Maytansinoid-based drug delivery system

文档序号：1102041 发布日期：2020-09-25 浏览：6次中文

阅读说明：本技术 基于美登木素生物碱的药物递送系统 (Maytansinoid-based drug delivery system ) 是由 F·克拉茨 K·阿布阿贾伊 A·瓦尔内克 F·I·诺尔曼 S·D·克斯特 J·加西亚费尔南于 2018-11-30 设计创作，主要内容包括：本主题提供了白蛋白结合前药、基于美登木素生物碱的化合物及其用途。(The present subject matter provides albumin binding prodrugs, maytansinoid-based compounds, and uses thereof.)

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

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

wherein:

R¹is selected from-H and C₁-C₄An alkyl group;

the spacer is selected from:

v is absent or selected from-CH₂-, -O-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

each R²Independently selected from-H, halo groups (e.g., -F, -Cl, -Br or-I) and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

n is 0 to 3;

x is absent or selected from-CH₂-, -O-, -S-, -Se-and-NR-⁴-, wherein R⁴is-H or C₁-C₄An alkyl group;

y is selected from ═ CH-and ═ N-;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、C₁-C₄Alkyl and C₂-C₄An alkoxy group;

AA is an amino acid selected from the group consisting of glycine, D or L proline, sarcosine, N-ethylglycine, D or L alanine, D or L N-methylalanine, beta-alanine, N-methyl-beta-alanine, alpha-aminoisobutyric acid and N-methyl-alpha-aminoisobutyric acid;

r' is selected from O and

y' is absent or selected from optionally substituted C₁-C₆Alkyl, -NH-C (O) -and-C (O) -NH-; or Y' is selected from the group consisting of:

wherein n is 0 to 6;

R^1’absent or selected from the group consisting of:

wherein M is¹Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁–C₄Alkyl groups);

R^2’is optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substitution;

Z^1’、Z^2’、Z^3’and Z^4’Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、-SO₃M²And C₁-C₄Alkyl radical, wherein M²Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca²⁺、Mg² ⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁–C₄Alkyl groups);

TBG is a thiol binding group selected from: optionally substituted maleimido group, optionally substituted haloacetamido group, optionally substituted haloacetate group, optionally substituted pyridylthio group, optionally substituted isothiocyanate group, optionally substituted vinylcarbonyl group, optionally substituted aziridine group, optionally substituted disulfide group, optionally substituted ethynyl group and optionally substituted N-hydroxysuccinimide ester group;

wherein the TBG is optionally bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

2. The compound of claim 1, wherein the compound has the structure of formula (II):

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

wherein:

each R²Independently selected from-H and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

x is absent or selected from-CH₂-, -O-, -S-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃。

3. The compound of claim 1, wherein the compound has the structure of formula (III):

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

wherein:

each R²Independently selected from-H and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

x is absent or selected from-CH₂-, -O-, -S-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃。

4. The compound of claim 1, wherein the compound has the structure of formula (IV):

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

wherein:

x is absent or selected from-CH₂-and-NH-;

y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each independently selected from-H, halo groups (e.g., -F, -Cl)-Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

5. A compound according to any one of claims 1 to 4, wherein R¹is-H.

6. The compound according to any one of claims 1 to 4, wherein Z¹、Z²、Z³And Z⁴Is not H.

7. The compound according to any one of claims 1 to 4, wherein Z¹、Z²、Z³And Z⁴At least one of which is-F or-NO₂。

8. The compound of any one of claims 1 to 4, wherein n is 0 and X is absent.

9. The compound according to any one of claims 1 to 4, wherein n is 0 and X is-CH₂-。

10. The compound according to any one of claims 1 to 4, wherein n is 0 and X is-O-or-S-.

11. The compound of claim 1, wherein the compound is selected from the group consisting of:

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

12. The compound of any one of claims 1 to 11, wherein the pharmaceutically acceptable counterion is selected from H⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁–C₄An alkyl group.

13. The compound according to any one of claims 1 to 11, wherein R' is O.

14. The compound according to any one of claims 1 to 12, wherein R' is:

15. the compound of claim 14, wherein the compound is not bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

16. The compound of claim 14, wherein the compound is conjugated to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

17. The compound of claim 16, wherein the thiol-bearing macromolecular carrier or thiol-bearing tumor-specific carrier is selected from endogenous albumin, exogenous albumin, antibodies, antibody fragments, peptides, natural or synthetic polymers, liposomes, and nanoparticles.

18. The compound of any one of claims 14 to 17, wherein TBG is an optionally substituted maleimide group.

19. The compound according to any one of claims 14 to 18, wherein Z^1’Is selected from-NO₂or-SO₃M²；

And Y' is selected from-NHC (O) -, or

20. The compound according to any one of claims 14 to 19, wherein R^1’The method comprises the following steps:

21. the compound according to any one of claims 14 to 19, wherein R' is:

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

wherein

R^2’Selected from optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substitution.

22. The compound according to any one of claims 14 to 21, wherein R' is:

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

23. The compound of claim 22, wherein the compound is selected from the group consisting of:

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

24. The compound according to any one of claims 14 to 20, wherein R' is:

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

wherein M is a pharmaceutically acceptable counterion.

25. The compound of claim 24, wherein the compound is selected from the group consisting of:

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

26. The compound according to any one of claims 14 to 20, wherein R' is:

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

wherein M is¹Is a pharmaceutically acceptable counterion.

27. The compound of claim 26, wherein the compound is:

28. a pharmaceutical composition comprising a compound according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. A method for treating a disease or condition selected from: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases and diseases caused by bacteria, fungi or other microorganisms, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 28.

30. The method of claim 29, wherein the disease is cancer.

31. The method of claim 30, wherein the cancer is selected from adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampulla, anal, astrocytoma, basal cell carcinoma, pancreatic carcinoma, connective tissue tumor, bladder cancer, bronchial cancer, non-small cell bronchial cancer, breast cancer, burkitt's lymphoma, uterine cancer, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial cancer, gallbladder cancer, cystic carcinoma, uterine cancer, cervical cancer, neck, nose, ear tumor, hematologic tumor, hairy cell leukemia, urinary tract cancer, skin cancer, glioma, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, large intestine cancer, head and neck cancer, colon cancer, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, stomach cancer, colon cancer, breast cancer, colon cancer, Medulloblastoma, melanoma, meningioma, renal cancer, renal cell carcinoma, oligodendroglioma, esophageal cancer, osteolytic and bone cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, tongue cancer, ovarian cancer, and lymphoma.

32. A method of reducing cytotoxicity of a compound, the method comprising administering to a patient in need thereof a compound of any one of claims 1-27 or a pharmaceutical composition of claim 28, wherein the administration results in reduced cytotoxicity when compared to an equivalent dose of unmodified active agent.

33. A method of increasing the concentration of a metabolite of a compound in a tumor, the method comprising administering to a patient in need thereof a compound according to any one of claims 1-27 or a pharmaceutical composition of claim 28, wherein the increase is compared to an equivalent dose of the unmodified active agent.

34. A compound according to any one of claims 1 to 27 for use as a medicament.

35. A compound according to any one of claims 1 to 27 for use in the treatment of a disease or condition selected from the group consisting of: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms.

36. Use of a compound according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 28 in the manufacture of a medicament for the treatment of a disease or condition selected from: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms.

Background

Many drugs, particularly cancer therapeutics, have a narrow therapeutic window in which their side effects limit their beneficial effects. Systemic administration of such drugs often results in limited therapeutic effect because the doses required to elicit a stronger effect can cause the patient to experience unacceptable side effects. This is particularly critical for those drugs that have a high cytotoxic potential, such as cytostatics, viral inhibitors or immunosuppressants. This is even more critical for certain cytotoxic agents that inhibit tumor cell growth in the picomolar range. These agents are generally too toxic to be used as chemotherapeutic agents. For example, tubulin binding to maytansine is very effective in inhibiting tumor cell growth, but fails in various clinical trials due to an unacceptable toxicity profile.

Many research efforts have been directed to the delivery of specific drugs at specific sites of action. Generally, this method produces higher concentrations of drug at the site of action than can be achieved by systemic administration, while limiting side effects.

Drug delivery in oncology is of particular interest due to the narrow therapeutic window of the agents used in this indication. Much research has focused on the conjugation of anticancer drugs to a variety 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 incorporates a predetermined breakpoint that allows the bound drug to be released at the cellular target site (Kratz et al, ChemMedChem, 3: 20-53 (2008)).

Conjugates are known in which cytostatics bind to serum proteins, mainly to specific carrier molecules, such as human serum albumin and human serum transferrin, and are then administered. In other cases, the conjugate comprising the therapeutically effective substance, the spacer molecule and the protein binding molecule is covalently bound to circulating serum albumin after administration, which results in transport of the therapeutically effective substance to the target site where it is released (US 7,387,771). 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-.

However, in designing a drug delivery system, an appropriate balance should be struck between maintaining the targeting properties of the drug carrier and controlling drug release. Drug delivery systems should have sufficient stability in the bloodstream and allow efficient drug release at the tumor site by enzymatic cleavage, reduction or in a pH dependent manner (Kratz et al, chemed chem, 3: 20-53 (2008)). For highly potent cytotoxic agents from the class of maytansinoids (derived from maytansinoids), only drug delivery systems in which maytansinoid-based active species are released in a non-specific or reductive manner have been reported. In theseOnly those antibodies that use monoclonal antibodies as carrier molecules are in clinical development and only one antibody-maytansinoid conjugate, namely T-DM1Market acceptance for certain breast cancer subtypes is obtained. Thus, there remains a need for effective and less complex drug delivery and release systems that release highly potent cytotoxic agents based on maytansinoids in an effective manner.

Disclosure of Invention

The present disclosure provides a compound having the structure of formula (I):

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

R¹is selected from-H and C₁-C₄An alkyl group;

the spacer is selected from:

v is absent or selected from-CH₂-, -O-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

each R²Independently selected from-H, halo groups (e.g., -F, -Cl, -Br or-I) and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

n is 0 to 3;

x is absent or selected from-CH₂-, -O-, -S-, -Se-and-NR-⁴-, wherein R⁴is-H or C₁-C₄An alkyl group;

y is selected from ═ CH-and ═ N-;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, haloAn elemental group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、C₁-C₄Alkyl and C₂-C₄An alkoxy group;

r' is selected from O and

y' is absent or selected from optionally substituted C₁-C₆Alkyl, -NH-C (O) -and-C (O) -NH-; or Y' is selected from the group consisting of:

wherein n is 0 to 6;

R^1’absent or selected from the group consisting of:

wherein M is¹Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

R^2’is optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substitution;

Z^1’、Z^2’、Z^3’and Z^4’Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、-SO₃M²And C₁-C₄Alkyl radical, wherein M²Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca² ⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

wherein the TBG is optionally bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

In some embodiments, the present disclosure provides a compound having the structure of formula (I):

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

R¹is selected from-H and C₁-C₄An alkyl group;

the spacer is selected from:

v is absent or selected from-CH₂-, -O-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

each R²Independently selected from-H, halo groups (e.g., -F, -Cl, -Br or-I) and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

n is 0 to 3;

x is absent or selected from-CH₂-, -O-, -S-, -Se-and-NR-⁴-, wherein R⁴is-H or C₁-C₄An alkyl group;

y is selected from ═ CH-and ═ N-;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、C₁-C₄Alkyl and C₂-C₄An alkoxy group;

AA is an amino acid selected from the group consisting of glycine, D or L proline, sarcosine, D or L alanine, D or L N-methylalanine, β -alanine, N-methyl- β -alanine, α -aminoisobutyric acid and N-methyl- α -aminoisobutyric acid;

r' is selected from O and

y' is absent or selected from optionally substituted C₁-C₆Alkyl, -NH-C (O) -and-C (O) -NH-; or Y' is selected from the group consisting of:

wherein n is 0 to 6;

R^1’absent or selected from the group consisting of:

wherein M is¹Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

R^2’is optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl group are each independentlyStereo quilt-OCH₂CH₂-substitution;

Z^1’、Z^2’、Z^3’and Z^4’Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、-SO₃M²And C₁-C₄Alkyl radical, wherein M²Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca² ⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

wherein the TBG is optionally bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

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

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

wherein:

each R²Independently selected from-H and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

x is absent or selected from-CH₂-, -O-, -S-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃。

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

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

wherein:

each R²Independently selected from-H and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

x is absent or selected from-CH₂-, -O-, -S-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃。

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

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

wherein:

x is absent or selected from-CH₂-and-NH-;

y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

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

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

wherein:

x is absent or selected from-CH₂-and-NH-;

y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

AA is an amino acid selected from the group consisting of glycine, D or L proline, sarcosine, D or L alanine, D or L N-methylalanine, beta-alanine, N-methyl-beta-alanine, alpha-aminoisobutyric acid and N-methyl-alpha-aminoisobutyric acid.

In some embodiments, R¹is-H. In some embodiments, Z¹、Z²、Z³And Z⁴Is not H. In some embodiments, Z¹、Z²、Z³And Z⁴At least one of which is-F or-NO₂. In some embodiments, n is 0 and X is absent. In some embodiments, n is 0 and X is-CH₂-. In some embodiments, n is 0 and X is-O-, NHMe, or-S-. In some embodiments, the compound is selected from:

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

In some embodiments, the pharmaceutically acceptable counterion is selected from H⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄An alkyl group.

In some embodiments, R' is O. In some embodiments, R' is:

in some embodiments, the compound is not bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier. In some embodiments, the compound is conjugated to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier. In some embodiments, the thiol-bearing macromolecular carrier or thiol-bearing tumor-specific carrier is selected from endogenous albumin, exogenous albumin, antibodies, antibody fragments, peptides, natural or synthetic polymers, liposomes, and nanoparticles. In some embodiments, TBG is an optionally substituted maleimide group. In some embodiments, Z^1’Is selected from-NO₂or-SO₃M²；

And Y' is selected from-NHC (O) -, or

In some embodiments, R^1’Is that

In some embodiments, R' is:

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

wherein R is^2’Selected from optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substitution.

