RU2783076C2 - Drug delivery systems based on maytansinoid - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to albumin-binding prodrugs, compounds based on maytansinoid, and their use in a drug delivery system for the treatment of cancer.

EFFECT: obtaining drug delivery systems.

25 cl, 16 dwg, 8 tbl, 19 ex

Description

[1] This application claims priority from Provisional Application US 62/593184, filed Nov. 30, 2017, the entire contents of which are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

[2] Many drugs, particularly cancer drugs, have a narrow therapeutic window in which their side effects limit their beneficial effects. Systemic administration of such drugs often results in a limited therapeutic effect, since the dose required to obtain a stronger effect leads to unacceptable side effects for the patient. This is especially important in the case of those drugs that have a high cytotoxic potential, such as cytostatic agents, virusostatic agents or immunosuppressants. This is even more important in the case of certain cytotoxic agents that inhibit the growth of tumor cells in the picomolar range. These agents are generally too toxic to be used as chemotherapeutic agents. For example, the tubulin-binding maytansine is highly effective in inhibiting tumor cell growth, but has failed in various clinical trials due to an unacceptable toxicity profile.

[3] Numerous studies aim to deliver a specific drug to a specific site of action. Often this approach results in a higher concentration of the drug at the site of action than would be achieved by systemic administration, while limiting side effects.

[4] Drug delivery in oncology is of particular interest because of the narrow therapeutic window of agents used in this indication. Numerous research efforts have focused on the conjugation of anticancer drugs to a wide range of low and high molecular weight carriers, including sugars, growth factors, vitamins, peptides, antibodies, polysaccharides, lectins, serum proteins, and synthetic polymers. In most of these drug delivery systems, the drug is bound to the carrier via a spacer that includes a predetermined breakpoint that allows the bound drug to be released at the cellular target site (Kratz et al., ChemMedChem, 3:20-53 (2008)).

[5] Conjugates are known in which cytostatic agents bind to serum proteins, preferably specific carrier molecules such as human serum albumin and human serum transferrin, and then administered. In other cases, conjugates containing a therapeutically effective substance, a spacer molecule, and a protein-binding molecule covalently bind to circulating serum albumin upon administration, resulting in transport of the therapeutically effective substance to the target site where it is released (US 7387771). In other cases, antibody drug conjugates (ADCs) can transport the drug to the target site for local release (Kratz et al., ChemMedChem, 3:20-53 (2008); Panowski et al., mAbs, 6, 34-45 (2014); Chari et al., Angewandte Chem. Int. Ed., 53, 3796-3827 (2014)).

[6] However, when designing drug delivery systems, a proper balance should be struck between maintaining the targeting properties of the drug carrier while providing controlled drug release. The drug delivery system must have sufficient stability in the circulation while still providing efficient drug release at the tumor site by enzymatic degradation, reduction, or pH dependence (Kratz et al., ChemMedChem, 3:20-53 (2008)) . With respect to highly potent cytotoxic agents from the maytansinoid class (derived from maytansine), only drug delivery systems have been reported in which maytansinoid-based active substances are released non-specifically or reductively. Among them, only those using a monoclonal antibody as a carrier molecule have entered the clinical stage of development, and only one antibody-maytansinoid conjugate, namely T-DM1 (Kadcyla®), has received market approval against certain subtypes of breast cancer. Therefore, there is still a need for efficient and less complex drug delivery and release systems that efficiently release highly potent maytansinoid-based cytotoxic agents.

DISCLOSURE OF THE INVENTION

[7] The present invention provides a compound with the structure of formula (I):

Formula (I)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

R ¹ is selected from -H and C ₁ -C ₄ alkyl;

Spacer selected from:

и

and

V is absent or selected from -CH ₂ -, -O- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

each R ² is independently selected from -H, halogen (eg -F, -Cl, -Br or -I) and C ₁ -C ₄ alkyl or the two R ² together form C ₃ -C ₆ , cycloalkyl;

n is 0-3;

X is absent or selected from -CH ₂ -, -O-, -S-, -Se- and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl;

Y is selected from =CH- and =N-;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ . C ₁ -C ₄ alkyl and C ₂ -C ₄ alkoxy;

AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethyl-glycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α-aminoisobutanoic acid;

R' is chosen from O and

Y' is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)- and -C(O)-NH-; or Y' is selected from the group consisting of:

и

где n=0-6;

and

where n=0-6;

R ¹ ' is absent or selected from the group consisting of:

и

and

where M ¹ represents a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ ; where R represents H or C ₁ -C ₄ alkyl);

R ² ' is an optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -;

Z ¹ ', Z ² ', Z ³ ' and Z ⁴ ' are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I) -CF ₃ , -OCH ₃ , -CN, - NO ₂ , -SO ₃ M ² and C ₁ -C ₄ alkyl, where M ² is a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ where R is H or C ₁ -C ₄ alkyl);

TBG is a thiol-binding group selected from an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group a group and an optionally substituted N-hydroxysuccininide ester group;

wherein said TBG is optionally linked to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier.

[8] In some embodiments, the present invention provides a compound with the structure of formula (I):

Formula (I)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein: R ¹ is selected from -H and C ₁ -C ₄ alkyl;

Spacer selected from:

и

and

V is absent or selected from -CH ₂ -, -O- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

each R ² is independently selected from -H, halogen (eg -F, -Cl, -Br or -I) and C ₁ -C ₄ alkyl or the two R ² together form C ₃ -C ₆ , cycloalkyl;

n is 0-3;

X is absent or selected from -CH ₂ -, -O-, -S-, -Se-, and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl;

Y is selected from =CH- and =N-;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ . C ₁ -C ₄ alkyl and C ₂ -C ₄ alkoxy;

AA is an amino acid selected from glycine, D or L proline, methylglycine, D or L alanine, D or L N -methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α -aminoisobutanoic acid;

R' is chosen from O and

Y' is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)-, and -C(O)-NH-; or Y' is selected from the group consisting of:

и

, где n=0-6;

and

, where n=0-6;

R ¹ ' is absent or selected from the group consisting of:

и

and

where M ¹ represents a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ ; where R represents H or C ₁ -C ₄ alkyl);

R ² ' is an optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -;

Z ¹ ', Z ² ', Z ³ ' and Z ⁴ ' are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I) -CF ₃ , -OCH ₃ , -CN, - NO ₂ , -SO ₃ M ² and C ₁ -C ₄ alkyl, where M ² is a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ where R is H or C ₁ -C ₄ alkyl);

TBG is a thiol-binding group selected from an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group a group and an optionally substituted N-hydroxysuccininide ester group;

wherein said TBG is optionally linked to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier.

[9] In some embodiments, the compound has the structure of formula (II):

Formula (II)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

each R ² is independently selected from -H and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ , cycloalkyl;

X is absent or selected from -CH ₂ -, -O-, -S- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ .

[10] In some embodiments, the compound has the structure of formula (III):

Formula (III)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

each R ² is independently selected from -H and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ , cycloalkyl;

X is absent or selected from -CH ₂ -, -O-, -S- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I),

-CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ .

[11] In some embodiments, the compound has the structure of formula (IV):

Formula (IV)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof,

where:

X is absent or selected from -CH ₂ - and -NH-;

Y is ═CH- or ═N-;

Z ¹ , Z ² , Z ³ and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I),

-CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethyl-glycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N -methyl-α-aminoisobutanoic acid.

[12] In some embodiments, the compound has the structure of formula (IV):

Formula (IV)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

X is absent or selected from -CH ₂ - and -NH-;

Y is ═CH- or ═N-;

Z ¹ , Z ² , Z ³ and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

AA is an amino acid selected from glycine, D or L proline, methylglycine, D or L alanine, D or L N -methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α -aminoisobutanoic acid.

[13] In some embodiments, R ¹ is -H. In some embodiments, at least one of Z ¹ , Z ² , Z ³ and Z ⁴ is not H. In some embodiments, at least one of Z ¹ , Z ² , Z ³ and Z ⁴ is -F or - _NO2 . In some embodiments, n is 0 and X is absent. In some embodiments, n is 0 and X is -CH ₂ -. In some embodiments, n is 0 and X is -O-, NHMe, or -S-. In some embodiments, the compound is selected from:

and

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[14] In some embodiments, the pharmaceutically acceptable counterion is selected from H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ ; where R represents H or C ₁ -C ₄ alkyl.

[15] In some embodiments, R' is O. In some embodiments, R' is:

[16] In some embodiments, the compound does not bind to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier. In some embodiments, the compound is bound to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier. In some embodiments, the thiol-containing macromolecular carrier or tumor-specific thiol-containing carrier is selected from endogenous albumin, exogenous albumin, antibody, antibody fragment, peptide, natural or synthetic polymer, liposome, and nanoparticle. In some embodiments, TBG is an optionally substituted maleimide group. In some embodiments, Z ^1' is selected from -NO ₂ or -SO ₃ M ² ;

and Y' is selected from -NHC(O)- or

[17] In some embodiments, R ^1' is

[18] In some embodiments, R' is:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof,

where R ^2' is selected from optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -.

или его фармацевтически приемлемую соль, гидрат, сольват или изомер.[19] In some embodiments, R' is:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[20] In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[21] In some embodiments, R' is:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; where M ¹ is a pharmaceutically acceptable counterion.

[22] In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[23] In some embodiments, a compound according to any one of claims 14-20, wherein R' is:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof; wherein M ¹ is a pharmaceutically acceptable counterion.

[24] In some embodiments, the compound of claim 26 is:

[25] Other embodiments include a pharmaceutical composition containing a compound as described herein and a pharmaceutically acceptable carrier.

[26] Other embodiments include a method of treating a disease or condition selected from cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms, comprising administering to a patient, if necessary, therapeutically an effective amount of a compound or pharmaceutical composition as described herein. In some embodiments, the disease is a cancer, e.g., the cancer is selected from adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampullar colon cancer, anal carcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, non-small cell bronchial carcinoma, breast cancer, Burkitt's lymphoma, body carcinoma, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial carcinoma, gallbladder cancer, gallbladder carcinoma, uterine cancer, cervical cancer, tumor neck, nose and ear, hematologic neoplasia, hairy cell leukemia, urethral cancer, skin cancer, glioma, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal cancer, head/neck tumors, colon carcinoma, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer , colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer, renal cell carcinoma, oligodendroglioma, esophageal cancer, osteolytic and osteoplastic carcinoma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, penile cancer, prostate cancer, tongue cancer, ovarian carcinoma and cancer of the lymph node.

[27] Other embodiments include a method of reducing the cytotoxicity of a compound, comprising administering a compound or pharmaceutical composition as described herein to a patient in need thereof, wherein administration results in a reduction in cytotoxicity compared to an equivalent dose of the unmodified active agent.

[28] Other embodiments include a method of increasing the concentration of a metabolite of a compound in a tumor, comprising administering a compound or pharmaceutical composition as described herein to a patient in need thereof, wherein the increase is compared to an equivalent dose of the unmodified active agent.

[29] Other embodiments include the compound disclosed herein for use as a drug.

[30] Other embodiments include a compound described herein for use in the treatment of a disease or condition selected from the group consisting of cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms.

[31] Other embodiments include the use of a compound or composition as described herein for the preparation of a medicament for the treatment of a disease or condition selected from cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi or other microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[32] Figure 1 shows the stability of various c 4 linkers in CD1 mouse plasma.

[33] Figure 2 shows a heatmap of geometric mean IC ₅₀ values across a panel of different cell lines.

[34] Figure 3 shows tumor growth curves of the control group, the maytansine group, and the groups treated with compounds 30, 42, 31, and 35 in the RXF631 renal cell tumor model.

[35] Figure 4 shows body weight curves in the control group, the maytansine group and the groups treated with compounds 30, 42, 31 and 35 in the RXF631 renal cell tumor model.

[36] Figure 5 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compounds 32, 30 and 31 in the LXFE 937 squamous cell lung carcinoma model.

[37] Figure 6 shows body weight curves in the control group, the maytansine group and the groups treated with compounds 32, 30 and 31 in the LXFE 937 squamous cell lung carcinoma model.

[38] Figure 7 shows the tumor growth curves of the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the LXFE 937 squamous cell lung carcinoma model.

[39] Figure 8 shows body weight curves for the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the LXFE 937 squamous cell lung carcinoma model.

[40] Figure 9 shows the tumor growth curves of the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the LXFA 737 lung adenocarcinoma model.

[41] Figure 10 shows body weight curves for the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the LXFA 737 lung adenocarcinoma model.

[42] Figure 11 shows the tumor growth curves of the control group, the maytansine group, and the groups treated with compounds 32, 30 and 31 in the MDA-MB 231 breast cancer model.

[43] Figure 12 shows body weight curves for the control group, the maytansine group, and the groups treated with compounds 32, 30 and 31 in the MDA-MB 231 breast tumor model.

[44] Figure 13 shows the tumor growth curves of the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the A2780 ovarian cancer model.

[45] The figure 14 shows the curves of changes in body weight in the control group, the maytansine group and the groups treated with compounds 30 and 31 in the model of ovarian cancer A2780.

[46] Figure 15 shows the tumor growth curves of the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the MDA-MB 468 breast cancer model.

[47] Figure 16 shows body weight curves for the control group, the maytansine group, and the groups treated with compounds 30 and 31 in the MDA-MB 468 breast tumor model.

DETAILED DESCRIPTION

[48] Unless otherwise indicated, scientific and technical terms used in this application should have the meanings that are commonly understood by experts in this field of technology. Generally, the nomenclature that refers to the methods of chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein chemistry described herein is well known and widely used in the art.

[49] All publications, patents and published patent applications mentioned in this application are expressly incorporated herein by reference. In the event of a conflict, the present description, including its specific definitions, will govern. Unless otherwise indicated, it is to be understood that each embodiment disclosed herein may be used alone or in combination with any one or more other embodiments of the invention.

Definitions

[50] As used herein, the word "comprise" or variations thereof such as "comprises" or "comprising" should be understood to mean the inclusion of a specified integer (or components) or group of integers (or components), but not the exclusion of any or another integer (or components) or a group of integers (or components).

[51] Throughout this application, where a compound or composition is described as having, including, or containing specific components, it is contemplated that such compound or composition may also consist substantially of, or consist of, the recited components. Similarly, when methods or processes are described as having, including, or containing specific process steps, the processes may also consist essentially of, or consist of, the listed process steps. In addition, it should be understood that the order of the steps or the order in which certain actions are performed is not essential, as long as the compounds, compositions, and methods described herein remain operable. In addition, two or more steps or actions may be performed at the same time.

[52] Singular noun forms include plural forms unless the context clearly specifies otherwise.

