N-substituted indole derivatives and conjugates thereof for treatment of cancer

Abstract

The present invention relates to N-substituted derivatives of indoles of formula (I) and their use in the treatment of cancer. The present invention also provides protein-drug conjugates, more particularly antibody-drug conjugates, from the compounds of Formula (I). # imgabs0 #

Description

Field of the Invention

[0001] The present invention relates to the field of medicine, and more specifically to N-substituted indole derivatives and their use as therapeutic compounds. Specifically, such derivatives are used to treat cancer. Some of them are also used to prepare conjugates with proteins or antibodies. Background of the Invention

[0003] Kinesins are a superfamily of motor proteins with ATPase activity. They are involved in the normal biological activities of various cells, including mitosis, meiosis and intracellular vesicle transport. Kinesin family member 20A (KIF20A, also known as MKlp2) is located on chromosome 5q31.2 and plays an important role in the occurrence and development of tumors. Recently, multiple studies have shown that KIF20A may play an important role in the development and progression of many different types of cancers such as melanoma, breast cancer, nasopharyngeal carcinoma, pancreatic cancer, hepatocellular carcinoma, lung cancer and colorectal cancer.

[0004] In the past few years, a class of indole derivatives of compounds targeting MKlp2 (KIF20A) has been developed, such as the compounds described in International Patent Application WO 2014 / 086964. Although these compounds are poorly soluble in water at neutral and acidic pH, they are nanomolar-efficient selective inhibitors of MKlp2 (KIF20A).

[0005] It is well known that solubility is one of the most important parameters for achieving the desired concentration of the drug in the systemic circulation to achieve a pharmacological response, regardless of the route of administration. In fact, low aqueous solubility is a key issue encountered during the development of pharmaceutical chemical entities and formulations containing them.

[0006] Therefore, it is necessary to enhance the water solubility of such indole derivatives to ensure the solubility of such derivatives at the absorption site. Summary of the invention

[0007] In this context, the inventors provide highly soluble prodrugs in water derived from indole derivatives (particularly indole derivatives reported in WO 2014 / 086964). More specifically, the nitrogen atom of indole is either connected to a promoieties containing a phosphonic acid or to a cleavable group (e.g., an easily metabolizable group). The cleavable group may also include a protein-binding connector to provide a protein conjugate (e.g., a conjugate with albumin) for increasing the internalization of these drugs in cancer cells, for example, by antibody conjugates to increase the circulation half-life of the drug and promote the intracellular and / or extracellular release of these drugs to the tumor.

[0008] Therefore, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] in:

[0011] -X represents a nitrogen atom, a C-CN unit or a N+-O- unit;

[0012] - R1 and R1' independently represent hydrogen, halogen, (C1-C6)alkoxy or a -SO2-CH3 group, provided that if R1 or R1' is (C1-C6)alkoxy, R2 is not halogen;

[0013] -R2 stands for:

[0014] a (C1-C6)alkoxy, (C3-C6)cycloalkoxy, (C3-C6)heterocycloalkoxy, aryloxy, heteroaryloxy, (C1-C6)alkyl-aryloxy, (C1-C6)alkyl-heteroaryloxy group, which is optionally substituted by at least one halogen, or a thio-(C1-C6)alkyl, thio-aryl, thio-heteroaryl, thio-(C1-C6)alkyl-aryl or thio-(C1-C6)alkyl-heteroaryl group, which is optionally substituted by at least one halogen or (C1-C6)alkoxy group,

[0015] ■-NR4R5 unit, O-(C1-C6)alkyl-NR4R5 unit or S-(C1-C6)alkyl-NR4R5 unit, wherein R4 and R5 represent H, (C1-C6)alkyl, or R4 and R5 together form a 3- to 7-membered ring, which is optionally interrupted by one or more heteroatoms, provided that at least one of R4 and R5 is not H,

[0016] ■NHCOR6 unit, wherein R6 represents (C1-C6) alkyl,

[0017] ■ aryl or heteroaryl, which is optionally substituted by at least one halogen, trifluoromethyl or (C1-C3)alkoxy, or

[0018] ■Halogen,

[0019] - R3 represents hydrogen, (C1-C3)alkyl, (C1-C3)alkoxy or halogen; and

[0020] -Ra is a group selected from the following:

[0021] ■Groups of formula (A): or a group of formula (A'): wherein n is an integer between 1 and 12; and

[0022] ■Groups of formula (B): in:

[0023] ○ L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group;

[0024] ○ L2 is tert-butyloxycarbonyl or a protein-binding linker, and

[0025] ○m is an integer equal to 0 or 1.

