation of the basic functional group, or aThe salt forms are obtained when an acid functional group (e.g., carboxylic acid) of a compound having the formula (I) interacts with a hydroxide salt (e.g., NaOH or KOH) or an organic base (e.g., triethylamine) having sufficient strength to deprotonate the acid functional group. In some aspects, the salt form of the compound contains at least one basic amine functional group, and therefore, an acid addition salt can be formed with this amine group, including the basic amine functional group of a cyclic or acyclic basic unit. Suitable salt forms in the context of a drug-linker compound are those that do not unduly interfere with the condensation reaction between the targeting agent and the drug-linker compound to provide a ligand-drug conjugate.

[0278] "Pharmaceutically acceptable salt," as used herein, unless otherwise indicated by context, refers to a salt form of a compound whose counterions are acceptable for administration of the salt form to an intended subject and include inorganic and organic countercations and anions. Exemplary pharmaceutically acceptable counteranions for basic amine functional groups (e.g., basic amine functional groups in cyclic or acyclic basic units) include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, mesylate, besylate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)).

[0279] Typically, pharmaceutically acceptable salts are selected from those described in P.H. Stahl and C.G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zuerich: Wiley-VCH / VHCA, 2002. The choice of salt depends on the properties that the drug product must exhibit, including suitable aqueous solubility at various pH values ​​depending on the intended route of administration, crystallinity with flow properties suitable for handling and low hygroscopicity (i.e., water absorption versus relative humidity), and the required shelf life as measured by chemical stability and solid-state stability, as in lyophilized formulations, under accelerated conditions (i.e., conditions to measure degradation or solid-state changes when stored at 40° C. and 75% relative humidity).

[0280] Terms such as "inhibit" and "inhibition of," unless otherwise indicated or implied by context, mean reducing by a measurable amount or preventing an undesired activity or result altogether. In some aspects, the undesired result or activity is associated with abnormal cells and includes hyperproliferation or overstimulation or other dysregulated cellular activity underlying a disease state. Inhibition of such dysregulated cellular activity by a ligand-drug conjugate is typically measured relative to untreated cells (cells mock-treated with vehicle) in a suitable test system, such as in cell culture (in vitro) or a xenograft model (in vivo). Typically, a ligand-drug conjugate targets an antigen that is not present on the abnormal cells of interest or that is present at low copy number on the abnormal cells of interest, or a ligand-drug conjugate genetically engineered not to recognize any known antigen is used as a negative control.

[0281] Unless otherwise indicated by context, the terms "treat," "treatment," and the like refer to therapeutic treatment, including prophylactic measures to prevent recurrence, with the purpose of inhibiting or slowing (alleviating) an undesired physiological change or disorder, such as the onset or spread of cancer or tissue damage resulting from chronic inflammation. Typically, beneficial or desired clinical outcomes of such therapeutic treatment include, but are not limited to, alleviation of symptoms, a decrease in the extent of disease, a stabilized (i.e., not worsening) disease state, a delay or slowing of disease progression, amelioration or palliation of the pathology, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival or quality of life compared to expected survival or quality of life if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to developing the condition or disorder.

[0282] In the context of cancer, the term "treating" includes any or all of inhibiting the growth of tumor cells, cancer cells, or tumors, inhibiting the replication of tumor cells or cancer cells, inhibiting the spread of tumor cells or cancer cells, alleviating the tumor burden or reducing the number of cancerous cells, or ameliorating one or more symptoms associated with cancer.

[0283] "Therapeutically effective amount," as used herein, unless otherwise indicated or implied by context, refers to an amount of free drug or a Ligand Drug Conjugate having a drug unit (released as free drug) effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of free drug or Ligand Drug Conjugate can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow and preferably stop) the infiltration of cancer cells into peripheral organs, inhibit (i.e., slow and preferably stop) the progression to some extent, inhibit tumor metastasis (i.e., slow and preferably stop) the progression to some extent, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that the free drug or Ligand Drug Conjugate can inhibit the growth and / or kill existing cancer cells, it can be cytostatic or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and determining the response rate (RR) and / or overall survival (OS).

[0284] In the case of immune disorders resulting from overstimulated immune cells, a therapeutically effective amount of a drug can reduce the number of overstimulated immune cells, the degree of their stimulation, and / or their infiltration into otherwise normal tissues, and / or alleviate to some extent one or more symptoms associated with a dysregulated immune system caused by overstimulated immune cells. For immune disorders caused by overstimulated immune cells, efficacy can be measured by assessing one or more inflammatory surrogates, including, for example, one or more cytokine levels, such as cytokine levels for IL-1β, TNFα, INFγ, and MCP-1, or the number of classically activated macrophages.

[0285] In some aspects of the present invention, a ligand drug conjugate compound associates with an antigen on the surface of a targeted cell (i.e., an abnormal cell, such as a hyperproliferative cell or an overstimulated immune cell), and the conjugate compound is internalized by receptor-mediated endocytosis. Once inside the cell, one or more cleavage units within the linker unit of the conjugate are cleaved, resulting in the release of the drug unit (D) as a free drug. The free drug thus released can then translocate into the cytosol and induce cytotoxic or cytostatic activity, or alternatively, in the case of overstimulated immune cells, inhibit proinflammatory signaling. In another aspect of the present invention, the drug unit (D) is released from the ligand drug conjugate compound outside but near the targeted cell, such that the free drug resulting from this release can localize to the desired site of action and subsequently enter the cell rather than being prematurely released at a distant site.

[0286] 2. Embodiment

[0287] Various embodiments of the invention are described below, followed by a more detailed discussion of their components (e.g., groups, reagents, and steps useful in the processes of the invention). Any of the selected embodiments for the components of these processes may apply to each and every aspect of the invention as described herein, or they may relate to one aspect. In some aspects, selected embodiments may be combined in any combination suitable to describe an auristatin ligand drug conjugate having a hydrophobic auristatin F drug unit, a drug linker compound, or an intermediate thereof.