In some embodiments, R' is:

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

In some embodiments, the compound is selected from:

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

In some embodiments, R' is:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof; wherein M is¹Is a pharmaceutically acceptable counterion.

In some embodiments, the compound is selected from:

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

In some embodiments, the compound of any one of claims 14 to 20, wherein R' is:

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

wherein M is¹Is a pharmaceutically acceptable counterion.

In some embodiments, the compound of claim 26, wherein the compound is:

other embodiments include a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable carrier.

Other embodiments include a method for treating a disease or condition selected from: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms, comprising administering to a patient in need thereof a therapeutically effective amount of a compound or pharmaceutical composition disclosed herein. In some embodiments, the disease is a cancer, e.g., the cancer is selected from adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampulla, anal, astrocytoma, basal cell carcinoma, pancreatic carcinoma, connective tissue tumors, bladder cancer, bronchial cancer, non-small cell bronchial cancer, breast cancer, burkitt's lymphoma, uterine cancer, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial cancer, gallbladder cancer, cystic carcinoma, uterine cancer, cervical cancer, neck, nose, ear tumor, hematologic tumor, hairy cell leukemia, urinary tract cancer, skin cancer, glioma, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, large intestine cancer, head and neck cancer, colon cancer, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, stomach cancer, colon cancer, breast, Medulloblastoma, melanoma, meningioma, renal cancer, renal cell carcinoma, oligodendroglioma, esophageal cancer, osteolytic and bone cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, tongue cancer, ovarian cancer, and lymphoma.

Other embodiments include a method of reducing the cytotoxicity of a compound, comprising administering to a patient in need thereof a compound or pharmaceutical composition disclosed herein, wherein the administration results in reduced cytotoxicity when compared to an equivalent dose of an unmodified active agent.

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

Other embodiments include a compound disclosed herein for use as a medicament.

Other embodiments include a compound disclosed herein for use in treating a disease or condition selected from the group consisting of: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms.

Other embodiments include the use of a compound or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a disease or condition selected from: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms.

Drawings

Figure 1 shows the stability of the different linkers of 4 in the plasma of CD1 mice.

FIG. 2 shows the geometric mean IC in a set of different cell lines₅₀Heat map of values.

Figure 3 shows tumor growth curves for the control group, maytansine group, and the groups treated with compounds 30, 42, 31, and 35 in RXF631 kidney cell tumor model.

Figure 4 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30, 42, 31 and 35 in RXF631 kidney cell tumor model.

Fig. 5 shows tumor growth curves of the control group, maytansine group and the group treated with compounds 32, 30 and 31 in the LXFE 937 squamous cell lung cancer model.

Fig. 6 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 32, 30 and 31 in the LXFE 937 squamous cell lung cancer model.

Fig. 7 shows tumor growth curves of the control group, maytansine group, and the group treated with compounds 30 and 31 in the LXFE 937 squamous cell lung cancer model.

Fig. 8 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30 and 31 in the LXFE 937 squamous cell lung cancer model.

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

Fig. 10 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30 and 31 in LXFA 737 lung adenocarcinoma model.

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

Figure 12 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 32, 30 and 31 in the MDA-MB 231 breast cancer model.

Figure 13 shows tumor growth curves for the control group, maytansine group, and the groups treated with compounds 30 and 31 in the a2780 ovarian cancer model.

Fig. 14 shows the body weight change curves of the control group, maytansine group, and the group treated with compounds 30 and 31 in the a2780 ovarian cancer model.

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

Figure 16 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30 and 31 in the MDA-MB 468 breast cancer model.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in the present application shall have the meanings that are commonly understood by one of ordinary skill in the art. Generally, the nomenclature described herein that relates to chemistry, molecular biology, cellular and cancer biology, immunology, microbiology, pharmacology, and protein chemistry is well known and commonly employed in the art.

All publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. Unless otherwise specified, it is to be understood that each embodiment disclosed herein can be used alone or in combination with any one or more other embodiments of the present invention.

Definition of

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer (or component) or group of integers (or components) but not the exclusion of any other integer (or component) or group of integers (or components).

Throughout this application, where a compound or composition is described as having, including, or comprising a particular component, it is contemplated that such compound or composition can also consist essentially of, or consist of, the recited component. Similarly, where a method or process is described as having, including, or comprising specific process steps, the process may also consist essentially of, or consist of the recited process steps. In addition, it is to be understood that the order of steps or order of performing certain actions is immaterial so long as the compounds, compositions, and methods described herein remain operable. Also, two or more steps or actions may be performed simultaneously.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "including" is used to mean "including but not limited to". "include" and "include but are not limited to" may be used interchangeably.

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

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 mean any compound which, by itself or following transformation in the relevant organism, produces a pharmacological effect and therefore also includes derivatives from such transformations. The pharmacological effect of the drug of the composition according to the present disclosure can only be a single effect, for example: cytostatic and/or cytotoxic effects, or a broad spectrum of pharmacological effects, such as having both immunosuppressive and anti-inflammatory effects.

The terms "patient," "subject," or "individual" are used interchangeably and refer to a human or non-human animal. These terms include mammals such as humans, primates, livestock (e.g., cows, pigs), companion animals (e.g., dogs, cats) and rodents (e.g., mice and rats). In certain embodiments, the patient or subject is a human patient or subject, such as a human patient having a condition in need of treatment.

The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, e.g., in combination with one or more pharmaceutically acceptable carriers, excipients, or solvents, for a subject animal, including humans and mammals. Such compositions may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, protectants, and other materials well known in the art. In certain embodiments, pharmaceutical compositions encompass compositions comprising an active ingredient, and an inert ingredient consisting of an excipient, carrier, or diluent, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from decomposition of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and one or more pharmaceutically acceptable excipients, carriers, and/or diluents.

The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that can be administered to a patient with a therapeutically effective substance disclosed herein without destroying the pharmaceutical activity of the agent. The term "excipient" refers to an additive that is not a pharmaceutically active ingredient in a formulation or composition. In certain embodiments, a "pharmaceutically acceptable" substance is suitable for contact with cells, tissues or organs of an animal or human without undue toxicity, irritation, allergic response, immunogenicity, or other adverse effect, in a dosage form of: according to the dosing schedule and with a reasonable benefit/risk ratio. In certain embodiments, a "pharmaceutically acceptable" substance that is a component of a pharmaceutical composition is additionally compatible with other ingredients of the composition. In certain embodiments, the terms "pharmaceutically acceptable excipient", "pharmaceutically acceptable carrier" and "pharmaceutically acceptable diluent" include, but are not limited to, pharmaceutically acceptable inactive ingredients, materials, compositions and vehicles, such as liquid fillers, solid fillers, diluents, excipients, carriers, solvents and encapsulating materials. Carriers, diluents and excipients also include all pharmaceutically acceptable dispersion media, coatings, buffers, isotonic agents, stabilizers, absorption delaying agents, antimicrobial agents, antibacterial agents, antifungal agents, adjuvants and the like. The present disclosure encompasses the use of conventional excipients, carriers and diluents in pharmaceutical compositions, except that any conventional excipient, carrier or diluent is incompatible with the active ingredient. See, e.g., Remington: the Science and Practice of Pharmacy, 21 st edition, Lippincott Williams & Wilkins (Philadelphia, Pennsylvania, 2005); handbook of Pharmaceutical Excipients, 5 th edition, written by Rowe et al, The Pharmaceutical Press and The American Pharmaceutical Association (2005); handbook of pharmaceutical additives, 3 rd edition, written by Ash and Ash, Gower Publishing Co. (2007); and pharmaceutical compression and Formulation, written by Gibson, CRC Press LLC (Boca Raton, Florida, 2004).

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, e.g., to effect a beneficial and/or desirable change in the treatment, cure, inhibition, or amelioration, etc., of the overall health, physiological response or condition of a patient having a disease (e.g., cancer) or condition. The full therapeutic effect does not necessarily occur by one dose administration, and may only occur after a series of doses. Thus, a therapeutically effective amount may be administered by one or more administrations. The exact effective amount required by a subject will depend, for example, on the size, health and age of the subject, the nature and extent of the disease, the therapy or combination of therapies selected for administration, and the mode of administration. The skilled person can readily determine the effective amount for a given situation by routine experimentation. The skilled artisan will recognize that treating cancer includes, but is not limited to, killing cancer cells, preventing the growth of new cancer cells, causing tumor regression (reduction in tumor size), causing reduced metastasis, improving the patient's vital functions, improving the patient's health, 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 needed to improve the clinical symptoms of a patient. The one or more methods of treating cancer described herein should not be interpreted as, or otherwise limited to, "curing" cancer.

As used herein, the term "treating" or "treatment" includes reversing, alleviating or preventing symptoms, clinical signs and underlying pathological conditions in a manner to improve or stabilize the condition of the subject. As used herein, and as is well known in the art, "treatment" is a route for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation, amelioration, or slowing of one or more symptoms or progression of the condition associated with the condition, e.g., reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable with respect to cancer. "treatment" may also mean an extended survival period as compared to the expected survival period without treatment. Exemplary beneficial clinical results are described herein.

A subject may be "administered" or "administered" a substance, compound, or agent using one of a variety of methods known to those of skill in the art. For example, the compound or agent may be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a dermal catheter). The compound or agent may also be suitably introduced by rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide for prolonged, slow or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and/or over one or more extended periods of time. In certain aspects, administration includes both direct administration (including self-administration) and indirect administration (including the act of prescribing a drug). For example, as used herein, a physician who instructs a patient to self-administer a drug or to administer a drug by another person, and/or a physician who provides a prescription for a drug to a patient is administering a drug to a patient. When a method is part of a treatment regimen involving more than one agent or modality, the present disclosure contemplates that the agents may be administered at the same or different times and by the same or different routes of administration. Suitable methods of administering a substance, compound, or agent to a subject also depend on, for example, the age of the subject, whether the subject is active or inactive at the time of administration, whether the cognitive ability of the subject is impaired at the time of administration, the degree of injury, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity).

The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbon atoms of the backbone of a chemical compound. It is understood that "substituted" or "substituted.. includes the implicit proviso that such substitution is according to the allowed valencies of the substituting atoms and substituents, and that the substitution results in a stable compound, e.g., that no spontaneous transformations, such as by rearrangement, cyclization, elimination, etc., occur. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For suitable organic compounds, the permissible substituents can be one or more and can be the same or different. For purposes of this disclosure, a heteroatom (such as nitrogen) may have a hydrogen substituent, and/or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatom. Substituents may include any of the substituents described herein, for example, halogen, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amino, imino, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic (e.g., C₆-C₁₂Aryl) or heteroaromatic (e.g., heteroaryl) moieties.

"optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the present application includes instances where such circumstance occurs and instances 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, this application includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.

Unless specifically stated as "unsubstituted," chemical moieties referred to herein are understood to include substituted variants. For example, reference to an "alkyl" group or moiety implicitly includes both substituted and unsubstituted variants. Examples of substituents on a chemical moiety include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amide, amino, imino, cyano, nitro, azido, thiol, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aryl or heteroaryl moiety.

"aryl" indicates an aromatic carbocyclic ring having the specified number of carbon atoms in the ring, for example 6 to 12 or 6 to 10 carbon atoms. The aryl group can be monocyclic or polycyclic (e.g., bicyclic, tricyclic). In some cases, both rings of the polycyclic aryl are aromatic (e.g., naphthyl). In other instances, the polycyclic aryl can include a non-aromatic ring (e.g., cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl) fused to an aromatic ring, provided that the polycyclic aryl is bonded to the parent structure via an atom in the aromatic ring. Thus, 1, 2, 3, 4-tetrahydronaphthalen-5-yl (wherein the moiety is bound to the parent structure through an aromatic carbon atom) is considered an aryl group, while 1, 2, 3, 4-tetrahydronaphthalen-1-yl (wherein the moiety is bound to the parent structure through a non-aromatic carbon atom) is not considered an aryl group. Similarly, 1, 2, 3, 4-tetrahydroquinolin-8-yl (wherein the moiety is bonded to the parent structure through an aromatic carbon atom) is considered an aryl group, and 1, 2, 3, 4-tetrahydroquinolin-1-yl (wherein the moiety is bonded 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 (e.g., quinolin-5-yl and quinolin-2-yl are both heteroaryl).

"heteroaryl" indicates an aromatic ring containing the specified number of ring atoms (e.g., 5 to 12 or 5 to 10 membered heteroaryl) consisting of one or more heteroatoms selected from N, O and S (e.g., 1, 2, 3, or 4 heteroatoms), the remaining ring atoms being carbon. A 5-membered heteroaryl is a heteroaryl having 5 ring atoms. A 6-membered heteroaryl is a heteroaryl having 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 the heteroaryl group is no more than 2. In some embodiments, the total number of S and O atoms in the heteroaryl group is no more than 1. Unless otherwise indicated, heteroaryl groups may be bonded to the parent structure through a carbon or nitrogen atom, where valency permits. For example, "pyridyl" includes 2-pyridyl, 3-pyridyl, and 4-pyridyl, and "pyrrolyl" includes 1-pyrrolyl, 2-pyrrolyl, and 3-pyrrolyl. When nitrogen is present in a heteroaryl ring, the nitrogen may be in the oxidation state (i.e., N) as the nature of the adjacent atoms and groups permits⁺-O^-) Are present. In addition, when sulfur is present in a heteroaryl ring, it may be in the oxidation state (i.e., S) as the nature of the adjacent atoms and groups permits⁺-O^-Or SO₂) Are present. Heteroaryl groups can be monocyclic or polycyclic (e.g., bicyclic, tricyclic).

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 (e.g., 1, 2, 4-triazine, 1, 3, 5-triazine), and tetrazine.

The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbyl-C (O) -such as alkyl-C (O) -.