[0026] In a specific embodiment, R2 represents:

[0027] ■(C1-C6) alkoxy, preferably methoxy,

[0028] ■(C3-C6)heterocycloalkoxy, preferably oxetanyloxy, or

[0029] ■ Heteroaryl, preferably furyl or triazolyl.

[0030] In a preferred embodiment, the compound of formula (I) is:

[0031] -X represents C-CN unit,

[0032] -R1 represents halogen, preferably bromine,

[0033] -R1' represents hydrogen,

[0034] - R2 represents (C1-C6) alkoxy, preferably methoxy, and

[0035] -R3 represents hydrogen.

[0036] In a further preferred embodiment, the compound of formula (I) is:

[0037] -X represents C-CN unit,

[0038] -R1 represents hydrogen,

[0039] -R1' represents (C1-C6) alkoxy, preferably methoxy,

[0040] - R2 represents (C1-C6) alkoxy, preferably methoxy, and

[0041] -R3 represents hydrogen.

[0042] In an embodiment of the present invention, in the compound of formula (I) or a pharmaceutically acceptable salt thereof, Ra is a group of formula (A): or a group of formula (A'): wherein n is an integer between 1 and 12. Preferably, n is an integer between 2 and 10. More preferably, n is an integer between 2 and 8, advantageously an integer between 2 and 6. Even more preferably, n is 2, 3, 4, 5 or 6.

[0043] In a specific embodiment, the pharmaceutically acceptable salt of the compound of formula (I) is selected from sodium salt, disodium salt, lysine salt, dilysine salt, arginine salt or diarginine salt, preferably sodium salt or disodium salt, more preferably sodium salt.

[0044] In a preferred embodiment, the compound of formula (I) is selected from:

[0045] - Example 1: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid;

[0046] - Example 2: (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid;

[0047] - Example 3: (Z)-7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonic acid;

[0048] - Example 4: (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonic acid;

[0049] - Example 5: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid;

[0050] - Example 6: (Z)-sodium hydrogen 5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonate;

[0051] - Example 7: (Z)-sodium hydrogen 3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonate;

[0052] - Example 8: (Z)-sodium hydrogen 4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonate;

[0053] - Example 9: (Z)-sodium hydrogen 6-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxohexylphosphonate;

[0054] - Example 10: (Z)-sodium hydrogen 7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonate;

[0055] - Example 11: Sodium hydrogen (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonate;

[0056] - Example 12: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid sodium hydrogen;

[0057] - Example 13: (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid disodium salt;

[0058] - Example 14: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid disodium salt;

[0059] - Example 15: (S)-2,6-diaminohexanoic acid compound and (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid (2:1);

[0060] - Example 16: (S)-2,6-diaminohexanoic acid compound and (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid (2:1);

[0061] - Example 17: 5-bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[hydroxy(dioxo)-λ6-phosphino]propyl ester; and

[0062] - Example 18: 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[hydroxy(dioxo)-λ6-phosphino]butyl ester.

[0063] In another embodiment of the present invention, in the compound of formula (I) or a pharmaceutically acceptable salt thereof, Ra is a group of formula (B): in:

[0064] o L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group;

[0065] o L2 is tert-butyloxycarbonyl or a protein-binding linker, and

[0066] om is an integer equal to 0 or 1.

[0067] In specific embodiments, L1 is an enzymatically cleavable group that is cleaved by a protease, peptidase, esterase, β-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, tubulin tyrosine ligase, or lipase.