[0288] 2.1 Ligand-drug conjugates

[0289] Ligand Drug Conjugate (LDC) compounds of the invention are compounds having a Drug unit connected to a Ligand unit via an intervening Linker Unit (LU) (LU comprising a peptide cleavable unit that is more susceptible to proteolytic cleavage by tumor tissue homogenates compared to normal tissue homogenates to result in release of D as the free drug), and typically have the formula 1: [ka]

[0290] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, wherein L is a Ligand unit; LU is a Linker unit; D' represents 1 to 4 Drug units, incorporating or corresponding in structure to the same free drug for each Drug Linker moiety of the formula -LU-(D)'; and the subscript p' is an integer ranging from 1 to 24, wherein the Ligand units are capable of selectively binding to antigens of targeted abnormal cells, and wherein the targeted antigens are capable of internalization along with the attached conjugate compound for subsequent intracellular release of the free drug, and wherein each Drug Linker moiety in the Ligand Drug Conjugate compound has the structure of Formula 1A: [ka]

[0291] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein -L of the drug linker moiety of Formula 1A is B -A a -B b The - moiety generally comprises a primary linker (L R ) and

[0292] where the wavy line indicates a covalent bond to L; Bis a Ligand covalent binding moiety; A is a first optional Stretcher unit; the subscript a is 0 or 1, indicating the absence or presence of A, respectively; B is an optional Branching unit; the subscript b is 0 or 1, indicating the absence or presence of B, respectively; D is a Drug unit; and the subscript q is an integer ranging from 1 to 4; and L O teeth, [ka]

[0293] where the wavy line adjacent to A' is L O indicates the site of covalent attachment of Y to the primary linker; the wavy line adjacent to Y indicates the site of covalent attachment of L O represents the site of covalent attachment to the Drug unit; A' is a second optional Spacer unit, the subscript a' is 0 or 1, indicating the absence or presence of A', respectively, W is a peptide cleavable unit, Y is a Spacer unit, and y is 0, 1, or 2, indicating the absence of a Spacer unit or the presence of one or two Spacer units, respectively.

[0294] The ligand drug conjugate composition comprises a distribution or collection of ligand drug conjugate compounds and is represented by the structure of Formula 1, where the subscript p' is replaced by the subscript p, where the subscript p is a number ranging from about 2 to about 24.

[0295] Conventional ligand drug conjugates, also represented by Formula 1, include a peptide cleavable unit (W) comprising a dipeptide covalently attached to D directly or indirectly via Y, where the dipeptide is designed to be selective for a particular intracellular protease whose activity is upregulated in diseased cells relative to that of normal cells. In contrast, the conjugates of the present invention are based on the unexpected finding that a suitably designed cleavable unit can differentiate overall protease activity in tissues containing diseased cells from that in normal tissues containing normal cells, while remaining resistant to cleavage by freely circulating proteases. For the conjugates of the present invention, that distinction is achieved by peptide cleavable units that incorporate specific tripeptides, where these peptides were identified by the screening methods described herein that compare protease activity from tissue homogenates containing abnormal cells with protease activity from normal tissue homogenates, where the normal tissue is known to be the source of on-target and / or off-target adverse event(s) that occur when a therapeutically effective amount of a conventional ligand drug conjugate is administered to a mammalian subject.

[0296] Thus, in a primary embodiment of the present invention, W is a peptide cleavable unit comprising a tripeptide that provides a recognition site that is selectively acted upon by one or more intracellular proteases of the targeted diseased cells relative to freely circulating proteases, and also selectively acted upon by proteases in tumor tissue homogenates relative to proteases in normal tissue homogenates. For cancer treatment, the tripeptide sequence of the peptide cleavable unit is selected so that normal tissue proteases, known to be the source of on-target and / or off-target adverse events resulting from the administration of therapeutically effective amounts of conventional ligand-drug conjugates, are less likely to act on a conjugate bearing this tripeptide-based cleavable unit than tumor tissue proteases, providing greater selectivity for targeting cancer cells. This selection is based on the lower overall protease activity in normal tissue homogenates compared to cancer tumor tissue homogenates. In contrast to the improved conjugates of the present invention, conventional ligand-drug conjugates containing dipeptide cleavable units are designed to be selectively acted upon by cathepsin B, an intracellular protease whose activity is upregulated in cancer cells, and rely primarily on immunological specificity for selectively targeting cancer cells relative to normal cells. The improved conjugates of the present invention have an additional level of selectivity in that they are less susceptible to protease action in normal tissue compared to tumor tissue in which the targeted cancer cells reside.

[0297] In some embodiments, the drug linker moiety of Formula 1A has Formula 1B: [ka]

[0298] wherein L B is the primary linker (L) in the linker unit (LU) of the drug-linker moiety or drug-linker compound. R A and B are ligand covalent binding moieties as defined herein for L Rthe first optional stretcher unit and optional branching unit are of the formula: O is as defined herein.

[0299] In some of these embodiments, W contains a tripeptide directly attached to the Drug unit, such that the subscript y is 0. When the subscript y is 1, the tripeptide is attached to a self-immolative spacer unit such that, upon cleavage by a protease, a drug linker fragment of the formula Y-D (Y undergoes self-immolation, completing the release of the free drug). When the subscript y is 2, the tripeptide is attached to a first self-immolative spacer unit (Y) such that, upon cleavage by a protease, a first drug linker fragment of the formula Y-Y'-D (Y' is the second spacer unit), which then self-immolates to provide a second drug linker fragment of the formula Y'-D, which degrades to complete the release of the free drug.

[0300] An exemplary ligand drug conjugate compound having a drug linker moiety of Formula 1B in which the tripeptide of the peptide cleavable unit (W) is attached directly to the drug unit or to an intervening spacer unit has the structure of Scheme 1a, where P1, P2, and P3 are amino acid residues of the tripeptide sequence, and D is a Y group where the subscript y is 2. yIn these exemplary ligand-drug conjugate compounds, the carbonyl functionality of the amide bond adjacent to P1 is derived from the C-terminus of the tripeptide sequence, where this amide bond is the site of protease cleavage (indicated by the arrow), and the amino group of the amide bond adjacent to P3 is derived from the N-terminus of the tripeptide sequence. Cleavage of the amide functionality to P1 yields a first drug-linker fragment having the structure shown in Scheme 1a, which undergoes self-immolation to provide a second drug-linker fragment, which spontaneously decomposes with the release of CO2, completing the release of D as a free drug of the formula HT*-D* (where the oxygen atom or nitrogen moiety of the hydroxyl or amine group, -NH-, is represented by T*, and D* represents the remainder of the free drug) bearing a hydroxyl or amine group.