[53] The term "including" is used to mean "including, but not limited to". "Including" and "including but not limited to" are used interchangeably.

[54] As used herein, the term "or" should be understood to mean "and/or" unless the context clearly indicates otherwise.

[55] The terms "drug", "agent", "therapeutic agent", "therapeutically active agent", "cytotoxic agent or drug", "highly cytotoxic agent or drug", or "therapeutically effective substance" are used to refer to any a compound which produces a pharmacological effect, either by itself or after its transformation in the organism in question, and therefore also includes derivatives of these transformations. The pharmacological effect of drugs according to the composition according to the present invention can be only a single one, for example, a cytostatic and/or cytotoxic effect, or a wide pharmacological spectrum of action, such as an immunosuppressive and anti-inflammatory effect at the same time.

[56] The terms "patient", "subject" or "individual" are used interchangeably and refer to a non-human human or animal. These terms include mammals such as humans, primates, farm animals (eg cows, pigs), domestic animals (eg dogs, cats) and rodents (eg mice and rats). In certain embodiments, the patient or subject is a patient or human subject, such as a human patient, having a condition to be treated.

[57] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal subject, including humans and mammals, for example, in combination with one or more pharmaceutically acceptable carriers, excipients or diluents. Such a composition may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, protectants, and other materials well known in the art. In some embodiments, a pharmaceutical composition includes a composition containing an active ingredient(s) and an inert ingredient(s) that form an excipient, carrier, or diluent, as well as any product that results directly or indirectly from the combination, complexation, or aggregation of any two or more ingredients, or dissociation of one or more ingredients, or other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition obtained by mixing a compound of the present invention and one or more pharmaceutically acceptable excipients, carriers and/or diluents.

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

[59] The terms "pharmaceutically effective amount", "therapeutically effective amount", or "therapeutically effective dose" refer to an amount effective to treat a disease or condition in a patient, for example, to bring about a beneficial and/or desirable change in the general health of a patient suffering from a disease (eg, cancer) or condition, treatment, healing, inhibition or attenuation of a physiological response or condition, and so on. The full therapeutic effect does not necessarily occur with the introduction of a single dose and may occur only after a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for a subject will depend, for example, on the size, health and age of the subject, the nature and extent of the disease, the therapeutic agents or combination of therapeutic agents chosen to be administered, and the route of administration. One skilled in the art can readily determine the effective amount for a given situation through routine experimentation. The skilled artisan will appreciate that treating cancer includes, but is not limited to, killing cancer cells, preventing the growth of new cancer cells, inducing tumor regression (reducing the size of the tumor), inducing a reduction in metastasis, improving the patient's vital functions, improving the patient's well-being, reducing pain, improving appetite. , improving the patient's weight, and any combination thereof. The terms "pharmaceutically effective amount", "therapeutically effective amount" or "therapeutically effective dose" also refer to the amount necessary to improve the clinical symptoms of the patient. Therapeutic methods or methods of treating cancer described herein should not be interpreted or otherwise limited to the "treatment" of cancer.

[60] As used herein, the term "treatment" or "treatment" includes changing, reducing, or stopping the symptoms, clinical signs, and pathology underlying the condition, so as to improve or stabilize the condition of the subject. As used herein and well understood in the art, "treatment" is an approach to obtain beneficial or desirable results, including clinical results. Beneficial or desirable clinical outcomes may include, but are not limited to, reduction, improvement, or slowing of the progression of one or more symptoms or conditions associated with a condition, such as cancer, reduction in disease severity, a stabilized disease state (i.e., not worsening), delaying or slowing down the progression of the disease, improvement or mitigation of the disease state and remission (partial or complete), detectable or not detectable. "Treatment" can also mean an increase in survival compared to expected survival if no treatment is given. Typical useful clinical results are described in this document.

[61] "Administration" or "administration" of a substance, compound, or agent to a subject can be accomplished using one of a variety of methods known to those skilled in the art. For example, a compound or agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, through the eyes, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebral, and transdermally (by absorption, for example, through the skin). duct). The compound or agent can also be appropriately administered using rechargeable or biodegradable polymeric devices or other devices, such as patches and pumps, or formulations that provide sustained, slow, or controlled release of the compound or agent. The administration may also be performed, for example, once, multiple times, and/or over one or more extended periods. In some aspects, administration includes both direct administration, including self-administration, and indirect administration, including the effect of the prescribed drug. For example, as used herein, a physician who instructs a patient to self-administer a drug or prescribes a drug to another person and/or who provides a patient with a prescription for a drug administers the drug to the patient. When the method is part of a therapeutic regimen that includes more than one agent or treatment, it is contemplated in the disclosure that the agents may be administered at the same or different times and by the same or different routes of administration. Suitable routes for administering a substance, compound, or agent to a subject will also depend on, for example, the age of the subject, whether the subject is active or inactive at the time of administration, whether the subject has cognitive impairment at the time of administration, the degree of impairment, and the chemical and biological properties of the compound or agent (e.g. , solubility, digestibility, bioavailability, stability and toxicity).

[62] The term "substituted" refers to moieties having substituents replacing hydrogen on one or more of the carbon atoms of the backbone of the chemical compound. It is understood that "substitution" or "substituted" includes the implicit condition that such substitution corresponds to the allowable valency of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as rearrangement, cyclization, deletion, etc. e. As used herein, the term "substituted" is intended to include all permissible organic substituents. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and straight, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Allowable substituents may be one or more, the same or different for the respective organic compounds. For the purposes of the present invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any valid substituents of the organic compounds described herein that satisfy the valency of the heteroatoms. The substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfonyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic (e.g., C ₆ -C ₁₂ aryl) or heteroaromatic (e.g., heteroaryl) fragment.

[63] "Optional" or "optional" means that the following circumstance may or may not occur, so the application includes cases where the circumstance occurs and cases where it does not. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus the application includes structures in which a non-hydrogen substituent is present and structures in which a non-hydrogen substituent is not present.

[64] Unless specifically stated that they are "unsubstituted", references to chemical moieties in this document are understood to include substituted variants. For example, reference to an "alkyl" group or moiety is implicitly inclusive of both substituted and unsubstituted variants. Examples of substituents on chemical moieties include, but are not limited to, moieties: halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aryl or heteroaryl.

[65] "Aryl" means an aromatic carbon ring having the specified number of carbon atoms, for example, 6 to 12 or 6 to 10 carbon atoms in the ring. Aryl groups may be monocyclic or polycyclic (eg bicyclic, tricyclic). In some instances, both rings of a polycyclic aryl group are aromatic (eg, naphthyl). In other instances, polycyclic aryl groups may include a non-aromatic ring (eg, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl) fused to an aromatic ring, provided that the polycyclic aryl group is bonded to the parent structure via an atom on the aromatic ring. Thus, the 1,2,3,4-tetrahydronaphthalen-5-yl group (where the moiety is linked to the parent structure through an aromatic carbon atom) is considered an aryl group, while 1,2,3,4-tetrahydronaphthalen-1-yl (where the moiety is linked to the parent structure through a non-aromatic carbon atom) is not considered an aryl group. Similarly, l,2,3,4-tetrahydroquinolin-8-yl group (where the moiety is linked to the parent structure via an aromatic carbon atom) is considered an aryl group, while 1,2,3,4-tetrahydroquinolin-1-yl group (where the moiety is linked to the parent structure through a non-aromatic nitrogen atom) is not considered an aryl group. However, the term "aryl" does not encompass or overlap with "heteroaryl" as defined herein, regardless of the point of attachment (eg, both quinolin-5-yl and quinolin-2-yl are heteroaryl groups).

[66] "Heteroaryl" means an aromatic ring containing the specified number of ring atoms (for example, 5-12- or 5-10-membered heteroaryl), consisting of one or more heteroatoms (for example, 1, 2, 3 or 4 heteroatoms), selected from N, O and S, and with the rest of the ring atoms being carbon. A 5-membered heteroaryl is a heteroaryl with 5 ring atoms. A 6-membered heteroaryl is a heteroaryl with 6 ring atoms. Heteroaryl groups do not contain adjacent S and O atoms. In some embodiments, the total number of S and O atoms in a heteroaryl group is no more than 2. In some embodiments, the total number of S and O atoms in a heteroaryl group is no more than 1. Unless otherwise noted , heteroaryl groups may be linked to the parent structure by a carbon or nitrogen atom, if valence permits. For example, "pyridyl" includes 2-pyridyl, 3-pyridyl, and 4-pyridyl groups, and "pyrrolyl" includes 1-pyrrolyl, 2-pyrrolyl, and 3-pyrrolyl groups. When nitrogen is present in the heteroaryl ring, it may, where the nature of adjacent atoms and groups permits, exist in an oxidized state (ie, N ⁺ -O ^- ). In addition, when sulfur is present on the heteroaryl ring, it may, where the nature of adjacent atoms and groups permits, exist in an oxidized state (ie S ⁺ -O ^- or SO ₂ ). Heteroaryl groups may be monocyclic or polycyclic (eg bicyclic, tricyclic).

[67] In some cases, the heteroaryl group is monocyclic. Examples include pyrrole, pyrazole, imidazole, triazole (e.g. 1,2,3-triazole, 1,2,4-triazole, 1,3,4-triazole), tetrazole, furan, isoxazole, oxazole, oxadiazole (e.g. 1 ,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole), thiophene, isothiazole, thiazole, thiadiazole (e.g. 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole), pyridine, pyridazine, pyrimidine, pyrazine, triazine (eg 1,2,4-triazine, 1,3,5-triazine) and tetrazine.

[68] The term "acyl" is known in the art and refers to the group represented by the general formula hydrocarbyl-C(O)-, eg alkyl-C(O)-.

[69] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups and branched chain alkyl groups. In some embodiments, a straight or branched alkyl has 30 or fewer carbon atoms in its backbone (e.g., C ₁ -C ₃₀ for straight chains, C ₄ -C ₃₀ for branched chains), and in other embodiments, 20 or less . In some embodiments, the alkyl groups are lower alkyl groups, such as methyl, ethyl, n -propyl, i -propyl, n -butyl, and n -pentyl. In addition, the term "alkyl" as used in the specification, examples and claims is intended to mean both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl groups having substituents replacing a hydrogen on one or more carbon atoms In some embodiments, straight or branched alkyl has 30 or fewer carbon atoms in its backbone (eg, C ₁ -C ₃₀ for straight chains, C ₃ -C ₃₀ for branched chains). In some embodiments, the chain has ten or fewer carbon atoms (C ₁ -C ₁₀ ) in its backbone. In other embodiments, the chain has six or fewer carbon atoms (C ₁ -C ₆ ) in its backbone.

или

Стереохимия гидразонового фрагмента может быть E или Z. Термин гидразон, используемый в данном документе, включает как E, так и Z-изомеры. Гидразоновые фрагменты, раскрытые в данном документе, в общем, нарисованы в одной конфигурации, но понятно, что это изобретение может включать как E, так и/или Z.[70] The terms "hydrazone fragment" or "hydrazone" refer to E and/or Z hydrazones, for example,

or

The stereochemistry of the hydrazone moiety can be E or Z. The term hydrazone as used herein includes both the E and Z isomers. The hydrazone moieties disclosed herein are generally drawn in one configuration, but it is understood that this invention may include both E and/or Z.

[71] At various places in the present description, the substituents of the compounds according to the invention are disclosed in groups or in ranges. In particular, the present invention is intended to include every single sub-combination of members of such groups and ranges. For example, the term "C ₁ -C ₆ alkyl" is specifically intended to cover individually methyl, ethyl, propyl, isopropyl, n -butyl, sec -butyl, isobutyl, and so on.

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

[73] The present invention further provides pharmaceutical compositions comprising one or more compounds of the invention together with a pharmaceutically acceptable carrier or excipient. The compounds or pharmaceutical compositions of the invention may be used in vitro or in vivo .

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

[75] The present invention further includes isotopically labeled or enriched compounds of the present invention. An "isotopically" or "radioactively labeled" compound is a compound of the present invention in which one or more atoms are replaced or replaced by an atom whose atomic mass or mass number is different from the atomic mass or mass number normally found in nature (i.e. , of natural origin). Suitable radionuclides that may be included in the compounds of the present invention include, but are not limited to, ² H (also referred to as D for deuterium), ³ H (also referred to as T for tritium), ¹¹ C, ¹³ C, ¹⁴ C , ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ¹⁸ F, ³⁵ S, ³⁶ Cl, ⁸² Br, ⁷⁵ Br, ⁷⁶ Br, and ⁷⁷ Br. The radionuclide that is included in the present radiolabeled compounds will depend on the specific application of that radiolabeled compound. For example, for labeling metalloproteases and in vitro competitive assays, compounds that include ³ H, ¹⁴ C, ⁸² Br, ³⁵ S or more will generally be most used. For radio imaging applications, ¹¹ C, ¹⁸ F, ⁷⁵ Br, ⁷⁶ Br or ⁷⁷ Br will be the most used. Tritium ( ³ H) and ¹⁴ C can be used for ADME studies. In some embodiments, each alkyl, cycloalkyl, alkene, alkylene, and alkoxy is optionally substituted with one or more -D or -F.

Compounds of the present invention

[76] Embodiments of the present invention provide for the connection with the structure of the presented formula (I):

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

R ¹ is selected from -H and C ₁ -C ₄ alkyl;

Spacer selected from:

и

and

V is absent or selected from -CH ₂ -, -O- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

each R ² is independently selected from -H, halogen (eg -F, -Cl, -Br or -I) and C ₁ -C ₄ alkyl or the two R ² together form C ₃ -C ₆ , cycloalkyl;

n is 0-3;

X is absent or selected from -CH ₂ -, -O-, -S-, -Se-, and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl;

Y is selected from =CH- and =N-;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ . C ₁ -C ₄ alkyl and C ₂ -C ₄ alkoxy;

AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethyl-glycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N -methyl-α-aminoisobutanoic acid;

R' is chosen from O and

Y' is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)- and -C(O)-NH-; or Y' is selected from the group consisting of:

и

and

where n=0-6; R ¹ ' is absent or selected from the group consisting of:

и

and

where M ¹ represents a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ ; where R represents H or C ₁ -C ₄ alkyl);

R ² ' is an optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -;

Z ¹ ', Z ² ', Z ³ ' and Z ⁴ ' are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ , -SO ₃ M ² and C ₁ -C ₄ alkyl, where M ² is a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ , where R is H or C ₁ -C ₄ alkyl);

TBG is a thiol-binding group selected from an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group a group and an optionally substituted N-hydroxysuccininide ester group;

wherein said TBG is optionally linked to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier.