[0068] In a preferred embodiment, L1 is p-aminobenzyloxycarbonyl-AA1 w -AA2 x -AA3 y -AA4 z A group wherein AA1, AA2, AA3 and AA4 independently represent an amino acid selected from alanine, valine, citrulline, phenylalanine, lysine, glycine, aspartic acid, asparagine, glutamic acid and derivatives thereof, preferably citrulline, valine, cysteic acid, glycine and glutamic acid, more preferably citrulline, valine and cysteic acid, and w, x, y and z independently represent an integer equal to 0 or 1.

[0069] According to a specific embodiment, m is 1, and L2 is a binding protein linker having the formula:

[0070] wherein r is an integer between 1 and 36, preferably an integer between 1 and 24, more preferably equal to 2, 4, 8, 12, 16, 20 or 24.

[0071] In a preferred embodiment, the compound of formula (I) is selected from:

[0072] - Example 19: (2S,3S,4S,5R,6S)-6-[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid;

[0073] - Example 20: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester;

[0074] - Example 21: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropionyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester hydrochloride;

[0075] - Example 22: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy](peg4)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester;

[0076] - Example 23: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino](peg24)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester;

[0077] - Example 24: 3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-(tert-butoxycarbonylamino)-3-oxo-propane-1-sulfonic acid;

[0078] - Example 25: 2-amino-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propane-1-sulfonic acid; and

[0079] -Example 26: (2R)-3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy]propionylamino]-3-oxo-propane-1-sulfonic acid.

[0080] Another object of the present invention is a pharmaceutical composition comprising a compound according to the invention and a pharmaceutically acceptable excipient.

[0081] Specifically, the pharmaceutical composition is used to treat cancer. Preferably, the cancer is selected from leukemia, acute myeloid leukemia, lymphoma, breast cancer, pancreatic cancer, lung cancer or colon cancer.

[0082] Another object of the present invention is a conjugate of formula (II):

[0083]

[0084] in:

[0085] -X, R1, R 1' , R2, R3 and L1 are as defined herein;

[0086] -L2 is the binding protein linker;

[0087] - P is a peptide or protein, preferably an antibody, antibody fragment or antigen-binding fragment, which is capable of binding to a target of interest; and

[0088] -v is an integer from 1 to 10. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 : Cathepsin B-mediated drug release assay

[0090] Drug concentration (μM) of quenched mal prodrug 20 μg / mL released after 0-5-10-15-30-60 min incubation. DETAILED DESCRIPTION OF THE INVENTION

[0092] definition

[0093] According to the present invention, the following terms have the following meanings:

[0094] The prefixed terms mentioned herein, such as C1-C3, C1-C6 or C2-C6, can also be used with a lesser number of carbon atoms, such as C1-C2, C1-C5 or C2-C5. For example, if the term C1-C3 is used, it means that the corresponding hydrocarbon chain can contain 1 to 3 carbon atoms, especially 1, 2 or 3 carbon atoms. For example, if the term C1-C6 is used, it means that the corresponding hydrocarbon chain can contain 1 to 6 carbon atoms, especially 1, 2, 3, 4, 5 or 6 carbon atoms. For example, if the term C2-C6 is used, it means that the corresponding hydrocarbon chain can contain 2 to 6 carbon atoms, especially 2, 3, 4, 5 or 6 carbon atoms.

[0095] The term "alkyl" refers to a saturated, straight or branched aliphatic group. The term "(C1-C3)alkyl" more specifically refers to methyl, ethyl, propyl or isopropyl. The term "(C1-C6)alkyl" more specifically refers to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl or hexyl. In a preferred embodiment, "alkyl" is methyl, ethyl, propyl, isopropyl or tert-butyl, more preferably methyl.

[0096] The term "alkoxy" or "alkyloxy" corresponds to an alkyl group as defined above bonded to the molecule via an -O- (ether) bond. (C1-C3) alkoxy includes methoxy, ethoxy, propoxy and isopropoxy. (C1-C6) alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy and hexyloxy. In a preferred embodiment, "alkoxy" or "alkyloxy" is methoxy.

[0097] The term "thio" corresponds to an alkyl group as defined above bonded to the molecule via an -S- (thioether) bond. Thio-(C1-C6)alkyl includes thio-methyl, thio-ethyl, thio-propyl, thio-butyl, thio-pentyl and thio-hexyl.

