RU2795101C2 - Albumin-binding prodrugs based on auristatin e derivatives - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: albumin-binding prodrugs derived from auristatin E, which can be used for the treatment of malignant diseases.

EFFECT: reduction in cytotoxicity compared to an equivalent dose of the unmodified active agent.

18 cl, 25 dwg, 5 ex

Description

[0001 ] This application claims priority from U.S. Provisional Application No. 62/592,721, filed Nov. 30, 2017, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002 ] Small molecule anticancer drugs often have a narrow therapeutic window ("therapeutic window"), which limits their clinical efficacy. These low molecular weight compounds show a high tendency to penetrate into the tissues of the body by diffusion, which leads to a uniform biodistribution. As a consequence, only small amounts of the drug reach the desired site of action, and as a result of penetration into healthy tissues of the body, these drugs cause significant side effects.

[0003 ] These disadvantages are especially significant for those drugs that have high cytotoxicity and have a very small breadth of therapeutic effects. Auristatins are drugs based on tubulin-binding peptides, and typical representatives, such as dolastatin 10, dolastatin 15, auristatin PE, auristatin E or auristatin F, show significant cytotoxic effects. In phase I and II clinical studies, dolastatin 10, dolastatin 15, and auristatin PE showed unacceptable systemic toxicity, while their antitumor activity was absent, so further studies were discontinued (EA Perez et al., Invest. New Drugs, 23:257- 261 (2005); M. von Mehren et al., Sarcoma, 8:107-111 (2004); RS Marks et al., Am. J. Clin. Oncol., 26:336-337 (2003)).

[0004 ] Drug delivery in oncology is an approach that can potentially increase the low therapeutic index of highly cytotoxic agents. In most drug delivery systems, the cytotoxic drug is bound to the carrier via a spacer that includes a predetermined breakpoint to allow the bound drug to be released at the cellular target site (F. Kratz et al., ChemMedChem, 3:20-53 (2008)).

[0005 ] Albumin or its drug conjugates have a markedly long half-life in the systemic circulation of up to 19 days. A particularly promising approach is the development of prodrugs of highly cytotoxic agents that covalently bind to circulating serum albumin, which serves as a macromolecular carrier upon administration. Due to 1.) increased permeability of macromolecules through the walls of vessels of malignant, infected or inflamed tissue in combination with an intact lymphatic drainage system and 2.) expression of albumin-binding proteins on the tumor endothelium and in the tumor interstitium, albumin-drug conjugates transport a therapeutically effective substance to target site (i.e. tumor) where a highly cytotoxic agent is released (US 7387771; F. Kratz, J. Control. Release, 132:171-183 (2008); F. Kratz, U. Beyer, Drug Delivery, 5 :281-299 (1998)).

[0006 ] However, when designing drug delivery systems with highly cytotoxic agents, a critical balance must be struck between maintaining the targeting properties of the drug carrier while providing controlled release of the cytotoxic drug and preventing its premature release into the bloodstream or systemically. Acid-sensitive drug delivery systems must be sufficiently stable in the bloodstream and at the same time provide effective drug release at the tumor site depending on pH (F. Kratz et al., ChemMedChem, 3: 20-53 (2008)).

[0007 ] For highly cytotoxic drugs with IC ₅₀ values against tumor cells in the picomolar range, such as the small molecular weight peptide class of auristatins, which cannot be administered due to their water insolubility and very narrow therapeutic window, there is a need for efficient delivery systems. and drug release to achieve more efficient and controlled delivery and release of such potent agents. In consequence of the foregoing, the present invention provides more effective and/or better tolerated pharmaceutical compositions of albumin-binding prodrugs based on auristatin E derivatives, which can be used for the treatment of malignant diseases.

DISCLOSURE OF THE INVENTION

[0008 ] The present invention provides a compound having the structure described by formula I or II:

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

wherein:

R' is H or -CH ₃ ,

M is H or a pharmaceutically acceptable counterion;

Y is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -C(O)-O - And

-O-C(O)-;

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

X is H or selected from the group consisting of halogen (for example, -F, -Cl, -Br or -I), -NO ₂ , -NR ² R ³ , -OR ² , -NHCOR ² and -OCOR ² where R ² and R ³ are each independently selected from H and C ₁ -C ₄ alkyl;

TBG is a thiol-binding group selected from the following: an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, and an optionally substituted acetylene group.

[0009 ] In some embodiments, R' is -CH ₃ . In other embodiments, R' is H.

[0010 ] In some embodiments, TBG is selected from an optionally substituted maleimide group.

[0011 ] In some embodiments, TBG is a maleimide group having the formula:

.

.

[0012 ] In some embodiments, Y is -NH-C(O)-.

[0013 ] In some embodiments, M is H ⁺ or Na ⁺ .

[0014 ] In some embodiments, R ¹ is optionally substituted with C ₁ -C ₅ alkyl. In other embodiments, R ¹ is C ₁ -C ₅ alkyl.

[0015 ] In some embodiments, the invention relates to a compound having the structure described by formula III:

[0016 ] In other embodiments, the present invention relates to a compound having the structure described by formula IV:

[0017 ] The present invention also relates to a pharmaceutical composition containing the compound provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of one or more of the following: a solubilizing agent, an encapsulating agent, and a lyoprotectant. In other embodiments, the pharmaceutically acceptable carrier comprises one or more of the following: dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and trimethyl-β-cyclodextrin.

[0018 ] In some embodiments, the pharmaceutical composition is for intravenous administration. In some embodiments, the composition, when administered intravenously to a patient, covalently and selectively rapidly binds in situ to endogenous albumin in the bloodstream. In other embodiments, the composition, when administered intravenously to a patient, covalently and selectively rapidly binds in situ to the cysteine-34 thiol group of endogenous albumin in the circulation.

[0019 ] The present invention also provides a method of treating a patient suffering from a disease selected from cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, and other microorganisms, comprising administering to a patient in need thereof therapeutically an effective amount of the compound or pharmaceutical composition in the manner described herein. In some embodiments, the disease is cancer and is selected from the following: carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, and melanoma. In some embodiments, the route of administration is intravenous.

[0020 ] The present invention also relates to a method for reducing the cytotoxicity of a compound, comprising administering to a patient in need thereof a compound or pharmaceutical composition as described herein, wherein the administration results in a reduction in cytotoxicity compared to an equivalent dose of the unmodified active agent.

[0021 ] The present invention further provides a method of increasing the concentration of a metabolite of a compound in a tumor, comprising administering a compound or pharmaceutical composition to a patient in need thereof as described herein, wherein the increase is compared to an equivalent dose of the unmodified active agent.

[0022 ] The present invention relates to a compound as provided herein for use as a medicine.

[0023 ] The present invention also provides a compound as described herein for use in the treatment of a disease or condition selected from the group consisting of cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms.

[0024 ] The present invention further provides the use of a compound or composition described herein for the preparation of a medicament for the treatment of a disease or condition selected from the following: cancer, viral disease, autoimmune disease, acute or chronic inflammatory disease, and disease caused by bacteria, fungi or other microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025 ] In FIG. 1 shows a comparison of the stability of AE-Keto-Sulf07 (panel (a)) and AE-Keto-EMCH (panel (b)) in recovery buffer (50 mM sodium phosphate buffer pH 7.65, 5% sucrose (w/v ) and 2% 2-hydroxypropyl-β-cyclodextrin (2-HPβCD (w/v)).

[0026 ] In FIG. 2 shows a comparison of the stability of AE-Ester-Sulf07 (panel (a)) and AE-Ester-EMCH (panel (b)) in recovery buffer (50 mM sodium phosphate buffer, pH 7.65, 5% sucrose (w/ v) and 4% 2-hydroxypropyl-β-cyclodextrin (2-HPβCD, (w/v)).

[0027 ] In FIG. 3 shows the conjugation of AE-Keto-Sulf07 to albumin in human plasma.

[0028 ] In FIG. 4 shows the conjugation of AE-Ester-Sulf07 to albumin in human plasma.

[0029 ] In FIG. 5 shows the kinetics of AE-Keto-Sulf07 binding to albumin in human plasma.

[0030 ] In FIG. 6 shows the kinetics of AE-Keto-Sulf07 binding to albumin in mouse plasma.

[0031 ] In FIG. 7 shows the kinetics of AE-Keto-Sulf07 binding to albumin in rat plasma.

[0032 ] In FIG. 8 shows the binding kinetics of AE-Ester-Sulf07 to albumin in human plasma.

[0033 ] In FIG. 9 shows the kinetics of binding of AE-Ester-Sulf07 to albumin in mouse plasma up to the limit of quantitation (LOQ) for the drug.

[0034 ] In FIG. 10 shows the kinetics of binding of AE-Ester-Sulf07 to albumin in rat plasma before reaching the drug LOQ.

[0035 ] In FIG. 11 shows the pH dependent release of AE-Keto (pH 7.4 (panel (a)) and pH 4.1 (panel (b)) from human albumin-bound AE-Keto-Sulf07.

[0036 ] In FIG. 12 shows the pH dependent release of AE-Ester (pH 7.4 (panel (a)) and pH 4.1 (panel (b)) from human albumin-bound AE-Ester-Sulf07.

[0037 ] In FIG. 13 shows the antitumor effect of auristatin E and AE-Keto-Sulf07 compared with the control group in the A375 malignant melanoma cancer model, mean median initial tumor volume ~134 mm ³ .

[0038 ] In FIG. 14 shows the antitumor effect of auristatin E and AE-Keto-Sulf07 compared with the control group in the A375 malignant melanoma cancer model, mean median initial tumor volume ~332 mm ³ .

[0039 ] In FIG. 15 shows the antitumor effect of auristatin E and AE-Keto-Sulf07 compared to the control group in the NSCLC LXFA737 xenograft model, mean median initial tumor volume ~132 mm ³ .

[0040 ] In FIG. 16 shows the antitumor effect of auristatin E and AE-Keto-Sulf07 compared with the control group in the NSCLC LXFA737 xenograft model, mean median initial tumor volume ~330 mm ³ .

[0041 ] In FIG. 17 shows the antitumor effect of auristatin E and AE-Keto-Sulf07 compared with the control group in the A2780 human ovarian carcinoma model, mean median initial tumor volume ~148 mm ³ .

[0042 ] In FIG. 18 shows the antitumor effect of auristatin E and AE-Keto-Sulf07 compared with the control group in the A2780 human ovarian carcinoma model, mean median initial tumor volume ~351 mm ³ .

[0043 ] In FIG. 19 shows the antitumor effect of auristatin E and AE-Ester-Sulf07 compared to the control group in the RXF631 renal cell carcinoma cancer model, initial tumor volume ~140 mm ³ .

[0044 ] In FIG. 20 shows the antitumor effect of auristatin E and AE-Ester-Sulf07 compared with the control group in the A375 malignant melanoma cancer model, mean median initial tumor volume ~332 mm ³ .

[0045 ] In FIG. 21 shows the antitumor effect of auristatin E and AE-Ester-Sulf07 compared to the control group in the NSCLC LXFA737 xenograft model, mean median initial tumor volume ~132 mm ³ .

[0046 ] In FIG. 22 shows the antitumor effect of auristatin E and AE-Ester-Sulf07 compared with the control group in the A2780 human ovarian carcinoma model, initial tumor volume ~351 mm ³ .

[0047 ] In FIG. 23 shows the antitumor effect of AE-Ester-Sulf07 compared to the control group in the A2780 human ovarian carcinoma model, initial tumor volume ~148 mm ³ .

[0048 ] In FIG. 24 shows accelerated degradation of lyophilized auristatin formulations to compare the stability of AE-Keto-Sulf07 and AE-Keto-EMCH.

[0049 ] In FIG. 25 shows accelerated degradation of lyophilized auristatin formulations to compare the stability of AE-Ester-Sulf07 and AE-Ester-EMCH.

DETAILED DESCRIPTION OF THE INVENTION

[0050 ] Unless otherwise indicated, scientific and technical terms used in this application have meanings that are commonly understood by those skilled in the art. Generally, the nomenclature relating to methods of chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein chemistry described herein is well known and widely used in the art.

[0051 ] All publications, patents and published patent applications mentioned in this application are expressly incorporated herein by reference. In the event of a conflict, the present description, including its specific definitions, will govern. Unless otherwise indicated, it is to be understood that each embodiment presented herein may be used alone or in combination with any one or more of the other embodiments presented herein.

Definitions

[0052 ] As used herein, the word "comprise" or variations thereof such as "comprises" or "comprising" should be understood to mean the inclusion of a specified integer (or components) or group of integers (or components), but not the exclusion of any or another integer (or components) or a group of integers (or components).

[0053 ] Throughout this application, where a compound or composition is described as having, including, or containing specific components, it is contemplated that such compound or composition may also consist essentially of or consist of the recited components. Similarly, when methods or processes are described as having, including, or containing specific process steps, those processes may also consist essentially of, or consist of, the listed process steps. In addition, it should be understood that the order of the steps or the order in which certain actions are performed is irrelevant as long as the compounds, compositions, and methods described herein remain usable. In addition, two or more steps or actions may be performed at the same time.

[0054 ] Forms in the singular include the plural, unless the context clearly requires otherwise.

[0055 ] The term "including" has the meaning "including without limitation". The terms "including" and "including without limitation" are used interchangeably.

[0056 ] As used herein, the term "or" should be understood to mean "and/or" unless the context clearly indicates otherwise.

[0057 ] The terms "drug", "agent", "therapeutic agent", "therapeutically active agent", "cytotoxic agent or drug", "highly cytotoxic agent or drug", or "therapeutically effective substance" are used to refer to any compound, which produces a pharmacological effect by itself or after its transformation in the organism in question, and thus also includes derivatives of these transformations. The pharmacological effect of the drugs of the composition according to the description of the present invention can be only a single one, for example, a cytostatic and/or cytotoxic effect, or a wide pharmacological spectrum of action, such as an immunosuppressive and anti-inflammatory effect at the same time.

[0058] The terms "patient", "subject", or "individual" are used interchangeably and refer to a non-human human or animal. These terms include mammals such as humans, primates, livestock (eg, cattle, porcine), pets (eg, canines, felines), and rodents (eg, mice and rats). In certain embodiments, the patient or subject is a human having a condition in need of treatment.

[0059 ] The term "pharmaceutical composition" refers to a composition intended for pharmaceutical use in a living subject, including humans and mammals, for example, in combination with one or more pharmaceutically acceptable carriers, excipients or diluents. Such a composition may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, protectants, and other materials well known in the art. In some embodiments, the pharmaceutical composition includes a composition containing the active ingredient(s) and the inert ingredient(s) that constitute the excipient, carrier or diluent, as well as any product that results directly or indirectly from the combination, complexation or aggregation of any two or more ingredients, or as a result of dissociation of one or more ingredients, or as a result of other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical compositions of the present invention include any composition obtained by mixing a compound of the present invention and one or more pharmaceutically acceptable excipient(s), carrier(s) and/or diluent(s).

[0060 ] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that can be administered to a patient along with a therapeutically effective substance provided herein, and which does not inhibit the pharmacological activity of the agent. The term "filler" refers to an additive in a formulation or composition that is not a pharmaceutically active ingredient. In certain embodiments, the "pharmaceutically acceptable" substance is for use in contact with cells, tissues, or organs of animals or humans without undue toxicity, irritation, allergic response, immunogenicity, or other undesirable reactions in the amount used in the dosage form according to the dosage regimen and commensurate with reasonable benefit/risk ratio. In some embodiments, the "pharmaceutically acceptable" substance that is a component of the pharmaceutical composition is also compatible with the other ingredient(s) of the composition. In some embodiments, the terms "pharmaceutically acceptable excipient", "pharmaceutically acceptable carrier", and "pharmaceutically acceptable diluent" encompass, without limitation, pharmaceutically acceptable inactive ingredients, materials, compositions, and carriers such as liquid excipients, solid excipients, diluents, excipients, carriers, solvents and sealing materials. Carriers, diluents and excipients also include all pharmaceutically acceptable dispersion media, coatings, buffers, isotonic agents, stabilizers, absorption retarding agents, antimicrobial agents, antibacterial agents, antifungal agents, adjuvants, and the like. Except where any conventional excipients, carriers or diluents incompatible with the active ingredient are used, the scope of the present invention includes the use of conventional pharmaceutical excipients, carriers and diluents in pharmaceutical compositions. See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania, 2005); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); and Pharmaceutical Preformulation and Formulation, Gibson, Ed., CRC Press LLC (Boca Raton, Florida, 2004).

[0061 ] The terms "pharmaceutically effective amount", "therapeutically effective amount", or "therapeutically effective dose" refer to an amount effective to treat a disease in a patient, for example, to effect a beneficial and/or desirable change in the general health of a patient suffering from a disease (eg, cancer), treatment, healing, suppression or attenuation of a physiological response or condition, and so on. The full therapeutic effect does not necessarily occur with the introduction of a single dose and may occur only after a series of doses. Thus, a therapeutically effective amount can be delivered in one or more administrations. The precise effective amount required for a subject will depend, for example, on body size, the condition and age of the subject, the nature and extent of the disease, the therapeutic agents or combination of therapeutic agents chosen to be administered, and the route of administration. One skilled in the art will readily be able to determine the effective amount for a given situation by routine experimentation. One of skill in the art will appreciate that treating cancer includes, without limitation, killing cancer cells, preventing the growth of new cancer cells, initiating tumor regression (reducing tumor size), inducing a reduction in metastasis, improving a patient's vital functions, improving a patient's well-being, reducing pain, improvement in appetite, improvement in patient weight, and any combination thereof. The terms "pharmaceutically effective amount", "therapeutically effective amount" or "therapeutically effective dose" also refer to the amount necessary to improve the clinical symptoms of the patient. Therapeutic methods or methods of treating cancer described herein should not be construed or otherwise limited to the term "treatment" of cancer.

[0062 ] As used herein, the term "treatment" or "therapy" includes the change, reduction or disappearance of the symptoms, clinical signs and pathological processes that cause the condition of the patient, in order to improve or stabilize the condition of the subject. As used herein and well understood by those skilled in the art, the term "treatment" refers to a method of obtaining favorable or optimal results, including clinical results. Beneficial or optimal clinical outcomes may include, but are not limited to, reduction or resolution of one or more symptoms, improvement or slowing of the progression of one or more conditions associated with a disease, such as cancer, reduction in the extent of a disease, a stabilized (i.e., no worsening) condition disease, delaying or slowing down the progression of the disease, improvement or alleviation of the condition of the disease and remission (partial or complete), detectable or not detectable. The term "treatment" can also mean an increase in survival compared to expected survival if no treatment is given. Exemplary favorable clinical results are described in this document.