[0301] Scheme 1a. [ka]

[0302] In these embodiments, one or more amino acids designated P4, P5, etc., are of the formula -L B -A' a’ - may be present between the primary linker and P3 as part of a peptide sequence containing a tripeptide that confers selectivity for intracellular proteolysis over proteolysis by freely circulating proteases and for proteolysis by tumor tissue homogenates over proteolysis by normal tissue homogenates. The mechanism of free drug release from ligand-drug conjugates with such extended peptide sequences is similar to that in Scheme 1a.

[0303] In another embodiment, the amino acid residue designated P-1 is a tripeptide conferring specificity to W and a D or -Y yThe D or drug-linker fragment initially released from protease action on the tripeptide intervening between P1 and P-D, and thus conferring specificity, contains this amino acid and therefore requires further processing by an intracellular endopeptidase to allow self-immolation of the spacer unit(s). For these embodiments, an exemplary ligand-drug conjugate compound having a drug-linker moiety of Formula 1B, in which the tripeptide conferring specificity of the peptide-cleavable unit is not directly attached to the drug unit or to an intervening spacer unit, has the structure shown in Scheme 1b. Protease cleavage of the susceptible amide bond (indicated by the arrow) between P1 and P-1 provides a drug-linker fragment in which the first self-immolative spacer unit (Y) is present as an amino acid residue that provides a substrate for an endopeptidase with a bond to the self-immolative moiety of Y, which is a para-aminobenzyl alcohol residue with a bond to D via a carbamate or carbonate functionality. The amino acid-para-aminobenzyl alcohol residue and the carbamate or carbonate functionality together form Y. y where the subscript y is 2. Endopeptidase removal of P-1 is followed by self-immolation to release D of formula HT*-D* as the free drug, as in Scheme 1a.

[0304] Scheme 1b [ka] [ka]

[0305] As before, one or more amino acids designated P4, P5, etc. are of the formula -L B -A' a’- may be present between the primary linker and P3 as part of a peptide sequence containing a tripeptide that confers selectivity for intracellular proteolysis over proteolysis by freely circulating proteases, and for tumor tissue homogenate over normal tissue homogenate. Although P-1 in Scheme 1b is formally part of the first self-immolative spacer unit (Y), for convenience it is attached to a tripeptide sequence, and thus W is a tetrapeptide in the SEQ ID NO: describing such a peptide cleavable unit. These units and other components of the ligand drug conjugates of the invention are further discussed below.

[0306] 2.2.1 Ligand unit

[0307] The Ligand unit (L) of the Ligand Drug Conjugate is the targeting moiety of the conjugate that selectively binds to the targeted moiety. In some embodiments, the Ligand unit selectively binds to a cellular component that serves as the targeted moiety (cell-binding agent) or other target molecule of interest. The Ligand unit acts to target and present the Drug unit of the Ligand Drug Conjugate to a specific target cell population with which it interacts to selectively release D as free drug. Targeting agents that provide the Ligand unit include, but are not limited to, proteins, polypeptides, and peptides. Exemplary Ligand units include, but are not limited to, those provided by proteins, polypeptides, and peptides such as antibodies, e.g., full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors, and colony-stimulating factors. Other suitable Ligand units are derived from vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the Ligand unit is derived from a non-antibody protein targeting agent. In other embodiments, the Ligand unit is derived from a protein targeting agent such as an antibody. Preferred targeting agents are large molecular weight proteins, eg, cell binding agents having a molecular weight of at least about 80 Kd.

[0308] The targeting agent is a primary linker precursor (L R Ligand covalent precursor (L B ') moiety of the drug-linker moiety of Formula 1A to form the primary linker (L R ) covalent ligand bond (L B ) moiety to form a Ligand unit covalently linked to the Drug-Linker moiety. The targeting agent has, or is modified to have, an appropriate number of binding sites to accommodate the required number of Drug-Linker moieties (whether naturally occurring or non-naturally occurring (e.g., engineered)), as defined by the subscript p. For example, to have a value of 6 to 14 for the subscript p, the targeting agent must be capable of forming bonds to 6 to 14 Drug-Linker moieties. The binding sites can be naturally occurring or engineered into the targeting agent. The targeting agent is characterized by the L of the linker unit of the Drug-Linker compound. SS Bonding to moiety can be formed through the reactive or activatable heteroatom or heteroatom-containing functional group of targeting agent.Reactive or activatable heteroatom or heteroatom-containing functional group that can be present in targeting agent includes sulfur (in one embodiment, derived from the thiol functional group of targeting agent), C=O (in one embodiment, derived from the carbonyl group, carboxyl group or hydroxyl group of targeting agent) and nitrogen (in one embodiment, derived from the primary or secondary amino group of targeting agent).These heteroatoms can be present in targeting agent in the natural state of targeting agent (for example, naturally occurring antibody), or can be introduced into targeting agent by chemical modification or genetic engineering.

[0309] In one embodiment, the targeting agent has a thiol functional group (e.g., of a cysteine ​​residue) and the Ligand unit derived therefrom is attached to the Drug Linker portion of the Ligand Drug Conjugate compound via the sulfur atom of the thiol functional group.

[0310] In another embodiment, the targeting agent is selected from the group consisting of L of the linker units of the drug-linker compound, including but not limited to N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters. R The Drug-Linker Compound has a lysine residue that can react with an activated ester of the formula: thus creating an amide bond between the nitrogen atom from the Ligand unit and the C=O functionality from the Linker unit of the Drug-Linker Compound.