[77] In some embodiments, in a compound of formula (I), R ¹ is selected from -H and C ₁ -C ₄ alkyl;

Spacer selected from:

и

and

V is absent or selected from -CH ₂ -, -O- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

each R ² is independently selected from -H, halogen (eg -F, -Cl, -Br or -I) and C ₁ -C ₄ alkyl or the two R ² together form C ₃ -C ₆ , cycloalkyl;

n is 0-3;

X is absent or selected from -CH ₂ -, -O-, -S-, -Se-, and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl;

Y is selected from =CH- and =N-;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ . C ₁ -C ₄ alkyl and C ₂ -C ₄ alkoxy;

AA is an amino acid selected from glycine, D or L proline, methylglycine, D or L alanine, D or L N -methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α -aminoisobutanoic acid; R' is chosen from O and

Y' is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)- and -C(O)-NH-; or Y' is selected from the group consisting of:

и

and

where n=0-6;

R ¹ ' is absent or selected from the group consisting of:

и

and

where M ¹ represents a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ ; where R represents H or C ₁ -C ₄ alkyl);

R ² ' is an optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -;

Z ¹ ', Z ² ', Z ³ ' and Z ⁴ ' are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ , -SO ₃ M ² and C ₁ -C ₄ alkyl, where M ² is a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ , where R is H or C ₁ -C ₄ alkyl);

TBG is a thiol-binding group selected from an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group a group and an optionally substituted N-hydroxysuccininide ester group;

[78] wherein said TBG is optionally associated with a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier.

[79] In some embodiments, R' is O. These novel compounds may be active agents and may be, for example, an active component of a drug delivery system or an active metabolite that is released from a drug delivery system.

[80] In some embodiments, a compound of formula (I) wherein R' is O has the structure of any of formulas (II'), (III'), and (IV'):

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

each R ² is independently selected from -H and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ , cycloalkyl;

X is absent or selected from -CH ₂ -, -O-, -S- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

Formula (IV')

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

X is absent or selected from -CH ₂ - and -NH-;

Y is ═CH- or ═N-;

Z ¹ , Z ² , Z ³ and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I),

-CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethyl-glycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α-aminoisobutanoic acid.

[81] In other embodiments, in compounds of formula (IV'):

X is absent or selected from -CH ₂ - and -NH-; Y is ═CH- or ═N-;

Z ¹ , Z ² , Z ³ and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I),

-CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

AA is an amino acid selected from glycine, D or L proline, methylglycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α -aminoisobutanoic acid.

[82] In some embodiments, the compound is selected from the following specific compounds:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[83] Other embodiments include prodrugs, such as prodrugs represented by formula (I), where R' is:

These new compounds may be a drug delivery system whereby the active metabolite is selectively released from the drug delivery system. They include, for example, albumin-binding prodrugs.

[84] In certain embodiments, these compounds have the structure of any of formulas (II), (III) and (IV):

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

each R ² is independently selected from -H, and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ -cycloalkyl;

X is absent or selected from -CH ₂ -, -O-, -S- and -NR ³ -, where R ³ represents -H or C ₁ -C ₄ alkyl;

Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

Formula (IV)

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof, wherein:

X is absent or selected from -CH ₂ - and -NH-;

Y is ═CH- or ═N-;

Z ¹ , Z ² , Z ³ and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethyl-glycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N -methyl-α-aminoisobutanoic acid; and

where R' is:

[85] In other embodiments, in compounds of formula (IV'):

X is absent or selected from -CH ₂ - and -NH-;

Y is ═CH- or ═N-; Z ¹ , Z ² , Z ³ and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ ;

AA is an amino acid selected from glycine, D or L proline, methylglycine, D or L alanine, D or L N-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutanoic acid and N-methyl-α -aminoisobutanoic acid; and

where R' is:

[86] In some embodiments, R ¹ is -H. In other embodiments, at least one of Z ¹ , Z ² , Z ³ and Z ⁴ is not H and/or at least one of Z ¹ , Z ² , Z ³ and Z ⁴ is -F or -NO ₂ . In some embodiments, when n is 0, X is absent. In other embodiments, when n is 0, X is -CH ₂ -. In some embodiments, n is 0 and X is -O- or -S-. Additional embodiments include pharmaceutically acceptable salts, solvates, hydrates, tautomers, and solid forms of the compounds of the present invention.

[87] In some embodiments, the compound is selected from the following specific compounds:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

[88] In certain embodiments, the compound does not bind to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier. In other embodiments, the compound is bound to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier. For example, a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier is selected from endogenous albumin, exogenous albumin, antibody, antibody fragment, peptide, natural or synthetic polymer, liposome, and nanoparticle.

[89] In some embodiments, TBG is an optionally substituted maleimide group, eg, an unsubstituted maleimide group. In some embodiments, the maleimide group binds rapidly and selectively to the cysteine-34 of albumin after administration to a subject, such as a human.

. В некоторых вариантах воплощения R¹’ представляет собой

. В некоторых вариантах воплощения R²’ выбран из необязательно замещенного C₁-C₁₈ алкила, где необязательно до шести атомов углерода в указанном C₁-C₁₈ алкиле каждый независимо замещены с -OCH₂CH₂- (например, 1, 2, 3, 4, 5 или 6 атомы углерода заменены с -OCH₂CH₂-).[90] In some embodiments, Z ¹ ' is selected from -NO ₂ or -SO ₃ M ² and/or and Y' is selected from -NHC(O)- or

. In some embodiments, R ¹ ' is

. In some embodiments, R ² ' is selected from an optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ - (e.g., 1, 2, 3 , 4, 5 or 6 carbon atoms are replaced with -OCH ₂ CH ₂ -).

[91] In some embodiments, R' is:

или

For example, R' might be:

or

[92] Specific compounds within the scope of the present invention include the following:

or a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof.

Pharmaceutical compositions

, или различные варианты осуществления, раскрытые в данном документе.[93] In some embodiments, the present invention relates to a pharmaceutical composition containing the compound described herein. In some embodiments, the composition comprises a compound of formula (I) wherein R' is:

, or various embodiments disclosed herein.

[94] The total amount of the compound in the composition for administration to a patient is that which is appropriate for that patient. One skilled in the art will appreciate that different individuals may require different total amounts of a therapeutically effective substance. In some embodiments, the amount of the compound is a pharmaceutically effective amount. One skilled in the art will be able to determine the amount of a compound in a composition needed to treat a patient based on factors such as, for example, the age, weight, and physical condition of the patient. The concentration of the compound depends on its solubility in the intravenous solution and on the volume of liquid that can be administered. For example, the concentration of the compound may be from about 0.1 mg/ml to about 50 mg/ml in an injectable composition. In some embodiments, the concentration of the compound may range from about 0.1 to about 40 mg/mL.

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

[96] Compositions can be administered in various conventional ways. Typical routes of administration that can be used include oral, parenteral, intravenous, intraarterial, dermal, subcutaneous, intramuscular, topical, intracranial, intraorbital, ophthalmic, intravitreal, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, aerosol, administration through the central nervous system (CNS) or suppository. In some embodiments, the compositions are suitable for parenteral administration. These compositions can be administered, for example, intraperitoneally, intravenously or intrathecally. In some embodiments, the compositions are administered intravenously. In some embodiments, a reconstituted composition can be prepared by reconstituting a lyophilized compound composition in a reconstituting liquid containing, for example, alcohol, DMSO and/or polyethylene glycol and water and/or saline buffer. Such recovery may include the addition of a recovery liquid and mixing, for example, by stirring or shaking the mixture. The reconstituted composition can then be made suitable for injection by mixing, for example, lactated Ringer's solution, 5% glucose solution, isotonic saline, or a suitable saline buffer with the composition to create an injectable composition. One of skill in the art will appreciate that the manner in which a therapeutically effective formulation or composition of substance is administered will depend on such factors as the age, weight, and physical condition of the patient being treated and the disease or condition being treated. Thus, a qualified specialist will be able to choose the method of administration that is optimal for the patient, in each case.

[97] In some embodiments, the compounds and compositions disclosed herein are for use in the treatment of cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms.

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

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

[100] In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, left ventricular carcinoma, anal carcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, non-small cell bronchial carcinoma, cancer breast, Burkitt's lymphoma, body carcinoma, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial carcinoma, gallbladder cancer, gallbladder carcinoma, uterine cancer, cervical cancer, tumors of the neck, nose and ears, hematologic neoplasias , hairy cell leukemia, urethral cancer, skin cancer, gliomas, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal cancer, head/neck tumors, colon carcinoma, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer , Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer, renal cell carcinoma a, oligodendroglioma, esophageal cancer, osteolytic and osteoplastic carcinoma, osteosarcoma, ovarian cancer, pancreatic carcinoma, penile cancer, prostate cancer, tongue cancer, ovarian cancer or lymph node cancer.

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

[102] In some embodiments, one or more excipients may be included in the composition. One skilled in the art will appreciate that the choice of any one excipient may influence the selection of any other excipient. For example, the choice of excipient may preclude the use of one or more additional excipients because the combination of excipients will cause undesirable effects. One skilled in the art will be able to empirically determine which excipients, if any, to include in the compositions. Fillers may include, but are not limited to, cosolvents, solubilizing agents, buffers, pH adjusters, bulking agents, surfactants, encapsulating agents, tonicity adjusters, stabilizers, protectants, and viscosity modifiers. In some embodiments, it may be advantageous to include a pharmaceutically acceptable carrier in the compositions.

[103] In some embodiments, a solubilizing agent may be included in the composition. Solubilizing agents can be used to increase the solubility of any of the components of the composition, including the compound or excipient. The solubilizing agents described herein are not intended to be an exhaustive list, but are provided only as exemplary solubilizing agents that can be used in the compositions. In some embodiments, solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerin, propylene glycol, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, cyclodextrins such as dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin , hydroxypropyl-β-cyclodextrin and trimethyl-β-cyclodextrin and combinations thereof, as well as any pharmaceutically acceptable salts and/or combinations thereof

[104] The pH of the compositions can be any pH that provides the desired properties for the composition or composition. Desirable properties may include, for example, compound stability, increased compound retention compared to formulations at other pHs, and improved filtration efficiency. In some embodiments, the pH of the compositions may be from about 3.0 to about 9.0, for example, from about 5.0 to about 7.0. In specific embodiments, the pH value of the compositions may be 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1.6 .0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, 6.5±0.1.6.6 ±0.1, 6.7±0.1, 6.8±0.1, 6.9±0.1, 7.0±0.1, 7.1±0.1 and 7.2±0 ,one.

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

[106] In some embodiments, a pH adjuster may be included in the composition. Changing the pH of the composition may have a beneficial effect, for example, on the stability or solubility of the compound, or may be useful in obtaining a composition suitable for parenteral administration. pH adjusters are well known in the art. Accordingly, the pH adjusters described herein are not intended to be an exhaustive list, but are provided only as exemplary pH adjusters that may be used in the compositions. pH adjusters may include, for example, acids and bases. In some embodiments, the pH adjuster includes, but is not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

[107] In some embodiments, a bulking agent may be included in the composition. Bulking agents are commonly used in lyophilized compositions to add extra volume to the composition and to allow visualization of the composition, especially in cases where the lyophilized pellet would be difficult to see. Bulking agents can also help prevent release of the active ingredient(s) of the pharmaceutical composition and/or impart cryoprotection to the composition. Bulking agents are well known in the art. Accordingly, the bulking agents described herein are not intended to be an exhaustive list, but are provided only as exemplary bulking agents that may be used in the compositions.

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

[109] In some embodiments, a surfactant may be included in the composition. Surfactants generally lower the surface tension of a liquid composition. This may provide useful properties such as improved ease of filtering. Surfactants can also act as emulsifying agents and/or solubilizing agents. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to be an exhaustive list, but are presented only as exemplary surfactants that may be used in the formulations or compositions of the present invention. Surfactants that may be included include, but are not limited to, sorbitol esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (tho there are pluronics), ethylene oxides and polyethylene oxides (eg Triton X-100), saponins, phospholipids (eg lecithin), and combinations thereof.

[110] In some embodiments, an encapsulating agent may be included in the composition. Encapsulating agents can isolate molecules and help stabilize or dissolve them. Encapsulating agents are well known in the art. Accordingly, the encapsulating agents described herein are not intended to be an exhaustive list, but are provided only as exemplary encapsulating agents that may be used in the compositions. Encapsulating agents that may be included in the compositions include, but are not limited to, α-cyclodextrins, β-cyclodextrins, γ-cyclodextrin, and combinations thereof (e.g., α-cyclodextrin, dimethyl-α-cyclodextrin, hydroxyethyl-α-cyclodextrin, hydroxypropyl-α-cyclodextrin, trimethyl-α-cyclodextrin, β-cyclodextrin, dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, trimethyl-β-cyclodextrin, γ-cyclodextrin, dimethyl-γ-cyclodextrin, hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, trimethyl-γ-cyclodextrin and combinations thereof.

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

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

[113] In some embodiments, a protective agent may be included in the composition. Protectants are agents that protect a pharmaceutically active ingredient (eg, a therapeutically effective substance or compound) from an undesirable condition (eg, instability caused by freezing or lyophilization or oxidation). Protective agents may include, for example, cryoprotectants, lyoprotectants and antioxidants. Cryoprotectants are used to prevent the loss of activity of an active pharmaceutical ingredient (eg an anthracycline compound) when the composition is exposed to temperatures below its freezing point. For example, a cryoprotectant may be included in the reconstituted lyophilized composition such that the composition may be frozen prior to dilution for intravenous administration. Cryoprotectants are well known in the art. Accordingly, the cryoprotectants described herein are not intended to be an exhaustive list, but are provided only as exemplary cryoprotectants that may be used in the compositions. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Cryoprotectants may include, for example, disaccharides (eg sucrose, lactose, maltose and trehalose), polyols (eg glycerol, mannitol, sorbitol and dulcitol), glycols (eg ethylene glycol, polyethylene glycol and propylene glycol).

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

[115] Antioxidants are used to prevent oxidation of the components of the composition. Oxidation can lead to drug product aggregation or other detrimental effects on drug product purity or potency. Antioxidants are well known in the art. Accordingly, the antioxidants described herein are not intended to be an exhaustive list, but are provided only as exemplary antioxidants that may be used in the compositions. Antioxidants can be, for example, sodium ascorbate, citrate, thiols, metabisulphite, and combinations thereof.

[116] In some embodiments, a viscosity modifier may be included in the composition. Viscosity modifiers change the viscosity of liquid compositions. This can be advantageous because viscosity plays an important role in the ease of filtering of a liquid composition. The composition may be filtered before lyophilization and reconstitution or after reconstitution. Viscosity modifiers are well known in the art. Accordingly, the viscosity modifiers described herein are not intended to be an exhaustive list, but are provided only as exemplary viscosity modifiers that may be used in compositions. Viscosity modifiers include solvents, solubilizing agents, surfactants and encapsulating agents. Typical viscosity modifiers that may be included in the compositions include, but are not limited to, N-acetyl-DL-tryptophan and N-acetyl-cysteine.