[0063 ] The "administration" or "use" of a substance, compound, or agent to a subject may be accomplished using one of many methods known to those skilled in the art. For example, a compound or agent can be administered intravenously, intra-arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebral, and transdermally (by absorption, for example, through the skin ducts ). The compound or agent may also be appropriately administered using rechargeable or biodegradable polymeric devices or other devices such as patches and pumps or formulations that provide sustained, slow or controlled release of the compound or agent. The administration may also be performed, for example, once, multiple times, and/or over one or more extended periods. In some aspects, administration includes both direct administration, including self-administration, and indirect administration, including administration of a drug. For example, as set forth herein, a physician who instructs a patient to self-administer a drug, or directs another person to administer a drug to a patient, and/or provides the patient with a prescription for a drug, is administering the drug to the patient. When the method is part of a treatment regimen that includes more than one agent or method of treatment, the present invention contemplates that the agents may be administered at the same or different times and by the same or different routes of administration. Acceptable routes of administering a substance, compound, or agent to a subject will also depend on, for example, the age of the subject, whether the subject is active or inactive at the time of administration, whether the subject has impaired perception during administration, the degree of impairment, and the chemical and biological properties of the compound. or agent (eg, solubility, digestibility, bioavailability, stability, and toxicity).

[0064 ] The term "substituted" refers to moieties having substituents replacing hydrogen on one or more carbon atoms of the backbone of a chemical compound. One skilled in the art will appreciate that the terms "substitution" or "substituted" include the optional condition that such substitution corresponds to the allowable valency of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., one that is not subject to spontaneous transformation, such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible organic substituents. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and straight, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Allowable substituents may be one or more and the same or different for the respective organic compounds. In the description of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any valid substituents of the organic compounds described herein, which correspond to the valency of the heteroatoms. Substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate , phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aromatic (for example, C ₆ -C ₁₂ aryl) or heteroaromatic (for example , heteroaryl) groups.

[0065 ] The terms "optional(s)" or "optional" mean that the circumstance described subsequently occurring may or may not occur, thus this application includes cases where that circumstance occurs and cases where it does not. happening. 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 in which a non-hydrogen substituent is present and structures in which a non-hydrogen substituent is not present.

[0066 ] Unless the term "unsubstituted" is specifically indicated, this means that references herein to chemical groups include substituted variants. For example, reference to an "alkyl" group or moiety is implied to include both substituted and unsubstituted variants. Examples of substituents on chemical groups include, without limitation, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aryl or heteroaryl groups.

[0067 ] The term "aryl" means an aromatic carbon ring having the specified number of carbon atoms in the ring, for example 6 to 12 or 6 to 10 carbon atoms. Aryl groups may be monocyclic or polycyclic (eg bicyclic, tricyclic). In some instances, both rings of a polycyclic aryl group are aromatic (eg, naphthyl). In other instances, polycyclic aryl groups may include a non-aromatic ring (eg, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl) fused to an aromatic ring, provided that the polycyclic aryl group is bonded to the parent structure via an atom on the aromatic ring. Thus, the 1,2,3,4-tetrahydronaphthalene-5-yl group (in which the fragment is linked to the parent structure via an aromatic carbon atom) is considered an aryl group, while 1,2,3,4-tetrahydronaphthalene-1- yl (in which the moiety is linked to the parent structure through a non-aromatic carbon atom) is not considered an aryl group. Similarly, the 1,2,3,4-tetrahydroquinolin-8-yl group (in which the moiety is linked to the parent structure through an aromatic carbon atom) is considered an aryl group, while 1,2,3,4-tetrahydroquinolin-1- an yl group (in which the moiety is linked to the parent structure through a non-aromatic nitrogen atom) is not considered an aryl group. However, the term "aryl" is not encompassed or overlapped by the term "heteroaryl" as defined herein, regardless of the point of attachment (eg, both quinolin-5-yl and quinolin-2-yl are heteroaryl groups).

[0068 ] The term "heteroaryl" means an aromatic ring containing the specified number of ring atoms (for example, 5-12 or 5-10 membered heteroaryl), and consisting of one or more heteroatoms (for example, 1, 2, 3 or 4 heteroatoms) selected from N, O and S, and the rest of the ring atoms being carbon atoms. 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 a heteroaryl group is no more than 2. In some embodiments, the total number of S and O atoms in a heteroaryl group is no more than 1. Unless otherwise indicated , heteroaryl groups may be bonded to the base structure by a carbon or nitrogen atom if valence permits. For example, the term "pyridyl" includes 2-pyridyl, 3-pyridyl, and 4-pyridyl groups, and the term "pyrrolyl" includes 1-pyrrolyl, 2-pyrrolyl, and 3-pyrrolyl groups. If nitrogen is present in the heteroaryl ring, it may, if the nature of the neighboring atoms and groups permit, exist in an oxidized state (ie, N ⁺ -O ^- ). In addition, if sulfur is present in the heteroaryl ring, it may, if the nature of the neighboring atoms and groups permits, exist in an oxidized state (ie, S ⁺ -O ^- or SO ₂ ). Heteroaryl groups may be monocyclic or polycyclic (eg bicyclic, tricyclic).

[0069 ] In some instances, the heteroaryl group is monocyclic. Examples include pyrrole, pyrazole, imidazole, triazole (e.g. 1,2,3-triazole, 1,2,4-triazole, 1,3,4-triazole), tetrazole, furan, isoxazole, oxazole, oxadiazole (e.g. 1 , 2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole), thiophene, isothiazole, thiazole, thiadiazole (e.g. 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole), pyridine, pyridazine, pyrimidine, pyrazine, triazine (eg 1,2,4-triazine, 1,3,5-triazine) and tetrazine.

[0070 ] The term "acyl" is known in the art and refers to the group represented by the general formula hydrocarbyl-C(O)-, for example,

alkyl-C(O)-.

[0071 ] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups and branched chain alkyl groups. In some embodiments, a straight or branched alkyl has 30 or fewer carbon atoms in its backbone (e.g., C ₁ -C ₃₀ for straight chains, C ₄ -C ₃₀ for branched chains), and in other embodiments, 20 or less . In some embodiments, the alkyl groups are lower alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, and n-pentyl. In addition, the term "alkyl" as used in the description, examples and claims is used to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl groups having substituents replacing hydrogen on one or more carbon atoms of the hydrocarbon backbone. In some embodiments, straight or branched alkyl has 30 or fewer carbon atoms in its backbone (eg, C ₁ -C ₃₀ for straight chains, C ₃ -C ₃₀ for branched chains). In some embodiments, the chain has ten or fewer carbon atoms (C ₁ -C ₁₀ ) in its backbone. In other embodiments, the chain has six or fewer carbon atoms (C ₁ -C ₆ ) in its backbone.

[0072 ] The terms "hydrazone moiety" or "hydrazone" refer to E- and/or Z-isomers of hydrazones, for example,

или

.

or

.

The stereochemistry of the hydrazone moiety may include E or Z isomers. The term "hydrazone" as used herein includes both the E and Z isomers. The hydrazone moieties presented herein are generally depicted in one configuration, but it is understood that the description herein may include both the E and/or Z isomers.

[0073 ] In various paragraphs of the description of the present invention, the substituents of the compounds of the invention are described in groups or in ranges. In particular, the description is intended to include every single sub-combination of members of such groups and ranges. For example, the term "C ₁ -C ₆ alkyl" is specifically intended to describe individually methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, etc.

[0074] The term "pharmaceutically acceptable salt" means a salt of a compound intended for pharmaceutical use, including, without limitation, metal salts (e.g., sodium, potassium, magnesium, calcium, etc.), acid addition salts (e.g., mineral acids, carboxylic acids, etc.) and base addition salts (eg, ammonia, organic amines, etc.). An acid addition salt of a compound which occurs in its free base form can be prepared by treating said free base form with an appropriate acid, such as an inorganic acid, for example hydrohalic, including hydrochloric or hydrobromic, sulfuric, nitric. phosphoric acid and the like; or such as an organic acid such as acetic, hydroxyacetic, propionic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclic, salicylic, p-aminosalicylic, pamoic and the like (see, for example, application WO 01/062726. Some pharmaceutically acceptable salts listed by Berge et al., J. Pharm. Sci., 66: 1-19 (1977) are incorporated herein in their entirety via link). Compounds containing acidic protons can be converted to their therapeutically active, non-toxic base addition salt form, such as metal or amine salts, by treatment with appropriate organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal or ion salts, for example lithium, sodium, potassium, magnesium, calcium and the like salts, organic base salts, for example N-methyl-D-glucamine, hydrabamine, salts of amino acids such as, for example, arginine, lysine and the like. Conversely, said salt forms can be converted to free forms by treatment with an appropriate base or acid. The compounds and their salts may be in the form of solvates, which is within the scope of the present invention. Such solvates include, for example, hydrates, alcoholates, and the like (see, for example, WO 01/062726).

[0075 ] The present invention also provides pharmaceutical compositions comprising one or more compounds of the invention together with a pharmaceutically acceptable carrier or excipient. The compounds or pharmaceutical compositions of the invention may be used in vitro or in vivo.

[0076 ] As used herein, the term "isomer" includes, without limitation, tautomers, cis and trans isomers (E (entgegen), Z (zusammen)), R and S enantiomers (referred to as R and S are used in according to the rules described in Pure Appl Chem (1976), 45, 11-30), diastereomers, (D)-isomers, (L)-isomers, stereoisomers, respectively, their racemic mixtures and, respectively, other mixtures. All such isomers, as well as mixtures thereof, are intended to be included within the scope of this invention. Tautomers, although not explicitly indicated in the formulas described herein, are intended to be included within the scope of the present invention.

Compounds of the Invention

[0077 ] Embodiments of the present invention provide a compound having the structure described by Formula I or Formula II:

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

R' is -H or -CH ₃ ,

M is selected from H and a pharmaceutically acceptable counterion such as Na ⁺ , K ⁺ , NR ₄ ⁺ or NHR ₃ ⁺ , where R is selected from H and C ₁ -C ₄ alkyl,

Y is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -C(O)-O - And

-OC(O)-,

R ¹ is absent or is optionally substituted with C ₁ -C ₁₈ -alkyl, where optionally up to six carbon atoms in the specified C ₁ -C ₁₈ alkyl, each independently, is substituted with -OCH ₂ CH ₂ -,

X is H or selected from halogen groups (for example, -F, -Cl, -Br or -I), -NO ₂ , -NR ² R ³ , -OR ² , -NHCOR ² and -OCOR ² where R ² and R ³ is each independently selected from H and C ₁ -C ₄ alkyl, and

TBG is a thiol-binding group selected from the following: an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, and an optionally substituted acetylene group.

[0078 ] In some embodiments, a compound having the structure described by formula I or II is formulated as a pharmaceutical composition containing an optional pharmaceutically acceptable carrier that is administered to the body and covalently, selectively and rapidly binds in situ to the thiol group of endogenous cysteine-34 albumin in the bloodstream.

[0079 ] In some embodiments, R' is -CH ₃ . In other embodiments, R' is H.

[0080 ] In some embodiments, the thiol-binding group (TBG) is a maleimide group:

.

.

[0081 ] In some embodiments, the highly cytotoxic peptide drug is auristatin E derivatized to result in a carbonyl group allowing an acid sensitive hydrazone bond to be formed with a maleimide water soluble linker containing a hydrazide group.

[0082 ] In some embodiments, the present invention relates to a pharmaceutical composition comprising a compound described herein, and optionally a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered intravenously and covalently, selectively and rapidly binds in situ to a thiol group of cysteine-34 endogenous albumin in the bloodstream.

[0083 ] In some embodiments, in the albumin-binding compounds provided herein, the highly cytotoxic drug is a carbonyl-containing pentapeptide derivative of auristatin E, the albumin-binding moiety is a thiol-binding group (TBG), e.g., a maleimide group, which upon administration rapidly and selectively binds to cysteine -34 albumin, and the acid-sensitive bond is a derivatized benzoylhydrazone bond of a compound of general formula I and II:

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

R' is H or -CH ₃ ,

M is selected from H and a pharmaceutically acceptable counterion such as Na ⁺ , K ⁺ , NR ₄ ⁺ or NHR ³ where R is selected from H and C ₁ -C ₄ alkyl,

Y is absent or selected from optionally substituted C ₁ -C ₆ alkyl, -NH-C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -C(O)-O - And

-OC(O)-,

R ¹ is absent or is optionally substituted with C ₁ -C ₁₈ alkyl, in which optionally up to six carbon atoms are each independently substituted

-OCH ₂ CH ₂ -,

X is H or selected from halogen groups (for example, -F, -Cl, -Br or -I), -NO ₂ , -NR ² R ³ , -OR ² , -NHCOR ² and -OCOR ² where R ² and R ³ each independently selected from H and C ₁ -C ₄ alkyl,

TBG is a thiol-binding group selected from the following: an optionally substituted maleimide group, an optionally substituted haloacetamide group, an optionally substituted haloacetate group, an optionally substituted pyridylthio group, an optionally substituted isothiocyanate group, an optionally substituted vinylcarbonyl group, an optionally substituted aziridine group, an optionally substituted disulfide group, and an optionally substituted acetylene group.

[0084 ] In some embodiments, TBG is substituted with C ₁ -C ₆ alkyl or halo. In some embodiments, TBG is substituted with methyl, -Cl, or -Br.

[0085 ] The disulfide group can be activated with thionitrobenzoic acid (eg, 5'-thio-2-nitrobenzoic acid) as an exchange group. The maleimide or pyridyldito group may optionally be substituted with an alkyl group or the above water-soluble groups. Typically, a thiol-binding group has protein-binding properties, that is, it covalently binds ("covalent protein-binding group") in a physiological environment to specific amino acids on the surface of the protein. In some embodiments, a maleimide group, a haloacetamide group, a haloacetate group, a pyridyldithio group, a disulfide group, a vinylcarbonyl group, an aziridine group, and/or an acetylenic group are reacted with thiol (-SH) groups of cysteines. In some embodiments, the protein binding group is a maleimide group

,

,

which binds to cysteine-34 of albumin.

[0086 ] In some embodiments, the drug delivery system contains an acid-sensitive cleavable hydrazone moiety. Cleavage of the hydrazone moiety and drug release half-life vary depending on the structure of the carbonyl derivative.

[0087 ] In some embodiments, the half-life of albumin-bound drug release in the pH range of 4.0-6.5 ranges from about 1.5 hours to about 80 hours.

[0088 ] Without being bound by theory, we believe that a phenyl ring containing one electron withdrawing group such as a sulfonic acid (-SO ₃ H) or a sulfonate group (-SO ₃ ^- ) attached ortho to the hydrazone bond stabilizes hydrazone fragment, which leads to a slow and prolonged release of the drug in an acidic environment.

[0089 ] In some embodiments, R' is -CH ₃ . In other embodiments, R' is H.

[0090 ] In some embodiments, Y and/or R ¹ are present. In some embodiments, Y is absent. In some embodiments, R ¹ is absent. In some embodiments, both Y and R ¹ are absent.

[0091 ] In some embodiments, Y is selected from the following: methyl, ethyl, -NH-C(O)-, -C(O)-NH-, -C(O)-O- and -OC(O)-.

[0092 ] In some embodiments, R ¹ is optionally substituted with C ₁ -C ₁₈ alkyl. In some embodiments, R ¹ is an optionally substituted C ₁ -C ₁₈ alkyl wherein one carbon atom is substituted with -OCH ₂ CH ₂ -. In some embodiments, R ¹ is an optionally substituted C ₁ -C ₁₈ alkyl wherein two carbon atoms are substituted with -OCH ₂ CH ₂ -. In some embodiments, R ¹ is an optionally substituted C ₁ -C ₁₈ alkyl wherein three carbon atoms are substituted with -OCH ₂ CH ₂ -. In some embodiments, R ¹ is an optionally substituted C ₁ -C ₁₈ alkyl wherein four carbon atoms are substituted with -OCH ₂ CH ₂ -. In some embodiments, R ¹ is an optionally substituted C ₁ -C ₁₈ alkyl wherein five carbon atoms are substituted with -OCH ₂ CH ₂ -. In some embodiments, R ¹ is an optionally substituted C ₁ -C ₁₈ alkyl wherein six carbon atoms are substituted with -OCH ₂ CH ₂ -.

[0093 ] US6884869-B2 (Application No: 10/001191, filed Nov. 1, 2001) describes antibody drug conjugates in which pentapeptide derivatives having the chemical structure described by Formula A and Formula B are shown below:

Formula B

[0094 ] These compounds were conjugated with thiol-containing antibodies. Both compounds contain an aliphatic 6-maleimidocaproylhydrazone moiety providing minimal water solubility for the two compounds having formula A and formula B depicted above. Indeed, the conjugation of both of the above compounds with antibodies was achieved by dissolving the compounds only with organic solvents, ie a mixture of acetonitrile:DMSO in a ratio of 9:1. The use of exclusively organic solvents in the formulation of the used pharmaceutical composition for in situ binding to cysteine-34 of circulating albumin, namely the approach to drug delivery described in this document, is not possible. Accordingly, in some embodiments, the compositions of the present invention do not include an organic solvent.

[0095 ] Thus, the invention includes aromatic maleimide linkers containing a sulfonic acid moiety, providing sufficient water solubility of albumin-binding prodrugs for the preparation of a pharmaceutical composition for intravenous administration. One such linker is 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazinecarbonyl)-benzenesulfonic acid, abbreviated as Sulf07, which has the chemical structure , represented by the formula:

[0096 ] The Sulf07 linker, 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazinecarbonyl)-benzenesulfonic acid, was prepared according to the method Synthesis A and/or Synthesis Method B, as shown in the following Synthesis Route Schemes:

[0097 ] A novel synthetic route and characterization of a new Sulf07 linker, 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazinecarbonyl)-benzenesulfonic acid, which was obtained by the synthesis method A and B are described in Example 1.

[0098 ] Linker Sulf07 reacted with:

AE-Keto

or

AE-Ester in order to obtain compounds described by formula III (abbreviated as AE-Keto-Sulf07) and formula IV (abbreviated as AE-Ester-Sulf07), respectively:

Formula III (AE-Keto-Sulf07)

Formula IV (AE-Ester-Sulf07)

[0099 ] Synthesis and characterization of AE-Keto-Sulf07 (Formula III) and AE-Ester-Sulf07 (Formula IV) are described in Examples 2 and 3.

[00100 ] Analysis of the structures of AE-Keto-Sulf07 and AE-Ester-Sulf07 shows that in both molecules, two groups (-SO ₃ H and -N(CH ₃ ) ₂ groups) exist as an acid-base pair, thus forming zwitterions shown in Scheme 3 and Scheme 4.

[00101 ] The sulfonic acid moiety integrated into the Sulf07 linker, as well as the zwitterionic property of the two auristatin prodrugs AE-Keto-Sulf07 and AE-Ester-Sulf07, provide sufficient water solubility for the preparation of a pharmaceutical composition and, more importantly, high stability of the maleimide group , which is significantly improved compared to formula A (AE-Keto-EMCH) and B (AE-Ester-EMCH):

Formula A

Formula B

presented in US 6884869-B2 (application no: 10/001191, filed 11/01/2001).