[0098] The term "halogen" corresponds to a fluorine, chlorine, bromine or iodine atom, preferably fluorine, chlorine or bromine, more preferably chlorine or bromine.

[0099] The term "aryl" corresponds to a monocyclic or bicyclic aromatic hydrocarbon having 6 to 12 carbon atoms. For example, the term "aryl" includes phenyl, biphenyl or naphthyl. In a preferred embodiment, aryl is phenyl.

[0100] The term "heteroaryl" as used herein corresponds to an aromatic monocyclic or polycyclic group containing 5 to 14 atoms and containing at least one heteroatom, such as a nitrogen, oxygen or sulfur atom. Examples of such monocyclic and polycyclic heteroaryl groups may be: pyridyl, thiazolyl, thienyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolinyl, quinolyl, isoquinolyl, benzimidazolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, triazinyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthene, phenoxanthinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, quinolizinyl, phthalazinyl (p In some embodiments, the heteroaryl group is pyridyl, furanyl, pyrazolyl, pyrazolyl, pyrazolyl, indolyl, isoindolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, benzothiophenyl, benzothiazolyl, isatinyl, dihydropyridyl, pyrimidinyl, s-triazinyl, oxazolyl or thiofuranyl. In a preferred embodiment, the heteroaryl group is pyridyl, furanyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl and isoxazolyl. Preferably, heteroaryl is pyridyl, thiazolyl, furyl, pyranyl, pyrrolyl, imidazolyl, tetrazolyl, benzofuranyl, pyrrolinyl, triazine, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl. More preferably, heteroaryl is furyl or triazolyl.

[0101] The term "cycloalkyl" corresponds to a saturated or unsaturated monocyclic, bicyclic or tricyclic alkyl group containing 3 to 20 carbon atoms. It also includes fused, bridged or spiro-connected cycloalkyl groups. The term "cycloalkyl" includes, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In a preferred embodiment, "cycloalkyl" is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0102] The term "heterocycloalkyl" corresponds to a saturated or unsaturated cycloalkyl as defined above, further comprising at least one heteroatom such as nitrogen, oxygen or sulfur atom. It also includes fused, bridged or spirally connected heterocycloalkyl. Representative heterocycloalkyl includes but is not limited to 3-dioxolane, benzo [1,3] dioxolyl, azetidinyl, oxetanyl, pyrazolyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidinyl, piperazinyl, 1,4-dioxane, imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, oxazolinyl (oxozoliny), oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrofuranyl and tetrahydrothienyl.

[0103] The terms "cycloalkoxy", "heterocycloalkoxy", "aryloxy" and "heteroaryloxy" correspond to "cycloalkyl", "heterocycloalkyl", "aryl" and "heteroaryl" as defined above, which are bonded to the molecule via an -O- (ether) bond. For example, "cycloalkoxy", "heterocycloalkoxy", "aryloxy" and "heteroaryloxy" correspond to -O-cycloalkyl, -O-heterocycloalkyl, -O-aryl and -O-heteroaryl. A preferred embodiment of "heterocycloalkoxy" is oxetanyloxy or oxetanyloxy (-O-oxetanyl).

[0104] The term "Boc" corresponds to the following tert-butoxycarbonyl group "-C(=O)-OC(CH3)3".

[0105] The expression "at least substituted" means that the group is substituted by one or more groups in the list.

[0106] "Pharmaceutical salts" or "pharmaceutically acceptable salts" include inorganic acid salts and organic acid salts. Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, maleic acid, methanesulfonic acid, etc. Other examples of pharmaceutical inorganic or organic acid addition salts include pharmaceutical salts listed in J. Pharm. Sci. 1977, 66, 2 and Handbook of Pharmaceutical Salts: Properties, Selection, and Use edited by P. Heinrich Stahl and Camille G. Wermuth 2002. In a preferred embodiment, the salt is selected from maleate, hydrochloride, hydrobromide and methanesulfonate. "Pharmaceutical salts" also include inorganic and organic base salts. Representative examples of suitable inorganic bases include sodium or potassium salts, alkaline earth metal salts (e.g., calcium or magnesium salts) or ammonium salts. Representative examples of suitable salts formed with organic bases include, for example, salts formed with methylamine, dimethylamine, trimethylamine, piperidine, morpholine, tris-(2-hydroxyethyl)amine. The "pharmaceutical salts" or "pharmaceutically acceptable salts" also include salts formed with amino acids such as aspartic acid, lysine and arginine. In a preferred embodiment, the salt is selected from sodium salts, lysine salts, arginine salts. It also includes disodium salts, diarginine salts and dilysine salts.