[0311] In yet another embodiment, the targeting agent has one or more lysine residues that can be chemically modified to introduce one or more thiol functional groups.The ligand unit derived from this targeting agent is linked to the linker unit via the sulfur atom of the introduced thiol functional group.Reagents that can be used to modify lysine include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0312] In another embodiment, the targeting agent may have one or more carbohydrate groups that can be chemically modified to have one or more thiol functional groups. The ligand unit derived from this targeting agent is bonded to the linker unit via the sulfur atom of the introduced thiol functional group. Alternatively, the targeting agent may have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, for example, Laguzza, et al., 1989, J. Med. Chem. 32 (3): 548-55). The corresponding aldehyde is then oxidized to the L-terminal of a drug-linker compound having a nucleophilic nitrogen. SS A L moiety that can react with a carbonyl group of a targeting agent. ROther reactive sites include, but are not limited to, hydrazine and hydroxylamine. Other protocols for modifying proteins for attachment of drug linker moieties are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0313] In a preferred embodiment, L of the drug linker compound R The reactive group of 1 ) part of L R Covalent attachment to the targeting agent is achieved via the thiol functional group of the targeting agent, resulting in the formation of a thio-substituted succinimide (M 2 The thiol functional group can be present on the targeting agent in its native state (e.g., as a naturally occurring residue) or can be introduced into the targeting agent by chemical modification and / or genetic engineering.

[0314] It has been observed that the site of drug conjugation for bioconjugates can affect several parameters, including the ease of conjugation, the stability of the drug-linker, the effect on the biophysical properties of the resulting bioconjugate, and in vitro cytotoxicity. With regard to drug-linker stability, the site of conjugation of the drug-linker to the ligand can affect the ability of the conjugated drug-linker moiety to undergo a cleavage reaction and, for the drug-linker moiety, its ability to be transferred from the ligand unit of the bioconjugate to an alternative reactive thiol present in the bioconjugate's environment, such as the reactive thiol of albumin, free cysteine, or glutathione in plasma. Such sites include, for example, interchain disulfides and selected engineered cysteine ​​sites. The ligand-drug conjugates described herein can be conjugated to thiol residues at sites less susceptible to cleavage reactions (e.g., position 239 according to the EU index as described in Kabat) in addition to other sites.

[0315] In a preferred embodiment, the Ligand unit (L) is of an antibody or antigen-binding fragment thereof, thereby defining the antibody Ligand unit of an antibody drug conjugate (ADC), wherein the antibody Ligand unit is capable of selectively binding to a targeted antigen of a cancer cell for subsequent release of D as a free drug, wherein the targeted antigen, upon said binding, is capable of internalization into said cancer cell to initiate intracellular release of the free drug.

[0316] Useful antibodies include polyclonal antibodies, which are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Other useful antibodies are monoclonal antibodies, which are homogeneous populations of antibodies against a specific antigenic determinant (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against a target antigen can be prepared using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture.

[0317] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be produced using any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80: 7308-7312; Kozbor et al., 1983, Immunology Today 4: 72-79; and Olsson et al., 1982, Meth. Enzymol. 92: 3-16).

[0318] The antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a target cell (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen), or other antibody that binds to a tumor cell or matrix. In this regard, "functionally active" means that the fragment, derivative, or analog can immunospecifically bind to a target cell. To determine which CDR sequence binds to an antigen, synthetic peptides containing the CDR sequence can be used in binding assays with the antigen by any binding assay method known in the art (e.g., BIAcore assay) (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Kabat E et al., 1980, J. Immunology 125 (3): 961-969).

[0319] Other useful antibodies include fragments of antibodies, such as, but not limited to, F(ab')2 fragments, Fab fragments, Fv, single chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-Fv, or any other molecule with the same specificity as an antibody.

[0320] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions, which can be produced using standard recombinant DNA techniques, are useful. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region (see, e.g., U.S. Pat. Nos. 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules derived from non-human species that have one or more complementarity-determining regions (CDRs) from the non-human species and a framework region derived from a human immunoglobulin molecule (see, e.g., U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety).Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, as described in International Publication No. WO 87 / 02671; European Patent Application Publication No. 0 184 187; European Patent Application Publication No. 0 171 496; European Patent Application Publication No. 0 173 494; International Publication No. WO 86 / 01533; U.S. Pat. No. 4,816,567; European Patent Application Publication No. 012 023; Berter et al., Science (1988) 240: 1041-1043; Liu et al., Proc. Natl. Acad. Sci. (USA) (1987) 84: 3439-3443; Liu et al., J. Immunol. (1987) 139: 3521-3526; Sun et al. Proc. Natl. Acad. Sci. (USA) (1987) 84: 214-218; Nishimura et al. Cancer. Res. (1987) 47: 999-1005; Wood et al., Nature (1985) 314: 446-449; Shaw et al., J. Natl. Cancer Inst. (1988) 80: 1553-1559; Morrison, Science (1985) 229: 1202-1207; Oi et al. BioTechniques (1986) 4: 214; US Patent No. 5,225,539; Jones et al., Nature (1986) 321: 552-525; Verhoeyan et al. al., Science (1988) 239: 1534; and Beidler et al., J. Immunol. (1988) 141: 4053-4060.

[0321] Completely human antibodies are particularly preferred, which can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.

[0322] Antibodies also include analogs and derivatives modified by the covalent attachment of any type of molecule, provided that such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, without limitation, antibody derivatives and analogs include those further modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular antibody entities or other proteins, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, analogs or derivatives may contain one or more unnatural amino acids.

[0323] The antibody may have modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors. In particular, the antibody may have modifications in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97 / 34631, incorporated herein by reference in its entirety).

[0324] In certain embodiments, known antibodies for treating cancer are used, hi some embodiments, the antibodies selectively bind to cancer antigens of hematological malignancies.