[00102 ] For in situ binding to cysteine-34 of endogenous albumin, the stability of the maleimide group under physiological conditions in the pH range of 7.4-7.6 is a criterion for intravenous administration and effective binding to circulating albumin.

[00103 ] As shown in FIG. 1 and FIG. 2, the hydrolysis stability of the maleimide group for both AE-Keto-Sulf07 and AE-Ester-Sulf07 is significantly improved compared to the two compounds described in US6884869-B2, ie. AE-Keto-EMCH (compound described by formula A) and AE-Ester-EMCH (compound described by formula B). After 4 hours incubation at room temperature in reconstitution buffer (50 mM sodium phosphate, pH 7.65 containing 4% HPβCD and 5% sucrose), 2.2% of AE-Keto-Sulf07 maleimide was hydrolyzed compared to 5.4% hydrolysis of AE-Keto-EMCH maleimide and similarly 2.5% of AE-Ester-Sulf07 maleimide were hydrolyzed compared to 11.0% for AE-Ester-EMCH. In addition, the active pharmaceutical ingredient (API) formulations of the Sulf07 derivatives showed a high level of stability (FIG. 24 and FIG. 25) under accelerated degradation conditions (e.g., at 55°C for up to 264 hours), while the maleimide groups of the EMCH derivatives quickly hydrolyzed. A minimum level of maleimide hydrolysis is required for product development and manufacture to allow quantitative binding of endogenous albumin and thus limit any premature free drug release into the bloodstream and maximize clinical efficacy.

[00104 ] Another advantage of aqueous solutions of AE-Keto-Sulf07 and AE-Ester-Sulf07 is that they have a physiological pH in the range of 6.8 to 7.5.

[00105 ] In addition, the solubility and stability of zwitterionic APIs are improved when used in combination with approved pharmaceutical carriers such as Tween 80, 2-hydroxypropyl-β-cyclodextrin, and this may facilitate pharmaceutical formulation.

[00106 ] The pharmaceutical formulations AE-Keto-Sulf07 and AE-Ester-Sulf07 achieved very rapid binding to [human, mouse and rat (FIG. 5-10)] plasma albumin. For both therapeutic agents, the specificity of binding to albumin in human plasma was also demonstrated (Fig. 3-4).

Pharmaceutical compositions

[00107 ] In some embodiments, the invention relates to a pharmaceutical composition containing a compound described herein.

[00108 ] The total amount of the compound in the composition for administration to a patient is one acceptable for that patient. One skilled in the art will appreciate that different patients may require different total amounts of a therapeutically effective substance. In some embodiments, the amount of the compound is a pharmaceutically effective amount. One skilled in the art will be able to determine the amount of a compound in a composition needed to treat a patient based on factors such as, for example, the age, weight, and physical condition of the patient. The concentration of the compound depends on its solubility in the intravenous solution and on the volume of liquid that can be administered. For example, the concentration of the compound may be from about 0.1 mg/mL to about 50 mg/mL in an injectable composition. In some embodiments, the concentration of the compound may range from about 0.1 mg/mL to about 40 mg/mL.

[00109 ] The pharmaceutical compositions and kits of the present invention may also contain diluents, excipients, salts, buffers, stabilizers, solubilizers, protectants, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not affect the degree of biological activity of the active(s) ingredient(s). The characteristics of the carrier will depend on the route of administration.

[00110 ] The compositions can be administered in a variety of conventional ways. Typical routes of administration that can be used include oral, parenteral, intravenous, intraarterial, dermal, subcutaneous, intramuscular, topical, intracranial, intraorbital, intraocular, intravitreal, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, central nervous system (CNS) or administration via a suppository. In some embodiments, the compositions are suitable for parenteral administration. These compositions can be administered, for example, intraperitoneally, intravenously or intrathecally. In some embodiments, the compositions are administered intravenously. In some embodiments, a redissolved composition can be prepared by redissolving a lyophilized composition compound in a redissolving liquid containing, for example, alcohol, DMSO and/or polyethylene glycol and water and/or saline buffer. Such redissolution may include the addition of a redissolve liquid and mixing, for example by agitating or agitating the mixture. The reconstituted composition can then be made suitable for injection by mixing, for example, lactated Ringer's solution, 5% glucose solution, isotonic saline, or a suitable saline buffer with an injectable dosage form. One skilled in the art will appreciate that the manner in which a therapeutically effective formulation or composition of substance is administered will depend on such factors as the age, weight, and physical condition of the patient being treated and the disease or condition being treated. Thus, a person skilled in the art will be able to select the optimal route of administration for the patient in each particular case.

[00111 ] In some embodiments, the compounds and compositions provided herein are for use in the treatment of cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, or other microorganisms.

[00112 ] In some embodiments, a compound provided herein may be used in the manufacture of a medicament for the treatment of a disease selected from the following: cancer, viral disease, autoimmune disease, acute or chronic inflammatory disease, and disease caused by bacteria, fungi, or other microorganisms.

[00113 ] In some embodiments, the cancer is a blood cancer or a cancer that is a solid tumor. In some embodiments, the cancer is selected from carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, and melanoma.

[00114 ] In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, anal carcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, non-small cell bronchial cancer, cancer breast, Burkitt's lymphoma, carcinoma of the main part of the organ, cancer without an identified primary focus (CUP syndrome), colon cancer, small intestine cancer, ovarian cancer, endometrial cancer, gallbladder cancer, gallbladder carcinomas, uterine cancer, cervical cancer, neck, nose and ear tumors, hematologic neoplasms, hairy cell leukemia, urethral cancer, skin cancer, gliomas, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal cancer, head and/or neck tumors, colon cancer, craniopharyngioma, cancer liver, leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer, renal cell carcinomas, oligodendroglioma, esophageal cancer, osteolytic carcinomas and osteoplastic carcinomas, osteosarcoma , ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, tongue cancer, ovarian cancer or lymph node cancer.

[00115 ] In some embodiments of the invention, a kit is provided that contains a compound described herein and a pharmaceutically acceptable excipient, carrier, and/or diluent.

[00116 ] In some embodiments, one or more excipients may be included in the composition. The person skilled in the art will understand 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 of the additional excipients because the combination of excipients will cause undesirable effects. One skilled in the art will be able to empirically determine which excipients, if any, should be included in the compositions. Adjuvants may include, without limitation, co-solvents, solubilizing agents, buffers, pH adjusting agents, bulking agents, surfactants, encapsulating agents, tonicity adjusting agents, stabilizers, protectants, and viscosity modifiers. In some embodiments, it may be useful to include a pharmaceutically acceptable carrier in the compositions.

[00117 ] In some embodiments, a solubilizing agent may be included in the composition. Solubilizing agents may be used to increase the solubility of any of the components of the composition, including the compound or excipient. The solubilizing agents described herein are not intended to be an exhaustive list, but only as examples of solubilizing agents that can be used in the compositions. In some embodiments, solubilizing agents include, without limitation, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerin, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, cyclodextrins such as dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β -cyclodextrin and trimethyl-β-cyclodextrin and combinations thereof, as well as any pharmaceutically acceptable salts and/or combinations thereof.

[00118 ] The pH of the compositions can be any pH that provides optimal properties of the compound or composition. Optimal properties may include, for example, compound stability, increased compound stability over compositions at other pHs, and improved filtration efficiency. In some embodiments, the pH of the compositions may be from about 3.0 to about 9.0, for example, from about 5.0 to about 7.0. In specific embodiments, the pH value of the compositions may be 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1.6 .0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, 6.5±0.1, 6.6 ±0.1, 6.7±0.1, 6.8±0.1, 6.9±0.1, 7.0±0.1, 7.1±0.1 and 7.2±0 ,1.

[00119 ] In some embodiments, it may be preferable to buffer the pH by including one or more buffers in the compositions. In some embodiments, the buffer may have a pKa, such as about 5.5, about 6.0, or about 6.5. One of skill in the art will appreciate that a suitable buffer may be selected for inclusion in compositions based on its pKa and other properties. Buffers are well known to the person skilled in the art. Accordingly, the buffers described herein do not constitute an exhaustive list, but are listed only as examples of buffers that can be used in the formulations or compositions of the present invention. In some embodiments, the buffer includes, without limitation, Tris, Tris-HCl, Potassium Phosphate, Sodium Phosphate, Sodium Citrate, Sodium Ascorbate, Sodium and Potassium Phosphate Combinations, Tris/Tris-HCl, Sodium Bicarbonate, Arginine Phosphate, Arginine Hydrochloride, Hydrochloride histidine, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof.

[00120 ] In some embodiments, a pH adjusting agent may be included in the composition. Changing the pH of the composition may have a positive effect, for example, on the stability or solubility of the compound, or may be useful in obtaining a composition suitable for parenteral administration. pH adjusting agents are well known to those skilled in the art. Accordingly, the pH adjusting agents described herein are not intended to be an exhaustive list, but only as examples of pH adjusting agents that may be used in the compositions. pH adjusting agents may include, for example, acids and bases. In some embodiments, the pH adjusting agent includes, without limitation, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

[00121 ] In some embodiments, a filler may be included in the composition. Excipients are commonly used in lyophilized compositions to add bulk to the composition and to facilitate visualization of the composition, especially in cases where the lyophilized pellet would be difficult to see without their use. Excipients can also help prevent release of the active ingredient(s) of the pharmaceutical composition and/or help cryoprotect the composition. Fillers are well known to the person skilled in the art. Accordingly, the excipients described herein do not constitute an exhaustive list, but are listed only as examples of excipients that can be used in the compositions.

[00122 ] Exemplary excipients may include carbohydrates, monosaccharides, disaccharides, polysaccharides, sugar alcohols, amino acids, and sugar acids, and combinations thereof. Carbohydrate excipients include, without limitation, mono-, di-, or polycarbohydrates, starches, aldoses, ketoses, amino sugars, glyceraldehyde, arabinose, lyxose, pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose, talose, heptose, glucose , fructose, methyl-α-D-glucopyranoside, maltose, lactone, sorbose, erythrose, threose, arabinose, allose, altrose, gulose, idose, talose, erythrulose, ribulose, xylulose, psicose, tagatose, glucosamine, galactosamine, arabinans, fructans , fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galactocarolose, pectins, amylose, pullulan, glycogen, amylopectin, cellulose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid , xanthine gum, sucrose, trehalose, dextran and lactose. Sugar alcohol fillers include, without limitation, alditols, inositols, sorbitol, and mannitol. Sugar acid excipients include, without limitation, aldonic acids, uronic acids, aldaric acids, gluconic acid, isoascorbic acid, ascorbic acid, glucaric acid, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, neuraminic acid, pectin acids and alginic acid. Amino acid excipients include, without limitation, glycine, histidine, and proline.

[00123 ] In some embodiments, a surfactant may be included in the composition. Surfactants generally lower the surface tension of a liquid composition. This may provide useful properties such as improved ease of filtering. Surfactants can also act as emulsifying agents and/or solubilizing agents. Surfactants are well known to the person skilled in the art. Accordingly, the surfactants described herein do not constitute an exhaustive list, but are presented only as examples of surfactants that can be used in the formulations or compositions of the present invention. Surfactants that can be used include, without limitation, 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., pluronics ), ethylene oxides and polyethylene oxides (eg Triton X-100), saponins, phospholipids (eg lecithin), and combinations thereof.

[00124 ] In some embodiments, an encapsulating agent may be included in the composition. Encapsulating agents can isolate molecules and help stabilize or dissolve them. Encapsulating agents are well known to the person skilled in the art. Accordingly, the encapsulating agents described herein do not constitute an exhaustive list, but are listed only as examples of encapsulating agents that can be used in the compositions. Encapsulating agents that may be included in the compositions include, without limitation, α-cyclodextrins, β-cyclodextrins, γ-cyclodextrin, and combinations thereof (e.g., α-cyclodextrin, dimethyl-α-cyclodextrin, hydroxyethyl-α-cyclodextrin, hydroxypropyl- α-cyclodextrin, trimethyl-α-cyclodextrin, β-cyclodextrin, dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, trimethyl-β-cyclodextrin, γ-cyclodextrin, dimethyl-γ-cyclodextrin, hydroxyethyl- γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, trimethyl-γ-cyclodextrin and combinations thereof.

[00125 ] In some embodiments, a tonicity agent may be included in the composition. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example by parenteral administration. Thus, tonicity agents can be used to create a composition suitable for administration. Tonicity agents are well known to those skilled in the art. Accordingly, the tonicity agents described herein are not intended to be an exhaustive list, but only as examples of tonicity agents that may be used in the compositions. Tonicity agents may be ionic or non-ionic and include, without limitation, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Representative inorganic salts may include sodium chloride, potassium chloride, sodium sulfate and potassium sulfate. An example of an amino acid is glycine. Representative sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, dextrose, and mannitol.

[00126 ] In some embodiments, a stabilizing agent may be included in the composition. Stabilizing agents help to increase the stability of the compound in the compositions. This can occur, for example, by reducing degradation or preventing compound aggregation. Without wishing to be bound by theory, stability mechanisms may include sequestration of a compound from a solvent or inhibition of free radical oxidation of a therapeutically effective substance. Stabilizing agents are well known to the person skilled in the art. Accordingly, the stabilizing agents described herein do not constitute an exhaustive list, but are listed only as examples of stabilizing agents that can be used in the compositions. Stabilizing agents may include, without limitation, emulsifiers and surfactants.

[00127 ] In some embodiments, a protective agent may be included in the composition. Protective agents are agents that protect a pharmaceutically active ingredient (eg, a therapeutically effective substance or compound) from an undesirable condition (eg, instability caused by freezing, lyophilization, or oxidation). Protective agents may include, for example, cryoprotectants, lyoprotectants and antioxidants. Cryoprotectants are useful in preventing the loss of activity of an active pharmaceutical ingredient (eg an anthracycline compound) when the composition is exposed to a temperature below its freezing point. For example, a cryoprotectant may be included in the reconstituted lyophilized composition such that the composition may be frozen prior to dilution for intravenous administration. Cryoprotectants are well known to the person skilled in the art. Accordingly, the cryoprotectants described herein do not constitute an exhaustive list, but are listed only as examples of cryoprotectants that can be used in the compositions. Cryoprotectants include, without limitation, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Cryoprotectants may include, for example, disaccharides (eg sucrose, lactose, maltose and trehalose), polyols (eg glycerol, mannitol, sorbitol and dulcitol), glycols (eg ethylene glycol, polyethylene glycol and propylene glycol).

[00128 ] Lyoprotectants are useful for stabilizing the components of a lyophilized composition. For example, a therapeutically effective substance may be lyophilized with a lyoprotectant prior to reconstitution. Lyoprotectors are well known to the person skilled in the art. Accordingly, the lyoprotectants described herein are not intended to be an exhaustive list, but are provided only as examples of lyoprotectants that can be used in compositions. Lyoprotectants include, without limitation, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Examples of lyoprotectants are, for example, sugars and polyols. Trehalose, sucrose, dextran, and hydroxypropyl-beta-cyclodextrin are non-limiting examples of lyoprotectants.

[00129 ] Antioxidants are useful in preventing oxidation of the components of the composition. Oxidation can lead to drug product aggregation or other detrimental effects on drug product purity or potency. Antioxidants are well known to the person skilled in the art. Accordingly, the antioxidants described herein do not constitute an exhaustive list, but are listed only as examples of antioxidants that can be used in the compositions. Antioxidants can be, for example, sodium ascorbate, citrate, thiols, metabisulphite, and combinations thereof.

[00130 ] In some embodiments, a viscosity modifier may be included in the composition. Viscosity modifiers change the viscosity of liquid compositions. This can be useful since viscosity plays an important role in the ease of filtering of a liquid composition. The composition may be filtered before lyophilization and reconstitution or after reconstitution. Viscosity modifiers are well known to the person skilled in the art. Accordingly, the viscosity modifiers described herein are not intended to be an exhaustive list, but are provided only as exemplary viscosity modifiers that can be used in compositions. Viscosity modifiers include solvents, solubilizing agents, surfactants and encapsulating agents. Typical viscosity modifiers that may be included in the compositions include, without limitation, N-acetyl-DL-tryptophan and N-acetyl-cysteine.

Antitumor activity in murine xenogeneic models of human tumors

[00131 ] The albumin-binding prodrugs AE-Keto-Sulf07 and AE-Ester-Sulf07 demonstrated exceptional antitumor activity in tumor cell lines with _IC50 of the free drugs AE-Keto and AE-Ester in the picomolar range (259 and 339 pM, respectively, compared to IC ₅₀ 130 pM parent compound AE (see example 4), as well as in some xenogeneic human tumor models in nude mice, causing partial and complete remission in all xenogeneic human tumor models evaluated (see examples in Fig. 13-23 These cases included small tumors with initial volumes in the range of approximately 130-170 mm ³ as well as large tumors with initial volumes up to approximately 380 mm ^3. In addition, in most cases, therapy with albumin-binding prodrugs AE-Keto-Sulf07 and AE α-ester-Sulf07 induced long-term remissions and a reduction in relative tumor volume (RTV) The parent compound auristatin E (AE) was fundamentally inactive or showed only marginal tumor inhibition in the models tested. The experimental procedure and results in mouse tumor models are described in detail in Example 5 and in FIG. 13-23.

Methods of treatment

[00132 ] The compounds and compositions described herein may be used in a variety of clinical applications.

[00133 ] The compounds and compositions described herein can cause prolonged or delayed inhibition of tumor growth. In some embodiments, prolonged or delayed tumor growth inhibition occurs without any loss of body weight, or without any or only minor bone marrow toxicity.

[00134 ] In some embodiments, the present invention relates to a method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition containing a compound described herein. For example, some embodiments include a method of treating a patient suffering from a disease selected from the group including cancer, a viral disease, an autoimmune disease, an acute or chronic inflammatory disease, and a disease caused by bacteria, fungi, and other microorganisms, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.

[00135 ] The present invention relates to methods for treating a condition or disease in a patient selected from the group including cancer, viral disease, autoimmune disease, acute or chronic inflammatory disease, and disease caused by bacteria, fungi or other microorganisms, comprising administering to the patient a compound or a pharmaceutical compositions in the manner described herein.

[00136 ] In some embodiments, the cancer is a blood cancer or a cancer that is a solid tumor. In some embodiments, the cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, or melanoma.