Antitumor activity in mouse models of human tumor xenografts

[117] The albumin-binding maytansinoids 30 and 31 demonstrated exceptional antitumor activity in five human tumor xenograft models in nude mice, inducing partial and complete tumor regression in all human tumor xenografts evaluated (see Figures 3-16). This included initial tumor volumes in the range of approximately 80-110 mm ³ , but also initial initial tumor volumes up to approximately 400 mm ³ . In addition, in most cases, therapy with albumin-binding maytansinoids 30 and 31 caused long-term remissions and a decrease in relative tumor volume (RVR). The parent compound maytansine was largely inactive in the models tested or showed only marginal tumor inhibition. The experimental procedure and results in mouse tumor models are detailed in Examples 11-19 and Figures 3-16.

Methods of treatment

[118] The compounds and compositions described herein are used in a variety of clinical applications. In embodiments, the methods of treatment use a compound of the composition that includes a compound of formula (I), where R' is:

or various embodiments disclosed herein.

[119] Described in this document, the compounds and compositions can cause prolonged or prolonged inhibition of tumor growth. In certain embodiments, prolonged or prolonged inhibition of tumor growth occurs without any loss of body weight, or any or only minor bone marrow toxicity.

[120] In some embodiments, the present invention relates to a method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition containing a compound described herein. For example, some embodiments include a method of treating a patient suffering from a disease or condition selected from cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms, comprising administering to the patient, if necessary, this, a therapeutically effective amount of a compound or pharmaceutical composition according to the present invention.

[121] The present invention provides methods for treating a condition or disease in a patient, said condition or disease selected from cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms, comprising administering to the patient a compound or a pharmaceutical composition as described herein.

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

[123] In some embodiments, the cancer is selected from adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampullar colon cancer, anal carcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, non-small cell bronchial carcinoma, breast cancer, Burkitt's lymphoma, body carcinoma, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial carcinoma, gallbladder cancer, uterine cancer, cervical cancer, tumors of the neck, nose and ears, hematologic neoplasia , hairy cell leukemia, urethral cancer, skin cancer, glioma, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal cancer, head/neck tumors, colon carcinoma, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer , Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, colon cancer, medulloblastoma, melanoma, meningioma, cancer kidney, renal cell carcinoma, oligodendroglioma, esophageal cancer, osteolytic and osteoplastic carcinoma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, penile cancer, prostate cancer, tongue cancer, ovarian carcinoma and lymph node cancer.

[124] Some embodiments include a method of increasing the concentration of a metabolite of a compound in a tumor, comprising administering a compound according to the present invention. In embodiments, the compound is a compound of formula (I) where R' is:

,

,

or various embodiments disclosed herein. In some embodiments, the increase is compared to an equivalent dose of the unmodified active agent, for example, "unmodified active agent" can be the same compound of formula (I) where R' is O.

[125] Some embodiments include a method of reducing the cytotoxicity of a compound, comprising administering a compound or pharmaceutical composition of the present invention to a patient in need thereof, wherein the administration results in a reduction in cytotoxicity compared to an equivalent dose of the unmodified active agent. For example, in some embodiments, a method for reducing cytotoxicity comprises administering a compound of formula (I) where R' is:

or various embodiments disclosed herein and "unmodified active agent" is the same compound of formula (I) where R' is O.

Explanation with an example

[126] Now that aspects of the present disclosure have been generally described, they will be better understood with reference to the following examples, which are included for the purpose of illustrating certain features and embodiments of the present invention only and are not intended to be limiting.

Equivalents

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

Examples

[128] The following examples demonstrate various embodiments and aspects of the invention.

Abbreviations

[129] The following is a list of abbreviations used in the examples, with their full chemical names. If they are not defined, the terms have their conventional meanings.

aq. = water

Boc= N -tert-butoxycarbonyl

calculated=calculated

DXM=dichloromethane

DIC= N, N' -diisopropylcarbodiimide

DMAP= N, N -dimethyl-4-aminopyridine

DMF= N, N -dimethylformamide

DMSO=dimethyl sulfoxide

vol.column=column volume

EDK=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

equiv.=equivalent

ESI=electrospray ionization

Fmoc=fluorenylmethyloxycarbonyl

HATU=(l-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)

HOAt=1-hydroxy-7-azabenzotriazole

HOBt=N-hydroxybenzotriazole

HPLC = high performance liquid chromatography

HRMS = high resolution mass spectrometry

HSA=human serum albumin

IC = ion chromatography

LC-MS=liquid chromatography - mass spectroscopy

MSNR = low resolution mass spectrometry.

MeCN=acetonitrile

MeOH=methanol

NMR = nuclear magnetic resonance spectroscopy

NF=normal phase

Np=4-nitrophenoxycarbonyl

nd=not found

na= not available

PBS=phosphate buffered saline, pH 7

RP=reverse phase

TFA=trifluoroacetic acid

Tris=2-amino-2-(hydroxymethyl)-1,3-propandio

Example 1

Obtaining linker 1

[130] Linker 1 can be prepared as described below and shown in Scheme 1.

Scheme 1

Synthesis of 4-nitro-2-sulphobenzoic acid ( A )

[131] To a stirred solution of potassium permanganate (72 g, 460 mmol, 4.5 eq.) in water (450 ml) was added a solution of 4-nitro-2-sulfonic acid hydrate (26 g, 102 mmol, 1 0 eq.) in Millipore water (100 ml). The resulting purple mixture was stirred at 115°C for 5 hours and after this time it turned brown. HPLC analysis (PDA 220 nm) confirmed that the reaction was complete after 5 hours (more than 90% conversion). The reaction mixture was cooled to room temperature. The brown solid formed during the reaction was removed by suction filtration on a Celite pad, washed with Millipore water (300 ml) and the brown/yellow filtrate solution was concentrated to approximately 125 ml using a rotary evaporator at 40°C, slowly acidified with a 5 M solution HCl (about 2 ml) to form a white suspension (pH about 1.0). The white suspension was then heated at 100° C. until a clear solution was obtained, which was then left in an ice bath for 10 minutes until a white solid formed. A white solid was obtained by suction filtration using a fritted filter. The white solid was then dried under high vacuum to give 4-nitro-2-sulphobenzoic acid. Yield: 18 g (72%). Purity by RP-HPLC, 220 nm, over 95%. MSNR-ER (m/z) calc. for C7H4NO7S [MH] ^- : 245.98. Found: 245.83.

Synthesis of 4-amino-2-sulphobenzoic acid ( B )

[132] A stirred suspension of 4-nitro-2-sulphobenzoic acid (13 g, 51 mmol, 1.0 eq.) in water (75 ml) was heated under reflux until the 4-nitro-2-sulphobenzoic acid was completely dissolved. At this temperature, acetic acid (7.2 ml) was then added, followed by iron powder (9.5 g, 180 mmol, 3.5 eq.), which was added in portions (~ 1 g/min) over 10 minutes to avoid exothermic reaction. The reaction mixture was then allowed to stir under reflux for 1 hour. During this time, a brown solid formed and HPLC analysis (PDA 220 nm) confirmed that the reaction was complete (over 95% conversion). The brown solid was removed by suction filtration directly on the celite pad (while still hot) and then washed with hot water. The filtrate was re-filtered. The resulting filtrate was concentrated on a rotary evaporator at 40° C. to a final volume of 100 ml. Concentrated HCl was added dropwise until pH 1 was reached, after which a white/yellow precipitate formed. The suspension was left at 4°C for 1 hour. The solid was collected by suction filtration using a fritted filter and dried under high vacuum to give 4-amino-2-sulfobenzoic acid as a white solid. Yield: 9 g (81%). Purity by RP-HPLC, 220 nm, over 95%. MSNR-ER (m/z) calc. for C7H4NO5S [MH] ^- : 216.00. Found: 216.16.

Synthesis of 6-maleimidohexanoyl chloride or EMC-Cl ( C )

[133] To a stirred yellow solution of 6-maleimidocaproic acid (EMC) (33 g, 156 mmol, 1.0 eq.) in dry DCM (150 ml) at room temperature and N ₂ atmosphere for 30 min (~0, 5 ml/min) oxalyl chloride (15 ml, 171 mmol, 1.1 eq.) was added using an addition funnel. The reaction mixture was stirred at room temperature for 5 hours. The color of the reaction solution changed to dark yellow during the reaction time, and HPLC analysis (PDA 220 nm) confirmed that the reaction was complete after 5 hours (more than 95% conversion). The solvent was removed using a rotary evaporator at 40°C to obtain an oil. This residual oil was dried under high vacuum overnight (solidified overnight). The resulting light brownish solid was crushed and dried for an additional 20 hours under high vacuum to give 6-maleimidohexanoyl chloride as a yellow microcrystalline solid. The compound was used in the next reaction without further purification. Yield: 34 g (95%). Purity by RP-HPLC, 220 nm, over 95% as methyl ester. MSNR-ER (m/z) calc. for C ₁₁ H ₁₆ NO ₄ (as methyl ester) [M+H] ⁺ : 226.10. Found: 225.97.

Synthesis of 4-(6-maleimidohexamido)-2-sulphobenzoic acid ( D )

[134] 4-Amino-2-sulphobenzoic acid (18.5 g, 85.0 mmol, 1.0 eq.) was dissolved in anhydrous DMF (300 mL) under N ₂ atmosphere. The solution was cooled to 4°C and left to stir for 10 minutes. Then, 4-N-methylmorpholine (18.7 ml, 170 mmol, 2.0 eq.) (~0.3 ml/min) was added dropwise to the cooled solution over 1 hour using an addition funnel. To this dark brown mixture was added dropwise (~0.5 g/min) a solution of EMC-Cl (29.3 g, 127 mmol, 1.5 eq.) in anhydrous DMF (200 ml). The reaction mixture was stirred overnight and then allowed to warm to room temperature over 10 hours. After completion of the reaction, as shown by HPLC analysis (PDA 220 nm, >95% conversion), the reaction solution was distributed into 8 x 50 ml tubes from Falcon.

[135] Samples were centrifuged for 20 minutes at 10°C and 4000 rpm. The supernatants were removed by decantation and the solids were resuspended in 10 ml DMF per tube and centrifuged again for 20 min at 10° C. and 4000 rpm. All DMF supernatants were combined and concentrated under reduced pressure at 50° C. for 3 hours to give a light orange solid. The solid was resuspended in methanol (250 ml) and transferred to 8 x 50 ml Falcon tubes. Samples were centrifuged for 20 minutes at 10°C and 4000 rpm. Supernatants were removed by decantation and the solids were resuspended in 5 ml of methanol per tube and centrifuged again for 20 minutes at 10° C. and 4000 rpm. All solids were combined and dried under high vacuum for 24 hours to give a crystalline yellow solid. Yield: 17 g (48%). Reverse phase RP-HPLC purity, 220 nm, 80%. MSNR-ER (m/z) calc. for C ₁₇ H ₁₇ N ₂ O ₈ S [MH] ^- : 409.08. Found: 409.13.

Synthesis of 2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-maleimidohexaneamido)benzenesulfonic acid or Boc-protected linker 1 ( E )

[136] _EBA -HCl ( 8.72 g, 45.5 mmol, 1.1 eq.) and 1HOBt (6.15 g, 45.5 mmol, 1.1 eq.). The reaction mixture was allowed to stir for 30 minutes at room temperature and then tert-butylcarbazate (7.12 g, 53.9 mmol, 1.3 eq) was added and the solution turned clear yellow to reddish. The reaction mixture was stirred at room temperature overnight. After this time, the completion of the reaction was confirmed by HPLC (PDA 220 nm, more than 95% conversion). The solvent was removed using a rotary evaporator at 40°C and under high vacuum for 1 hour to give a purplish brown oil, which was purified using the Biotage Isolera One Rapid Clear System with two pre-packaged SNAP ULTRA 340 g cartridges, with spherical silica Biotage® HP-Sphere™. Tubes containing the desired product were combined and dried on a rotary evaporator and under high vacuum for 10 hours to give 2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-maleimidohexanamido)benzenesulfonic acid as a foamy yellow solid. Yield: 9 g (42%). RP-HPLC (220nm) over 95%. MSNR-ER (m/z) calc. for C22H27N4O9S [MH] ^- : 523.16. Found: 523.15.

Synthesis of linker 1

[137] To a chilled (4-5°C) solution of Boc-protected linker 1 (10.2 g, 19.4 mmol, 1.0 eq.) in anhydrous DCM (30 ml) was added dropwise (~0.5 ml/min) TFA (15 ml) for 30 minutes. Thereafter, the cold bath was removed and the reaction mixture was allowed to stir at room temperature for 3 hours. After this time, the completion of the reaction was confirmed by HPLC analysis (PDA 220 nm). The reaction mixture was poured dropwise into six Falcon tubes, each containing approximately 35 ml of cold diethyl ether. A white precipitate formed immediately. The tubes were left at 4°C for 3 hours. After centrifugation of the Falcon tubes (4000 rpm, 20 min, 10°C), the supernatants were removed by decantation and the solids were resuspended in 5 ml of diethyl ether per tube and centrifuged again (4000 rpm, 20 min, 10°C. The supernatants were again removed by decantation and the solids were collected and dried under high vacuum to give linker 1 as a white microcrystalline solid as TFA salt Yield: 10 g (96%) RP-HPLC (220 nm) over 95% MSNR -ESI (m/z) calcd for C ₁₇ H ₂₁ N ₄ O ₇ S [M+H]+: 425.11 Found: 425.07 MSHF-ESI (m/z) calcd for C ₁₇ H ₁₉ N ₄ O ₇ S [MH]-: 423.11 Found: 423.12 HRMS-ESI (m/z) calcd for C ₁₇ H ₂₁ N ₄ O ₇ S [M+H]+: 425, 1125 Found: 425.1125 HRMS-ESI (m/z) calc for C ₁₇ H ₁₉ N ₄ O ₇ S [MH]-: 423.0978 Found: 423.0980

[138] The structure was confirmed by ¹ H NMR and ¹³ C NMR: ¹ H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H; Cl-NH), 10.27 (s, 1H; C8 -NH), 8.01 (d, J= 2.2 Hz, 1H; C4-CH), 7.93 (dd, J= 8.5, 2.2 Hz, 1H; C6-CH), 7, 68 (d, J= 8.4 Hz, 1H; C7-CH), 7.00 (s, 2H; C15-CH, C16-CH), 3.40 (t, J= 7.0 Hz, 2H; C13- CH2), 2.32 (t, J= 7.4 Hz, 2H; C9-CH2), 1.60 (p, J= 7.5 Hz, 2H; C10-CH2), 1.52 (p, J = 7.2 Hz, 2H; C12-CH2), 1.26 (q, J= 8.8 Hz, 2H; C11-CH2); 13C NMR (101 MHz, DMSO-d6) δ 172.20 (C8), 171.54 (C14, C17), 167.59 (C1), 145.73 (C5), 142.09 (C3), 134, 90 (C15, C16), 132.09 (C7), 123.79 (C2), 119.44 (C6), 117.47 (C4), 37.42 (C13), 36.65 (C9), 28 .22 (C12), 26.21 (C11), 24.90 (C10). Anal. calc. for C ₁₇ H ₂₁ N ₄ O ₇ S ^1/2 _TFA C, 45.71; H, 4.26; N, 11.85; S, 6.78. Found: C, 46.2917; H, 4.4836; N, 12.8879; S, 6.7886. The content of TFA is 0.51%.