[0325] 2.2.2 Primary Linker

[0326] In one group of embodiments, the Ligand Drug Conjugate has the formula -L R -L O -D, wherein L O is, as described herein, -A' a’ -WY y -, where L Ris a primary linker, A' is a second optional stretcher unit, a' is 0 or 1, indicating the absence or presence of A', respectively, Y is a spacer unit, and the subscript y is 0, 1, or 2, indicating the absence of a spacer unit or the presence of one or two spacer units, respectively, D is a drug unit, and W is a peptide cleavable unit, wherein the peptide cleavable unit is a sequence of up to 12 (e.g., 3-12 or 3-10) consecutive amino acids, wherein the sequence is selected to increase the cleavability of the peptide cleavable unit in tumor tissue homogenate compared to normal tissue homogenate to initiate the release of D as free drug. In some of these embodiments, the -L comprises a tripeptide that is more susceptible to proteolytic cleavage by -L, wherein cytotoxicity to normal tissue cells due to unintended release of free drug within and / or near these cells is associated with adverse events from administering an effective amount of a comparative Ligand Drug Conjugate (the sequence of amino acids in the peptide cleavable unit of which is dipeptide-valine-citrulline) to a subject in need thereof, and / or the tripeptide increases the bioavailability of the Ligand Drug Conjugate relative to the comparative conjugate so as to prevent its bioavailability to normal tissues. R -L B -A a -B b -, where L B is a ligand covalent binding moiety, A is a first optional Stretcher unit, the subscript a is 0 or 1, indicating the absence or presence of A, respectively, B is an optional Branching unit, and the subscript b is 0 or 1, indicating the absence or presence of B, respectively.

[0327] In some embodiments, the drug linker moiety is

[0328] [ka]

[0329] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein L R , A', a', Y, y and D retain their previous meanings, and P1, P2 and P3 are amino acid residues that together provide selectivity for proteolysis by tumor tissue homogenates over proteolysis by normal tissue homogenates and / or provide increased bioavailability to tumor tissue to the detriment of normal tissue compared to a comparative ligand-drug conjugate in which the amino acid sequence of the peptide cleavable unit is dipeptide-valine-citrulline-, where proteolytic cleavage occurs at the covalent bond between P1 and Y when subscript y is 1 or 2, or at the covalent bond between P1 and D when subscript y is 0, and where the tumor tissue and normal tissue are of the same species.

[0330] As described elsewhere, other embodiments contain an additional amino acid residue (designated P-1) between P1 and Y or D depending on the value of the subscript y, such that selective endopeptidase action by the proteolytic enzyme(s) of the tumor tissue homogenate occurs at the amide bond between P1 and P-1 to produce a compound of the formula -[P-1]-Y y -D drug-linker fragment is released. When the subscript y is 0 (i.e., Y is absent), release of the free drug from this fragment occurs from the exopeptidase action of the proteolytic enzyme removing the P-1 amino acid residue to provide the free drug directly.

[0331] In some embodiments where an additional amino acid residue is present between P1 and Y or D, the drug linker moiety is

[0332] [ka]

[0333] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein L R, A', a', Y, y, and D retain their previous meanings, and P1, P2, and P3 are amino acid residues that, optionally with P-1, together provide selectivity for proteolysis by tumor tissue homogenates over proteolysis by normal tissue homogenates, where proteolytic cleavage occurs at the covalent bond between P1 and P-1 to form [P-1]-Y y The linker fragment having the structure -D is released.

[0334] In some of these embodiments, when the subscript y is 0, the [P-1]-D residue resulting from endopeptidase cleavage of the amide bond between the P1 and P-1 amino acids also exerts cytotoxic activity. In other embodiments, the subscript y is 1 or 2, such that exopeptidase action of removing the P-1 amino acid residue results in a [P-1]-D residue of the formula -Y y An alternative drug linker fragment of -D is provided, which spontaneously fragments to provide the free drug.

[0335] In other embodiments, P4, P5 ··· P n (where the subscript n ranges up to 12 (e.g., 3 to 12 or 3 to 10)) is expressed as L R or A', which in some embodiments is additional to the peptide cleavable unit containing the P-1 amino acid residue. In either instance, additional P4, P5 ··· P n The amino acid residue is -Y y -D or -[P-1]-Y y The cleavage site providing the -D fragment is selected so that it is not altered, but instead is selected so that it confers desirable physiochemical and / or pharmacokinetic properties to the ligand drug conjugate, such as improved solubility to reduce aggregation.

[0336] In some embodiments having an additional amino acid residue(s) N-terminal to P3, or further having P-1 between P1 and Y or D, the drug linker moiety is

[0337] [ka]

[0338] [ka]

[0339] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein L R , A', a', Y, y and D retain their previous meanings, and P-1 and P1, P2, P3 ··· P n are amino acid residues, where the subscript n ranges up to 12 (e.g., 3 to 12 or 3 to 10), and P1, P2, and P3, optionally with P-1, together provide selectivity for proteolysis by tumor tissue homogenate over proteolysis by normal tissue homogenate, where P1 and Y y Proteolytic cleavage occurs at the covalent bond between P1 and Y-D or between P1 and P-1, respectively. y -D or [P-1]-Y y In the latter case, a linker fragment having the structure Y-D is released, and in the latter case, subsequent exopeptidase cleavage occurs to give Y y In both instances, a linker fragment having the structure Y -D is released. y The -D linker fragment undergoes spontaneous decomposition, resulting in the release of D as the free drug.

[0340] In any one of these embodiments, when the subscript b is 0, the L R Ha-L B -A a where L Bis a Ligand Covalent Binding Moiety, and A is a First Optional Stretcher Unit. In such an embodiment, when a is 1 and the subscript a' is 1, A' exists as a subunit of A and is therefore considered a component of the Primary Linker.

[0341] In some preferred embodiments where subscript b is 0 and subscript a is 1, the formula -L B -A-L R is a self-stabilizing linker (L SS ) part, or L SS of succinimide (M 2 The self-stabilizing linker (L) obtained from the controlled hydrolysis of the S ) moiety. Exemplary L of the drug linker portion of a ligand drug conjugate composition or conjugate compound thereof having either type of primary linker are: SS and L S The primary linkers are, respectively: [ka]

[0342] or a salt thereof, particularly a pharmaceutically acceptable salt, where the wavy line indicates the site of covalent attachment to A' or W depending on the value of the subscript a'; A' is an optional subunit of A; [HE] is an optional hydrolysis enhancing unit that is a component provided by A; BU is a basic unit; and R a2 are replaced as necessary by C1 to C 12 is an alkyl group; and the dashed curve indicates optional cyclization, such that in the absence of said cyclization, BU is an acyclic basic unit having a primary, secondary, or tertiary amine functionality as the basic functionality of the acyclic basic unit, or in the presence of said cyclization, BU is a cyclized basic unit, where R a2and BU, together with the carbon atom to which they are both attached, form an optionally substituted spiro C-C ring containing a backbone basic nitrogen atom of a secondary or tertiary amine functional group as the basic functional group of the cyclic basic unit. 20 defines heterocyclo,

[0343] wherein the basic nitrogen atom of the acyclic basic unit or cyclic basic unit is suitably protected by a nitrogen protecting group, or optionally protonated, depending on the degree of substitution of the basic nitrogen atom.