The term "alkyl" denotes saturated aliphatic radicalsGroups, including straight chain alkyl and branched chain alkyl. In some embodiments, the linear or branched alkyl group has 30 or fewer carbon atoms in its backbone (e.g., for linear, C₁-C₃₀For the branch C₄-C₃₀) And in other embodiments, 20 or less. In certain embodiments, alkyl is lower alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, and n-pentyl. Also, the term "alkyl" as used throughout the specification, examples and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbon atoms of the hydrocarbon backbone. In certain embodiments, the linear or branched alkyl group has 30 or fewer carbon atoms in its backbone (e.g., for linear, C₁-C₃₀For the branch C₃-C₃₀). In some embodiments, the chain has ten or fewer carbon atoms (C) in its backbone chain₁-C₁₀). In other embodiments, the chain has six or fewer carbon atoms (C) in its backbone chain₁-C₆)。

The term "hydrazone moiety" or "hydrazone" refers to E and/or Z hydrazones, for example,

the stereochemical configuration of the hydrazone moiety may 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 the disclosure may include both E and/or Z.

In various places in the specification, substituents of the compounds of the present disclosure are disclosed in groups or ranges. It is specifically intended that the present disclosure includes each and every individual subcombination of the members of such groups and ranges. For example, the term "C₁-C₆Alkyl "is specifically intended to individually disclose methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and the like.

"pharmaceutically acceptable salts" are salts of compounds suitable for pharmaceutical use, including, but not limited to, metal salts (e.g., sodium, potassium, magnesium, calcium, and the like), acid addition salts (e.g., inorganic acids, carboxylic acids, and the like), and base addition salts (e.g., aqueous ammonia, organic amines, and the like). The acid addition salt forms of the compounds present in free form as bases may be obtained by treating the free base form with a suitable acid, such as an inorganic acid, for example a hydrohalic acid, such as hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as, for example, acetic acid, glycolic acid, propionic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclic acids, salicylic acid, p-aminosalicylic acid, palmitic acid, and the like (see, e.g., WO 01/062726. Berge et al, some of the pharmaceutically acceptable salts listed, Journal of pharmaceutical Sciences, 66: 1-19(1977), which are incorporated herein by reference in their entirety). The compounds containing an acidic proton may be converted into their therapeutically active non-toxic base addition salt forms, such as metal or amine salts, by treatment with appropriate organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali metal and alkaline earth metal salts or ions, e.g., lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g., N-methyl-d-glucamine, hydrabamine salts, and salts with amino acids, such as, for example, arginine, lysine and the like. Instead, the salt form may be converted to the free form by treatment with a suitable base or acid. The compounds and salts thereof may be in the form of solvates which are included within the scope of the present disclosure. Such solvates include, for example, hydrates, alcoholates and the like (see, for example, WO 01/062726).

The present disclosure also provides pharmaceutical compositions comprising one or more compounds of the present disclosure and a pharmaceutically acceptable carrier or excipient. The compounds or pharmaceutical compositions of the present disclosure may be used in vitro or in vivo.

The term "isomer" as used herein includes, but is not limited to, tautomers, cis and trans isomers (E (ipsilateral), Z (ipsilateral)), R-and S-enantiomers (R and S notation is used consistent with the rules set forth in pureepl. chem. (1976), 45, 11-30), diastereomers, (D) -isomers, (L) -isomers, stereoisomers, racemic mixtures thereof, and other mixtures thereof. All such isomers and mixtures thereof are intended to be included in this disclosure. Tautomers, while not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

The present disclosure also includes isotopically labeled or enriched compounds of the present disclosure. An "isotopically labeled" or "radio-labeled" compound is a compound of the present disclosure in which one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found (naturally occurring) in nature. Suitable radionuclides that may be incorporated into the compounds of the present disclosure include, but are not limited to²H (also written as D for deuterium),³H (also written as T for tritium)¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、¹⁸F、³⁵S、³⁶Cl、⁸²Br、⁷⁵Br、⁷⁶Br and⁷⁷br is added. The radionuclide that is incorporated into the radiolabeled compounds of the present invention will depend on the particular application for which the radiolabeled compound is used. For example, for in vitro metalloproteinase labeling and competition assays, incorporation is common³H、¹⁴C、⁸²Br、³⁵S or the compound of (a). For the application of radio imaging,¹¹C、¹⁸F、⁷⁵Br、⁷⁶br or⁷⁷Br is usually the most useful. Tritium (A)³H) And¹⁴c may be useful for ADME studies. In some embodiments, each of the alkyl, cycloalkyl, alkene, alkylene, and alkoxy groups is optionally substituted with one or more-D or-F.

Compounds of the present disclosure

Embodiments of the present disclosure provide a compound having a structure represented by formula (I):

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

wherein:

R¹is selected from-H and C₁-C₄An alkyl group;

the spacer is selected from:

v is absent or selected from-CH₂-, -O-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

each R²Independently selected from-H, halo groups (e.g., -F, -Cl, -Br or-I) and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

n is 0 to 3;

x is absent or selected from-CH₂-, -O-, -S-, -Se-and-NR-⁴-, wherein R⁴is-H or C₁-C₄An alkyl group;

y is selected from ═ CH-and ═ N-;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、C₁-C₄Alkyl and C₂-C₄An alkoxy group;

r' is selected from O and

y' is absent or selected from optionally substituted C₁-C₆Alkyl, -NH-C (O) -and-C (O) -NH-; or Y' is selected fromThe group consisting of:

wherein n is 0 to 6;

R^1’absent or selected from the group consisting of:

wherein M is¹Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

R^2’is optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substitution;

Z^1’、Z^2’、Z^3’and Z^4’Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、-SO₃M²And C₁-C₄Alkyl radical, wherein M²Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca² ⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

wherein the TBG is optionally bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

In some embodiments, in the compound of formula (I), R¹Is selected from-H and C₁-C₄An alkyl group;

the spacer is selected from:

v is absent or selected from-CH₂-, -O-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

each R²Independently selected from-H, halo groups (e.g., -F, -Cl, -Br or-I) and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

n is 0 to 3;

x is absent or selected from-CH₂-, -O-, -S-, -Se-and-NR-⁴-, wherein R⁴is-H or C₁-C₄An alkyl group;

y is selected from ═ CH-and ═ N-;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、C₁-C₄Alkyl and C₂-C₄An alkoxy group;

r' is selected from O and

y' is absent or selected from optionally substituted C₁-C₆Alkyl, -NH-C (O) -and-C (O) -NH-; or Y' is selected from the group consisting of:

wherein n is 0 to 6;

R^1’absent or selected from the group consisting of:

wherein M is¹Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca²⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

R^2’is optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substitution;

Z^1’、Z^2’、Z^3’and Z^4’Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-CN、-NO₂、-SO₃M²And C₁-C₄Alkyl radical, wherein M²Is a pharmaceutically acceptable counterion (e.g., H)⁺、Na⁺、K⁺、Ca² ⁺、Mg²⁺、NR₄ ⁺And NHR₃ ⁺(ii) a Wherein R is H or C₁-C₄Alkyl groups);

wherein the TBG is optionally bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier.

In some embodiments, R' is O. These novel compounds may represent active substances and may be, for example, the active component of a drug delivery system or an active metabolite released from a drug delivery system.

In certain embodiments, compounds of formula (I) wherein R 'is O have the structure of any one of formulae (II'), (III '), and (IV'):

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

wherein:

each R²Independently selected from-H and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

x is absent or selected from-CH₂-, -O-, -S-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

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

wherein:

x is absent or selected from-CH₂-and-NH-;

y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each of which isIndependently selected from-H, halo groups (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

In still other embodiments, in the compound of formula (IV'):

x is absent or selected from-CH₂-and-NH-; y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

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

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

Other embodiments include prodrugs, such as those represented by formula (I), wherein R' is:

these novel compounds may represent drug delivery systems whereby the active metabolites are selectively released from the drug delivery system. These prodrugs include, for example, albumin binding prodrugs.

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

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

wherein:

each R²Independently selected from-H and C₁-C₄Alkyl, or two R²Together form C₃-C₆A cycloalkyl group;

x is absent or selected from-CH₂-, -O-, -S-and-NR³-, wherein R³is-H or C₁-C₄An alkyl group;

Z¹、Z²、Z³and Z⁴Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

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

wherein:

x is absent or selected from-CH₂-and-NH-;

y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

Wherein R' is:

in still other embodiments, in the compound of formula (IV'):

x is absent or selected from-CH₂-and-NH-;

y is ═ CH-or ═ N-;

Z¹、Z²、Z³and Z³Each independently selected from-H, a halogen group (e.g., -F, -Cl, -Br or-I), -CF₃、-OCH₃、-NO₂and-CH₃；

Wherein R' is:

in some embodiments, R¹is-H. In other embodiments, Z¹、Z²、Z³And Z⁴Is not H and/or Z¹、Z²、Z³And Z⁴At least one of which is-F or-NO₂. In some embodiments, when n is 0, X is absent. In other embodiments, when n is 0, X is-CH₂-. 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 disclosed compounds.

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

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

In certain embodiments, the compound is not bound to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier. In other embodiments, the compound is conjugated to a thiol-bearing macromolecular carrier or a thiol-bearing tumor-specific carrier. For example, the thiol-bearing macromolecular carrier or thiol-bearing tumor-specific carrier is selected from endogenous albumin, exogenous albumin, antibodies, antibody fragments, peptides, natural or synthetic polymers, liposomes, and nanoparticles.

In some embodiments, TBG is an optionally substituted maleimide group, e.g., an unsubstituted maleimide group. In some embodiments, the maleimide group rapidly and selectively binds to cysteine 34 of albumin following administration to a subject (such as a human).

In some embodiments, Z^1’Is selected from-NO₂or-SO₃M²And/or Y' is selected from-NHC (O) -or

In some embodiments, R^1’Is thatIn some embodiments, R^2’Selected from optionally substituted C₁-C₁₈Alkyl, wherein at said C₁-C₁₈Optionally up to six carbon atoms in the alkyl radical are each independently-OCH₂CH₂-substituted (e.g. 1, 2, 3, 4, 5 or 6 carbon atoms by-OCH₂CH₂-substitution).

In some embodiments, R' is:

for example, R' may be:

specific compounds within the disclosure include the following:

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

Pharmaceutical composition

In some embodiments, the present disclosure provides pharmaceutical compositions comprising a compound described herein. In some embodiments, the composition comprises a compound of formula (I), wherein R' is:

or various embodiments disclosed herein.

The total amount of compound in the composition administered to a patient is the amount best suited for that patient. One skilled in the art will appreciate that different individuals may require different total amounts of therapeutically effective substances. In some embodiments, the amount of the compound is a pharmaceutically effective amount. The skilled artisan will be able to determine the amount of compound in the composition required 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 the volume of liquid that can be administered. For example, the concentration of the compound in the injectable composition can be from about 0.1mg/ml to about 50 mg/ml. In some embodiments, the concentration of the compound may be in the range of about 0.1mg/mL to about 40 mg/mL.

The pharmaceutical compositions and kits of the present disclosure may also comprise diluents, fillers, salts, buffers, stabilizers, solubilizers, protective agents, and other materials well known in the art. The term "pharmaceutically acceptable" refers to non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration.

The compositions can be administered in a variety of conventional ways. Exemplary routes of administration that can be used include oral, parenteral, intravenous, intraarterial, cutaneous, subcutaneous, intramuscular, topical, intracranial, orbital, ophthalmic, intravitreal, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, Central Nervous System (CNS) administration or administration by suppository. In some embodiments, the composition is suitable for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously, or intrathecally. In some embodiments, the composition is injected intravenously. In some embodiments, a reconstituted formulation can be prepared by reconstituting the lyophilized compound composition in a reconstitution fluid including, for example, an alcohol, DMSO, and/or polyethylene glycol, and water and/or a salt buffer. Such reconstitution can include adding a reconstitution fluid and mixing, for example, by swirling or vortexing the mixture. The reconstituted formulation may then be made suitable for injection by mixing, for example, ringer's lactate solution, 5% dextrose solution, isotonic saline, or a suitable salt buffer with the formulation to make an injectable composition. One skilled in the art will appreciate that the method of administering a therapeutically effective agent formulation or composition will depend on factors such as the age, weight, and physical condition of the patient being treated, as well as the disease or condition being treated. Thus, the skilled person will be able to select the optimal method of administration for the patient on a case-by-case basis.

In some embodiments, the compounds and compositions disclosed herein are used to treat cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms.

In some embodiments, the compounds disclosed herein may be used in the manufacture of a medicament for treating a disease selected from: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms.

In some embodiments, the cancer is a hematologic cancer or a solid tumor cancer. In some embodiments, the cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, and melanoma.

In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampulla cancer, anal cancer, astrocytoma, basal cell carcinoma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial cancer, non-small cell bronchial cancer, breast cancer, burkitt's lymphoma, uterine corpus cancer, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial cancer, gall bladder tumor cancer, uterine cancer, cervical cancer, neck, nose, ear tumor, hematologic tumor, hairy cell leukemia, urinary tract cancer, skin cancer, glioma, testicular cancer, kaposi's sarcoma, laryngeal cancer, bone cancer, large intestine cancer, head and neck cancer, colon cancer, craniopharyngioma, liver cancer, leukemia, lung cancer, non-small cell lung cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, stomach cancer, colon cancer, medulloblastoma, melanoma, cervical cancer, meningioma, renal cancer, renal cell carcinoma, oligodendroglioma, esophageal cancer, osteolytic and bony cancers, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, tongue cancer, ovarian cancer, and lymphatic cancer.

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

In some embodiments, one or more excipients may be included in the composition. It will be appreciated by those skilled in the art that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of excipient may preclude the use of one or more other excipients because the combination of excipients may produce undesirable effects. One skilled in the art will be able to empirically determine which excipients, if any, to include in the composition. Excipients may include, but are not limited to, cosolvents, solubilizers, buffers, pH adjusters, bulking agents, surfactants, encapsulating agents, tonicity adjusters, stabilizers, protectants, and viscosity modifiers. In some embodiments, it may be beneficial to include a pharmaceutically acceptable carrier in the composition.