[0107] The present invention provides highly water-soluble indole derivatives which are substituted on the nitrogen of the indole nucleus (ie, N-substituted indole derivatives).

[0108] As used herein, derivatives or compounds that are not substituted on the nitrogen of the indole nucleus are also referred to as "indole derivatives", "payloads", "active ingredients", "active compounds", "pharmaceutically active ingredients", "biologically active ingredients", "biologically active forms", "drugs" or "molecules", but are not limited thereto. Such "indole derivatives" and the like have the following structural formula:

[0109] Among them, X, R1, R 1' , R2, R3 are defined in this disclosure.

[0110] As used herein, N-Substituted Indole Derivatives are also referred to as "prodrugs."

[0111] Generally speaking, the "prodrug" used in this article is an "N-substituted indole derivative" of formula (I), which itself may have low activity or even no activity, but after administration, it is converted into a corresponding biologically active form under physiological conditions, such as by metabolism, solvolysis or other means.

[0112] Specifically, the prodrug of the indole derivative defined herein can be a compound of formula (I) obtained by acylation, alkylation or phosphorylation of the amino group of the indole derivative (for example, a compound of formula (I) obtained by eicosanoylation, alanylation, pentanylaminocarbonylation, (5-methyl-2-oxo-1,3-dioxol-4-yl)methoxycarbonylation, tetrahydrofuranylation, pyrrolidinylmethylation, pivaloyloxymethylation or tert-butylation of the amino group of the indole derivative).

[0113] In one embodiment, the prodrugs of indole derivatives defined herein include, but are not limited to, any prodrugs suitable for drugs containing an amino group, such as indole derivative drugs. For example, it may include prodrugs such as amides, carbamates and thiocarbamates (O-thiocarbamates or S-thiocarbamates), N-acyloxyalkyl derivatives, N-acyloxyalkoxycarbonyl derivatives, β-aminoketones, (oxydioxolyl) methyl derivatives, N-Mannich bases, imines (Schiff bases), enamines and enamine ketones, azo compounds, lactonization systems, tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (THTT), redox systems, and PEG disclosed by Simplicio et al. (Molecules 2008, 13, 519-547).

[0114] In one embodiment, the prodrug of the indole derivative defined herein is an amide prodrug. More specifically, the nitrogen atom of the indole core is substituted by a -CO-R' group, wherein R' can represent H, (C1-C6) alkyl, and the (C1-C6) alkyl is optionally substituted by halogen, cycloalkyl, aryl or heteroaryl (which can be substituted or unsubstituted). In a further embodiment, the prodrug of the indole derivative defined herein is a carbamate prodrug. More specifically, the nitrogen atom of the indole core is substituted by a -CO2-R' group, wherein R' can represent H, (C1-C6) alkyl, and the (C1-C6) alkyl is optionally substituted by halogen, cycloalkyl, aryl or heteroaryl (which can be substituted or unsubstituted). In a further embodiment, the prodrug of the indole derivative defined herein is an N-acyloxyalkylamine. More specifically, the nitrogen atom of the indole core is substituted with a -CH(R")-CO2-R' group, wherein R' may represent H, (C1-C6)alkyl, which (C1-C6)alkyl is optionally substituted with halogen, cycloalkyl, aryl or heteroaryl (which may be substituted or unsubstituted), and R" may represent H, (C1-C6)alkyl optionally substituted with halogen. Amide, carbamate and N-acyloxyalkylamine prodrugs may release the drug by amidases, peptidases or esterases.