[00137 ] In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, anal carcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissue tumor, bladder cancer, bronchial carcinoma, non-small cell bronchial cancer, cancer breast, Burkitt's lymphoma, body organ carcinoma, cancer without identified primary site (CUP syndrome), colon cancer, small intestine cancer, ovarian cancer, endometrial cancer, gallbladder cancer, gallbladder carcinomas, uterine cancer, cervical cancer, tumors neck, nose and ear, hematologic neoplasms, hairy cell leukemia, urethral cancer, skin cancer, gliomas, testicular cancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal cancer, head and/or neck tumors, colon cancer, craniopharyngioma, liver cancer , leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer, renal cell carcinomas, oligodendroglioma, esophageal cancer, osteolytic carcinomas and osteoplastic carcinomas, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, tongue cancer, ovarian cancer, or cancer of the lymph node.

[00138 ] Some embodiments include a method of increasing the concentration of a metabolite of a compound in a tumor, comprising administering a compound in accordance with the present invention.

Variants and modifications

[00139 ] The variations, modifications, and other embodiments described herein will be considered by those skilled in the art without deviating from the spirit and scope of the present invention. Accordingly, the present invention is not to be limited by the foregoing description or the following examples.

Illustrative example

[00140 ] Aspects generally described in the present invention will be better understood when considered with reference to the following examples, which are included only for the purpose of illustrating certain features and embodiments of the present invention, but are not limited to them.

Equivalents

[00141 ] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds, compositions, and methods of use described herein. Such equivalents are considered to be within the scope of the present invention.

Examples

Materials and methods for the preparation and analysis of compounds

[00142 ] All reactions were carried out under nitrogen, unless otherwise noted. Commercially available reagents were used without further purification unless otherwise noted. Anhydrous solvents were purchased in anhydrous form (dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, etc.) and all other solvents used were high performance liquid chromatography (HPLC) or liquid chromatography coupled mass spectrometry reagents. (LCMS).

[00143 ] Glassware and stirrers were typically oven dried at 110° C. for at least 12 hours and then cooled under nitrogen prior to use, where applicable. All other reactions were carried out in round bottom flasks sealed with rubber septa. Plastic syringes or glass tubes were used to transfer liquid reagents. The reaction mixtures were stirred using Teflon-coated magnetic stirrers. The organic solutions were concentrated under reduced pressure using a KNF RC 600 rotary evaporator and a Heidolph Hei-VAP.

[00144 ] Flash column chromatography was performed using prepackaged Biotage® SNAP Ultra and SNAP Ultra C18 flash silica gel cartridges using Biotage® IsoleraTM One and Biotage® IsoleraTM SL flash purification systems (large scale).

[00145 ] The pH value of the solution was measured at room temperature using a WTW Inolab 7310 pH meter with SenTix® mic-D electrodes.

[00146 ] High performance liquid chromatography (HPLC) was performed using a Shimadzu Nexera XR HPLC system equipped with an SPD-M20A photodiode array detector and monitored at 220 nm unless otherwise noted.

[00147 ] Low resolution mass spectra (LRMS) were collected using liquid chromatography combined with mass spectrometry (LCMS) on preferred Bruker Amazon SL or Thermo Fisher LCQ spectrometers (electrospray ionization, ESI). High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF instrument using liquid chromatography, electrospray ionization and time-of-flight mass spectrometer (ESI-TOF). Elemental analysis was performed on a Leco TruSpec® CHNS Macro with an IR detector for C, H, and S; and with thermal conductivity detector (TCD) for N.

[00148 ] Lyophilization was performed using a Martin Christ Alpha or Epsilon 2-4 LSCplus freeze dryer.

[00149 ] Centrifugation was performed using an Eppendorf 5810 R centrifuge, refrigerated, with an A-4-81 rotor, 230 V/50-60 Hz.

[00150 ] Trifluoroacetic acid (TFA) content was measured by ion chromatography and water content was measured by Karl Fischer (KF) coulometry.

[00151 ] Nuclear magnetic resonance (NMR) spectra were recorded at ambient temperature on a 400 MHz spectrometer: Bruker Avance 400 Ultrashield (400 MHz at ¹ H, 100 MHz at ¹³ C). All proton chemical shift values are in parts per million (δ) and refer to deuterated protons in DMSO-d ₆ (δ 2.50). All carbon chemical shift values are in parts per million (δ) and refer to carbon resonances in DMSO-d ₆ (δ 39.52). NMR data are presented as follows: chemical shift, fold (s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, m=multiplet, br=wide, dd=doublet of doublets, td=triplet of doublets), coupling constant = J (Hz=Hertz) and integration.

HPLC methods

[00152 ] Method 1: HPLC method for linker intermediates and final Sulf07 product purity. Column: Phenomenex Kinetex Polar C18 (150 x 4.6 mm, 2.6 µm, 100 Å), gradient: mobile phase A: 95: 5 ammonium acetate, 10 mM, pH 7.0: acetonitrile, mobile phase B: 95 : 5, acetonitrile: ammonium acetate 10 mM, pH 7.0. Phase B elution gradient: 0-2.5 min: 0%, 2.5-18 min: 0-45%, 18-20 min: 45-75%, 20-24 min: 75%, 24-26 min: 75% - 0%, 26-30 minutes: 0%, 30 minutes: end of process, flow rate = 1.0 ml/min.

[00153 ] Method 2: HPLC method for example 2 and example 3. Column: Phenomenex Kinetex Polar C18 (150×4.6 mm, 2.6 μm, 100 Å), gradient: mobile phase A: 95:5 ammonium acetate, 10 mM, pH 7.0: acetonitrile, mobile phase B: 95:5, acetonitrile: ammonium acetate 10 mM pH 7.0. Phase B elution gradient: 0-0.5 min: 30%, 0.5-9 min: 30-95%, 9-11 min: 95%, 11-12.5 min: 95-30%, 12.5 -15 min: 30%, 15 min: end of process, flow rate = 1.0 ml/min.

[00154 ] Method 3: LCMS method for intermediate linkers and end product Sulf07. Column: Phenomenex Kinetex Polar C18 (150 x 2.1 mm, 2.6 µm, 100 Å), gradient: mobile phase A: 95: 5 ammonium acetate 10 mM, pH 7.0: acetonitrile, mobile phase B: 95: 5, acetonitrile: ammonium acetate 10 mM, pH 7.0. Phase B elution gradient: 0-2.5 min: 0%, 2.5-18 min: 0-45%, 18-20 min: 45-75%, 20-24 min: 75%, 24-26 min: 75% - 0%, 26-30 minutes: 0%, 30 minutes: end of process. Flow rate = 0.4 ml/min.

[00155 ] Method 4: LCMS method for example 2 and example 3. Column: Phenomenex Luna Omega Polar C18 (50 x 2.1 mm, 1.6 μm, 100 Å), gradient: mobile phase A: 99.9: 0 .1 water: formic acid, mobile phase B: 99.9: 0.1 acetonitrile: formic acid. Phase B elution gradient: 0-0.5 min: 20%, 0.5-3.5 min: 20-100%, 3.5-5.0 min: 100%, 5.0-5.5 min: 100-20%, 5.5-7.0 min: 20%, 7 min: end of process. Flow rate = 0.4 ml/min.

[00156 ] Method 5: LCMS method for plasma binding experiments. Column: Phenomenex Kinetex Polar C18 (50×2.1 mm, 2.6 µm, 100 Å), gradient: mobile phase A: 95: 5 ammonium acetate, 10 mM, pH 7.0: acetonitrile, mobile phase B: 95 : 5, acetonitrile: ammonium acetate 10 mM, pH 7.0. Phase B elution gradient: 0-1.0 min: 0%, 1.0-7.0 min: 0-75%, 7.0-7.5 min: 75-90%, 7.5-8.5 min: 90-0%, 8.5-10.0 min: 0%, 10 minutes: end of process, flow rate = 0.4 ml/min.

[00157 ] Method 6: Hydrophobic interaction chromatography (HIC) method for plasma binding. Column: MabPac HIC-20 (250×4.6 mm, 5 μm, 100 Å), gradient: mobile phase A: 1.5 M (NH ₄ ) ₂ SO ₄ , 20 mM Na ₂ HPO ₄ , pH 8.0, mobile phase B: 20 mM Na ₂ HPO ₄ pH 8.0 and 20% isopropyl alcohol. Phase B elution gradient: 0-2.0 min: 0%, 2.0-10.0 min: 0-50%, 10.0-13.0 min: 50-60%, 13.0-17.0 min: 60-100%, 17.0-20.0 min: 100%, 20.0-25.0 min: 100-0%, 25.0-30.0 min: 0%, 30 min: end of process , flow rate = 1 ml/min.

[00158 ] Method 7: HPLC method for albumin drug conjugate pH stability profile. Column: Phenomenex Aeris WP C18 (250 x 4.6 mm, 3.6 µm, wide pore form), gradient: mobile phase A: 20 mM Tris buffer, pH 8.0, mobile phase B: 90% UHPLC acetonitrile and 10 % water. Phase B elution gradient: 0-0.5 min: 25%, 0.5-2.5 min: 25-35%, 2.5-16.0 min: 35-85%, 16.0-17.0 min: 85-95%, 17.0-20.0 min: 95%, 20.0-25.0 min: 95-25%, 25.0-30.0 min: 25%, 30 min: end of process , flow rate=1.0 ml/min.

[00159 ] Method 8: HPLC method for accelerated degradation of API formulations. Column: Phenomenex Kinetex Polar C18 (150 x 4.6 mm, 2.6 µm, 100 Å), gradient: mobile phase A: 95: 5 ammonium acetate, 10 mM, pH 7.0: acetonitrile, mobile phase B: 95 : 5, acetonitrile: ammonium acetate 10 mM, pH 7.0. Phase B elution gradient: 0-2.5 min: 20%, 0.5-9 min: 20-75%, 9-11 min: 75%, 11-12.5 min: 75-20%, 12.5 -15 min: 20%, 15 min: end of process, flow rate=1.0 ml/min.

[00160 ] Method 9: HPLC method for intermediate linkers and final product of purity Sulf07 obtained according to synthetic route C. Column: Phenomenex Kinetex Polar C18 (150×4.6 mm, 2.6 μm, 100 Å), gradient: mobile phase A: 0.1% trifluoroacetic acid in H ₂ O, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. Phase B elution gradient: 0-5.5 min: 1%, 5.5-20 min: 1-40%, 20-22 min: 40-65%, 22-24 min: 65%, 24-27 min: 65% - 1%, 27-30 minutes: 1%, 30 minutes: end of process, flow rate = 1.0 ml/min.

[00161 ] Method 10: HPLC Method to Determine the Purity of AE-Keto-Sulf07 Derived from Sulf07 Linker According to Synthesis Route C. Kinetex Polar C18 Column (2.6 µm, 100 Å, 150 mm×4.6 mm), Gradient: mobile phase A: 95: 5 ammonium acetate 5 mM pH 7.0: methanol, mobile phase B: 95: 5 methanol: ammonium acetate 5 mM pH 7.0. Phase B elution gradient: 0-2.5 min: 60%, 2.5-18 min: 60-80%, 18-22 min: 80-95%, 22-26 min: 95%, 26-30 min: end of the way. Flow rate = 1.0 ml/min. Column thermostat: 37°C.

Example 1

[00162 ] Sulf07, 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazine-carbonyl)benzenesulfonic acid) was prepared as described below and shown in Scheme 1, respectively, the route of synthesis A.

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

[00163 ] To a stirred solution of potassium permanganate (72 g, 460 mmol, 4.5 eq., Sigma Aldrich) in Millipore water (450 ml) was added a solution of 4-nitro-2-sulfonic acid hydrate (26 g, 102 mmol, 1.0 eq., Abcr) in Millipore water (100 ml). The resulting purple mixture was stirred at 115° C. for 5 hours, after which time the mixture turned brown. HPLC (method 1, 220 nm) confirmed that the reaction was complete after 5 hours. The reaction mixture was cooled to room temperature. The brown solid formed during the reaction was removed by suction filtration on a Celite pad, washed with Millipore water (300 mL) and the brown-yellow filtrate solution concentrated to ~125 mL under reduced pressure at 40°C, slowly acidified with 5 M HCl until a white suspension is formed (~pH 1). The white suspension was then heated at 100° C. 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 fritted filter (pore size 4). The white solid was then dried under high vacuum for 10 hours to give product A. Yield: 18 g, 72%. HPLC purity (method 1, 220 nm) over 95%. LRMS-ESI (m/z) calc. for C ₇ H ₄ NO ₇ S [ M-H] ^- : 245.98. Result: 245.83.

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

[00164 ] A stirred suspension of 4-nitro-2-sulphobenzoic acid (Product A ) (13 g, 51 mmol, 1.0 equiv.) in Millipore water (75 ml) was refluxed (120° C.) until complete dissolution. At this temperature, acetic acid (7.2 ml, Sigma Aldrich) was added, followed by iron powder (9.5 g, 180 mmol, 3.5 equiv., Sigma Aldrich), which was added in portions (~ 1 g/1.0 min) for 10 min to avoid an exothermic reaction. The reaction mixture was then allowed to stir under reflux for 1 hour. During this time, a brown solid formed and HPLC analysis (method 1, 220 nm) confirmed that the reaction was complete. The brown solid was removed by suction filtration directly on the celite pad (when it was still hot) and then washed with hot water (3×50 ml). The filtrate was then filtered through Whatman filter paper (11 µm). The resulting filtrate was concentrated under reduced pressure at 50° C. to a volume of ~100 ml. Concentrated HCl was added dropwise (~1 ml/2.0 min) until a pH of 1 was reached, resulting in a white/yellow solid precipitated. The suspension was left at 4°C for 1 hour. The solid was collected by suction filtration using a frit filter (pore size 4) and dried under high vacuum for 10 hours to give product B as a white solid. Yield: 9 g, 81%. HPLC purity (method 1, 220 nm) over 95%. LRMS-ESI (m/z) calc. for C ₇ H ₄ NO ₅ S [ M-H] ^- : 216.00. Result: 216.16.

Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride or EMC-Cl (product C )

[00165 ] To a stirred solution of 6-maleimidocaproic acid (EMC) (33 g, 156 mmol, 1.0 eq., Alfa Aesar) in dry dichloromethane (150 ml) at room temperature and under nitrogen was added over 30 minutes (~ 1 ml/2 min) oxalyl chloride (15 ml, 171 mmol, 1.1 equiv., Sigma Aldrich) using an addition funnel. Caution: Gas evolution was observed during the addition process. The reaction mixture was stirred at room temperature for 5 hours. The color of the reaction solution changed to dark yellow during the reaction time, and HPLC analysis (method 1, 220 nm) confirmed that the reaction was complete after 5 hours. The solvent was removed under reduced pressure at 40° C. to give an oil which was dried under high vacuum overnight to give a solidified compound. The resulting light brownish solid was ground with a spatula and dried for an additional 20 hours under high vacuum to give product C as a yellow microcrystalline solid. The compound was used in the next reaction without further purification. Yield: 34 g, 95%. HPLC purity (method 1, 220 nm) >95% as methyl ester. LRMS-ESI (m/z) calc. for C ₁₁ H ₁₆ NO ₄ (as methyl ester) [ M+H] ⁺ : 226.10. Result: 225.97.

Synthesis of 4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-sulphobenzoic acid (product D )

[00166 ] Product B (18.5 g, 85 mmol, 1.0 equiv.) was dissolved in anhydrous N,N-dimethylformamide (300 mL) under nitrogen. The solution was cooled in an ice bath and allowed to stir for 10 min until reaching 4°C. Then 4-methylmorpholine (18.69 ml, 170 mmol, 2.0 eq., Sigma Aldrich) was added dropwise (~1 ml/3.5 min) to the cooled solution over 1 h using an addition funnel. After the addition was complete, the mixture turned dark brown, and to this dark brown mixture was added dropwise (~0.5 g/min) over 1 hour, using an addition funnel, a solution of product C (29.28 g, 127 mmol, 1 .5 eq.) in anhydrous N,N-dimethylformamide (200 ml). The reaction mixture was allowed to gradually warm to room temperature overnight and then allowed to stir at room temperature for 10 hours. After complete conversion of the reaction, as determined by HPLC (method 1, 220 nm), the reaction solution was dispensed into 8×50 ml Falcon tubes and centrifuged for 20 minutes at 10°C and 4000 rpm. The supernatants were removed and the solids were resuspended in 10 ml of N,N-dimethylformamide per tube and centrifuged again for 20 minutes at 10° C. and 4000 rpm. All supernatants were combined and concentrated under reduced pressure at 50° C. for 3 hours to give a light orange solid (yield: 34 g, HPLC purity (method 1, 220 nm) 66%). This solid was resuspended in methanol (250 ml), transferred to 8×50 ml Falcon tubes and centrifuged for 20 minutes at 10° C. and 4000 rpm. The supernatants were removed and the solids were resuspended in 5 ml of methanol per tube and centrifuged again for 20 minutes at 10° C. and 4000 rpm. All solids were combined and dried under high vacuum for 24 hours to give product D as a crystalline yellow solid. Yield: 24 g, 37%. HPLC purity (method 1, 220 nm) 97%. LRMS-ESI (m/z) calc. for C ₁₇ H ₁₇ N ₂ O ₈ S [ M-H] ^- : 409.08. Result: 409.13.

Synthesis of 2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)benzenesulfonic acid or BOC -protected linker (Product E )

[00167 ] To a solution of product D (17 g, 41.4 mmol, 1.0 equiv.) in anhydrous NN-dimethylformamide (350 mL) under nitrogen was added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ( EDC-HCl) (8.72 g, 45.5 mmol, 1.1 eq., Roth) and hydroxybenzotriazole (HOBt) (6.15 g, 45.5 mmol, 1.1 eq., Sigma Aldrich). The reaction mixture was allowed to stir for 30 minutes at room temperature and then tert-butylcarbazate (7.12 g, 53.9 mmol, 1.3 eq., Sigma Aldrich) was added and the solution turned clear yellow to reddish. The reaction mixture was stirred at room temperature overnight. After this time, complete conversion of the reaction was confirmed by HPLC (method 1, 220 nm). The solvent was removed under reduced pressure at 40° C. and then under high vacuum at room temperature for 1 hour to give a purplish brown oil which was purified using a quick cleanup system with two prepackaged SNAP Ultra 340 g cartridges. Purification was carried out using a linear gradient system from 100% dichloromethane to 90%/10% dichloromethane/methanol in 60 column volumes. The tubes containing the desired product were combined and dried under reduced pressure at 40° C. for 1 hour and under high vacuum for an additional 6 hours to give product E as a foamy yellow solid. Yield: 9 g, 17.1 mmol, 42%. HPLC (method 1, 220 nm) over 95%. LRMS-ESI (m/z) calc. for C ₂₂ H ₂₇ N ₄ O ₉ S [ M-H] ^- : 523.16. Result: 523.15.