[139] Linker 1 can also be prepared as described below and shown in Scheme 2.

Scheme 2

Linker 1

Synthesis of 5-amino-2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)benzenesulfonic acid ( F )

[140] Triethylamine (41.93 g, 57.76 ml, 414.37 mmol, 3, 00 eq.) and the mixture was stirred for 10 minutes. Then tert-butylcarbazate (27.38 g, 207.19 mmol, 1.50 eq) was added and the mixture was cooled to minus 35°C. At this temperature, a solution of propylphosphonic anhydride, T3P, (114.27 g, 106.79 ml, 179.56 mmol, 50% solution in ethyl acetate, 1.3 eq.) was added dropwise over 1 hour. The reaction mixture was stirred at minus 35°C for 2 hours. The mixture was allowed to warm to room temperature and filtered through Celite® 545 (100 g). Celite® was further washed with acetonitrile (500 ml). Both filtrates were combined and concentrated to 250 ml. The solution was divided equally into 6 portions and the solvent was removed under reduced pressure. Each batch was dissolved in dichloromethane containing 1% Et ₃ N (50 mL) and purified by NF flash chromatography on a Biotage Isolera™ One Rapid Purification System with a pre-packed SNAP Ultra 340 g column using a step gradient from 2% to 12% methanol (containing 1% NEt ₃ ) in DCM (containing 1% NEt ₃ ) in 7 column volumes. The purified fractions from all parts were then combined, the solvent was removed under reduced pressure, and the solid was dried under high vacuum to give the title compound F as an off-white solid. Yield: 53.25 g, 108.0 mmol, 78.2% (NMR in DMSO-d6 showed the presence of 1.6 eq. triethylamine). HPLC (method 9, 220 nm) over 99%. MSNR-ER (m/z) calc. for C ₁₂ H ₁₆ N ₃ O ₆ S [MH] ^- : 330.08. Found: 330.08.

Synthesis of N-ethyl-N-isopropylpropane-2-aminium 2-(2-(tert-butoxycarbonyl)hydrazine-1-cargonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1-)hexanamido)benzenesulfonate ( E )

[141] Acetonitrile (450 ml ), triethylamine (13.85 g, 19.08 ml, 136.86). mmol) and T3P (43.55 g, 40.70 ml, 136.86 mmol, 50% solution in ethyl acetate) were added in one portion at room temperature. The solution was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure. The crude product was then purified by a flash purification system using seven prepackaged 340 g SNAP Ultra cartridges with a linear gradient from 2% methanol to 15% methanol in dichloromethane to give the title compound E as an off-white solid. Yield: 30.55 g, 53.5% (NMR in DMSO-d6 indicated the presence of 1.1 eq. triethylamine). HPLC (method 9, 220 nm) over 99%. MSNR-ER (m/z) calc. for C ₂₂ H ₂₇ N ₄ O ₉ S [MH] ^- : 523.15. Found: 523.26.

Synthesis of 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazinecarbonyl)benzenesulfonic acid (Linker one )

[142] Trifluoroacetic acid ( 18.22 g, 12.31 ml, 159 .81 mmol, 10.17 eq.) for 15 min. The mixture was further stirred at 4°C for 15 minutes and then allowed to gradually warm to room temperature and stirred for 150 minutes. The reaction mixture was added dropwise via a separating funnel to a stirred solution of methyl tert-butyl ether, MTBE (400 ml) and dichloromethane (200 ml). The resulting white solid was filtered through a 4 A funnel and washed successively with dichloromethane (2 x 150 ml) and MTBE (1 x 50 ml), MeOH (1 x 50 ml) and again with MTBE (2 x 150 ml). The solid was allowed to dry on a fritted funnel overnight at room temperature for 10 minutes. Further drying was carried out in high vacuum at 25° C. for 18 hours. The final product Linker 1 was obtained as a yellow solid. Yield: 5.786 g, 13.63 mmol, 98.7%, HPLC (method 9, 220 nm) over 96%. MSNR-ER (m/z) calc. for C ₁₇ H ₁₉ N ₄ O ₇ S [MH] ^- : 423.10. Found: 422.95.

Example 2

Production of keto-maytansinoids by direct esterification of maytansinol with keto acid

[143] General Method A : Maytansinol (500 mg, 0.88 mmol, 1 eq.) and the corresponding keto acid (3.52 mmol, 4 eq.) were dissolved in anhydrous DCM (40 ml) in the presence of activated molecular sieves and cooled to 4-5°C using an ice bath. To this solution was then added a solution of zinc (II) chloride in diethyl ether (2.64 ml, 2.64 mmol, 3.0 eq., 1 M solution) over 10 seconds. The resulting solution was stirred for 40 minutes at 4-5°C followed by the addition of N,N'-diisopropylcarbodiimide (0.55 ml, 3.52 mmol, 4 eq.). The mixture, immersed in an ice bath, was stirred at 4° C. and then allowed to warm slowly to room temperature overnight. Conversion was monitored by LC-MS and after reaching 60% the reaction mixture was concentrated under reduced pressure at 40°C to half volume, filtered through a 0.45 μm syringe filter (Macherey-Nagel, Chromafil® PTFE-O-45/25), and the filtrate was evaporated. The final product was purified using a Biotage Isolera One Rapid Cleanup System with a pre-packaged 50 g SNAP ULTRA cartridge, Biotage® HP-Sphere™ silica spherical (linear gradient from 100% DCM to 90/10 DCM/methanol at 25 vol. col. ). The tubes containing the product were combined and dried for 1 hour using a rotary evaporator and under high vacuum to give the corresponding ketomaytansinoid.

[144] General Method B : Maytansinol (758 mg, 1.34 mmol, 1.0 eq.), the corresponding keto acid (1.47 mmol, 1.1 eq.) and DMAP (181 mg, 1.47 mmol, 1 1 eq.) was dissolved under N2 in anhydrous DCM (30 mL) in the presence of activated molecular sieves (0.8 g, 4 Å, 325 mesh, Sigma Aldrich). The mixture was cooled over 10 minutes to 4°C using an ice bath. A solution of EBA-HCl (283 mg, 1.47 mmol, 1.1 eq.) in dry DCM (15 ml) was added over 30 min (~0 mg/min) to the cooled mixture and the reaction mixture was stirred at 4°C for over 2 hours. After this time, another portion of keto acid (1.47 mmol, 1.1 eq.) was added, followed by the addition of a solution of EBA-HCl (283 mg, 1.47 mmol, 1.1 eq.) in dry DCM (10 ml) for 30 min (~10 mg/min) to the cooled mixture, and the reaction mixture was stirred in an inert atmosphere at 4°C for 2 hours. After this time, the addition of reagents was repeated once more, and the reaction mixture was left to stir overnight at 4°C until the mixture gradually reached room temperature. The mixture was filtered (Macherey-Nagel, Chromafil® PTFE-O-45/25) and the solvent was removed using a rotary evaporator at 40°C to a final volume of approximately 10 ml. The crude product was purified using the Biotage Isolera One Rapid Cleanup System, pre-packaged SNAP ULTRA 100 g cartridge, with Biotage® HP-Sphere™ silica (linear gradient from 100% DCM to 90/10 DCM/methanol at 25 vol.col. .). The tubes containing the product were combined and the solvent was removed using a rotary evaporator to obtain a solid. The solid was dried under high vacuum to give the corresponding ketomaytansinoid.

Obtaining maytansinoid 2

[145] From the reaction of maytansinol with 2-(4-acetyl-2-fluorophenyl)acetic acid using method A: Maytansinoid 2 was obtained as a yellowish solid. Yield: 47%. Purity by RP-HPLC, 220 nm, 96%. MSNR-ER (m/z) calc. for: C ₃₈ H ₄₅ C1FN ₂ O ₁₀ [M+H] ⁺ : 743.22. Found: 743.25. MSNR-ER (m/z) calc. for: C ₃₈ H ₄₃ C1FN ₂ O ₁₀ [MH]-: 741.22. Found: 741.41.

[146] The structure was confirmed by ¹ H NMR and ¹³ C NMR: ¹ H NMR (400 MHz, CDCl3) δ 7.73 (dd, J=7.9, 1.6 Hz, 1H; C31-CH), 7.66 (dd,7=10.5, 1.6Hz, 1H; C27-CH), 7.46 (t, J=7.6Hz, 1H; C32-CH), 6.82 (d, J= 1.8 Hz, 1H; C17-CH), 6.59 (d, J= 1.8 Hz, 1H; C21-CH), 6.48 (dd, J= 15.5, 11.0 Hz , 1H; C12-CH), 6.43 (s, 1H; C9-NH), 6.25 (d, J= 10.9 Hz, 1H; C13-CH), 5.63 (dd, J= 15 .4, 8.8 Hz, 1H; C11-CH), 4.99 (dd, J=11.8, 2.7 Hz, 1H; C3-CH), 4.27 (td, J=11.2 , 10.4, 1.8 Hz, 1H; C7-CH), 3.97 (s, 3H; C20-OCH3), 3.90 (d, J= 15.7 Hz, 1H; C24-CH2), 3.76 (d, J= 15.5 Hz, 1H; C24-CH2), 3.54 (d, J= 8.8 Hz, 1H; C10-CH), 3.46 (d, J= 12, 8 Hz, 1H; C15-CH2), 3.38 (s, 3H; C10-OCH3), 3.19 (d, J= 12.8 Hz, 1H; C15-CH2), 3.00 (s, 3H ; Cl-NCH3), 2.87 (d, J= 9.7 Hz, 1H; C5-CH), 2.57 (s, 3H; C29-CH3), 2.51 (dd, J= 14.1 , 11.9 Hz, 1H; C2-CH ₂ ), 2.17 (dd, J= 13.9, 2.6 Hz, 1H; C2-CH ₂ ), 1.72 (d, J= 13.6 Hz, 1H; C8-CH2), 1.68 (s, 3H; CM-CH3), 1.50 (m, 1H; C6-CH), 1.28 (d, J= 6.3 Hz, 4H; C6-CH3, C8-CH2), 0.87 (d, J= 1 .4 Hz, 3H; C4- _CH3 ); ¹³ C NMR (101 MHz, CDCl ₃ ) δ 196.50 (C28), 168.86 (C23), 168.30 (Cl), 160.78 (d, ¹ J _CF =247.0 Hz; C26), 156.13 (C20), 152.46 (C22), 142.53 (C18), 140.30 (C19), 140.24 (C14), ^138.49 (d, 3J _CF = 6.4Hz; C30 ), 132.58 (C12), 131.68 (d, ³ J _CF = 3.6 Hz; C32), 128.32 (C11), 126.38 (d, ² J _CF = 16.1 Hz; C25 ), 124.82 (d, ⁴ J _CF = 3.3 Hz; C31), 124.56 (C13), 122.04 (C21), 119.52 (C16), 114.73 (d, ² J _CF = 23.2 Hz; C27), 113.13 (C17), 88.23 (C10), 81.25 (C9), 77.95 (C3), 74.37 (C7), 66.24 (C5) , 60.39 (C4), 56.91 (C20-OCH3), 56.71 (C10-OCH3), _47.26 (C15), 38.37 (C6), 36.01 (C8), 35, 46 (C1-NCH3), 33.86 (C24), 32.76 (C2), 26.78 (C29), 15.88 (C14-CH3), 14.60 (C6-CH3), 12.35 ( C4- _CH3 ).

Obtaining maytansinoid 3

[147] From the reaction of maytansinol with 2-(3-acetylphenoxy)acetic acid using method B: Maytansinoid 3 was obtained as a yellowish solid. Yield: 49%. Purity by RP-HPLC, 220 nm, 98%. MSNR-ER (m/z) calc. for: C ₃₈ H ₄₆ ClN ₂ O ₁₁ [M+H] ⁺ : 741.23. Found: 741.23.

[148] The structure was confirmed by ¹ H NMR and ¹³ C NMR: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.58 (dt, J= 7.8, 1.1 Hz, 1H; C30-CH) , 7.55 (s, 1H; C26-CH), 7.44 (d, J= 7.9 Hz, 1H; C31-CH), 7.12 (dd, J= 8.3, 2.8 Hz , 1H; C32-CH), 6.79 (d, J= 1.8 Hz, 1H; C17-CH), 6.65 (d, J= 1.8 Hz, 1H; C21-CH), 6, 46 (dd, J= 15.4, 11.0 Hz, 1H; C12-CH), 6.37 (s, 1H; C9-NH), 6.26 (d, J= 11.0 Hz, 1H; C13-CH), 5.62 (dd, J= 15.4, 8.9 Hz, 1H; C11-CH), 5.08 (dd, J= 11.9, 2.7 Hz, 1H; C3- CH), 4.90 (d, J =15.9 Hz, 1H; C24-CH ₂ ), 4.66 (d, J= 15.9 Hz, 1H; C24-CH ₂ ), 4.28 (t , J= 10.6 Hz, 1H; C7-CH), 3.96 (s, 3H; C20-OCH ₃ ), 3.68 (s, 1H; C9-OH), 3.53 (d, J= 8.9 Hz, 1H; C10-CH), 3.47 (d, J= 12.9 Hz, 1H; C15-CH ₂ ), 3.36 (s, 3H; C10-OCH3), 3.15 ( d, J= 12.8 Hz, 1H; C15-CH2), 2.92 (d, J= 9.7 Hz, 1H; C5-CH), 2.87 (s, 3H; C1-NCH3), 2 .59 (s, 3H; C29-CH ₃ ), 2.55 (d, J= 11.9 Hz, 1H; C2-CH ₂ ), 2.22-2.14 (m, 1H; C2-CH ₂ ), 1.71 (d, J= 1.8 Hz, 1H; C8-CH ₂ ), 1.67 (s, 3H; C14-CH ₃ ), 1.55-1.45 (m, 1H; C6 -CH), 1.28 (d, J= 6.4 G c, 3H; C6-CH3), 1.28-1.25 (m, 1H; C8-CH2), 0.86 (s, _3H ; C4-CH3); ¹³ C NMR (101 MHz, CDCl ₃ ) δ 197.99 (C28), 168.09 (C1), 168.07 (C23), 158.27 (C25), 156.09 (C20), 152.36 ( C22), 142.46 (C18), 140.38 (C19), 139.83 (C14), 138.84 (C27), 132.85 (C12), 130.48 (C31), 128.22 (C11 ), 124.95 (C13), 122.41 (C30), 122.12 (C21), 119.67 (C32), 119.25 (C16), 113.73 (C26), 113.02 (C17) , 88.18 (C1O), 81.16 (C9), 78.13 (C3), 74.18 (C7), 66.37 (C24), 66.26 (C5), 60.26 (C4), 56.87 (C20-OCH ₃ ), 56.68 (C10-OCH ₃ ), 47.05 (C15), 38.41 (C6), 36.34 (C8), 35.31 (C1-NCH ₃ ) , 32.67 (C2), 26.88 (C29), 15.86 (C14-CH ₃ ), 14.58 (C6-CH ₃ ), 12.50 (C4-CH ₃ ).