[0344] In another preferred embodiment where the subscript b is 0 and the subscript a is 1, the formula -L B the primary linker of -A- does not contain a basic unit, [ka]

[0345] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, wherein the variable group is L SS or L S As described above for the primary linker.

[0346] L R A representative LL in which is covalently bound to the ligand unit (L) of LDC. R -The structure is as follows: [ka]

[0347] and salts thereof, particularly pharmaceutically acceptable salts, and structures where the succinimide ring system has been hydrolyzed to the open form, where the indicated (#) sulfur atom is from the Ligand unit; and where the wavy line indicates the site of covalent attachment to the remainder of the Conjugate structure.

[0348] Other representative LL R -The structure is as follows: [ka]

[0349] where the indicated (#) nitrogen, carbon, or sulfur atom is from the Ligand unit; and where the wavy line indicates the site of covalent attachment to the remainder of the binder structure.

[0350] In another group of embodiments, drug linker compounds useful in the preparation of the ligand drug conjugates described in the previous group of embodiments are L R '-A' a’ -WY y -D, where L R ' is the primary linker of the drug-linker compound, which is the primary linker L of the drug-linker portion of the ligand-drug conjugate R (when the Drug Linker Compound is used to prepare the Conjugate), A' is a second optional Stretcher unit, and a' is 0 or 1, indicating the absence or presence of A', respectively, where L R If A' does not contain a branching unit and the subscript a' is 1, then A' is L R 'A exists as a component of L as a subunit R ', Y is a spacer unit, and the subscript y is 0, 1, or 2, indicating the absence of a spacer unit or the presence of one or two spacer units, respectively; D is a drug unit; and W is a peptide cleavable unit comprising a tripeptide that is more susceptible to proteolytic cleavage by homogenates of tumor tissue compared to homogenates of normal tissue, wherein cytotoxicity to cells of normal tissue due to the unintended release of D as free drug within and / or near these cells is associated with adverse events from administration of a Ligand Drug Conjugate intended for targeting cancer cells of tumor tissue. In some of these embodiments, L R '-L B -A a -B b -, where LB ' is the ligand covalent binding portion of the primary linker of a drug-linker compound, which is sometimes referred to as the primary linker (L) of the drug-linker portion of a ligand-drug conjugate when the drug-linker compound is used to prepare the conjugate. R ) the ligand covalent binding portion (L B ), and is therefore referred to as a Ligand Covalent Precursor Moiety, where A is a first optional Stretcher unit, the subscript a is 0 or 1, indicating the absence or presence of A, respectively, B is an optional Branching unit, and the subscript b is 0 or 1, indicating the absence or presence of B, respectively.

[0351] In some embodiments, the drug linker compound is

[0352] [ka]

[0353] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein L R ', A', a', Y, y, and D retain their previous meanings, and P1, P2, and P3 are amino acid residues that together provide selectivity for proteolysis by tumor tissue homogenates over proteolysis by normal tissue homogenates, where proteolytic cleavage occurs at the covalent bond between P1 and Y when subscript y is 1 or 2, or at the covalent bond between P1 and D when subscript y is 0.

[0354] As described elsewhere, other embodiments contain an additional amino acid residue (designated P-1) between P1 and Y or D depending on the value of the subscript y, such that selective endopeptidase action by the proteolytic enzyme(s) of the tumor tissue homogenate occurs at the amide bond between P1 and P-1 to produce a compound of the formula -[P-1]-Y y-D drug-linker fragment is released. When the subscript y is 0 (i.e., Y is absent), release of the free drug from this fragment occurs from the exopeptidase action of the proteolytic enzyme removing the P-1 amino acid residue to provide the free drug directly.

[0355] In some embodiments where there is an additional amino acid residue between P1 and Y or D, the drug linker compound is

[0356] [ka]

[0357] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein L R ', A', a', Y, y, and D retain their previous meanings, and P1, P2, and P3 are amino acid residues that, optionally with P-1, together provide selectivity for proteolysis by tumor tissue homogenates over proteolysis by normal tissue homogenates, where proteolytic cleavage occurs at the covalent bond between P1 and P-1 to form [P-1]-Y y The linker fragment having the structure -D is released.

[0358] In some of these embodiments, when the subscript y is 0, the [P-1]-D residue resulting from endopeptidase cleavage of the amide bond between the P1 and P-1 amino acids also exerts cytotoxic activity. In other embodiments, the subscript y is 1 or 2, such that exopeptidase action of removing the P-1 amino acid residue results in a [P-1]-D residue of the formula -Y y An alternative drug linker fragment of -D is provided, which spontaneously fragments to provide the free drug.

[0359] In other embodiments, P4, P5 ··· P n(where the subscript n ranges up to 12 (e.g., 3 to 12 or 3 to 10)) is expressed as L R or A', which in some embodiments is additional to the peptide cleavable unit containing the P-1 amino acid residue. In either instance, additional P4, P5 ··· P n The amino acid residue is -Y y -D or -[P-1]-Y y The cleavage site providing the -D fragment is selected so that it is not altered, but instead is selected so that it confers desirable physiochemical and / or pharmacokinetic properties to the ligand drug conjugate, such as improved solubility to reduce aggregation.