In some embodiments, a solubilizing agent may be included in the composition. The solubilizing agent may be used to increase the solubility of any component of the composition, including the compound or excipient. The solubilizing agents described herein are not intended to constitute an exhaustive list, but are provided only as exemplary solubilizing agents that may be used in the compositions. In certain embodiments, the solubilizing agent includes, but is not limited to, ethanol, t-butanol, polyethylene glycol, glycerol, methyl paraben, propyl paraben, polyethylene glycol, polyvinylpyrrolidone, cyclodextrins, such as, for example, dimethyl- β -cyclodextrin, hydroxyethyl- β -cyclodextrin, hydroxypropyl- β -cyclodextrin, and trimethyl- β -cyclodextrin, and combinations thereof, and any pharmaceutically acceptable salts and/or combinations thereof.

The pH of the composition can be any pH that provides the desired characteristics to the formulation or composition. Desirable characteristics may include, for example, compound stability, increased retention of the compound compared to compositions at other pH values, and improved filtration efficiency. In some embodiments, the pH of the composition may be from about 3.0 to about 9.0, for example from about 5.0 to about 7.0. In particular embodiments, the pH of the composition can 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 + -0.1.

In some embodiments, it may be beneficial to buffer the pH by including one or more buffering agents in the composition. In certain embodiments, the buffer may have a pKa of, for example, about 5.5, about 6.0, or about 6.5. One skilled in the art will appreciate that an appropriate buffer may be selected for inclusion in the composition based on its pKa and other characteristics. Buffers are well known in the art. Thus, the buffers described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary buffers that may be used in the formulations or compositions of the present disclosure. In certain embodiments, the buffering agent includes, but is not limited to, Tris-HCl, potassium phosphate, sodium citrate, sodium ascorbate, a combination of sodium and potassium phosphates, Tris/Tris-HCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cocoate, succinate, 2- (N-morpholino) ethanesulfonic acid (MES), maleate, bis-Tris, phosphate, carbonate, and any pharmaceutically acceptable salt and/or combination thereof.

In some embodiments, a pH adjuster may be included in the composition. Altering the pH of the composition may have a beneficial effect, for example, on the stability or solubility of the compound, or be useful in preparing compositions suitable for parenteral administration. pH adjusting agents are well known in the art. Thus, the pH adjusting agents described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary pH adjusting agents that may be used in the compositions. The pH adjusting agent may include, for example, an acid and a base. In some embodiments, pH adjusters include, but are not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

In some embodiments, a bulking agent may be included in the composition. Bulking agents are commonly used in lyophilized compositions to increase the volume of the composition and aid in visualization of the composition, particularly where the lyophilized particles are difficult to see. The bulking agent may also help prevent the expulsion of the active components of the pharmaceutical composition and/or help with the cryoprotection of the composition. Bulking agents are well known in the art. Thus, the bulking agents described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary bulking agents that may be used in the compositions.

Exemplary bulking agents may include carbohydrates, monosaccharides, disaccharides, polysaccharides, sugar alcohols, amino acids, sugar acids, and combinations thereof. Carbohydrate bulking agents include, but are not limited to, monosaccharides, disaccharides, polysaccharide carbohydrates, starches, aldoses, ketoses, amino sugars, glyceraldehydes, arabinose, lyxose, pentoses, ribose, xylose, galactose, glucose, hexoses, idose, mannose, talose, heptose, glucose, fructose, methyl alpha-D-glucopyranoside, maltose, lactone, sorbose, erythrose, threose, arabinose, allose, altrose, gulose, idose, talose, erythrose, ribose, xylulose, psicose, tagatose, galactosamine, arabinoglycans, fructans, fucans, galactans, galacturonic acids, glucans, mannans, xylans, inulin, fructans, fucoidans, carrageenans, galacturone, pectins, amylose, and the like, Pullulan, glycogen, amylopectin, cellulose, umbilicaria polysaccharide, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, xanthan gum, sucrose, trehalose, dextran, and lactose. Sugar alcohol bulking agents include, but are not limited to, sugar alcohols, inositol, sorbitol, and mannitol. Sugar bulking agents include, but are not limited to, aldonic acids, uronic acids, aldonic acids, gluconic acids, erythorbic acids, ascorbic acids, glucaric acids, glucuronic acids, gluconic acids, glucaric acids, galacturonic acids, mannuronic acids, neuraminic acids, pectic acids, and alginic acids. Amino acid swelling agents include, but are not limited to, glycine, histidine, and proline.

In some embodiments, a surfactant may be included in the composition. Surfactants generally lower the surface tension of the liquid composition. This may provide beneficial characteristics, such as improved ease of filtration. Surfactants may also be used as emulsifiers and/or solubilizers. Surfactants are well known in the art. Thus, the surfactants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary surfactants that may be used in the formulations or compositions of the present disclosure. 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 (i.e., pluronic), ethylene oxide and polyethylene oxide (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

In some embodiments, an encapsulant may be included in the composition. The encapsulating agent can sequester the molecule and help stabilize or solubilize the molecule. Encapsulants are well known in the art. Thus, the encapsulants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary encapsulants that can be used in the compositions. The encapsulating agent that may be included in the composition includes, but is not limited to, alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, and combinations thereof (e.g., alpha-cyclodextrin, dimethyl-alpha-cyclodextrin, hydroxyethyl-alpha-cyclodextrin, hydroxypropyl-alpha-cyclodextrin, trimethyl-alpha-cyclodextrin, beta-cyclodextrin, dimethyl-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, trimethyl-beta-cyclodextrin, gamma-cyclodextrin, dimethyl-gamma-cyclodextrin, hydroxyethyl-gamma-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, trimethyl-gamma-cyclodextrin, and combinations thereof).

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

In some embodiments, a stabilizer may be included in the composition. The stabilizing agent helps to increase the stability of the compound in the composition. This may occur, for example, by reducing degradation of the compound or preventing its aggregation. Without wishing to be bound by theory, the mechanism of enhancing stability may include isolating the compound from the solvent or inhibiting free radical oxidation of the therapeutically effective substance. Stabilizers are well known in the art. Thus, the stabilizers described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary stabilizers that may be used in the compositions. Stabilizers may include, but are not limited to, emulsifiers and surfactants.

In some embodiments, a protectant may be included in the composition. A protective agent is an agent that protects a pharmaceutically active ingredient (e.g., a therapeutically effective substance or compound) from an adverse condition (e.g., instability caused by freezing or lyophilization or oxidation). Protectants may include, for example, cryoprotectants, lyoprotectants, and antioxidants. Cryoprotectants may be used to prevent loss of efficacy of the active pharmaceutical ingredient (e.g., anthracyclines) when the composition is exposed to temperatures below its freezing point. For example, a cryoprotectant may be included in a reconstituted lyophilized formulation, such that the formulation may be frozen prior to dilution for intravenous administration. Cryoprotectants are well known in the art. Thus, the cryoprotectants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary cryoprotectants that may be used in the compositions. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulants, stabilizers, viscosity modifiers, and combinations thereof. Cryoprotectants may include, for example, disaccharides (e.g., sucrose, lactose, maltose, and trehalose), polyols (e.g., glycerol, mannitol, sorbitol, and diethylene glycol), glycols (e.g., ethylene glycol, polyethylene glycol, and propylene glycol).

Lyoprotectants can be used to stabilize components of a composition undergoing lyophilization. For example, the therapeutically effective substance may be lyophilized with a lyoprotectant prior to reconstitution. Lyoprotectants are well known in the art. Thus, the lyoprotectants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary lyoprotectants that can be used in the compositions. Lyoprotectants include, but are not limited to, solvents, surfactants, encapsulants, stabilizers, viscosity modifiers, and combinations thereof. Exemplary lyoprotectants can be, for example, sugars and polyols. Trehalose, sucrose, dextran, and hydroxypropyl- β -cyclodextrin are non-limiting examples of lyoprotectants.

Antioxidants can be used to prevent oxidation of the components of the composition. Oxidation may lead to aggregation of the drug or other deleterious effects on the purity of the drug or its efficacy. Antioxidants are well known in the art. Thus, the antioxidants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary antioxidants that can be used in the compositions. The antioxidant can be, for example, sodium ascorbate, citrate, thiol, metabisulfite, and combinations thereof.

In some embodiments, a viscosity modifier may be included in the composition. The viscosity modifier changes the viscosity of the liquid composition. This may be beneficial because viscosity plays an important role in ease of filtering the liquid composition. The composition may be filtered prior to lyophilization and reconstitution or after reconstitution. Viscosity modifiers are well known in the art. Thus, the viscosity modifiers described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary viscosity modifiers that may be used in the compositions. Viscosity modifiers include solvents, solubilizers, surfactants, and encapsulating agents. Exemplary viscosity modifiers that may be included in the composition include, but are not limited to, N-acetyl-DL-tryptophan and N-acetyl-cysteine.

Antitumor Activity of human tumor xenograft mouse model

Albumin-bound maytansinoids 30 and 31 exhibited excellent anti-tumor activity in five nude mouse human tumor xenograft models, thereby causing partial and complete tumor regression in all human tumor xenografts evaluated (see fig. 3-16). This includes about 80 to 110mm³Initial tumor volume within the range, but also including up to about 400mm³The starting tumor volume of (a). Furthermore, in most cases, treatment with albumin-bound maytansinoids 30 and 31 results in long-term remission and a reduction in Relative Tumor Volume (RTV). The parent compound maytansine is mainly inactive in the test model or shows only minimal tumor suppression. The experimental procedures and results of the tumor-bearing mouse model are described in detail in examples 11 to 19 and fig. 3 to 16.

Method of treatment

The compounds and compositions described herein can be used in a variety of clinical applications. In embodiments, the methods of treatment utilize compounds of the compositions comprising compounds of formula (I) wherein R' is:

or various embodiments disclosed herein.

The compounds and compositions described herein can induce prolonged or long-term inhibition of tumor growth. In certain embodiments, the prolonged or long-term inhibition of tumor growth is free of any weight loss or any or only minimal bone marrow toxicity.

In some embodiments, the present disclosure provides a method for treating a malignant disease comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound described herein. For example, some embodiments include a method of treating a patient having a disease or condition selected from: cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to the present disclosure.

The present disclosure provides methods of treating a condition or disease selected from cancer, viral diseases, autoimmune diseases, acute or chronic inflammatory diseases, and diseases caused by bacteria, fungi, or other microorganisms in a patient comprising administering to the patient a compound or pharmaceutical composition disclosed herein.

In some embodiments, the cancer comprises a vascularized tumor. In some embodiments, the cancer is a hematologic cancer or a solid tumor cancer. In some embodiments, the cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, and melanoma.

In some embodiments, the cancer is selected from adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampulla cancer, anal cancer, astrocytoma, basal cell carcinoma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial cancer, non-small cell bronchial cancer, breast cancer, burkitt's lymphoma, uterine corpus cancer, CUP syndrome, colon cancer, small intestine cancer, ovarian cancer, endometrial cancer, gallbladder cancer, uterine cancer, cervical cancer, neck, nose, ear tumor, blood tumor, hairy cell leukemia, urinary tract cancer, skin cancer, glioma, testicular cancer, kaposi's sarcoma, laryngeal cancer, bone cancer, large intestine cancer, head and neck cancer, colon cancer, craniopharyngeal tumor, liver cancer, leukemia, lung cancer, non-small cell lung cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, stomach cancer, colon cancer, medulloblastoma, melanoma, meningioma, cervical cancer, renal cancer, renal cell carcinoma, oligodendroglioma, esophageal cancer, osteolytic and osteocarcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, tongue cancer, ovarian cancer, and lymph gland cancer

Some embodiments include methods of increasing the metabolite concentration of a compound in a tumor, the method comprising administering a compound according to the present disclosure. In an embodiment, the compound is a compound of formula (I) wherein R' is:

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

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

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

Illustration of

Having now generally described aspects of the present disclosure, these aspects will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain features and embodiments of the present disclosure, and are not intended to be limiting.

Equivalents of

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the compounds, compositions, and methods of use thereof described herein. Such equivalents are considered to be within the scope of this disclosure.

Examples of the invention

The following examples illustrate various embodiments and aspects of the present invention.

Abbreviations

The following is a list of abbreviations used in the examples, along with their full chemical names. These terms have their commonly accepted meanings if not defined.

aq. is water

Boc ═ N-tert-butoxycarbonyl

calcd (calculated value)

DCM ═ dichloromethane

DIC is N, N' -diisopropylcarbodiimide

DMAP ═ N, N-dimethyl-4-aminopyridine

DMF ═ N, N-dimethylformamide

DMSO ═ dimethyl sulfoxide

CV is the column volume

EDC ═ 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

eq ═ equivalent

ESI-electrospray ionization

Fmoc ═ fluorenylmethoxycarbonyl

HATU ═ 1- [ 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 spectrum

LRMS (low resolution mass spectrometry)

MeCN ═ acetonitrile

MeOH ═ methanol

NMR spectrum

NP is positive phase

4-Nitrophenoxycarbonyl

nd is not detectable

na is not usable

PBS ═ phosphate buffered saline, pH7

RP-inverse

TFA ═ trifluoroacetic acid

TRIS ═ 2-amino-2- (hydroxymethyl) -1, 3-propanediol

Example 1

Preparation of linker 1

Linker 1 can be prepared as described below and shown in scheme 1.