Example 3

[149] Three-step synthesis of ketomaytansinoids by esterification with an Fmoc-protected amino acid, cleavage of the Fmoc group, and condensation with a keto acid.

[150] General Method C - Step 1: Reaction of maytansinol with an Fmoc-protected amino acid. Maytansinol (565 mg, 1.00 mmol, 1.0 eq), Fmoc protected amino acid (3.00 mmol, 3.0 eq), DMAP (982 mg, 8.00 mmol, 8.0 eq) and scandium(III) trifluoromethanesulfonate (541 mg, 1.1 mmol). 1.1 eq.) was dissolved under N ₂ in anhydrous DCM (10 ml) in the presence of activated molecular sieves (1 g, 4 Å, particle size 325 mesh, Sigma Aldrich). The mixture was cooled to 4°C using an ice/water bath and allowed to stir for 30 minutes to reach this temperature. After this time, DIC (2.47 ml, 16.0 mmol, 16.0 eq.) (~0.25 ml/min) was added over 10 minutes and the reaction mixture was stirred at 4°C for 2 hours and left gradually reach room temperature during this time. The mixture was filtered by gravity. The filtrate was diluted with DCM (50 ml) and then washed with sodium phosphate buffer (50 ml x 3, pH 7.5) and brine (50 ml). The organic layer was then dried over anhydrous sodium sulfate, filtered under gravity and the solvent was removed on a rotary evaporator at 40°C to a final volume of approximately 10 ml. The crude material was purified on a Biotage Isolera One Rapid System with a pre-packaged Biotage® HP-Sphere™ 50 g SNAP ULTRA Silica Spherical Cartridge (linear gradient from 100% DCM to 90/10 DCM/methanol at 25 vol.col.). The solvent was removed using a rotary evaporator at 40° C. for 2 hours to give the corresponding product as a yellowish to yellow solid.

[151] General Method C - Step 2: deprotection of the Fmoc group. The Fmoc protected intermediate (0.53 mmol, 1.0 eq.) was dissolved in DCM (5 ml) and to this solution was added tris-(2-aminoethyl)amine (0.320 ml, 2.13 mmol, 4.0 equiv.) for 10 s. The reaction mixture was allowed to stir at room temperature for 1 hour. The resulting white precipitate was filtered through a pad of celite, washed with DCM (20 ml) and the yellow filtrate was evaporated to dryness on a rotary evaporator at 40° C. for 2 hours and then dried under high vacuum for 4 hours to give the free amine, which was immediately used on next step without further purification.

[152] General Method C - Step 3: Reaction of an amine-maytansinoid with a keto acid. Intermediate free amine (77.0 µmol, 1.0 eq), keto acid (150 µmol, 2.0 eq), HATU (35 mg, 94.0 µmol, 1.2 eq), HOAt (13 mg, 94.0 μmol, 1.2 eq.) and N -methylmorpholine (17 μl, 150 μmol, 2.0 eq.) were dissolved under N ₂ in anhydrous DMF (1 ml). The mixture was allowed to stir at room temperature overnight. The reaction mixture was diluted with DCM (5 ml) and washed with saturated ammonium chloride solution (5 ml × 5) and brine (5 ml). The organic phase was then dried over anhydrous sodium sulfate, filtered under gravity and the solvent was removed on a rotary evaporator at 40°C. The crude material was purified in a Biotage Isolera One Rapid Purification System using a 12 g prepackaged SNAP ULTRA C-18 cartridge, Biotage® HP-Sphere™ C18, 25 µm spherical silica (linear gradient system from 80/20 water/MeCN to 100% MeCN in 20 volumes). Fractions containing product were pooled, frozen in liquid nitrogen and lyophilized for 24 hours to give the corresponding keto-maytansinoid.

Example 4

Obtaining maytansinoid 28

Maytansinol (56.5 mg, 0.10 mmol, 1.0 eq.) was dissolved in dry DCM (10 ml), a solution of zinc (II) chloride in diethyl ether (0.3 ml, 0.30 mmol, 3.0 equiv., 1 M solution) was added at room temperature under N2 and allowed to stir for 10 min. 4-Acetylphenylisocyanate (48.3 mg, 0.30 mmol, 3.0 eq.) was added over 10 seconds and the resulting solution was stirred at room temperature for 5 hours. After this time, the completion of the reaction was confirmed by HPLC analysis (PDA 220 nm). Volatiles were removed using a rotary evaporator at 40°C. The crude product was purified by NP chromatography using the Biotage Isolera One Rapid Purification System with a pre-packaged 10 g SNAP ULTRA cartridge containing Biotage® HP-Sphere™ silica spherical (linear gradient system from 100% DCM to 90/10 DCM/ methanol at 13 vol.) followed by RP-chromatography using a pre-packaged SNAP ULTRA C-18 12 g Biotage® HP-Sphere ™ C18 cartridge, 25 µm spherical silica (linear gradient from 80/20 water/MeCN to 100 % MeCN in 30 vol.col.). Fractions containing product were combined, frozen in liquid nitrogen and lyophilized for 24 hours to give compound 28 as a white solid. Yield: 20 mg (28%). Purity by RP-HPLC (220 nm) over 95%. MSNR-ER ( m/z ) calc. for: C ₃₇ H ₄₅ ClN ₃ O ₁₀ [M+H] ⁺ : 726.27. Found: 726.25. MSNR-ER ( m/z ) calc. for: C ₃₇ H ₄₃ ClN ₃ O ₁₀ [MH] ^- : 724.27. Found: 724.43.

Example 5

Obtaining maytansinoid 29

[153] Synthesis of May-ONp : To a solution of maytansinol (88 mg, 155 µmol, 1.0 eq.) in DCM (8 ml) was added pyridine (25 µl, 310 µmol, 2.0 eq.) at room temperature. The resulting clear solution was stirred for 15 min, then cooled to 4° C. and p-nitrophenyl chloroformate (219 mg, 1.08 mmol, 7.0 eq.) in DCM (4 ml) was added, after which a white precipitate immediately formed. The reaction mixture was stirred for 24 hours at room temperature. The crude material was concentrated using a rotary evaporator at 40° C. for 1 hour and purified using the Biotage Isolera One Rapid Purification System using a pre-packaged 25 g SNAP ULTRA cartridge with Biotage® HP-Sphere™ spherical silica (linear gradient from 100% ethyl acetate to 40/60 ethyl acetate/DCM at 50 vol.). Fractions containing the product were dried on a rotary evaporator at 40° C. for 30 minutes and under high vacuum for an additional 30 minutes to give intermediate May-ONp as a white solid. Yield: 102 mg (90%). MSNR-ER (m/z) calc. for C ₃₅ H ₄₁ ClN ₃ O ₁₂ [M+H] ⁺ : 730.23. Found: 730.01.

[154] Synthesis of maytansinoid 29 : To a solution of compound May-ONp (3.7 mg, 5.00 µmol, 1.0 eq.) in DCM (1 ml) was added a solution of 4-(aminomethyl)acetophenone (1.1 mg, 7.50 µmol, 1.5 eq.) in DCM/DMF (2:0.1 v/v) at room temperature and stirred for 5 minutes before adding triethylamine (2 µl, 10.0 µmol, 2.0 eq. ). The reaction mixture was stirred at room temperature for 2 hours and at 60° C. overnight. The crude material was concentrated at 40° C. for 1 hour and purified with the Biotage Isolera One Rapid Purification System using a prepackaged 10 g SNAP ULTRA cartridge with Biotage® HP-Sphere™ silica spherical (linear gradient from 100% chloroform to 90/ 10 chloroform/methanol in 20 vols). Fractions containing the product were dried on a rotary evaporator at 40°C for 30 minutes and in high vacuum for another 30 minutes to obtain compound 29 as a white solid. Yield: 1 mg (27%). Purity by RP-HPLC (220 nm) over 95%. MSNR-ER (m/z) calc. for C ₃₈ H ₄₆ ClN ₃ O ₁₀ [M+H] ⁺ : 739.28. Found: 739.96.

Example 6

Production of albumin-binding maytansinoids

[155] General method D for the synthesis of albumin-binding maytansinoids from ketomaytansinoid and maleimido-hydrazide linker: To a stirred solution of ketomaytansinoid (1.0 eq.) in dry solvent (suitable solvents are DCM, DMSO, dioxane, 2-methyltetrahydrofuran) at room temperature under N2, molecular sieves (1:1 to 5:1 v/v) and catalyst (TFA, p-toluenesulfonic acid, Amberlist-H form, Amberlite-Na form) were added, followed by the hydrazide linker solution (from 1.0-5.0 eq.) in dry DMSO. The reaction mixture was stirred at room temperature and the conversion was confirmed by HPLC analysis (PDA 220 nm) (more than 90% conversion). The reaction mixture was filtered, concentrated on a rotary evaporator at 30° C. and purified by chromatography using a Biotage Isolera One Rapid Purification System using prepackaged SNAP ULTRA Biotage® HP-Sphere™ silica spheres (linear gradient from 100% DCM to 90/10 DCM /methanol), dried in a rotary evaporator at 30°C for 30 minutes and in high vacuum for another 30 minutes to obtain the free acid. The product was redissolved in a suitable solvent (methanol, acetone, 2-methyltetrahydrofuran or other organic polar solvents) and then neutralized using salt solutions (sodium salts, potassium salts, triethylammonium salts) until a pH in the range of 5.5-7.5 was reached. The solution was frozen in liquid nitrogen and lyophilized for 24 hours to give the compound as a salt. The counterion content was determined by IC. The content of sodium ions was in the range from 2 to 1.0 eq. (approximately 0.2-0.9%), and the content of trimethylammonium ions - from 0.8 to 2.0 eq. (about 6-16%).

Preparation of albumin-binding maytansinoid thirty

[156] From the reaction of maytansinoid 2 with linker 1 : To a stirred solution of maytansinoid 2 (170 mg, 0.23 mmol, 1.0 eq.), molecular sieves (0.2 g, powder, activated, 4 Å, particle size 325 mesh) and Amberlyst®-H (20 mg, macroporous, 30-60 mesh) in dry DCM (2 ml) at room temperature under N2 was added a solution of linker 1 (51 mg, 0.12 mmol, 2.0 eq.) in dry DMSO (0.6 ml). The reaction mixture was stirred at room temperature and after 3 h HPLC analysis (PDA 220 nm) confirmed the completion of the reaction (over 98% conversion). The reaction mixture was concentrated on a rotary evaporator at 30° C., filtered through a 0.45 μm syringe filter (Macherey-Nagel, Chromafil® PTFE-O-45/25). The filtrate was diluted with dry DCM (8 mL) and purified using the Biotage Isolera One Rapid Cleanup System using a pre-packaged SNAP ULTRA 10 g Biotage® HP-Sphere™ silica spherical cartridge (linear gradient from 100% DCM to 90/10 DCM/ MeOH in 22 vol.). The combined product containing fractions were rotary dried at 30° C. for 30 minutes and under high vacuum for an additional 30 minutes to give the free acid form 30 as a white-yellow solid. The solid was dissolved in MeOH/acetone (50:50 v/v, approximately 4 mL total) and then neutralized with 5 mM NaHCO ₃ in Millipore water to pH 6.8-7.1 (pH measured with pH -meter). The solution was frozen in liquid nitrogen and lyophilized for 24 hours to give the maytansinoid 30 prodrug as a white/yellow foam. Yield: 161 mg (60%). MSNR-ER (m/z) calc. for: C ₅₅ H ₆₁ ClFN ₆ O ₁₆ S [MH] ^- : 1147.36. Found: 1147.84. The content of Na ⁺ was in the range of 0.2-0.8%.

Preparation of albumin-binding maytansinoid 31

[157] From the reaction of maytansinoid 3 with linker 1 : To a stirred solution of maytansinoid 3 (186 mg, 0.25 mmol, 1.0 eq.), molecular sieves (0.4 g, powder, activated, 4 Å, particle size 325 mesh, Sigma Aldrich) and Amberlite® (form IR120 Na, 484 mg, 2.1 mmol/ml, 4.0 eq.) in dry dichloromethane (4 ml) at room temperature under N ₂ was added a solution of linker 1 (329 mg, 0 .77 mmol, 2.5 eq.) in dry DMSO (4 ml). The reaction mixture was stirred at room temperature and after 6 h HPLC analysis (PDA 220 nm) confirmed the completion of the reaction (more than 98% conversion). The reaction mixture was concentrated on a rotary evaporator at 30° C., filtered through a 0.45 μm syringe filter (Macherey-Nagel, Chromafil® PTFE-O-45/25). The filtrate was neutralized with sodium hydroxide (318 μl of 1 M NaOH solution, 1.3 eq.). The neutralized mixture was added dropwise to a 50 ml Falcon tube containing a chilled mixture (ice bath at 4°C) of methyl t -butyl ether (27 ml) and isopropyl alcohol (14 ml) and centrifuged at 4°C for 5 minutes . The supernatant was decanted and the pellet was resuspended in dichloromethane (10 mL) and purified using the Biotage Isolera One Rapid Cleanup System using a pre-packaged Biotage® HP-Sphere™ SNAP ULTRA 10 g silica spherical cartridge (linear gradient from 100% DCM to 90/10 DCM/MeOH in 22 vols). The combined fractions containing the product were dried on a rotary evaporator at 30° C. for 30 minutes and under high vacuum for an additional 10 hours to give 31 as a white-yellow solid. Yield: 156 mg (52%). Purity by RP-HPLC (220 nm) over 95%. MSNR-ER (m/z) calc. for: C ₅₅ H ₆₂ ClN ₆ O ₁₇ S [MH] ^- : 1145.36. Found: 1145.87. The Na content has a window of 0.2-0.8%.