Synthesis of the linker Sulf07, 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazinecarbonyl)benzenesulfonic acid

[00168 ] To a solution of the yellow product E (10.2 g, 19.4 mmol, 1.0 equiv.) in anhydrous dichloromethane (30 ml), which was cooled to 4-5°C, was added dropwise (~1 ml /2.0 min) for 30 minutes trifluoroacetic acid (15 ml, Roth.). After the addition, the cold bath was removed and the reaction mixture was allowed to stir at room temperature for 3 hours. After this time, the completion of the reaction was confirmed by HPLC (method 1, 220 nm). The reaction mixture was poured dropwise into 6 Falcon tubes, each containing ~35 ml of cold diethyl ether. A white precipitate formed immediately. The tubes were left to stand at 4°C for 3 hours. After centrifuging the Falcon tubes for 20 minutes at 10°C and 4000 rpm, the supernatants were removed and the solids were resuspended in 5 ml of diethyl ether per tube and centrifuged again (4000 rpm, 20 min, 10°C). The supernatants were again removed and the solids were collected and dried under high vacuum for 20 hours to give the Sulf07 linker as a white microcrystalline solid. Yield: 10 g, 23.6 mmol, 96%. HPLC (method 1, 220 nm) over 95%. LRMS-ESI (m/z) calc. for C ₁₇ H ₂₁ N ₄ O ₇ S [ M+H] ⁺ : 425.11. Result: 425.07. LRMS-ESI (m/z) calc. for C ₁₇ H ₁₉ N ₄ O ₇ S [ M-H] ^- : 423.11. Result: 423.12. HRMS-ESI (m/z) calc. for C ₁₇ H ₂₁ N ₄ O ₇ S [ M+H] ⁺ : 425.1125. Result: 425.1125. LRMS-ESI (m/z) calc. for C ₁₇ H ₁₉ N ₄ O ₇ S [ M-H] ^- : 423.0978. Result: 423.0980.

[00169 ] The structure of 5-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexaanamido)-2-(hydrazinecarbonyl)benzenesulfonic acid, Sulf07, was confirmed by 1H NMR: ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 11.94 (s, 1H; C1-NH), 10.27 (s, 1H; C8-NH), 8.01 (d, J=2.2 Hz , 1H; C4-CH), 7.93 (dd, J=8.5, 2.2 Hz, 1H; C6-CH), 7.68 (d, J=8.4 Hz, 1H; C7-CH ), 7.00 (s, 2H; C15-CH, C16-CH) 3.40 (t, J=7.0 Hz, 2H; C13-CH ₂ ), 2.32 (t, J=7.4 Hz, 2H; C9-CH ₂ ), 1.60 (p, J=7.5 Hz, 2H; C10-CH ₂ ), 1.52 (p, J=7.2 Hz, 2H; C12-CH2) , 1.26 (q, J=8.8 Hz, 2H; C11-CH ₂ ); ¹³ C NMR (101 MHz, DMSO- d ₆ ) δ 172.20 (C8), 171.54 (C14, C17), 167.59 (C1), 145.73 (C5), 142.09 (C3), 134.90 (C15, C16), 132.09 (C7), 123.79 (C2), 119.44 (C6), 117.47 (C4), 37.42 (C13), 36.65 (C9) , 28.22 (C12), 26.21 (C11), 24.90 (C10). Elemental analysis calc. for C ₁₇ H ₂₁ N ₄ O ₇ S; C 45.71; H 4.26; N 11.85; S 6.78. Found: C 46.2917; H 4.4836; No. 12.8879; S 6.7886. TFA content < 2% (w/w).

[00170 ] Sulf07, 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazine-carbonyl)benzenesulfonic acid was prepared as described below, and shown in Scheme 2, respectively, of the route of synthesis of B.

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

N , N-diisopropylethylamine (DIPEA, 40.20 mL, 29.75 g, 230.20 mmol, 1.00 equiv.) for 5 minutes. Then EDC-HCl (48.54 mg, 253.21 mmol, 1.10 equiv.) was added and the mixture was stirred at 23° C. for 30 minutes. Then tert-butylcarbazate (33.46 g, 253.18 mmol, 1.10 eq.) was added and the mixture was heated to 70° C. and stirred for 2 hours. A second portion of EDC-HCl (24.31 g, 126.68 mmol, 0.55 eq.), tert-butylcarbazate (16.76 mg, 126.68 mmol, 0.55 eq.) was added and the reaction mixture was further stirred at 70°C for 14 hours. The reaction mixture was cooled to 23°C and filtered through Celite® 545 (100 g). Celite® 545 was further washed with 700 ml of methanol. The filtrate was concentrated under reduced pressure and purified using a fast cleaning system using seven prepackaged SNAP Ultra 340 g cartridges. Purification was carried out using a linear gradient system from 100% dichloromethane to 70% dichloromethane/30% methanol to give the title product F as a white solid. Yield: 32.50 g, 98.09 mmol, 42.6%, HPLC (method 1, 220 nm) over 97%. LRMS-ESI (m/z) calc. for C ₁₂ H ₁₆ N ₃ O ₆ S [ M-H] ^- : 330.08. Result: 330.17.

Synthesis of N-ethyl-N-isopropylpropan-2-amine 2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1-)hexanamido)benzenesulfonate (Product E )

[00172 ] To a solution of product F (26.97 g, 81.40 mmol, 1.00 eq.) in a mixture of anhydrous dimethylacetamide (110 ml) and anhydrous tetrahydrofuran (THF, 275 ml) was added a solution of 6-maleimidocaproic acid chloride (product C ) (26.60 g, 115.82 mmol, 1.42 eq.) in anhydrous tetrahydrofuran (165 ml) in one portion at room temperature. The clear reaction solution was stirred at room temperature for 10 minutes. The reaction mixture was then added with stirring to two 2 liter conical flasks, each containing diisopropyl ether (1100 ml) and DIPEA (10.15 ml). After settling the oil, the supernatant was carefully poured out and the oil was washed with diisopropyl ether (2×100 ml), dissolved in methanol, combined and the solvent was removed under high vacuum. The resulting oil was purified using a flash purification system using six prepackaged 340 g SNAP Ultra cartridges. Purification was carried out using a linear gradient system from 100% dichloromethane to 80% dichloromethane/20% methanol to give the title compound E as a white solid. substances. Yield: 24.00 g, 36.71 mmol, 45.1%, HPLC (method 1, 220 nm) over 95%. LRMS-ESI (m/z) calc. for C ₂₂ H ₂₇ N ₄ O ₉ S [ M-H] ^- : 523.15. Result: 523.30.

Synthesis of 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazine-carbonyl)benzenesulfonic acid ( Sulf07 )

[00173 ] To a cold solution (4°C) of product E (21.00 g, 32.12 mmol, 1.00 eq.) in dichloromethane (150 ml) was added trifluoroacetic acid (25.00 ml, 37.25 g, 326.70 mmol, 10.17 eq.) for more than 1 hour. The reaction mixture was then allowed to warm gradually (over 30 minutes) to room temperature and stirred for 1 hour. The reaction mixture was added dropwise via a separating funnel over 1 hour to a 2 L conical flask containing diisopropyl ether (1.4 L) at 0°C. The resulting white solid was filtered through a funnel with a porosity of 4 Å and washed successively with dichloromethane (2×1000 ml) and diisopropyl ether (2×1000 ml). The solid was left to dry on a fritted funnel overnight at room temperature. Further drying was carried out in high vacuum at 25° C. for 2 hours. The final product Sulf07 was obtained as a white solid. Yield: 13.58 g, 32.00 mmol, 99.6%, HPLC (method 1, 220 nm) > 96%. LRMS-ESI (m/z) calc. for C ₁₇ H ₁₉ N ₄ O ₇ S [ M-H] ^- : 423.10. Found: 423.21.

[00174 ] Sulf07, 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazine-carbonyl)benzenesulfonic acid was prepared as described below and shown in Scheme 2, respectively, the pathways for the synthesis of C.

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

[00175 ] Triethylamine (41.93 g, 57.76 ml , 414.37 mmol, 3 00 eq.) and the mixture was stirred for 10 minutes. Then tert-butylcarbazate (27.38 g, 207.19 mmol, 1.50 eq.) was added and the mixture was cooled to minus 35°C. At this temperature, a solution of propylphosphonic anhydride, T3P (114.27 g, 106.79 ml, 179.56 mmol, 50% solution in ethyl acetate, 1.3 eq.) was added dropwise over 1 hour. The reaction mixture was stirred at minus 35°C for 2 hours. The mixture was allowed to warm to room temperature and filtered through Celite® 545 (100 g). Celite® was further washed with acetonitrile (500 ml). Both filtrates were combined and concentrated to 250 ml. The solution was divided equally into 6 portions and the solvent was removed under reduced pressure. Each batch was dissolved in dichloromethane containing 1% Et ₃ N (50 mL) and purified by NP flash chromatography on a Biotage IsoleraTM One Rapid Purification System, pre-filled with a 340 g SNAP Ultra column, using a stepwise gradient from 2% to 12% methanol (containing 1% NEt3) in DCM (containing 1% NEt ₃ ) over 7 column volumes. The purified fractions from all portions were then combined, the solvent was removed under reduced pressure, and the solid was dried under high vacuum to give the title compound F as an off-white solid. Yield: 53.25 g, 108.0 mmol, 78.2% (NMR in DMSO-d6 showed the presence of 1.6 eq. triethylamine). HPLC (method 9, 220 nm) over 99%. LRMS-ESI (m/z) calc. for C ₁₂ H ₁₆ N ₃ O ₆ S [ M-H] ^- : 330.08. Result: 330.08.

Synthesis of N-ethyl-N-isopropylpropan-2-amine 2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1-)hexanamido)benzenesulfonate (Product E )

[00176 ] To a mixture of product F (45.00 g, 91.24 mmol, 1.00 eq.) and 6-maleimidocaproic acid (19.27 g, 91.24 mmol, 1.00 eq.) was added in one portion at room temperature acetonitrile (450 ml), triethylamine (13.85 g, 19.08 ml, 136.86 mmol) and T3P (43.55 g, 40.70 ml, 136.86 mmol, 50% solution in ethyl acetate). The solution was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure. The crude product was then purified with a flash purification system using seven prepackaged 340 g SNAP Ultra cartridges with a linear gradient from 2% methanol to 15% methanol in dichloromethane to give the title compound E as an off-white solid. Yield: 30.55 g, 53.5% (NMR in DMSO-d6 indicated the presence of 1.1 eq triethylamine). HPLC (method 9, 220 nm) over 99%. LRMS-ESI (m/z) calc. for C ₂₂ H ₂₇ N ₄ O ₉ S [ M-H] ^- : 523.15. Result: 523.26.

Synthesis of 5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazine-carbonyl)benzenesulfonic acid ( Sulf07 )

[00177 ] Trifluoroacetic acid (18.22 g, 12.31 ml, 159.81 mmol, 10.17 eq.) for 15 min. The mixture was further stirred at 4°C for 15 minutes and then allowed to gradually warm to room temperature and stirred for 150 minutes. The reaction mixture was added dropwise via a separating funnel to a stirred solution of methyl tert-butyl ether, MTBE (400 ml) and dichloromethane (200 ml). The resulting white solid was filtered through a 4 Å funnel and washed successively with dichloromethane (2 x 150 ml) and MTBE (1 x 50 ml), MeOH (1 x 50 ml) and again with MTBE (2 x 150 ml). The solid was allowed to dry on a fritted funnel overnight at room temperature for 10 minutes. Further drying was carried out in high vacuum at 25° C. for 18 hours. The final product Sulf07 was obtained as a yellow solid. Yield: 5.786 g, 13.63 mmol, 98.7%, HPLC (method 9, 220 nm) over 96%. LRMS-ESI (m/z) calc. for C ₁₇ H ₁₉ N ₄ O ₇ S [ M-H] ^- : 423.10. Result: 422.95.

Example 2

[00178 ] Compound AE-Keto-Sulf07 , 2-(2-((R)-2-((2R, 3R)-3-((S)-1-((3R, 4S, 5S)-4-( (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanamido)-1-phenylpropylidene)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)benzenesulfonic acid was synthesized as described below, and shown in Figure 5.

Synthesis of (S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2 -methyl-3-oxo-3-(((R)-1-oxo-1-phenylpropan-2-yl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl) (methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-N,N,3-trimethyl-1-oxobutan-2-amine 2,2,2-trifluoroacetate (AE-Keto TFA salt)

Synthesis Route A: Synthesis according to the improved procedure of US6884869-B2.

[00179 ] Pyridinium chlorochromate (PCC) (382 mg, 1.772 mmol, 1.5 eq., Sigma Aldrich) was added to a solution of auristatin E TFA salt (1000 mg, 1.183 mmol, Levena Biopharma) in anhydrous dichloromethane (30 ml) under atmosphere nitrogen. The reaction mixture was stirred at room temperature for 17 hours. LC-MS (method 4, positive mode) showed complete conversion of starting material. The reaction mixture was diluted with 25 ml dichloromethane and washed with brine (3×20 ml). The aqueous phase was extracted once with dichloromethane (20 ml). The combined organic phases were evaporated under reduced pressure at 40°C. The residue was dissolved in 7.5 ml acetonitrile and purified by RP flash chromatography with a prepackaged 30 g SNAP Ultra C18 cartridge using a step gradient from 2% to 40% acetonitrile (+0.1% TFA) in water (+0 .1% TFA) in more than 16 column volumes. The pure product fractions were combined, the acetonitrile was removed under reduced pressure at 40° C. and the residual solvent was lyophilized to give a white solid. Yield: 530 mg, 53%, RP-HPLC (method 2, 220 nm): over 95%. LRMS-ESI (m/z) calc. for C ₄₀ H ₆₈ N ₅ O ₇ [ M+H] ⁺ : 730.51. Result: 730.34.

Synthesis route B: Synthesis using a polymer-bound oxidant.

[00180 ] IBX-polystyrene (1.13 g, 4.5 eq., 1.34 mmol, loading 1.18 mmol/g, Merck Novabiochem) was suspended in anhydrous dichloromethane (7.5 ml) and stirred gently for 15 minutes at room temperature. TFA salt AE (250 mg, 295.51 µmol, Sage Chemical Co.) dissolved in anhydrous dichloromethane (5 ml) was added and the mixture was gently stirred at room temperature. After 21 hours, complete conversion of starting material to product was observed by HPLC (method 2, 220 nm). The resin was removed by filtration on folded filter paper (class 3, Sartorius), washed with dichloromethane and the combined filtrates were evaporated to dryness under reduced pressure at 40°C. The oily residue was triturated with diethyl ether to form an off-white precipitate which was dried under high vacuum to give a foamy off-white solid. Yield: 171 mg, 69%, HPLC (method 2, 220 nm) over 99%. LRMS-ESI (m/z) calc. for C ₄₀ H ₆₈ N ₅ O ₇ [ M+H] ⁺ : 730.51. Result: 730.69; calculated for C ₄₀ H ₆₆ N ₅ O ₇ [ M-H] ^- : 728.50. Result: 728.79.

Synthesis 2- (2-((R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S )-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-1-phenylpropylidene)hydrazin- 1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)benzenesulfonic acid ( AE-Keto-Sulf07 )

[00181 ] Synthesis 1 Under nitrogen, the AE-Keto salt of TFA (200 mg, 236.96 µmol) was dissolved in anhydrous ethanol (6 ml) followed by the addition of molecular sieves (0.3 nm beads, 3A). Sulf07 (201 mg, 473.92 µmol, 2 eq.) dissolved in anhydrous dimethyl sulfoxide (1.5 ml) was added. The yellow solution was stirred at room temperature. Samples for HPLC/LC-MS analyzes (methods 2 and 4, 220 nm) were taken after 24 and 44 hours. After conversion (85% after 44 hours), the mixture was filtered through a 0.2 μm PVDF syringe filter, most of the ethanol was removed under reduced pressure. 3 ml of 200 mM sodium phosphate pH 7.2 was added dropwise until the pH of the solution was ~6. The solution (˜5 ml) was purified by RP flash chromatography on a pre-packaged 30 g SNAP Ultra C18 cartridge using a stepwise gradient from 2% to 50% acetonitrile in water over 16 column volumes. Product fractions were analyzed by HPLC (method 2, 220 nm), pure fractions were pooled, acetonitrile was removed under reduced pressure and the remaining solution was lyophilized to give an off-white fluffy solid. Yield: 113 mg, 42%, melting point: above 105°C. HPLC (method 2, 220 nm) over 95%. LRMS-ESI (m/z) calc. for C ₅₇ H ₈₆ N ₉ O ₁₃ [ M+H] ⁺ : 1136.61. Result: 1136.76; calculated for C ₅₇ H ₈₄ N ₉ O ₁₃ [ M-H] ^- : 1134.59. Result: 1135.09. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 11.21 (2 x s, 1H), 10.09 (2 x s, 1H), 8.66 (d, J=8.3 Hz, 1H) , 8.20 (2 x d, J=8.4 Hz, 1H), 7.84-7.69 (m, 2H), 7.50-7.41 (m, 1H), 7.35-7 .21 (m, 5H), 6.98 (s, 2H), 4.96-4.84 (m, 1H), 4.77-4.60 (m, 1H), 4.56 (td, J =8.6, 2.2 Hz, 1H), 4.03-3.92(m, 1H), 3.61-3.52(m, 1H), 3.51-3.41(m, 2H ), 3.38 (t, J=7.0 Hz, 2H), 3.35-3.32 (m, 1H), 3.20 (m, 7H), 3.02 (2 x s, 3H) , 2.61 (2 x s, 6H), 2.47-2.37 (m, 1H), 2.26 (t, J=7.4 Hz, 2H), 2.22-2.04 (m , 3H), 2.04-1.91 (m, 1H), 1.88-1.63 (m, 3H), 1.62-1.43 (m, 7H), 1.37-1.27 (m, 4H), 1.27-1.19 (m, 2H), 1.06 (d, J=6.6Hz, 2H), 0.98 (d, J=6.6Hz, 1H) , 0.96-0.88 (m, 8H), 0.87-0.81 (m, 5H), 0.80-0.73 (m, 5H). ¹³ C NMR (101 MHz, DMSO- d ₆ ) δ 172.67, 172.38, 172.26, 171.47, 171.45, 171.13, 171.10, 168.76, 168.52, 166 .64, 166.23, 164.82, 164.61, 156.63, 155.75, 144.76, 144.68, 140, 27, 140.14, 134, 46.133.00, 131.89, 131 .79, 128.78, 128.56, 128.40, 128.31, 127.66, 127.61, 127.10, 120.25, 118.66, 118.56, 116.47, 116.34 , 85.45, 81.38, 77.69, 76.74, 71.91, 60.94, 60.20, 58.57, 58.16, 57.05 , 55.07, 54.48, 54 .39, 49.25, 48.86, 47.18, 46.24, 43.58, 43.12, 41.50, 41.44, 37.15, 36.96, 36.15, 35.01 , 31.83, 31.57, 30.04, 29.94, 27.77, 26.68, 26.64, 25.76, 25.39, 25.27 24.52, 24.50, 24, 29, 23.17, 19.30, 19.28, 18.85, 18.62, 18.57, 18.48, 18.20, 17.17, 16.92, 15.58, 15.51, 15.23, 14.96, 10.43, 10.25. Note: Some ¹ H-NMR signals split into two different signals due to the presence of conformers in solution. For this reason, the number of peaks in the ¹³ C-NMR spectrum is higher than the number of carbon atoms expected for AE-Keto-Sulf07.