Scheme 1

Synthesis of 4-Nitro-2-sulfobenzoic acid (A)

To a stirred solution of potassium permanganate (72g, 460mmol, 4.5 equiv.) in water (450mL) was added a solution of 4-nitro-2-sulfonic acid hydrate (26g, 102mmol, 1.0 equiv.) in microporous water (100mL) over 10 seconds. The resulting purple mixture was stirred at 115 ℃ for 5 hours, after which time it turned brown. HPLC analysis (PDA220nm) confirmed that the reaction was complete after 5 hours (> 90% conversion). The reaction mixture was cooled to room temperature. The brown solid formed during the reaction was removed by suction filtration on a pad of celite, washed with microporous water (300mL), and the brown/yellow filtrate solution was concentrated to about 125mL with a rotary evaporator at 40 ℃ and slowly acidified with a 5M HCl (about 2mL) solution until a white suspension (about ph1.0) formed. The white suspension was then heated at 100 ℃ until a clear solution was obtained, which was left to stand in an ice bath for 10 minutes until a white solid formed. A white solid was obtained by suction filtration using a sintered filter. The white solid was then dried under high vacuum to give 4-nitro-2-sulfobenzoic acid. Yield: 18g (72%). Purity by RP-HPLC, 220nm, > 95%. C₇H₄NO₇S[M-H]^-Calculated LRMS-ESI (m/z): 245.98. measured value: 245.83.

synthesis of 4-amino-2-sulfobenzoic acid (B)

A stirred suspension of 4-nitro-2-sulfobenzoic acid (13g, 51mmol, 1.0 equiv.) in water (75mL) was added back toHeating until 4-nitro-2-sulfobenzoic acid is completely dissolved. Acetic acid (7.2mL) was then added at this temperature followed by iron powder (9.5g, 180mmol, 3.5 equiv.) and added in portions (about 1g/min) over 10 minutes to avoid an exothermic reaction. The reaction mixture was then stirred at reflux for 1 hour. During this time, a brown solid formed and HPLC analysis (PDA220nm) confirmed the reaction was complete (> 95% conversion). The brown solid (when still hot) was removed by suction filtration directly on a pad of celite and further washed with hot water. The filtrate was re-filtered. The resulting filtrate was concentrated to a final volume of 100mL using a rotary evaporator at 40 ℃. Concentrated HCl was added dropwise until pH1 was reached and a white/yellow solid precipitated. The suspension was left at 4 ℃ for 1 hour. The solid was collected by suction filtration using a sintered filter and dried under high vacuum to give 4-amino-2-sulfobenzoic acid as a white solid. Yield: 9g (81%). Purity by RP-HPLC, 220nm, > 95%. C₇H₄NO₅S[M-H]^-Calculated LRMS-ESI (m/z): 216.00. measured value: 216.16.

synthesis (C) of 6-maleimidocaproyl chloride or EMC-Cl

At room temperature and in N₂To a yellow stirred solution of 6-maleimidocaproic acid (EMC) (33g, 156mmol, 1.0 equiv.) in anhydrous DCM (150mL) was added oxalyl chloride (15mL, 171mmol, 1.1 equiv.) under an atmosphere using a dropping funnel over 30 minutes (about 0.5 mL/min). The reaction was stirred at room temperature for 5 hours. During the reaction time, the color of the reaction solution turned dark yellow and HPLC analysis (PDA220nm) confirmed that the reaction was complete after 5 hours (> 95% conversion). The solvent was removed by rotary evaporator at 40 ℃ to give an oil. The residual oil was dried under high vacuum overnight (cured overnight). The resulting light brown solid was crushed and further dried under high vacuum for 20 hours to give 6-maleimidocaproyl chloride as a yellow microcrystalline solid. The compound was used in the next reaction without further purification. Yield: 34g (95%). Purity by RP-HPLC, 220nm, > 95%, as methyl ester. C₁₁H₁₆NO₄(as methyl ester) [ M + H]⁺Calculated LRMS-ESI (m/z): 226.10. measured value: 225.97.

synthesis (D) of 4- (6-maleimidohexaamido) -2-sulfobenzoic acid

In N₂4-amino-2-sulfobenzoic acid (18.5g, 85.0mmol, 1.0 equiv.) was dissolved in anhydrous DMF (300mL) under atmosphere, the solution was cooled to 4 ℃ and stirred for 10 minutes, then 4-N-methylmorpholine (18.7mL, 170mmol, 2.0 equiv.) was added dropwise (about 0.3mL/min) to the cooled solution over 1 hour using a dropping funnel, a solution of EMC-Cl (29.3g, 127mmol, 1.5 equiv.) in anhydrous DMF (200mL) was added dropwise (about 0.5g/min) to the dark brown mixture over 1 hour using a dropping funnel, the reaction mixture was stirred overnight, then allowed to reach room temperature over 10 hours, after the reaction was indicated by HPLC analysis (220nm, 95% conversion), the reaction solution was partitioned into 8mL falcon tubes of 8 × 50 mL.

The sample was centrifuged at 10 ℃ and 4.000rpm for 20 minutes, the supernatant was removed by decantation, and the solids were resuspended in 10mL of DMF per tube, and centrifuged again at 10 ℃ and 4.000rpm for 20 minutes, all DMF supernatants were combined, and concentrated at 50 ℃ under reduced pressure for 3 hours to obtain a light orange solid, the solid was resuspended in methanol (250mL) and transferred to 8 × 50mL falcon tubes, the sample was centrifuged at 10 ℃ and 4.000rpm for 20 minutes, the supernatant was removed by decantation, the solids were resuspended in 5mL of methanol per tube, and centrifuged again at 10 ℃ and 4.000rpm for 20 minutes, all solids were combined, and dried under high vacuum for 24 hours to obtain a crystalline yellow solid yield: 17g (48%). RP-HPLC reverse derived purity, 220nm, > 80%. C₁₇H₁₇N₂O₈S[M-H]^-Calculated LRMS-ESI (m/z): 409.08. measured value: 409.13.

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

In N₂To a solution of 4- (6-maleimidohexaamido) -2-sulfobenzoic acid (17.0g, 41.4mmol, 1.0 equiv.) in anhydrous DMF (350mL) under atmosphere was added EDC-HCl (8.72g, 45.5mmol, 1.1 equiv.) and 1HOBt (6.15g, 45.5mmol, 1.1 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, followed by the addition of tert-butyl carbazate (7.12g, 53.9mmol, 1.3 equivalents) and the solution changed from clear yellow to reddish. The reaction mixture was stirred at room temperature overnight. After this time, the reaction was complete as confirmed by HPLC (PDA220nm, > 95% conversion). The solvent was removed on a rotary evaporator at 40 ℃ under high vacuum for 1 hour to give a violet-brown oil which was prepared using two preloaded SNAP ULTRA340g cartridges andHP-Sphere^TMpurification was performed using a Biotage Isolera One rapid purification system on spherical silica. The tubes containing the desired product were combined and dried on a rotary evaporator under high vacuum for 10 hours to give 2- (2- (tert-butoxycarbonyl) hydrazine-1-carbonyl) -5- (6-maleimidohexa) benzenesulfonic acid as a foamy yellow solid. Yield: 9g (42%). RP-HPLC (220nm) > 95%. C₂₂H₂₇N₄O₉S[M-H]^-Calculated LRMS-ESI (m/z): 523.16. measured value: 523.15.

synthesis of linker 1

To a cooled (4 ℃ to 5 ℃) solution of Boc protected linker 1(10.2g, 19.4mmol, 1.0 equiv.) in anhydrous DCM (30mL) was added TFA (15mL) dropwise over 30 min (ca. 0.5 mL/min). After addition, the cooling bath was removed and the reaction mixture was stirred at room temperature for 3 hours. After this time, analysis was performed by HPLC (PDA220nm)) The completion of the reaction was confirmed. The reaction mixture was poured dropwise into six falcon tubes, and approximately 35mL of cold diethyl ether was added to each falcon tube. A white precipitate formed immediately. The tube was left at 4 ℃ for 3 hours. After centrifugation of falcon tubes (4000rpm, 20 min, 10 ℃), the supernatant was removed by decantation and the solids were resuspended in 5mL of ether per tube and centrifuged again (4000rpm, 20 min, 10 ℃). The supernatant was removed again by decantation, and the solid was collected and dried under high vacuum to provide linker 1 as a white microcrystalline solid as a TFA salt. Yield: 10g (96%). RP-HPLC (220nm) > 95%. C₁₇H₂₁N₄O₇S[M+H]⁺Calculated LRMS-ESI (m/z): 425.11. measured value: 425.07. c₁₇H₁₉N₄O₇S[M-H]^-Calculated LRMS-ESI (m/z): 423.11. measured value: 423.12. c₁₇H₂₁N₄O₇S[M+H]⁺Calculated HRMS-ESI (m/z): 425.1125. measured value: 425.1125. c₁₇H₁₉N₄O₇S[M-H]^-Calculated HRMS-ESI (m/z): 423.0978. measured value: 423.0980.

by passing¹H NMR and¹³c NMR confirmed the structure:¹H NMR(400MHz，DMSO-d₆)11.94(s，1H；C1-NH)，10.27(s，1H；C8-NH)，8.01(d，J＝2.2Hz，1H；C4-CH)，7.93(dd，J＝8.5，2.2Hz，1H；C6-CH)，7.68(d，J＝8.4Hz，1H；C7-CH)，7.00(s，2H；C15-CH，C16-CH)，3.40(t，J＝7.0Hz，2H；C13-CH₂)，2.32(t，J＝7.4Hz，2H；C9-CH₂)，1.60(p，J＝7.5Hz，2H；C10-CH₂)，1.52(p，J＝7.2Hz，2H；C12-CH₂)，1.26(q，J＝8.8Hz，2H；C11-CH₂)；¹³C NMR(101MHz，DMSO-d₆)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)。C₁₇H₂₁N₄O₇s. 1/2 analytical calculation: TFA C, 45.71; h, 4.26; n, 11.85; s, 6.78. Measured value: c, 46.2917; h, 4.4836; n, 12.8879; and S, 6.7886. TFA content 0.51%.

Linker 1 may also be prepared as described below and shown in scheme 2.

Scheme 2

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

To a suspension of B (30.00g, 138.12mmol, 1.00 equiv) in anhydrous acetonitrile (600mL) was added triethylamine (41.93g, 57.76mL, 414.37mmol, 3.00 equiv) and the mixture was stirred for 10 min. After this time, tert-butyl carbazate (27.38g, 207.19mmol, 1.50 equiv.) was added and the mixture was cooled to-35 ℃. At this temperature, a solution of propylphosphonic anhydride, T3P (114.27g, 106.79mL, 179.56mmol, 50% in ethyl acetate, 1.3 equiv.) was added dropwise over 1 hour. The reaction was stirred at-35 ℃ for 2 h. The mixture was allowed to warm to room temperature and passed

545(100g) and filtering. Additional washes with acetonitrile (500mL)

The two filtrates were combined and concentrated to 250 mL. The solution was divided equally into 6 parts, and the solvent was removed under reduced pressure. Each aliquot was dissolved in a solution containing 1% Et₃N in dichloromethane (50mL) and by NP flash chromatography on a Biotage Isolera equipped with a pre-assembled SNAP Ultra340g cartridge^TMPurification was performed on One rapid purification system using a step gradient with DCM (containing 1% NEt) over 7 column volumes₃) Methanol (containing 1% NEt)₃) The content is from 2% to 12%. Next, the purified fractions were combined, the solvent removed under reduced pressure, and the solid dried under high vacuum to give the title compound F as an off-white solidA colored solid. Yield: 53.25g, 108.0mmol, 78.2% (NMR in DMSO-d6 showed the presence of 1.6 equivalents of triethylamine). HPLC (method 9, 220nm) > 99%. C₁₂H₁₆N₃O₆S[M-H]^-Calculated LRMS-ESI (m/z): 330.08. measured value: 330.08.

synthesis of N-ethyl-N-isopropylpropan-2-ammonium 2- (2- (tert-butoxycarbonyl) hydrazine-1-carbonyl) -5- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-) hexa-amino) benzenesulfonate (E)

To a mixture of F (45.00g, 91.24mmol, 1.00 equiv.) and 6-maleimidocaproic acid (19.27g, 91.24mmol, 1.00 equiv.) was added acetonitrile (450mL), triethylamine (13.85g, 19.08mL, 136.86mmol) and T3P (43.55g, 40.70mL, 136.86mmol, 50% ethyl acetate solution) in portions at room temperature. The solution was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure. The crude material was then purified by a rapid purification system using seven pre-packed SNAP Ultra340g cartridges, running a linear gradient from 2% methanol to 15% methanol in dichloromethane, to afford the title compound E as an off-white solid. Yield: 30.55g, 53.5% (NMR in DMSO-d6 showed the presence of 1.1 equivalents triethylamine). HPLC (method 9, 220nm) > 99%. C₂₂H₂₇N₄O₉S[M-H]^-Calculated LRMS-ESI (m/z): 523.15. measured value: 523.26.

synthesis of 5- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -2- (hydrazinocarbonyl) benzenesulfonic acid (linker 1)

To a cold suspension (4 ℃) of E (10.00g, 15.98mmol, 1.00 equiv.) in dichloromethane (50mL) was added trifluoroacetic acid (18.22g, 12.31mL, 159.81mmol, 10.17 equiv.) over 15 minutes. The mixture was further stirred at 4 ℃ for 15 minutes, then allowed to gradually warm to room temperature and stirred for 150 minutes. Through division intoLiquid funnel the reaction mixture was added dropwise to a stirred solution of methyl tert-butyl ether, MTBE (400mL) and dichloromethane (200 mL). The obtained white solid was passed throughThe porous sintered funnel was filtered and washed sequentially with dichloromethane (2 × 150mL) and MTBE (1 × 50mL), MeOH (1 × 50mL), and (again) MTBE (2X 150 mL.) the solid was dried on the sintered funnel at room temperature overnight for 10 minutes, further dried under high vacuum at 25 ℃ for 18 hours to obtain the final product linker as a yellow solid 1. yield: 5.786g, 13.63mmol, 98.7%, HPLC (method 9, 220nm) > 96%. C₁₇H₁₉N₄O₇S[M-H]^-Calculated LRMS-ESI (m/z): 423.10. measured value: 422.95.

examples of the invention2

Preparation of keto-maytansinoids by direct esterification of maytansinol with keto acids

General procedure a: maytansinol (500mg, 0.88mmol, 1 equiv.) and the corresponding keto acid (3.52mmol, 4 equiv.) were dissolved in anhydrous DCM (40mL) in the presence of activated molecular sieves and cooled to 4 deg.C to 5 deg.C with an ice bath. To this solution was then added a solution of zinc (II) chloride in ether (2.64mL, 2.64mmol, 3.0 equiv., 1M solution) over 10 seconds. The resulting solution was stirred at 4 ℃ to 5 ℃ for 40 minutes, then N, N' -diisopropylcarbodiimide (0.55mL, 3.52mmol, 4 equiv.) was added. The mixture was stirred at 4 ℃ and then immersed in an ice bath overnight, slowly bringing it to room temperature. The conversion was monitored by LC-MS and after reaching 60%, the reaction mixture was concentrated to half the volume at 40 ℃ under reduced pressure, filtered through a 0.45 μm syringe filter (Macherey-Nagel,PTFE-O-45/25) and the filtrate was evaporated. The final product was assembled with pre-assembled SNAP ULTRA50g cylinders andHP-Sphere^TMpurification was performed on a Biotage Isolera One rapid purification system on spherical silica (linear gradient from 100% DCM to 90/10 DCM/methanol in 25 CV). The product-containing tubes were combined and dried under high vacuum for 1 hour on a rotary evaporator to give the corresponding ketone maytansinoid.