Example 7

Stability and release kinetics of maytansinoid-HSA conjugates in buffer solution at pH 4.0 and pH 7.4

[158] To prepare HSA conjugates of albumin-binding maytansinoids, HSA (200 μM: 361.8 μl, 1078 μM free Cys34; 100 μM: 180.9 μl, 1078 μM free Cys34) was diluted with PBS buffer (4 mM sodium phosphate, 150 mM NaCl, pH 7.4) (200 µM: 678.2 µl; 100 µM: 859.1 µl) and DMSO (200 µM: 130.0 µl; 100 µM: 195.0 µl) and incubated in a heating block at 37°C for 30 minutes. Albumin-binding maytansinoid was added as a 2 mM stock solution in DMSO (200 μM: 130.0 μl; 100 μM: 65.0 μl) to a pre-incubated HSA sample to prepare a 200 mM or 100 μM maytansinoid prodrug solution and 300 μM or 150 µM free Cys34. The mixture having pH 7.4 was allowed to react for 10 minutes at 37° C. and then analyzed every hour for 24 hours by RP-HPLC (Phenomenex Aeris WP XB-C18, 3.6 µm, 250 x 4.6 mm).

[159] To study the release kinetics at pH 4.0, the mixture was acidified with a mixture of 50 mM sodium acetate buffer pH 3.0 (200 μM: 119.3 μl; 100 μM: 125.2 μl) and 1 M HCl (200 μM: 12.7 µl; 100 µM: 6.8 µl) to reach pH 4.0. The mixture was then analyzed every hour for 24 hours by RP-HPLC (Phenomenex Aeris WP XB-C18, 3.6 μm, 250 x 4.6 mm).

[160] The following RP-HPLC conditions were used: Phenomenex Aeris WP XB-C18 3.6 µm, 250 x 4.6 mm; eluent A (100% 20 mM Tris buffer, pH 8.0) and eluent B (90:10, MeCN: water) eluting with a gradient of eluent B (25% 0-0.5 min, 25-35% 0.5 -2.5 min, 35-85% 2.5-16 min, 85-95% 16-17 min, 95% 17-20 min, 95-25% 20-25 min, 25% 25-30 min, speed flow 1.0 ml/min). The temperature of the column thermostat 37°C; autosampler temperature 37°C; 20 µl injection volume.

[161] To quantify the percentage of free drug released, standard curves of free maytansinoids were generated at various concentrations (200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM). The area under the curve (AUC) was determined at 250 nm.

Table 4: Release Kinetics of Maytansinoid-HSA Conjugates at pH 4.0 and pH 7.4 Maytansinoid rel. t _1/2 at pH 4.1 free
maytansinoid after 24 h at pH 7.4 36 4.5 h* 4.7%* 41 4.0 h* 4.6%* 31 4.5 h* 6.0 h** 4.0%*
3.7%** 33 7.0 h* 6.5%* 32 4.0 h* 9.5%* 37 5.5 h* 3.9%* thirty 3.5 hours** 9.6%** 40 3.5 h* 8.9%* 39 5.5 h* 9.6%* 38 8.5 h* 4.9%* 42 45.4 hours** 4.8%**

* measured at 200 µM

** measured at 100 µM

Example 8

[162] Stability of maytansinoids and maytansinoid-HSA conjugates in CD1 and human plasma mice: To study the stability of maytansinoids as well as maytansinoid-HSA conjugates in CD1 and human plasma, compounds were incubated at 37° C. for 24 hours. The remaining free maytansinoid or the release of the corresponding maytansinoid was quantified by LC-MS/MS (or HPLC) at specific time points.

[163] LC-MS quantitation procedure: Mixed mouse CD1 or human plasma (K ₂ EDTA, Innovative Research) was centrifuged (1 min at 12.044 g). The supernatant was first filtered through a filter needle (5 µm, sterile, Becton Dickinson) and then through a cellulose acetate membrane (0.45 µm, sterile, Carl Roth). The filtered plasma (1710 µl) was preincubated at 37°C for 30 min in a heating block. Free maytansinoid or albumin-binding maytansinoid as a stock solution at a concentration of 300 μM in DMSO (190 μl) was added to a pre-incubated plasma sample to obtain a 30 μM solution of the appropriate maytansinoid or maytansinoid-HSA conjugate. The mixture was allowed to react for 10 min at 37°C, and then at each time point (0, 1, 2, 3, 4, 5, 21 and 24 hours) three samples (70 μl) were taken into a 96-well plate, sealed plastic film, immediately frozen in liquid nitrogen and stored at minus 20°C. At the end of the experiment, the 96-well plate was allowed to thaw at room temperature. Samples (30 µl) were then directly transferred using a multichannel pipette to an Impact™ 96-well protein precipitation plate (Strata®), which was washed once with MeCN (150 µl) and then loaded with internal standard (120 µl, MeCN containing 5 µg /ml maytansine). The Impact™ deposition plate was covered with silicone film and shaken for 2 minutes at 420 rpm. The Impact™ precipitation plate was then placed in a 96-well sampler and the filtrate was collected in another 96-well plate using gentle vacuum. The 96-well plate was covered with silicone film and stored at room temperature until quantified by LC-MS/MS. The filtrate was injected into LC-MS for quantitation using MRM mode.

[164] LC-MS method: Phenomenex Luna® OmegaPolar C18, 1.6 μm, 100 Å, 50 x 2.1 mm, column; eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in MeCN) eluting with a gradient of eluent B (20% 0-0.5 min, flow 0.4 ml/min; 20- 60% 0.5-9.0 min 0.4 ml/min 60-100% 9.0-9.5 min flow 0.4 ml/min 100-20% 9.5-10.5 min, flow 0.4 ml/min; 20% 10.5-12 min, 0.6 ml/min). Column thermostat temperature 25°C; autosampler temperature room; 10 µl injection volume.

[165] The area under the curve (AUC) for each free drug at time 0 was set to 100% for the corresponding prodrug. The AUC for each sample was normalized based on the AUC of maytansine.

[166] HPLC Quantification Procedure : Samples were prepared as previously described using a 2 mM stock solution in DMSO of the free drug as well as the prodrug. After incubation at 37° C., samples were directly injected into HPLC every hour for 24 hours. The area under the curve (AUC) for each free maytansinoid (200 μM in 10% DMSO in PBS buffer) was used as the value of 100% for the corresponding albumin-binding maytansinoid.

[167] HPLC method : Phenomenex Aeris WP XB-C18, 3.6 µm, 250 x 4.6 mm,; column, eluent A (20 mM Tris buffer, pH 8.0) and eluent B (90:10, MeCN: water), eluting with a gradient of eluent B (25% 0-0.5 min, 25-35% 0.5 -2.5 min, 35-85% 2.5-16 min, 85-95% 16-17 min, 95% 17-20 min, 95-25% 20-25 min, 25% 25-30 min, speed flow=1.0 ml/min). The temperature of the column thermostat 37°C; autosampler temperature 37°C; 20 µl injection volume.

[168] The relative release of free maytansinoid as well as the conversion to maytansinol are listed in Table 5 below.

Table 5: Relative plasma release of free maytansinoid and/or maytansinol in mouse and human CD1 for maytansinoid-HSA conjugates Albumen
contact
maytansinoid
(Compound) % free
maytansinoid released after 24 h % maytansinol conversion after 24 h CD1
mouse human CD1
mouse human 31 3.0 6.8 2.9 2.1 45 6.5 na nd na 34* 4.3 na nd na 33 8.7 10.3 nd nd 32 7.0 7.8 nd 0.3 thirty 4.1 6.2 nd nd 42 2.7 7.4 2.1 1.8 35 na 9.0 na nd

*data obtained by HPLC

[169] The amount of free maytansinoid remaining, as well as the degree of conversion to maytansinol, are shown in Table 6 below.

Table 6: Remaining free maytansinoid and maytansinol after 24 h in albumin in human CD1 mice Maytansinoid
(Compound) % free maytansinoid remaining after 24h % conversion of maytansinol after 24h CD1
mouse human CD1
mouse human four 65.2 75.6 0.5 nd 2 71.8 74.8 nd nd 3 40.8 69.6 19.1 4.5

[170] The stability of various c (maytansinoid 4 ) linkers in mouse CD1 plasma is shown in Figure 1.

Example 9

[171] In Vitro Binding Kinetics of Albumin-Binding Maytansinoids to Albumin in Mixed Whole Blood and Human Plasma: To study the kinetics of binding of albumin-binding maytansinoids in mixed human plasma and mixed human whole blood, the compounds were incubated together with mixed human plasma at 37°C. and samples were taken at specific time points. The remaining albumin-binding maytansinoids were quantified by HPLC.

[172] HPLC Quantification Procedure: To study binding kinetics in human whole blood, blood (K ₂ EDTA, 36 donors, Zen-Bio; 900 µl) was pre-incubated at 37° C. for 30 minutes in a heating block. To study the binding kinetics in mixed human plasma, mixed whole blood was centrifuged (10 minutes, 1811 g), supernatant plasma was collected and then pre-incubated at 37° C. for 30 minutes.

[173] Albumin-binding maytansinoid was added to a pre-incubated whole blood/plasma sample at a 10-fold dilution in PBS of a 1.2 mM stock solution in 2.5% propylene glycol in 10 mM sodium phosphate buffer and 1.48 mM citrate (100 µl) to obtain a solution of 12.0 µM of the corresponding albumin-binding maytansinoid. Samples (190 µl) were taken after 15 s, 2 min, 4 min, 8 min and 15 min. Samples were directly added to 760 µl of MeCN containing maytansine (5 µg/ml) as an internal standard and shaken for 1 min. After centrifugation (1 min at 12.044 g), the supernatant (850 μl) was concentrated under high vacuum. The residue was dissolved in DMSO/water (1:1 v/v; 95 μl) and then analyzed by RP-HPLC. Percent binding was determined by comparing the area under the curve (AUC) at 220 nm of albumin-binding maytansinoid versus a control sample (100% value) in PBS buffer. All experiments were performed in triplicate.

[174] HPLC method: Phenomenex Kinetex Polar C18, 2.6 μM, 100 Å, 150 x 4.6 mm; eluent A (95:5 5 mM ammonium acetate: MeCN) and eluent B (5:95 5 mM ammonium acetate: MeCN) eluting with eluent B gradient (30% 0-0.5 min, 30-95% 0.5 -9.0 min, 95% 11.0 min, 95-30%, 11.0-14.5 min, 30%, 15.0 min, flow rate=1.0 ml/min). The temperature of the column thermostat corresponds to the ambient temperature; autosampler temperature 4°C; 50 µl injection volume.

[175] The relative amounts of bound albumin-binding maytansinoid at 0 and 8 minutes are shown in the table below:

Table 7: Amount of bound albumin-binding maytansinoid at 15 s and 8 min in mixed whole blood and human plasma Albumin-binding
maytansinoid % bound plasma whole blood 15 s 8 min 15 s 8 min 31 92.0±7.5 98.6±0.1 99.2±0.4 99.3±0.2 thirty 96.4±3.6 98.5±0.6 98.3±0.5 98.5±0.6

Example 10

In vitro cytotoxicity of maytansine, DM1, DM1-SMe, maytansinol, and maytansinoids in various cell lines using the CellTiter-Blue ® cell viability assay

[176] The study tested the efficacy of all compounds in vitro using the cell viability assay Promega CellTiter-Blue®. The cancer cell lines tested were: LXFL 529 (large cell lung cancer), RKO (colon cancer), SW-620 (colon cancer), CAL-27 (head and neck cancer), LXFL 1674L (large cell lung cancer), MDA- MB 468 (breast cancer), SK-OV-3 (ovarian cancer), PAXF 1657 (pancreatic cancer), MCF7 (breast cancer), COLO 205 (colon cancer), MDA-MB 231 (breast cancer) , BT-474 (breast cancer) and Hep G2 (liver cancer).

[177] Cells were harvested from exponential phase cultures, counted, and plated in 96-well flat-bottomed microtiter plates at cell density versus cell line growth rate (4,000 and 60,000 cells). 10 pL of culture medium (four control wells/plate) or of culture medium with the test compound are added.

[178] Compounds were applied at half-log dilution steps at 10 concentrations in duplicate and cells were treated continuously for 96 hours. After cell treatment and incubation, 20 μl/well of Cell Titer-Blue® reagent was added. After incubation for up to 4 hours, fluorescence (FU) was measured using an EnSpire multilayer reader (excitation λ=531 nm, emission λ=615 nm). Sigmoid concentration response curves are fitted to data points (T/C values) obtained for each cell line using 4-parameter non-linear fitting (Oncotest Warehouse Software). IC ₅₀ values are presented as relative IC ₅₀ values, which is the concentration of test compound that produces a response (inhibition of colony formation/viability) halfway between the upper and lower plateau of the sigmoid concentration response curve (curve inflection point), or as absolute IC ₅₀ values , which is the concentration of the test compound at the intersection of the concentration-response curves with T/C=50%. The geometric mean is used to calculate the average IC ₅₀ values. The results are presented as mean graphs or heat maps (individual IC ₅₀ values versus geometric mean IC ₅₀ ) across all cell lines as tested. See Table 8 and Figure 2. Figure 2 shows the IC ₅₀ heatmap for all compounds tested.

Table 8: Geom.av. IC ₅₀ [nM] n Cell lines DM1 3.22 6 DM1-SMe 1.30 6 eighteen 0.13 12 24 9.04 7 44 7.21 6 four 0.16 13 2 0.30 eleven 5 0.90 6 6 2.14 6 28 10.6 6 twenty 0.94 6 7 0.89 6 23 26.80 ten 21 1.67 6 22 25.00 3 19 1.12 6 9 0.19 7 17 0.45 6 fifteen 0.18 7 16 0.32 7 fourteen 1.54 6 3 0.10 6 eight 0.10 6 48 59.90 6 Maitanzine 0.22 12 Maytansinol 86.00 6 27 11.70 6 46 67.80 6 eleven 0.21 eight 25 8.47 7 ten 0.30 7 47 10.70 7 26 18.50 7 12 0.76 6 13 0.88 6

Example 11

General procedure for evaluating maytansine and albumin-binding maytansinoid compounds in a patient-derived tumor xenograft model

[179] NMRI immunodeficient female nude mice from Charles River received unilateral tumor implants subcutaneously on the left flank while under isoflurane anesthesia with human cancers until tumors became palpable and reached a volume of 80-200 mm ³ (if not otherwise stated).