[00182 ] Synthesis 2 In a 25 ml round bottom flask under nitrogen atmosphere, AE-Keto (150 mg, 0.177 mmol, 1.0 eq.) and Sulf07 prepared from Synthesis Route C (93 mg, 0.213 mmol, 1.2 eq. .), was dissolved in a mixture of anhydrous EtOH (225 μl) and anhydrous DMSO (225 μl). Complete dissolution was achieved after 5 minutes of sonication at room temperature. Activated molecular sieves were added and the resulting yellow slurry was stirred at room temperature and progress was checked by HPLC analysis. After 22 hours, the reaction mixture was transferred to a 2 ml Eppendorf tube, the reaction vessel was washed twice with 600 μl of DMSO-EtOH (1:1) and the pooled portions were centrifuged. The solid was separated from the supernatant. The product was precipitated by adding 45 µl of MTBE to the supernatant. The white suspension was centrifuged and the resulting supernatant was removed. Purification of the target compound was carried out on an Isolera One system (Biotage AB, Uppsala, Sweden) equipped with an AQ C18 20-35 µm 100A 40 g spherical cartridge with a 2% to 50% water/acetonitrile gradient. After lyophilization, the title compound was obtained as a white solid. Yield 80 mg, 40% HPLC (method 10, 220 nm) over 97%. The results of NMR and LC-MS analysis were as described above.

Example 3

[00183 ] Compound AE-Ester-Sulf07, 2-(2-(1(4-(((1S,2R)-2)-((2R,3R)-3-((S)-1-((3R ,4R,5S)-4-((S)-2-((R)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2- yl)-3-methoxy-2-methylpropanamido)-1-phenylpropoxy)carbonyl)phenyl)ethylidene)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1-yl)hexanamido)benzenesulfonic acid was synthesized as described below and shown in Scheme 6.

Synthesis of (1S,2R)-2-((3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino )-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-1-phenylpropyl-4-acetylbenzoate ( AE-Ester )

Synthesis according to the improved method of patent US6884869-B2

[00184 ] To a solution of the TFA salt of auristatin E (1.0 g, 1.18 mmol, 1.0 eq., Levena Biopharma) and 4-acetylbenzoic acid (198 mg, 1.42 mmol, 1.2 eq., Acros Organics) in anhydrous dichloromethane (18 ml). 4-dimethylaminopyridine (DMAP, 244 mg, 2.36 mmol, 2.0 eq., Carbolution) and N,N'-diisopropylcarbodiimide (DIC, 186 µl, 1.42 mmol, 1.2 eq., Sigma Aldrich). The mixture was stirred at room temperature for 5 hours. The progress of the reaction was monitored by HPLC (method 2, 220 nm) showing a conversion to the desired product of 81%. Then DIC (155 μl, 1.18 mmol, 1.0 eq., Sigma Aldrich) was added and the mixture was left to stir at room temperature for 18 hours, after which HPLC showed 98% conversion of the reaction to the desired product. During the synthesis of the various parts of the compound, it was noted that when using fresh DIC from a newly opened bottle, there was no need for a second addition. Molecular sieves and fine precipitate, a by-product of DIC, were filtered through folded filter paper (grade 3 hw, Sartorius) and the resulting clear yellow solution was washed once with HCl (0.1 M, 15 ml). The aqueous layer was back extracted once with dichloromethane (15 ml), then the organic layer was combined, washed once with brine (15 ml) and dried over sodium sulfate. The solution was concentrated under reduced pressure at 40° C. to give a yellow oil which was then dissolved in a minimum amount of dichloromethane (5 ml). An excess of diethyl ether (50 ml) was added to give a white solid. The mixture was left at 2-4°C for 30 min, and then the solid was separated by centrifugation. The precipitation procedure was repeated three times, and the white powder was dried under reduced pressure. Yield: 706 mg, 68% yield. HPLC purity (method 2, 220 nm) 98%. LRMS-ESI (m/z) calculated for C ₄₉ H ₇₅ FN ₅ O ₉ [ M+H] ⁺ : 879.20. Result: 878.70.

Synthesis 2- (2-(1-(4-(((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-) -((S)-2-((R)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy- 2-methylpropanamido)-1-phenylpropoxy)carbonyl)phenyl)ethylidene)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido) benzenesulfonic acid ( AE-Ester-Sulf07 )

[00185 ] A solution of Sulf07 (164 mg, 0.31 mmol, 1.1 equiv.) in anhydrous dimethyl sulfoxide (2 ml) was added to a solution of AE-Ester (243 mg, 0.28 mmol, 1.0 equiv.) in anhydrous ethanol (8 ml) in the presence of molecular sieves (beads 0.3 nm, 3 Å). Para-toluenesulfonic acid (10.5 mg, 0.05 mmol, 0.2 equiv.) was added to catalyze the reaction, and then the reaction mixture was stirred at room temperature. TFA can also be used in place of p-toluenesulfonic acid. The reaction was monitored by HPLC (method 2, 220 nm) showing complete conversion after 15 hours. Molecular sieves and the resulting fine precipitate were removed from the reaction solution by filtration through folded filter paper (grade 3 hw, Sartorius). The solution was precipitated with an excess of methyl tert-butyl ether (60 ml) and the mixture was left at 2-4° C. for 30 minutes. The precipitate was then separated by centrifugation to give a yellow sticky solid. The yellow sticky solid was dissolved in methanol/dichloromethane 3/7 (10 mL) and the solution was injected into a flash cleaning system with a pre-packaged SNAP Ultra 25 g cartridge. Purification was performed on normal phase using a step gradient from 100% dichloromethane to 60% dichloromethane/40% methanol in 17 column volumes. The tubes containing the product were pooled and dried under high vacuum. Yield: 162 mg, 45% yield. Purity by RP-HPLC (method 2, 220 nm) 96%. LRMS-ESI (m/z) calculated for C ₆₆ H ₉₃ N ₉ O ₁₅ S [ M+H] ⁺ : 1285.58. Result: 1284.20. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.24 (d, J=5.3 Hz, 1H), 10.21 (s, 1H), 9.20 (2 × br.s, 1H) , 8.32-7.90 (m, 8H), 7.74-7.71 (dd, J=8.5, 2.7 Hz, 1H), 7.41-7.27 (m, 5H) , 7.00 (s, 2H), 6.13-6.02 (2xd, J=5.0 Hz, 1H), 4.80-4.51 (m, 2H), 4.41-4, 23 (m, 1H), 4.09-3.55 (m, 3H), 3.54-3.37 (t, J=7.0 Hz, 3H), 3.27-3.17 (m, 8H), 3.00 (s, 2H), 2.88-2.63 (m, 5H), 2.37-2.30 (m, 6H), 2.22-1.80 (m, 4H) , 1.75-1.48 (m, 7H), 1.42-1.22 (m, 6H), 1.18. 1.11 (m, 3H), 1.10-1.04 (m, 3H), 1.00-0.74 (m, 20H). ¹³ C NMR (101 MHz, DMSO) δ 172.93, 172.65, 171.44, 170.94, 168.61, 168.49, 164.59, 164.52, 164.24, 164.16, 149.20, 144.83, 143.05, 142.96, 140.71, 137.97, 137.78, 134.30, 132.25, 129.43, 129.38, 129.31, 129, 21, 128, 39, 128, 31, 126, 49, 126.44, 126.35, 125.72, 118.77, 116.77, 85.20, 81.26, 77.65, 77.04, 76.84, 60.82, 60.12, 58.59, 58.03, 57.01, 48.35, 47.76, 47.15, 46.03, 43.60, 42.99, 36, 89, 36.11, 31.64, 29.89, 29.73, 27.70, 26.56, 25.70, 25.33, 25.04, 24.42, 24.04, 23.03, 19.16, 18.63, 18.44, 18.37, 15.50, 15.47, 15.29, 15.14, 13.88, 13.84, 10.19 10.04. Note: Some ¹ H-NMR signals split into two different signals due to the presence of conformers in solution. For this reason, the number of peaks in the ¹³ C-NMR spectrum is higher than the number of carbon atoms expected for AE-Ester-Sulf07.

pH dependent release of AE-Keto-Sulf07 and AE-Ester-Sulf07 human serum albumin conjugates at pH 7.4 and pH 4.1 (FIGS. 11 and 12)

[00186 ] Auristatin-Sulf07 albumin-binding derivatives were prepared as 2 mM stock solutions in anhydrous DMSO.

[00187 ] Release at pH 7.4: 442.4 µl of 1000 µM reconstituted human serum albumin (881 µM free cysteine-34) and 727.6 µl of PBS buffer (4 mM sodium phosphate pH 7.4 and 150 mM NaCl) were added into a sealed HPLC vial and incubated at 37°C for 30 minutes. After 30 minutes incubation, 130 μl of the appropriate DMSO drug stock solution was added to the pre-incubated vial to obtain a 200 μM auristatin solution and a 300 μM albumin (free cysteine-34) solution. The mixture was allowed to react for 10 minutes at 37° C. and then analyzed by HPLC (method 7, 20 μl injection). The injections were repeated after 1 hour, and then every hour until 24 hours.

[00188 ] Release at pH 4.1: 408.5 µl of 1000 µM reconstituted human serum albumin (881 µM free cysteine-34) and 539.4 µl of water were added to a sealed HPLC vial and incubated at 37°C for 30 minutes. In a separate vial, 190 μl of sodium acetate buffer 50 mM pH 3.0 and 24.7 μl of 1 M HCl were incubated at 37°C for 30 minutes. After 30 minutes of incubation, 120 µl of the appropriate DMSO drug stock solution was added to the pre-incubated vial to prepare an albumin drug conjugate. After 10 minutes of incubation, 132 μl sodium acetate buffer solution was added to obtain a 200 μM auristatin derivative solution and 300 μM albumin (free cysteine-34) solution. The resulting solution was analyzed directly by HPLC (method 7). The vial injection was repeated after 1 hour and then every hour until 24 hours.

[00189 ] The percentage of free drug released was determined by HPLC with a calibration curve (200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM) for free drugs, i. AE-Keto for AE-Keto-Sulf07 and AE-Ester for AE-Ester-Sulf07. Stock drug solutions were prepared using DMSO as solvent and diluted 10-fold with phosphate buffer (4 mM sodium phosphate, pH 7.4 and 150 mM NaCl) prior to HPLC analysis. Drug release calculations were based on AUC: for AE-Keto at 254 nm and AE-Ester at 310 nm (local ultraviolet maxima). The percentage of AE-Ester released was higher than the percentage of AE-Keto released when the pH changed from 7.4 to 4.1 (compare Figures 11 and 12).

Restoration of stability of AE-Keto-Sulf07 and AE-Keto-EMCH (Fig. 1)

[00190 ] AE-Keto albumin-binding drug derivatives were reconstituted in 50 mM sodium phosphate buffer, pH 7.6, which contained 5% sucrose (w/v) and 2% 2-hydroxypropyl-β-cyclodextrin (2-HPβCD ). Both preparations were reconstituted at a concentration of 1277 μM (equal to 4.5 mg/kg mouse xenograft dose) and dissolution of both preparations was confirmed by HPLC (method 2, 254 nM). The stability of the reconstituted drugs was monitored by HPLC at room temperature every 15 minutes for a period of 240 minutes for hydrolysis of the maleimide and loss of API. AE-Keto-Sulf07 was more stable than AE-Keto-EMCH (see Fig. 1).

Stability recovery of AE-Ester-Sulf07 and AE-Ester-EMCH (Fig. 2)

[00191 ] AE-Ester albumin-binding derivatives were reconstituted in 50 mM sodium phosphate buffer, pH 7.6, which contained 5% sucrose (w/v) and 4% 2-HPβCD. Both preparations were reconstituted at a concentration of 655 μM (equal to 2.4 mg/kg mouse xenograft dose), dissolution of both preparations was confirmed by HPLC (method 2, 310 nm). The stability of the reconstituted drugs was monitored by HPLC at room temperature every 15 minutes over a period of 240 minutes for maleimide hydrolysis and loss of API. AE-Ester-Sulf07 was more stable than AE-Ester-EMCH (see Fig. 2).

Binding kinetics of auristatin-Sulf07 derivatives in human plasma, rat plasma, and mouse CD1 plasma (FIGS. 5-10).

[00192 ] Mouse CD1 plasma and Sprague Dawley rat plasma were removed from storage at minus 80°C and allowed to reach room temperature. Thawed plasma was centrifuged at 13.6 kRPM for 60 seconds, the supernatant was filtered through a filter needle (5 µm) and then through a 0.45 µm CA membrane.

[00193 ] Whole human blood was taken from a healthy volunteer (EDTA collection tube), plasma was separated from erythrocytes by rotating it for 3 minutes at 3000 rpm. The plasma was then used directly within 2 hours of blood donation. 360 μl of appropriate plasma was used for each binding experiment.

[00194 ] All binding experiments were repeated three times. Plasma was incubated at 37° C. using an Eppendorf C thermomixer. After 30 minutes of incubation, 40 μl of the appropriate albumin-binding auristatin-Sulf07 derivative was dissolved in the reconstitution solution and added to the plasma. Samples (40 μl) were taken after 15 seconds, 2 minutes, 4 minutes, 8 minutes and 15 minutes (5 samples).

[00195 ] Samples were immediately added to 160 μl of acetonitrile (containing 4 μg/ml MMAE as an internal standard) and shaken for 1 minute.

[00196 ] Solutions were pipetted into an Impact plasma precipitation plate (shaken for 60 seconds) and vacuum filtered onto a 96-well plate. Remaining drug was quantified by LCMS (method 5, 30 μl injection) using MRM MS2 negative mode (multiple response monitoring). Parent ions: auristatin F (wt-744.8), AE-Keto-Sulf07 (wt-1135.1) and AE-Ester-Sulf07 (wt-1283.2). Percent binding was determined by comparing the AUC for AE-Keto-Sulf07 and AE-Ester-Sulf07 with calibration curves generated by LCMS for each compound.

Procedure: Binding specificity of auristatin-Sulf07 derivatives to cysteine-34 of human serum albumin (FIGS. 3-4).

[00197 ] Auristatin-Sulf07 albumin-binding derivatives were reduced in 50 mM sodium phosphate buffer pH 7.6 and 5% sucrose that contained 2-HPβCD. To restore AE-Keto-Sulf07, a 2% concentration of HPβCD and 4% for AE-Ester-Sulf07 were used, respectively. Both preparations were reconstituted at 1 mg/ml (AE-Ester-Sulf07 780 µM, AE-Keto-Sulf07 880 µM).

[00198 ] Human plasma was removed from storage at minus 80°C and allowed to reach room temperature. Thawed plasma was centrifuged at 13.6 kRPM for 60 seconds, the supernatant was filtered through a filter needle (5 µm) and then through a 0.45 µm CA membrane. 180 μl of appropriate plasma was used for each binding experiment. Plasma was incubated at 37° C. using an Eppendorf C thermomixer. After 30 minutes of incubation, 20 μl of the appropriate auristatin-Sulf07 albumin-binding derivative was added. The albumin-binding preparation was allowed to react in plasma for 5 minutes at 37°C. After 5 minutes, the solution was analyzed directly by HPLC with hydrophobic interaction (HIC, method 6, 250 nm) to confirm site-specific conjugation to albumin and formation of an albumin drug conjugate.

Accelerated degradation of lyophilized auristatin formulations: Stability comparison of EMCH and Sulf07 (FIGS. 24-25).

[00199 ] The API was dissolved in lyophilization buffer which contained 2-HPβCD, 10 mM sodium citrate, pH 6.2, 50% tert-butyl alcohol (V/V). For AE-Keto derivatives, 2% (w/v) 2-HPβCD was used to dissolve API AE-Keto at a concentration of 1.27 mM; for AE-ester derivatives, 4% (w/v) 2-HPβCD was used to dissolve the API at a concentration of 1.05 mM. The dissolved APIs were sterile filtered using an Acrodisc Fluorodyne II syringe filter (0.2 µm). Sterile solutions were pipetted into vials for lyophilization, which were then partially closed with a rubber stopper. The vials were then placed in a lyophilizer for lyophilization. The vials were frozen on the shelf of a freeze dryer at minus 40°C for 2 hours, after 2 hours the main drying was started. The main drying was carried out for 26 hours, parameters: shelf temperature minus 20°C, vacuum 0.0048 mbar. After the main drying, the final drying began. The final drying was carried out for 16 hours, parameters: shelf temperature 20°C, vacuum 0.0047 mbar. At the end of the final drying, the vials were closed in vacuum.

[00200 ] The sealed vials were incubated at 55°C using an Eppendorf C thermomixer with a plate liner (Smartblock) and cap. At selected time points (t: 0, 46, 94, 166 and 264 hours) the samples were removed and dissolved in anhydrous DMSO. API stability was determined by HPLC (method 8, 254 nm for AE-Keto derivatives, 310 nm for AE-Ester derivatives). Percent maleimide hydrolysis at each time point was determined by comparison with the AUC for each API at time 0. AE-Keto-Sulf07 and AE-Ester-Sulf07 were more stable than the corresponding EMCH derivatives AE-Keto-EMCH and AE-Ester-EMCH (see Fig. 24 and Fig. 25).

Example 4

General Procedure for Assessing Auristatin E, AE-Keto, and AE-Ester Against a Panel of Tumor Cell Lines

Definition of IC ₅₀

[00201 ] Samples were provided by Charles River Discovery Research Services Germany GmbH as pharmaceutical grade frozen stock solutions DMSO (Sigma-Aldrich, Taufkirchen, Germany). On each day of the experiment, a frozen aliquot of stock solution was thawed. Subsequent serial dilutions were made with RPMI 1640 complete culture medium using an intermediate dilution plate. 10 µl taken from the intermediate dilution plate was then transferred to 140 µl/well of the cell culture plate. Cells were treated with test compounds at 10 concentrations in triplicate in half log steps from 0.003 nM to 100 nM for 96 hours.

[00202 ] Compounds were tested in a panel of 6 selected human cancer cell lines using the CellTiter-Blue® cell viability assay (Promega, Mannheim, Germany). The IC values are presented as the absolute IC ₅₀ values obtained for the test compound as the geometric mean of the IC ₅₀ for all cell lines tested.