General procedure B: after activation of the molecular sieve (0.8g,

325 mesh particle size, Sigma Aldrich) maytansinol (758mg, 1.34mmol, 1.0 equiv), the corresponding keto acid (1.47mmol, 1.1 equiv) and DMAP (181mg, 1.47mmol, 1.1 equiv) were added under N₂Dissolved in anhydrous DCM (30mL) under atmosphere. The mixture was cooled to 4 ℃ over 10 minutes using an ice/water bath. A solution of EDC-HCl (283mg, 1.47mmol, 1.1 equiv.) in anhydrous DCM (15mL) was added to the cooled mixture over 30 minutes (ca. 10mg/min) and the reaction mixture was stirred at 4 ℃ for 2 hours. After this time, another portion of the keto acid (1.47mmol, 1.1 equiv) was added, then a solution of EDC-HCl (283mg, 1.47mmol, 1.1 equiv) in anhydrous DCM (10mL) was added to the cooled mixture over 30 minutes (about 10mg/min) and the reaction mixture was stirred under an inert atmosphere at 4 ℃ for 2 hours. After this time, the addition of the reagents was repeated again and the reaction mixture was stirred at 4 ℃ overnight, then allowed to gradually reach room temperature during this time. The mixture was filtered (Macherey-Nagel,PTFE-O-45/25) and the solvent was removed at 40 c using a rotary evaporator to a final volume of about 10 mL. Crude material utilization equipment pre-assembled SNAP ULTRA 100g cylinders and

HP-Sphere^TMpurification was performed on a Biotage IsoleraOne rapid purification system on spherical silica (linear gradient from 100% DCM to 90/10 DCM/methanol in 25 CV). The tubes containing the product were combined and the solvent was removed using a rotary evaporator to give a solid. Drying the solid under high vacuum to give the corresponding ketoneMaytansinoids.

Preparation of maytansinoid 2

From the reaction of maytansinol with 2- (4-acetyl-2-fluorophenyl) acetic acid, using method a: maytansinoid 2 was obtained as a pale yellow solid. Yield: and 47 percent. Purity by RP-HPLC, 220nm, 96%. C₃₈H₄₅ClFN₂O₁₀[M+H]⁺Calculated LRMS-ESI (m/z): 743.22. measured value: 743.25. c₃₈H₄₃ClFN₂O₁₀[M-H]^-Calculated LRMS-ESI (m/z): 741.22. measured value: 741.41.

by passing¹H NMR and¹³c NMR confirmed the structure:¹H NMR(400MHz，CDCl₃)7.73(dd，J＝7.9，1.6Hz，1H；C31-CH)，7.66(dd，J＝10.5，1.6Hz，1H；C27-CH)，7.46(t，J＝7.6Hz，1H；C32-CH)，6.82(d，J＝1.8Hz，1H；C17-CH)，6.59(d，J＝1.8Hz，1H；C21-CH)，6.48(dd，J＝15.5，11.0Hz，1H；C12-CH)，6.43(s，1H；C9-NH)，6.25(d，J＝10.9Hz，1H；C13-CH)，5.63(dd，J＝15.4，8.8Hz，1H；C11-CH)，4.99(dd，J＝11.8，2.7Hz，1H；C3-CH)，4.27(td，J＝11.2，10.4，1.8Hz，1H；C7-CH)，3.97(s，3H；C20-OCH₃)，3.90(d，J＝15.7Hz，1H；C24-CH₂)，3.76(d，J＝15.5Hz，1H；C24-CH₂)，3.54(d，J＝8.8Hz，1H；C10-CH)，3.46(d，J＝12.8Hz，1H；C15-CH₂)，3.38(s，3H；C10-OCH₃)，3.19(d，J＝12.8Hz，1H；C15-CH₂)，3.00(s，3H；C1-NCH₃)，2.87(d，J＝9.7Hz，1H；C5-CH)，2.57(s，3H；C29-CH₃)，2.51(dd，J＝14.1，11.9Hz，1H；C2-CH₂)，2.17(dd，J＝13.9，2.6Hz，1H；C2-CH₂)，1.72(d，J＝13.6Hz，1H；C8-CH₂)，1.68(s，3H；C14-CH₃)，1.50(m，1H；C6-CH)，1.28(d，J＝6.3Hz，4H；C6-CH₃，C8-CH₂)，0.87(d，J＝1.4Hz，3H；C4-CH₃)；¹³C NMR(101MHz，CDCl₃)196.50(C28)，168.86(C23)，168.30(C1)，160.78(d，¹J_C-F＝247.0Hz；C26)，156.13(C20)，152.46(C22)，142.53(C18)，140.30(C19)，140.24(C14)，138.49(d，³J_C-F＝6.4Hz；C30)，132.58(C12)，131.68(d，³J_C-F＝3.6Hz；C32)，128.32(C11)，126.38(d，²J_C-F＝16.1Hz；C25)，124.82(d，⁴J_C-F＝3.3Hz；C31)，124.56(C13)，122.04(C21)，119.52(C16)，114.73(d，²J_C-F＝23.2Hz；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)。

preparation of maytansinoid 3

From the reaction of maytansinol with 2- (3-acetylphenoxy) acetic acid, using method B: maytansinoid 3 was obtained as a pale yellow solid. Yield: 49 percent. Purity by RP-HPLC, 220nm, 98%. C₃₈H₄₆ClN₂O₁₁[M+H]⁺Calculated LRMS-ESI (m/z): 741.23. measured value: 741.23.

by passing¹H NMR and¹³c NMR confirmed the structure:¹H NMR(400MHz，CDCl₃)7.58(dt，J＝7.8，1.1Hz，1H；C30-CH)，7.55(s，1H；C26-CH)，7.44(d，J＝7.9Hz，1H；C31-CH)，7.12(dd，J＝8.3，2.8Hz，1H；C32-CH)，6.79(d，J＝1.8Hz，1H；C17-CH)，6.65(d，J＝1.8Hz，1H；C21-CH)，6.46(dd，J＝15.4，11.0Hz，1H；C12-CH)，6.37(s，1H；C9-NH)，6.26(d，J＝11.0Hz，1H；C13-CH)，5.62(dd，J＝15.4，8.9Hz，1H；C11-CH)，5.08(dd，J＝11.9，2.7Hz，1H；C3-CH)，4.90(d，J＝15.9Hz，1H；C24-CH₂)，4.66(d，J＝15.9Hz，1H；C24-CH₂)，4.28(t，J＝10.6Hz，1H；C7-CH)，3.96(s，3H；C20-OCH₃)，3.68(s，1H；C9-OH)，3.53(d，J＝8.9Hz，1H；C10-CH)，3.47(d，J＝12.9Hz，1H；C15-CH₂)，3.36(s，3H；C10-OCH₃)，3.15(d，J＝12.8Hz，1H；C15-CH₂)，2.92(d，J＝9.7Hz，1H；C5-CH)，2.87(s，3H；C1-NCH₃)，2.59(s，3H；C29-CH₃)，2.55(d，J＝11.9Hz，1H；C2-CH₂)，2.22-2.14(m，1H；C2-CH₂)，1.71(d，J＝1.8Hz，1H；C8-CH₂)，1.67(s，3H；C14-CH₃)，1.55-1.45(m，1H；C6-CH)，1.28(d，J＝6.4Hz，3H；C6-CH₃)，1.28-1.25(m，1H；C8-CH₂)，0.86(s，3H；C4-CH₃)；¹³C NMR(101MHz，CDCl₃)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(C10)，81.16(C9)，78.13(C3)，74.18(C7)，66.37(C24)，66.26(C5)，60.26(C4)，56.87(C20-OCH₃)，56.68(C10-OCH₃)，47.05(C15)，38.41(C6)，36.34(C8)，35.31(C1-NCH₃)，32.67(C2)，26.88(C29)，15.86(C14-CH₃)，14.58(C6-CH₃)，12.50(C4-CH₃)。

table 1: maytansinoids synthesized using methods A or B

Example 3

The keto-maytansinoid can be synthesized in three steps by esterification with Fmoc-protected amino acids, cleavage of Fmoc groups and condensation with keto acids.

General procedure C-step 1: maytansinol was reacted with Fmoc-protected amino acids. After the active molecular sieve (1g,325 mesh size, Sigma Aldrich) in the presence of N₂Under atmosphere maytansinol (565mg, 1.00mmol, 1.0 equiv.), Fmoc-protected amino acid (3.00mmol, 3.0 equiv.), DMAP (982mg, 8.00mmol, 8.0 equiv.), and scandium (III) trifluoromethanesulfonate (541mg, 1.1mmol, 1.1 equiv.) are dissolved in anhydrous DCM (10 mL.) after this time DIC (2.47mL, 16.0mmol, 16.0 equiv.) is added over 10 minutes (about 0.25mL/min) and the reaction mixture is stirred at 4 ℃ for 2 hours and allowed to reach room temperature gradually during this time the mixture is gravity filtered, the filtrate is diluted with DCM (50mL) and then washed with sodium phosphate buffer (50mL × 3, pH 7.5) and brine (50mL) followed by drying the organic layer with anhydrous sodium sulfate, gravity filtered, and the solvent is removed by rotary evaporation at 40 ℃ with approximately 10mL of SNAP, about 10mL of crude APs and final volume of ULTRA

HP-Sphere^TMPurification was performed on a Biotage Isolera One rapid purification system on spherical silica (linear gradient from 100% DCM to 90/10 DCM/methanol in 25 CV). The solvent was removed with a rotary evaporator at 40 ℃ for 2 hours to obtain the corresponding product as a light yellow to yellow solid.

General procedure C-step 2: deprotection of the Fmoc group. The Fmoc-protected intermediate (0.53mmol, 1.0 equiv.) was dissolved in DCM (5mL) and tris- (2-aminoethyl) amine (0.320mL, 2.13mmol, 4.0 equiv.) was added to this solution over 10 seconds. The reaction mixture was stirred at room temperature for 1 hour. The white precipitate formed was filtered off through a pad of celite, washed with DCM (20mL) and the yellow filtrate was evaporated to dryness at 40 ℃ for 2h with a rotary evaporator and further dried under high vacuum for 4 h to give the free amine which was used directly in the next step without further purification.

General procedure C-step 3: reacting the amine-maytansinoid with a keto acid. Free amine intermediate (77.0. mu. mol, 1.0 equiv), keto acid (150. mu. mol, 2.0 equiv), HATU (35mg, 94.0. mu. mol, 1.2 equiv), HOAt (13mg, 94.0. mu. mol, 1.2 equiv) and N-methylmorpholine (17. mu.L, 150. mu. mol, 2.0 equiv) in N₂Dissolve in anhydrous DMF (1mL) under atmosphere the mixture was stirred at room temperature overnight the reaction mixture was diluted with DCM (5mL) and washed with saturated ammonium chloride solution (5mL × 5) and brine (5mL) then the organic phase was dried over anhydrous sodium sulfate, filtered by gravity and the solvent removed with a rotary evaporator at 40 deg.CHP-Sphere^TMPurification was performed on a Biotage Isolera One rapid purification system of C1825 μm spherical silica (linear gradient from 80/20 water/MeCN to 100% MeCN in 20 CV). The product-containing fractions were combined, frozen in liquid nitrogen, and lyophilized for 24 hours to give the corresponding ketomaytansinoid.