[180] Animals were kept in cages, the temperature inside the cages was maintained at 25 ± 1°C with a relative humidity of 45-65% and an air exchange rate in the cage of 60 times per hour. They were kept 14 hours in the light: 10 hours in the dark, artificial light cycle. Animals were fed Teklad Global (T.2019S.12) autoclaved 19% protein extruded diet from Envigo RMS SARL and had access to sterile filtered and acidified (pH 2.5) tap water that was changed twice a week. Food and water were provided ad libitum . Prior to therapy, animals were randomized (7 mice per group unless otherwise indicated) based on comparable median and mean group tumor volume. Animals were regularly monitored twice a day on weekdays and once a day on Saturdays and Sundays. Starting from day 0, the animals were weighed twice a week. Relative body weight (BW) of individual animals was calculated by dividing the individual absolute body weight on day X (BMW) by the individual body weight on the day of randomization, multiplied by 100%. Tumor volume was determined by two-dimensional caliper measurement on the day of randomization (Day 0) and twice a week thereafter. Tumor volumes were calculated according to the following equation: Tumor volume [mm ³ ] = l [mm] xw ² [mm ² ] x 0.5, where "l" is the length and "w" is the width of the tumor. Relative individual tumor volume on day X (OOOx) was calculated by dividing the absolute individual tumor volume [mm ³ ] of the corresponding tumor on day X (Tx) by the absolute individual tumor volume of the same tumor on randomization day multiplied by 100%. Schedules have been applied to the extent that animal welfare procedures permit. The destruction of individual mice was carried out with a tumor volume greater than 2000 mm ³ (unilateral). All test compounds were administered on days 1, 8, 15 and 22 and supplied as lyophilized sucrose containing solids. On the day of treatment, freeze-dried samples were dissolved in 10 mM sodium phosphate buffer, pH 6.8, containing 20% propylene glycol, and injected intravenously (100 μl/20 g mouse) together with vehicle (10 mM sodium phosphate buffer, 20% propylene glycol and 5% sucrose - pH 6.8).

Example 12

General Procedure for the Evaluation of Maytansine and Albumin-Binding Maytansinoid Compounds in a Xenograft Model Derived from a Cancer Cell Line

[181] Immune-deficient NMRI female nude mice from Janvier France received 5×10 ⁶ transplant of cultured cancer cells in buffer/Matrigel (1:1) subcutaneously (unless otherwise indicated) until tumors became palpable and reached a volume of 80 -200 mm ³ (unless otherwise specified). The animals were kept in cages, the temperature inside the cages was maintained at 22°C ± 1°C with a relative humidity of 50±10% and an air exchange rate in the cage of 60 times per hour. They were kept 12 hours in the light: 12 hours in the dark, artificial light cycle. Animals were fed autoclaved Ssniff NM from Ssniff® and had access to sterile filtered and acidified (pH 4.0) tap water, which was changed twice a week. Food and water were provided ad libitum . Prior to therapy, animals were randomized (7 mice per group unless otherwise indicated) based on comparable median and mean group tumor volume. Animals were monitored regularly twice a day on weekdays and once a day on Saturdays and Sundays. Starting from day 0, the individual body weight of the mice was determined two or three times a week, and the average body weight per group was correlated with the initial value in percent to calculate the change in body weight (BWC). Tumor volume was determined by two-dimensional caliper measurement on the day of randomization (day 0) and then twice or thrice a week. Tumor volumes were calculated using the following equation:

[182] Tumor volume [mm ³ ] = l [mm] xw ² [mm ² ] x 0.5, where "l" is the length and "w" is the width of the tumor. Relative individual tumor volume on day X (OOOx) was calculated by dividing the absolute individual tumor volume [mm ³ ] of the corresponding tumor on day X (Tx) by the absolute individual tumor volume of the same tumor on randomization day multiplied by 100%. Schedules have been applied to the extent that animal welfare procedures permit. The destruction of individual mice was carried out when the tumor volume is greater than 1500 mm ³ (one-sided) or when ulceration was observed. All test compounds were administered on days 1, 8, 15 and 22 and supplied as lyophilized sucrose containing solids. On the day of treatment, freeze-dried samples were dissolved in 10 mM sodium phosphate buffer, pH 6.8, containing 20% propylene glycol, and injected intravenously (100 μl/20 g mouse) together with vehicle (10 mM sodium phosphate buffer, 20% propylene glycol and 5% sucrose - pH 6.8).

Example 13

Evaluation of maytansine and albumin-binding maytansinoids 30, 42, 31 and 35 in the PDX RXF 631 human renal cell carcinoma model

[183] Evaluation of maytansine and albumin-binding compounds30, 42, 31and35 in the RXF 631 model on renal cancer cells was performed according to the general procedure for the xenograft model obtained from the patient. Treatment was started after the tumors had reached an average size of 100 mm.³. Figure 3 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compoundsthirty,42,31 and35. Figure 4 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compoundsthirty,42,31 and35.

Example 14

Evaluation of maytansine and albumin-binding maytansinoids 32, 30 and 31 in LXFE 937 squamous cell lung carcinoma model

[184] Evaluation of maytansine and albumin-binding compounds 32, 30 and 31 in the LXFE 937 model of lung squamous cell carcinoma was performed in accordance with the general procedure for the xenograft model obtained from the patient. Treatment was started after the tumors reached an average size of 117 mm.³. Figure 5 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compounds32,thirty and31. Figure 6 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compounds32,thirty and31.

Example 15

Evaluation of maytansine and albumin-binding maytansinoids 30 and 31 in PDX human lung squamous cell carcinoma in the LXFE 937 tumor model (large tumors)

[185] Evaluation of maytansine and albumin-binding compounds31 and31 in the LXFE 937 model of lung squamous cell carcinoma was performed according to the general procedure for the xenograft model obtained from a patient. Treatment was started after the tumors had reached an average size of 270 mm.³. Figure 7 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compoundsthirty and31. Figure 8 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compoundsthirty and31.

Example 16

Evaluation of maytansine and albumin-binding maytansinoids 30 and 31 in the PDX LXFA 737 human lung adenocarcinoma model.

[186] Evaluation of maytansine and albumin-binding compoundsthirty and31 in the human lung adenocarcinoma model, LXFA 737 was performed according to the general procedure for the patient-derived xenograft model. Treatment was started after the tumors had reached an average size of 311 mm.³. Figure 9 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compoundsthirty and31. Figure 10 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compoundsthirty and31.

Example 17

Evaluation of maytansine and albumin-binding maytansinoids 32, 30 and 31 in the MDA-MB-231 human breast carcinoma xenograft model

[187] Evaluation of maytansine and albumin-binding compounds32.30 and31 in the breast carcinoma model, MDA-MB-231 was performed according to the general procedure for a xenograft model derived from a cancer cell line. Treatment was started after the tumors had reached an average size of 80 mm.³. Figure 11 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compounds32,thirty and31. Figure 12 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compounds32,thirty and31.

Example 18

Evaluation of maytansine and albumin-binding maytansinoids 30 and 31 in the human ovarian cancer xenograft model A2780

[188] Evaluation of maytansine and albumin-binding compoundsthirty and31 in the A2780 ovarian cancer model was performed according to the general procedure for a xenograft model derived from a cancer cell line. Treatment was started after the tumors reached an average size of 380 mm.³. Figure 13 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compoundsthirty and31. Figure 14 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compoundsthirty and31.

Example 19

Evaluation of maytansine and albumin-binding maytansinoids 30 and 31 in the MDA-MB 468 human breast carcinoma xenograft model

Evaluation of maytansine and albumin-binding compoundsthirty and31 in the breast carcinoma model, MDA-MB-468 was performed according to the general procedure for a xenograft model derived from a cancer cell line. Treatment was started after the tumors reached an average size of 94 mm.³. Figure 15 shows the tumor growth curves of the control group, the maytansine group and the groups treated with compoundsthirty and31. Figure 16 shows curves of change in body weight in the control group, the maytansine group and the groups treated with compoundsthirty and31.

Claims (100)

1. A compound having the structure of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is selected from -H and C ₁ -C ₄ alkyl; Spacer selected from:

and

V is absent or selected from -CH ₂ - and -NR ₃ -, where R ₃ represents -H or C ₁ -C ₄ alkyl; each R ² is independently selected from -H, halogen (eg -F, -Cl, -Br or -I) and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ , cycloalkyl; n is 0-2; X is absent or selected from -CH ₂ -, -O-, -S- and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl; Y is selected from =CH- and =N-; Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -CN, -NO ₂ . C ₁ -C ₄ alkyl and C ₂ -C ₄ alkoxy, with the proviso that when Y is ═N—, then Z ¹ , Z ² , Z ³ are each independently -H; AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethylglycine, D or L alanine, D or L N-methylalanine, β-alanine and N-methyl-β-alanine; R' is chosen from O and

Y' is absent or is selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)- and -C(O)-NH- or Y' is selected from the group consisting of:

and

, where n ^Y' =0-6; R ¹ ' is absent or selected from the group consisting of:

and

where M ¹ represents a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR4 ⁺ and NHR3 ⁺ ; where R represents H or C ₁ -C ₄ alkyl); R ² ' is an optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -; Z ¹ ', Z ² ', Z ³ ' and Z ⁴ ' are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ . -CN, -NO ₂ , -SO ₃ M ² and C ₁ -C ₄ alkyl, where M ² is a pharmaceutically acceptable counterion (for example, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ , where R is H or C ₁ -C ₄ alkyl); TBG is a thiol-binding group selected from an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, an optionally substituted acetylene group a group and an optionally substituted N-hydroxysuccinimide ester group; wherein said TBG is optionally linked to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier. 2. The compound according to claim 1, characterized in that it has the structure of formula (II):

or a pharmaceutically acceptable salt thereof, wherein: each R ² is independently selected from -H and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ , cycloalkyl; X is absent or selected from -CH ₂ -, -O-, -S- and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl; Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ . 3. The compound according to claim 1, characterized in that it has the structure of formula (III):

or a pharmaceutically acceptable salt thereof, wherein: each R ² is independently selected from -H and C ₁ -C ₄ alkyl or two R ² together form C ₃ -C ₆ , cycloalkyl; X is absent or selected from -CH ₂ -, -O-, -S- and -NR ⁴ -, where R ⁴ represents -H or C ₁ -C ₄ alkyl; Z ¹ , Z ² , Z ³ and Z ⁴ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ . 4. The compound according to claim 1, characterized in that it has the structure of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein: X is absent or selected from -CH ₂ - and -NH-; Y is ═CH- or ═N-; Z ¹ , Z ² and Z ³ are each independently selected from -H, halogen (eg -F, -Cl, -Br or -I), -CF ₃ , -OCH ₃ , -NO ₂ and -CH ₃ , provided that when Y is=N-, then Z ¹ , Z ² and Z ³ are each independently -H; AA is an amino acid selected from glycine, D or L proline, methylglycine, N-ethyl-glycine, D or L alanine, D or L N-methylalanine, β-alanine and N-methyl-β-alanine. 5. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that R ¹ is -H. 6. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that at least one of Z ¹ , Z ² , Z ³ and Z ⁴ a) is not H; or b) is -F or -NO ₂ . 7. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: a) n is 0 and X is absent; b) n is 0 and X is -CH ₂ -; or c) n is 0 and X is -O- or -S-. 8. A compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the compound is selected from:

and

, or from their pharmaceutically acceptable salt. 9. The compound or its pharmaceutically acceptable salt according to claims 1 to 8, characterized in that the pharmaceutically acceptable counterion is selected from H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , NR ₄ ⁺ and NHR ₃ ⁺ ; where R represents H or C ₁ -C ₄ alkyl. 10. A compound or a pharmaceutically acceptable salt thereof according to claims 1 to 9, characterized in that R' is: a) O; or b)

11. A compound or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that a) the compound does not bind to a thiol-containing macromolecular carrier or a thiol-containing tumor-specific carrier; or b) the compound binds to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier. 12. A compound or a pharmaceutically acceptable salt thereof according to claim 11, characterized in that the compound binds to a thiol-containing macromolecular carrier or a tumor-specific thiol-containing carrier selected from the group consisting of endogenous albumin, exogenous albumin, antibody, antibody fragment, peptide, natural or synthetic polymer, liposomes and nanoparticles. 13. A compound or pharmaceutically acceptable salt thereof according to claims 10-12, characterized in that TBG is an optionally substituted maleimide group. 14. A compound or a pharmaceutically acceptable salt thereof according to claims 10-13, characterized in that Z ¹ ' is selected from -NO ₂ or -SO ₃ M ² ; and Y' is selected from -NHC(O)- or

15. A compound or a pharmaceutically acceptable salt thereof according to claims 10-14, characterized in that R ¹ ' is

16. The compound according to claims 10-14, characterized in that R' is: a)

or a pharmaceutically acceptable salt thereof, wherein R ² ' is selected from optionally substituted C ₁ -C ₁₈ alkyl, where optionally up to six carbon atoms in said C ₁ -C ₁₈ alkyl are each independently substituted with -OCH ₂ CH ₂ -; or b)

or a pharmaceutically acceptable salt thereof. 17. The compound according to claim 16, characterized in that the compound is selected from:

and

or from their pharmaceutically acceptable salt. 18. The compound according to claims 10-15, characterized in that R' is: a)

or a pharmaceutically acceptable salt thereof; where M ¹ is a pharmaceutically acceptable counterion; or b)

or a pharmaceutically acceptable salt thereof; where M ¹ is a pharmaceutically acceptable counterion. 19. The compound according to claim 18, characterized in that the compound is selected from:

or from their pharmaceutically acceptable salt. 20. A compound or a pharmaceutically acceptable salt thereof according to claim 18, characterized in that the compound is:

21. A pharmaceutical composition for the treatment of cancer in a patient in need thereof, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier. 22. A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to claims 1 to 20 or a pharmaceutical composition according to claim 21. 23. The method of claim 22, wherein the cancer is selected from adenocarcinoma, ampullar colon cancer, anal carcinoma, bronchial carcinoma, non-small cell bronchial carcinoma, breast cancer, body carcinoma, ovarian cancer, endometrial carcinoma, gallbladder cancer, colorectal cancer, colon carcinoma, lung cancer, non-small cell lung cancer, ovarian carcinoma, pancreatic carcinoma and ovarian carcinoma. 24. A method for reducing the cytotoxicity of a compound, comprising administering a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 to a patient in need thereof, wherein the administration results in a decrease in cytotoxicity compared to an equivalent dose of an unmodified active agent . 25. A method of increasing the concentration of a metabolite of a compound in a tumor, comprising administering a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 to a patient in need thereof, the increase being compared with an equivalent dose of the unmodified active agent.

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