[0115] In one embodiment, the prodrug of the indole derivative defined herein is a sulfenamide prodrug. More specifically, the nitrogen atom of the indole core is substituted by a -S-R' group, wherein R' may represent H, (C1-C6) alkyl, which is optionally substituted by halogen, cycloalkyl, aryl or heteroaryl (which may be substituted or unsubstituted).

[0116] In one embodiment, the prodrug of the indole derivative as defined herein is a phosphate prodrug. More specifically, the nitrogen atom of the indole core is replaced by a (C1-C6)alkyl-OP(O)(OH)(O - The phosphate prodrug can release the drug in the presence of a phosphatase (e.g., alkaline phosphatase).

[0117] In one embodiment, the prodrug of the indole derivative defined herein is an N-Mannich base prodrug. More specifically, the nitrogen atom of the indole core is substituted by a (C1-C6) alkyl-NH-CO-R' group, wherein R' may represent H, (C1-C6) alkyl, which is optionally substituted by halogen, cycloalkyl, aryl or heteroaryl (which may be substituted or unsubstituted).

[0118] In a specific embodiment, the prodrug of the Indole Derivative as defined herein is selected from:

[0119]

[0120] Where R 1 It can be H, alkyl or especially C1-C8 alkyl;

[0121] R 2 may be an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group or a haloalkyl group, which may be substituted or unsubstituted;

[0122] R 3 It can be H, a metal, R2, or a substituted or unsubstituted primary, secondary or tertiary amine;

[0123] R 4 It can be H, metal, ammonium salt, or alkyl;

[0124] R 5 May be substituted or unsubstituted natural amino acids;

[0125] R a or R b It can be H, alkyl or aryl; or NR a or NR b It can be an amino acid;

[0126] X can be C or O;

[0127] k can be 1 or 2, m can be 2-22, or (CH k ) m It can be a saturated, unsaturated or conjugated hydrocarbon;

[0128] n can be 0-2; and

[0129] The metal may be Na, K, Li, Ca, Mg, Ag or Zn.

[0130] In another specific embodiment, the prodrug of the indole derivative defined herein is of the phosphorothioate prodrug type. More specifically, the nitrogen atom of the indole core is substituted by (C1-C6)alkyl-NH-P(O)(OR')-C(V)-XSR" or -P(O)(OR')-C(V)-XSR", wherein:

[0131] V represents (C1-C8) alkyl;

[0132] X represents CR3(R4);

[0133] R' independently represents an optionally substituted aliphatic or aromatic residue;

[0134] Optionally, R' represents (C1-C8)alkyl, which is optionally substituted by at least one of the following groups: (C1-C8)alkoxy) n , wherein n is 1, 2, 3, 4, 5 or 6; F, Cl, Br, I, -NO2, N(C1-C8)alkyl, -NH2, -N((C1-C8)alkyl)2, =O, C3-C8-cycloalkyl, -SS-(C1-C8)alkyl, hydroxy-((C1-C8)alkoxy) n , wherein n is 1, 2, 3, 4, 5 or 6; C2-C8-alkynyl or optionally substituted phenyl; or optionally, R' represents phenyl, which is optionally independently substituted by at least one of the following groups: (C1-C8)alkyl, (C1-C8)alkoxy) n (wherein n is 1, 2, 3, 4, 5 or 6), F, Cl, I, Br, NO2, N(C1-C8)alkyl, NH2 or -N((C1-C8)alkyl)2; or optionally, R' represents a 5- or 6-membered heteroaromatic system;

[0135] R3 represents H or (C1-C8) alkyl;

[0136] R4 represents H or (C1-C8) alkyl.

[0137] R" represents optionally substituted (C1-C8) alkyl, optionally substituted phenyl, optionally substituted aromatic 5- or 6-membered heterocyclic ring system, amino acid, peptide, protein, antibody, sugar, polysaccharide, nucleotide, oligonucleotide or polymer.

[0138] The phosphorothioate prodrug type was developed by Tubulis and is more specifically disclosed in patent US11,161,873B2 and patent application US2020 / 0390901 A1.