[00203 ] The cell lines used were LXFL 1674L (derived from a xenograft obtained from a patient at Charles River Discovery Research Services Germany GmbH), SW-620 (courtesy of NCI, Bethesda, MD, USA), CAL-27 (purchased from DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), RKO, MDA-MB-468 and SK-OV-3 (from ATCC, American Type Culture Collection, Rockville, MD, USA). All cell lines have been authenticated at the DSMZ by STR (short tandem repeat) analysis, a PCR-based DNA fingerprinting technique. The cell lines were usually passaged once or twice a week and maintained in culture for up to 20 passages. Cells were grown at 37° C. in a humidified atmosphere with 5% CO ₂ in RPMI 1640 medium (25 mM HEPES, with L-glutamine, Biochrom, Berlin, Germany) supplemented with 10% (v/v) fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany) and 0.1 mg/ml gentamicin (Life Technologies, Karlsruhe, Germany). CellTiter-Blue® cell viability assay was used according to the manufacturer's instructions. Briefly, cells were harvested from exponential phase cultures, counted, and plated in 96-well flat-bottomed microtiter plates at a cell density of 4,000-10,000 cells per well, depending on the growth rate of the cell line. After a 24 hour recovery period, 10 µl of culture medium (six control wells per plate) or test compound culture medium was added to allow the cells to resume exponential growth. Compounds were applied at 10 concentrations in triplicate in half log increments up to 100 nM and cells were treated continuously for 96 hours. After four days of cell treatment, 20 μl per well of CellTiter-Blue® reagent was added. After an incubation period of up to 4 hours, fluorescence (FU) was measured using an Enspire multimode plate reader (λ excitation = 531 nm, λ emission = 615 nm). IC ₅₀ values were determined using GraphPad Prism bioanalytical software (San Diego, CA, USA).

[00204 ] Concentration-dependent activities with sigmoidal concentration-effect curves were observed in the six tumor cell lines tested. Geometric mean IC ₅₀ values were 259±0.11 pM for AE-Keto and 399±0.19 pM for AE-Ester. Compared to the potent drugs MMAE (171±0.10 pM) and AE (130±0.05 pM), the AE-Keto and AE-Ester derivatives have similar cytotoxicity in the picomolar range.

Example 5

General procedure for evaluating the activity of auristatin E and auristatin E albumin-binding derivatives in patient-derived xenogeneic tumor models.

[00205 ] NMRI immunodeficient female nude mice from Charles River Discovery Research Services Germany GmbH were transplanted with unilateral human-derived tumor implants subcutaneously on the left flank, under isoflurane anesthesia, until the tumors engrafted and reached the desired volume.

[00206 ] Animals were kept in cages, the temperature inside the cages was maintained at 25°C ± 1°C with a relative humidity of 45-65% and an air exchange rate of 60 times per hour. Mice were maintained on a 14 hour light/10 hour dark cycle of artificial lighting. Animals were fed an autoclaved extruded diet with 19% protein Teklad Global (T.2019S.12) from Envigo RMS SARL, they had access to sterile filtered and acidified (pH 2.5) tap water, which was changed twice a week. The animals were provided with food and water ad libitum. Prior to therapy, animals were randomized (7-8 mice per group) based on comparable median and mean group tumor volume. Animals were regularly monitored twice a day on weekdays and daily on Saturdays and Sundays. Starting from day 0, the animals were weighed twice a week. Relative body weight (RBW) of individual animals was calculated by dividing the individual absolute body weight on day X (BWx) by the individual body weight on randomization day multiplied by 100%. Tumor volume was determined by two-dimensional caliper measurement on the day of randomization (Day 0) and twice a week thereafter. Tumor volumes were calculated using the following equation:

Tumor volume [ mm ³ ] = l [ mm] × w ² [ mm ² ] × 0.5, where "l" is the length and "w" is the width of the tumor. The relative individual tumor volume on day X (RTV _x ) was calculated by dividing the absolute individual tumor volume [ mm ³ ] of the corresponding tumor on day X (T _x ) by the absolute volume of the same individual tumor tumor on randomization day multiplied by 100%. Calculations have been applied to the extent that animal welfare policy allows. The destruction of individual mice was performed at a tumor volume> 2000 mm ³ (unilateral). To assess the statistical significance of antitumor efficacy, a non-parametric Kruskal-Wallis test was performed followed by Dunn's method for pairwise comparisons, whereby the individual RTVs of test and control groups were compared on days on which trough T/C values were reached in test groups where T/C [ %] = (median RTV _x treated group/median RTV _x control group) x 100. Statistical analysis was performed only if at least 50% of the originally randomized animals in the respective group were still alive. Comparisons between test groups were made on the same days. As a general rule, p≤0.05 values indicated the significance of tumor inhibition. Statistical calculations were performed using GraphPad Prism bioanalytical software (San Diego California, USA, www.graphpad.com).

General procedure for evaluating the efficacy of auristatin E and auristatin E albumin-binding derivatives in dose discovery studies in cell line-derived xenogeneic tumor models.

[00207 ] NMRI immunodeficient female nude mice, Janvier, France, were inoculated subcutaneously with 5 x 10 ⁶ -10 ⁷ cancer cells in buffer/Matrigel (1:1) until tumors were palpable and reached the desired volume (for xenograft studies).

[00208 ] Animals were housed in cages (Macrolon Type-II wire mesh) with temperature maintained at 22°C±1°C, relative humidity 50±10%, and air exchange rate 60 times per hour. Mice were maintained on a 12 hour light/12 hour dark cycle of artificial lighting. Animals were fed in a Ssniff NM autoclave (Soest, Germany) and had access to sterile filtered and acidified (pH 4.0) tap water, which was changed twice a week. The animals were provided with food and water ad libitum. Prior to therapy, animals were randomized (3-4 mice per group in dose discovery studies and 7-8 mice per group in xenograft studies) based on comparable median and mean tumor volume per group. The health status of the animals was checked at the beginning of the experiment and twice a day during the experiment. Identification used: ear mark and cage labels. Starting from day 0, animals were weighed two or three times a week and the mean body weight per group was linked to a baseline percentage to calculate body weight change (BWC). Tumor diameter was determined by two-dimensional caliper measurement on the day of randomization (Day 0) and then two or three times a week thereafter. Tumor volumes were calculated using the following equation:

Tumor volume [ mm ³ ] = l [ mm] × w ² [ mm ² ] × 0.5, where "l" is the length and "w" is the width of the tumor. The relative individual tumor volume on day X (RTV _x ) was calculated by dividing the absolute individual tumor volume [ mm ³ ] on day X (T _x ) by the absolute volume of the same individual tumor on randomization day multiplied by 100%. Calculations have been applied to the extent that animal welfare policy allows. The destruction of individual mice was performed when the tumor volume is greater than 1500 mm ³ (unilateral) or when ulceration is observed. Statistical comparison was performed using the Mann-Whitney U-test.

Dose-finding studies for auristatin E and auristatin E albumin-binding derivatives AE-Keto-Sulf07 and AE-Ester-Sulf07.

[00209 ] Stock solutions were prepared as follows:

1) 3 mice per group, 26 g average weight per randomization day: 0.3, 0.6, or 1.2 mg/kg auristatin E TFA salt (AE equivalent) administered once a week for 4 weeks per day 0, 6, 13, 20 ≡ 0.35-1.41 mg/kg ≡ 7.1-28.3 µg/20 g mouse. Sample preparation: 8.5 mg weighed into a 50 ml vial, dissolved in 30.0 ml 25/75 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 0.2-0.8 ml were divided into aliquots in 2 ml vials depending on the desired final dose. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 0.8 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 3 mice per group, 26 g average weight per day of randomization: 1.2 and 2.4 mg/kg AE-Keto-Sulf07 (AE equivalent) administered twice a week for 4 weeks ≡ 1.94-3 .87 mg/kg ≡ 38.7 and 77.4 g/20 g mouse. Sample preparation: 11.0 mg weighed into a 30 ml vial, dissolved in 14.2 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 0.4-0.8 ml were divided into aliquots in 2 ml vials depending on the desired final dose. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 0.8 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

3) 3 mice per group, 26 g mean weight per randomization day: 1.2, 1.6, or 2.0 mg/kg AE-Ester-Sulf07 (AE equivalent) administered twice a week for 4 weeks a day ≡ 2.18-3.64 mg/kg ≡ 43.7-72.8 µg/20 g mouse. Sample preparation: 19.2 mg were weighed into a 30 ml vial, dissolved in 26.3 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; aliquots of 0.48-0.8 ml were taken in 2 ml tubes depending on the desired final dose. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 0.8 ml of 50 mM sodium phosphate buffer, 5% Tween 80, pH 7.6.

4) 4 mice per group, mean weight 23 g per day of randomization: 2.0, 2.2 and 2.4 mg/kg AE-Ester-Sulf07 (AE equivalent) administered twice a week for 4 weeks a day ≡ 3.64-4.37 mg/kg ≡ 72.8-87.3 µg/20 g mice and 4.0, 4.4 and 4.8 mg/kg AE-Ester-Sulf07 (AE equivalent), which administered once a week for 4 weeks per day 0, 7, 14, 21≡7.28-8.73 mg/kg ≡ 145.6-174.7 μg/20 g mouse. Sample preparation: 56.0 mg weighed into a 50 ml vial, dissolved in 32.0 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 0.33-0.8 ml were aliquoted in 2 ml vials depending on the desired final dose. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 0.8 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

[00210 ] Auristatin E doses of 1.2 and 0.6 mg/kg showed high toxicity, and no animal survived the first and second injections, respectively. The dose of 0.3 mg/kg was well tolerated. In more recent studies, AE was found to be tolerated at 0.3 mg/kg administered twice a week for four weeks (eight injections in total), and therefore this dose was used as the maximum tolerated dose (MTD) in xenograft studies.

[00211 ] All doses tested for AE-Keto-Sulf07 administered eight times on a two-week schedule were well tolerated. In later xenograft studies, the dose for this drug was increased to 6.5 mg/kg at the same dosing schedule, causing weight loss in one mouse and general malaise in a second mouse at this maximum dose, and treatment had to be discontinued after five injections. and not eight as planned. Therefore, this dose was considered to be above the MTD and 4.5 mg/kg was chosen as the safe dose.

AE-Ester-Sulf07 was initially tested at 2.0 mg/kg twice a week for four weeks without any clear signs of toxicity. Because skin irritation was later observed in the xenograft studies, a repeat dose-finding study was repeated at doses of 2.0, 2.2, and 2.4 mg/kg twice a week for four weeks and compared with the corresponding doubling doses administered 4, 0, 4.4 and 4.8 mg/kg once a week only. No change in body weight was observed, but skin irritation appeared after 9-14 days in all groups. Early and more frequent skin irritation was observed at higher doses. The side effect seemed to be reversible as the irritations began to heal after the end of the treatment. The group receiving 4.0 mg/kg once a week showed the least side effect, while no significant difference was observed between the other groups.

Criteria for Evaluation of Auristatin E and Albumin-Binding Derivatives of Auristatin E in Tumor Xenograft Models

[00212 ] The effectiveness of each drug was determined based on the reduction in tumor volume (% of tumor volume on the selected day compared to day 0): complete remission less than 10%; partial remission more than 10-50%; insignificant remission more than 50-75%; stable disease more than 75-125%; progressive disease more than 125% compared with the initial volume of the tumor.

Evaluation of the efficacy of auristatin E and the auristatin E albumin-binding derivative AE-Keto-Sulf07 in the human malignant melanoma model A375 - small tumors (FIG. 13).

[00213 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the A375 human malignant melanoma model was performed as described in the general procedure for cell line-derived xenograft models.

[00214 ] Stock solutions were prepared as follows:

1) 7 mice, mean weight 29 g, mean median tumor volume 135 mm ³ on day of randomization: 0.3 mg/kg auristatin E TFA salt (AE equiv.) twice a week for 3 weeks a day 0, 6, 9 , 13, 16, 19 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 2 mg weighed into a 30 ml vial, dissolved in 28.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 h at minus 40°C and lyophilized at minus 20°C for approximately 36 h and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, mean weight 28 g, mean median tumor volume 137 mm ³ on day of randomization: 3.0 mg/kg AE-Keto-Sulf07 (AE equiv.) twice a week for 3 weeks on day 0.6, 9, 13, 16, 19 ≡ 5.32 mg/kg ≡ 106.3 µg/20 g mouse. Sample preparation: 19.2 mg were weighed into a 30 ml vial, dissolved in 19.0 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

[00215 ] Tumor growth development in a melanoma A 375 xenograft model showed statistically significant antitumor efficacy of compound AE-Keto-Sulf07 at a dose of 3.0 mg/kg compared with a dose of auristatin E 0.3 mg/kg, both administered six times twice a day. week (p<0.01 from days 27 to 37). Mice treated with auristatin E reached a state of stable disease until day 21, after which the tumor began to grow again. In contrast, mice in the AE-Keto-Sulf07 treated group achieved long-term tumor regression, showing complete remission from day 19 until the end of the study on day 37 (final tumor volume 1 mm ³ ).

[00216 ] Mice in the AE-Keto-Sulf07 treated group had only a slight reduction in body weight gain compared to controls, but no adverse effects were observed.

Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the A375 human malignant melanoma model - large tumors (Fig. 14).

[00217 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the A375 human malignant melanoma model was performed as described in the general procedure for cell line-derived xenograft models.

[00218 ] Stock solutions were prepared as follows:

1) 7 mice, mean weight 28 g, mean median tumor volume 310 mm ³ on day of randomization: 0.3 mg/kg TFA salt auristatin E (equiv. AE) twice a week for 3 weeks on days 1, 6, 9 , 13, 16 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 2 mg weighed into a 30 ml vial, dissolved in 28.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at -40°C and lyophilized at -20°C for approximately 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, mean weight 27 g, mean median tumor volume 331 mm ³ on the day of randomization: 6.5 mg/kg AE-Keto-Sulf07 (equiv. AE) twice a week for 3 weeks on days 1, 6, 9, 13, 16 ≡ 11.52 mg/kg ≡ 230.4 µg/20 g mouse. Sample preparation: 28.0 mg weighed into a 30 ml vial, dissolved in 12.2 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

[00219 ] The antitumor efficacy of the AE-Keto-Sulf07 compound was also tested in mice bearing large A375 melanoma malignancies (~320 mm ³ ) to prove that the efficacy of the drug is not limited to small tumors. Mice treated with AE-Keto-Sulf07 at 6.5 mg/kg showed a statistically significant improvement in antitumor effect compared to the group treated with auristatin E at 0.3 mg/kg, both administered five times twice a week (p <0.01 from day 9 to day 33). Auristatin E showed baseline stable disease until day 14 and then stable growth, while mice in the AE-Keto-Sulf07 treated group had complete remission from day 19 until the end of the study on day 37, reaching a minimum tumor volume of 1 mm ³ on day 28 (12 days after the last treatment) and ~14 ^mm3 at the end of the study, resulting in a long-term effect despite a relatively large average initial tumor volume.

[00220 ] AE-Keto-Sulf07 caused transient weight loss (13% achieved at day 21) and two mice had to be sacrificed due to general poor condition.

Evaluation of the efficacy of auristatin E and the auristatin E albumin-binding derivative AE-Keto-Sulf07 in the human non-small cell lung cancer LXFA737-small tumor model (Fig. 15).

[00221 ] Evaluation of the efficacy of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the LXFA737 human non-small cell lung cancer model was performed as described in the general procedure for patient-derived xenograft models.

[00222 ] Stock solutions were prepared as follows:

1) 7 mice, mean weight 27 g, mean median tumor volume 137 mm ³ on day of randomization: 0.3 mg/kg auristatin E TFA salt (AE equiv.) twice a week for 4 weeks on day 0, 4, 7 , 11, 14, 18, 21, 25 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 2 mg is weighed into a 30 ml vial dissolved in 28.3 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, mean weight 26 g, mean median tumor volume 128 mm ³ on day of randomization: 4.5 mg/kg AE-Keto-Sulf07 (AE equivalent) twice a week for 4 weeks on day 0, 4, 7 , 11, 14, 18, 21, 25 ≡ 7.98 mg/kg ≡ 159.5 µg/20 g mouse. Sample preparation: 38 mg were weighed into a 30 ml vial, dissolved in 23.8 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% 2-hydroxypropyl-β-cyclodextrin 2-HPβCD, pH 7.6.

3) 7 mice, mean weight 27g, mean median tumor volume ^130mm3 on randomization day: 4.5mg/kg AE-Keto-Sulf07 (AE equivalent), twice a week for 4 weeks on day 0.4, 7, 11, 14, 18, 21, 25 ≡ 7.98 mg/kg ≡ 159.5 µg/20 g mouse. Sample preparation: 38 mg were weighed into a 30 ml vial, dissolved in 23.8 ml of 50/50 t-butanol/10 mM sodium citrate buffer, 5% sucrose, pH 6; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

[00223 ] Tumor growth development in the NSCLC LXFE737 xenograft model showed superior antitumor efficacy of compound AE-Keto-Sulf07 at 4.5 mg/kg (in Tween 80 and 2-HPβCD buffers) compared to auristatin E at 0.3 mg/kg. kg eight times twice a week with statistical significance (p<0.01 on days 56 and 63). Tumors in the auristatin E group remained only temporarily in a stable disease state (from day 21 to day 42) and showed significant growth after the end of therapy. In contrast, mice treated with AE-Keto-Sulf07 achieved complete tumor remission on day 28, 3 days after the end of therapy, resulting in tumor volume reduction to an unmeasurable level until the end of the study on day 74 (regardless from the buffer used), resulting in a long-term antitumor effect.

[00224 ] The change in body weight compared to control showed a partial increase in toxicity in the group treated with AE-Keto-Sulf07. On day 32, maximum body weight loss (-7%) was recorded with no other signs of toxicity in the case of 2-HPβCD, while two mice had to be sacrificed due to general poor conditions when the drug was administered with Tween 80.

Evaluation of the efficacy of auristatin E and the auristatin E albumin-binding derivative AE-Keto-Sulf07 in the human non-small cell lung cancer LXFA737-large tumor model (Fig. 16).

[00225 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the LXFA737 human non-small cell lung cancer model was performed as described in the general procedure for patient-derived xenograft models.

[00226 ] Stock solutions were prepared as follows:

1) 8 mice, mean weight 26g, mean median tumor volume ^324mm3 on randomization day: 0.3mg/kg Auristatin E TFA salt (AE equivalent) twice a week for 4 weeks on days 1, 5, 8, 12, 15, 19, 22, 26 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 3 mg weighed into a 100 ml vial, dissolved in 43.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 2.4 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 2.4 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 8 mice, mean weight 28 g, mean median tumor volume 342 ^mm3 on randomization day: 4.5 mg/kg AE-Keto-Sulf07 (AE equivalent), twice a week for 4 weeks on days 1, 5, 8, 12, 15, 19, 22, 26 ≡ 6.99 mg/kg ≡ 139.7 µg/20 g mouse. Sample preparation: 51 mg were weighed into a 100 ml vial dissolved in 36.5 ml of 50/50 t-butanol/10 mM sodium citrate buffer, 3% 2-HPβCD, pH 6.2; 2.4 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 2.4 ml of 50 mM sodium phosphate buffer, 5% sucrose, pH 7.6.