[0360] In some embodiments where there is an additional amino acid residue(s) N-terminal to P3, or further having P-1 between P1 and Y or D, the drug linker compound is

[0361] [ka]

[0362] [ka]

[0363] or a salt thereof, particularly a pharmaceutically acceptable salt, wherein L R ', A', a', Y, y and D retain their previous meanings, and P-1 and P1, P2, P3 ··· P n are amino acid residues, where the subscript n ranges up to 12 (e.g., 3 to 12 or 3 to 10), and P1, P2, and P3, optionally with P-1, together provide selectivity for proteolysis by tumor tissue homogenates over proteolysis by normal tissue homogenates, where proteolytic cleavage occurs between P1 and Yy -D or P1 and P-1, respectively, y -D or [P-1]-Y y In the latter case, a linker fragment having the structure Y-D is released, and in the latter case, subsequent exopeptidase cleavage occurs to give Y y In both instances, a linker fragment having the structure Y -D is released. y The -D linker fragment undergoes spontaneous degradation (also called self-immolation) to complete the release of D as the free drug.

[0364] In any one of these embodiments, when the subscript b is 0, L of the Drug Linker Compound R ' is L B -A a where L B A' is a Ligand Covalent Precursor Moiety, and A is a First Optional Stretcher Unit. In such an embodiment, when the subscript a is 1 and the subscript a' is 1, A' exists as a subunit of A and is therefore considered a component of the Primary Linker.

[0365] In some preferred embodiments where subscript b is 0 and subscript a is 1, the drug linker compound has formula L B '-A-L R ' is a self-stabilizing linker precursor (L SS ') moiety, which acts as a self-stabilizing linker (L) of the Ligand Drug Conjugate when this Drug Linker Compound is used to prepare the conjugate. SS ) moiety. SS The primary linker is [ka]

[0366] or a salt thereof, particularly a pharmaceutically acceptable salt, where the wavy line indicates the site of covalent attachment to A' or W depending on the value of the subscript a'; A' is an optional subunit of A; [HE] is an optional hydrolysis enhancing unit that is a component provided by A; BU is a basic unit; and R a2 is C1 to C, substituted as necessary 12 is an alkyl group; and the dashed curve indicates optional cyclization such that, in the absence of said cyclization, BU is an acyclic basic unit having a primary, secondary, or tertiary amine functionality as the basic functionality of the acyclic basic unit, or in the presence of said cyclization, BU is a cyclized basic unit, where R a2 and BU, together with the carbon atom to which they are both attached, form an optionally substituted spiro C-C ring containing a backbone basic nitrogen atom of a secondary or tertiary amine functional group as the basic functional group of the cyclic basic unit. 20 Heterocyclos are defined where the basic nitrogen atom of the acyclic or cyclic basic unit is optionally appropriately protected by a nitrogen protecting group or optionally protonated, depending on the degree of substitution of the basic nitrogen atom.

[0367] In another preferred embodiment where subscript b is 0 and subscript a is 1, formula L B The primary linker of -A- is [ka]

[0368] or a salt thereof, particularly a pharmaceutically acceptable salt thereof, exemplified by the structure: SS or L S As described above for the primary linker.

[0369] Representative L of drug linker compounds R '-The structure is as follows: [ka]

[0370] and salts thereof, particularly pharmaceutically acceptable salts, where the wavy line indicates the site of covalent attachment to the remainder of LU' of the drug linker compound structure, and where the basic nitrogen atom in the second or third structure is optionally protonated as an acid addition salt or optionally protected. If protected, the protecting group is preferably an acid-labile protecting group such as BOC.

[0371] 2.2.3 Peptide Cleavable Units

[0372] In some embodiments, the peptide-cleavable unit (W) of the Ligand Drug Conjugate is a peptide sequence containing a tripeptide attached directly to D or indirectly via one or two self-immolative spacer units, wherein the tripeptide is recognized by at least one intracellular protease, preferably more than one intracellular protease, wherein at least one protease is upregulated in tumor cells compared to normal cells and is more susceptible to proteolysis by tumor tissue homogenates containing the tumor cells targeted by the Ligand Drug Conjugate compared to homogenates of normal tissue, wherein cytotoxicity to the normal tissue is associated with an adverse event from administration of the comparative Ligand Drug Conjugate. In other embodiments, the tripeptide improves biodistribution of the conjugate to tumor tissue so as to prevent biodistribution to normal tissue, which in some of these embodiments is in addition to selectivity for proteolysis by tumor tissue homogenates compared to normal tissue homogenates. In any one of these embodiments, the normal tissue is sometimes bone marrow, and the ameliorated adverse event is neutropenia. In another embodiment, the normal tissue is bone marrow, liver, kidney, esophagus, breast, or corneal tissue, and the adverse event ameliorated is neutropenia. In some embodiments, the tripeptide is directly attached to D or indirectly attached to D via one or two self-immolative spacer units. In other embodiments, the peptide cleavable unit (W) comprising a tripeptide described herein is directly attached to D or indirectly attached to D via one or two self-immolative spacer units through an amino acid that is not part of the tripeptide.

[0373] The peptide-cleavable unit (W) of the comparative conjugate is typically a dipeptide that confers selectivity over freely circulating proteases for specific intracellular proteases that are upregulated in cancer cells, where the specific protease is capable of cleaving the amide bond between the C-terminal amino acid of the dipeptide and the amino group of the self-immolative spacer unit (Y), initiating release of the Drug unit as a free drug.

[0374] In some embodiments, ligand drug conjugates comprising a tripeptide disclosed herein exhibit improved tolerability compared to a comparative ligand drug conjugate (wherein the peptide-cleavable unit is a dipeptide that confers selectivity for a specific intracellular protease upregulated in cancer cells over freely circulating proteases, where the specific protease is capable of cleaving the amide bond between the C-terminal amino acid of the dipeptide and the amino group of the self-immolative spacer unit (Y), initiating release of the drug unit as a free drug). In some embodiments, the dipeptide is known to be selectively cleavable by cathepsin B. In some embodiments, the dipeptide in the comparative ligand drug conjugate is -valine-citrulline- or -valine-alanine-. In some embodiments, the dipeptide in the comparative ligand drug conjugate is -valine-citrulline-. In some embodiments, the dipeptide in the comparative ligand drug conjugate is -valine-alanine-. In some embodiments, tolerability refers to the extent to which adverse events associated with administration of the ligand drug conjugate affect a patient's ability or desire to comply with the dose or intensity of treatment. Thus, improved tolerability may be realized by a reduction in the occurrence or severity of adverse events.