Table 2: maytansinoids synthesized using method C (Steps 1-3)

Example 4

Preparation of maytansinoid 28

Maytansinol (56.5mg, 0.10mmol, 1.0 equiv.) was dissolved in anhydrous DCM (10mL) at room temperature under N₂A solution of zinc (II) chloride in ether (0.3mL, 0.30mmol, 3.0 equiv., 1M solution) was added under atmosphere and stirring was maintained for 10 minutes. 4-Acetylphenyl isocyanate (48.3mg, 0.30mmol, 3.0 equiv.) 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 (PDA220 nm). Volatiles were removed using a rotary evaporator at 40 ℃. Crude material was chromatographed by NP using a preloaded SNAP ULTRA10g cartridge andHP-Sphere^TMpurification was performed on a Biotage Isolera One rapid purification system on spherical silica (linear gradient from 100% DCM to 90/10 DCM/methanol in 13 CV) followed by RP chromatography using a pre-packed SNAP ULTRA C-1812 g cartridge andHP-Sphere^TMc1825 μm spherical silica was purified (linear gradient from 80/20 water/MeCN to 100% MeCN in 30 CV). The product-containing fractions were combined, frozen in liquid nitrogen, and lyophilized for 24 hours to give compound 28 as a white solid. Yield: 20mg (28%). Purity by RP-HPLC (220nm) > 95%. C₃₇H₄₅ClN₃O₁₀[M+H]⁺Calculated LRMS-ESI (m/z): 726.27. measured value: 726.25. c₃₇H₄₃ClN₃O₁₀[M-H]^-Calculated LRMS-ESI (m/z): 724.27. measured value: 724.43.

example 5

Preparation of maytansinoid 29

Synthesis of May-ONp: to a solution of maytansinol (88mg, 155. mu. mol, 1.0 eq) in DCM (8mL) was added pyridine (25. mu.L, 310. mu. mol, 2.0 eq) at room temperature. The resulting clear solution was stirred for 15 minutes and then cooledTo 4 ℃ and p-nitrophenyl chloroformate (219mg, 1.08mmol, 7.0 equiv.) was added to DCM (4mL) and a white precipitate formed immediately thereafter. The reaction mixture was stirred at room temperature for 24 hours. The crude material was concentrated using a rotary evaporator at 40 ℃ for 1 hour and passed through a Biotage Isolera One rapid purification system using a pre-loaded SNAP ULTRA 25g cartridge andHP-Sphere^TMthe spherical silica was purified (linear gradient from 100% ethyl acetate to 40/60 ethyl acetate/DCM in 50 CV). The product containing fractions were dried at 40 ℃ for 30 min with a rotary evaporator and further dried under high vacuum for 30 min to give intermediate May-ONp as a white solid. Yield: 102mg (90%). C₃₅H₄₁ClN₃O₁₂[M+H]⁺Calculated LRMS-ESI (m/z): 730.23. measured value: 730.01.

synthesis of maytansinoids 29: to a solution of compound May-ONp (3.7mg, 5.00. mu. mol, 1.0 equiv) in DCM (1mL) was added a solution of 4- (aminomethyl) acetophenone (1.1mg, 7.50. mu. mol, 1.5 equiv) in DCM/DMF (2: 0.1v/v) at room temperature and stirred for 5 min, then triethylamine (2. mu.L, 10.0. mu. mol, 2.0 equiv) was added. The reaction mixture was stirred at room temperature for 2 hours and at 60 ℃ overnight. The crude material was concentrated at 40 ℃ for 1 hour and passed through a Biotage Isolera One rapid purification system using a pre-loaded SNAP ULTRA10g cartridge andHP-Sphere^TMthe spherical silica was purified (linear gradient from 100% chloroform to 90/10 chloroform/methanol in 20 CV). The product containing fractions were dried at 40 ℃ for 30 minutes with a rotary evaporator and further dried under high vacuum for 30 minutes to give compound 29 as a white solid. Yield: 1mg (27%). Purity by RP-HPLC (220nm) > 95%. C₃₈H₄₆ClN₃O₁₀[M+H]⁺Calculated LRMS-ESI (m/z): 739.28. measured value: 739.96.

example 6

Preparation of albumin-binding maytansinoids

General procedure D for the synthesis of albumin-bound maytansinoids from ketone-maytansinoids and maleimide-hydrazide linkers: at room temperature under N₂To a stirred solution of the keto-maytansinoid (1.0 equiv) in anhydrous solvent (suitable solvents are DCM, DMSO, dioxane, 2-methyltetrahydrofuran) under an atmosphere was added a molecular sieve (1: 1 to 5: 1w/w) and a catalyst (TFA, p-toluenesulfonic acid, amberlyst-H form, amberlite-Na form), followed by a solution of hydrazide linker (1.0 to 5.0 equiv) in anhydrous DMSO. The reaction mixture was stirred at room temperature and conversion (> 90% conversion) was confirmed using HPLC analysis (PDA220 nm). The reaction mixture was filtered, concentrated at 30 ℃ on a rotary evaporator, and purified by Biotage Isolera One rapid purification system using a pre-loaded SNAP ULTRAHP-Sphere^TMThe spherical silica was chromatographed (linear gradient from 100% DCM to 90/10DCM/MeOH) and dried on a rotary evaporator at 30 deg.C for 30 minutes and then under high vacuum for another 30 minutes to give the free acid. The product is redissolved in a suitable solvent (methanol, acetone, 2-methyltetrahydrofuran or other organic polar solvent) and then neutralized with a salt solution (sodium, potassium, triethylammonium) until a pH in the range of 5.5 to 7.5 is reached. The solution was frozen in liquid nitrogen and lyophilized for 24 hours to give the compound as a salt. The content of counter ions is determined by IC. Sodium ion content ranges from 0.2 to 1.0 equivalents (about 0.2% to 0.9%), while triethylammonium ion content ranges from 0.8 to 2.0 equivalents (about 6% to 16%).

Preparation of Albumin-binding maytansinoids 30

From the reaction of maytansinoid 2 with linker 1: at room temperature under N₂Maytansinoid 2(170mg, 0.23mmol, 1.0 equiv), molecular sieve (0.2g, powder, activated,

325 mesh size) and(20mg, macropore, 30 to 60 mesh) in anhydrous DCM (2mL) was added a solution of linker 1(51mg, 0.12mmol, 2.0 equiv.) in anhydrous DMSO (0.6 mL). The reaction mixture was stirred at room temperature and after 3 hours HPLC analysis (PDA220nm) confirmed the reaction was complete (> 98% conversion). The reaction mixture was concentrated at 30 ℃ using a rotary evaporator, filtered through a 0.45 μm syringe filter (Macherey-Nagel,

PTFE-O-45/25). The filtrate was diluted with anhydrous DCM (8mL) and passed through Biotage Isolera One rapid purification system using a pre-packed SNAP ULTRA10g cartridge andHP-Sphere^TMthe spherical silica was purified (linear gradient from 100% DCM to 90/10DCM/MeOH in 22 CV). The combined product-containing fractions were dried on a rotary evaporator at 30 ℃ for 30 minutes and under high vacuum for a further 30 minutes to give the free acid form 30 as a white yellow solid. The solid was dissolved in MeOH/acetone (50: 50v/v, ca. 4mL total), which was followed by 5mM NaHCO₃Neutralization was carried out in a solution of microporous water until a pH of 6.8 to 7.1 (pH measured using a pH meter). The solution was frozen in liquid nitrogen and lyophilized for 24 hours to provide maytansinoid prodrug 30 as an off-white foam. Yield: 161mg (60%). C₅₅H₆₁ClFN₆O₁₆S[M-H]^-Calculated LRMS-ESI (m/z): 1147.36. measured value: 1147.84. na (Na)⁺The content of (b) is in the range of 0.2 to 0.8%.

Preparation of albumin-binding maytansinoid 31

From the reaction of maytansinoid 3 with linker 1 it follows: at room temperature under N₂Maytansinoid 3(186mg, 0.25mmol, 1.0 equiv), molecular sieve (0.4g, powder, activated,325 mesh size, SigmaAldrich) and(IR120 Na form, 484mg, 2.1mmol/mL, 4.0 equiv.) to a stirred solution in anhydrous dichloromethane (4mL) was added a solution of linker 1(329mg, 0.77mmol, 2.5 equiv.) in anhydrous DMSO (4 mL). The reaction mixture was stirred at room temperature and after 6 hours HPLC analysis (PDA220nm) confirmed the reaction was complete (> 98% conversion). The reaction mixture was concentrated at 30 ℃ using a rotary evaporator, filtered through a 0.45 μm syringe filter (Macherey-Nagel,

PTFE-O-45/25). The filtrate was neutralized with sodium hydroxide (318 μ L of 1M NaOH solution, 1.3 equivalents). The neutralized mixture was added dropwise to 50mL falcon tubes containing a mixture of cooled methyl tert-butyl ether (27mL) and isopropanol (14mL) (4 ℃ ice bath) and centrifuged at 4 ℃ for 5 minutes. The supernatant was decanted and the pellet resuspended in dichloromethane (10mL) and purified by Biotage Isolera One rapid purification system using a pre-loaded SNAP ULTRA10g cartridge and

HP-Sphere^TMthe spherical silica was purified (linear gradient from 100% DCM to 90/10DCM/MeOH in 22 CV). The combined product-containing fractions were dried at 30 ℃ for 30 minutes using a rotary evaporator and further dried under high vacuum for 10 hours to give31 as a white-yellow solid. Yield: 156mg (52%). Purity by RP-HPLC (220nm) > 95%. C₅₅H₆₂ClN₆O₁₇S[M-H]^-Calculated LRMS-ESI (m/z): 1145.36. measured value: 1145.87. the Na content has a window of 0.2 to 0.8%.

Table 3: albumin-binding maytansinoids synthesized using general procedure D

Examples of the invention7

Stability and release kinetics of maytansinoid-HSA conjugates in buffer solutions at pH4.0 and pH7.4

To prepare an HSA conjugate of albumin-bound maytansinoid, HSA (200. mu.M: 361.8. mu.L, 1078. mu.M free Cys 34; 100. mu.M: 180.9. mu.L, 1078. mu.M free Cys34) was diluted with PBS buffer (4mM sodium phosphate, 150mM NaCl, pH7.4) (200. mu.M: 678.2. mu.L; 100. mu.M: 859.1. mu.L) and DMSO (200. mu.M: 130.0. mu.L; 100. mu.M: 195.0. mu.L) and incubated in a heat block at 37 ℃ for 30 minutes. Albumin-bound maytansinoids were added to the pre-incubated HSA samples as a 2mM stock solution in DMSO (200. mu.M: 130.0. mu.L; 100. mu.M: 65.0. mu.L) to produce 200. mu.M or 100. mu.M maytansinoid prodrug solutions and 300. mu.M or 150. mu.M free Cys 34. The mixture at pH7.4 was reacted at 37 ℃ for 10 minutes and then analyzed by RP-HPLC (Phenomenex America WP XB-C18, 3.6 μm, 250X 4.6mm) once per hour for 24 hours.

To investigate the release kinetics at pH4.0, the mixture was acidified with 50mM sodium acetate buffer pH 3.0 (200. mu.M: 119.3. mu.L; 100. mu.M: 125.2. mu.L) and 1M HCl (200. mu.M: 12.7. mu.L; 100. mu.M: 6.8. mu.L) to reach pH 4.0. The mixture was then analyzed by RP-HPLC (Phenomenex Aeris WP XB-C18, 3.6 μm, 250X 4.6mm) once per hour for 24 hours.

The following RP-HPLC conditions were used: phenomenex Aeris WP XB-C183.6 μm, 250X 4.6 mm; eluent A (100% 20mM Tris buffer pH 8.0) and eluent B (90: 10, MeCN: water) were eluted with a gradient of eluent B (25% 0 to 0.5 min, 25 to 35% 0.5 to 2.5 min, 35 to 85% 2.5 to 16 min, 85 to 95% 16 to 17 min, 95% 17 to 20 min, 95 to 25% 20 to 25 min, 25% 25 to 30 min, flow rate 1.0 mL/min). The temperature of the column box is 37 ℃; autosampler temperature 37 ℃; the amount of the sample was 20. mu.L.

To quantify the percentage of free drug released, standard curves of free maytansinoids were prepared at different concentrations (200 μ M, 100 μ M, 50 μ M, 25 μ M and 12.5 μ M). The area under the curve (AUC) was determined to be 250 nm.

Table 4: kinetics of release of maytansinoid-HSA conjugates at pH4.0 and pH7.4

Measured value of 200. mu.M

Measurement at 100. mu.M

Example 8

Stability of maytansinoids and maytansinoid-HSA conjugates in CD1 mouse and human plasma: to investigate the stability of maytansinoids and maytansinoid-HSA conjugates in CD1 and human plasma, the compounds were incubated at 37 ℃ for 24 hours. The release of the remaining free maytansinoids or the corresponding maytansinoids was quantified by LC-MS/MS (or HPLC) at certain time points.

LC-MS quantification procedure: pooled CD1 mouse or human plasma (K)₂EDTA, innovative study) centrifugation (1 min, 12,044 g). First by filtrationNeedle (5 μm, sterile, Becton Dickinson) and then filter the supernatant through a cellulose acetate membrane (0.45 μm, sterile, Carl Roth). The filtered plasma (1710. mu.L) was incubated in a heat block for 30 minutes at 37 ℃. Free maytansinoids or albumin-bound maytansinoids were added to the pre-incubated plasma samples as 300 μ M stock solutions in DMSO (190 μ L) to give 30 μ M solutions of the corresponding maytansinoids or maytansinoid-HSA conjugates. The mixture was allowed to react at 37 ℃ for 10 minutes, then three samples (70 μ L) were placed into 96-well plates at each time point (0, 1, 2, 3, 4, 5, 21 and 24 hours), sealed with a plastic pad, immediately frozen with liquid nitrogen and stored at-20 ℃. At the end of the experiment, 96-well plates were thawed at room temperature. Samples (30. mu.L) were then transferred directly to 96-well Impact by multichannel pipettor^TMProtein precipitation plate () It was washed once with MeCN (150 μ L) and then loaded with internal standard (120 μ L, MeCN containing 5 μ g/mL maytansine). Impact^TMThe precipitate plate was sealed with a silica gel pad and shaken at 420rpm for 2 minutes. Then Impact^TMThe precipitation plate was placed on a 96-well sample manifold and the filtrate was collected onto another 96-well plate by applying a moderate vacuum. The 96-well plate was sealed with a silica gel pad and kept at room temperature until LC-MS/MS quantification. The filtrate was injected into LC-MS for quantification using MRM mode.

LC-MS method: phenomenexOmega Polar C18，1.6μm，

50 × 2.1.1 mm, column, eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in MeCN), elution with a gradient of eluent B (20% 0 to 0.5 min, flow rate 0.4 mL/min; 20 to 60% 0.5 to 9.0 min, 0.4 mL/min; 60 to 100% 9.0 to 9.5 min, flow rate 0.4 mL/min; 100 to 20% 9.5 to 10.5 min, flow rate 0.4 mL/min; 20% 105 to 12 minutes, 0.6 mL/min). The temperature of the column box is 25 ℃; the temperature of the automatic sample injector is room temperature; sample size of 10. mu.L.