[00227 ] Tumor growth development in the NSCLC LXFE737 xenograft model showed superior antitumor efficacy of compound AE-Keto-Sulf07 at a dose of 4.5 mg/kg compared to auristatin E at a dose of 0.3 mg/kg, both doses were administered 8 times twice per week, with statistical significance (p < 0.01 on day 28). Tumors in the auristatin E group did not show any activity, while mice treated with AE-Keto-Sulf07 achieved partial tumor remission on day 15 until the end of the study on day 59, resulting in a long-term antitumor effect.

[00228 ] No side effects have been reported.

Evaluation of the efficacy of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in human ovarian carcinoma, model A2780 - small tumors (Fig. 17).

[00229] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the A2780 human ovarian carcinoma model was performed as described in the general procedure for cell line-derived xenograft models.

[00230 ] Stock solutions were prepared as follows:

1) 7 mice with an average weight of 27 g, a mean median tumor volume of 147 mm ³ on the day of randomization: 0.3 mg/kg of auristatin E TFA salt (AE equivalent) twice a week for 4 weeks on days 1, 4, 8, 11, 15, 18, 22, 25 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 2 mg weighed into a 30 ml vial, dissolved in 28.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, mean weight 27 g, mean median tumor volume 153 mm ³ on randomization day: 3.0 mg/kg AE-Keto-Sulf07 (AE equivalent) twice a week for 4 weeks on days 1, 4, 8 , 11, 15, 18, 22, 25 ≡ 5.32 mg/kg ≡ 106.3 µg/20 g mouse. Sample preparation: 51.0 mg was weighed into a 30 ml vial, dissolved in 28.8 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 0.72 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

3) 7 mice, mean weight 25 g, mean median tumor volume 141 mm ³ on randomization day: 5.0 mg/kg AE-Keto-Sulf07 (AE equivalent), twice a week for 4 weeks on days 1, 4, 8, 11, 15, 18, 22, 25 ≡ 8.86 mg/kg ≡ 177.2 μg/20 g mouse. Sample preparation: 51.0 mg was weighed into a 30 ml vial, dissolved in 28.8 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

[00231 ] The development of tumor growth in the A2780 ovarian carcinoma model showed a statistically significant superior antitumor efficacy of the compound AE-Keto-Sulf07 at doses of 3.0 and 5.0 mg/kg compared to auristatin E at a dose of 0.3 mg/kg administered eight times twice a week (p < 0.01 from days 7 to 29). In mice treated with auristatin E, no antitumor effect was detected, and tumors grew comparable to tumors in the control group. However, AE-Keto-Sulf07 therapy produced a partial remission from day 9 to the end of the study on day 60, with a short-term complete remission from day 23 to day 35 for the lower dose, and from day 25 to day 51. day for the higher dose, resulting in prolonged tumor regression, regardless of dose.

[00232 ] The groups treated with AE-Keto-Sulf07 showed a temporary loss of body weight of ~2% after the first treatment. One mouse from the higher dose group was found dead on day 35 and one from the lower dose group was sacrificed on day 42 due to general ill health.

Evaluation of Auristatin E and Auristatin E Albumin-Binding Derivative AE-Keto-Sulf07 in Human Ovarian Carcinoma Model A2780 - Large Tumors (FIG. 18).

[00233 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Keto-Sulf07 in the A2780 human ovarian carcinoma model was performed as described in the general procedure for cell line-derived xenograft models.

[00234 ] Stock solutions were prepared as follows:

1) 8 mice, mean weight 28 g, mean median tumor volume 341 mm ³ on day of randomization: 0.3 mg/kg auristatin E TFA salt (AE equivalent) administered twice a week for 4 weeks on days 1, 4, 8, 11, 15, 18, 22, 25 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 3.7 mg was weighed into a 100 ml vial, dissolved in 52.3 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.5 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.5 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 8 mice, mean weight 28 g, mean median tumor volume 378 mm ³ on randomization day: 4.5 mg/kg AE-Keto-Sulf07 (AE equivalent), twice a week for 4 weeks on days 1, 4, 8, 11, 15, 18, 22, 25 ≡ 6.99 mg/kg ≡ 139.7 µg/20 g mouse. Sample preparation: 40.0 mg weighed into a 30 ml vial, dissolved in 28.6 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.5 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.5 ml of 50 mM sodium phosphate buffer, 2% 2-HPβCD, pH 7.6.

[00235 ] The antitumor efficacy of compound AE-Keto-Sulf07 was also tested in mice with larger tumors (~360 mm ³ ) of the A2780 human ovarian cancer model. The drug showed a statistically significant superior antitumor effect at a dose of 4.5 mg/kg compared with auristatin E dosed at a dose of 0.3 mg/kg, both drugs were administered eight times twice a week (p<0.001 from 22 to 40 days). In mice treated with auristatin E, no antitumor effect was obtained, in contrast to mice treated with AE-Keto-Sulf07, where there was a complete remission from day 26 to the end of the study at day 103, and long-term tumor regression was achieved, as already observed with a smaller tumor volume.

[00236 ] Mice treated with AE-Keto-Sulf07 had only slightly reduced body weight gain compared to controls.

Initial Evaluation of the Efficacy of Auristatin E and the Auristatin E Albumin-Binding Derivative AE-Ester-Sulf07 in the Human Kidney Cancer Model RXF631 - Small Tumors (Fig. 19).

[00237 ] An initial evaluation of the efficacy of auristatin E and auristatin E albumin-binding derivative AE-Ester-Sulf07 in the human kidney cancer model RXF631 was performed as described in the general procedure for the patient-derived xenograft model.

[00238 ] For this in vivo experiment, stock solutions were prepared as follows:

1) 7 mice, 25 g mean weight, 138 mm ³ mean tumor volume on day of randomization: 0.3 mg/kg Auristatin E TFA salt (AE equivalent) administered twice a week for 4 weeks on days 1, 5, 8 , 12, 15, 19, 22, 26 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 2 mg weighed into a 30 ml vial, dissolved in 28.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, 26 g mean weight, 141 mm ³ mean tumor volume on day of randomization: 2.2-3.0 mg/kg AE-Ester-Sulf07 (AE equivalent) administered twice a week for 4 weeks a day 1, 5, 8, 12, 15, 19, 26 ≡ 4.00 mg/kg ≡ 80.1 µg/20 g mouse. Sample preparation: 15.7 mg were weighed into a 30 ml vial, dissolved in 19.6 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

[00239 ] Tumor growth in the RXF631 renal cell cancer model shows a statistically significant superior antitumor efficacy of AE-Ester-Sulf07 compound dosed at 2.2 mg/kg (dose was temporarily increased to 3.0 mg/kg at 15 and 19 day of injection) compared with auristatin E 0.3 mg/kg administered eight times and seven times, respectively, twice a week (p<0.05). Auristatin E caused the achievement of a state of stable disease up to approximately 40 days, after which it steadily increased until the end of the study. In contrast, AE-Ester-Sulf07 therapy caused a complete remission from day 24 to day 53 with a minimum tumor volume of 1 mm ³ and a long-term effect.

[00240 ] AE-Ester-Sulf07 caused transient weight loss with a minimum of 9% reached on day 31. Starting from the 17th day, bite and scratch wounds were also observed. Two mice died and one had to be sacrificed due to poor general condition (days 20, 43 and 66).

Evaluation of the efficacy of auristatin E and the auristatin E albumin-binding derivative AE-Ester-Sulf07 in the human malignant melanoma model A375 - large tumors (FIG. 20).

[00241 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Ester-Sulf07 in the A375 human malignant melanoma model was performed as described in the general procedure for cell line-derived xenograft models.

[00242 ] Based on dosing experience in the RXF631 xenograft model, stock solutions were prepared as follows:

1) 7 mice, mean weight 28 g, mean median tumor volume 310 mm ³ on day of randomization: 0.3 mg/kg auristatin E TFA salt (AE equivalent) twice a week for 3 weeks on days 1, 6, 9, 13, 16 ≡ 0.35 mg/kg ≡ 7.1 μg/20 g mouse. Sample preparation: 2 mg weighed into a 30 ml vial, dissolved in 28.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, mean weight 29 g, mean median tumor volume 371 mm ³ on day of randomization: 2.4 mg/kg AE-Ester-Sulf07 (AE equivalent) twice a week for 3 weeks on days 1, 6, 9 , 13, 16 ≡ 4.37 mg/kg ≡ 87.3 μg/20 g mice. Sample preparation: 12.6 mg was weighed into a 30 ml vial, dissolved in 14.4 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 2% Tween 80, pH 7.6.

3) 7 mice, mean weight 31 g, mean median tumor volume 355 mm ³ on day of randomization: 2.4 mg/kg AE-Ester-Sulf07 (AE equivalent) twice a week for 3 weeks on days 1, 6, 9 , 13, 16 ≡ 4.37 mg/kg ≡ 87.3 μg/20 g mice. Sample preparation: 12.6 mg was weighed into a 30 ml vial dissolved in 14.4 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 4% 2-HPβCD, pH 7.6.

[00243 ] Tumor growth development in the A375 melanoma xenograft model demonstrates a statistically significant antitumor effect of AE-Ester-Sulf07 compound dosed at 2.4 mg/kg (regardless of recovery buffer, 2% Tween 80 or 4% 2-HPβCD) by compared with auristatin E dosed at 0.3 mg/kg administered five times twice a week (p<0.01 from days 9 to 33). Treatment with auristatin E showed initial stable disease until day 14, after which the tumors grew steadily. However, mice treated with AE-Ester-Sulf07 achieved near complete remission from day 16 until the end of the study on day 37, reaching a minimum of 4 ^mm3 on day 23 and 1 ^mm3 on day 26 for recovered AE. -Ester-Sulf07 with 2% Tween 80 and 4% 2-HPβCD, respectively, despite a large median tumor volume at the start of the experiment.

[00244 ] There was no significant change in body weight compared to controls, but bite and scratch wounds were observed from day 9. Treatment with bepanthen improved the health of injured mice. Only one mouse in the AE-Ester-Sulf07 2% Tween 80 treated group had to be euthanized due to general poor condition.

Evaluation of the efficacy of auristatin E and the auristatin E albumin-binding derivative AE-Ester-Sulf07 in the LXFA737-small tumor human non-small cell lung cancer model (Fig. 21).

[00245 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Ester-Sulf07 in the LXFA737 human non-small cell lung cancer model was performed as described in the general procedure for patient-derived xenograft models.

[00246 ] Stock solutions were prepared as follows:

1) 7 mice, mean weight 27 g, mean median tumor volume 137 ^mm3 on randomization day: 0.3mg/kg Auristatin E TFA salt (AE equivalent) twice a week for 4 weeks on days 0, 4, 7, 11, 14, 18, 21, 25 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 2 mg weighed into a 30 ml vial, dissolved in 28.3 ml 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.2 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 7 mice, mean weight 27g, mean median tumor volume ^126mm3 on day of randomization: 2.4mg/kg AE-Ester-Sulf07 (AE equivalent), twice a week for 4 weeks on day 0.4, 7, 11, 14, 18, 21, 25 ≡ 4.37 mg/kg ≡ 87.3 µg/20 g mouse. Sample preparation: 31 mg were weighed into a 30 ml vial, dissolved in 28.5 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 0.96 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.2 ml of 50 mM sodium phosphate buffer, 4% 2-HPβCD, pH 7.6.

[00247 ] Tumor growth in the NSCLC LXFE737 xenograft model showed superior antitumor efficacy of compound AE-Ester-Sulf07 at 2.4 mg/kg compared to auristatin E at 0.3 mg/kg, both dosed eight times twice a week , with statistical significance (p < 0.05 at days 56 and 63). Tumors in the auristatin E group remained only temporarily in a stable disease state from days 21 to 42 and increased significantly after the end of therapy. In contrast, mice treated with AE-Ester-Sulf07 achieved complete tumor remission at day 21, resulting in a tumor volume reduction to an unmeasurable level by the end of the study at day 74, resulting in a long-term antitumor effect.

[00248 ] During the study, five mice were euthanized due to a combination of weight loss and skin lesions.

Efficacy Evaluation of Auristatin E and Auristatin E Albumin-Binding Derivative AE-Ester-Sulf07 in Human Ovarian Carcinoma Model A2780 - Large Tumors (FIG. 22).

[00249 ] Evaluation of auristatin E and auristatin E albumin-binding derivative AE-Ester-Sulf07 in the A2780 human ovarian carcinoma model was performed as described in the general procedure for cell line-derived xenograft models.

[00250 ] Stock solutions were prepared as follows:

1) 8 mice, mean weight 28 g, mean median tumor volume 341 mm ³ on day of randomization: 0.3 mg/kg auristatin E TFA salt (AE equivalent) administered twice a week for 4 weeks on days 1, 4, 8, 11, 15, 18, 22, 25 ≡ 0.35 mg/kg ≡ 7.1 µg/20 g mouse. Sample preparation: 3.7 mg was weighed into a 100 ml vial, dissolved in 52.3 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.5 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, lyophilized samples were reconstituted with 1.5 ml of 10 mM sodium phosphate buffer, 20% propylene glycol, pH 7.0.

2) 8 mice, mean weight 27 g, mean median tumor volume 403 mm ³ on day of randomization: 1.9 mg/kg AE-Ester-Sulf07 (AE equivalent) was administered once a week for 4 weeks on day 1, 4 , 8, 11, 15, 18, 22, 25 ≡ 3.46 mg/kg ≡ 69.2 µg/20 g mouse. Sample preparation: 36.5 mg were weighed into a 30 ml vial, dissolved in 26.4 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose - pH 7.0; 0.75 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.5 ml of 50 mM sodium phosphate buffer, 2% 2-HPβCD, pH 7.6.

[00251 ] AE-Ester-Sulf07 dosed at 1.9 mg/kg demonstrates superior antitumor efficacy in the A2780 ovarian carcinoma model compared to auristatin E dosed at 0.3 mg/kg, both administered eight times twice a week ( p<0.001 from day 37 to day 40). Mice treated with auristatin E did not show any antitumor effect. However, mice treated with AE-Ester-Sulf07 showed a partial remission from day 19 to day 51 and a complete remission from day 54 until the end of the study at day 103, resulting in long-term tumor regression.

[00252 ] Mice treated with AE-Ester-Sulf07 showed no evidence of weight loss compared to controls, but four animals had to be withdrawn from the study due to tumor necrosis.

Evaluation of the effectiveness of albumin-binding derivatives of auristatin E - compound AE-Ester-Sulf07 for human ovarian carcinoma, model A2780 - small tumors (Fig. 23).

[00253 ] Evaluation of the albumin-binding derivative of auristatin E - AE-Ester-Sulf07 in the A2780 human ovarian carcinoma model was performed as described in the general procedure for cell line derived xenograft models.

[00254 ] Stock solutions were prepared as follows:

1) 8 mice, mean weight 27 g, mean median tumor volume 174 mm ³ on randomization day: 3.8 mg/kg AE-Ester-Sulf07 (AE equivalent) once a week for 4 weeks on days 1, 8, 15, 22 ≡ 6.67 mg/kg ≡ 133.4 µg/20 g mouse. Sample preparation: 25.6 mg were weighed into a 30 ml vial, dissolved in 19.2 ml of 50/50 t-butanol/10 mM sodium phosphate buffer, 5% sucrose, pH 7.0; 1.5 ml were aliquoted into 4 ml vials. The vials were frozen for 1 hour at minus 40°C and lyophilized at minus 20°C for about 36 hours and then stoppered. On the day of injection, freeze-dried samples were reconstituted with 1.5 ml of 50 mM sodium phosphate buffer, 6% 2-HPβCD, pH 7.6.

[00255 ] AE-Ester-Sulf07 was also tested in the same A2780 ovarian carcinoma model with smaller tumors and a different dosing regimen to reduce side effects of the drug on the skin of mice. AE-Ester-Sulf07 was dosed at 3.8 mg/kg four times once a week, again showing a statistically significant improvement in antitumor effect compared to control (p<0.01 from days 7 to 14). Mice treated with AE-Ester-Sulf07 in the new dosing regimen achieved partial remission from day 12 to day 35 and complete remission from day 39 to the end of the study at day 70. Three mice treated with AE-Ester-Sulf07 showed weight loss and had to be euthanized on day 59.

Claims (36)

1. A compound having the structure described by formulas I or II:

or a pharmaceutically acceptable salt thereof; wherein: R' is H or -CH ₃ , M is H or Na ⁺ , K ⁺ , NR ₄ ⁺ or NHR ₃ ⁺ , where R is H; Y is -NH-C(O)-; R ¹ is absent or represents C ₁ -C ₁₈ alkyl; X is H; TBG is a maleimide group. 2. The compound according to claim 1, characterized in that R' represents -CH ₃ . 3. Connection according to claim 1 or 2, characterized in that TBG is a maleimide group having the formula

. 4. A compound according to any one of claims 1 to 3, characterized in that M is H ⁺ or Na ⁺ . 5. A compound according to any one of claims 1 to 4, characterized in that R ¹ is C ₁ -C ₅ alkyl. 6. The compound according to claim 1, characterized in that it has the structure represented by formula III

or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 1, characterized in that it has a structure represented by formula IV

or a pharmaceutically acceptable salt thereof. 8. A pharmaceutical composition for the treatment of cancer in a patient in need thereof, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 9. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier is selected from one or more of the following: a solubilizing agent, an encapsulating agent, and a lyoprotectant. 10. Pharmaceutical composition according to claim 9, characterized in that the pharmaceutically acceptable carrier contains one or more of dimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and trimethyl-β-cyclodextrin. 11. Pharmaceutical composition according to any one of claims 8 to 10, characterized in that the pharmaceutical composition is suitable for intravenous administration. 12. A pharmaceutical composition according to claim 11, characterized in that the composition, when administered intravenously to a patient, covalently and rapidly selectively binds in situ to: a) endogenous albumin in the bloodstream; or b) the thiol group of cysteine-34 of endogenous albumin in the bloodstream. 13. A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 8 to 12. 14. The method of claim 13 wherein the cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma and melanoma. 15. The method of claim 14, wherein the cancer is selected from human ovarian carcinoma, renal cell carcinoma, non-small cell lung cancer, and melanoma. 16. The method according to any one of claims 13 to 15, characterized in that the method of administration is intravenous administration. 17. A method for reducing the cytotoxicity of a compound, comprising administering a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 8 to 12 to a patient in need thereof, characterized in that the administration results in a reduction in cytotoxicity compared to with an equivalent dose of the unmodified active agent. 18. A method for increasing the concentration of a metabolite of a compound in a tumor, comprising administering a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 8 to 12 to a patient in need thereof, the increase being compared to an equivalent dose of unmodified active agent.

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