[0375] Without being bound by theory, aggregated ligand drug conjugate compounds are more likely to distribute to normal tissues (e.g., bone marrow), where normal tissues are known to be the source of on-target and / or off-target adverse event(s) in mammalian subjects when a therapeutically effective amount of the ligand drug conjugate is administered. In some embodiments, improved tolerability is demonstrated by a reduced aggregation rate of a ligand drug conjugate comprising a tripeptide compared to a comparative ligand drug conjugate. In some embodiments, the aggregation rate of a ligand drug conjugate comprising a tripeptide and a comparative ligand drug conjugate is determined by measuring the concentration of high molecular weight aggregates after incubating the conjugates in rat plasma, cynomolgus monkey plasma, or human plasma at the same concentration for 12, 24, 36, 48, 60, 72, 84, or 96 hours.

[0376] In some embodiments, the improved tolerability of the tripeptide-containing Ligand Drug Conjugate is demonstrated by improved selectivity of tumor tissue over normal tissue exposure to the free cytotoxic compound released from the tripeptide-containing Ligand Drug Conjugate compared to the cytotoxic compound released from the comparative Ligand Drug Conjugate. In some embodiments, the tumor tissue and normal tissue are derived from a rodent species (e.g., rat or mouse) or a primate species (e.g., cynomolgus monkey or human). In some embodiments, when the tumor tissue and normal tissue are derived from a species different from human, the normal tissue is of the same tissue type in humans, where cytotoxicity to cells of that tissue is at least partially responsible for adverse events in human subjects receiving a therapeutically effective amount of the comparative Ligand Drug Conjugate. In some embodiments, the normal tissue is bone marrow, liver, kidney, esophagus, breast, or corneal tissue. In some embodiments, the normal tissue is bone marrow.

[0377] In some embodiments, improved exposure selectivity is demonstrated by a reduction in the plasma concentration of free cytotoxic compound released from a tripeptide-containing Ligand Drug Conjugate compared to a comparable Ligand Drug Conjugate when the conjugates are administered at the same dose. In some embodiments, the tripeptide-containing Ligand Drug Conjugate retains efficacy (e.g., achieves substantially the same tumor volume reduction compared to the comparable Ligand Drug Conjugate) in tumor xenograft models when administered at the same effective amount and dose schedule as previously determined for the comparable Ligand Drug Conjugate.

[0378] In some embodiments, improved exposure selectivity is demonstrated by reduced non-target-mediated cytotoxicity or normal cell preservation in normal tissue compared to a comparative ligand-drug conjugate when the conjugate is administered at the same dose. In some embodiments, the normal tissue is bone marrow, liver, kidney, esophagus, breast, or corneal tissue. In some embodiments, the normal tissue is bone marrow. In some embodiments, reduced non-target-mediated cytotoxicity or normal cell preservation in normal tissue is demonstrated by bone marrow histology (e.g., reduced reduction in nuclear staining of mononuclear cells). In some embodiments, reduced non-target-mediated cytotoxicity or normal cell preservation is demonstrated by reduced neutrophil and / or reticulocyte reduction and / or a more rapid rebound from the reduction. In some embodiments, reduced non-target-mediated cytotoxicity or normal cell preservation is demonstrated by reduced neutropenia. In some embodiments, reduced non-target-mediated cytotoxicity or normal cell preservation is demonstrated by reduced reticulocyte reduction. In some embodiments, the tripeptide-containing Ligand Drug Conjugate retains efficacy in tumor xenograft models when administered at the same effective dose and dose schedule as previously determined for the comparative Ligand Drug Conjugate. In some embodiments, when comparing exposure selectivity between a tripeptide-containing Ligand Drug Conjugate and a comparative Ligand Drug Conjugate, the Ligand unit of both conjugates is replaced by a non-binding antibody.

[0379] In some embodiments, ligand drug conjugates (e.g., ADCs) are provided that are less active than a comparable ligand drug conjugate (e.g., a -val-cit-containing dipeptide ADC) either in vivo or in vitro, but also have significantly less toxicity. Without being bound by theory, the ligand drug conjugates do not need to be as active, as lower activity and lower toxicity can still increase the therapeutic window.

[0380] In a preferred embodiment, the amide bond between the carboxylic acid of the C-terminal amino acid of the tripeptide and the amino group of the self-immolative spacer...

Claims

【Request Item 1】 【Chemistry 223】 or a pharmaceutically acceptable salt thereof; A ligand drug conjugate compound wherein L is a ligand unit and the subscript p' is an integer from 1 to 12.

2. 2. The ligand drug conjugate compound of claim 1, wherein L is an antibody ligand unit of an intact antibody or an antigen-binding fragment thereof.

3. 3. The ligand drug conjugate compound of claim 2, wherein the intact antibody or fragment thereof is capable of selectively binding to a cancer cell antigen.

4. 3. The ligand drug conjugate compound of claim 2, wherein the intact antibody is a chimeric, humanized, or human antibody, wherein the antibody is capable of selectively binding to a cancer cell antigen, or the antibody is a non-binding control antibody, thereby defining a non-binding control conjugate compound.

5. 5. The ligand drug conjugate compound of any one of claims 1 to 4, wherein the subscript p' ranges from about 2 to about 12, or from about 2 to about 10, or from about 2 to about 8, or the subscript p' is about 2, about 4, or about 8.

6. A pharmaceutically acceptable formulation comprising an effective amount of a ligand drug conjugate compound or an equivalent amount of a non-binding control conjugate compound according to any one of claims 1 to 5 and at least one pharmaceutically acceptable excipient.

7. 7. The pharmaceutically acceptable formulation of claim 6, wherein the at least one pharmaceutically acceptable excipient is a liquid carrier that results in a liquid formulation, wherein the liquid formulation is suitable for lyophilization or administration to a subject in need thereof.

8. 8. The pharmaceutically acceptable formulation of claim 6, wherein the formulation is a lyophilized solid or a liquid formulation of claim 7, wherein at least one excipient of the solid formulation is a cryoprotectant. 【Request Item 9】 【Chemistry 231】 【change】 The drug linker compound has the structure: 【Request Item 10】 【Chemistry 235-1】 or 【Chemistry 235-2】 or a salt thereof, wherein RG is a reactive group.

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