The area under the curve (AUC) of each free drug at time point 0 was set as the 100% value of the corresponding prodrug. The AUC of each sample was normalized based on the AUC of maytansine.

HPLC quantitative procedure: samples were prepared as described before, before using a 2mM stock solution of the free drug and prodrug in DMSO. After incubation at 37 ℃, samples were injected directly into the HPLC every hour for 24 hours. The area under the curve (AUC) (200 μ M in 10% DMSO in PBS buffer) for each free maytansinoid was used as a 100% value for the corresponding albumin-bound maytansinoid.

HPLC method: phenomenex Aeris WP XB-C18, 3.6 μm, 250X 4.6mm, column; eluent a (20mm tris buffer pH 8.0) and eluent B (90: 10, MeCN: water) were eluted with a gradient of eluent B (25% 0 to 0.5 min, 25 to 35% 0.5 to 2.5 min, 35 to 85% 2.5 to 16 min, 85 to 95% 16 to 17 min, 95% 17 to 20 min, 95 to 25% 20 to 25 min, 25% 25 to 30 min, flow rate ═ 1.0 mL/min). The temperature of the column box is 37 ℃; autosampler temperature 37 ℃; the amount of the sample was 20. mu.L.

Table 5 below lists the relative release of free maytansinoids and conversion to maytansinol.

Table 5: relative release of free maytansinoids and/or maytansinol against maytansinoid-HSA conjugates in CD1 mouse and human plasma

Data obtained by HPLC

Table 6 below lists the amount of free maytansinoid remaining and the conversion to maytansinol.

Table 6: residual amounts of free maytansinoids and maytansinol in CD1 mouse and human serum albumin after 24 hours

The stability of the different linkers (maytansinoids 4) in CD1 murine plasma is depicted in figure 1.

Example 9

Kinetics of albumin binding of maytansinoids to albumin in pooled human whole blood and plasma: to study the binding kinetics of albumin-bound maytansinoids in pooled human plasma and pooled human whole blood, compounds were incubated with pooled human plasma at 37 ℃ and sampled at the indicated time points. The remaining albumin bound maytansinoids were quantified by HPLC.

HPLC quantitative procedure: to investigate the binding kinetics in human whole blood, blood (K) was used₂EDTA, 36 donors, Zen-Bio; 900 μ L) was preincubated in a heat block at 37 ℃ for 30 minutes. To study binding kinetics in pooled human plasma, pooled whole blood was centrifuged (10 min, 1811g), supernatant plasma was collected and then preincubated at 37 ℃ for 30 min.

Albumin-bound maytansinoids were added to pre-incubated whole blood/plasma samples diluted 10-fold in 1.2mM stock solution in 2.5% propylene glycol in 10mM sodium phosphate buffer and 1.48mM citrate (100 μ L) in PBS, yielding 12.0 μ M solutions of the corresponding albumin-bound maytansinoids. Samples (190 μ L) were taken after 15 seconds, 2 minutes, 4 minutes, 8 minutes and 15 minutes. Samples were added directly to 760 μ L of MeCN containing maytansine (5 μ g/mL) as an internal standard and vortexed for 1 min. After centrifugation (12,044g for 1 min), the supernatant (850 μ L) was concentrated under high vacuum. The residue was dissolved in DMSO/water (1: 1 v/v; 95. mu.L) and then analyzed by RP-HPLC. Percent binding was determined by comparing the area under the curve (AUC) at 220nm of albumin-bound maytansinoids relative to a control sample (100% value) in PBS buffer. All experiments were performed in triplicate.

HPLC method: phenomenex Kinetex Polar C18, 2.6 μm,

150 × 4.6.6 mM, eluent A (95: 55 mM ammonium acetate: MeCN) and eluent B (5: 955 mM ammonium acetate: MeCN), eluting with a gradient of eluent B (30% 0 to 0.5 min, 30 to 95% 0.5 to 9.0 min, 95% 11.0 min, 95 to 30% 11.0 to 14.5 min, 30% 15.0 min, flow rate 1.0 mL/min.) the ambient temperature of the column box, the autosampler temperature 4 ℃; 50. mu.L sample size.

The following table lists the relative amounts of albumin-bound maytansinoids at 0 and 8 minutes:

table 7: amounts of bound albumin-bound maytansinoids in pooled human whole blood and plasma after 15 seconds and 8 minutes

Example 10

Use of

Cell viability assay, in vitro cytotoxicity of maytansine, DM1, DM1-SMe, maytansinol and maytansinoids in different cell lines

The study used PromegaCell viability assay to test the in vitro efficacy of all compounds. 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), PAXF1657 (pancreatic cancer), MCF7 (breast cancer), COLO 205 (colon cancer), MDA-MB 231 (breast cancer), BT-474 (breast cancer) and Hep G2 (liver cancer).

Cells were harvested from exponential phase cultures, counted and plated in 96-well flat-bottomed microtiter plates at a cell density dependent on the growth rate of the cell line (4,000 and 60,000 cells). After a recovery period of 24 hours, the cells were allowed to resume exponential growth, then 10 μ Ι _ of medium (four control wells/plate) or medium containing the test compound was added.

Compounds were administered in duplicate at 10 concentrations in a semilog dilution procedure and cells were treated continuously for 96 hours. After treatment and incubation of the cells, 20. mu.L/well was addedAnd (3) a reagent. After incubation for up to 4 hours, Fluorescence (FU) was measured using an ensspire multiple label reader (excitation λ 531nm, emission λ 615 nm). Sigmoidal concentration response curves were fitted to the data points (T/C values) obtained for each cell line using a 4-parameter nonlinear curve fit (Oncotest ware Software). IC (integrated circuit)₅₀Values are reported as relative IC₅₀Values, which are the concentration of test compound (inflection point of the curve) that produces a response (inhibition of colony formation/viability) between the top plateau and the bottom plateau of the sigmoidal concentration response curve, or reported as absolute IC₅₀Values, which are the concentrations of the test compound at the intersection of the concentration-response curves at T/C-50%. To calculate the average IC₅₀Values, geometric means were used. The results presented as a mean graph or heat map (versus geometric mean IC) of all cell lines tested₅₀Single IC of value₅₀Value). See table 8 and fig. 2. FIG. 2 shows the IC of all tested compounds₅₀Heat map.

TABLE 8

Example 11

General procedure for the assessment of maytansine and albumin-binding maytansinoid compounds in patient-derived tumor xenograft models

Female immunodeficiency from Charles RiverNude mice with NMRI received unilateral tumor implantation under isoflurane anesthesia with human cancer tumor subcutaneously in the left flank until the cancer tumor was accessible and reached a volume of 80 to 200mm³Unless otherwise indicated.

Animals were housed in cages maintained at 25 + -1 deg.C and a relative humidity of 45 to 65% and the rate of aeration in the cages was 60 times per hour. Animals were left to light for 14 hours: a dark artificial photoperiod of 10 hours. Animals were fed autoclaved Teklad Global 19% protein extrusion feed (t.2019s.12) from Envigo RMSSARL and sterile filtered and acidified (pH 2.5) tap water was obtained, exchanged twice weekly. Feed and water were provided ad libitum. Prior to treatment, animals were randomly grouped (7 mice per group unless otherwise indicated) taking into account comparable median and mean values of tumor volumes in the groups. Animals were routinely monitored twice daily on weekdays, once daily on saturday and sunday. Animals were weighed twice weekly starting on day 0. Relative Body Weight (RBW) of individual animals absolute Body Weight (BW) of individual on day X_x) Divided by the individual body weight on the day of randomization times 100%. Tumor volume was determined on the day of randomization (day 0) and then twice weekly by two-dimensional measurement with calipers. Tumor volume was calculated according to the following equation: tumor volume [ mm³]＝1[mm]×w²[mm²]× 0.5.5, where "1" is the length of the tumor and "w" is the width of the tumor by comparing the absolute individual tumor volume of each tumor on day X (Tx) [ mm³]Relative volume of single tumor (RTVx) at day X was calculated by dividing by the absolute single tumor volume of the same tumor on the day of randomization times 100%. The schedule is applied to the extent permitted by animal welfare policies. At > 2000mm³Tumor volume (unilateral) of (c) terminated individual mice. All test compounds were administered on days 1, 8, 15 and 22 and were provided as lyophilized solids containing sucrose. On the day of treatment, the lyophilized samples were dissolved in 10mM sodium phosphate buffer pH 6.8 containing 20% propylene glycol and injected intravenously (100 μ L/20-g mouse) with vehicle (10mM 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 cancer cell line-derived xenograft models

Female immunodeficient NMRI nude mice from Yanvey, France received 5 × 10 from a buffer/matrigel (1: 1) subcutaneous transplant (unless otherwise indicated)⁶Cultured cancer cells (unless otherwise stated) until the tumor is accessible and has reached a volume of 80 to 200mm³(unless otherwise specified). Animals were housed in cages maintained at 22 + -1 deg.C and 50 + -10% relative humidity, with air exchange rates in the cages being 60 times per hour. Animals were left to light for 12 hours: a dark artificial photoperiod of 12 hours. By a gas coming fromThe animals were fed autoclaved ssnifffnm and sterile filtered and acidified (ph4.0) tap water was obtained, exchanged twice a week. Feed and water were provided ad libitum. Prior to treatment, animals were randomly grouped (7 mice per group unless otherwise indicated) taking into account comparable median and mean values of tumor volumes in the groups. Animals were routinely monitored twice daily on weekdays, once daily on saturday and sunday. The body weight of the mice was determined twice a week starting on day 0, and the average body weight of each group was correlated with the initial value (in percent) to calculate the Body Weight Change (BWC). Tumor volume was determined on the day of randomization (day 0), then two or three times a week, using two-dimensional measurements with calipers. Tumor volume was calculated according to the following equation:

tumor volume [ mm³]＝1[mm]×w²[mm²]× 0.5.5, where "1" is the length of the tumor and "w" is the width of the tumor by comparing the absolute individual tumor volume of each tumor on day X (Tx) [ mm³]Relative volume of single tumor (RTV) on day X was calculated by dividing by the absolute single tumor volume of the same tumor on the day of randomization times 100%_x). The schedule is applied to the extent permitted by animal welfare policies. At > 1500mm³Tumor volume (unilateral) of (c) terminated individual mice, or visible ulcers. All compounds tested are in the 1 st, 8 th,For 15 and 22 days and provided as a lyophilized solid containing sucrose. On the day of treatment, the lyophilized samples were dissolved in 10mM sodium phosphate buffer pH 6.8 containing 20% propylene glycol and injected intravenously (100 μ L/20-g mouse) with vehicle (10mM sodium phosphate buffer, 20% propylene glycol and 5% sucrose-pH 6.8).

Example 13

Assessment of maytansine and albumin-binding maytansinoids 30, 42, 31 and 35 in the human PDX renal cell carcinoma model RXF631

Maytansine and albumin binding compounds 30, 42, 31 and 35 in the renal cancer cell RXF631 model were evaluated according to the general procedure of a patient-derived xenograft model. When the tumor reaches 100mm³Begin treatment after the average size of (d). Fig. 3 shows tumor growth curves for the control group, maytansine group, and the groups treated with compounds 30, 42, 31, and 35. Fig. 4 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30, 42, 31 and 35.

Example 14

Assessment of maytansine and albumin-binding maytansinoids 32, 30 and 31 in the human PDX squamous cell lung carcinoma model LXFE 937

Maytansine and albumin binding compounds 32, 30 and 31 were evaluated in the squamous cell lung carcinoma LXFE 937 model according to the general procedure of a patient-derived xenograft model. Reach 117mm in tumor³Begin treatment after the average size of (d). Fig. 5 shows tumor growth curves for the control group, maytansine group, and the group treated with compounds 32, 30, and 31. Fig. 6 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 32, 30 and 31.

Example 15

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

Maytansine and albumin binding compounds 30 and 31 were evaluated in a squamous cell lung carcinoma LXFE 937 model according to the general procedure of a patient-derived xenograft model. Reaches 270mm in tumor³Average size ofAnd (5) starting treatment. Fig. 7 shows tumor growth curves for the control group, maytansine group, and the groups treated with compounds 30 and 31. Fig. 8 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30 and 31.

Example 16

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

Maytansine and albumin binding compounds 30 and 31 were evaluated in a lung adenocarcinoma LXFA 737 model according to the general procedure of a patient-derived xenograft model. When the tumor reaches 311mm³Begin treatment after the average size of (d). Fig. 9 shows tumor growth curves for the control group, maytansine group, and the groups treated with compounds 30 and 31. Fig. 10 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30 and 31.

Example 17

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

Maytansine and albumin binding compounds 32, 30 and 31 were evaluated in the MDA-MB 231 breast cancer model according to the general procedure of a cancer cell line-derived xenograft model. When the tumor reaches 80mm³Begin treatment after the average size of (d). Fig. 11 shows tumor growth curves for the control group, maytansine group, and the group treated with compounds 32, 30, and 31. Fig. 12 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 32, 30 and 31.

Example 18

Assessment of maytansine and albumin-binding maytansinoids 30 and 31 in the human xenograft ovarian cancer model a2780

Maytansine and albumin binding compounds 30 and 31 in the ovarian cancer a2780 model were evaluated according to the general procedure for the ovarian cancer cell line xenograft model. Reaches 380mm in tumor³Begin treatment after the average size of (d). Fig. 13 shows tumor growth curves for the control group, maytansine group, and the groups treated with compounds 30 and 31. FIG. 14 shows control group and AmericanBody weight change curves for the dendin group and the groups treated with compounds 30 and 31.

Example 19

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

Maytansine and albumin binding compounds 30 and 31 in breast cancer MDA-MB 468 were evaluated according to the general procedure of a cancer cell line-derived xenograft model. Reach 94mm in tumor³Begin treatment after the average size of (d). Fig. 15 shows tumor growth curves for the control group, maytansine group, and the group treated with compounds 30 and 31. Fig. 16 shows the body weight change curves of the control group, maytansine group and the group treated with compounds 30 and 31.

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