[0139] In a further specific embodiment, the prodrug of the indole derivative defined herein is a carbohydrate prodrug type. The carbohydrate prodrug type can release the drug in the presence of glycosidase. Specifically, the carbohydrate prodrug type includes the prodrug disclosed by H.Martin et al. (Chem.Soc.Rev., 2022, 51, 9694) or in application number US 2009 / 0227617. In a specific embodiment, it includes a sugar conjugate having the following structural formula:

[0140]

[0141] in:

[0142] X, R1, R 1' , R2 and R3 are defined in this disclosure,

[0143] Y is -O- or -NH-CO-O-,

[0144] R is H, -OH, -CH2OH, -CH3, -COOR' (wherein R' is H, -CH3, -CH2-CH=CH2, -CH2-CH2-OH, -CH2-CH2-OCH3) or a residue having the formula

[0145]

[0146] n is an integer from 1 to 40, preferably from 1 to 10, more preferably from 2 to 6;

[0147] x is an integer from 1 to 5, preferably from 1 to 3, more preferably 1 or 2;

[0148] p is an integer from 1 to 5, preferably from 1 to 3, more preferably 1 or 2; and

[0149] The alkyl group is a straight-chain or branched alkyl residue having 1 to 10 carbon atoms, preferably 1 to 3 carbon atoms.

[0150] In a further specific embodiment, it comprises a glycoconjugate having the formula:

[0151]

[0152] in:

[0153] X, R1, R 1' , R2 and R3 are defined in this disclosure,

[0154] Y is -O- or -NH-CO-O-,

[0155] R is H, -OH, -CH2OH, -CH3, -COOR' (wherein R' is H, -CH3, -CH2-CH=CH2, -CH2-CH2-OH, -CH2-CH2-OCH3) or a residue having the formula

[0156]

[0157] n is an integer from 1 to 40, preferably from 1 to 10, more preferably from 2 to 6;

[0158] x is an integer from 1 to 5, preferably from 1 to 3, more preferably 1 or 2;

[0159] p is an integer from 1 to 5, preferably from 1 to 3, more preferably 1 or 2; and

[0160] The alkyl group is a straight-chain or branched alkyl residue having 1 to 10 carbon atoms, preferably 1 to 3 carbon atoms.

[0161] In another embodiment according to the present invention, the term "N-substituted indole derivatives" corresponds to indole derivatives wherein the nitrogen atom of the indole core is substituted by a substituent Ra.

[0162] Therefore, the present invention provides a compound of formula (I) or one of its pharmaceutically acceptable salts:

[0163]

[0164] in:

[0165] -X represents a nitrogen atom, a C-CN unit or a N+-O- unit;

[0166] - R1 and R1' independently represent hydrogen, halogen, (C1-C6)alkoxy or a -SO2-CH3 group, provided that if R1 or R1' is (C1-C6)alkoxy, R2 is not halogen;

[0167] -R2 stands for:

[0168] a (C1-C6)alkoxy, (C3-C6)cycloalkoxy, (C3-C6)heterocycloalkoxy, aryloxy, heteroaryloxy, (C1-C6)alkyl-aryloxy, (C1-C6)alkyl-heteroaryloxy group, which is optionally substituted by at least one halogen, or a thio-(C1-C6)alkyl, thio-aryl, thio-heteroaryl, thio-(C1-C6)alkyl-aryl or thio-(C1-C6)alkyl-heteroaryl group, which is optionally substituted by at least one halogen or (C1-C6)alkoxy group,

[0169] ■-NR4R5 unit, O-(C1-C6)alkyl-NR4R5 unit or S-(C1-C6)alkyl-NR4R5 unit, wherein R4 and R5 represent H, (C1-C6)alkyl, or R4 and R5 together form a 3- to 7-membered ring, which is optionally interrupted by one or more heteroatoms, provided that at least one of R4 and R5 is not H,

[0170] ■NHCOR6 unit, wherein R6 represents (C1-C6) alkyl,

[0171] ■ aryl or heteroaryl, which is optionally substituted by at least one halogen, trifluoromethyl or (C1-C3)alkoxy, or

[0172] ■Halogen,

[0173] - R3 represents hydrogen, (C1-C3)alkyl, (C1-C3)alkoxy or halogen; and

[0174] -Ra is a group selected from the following:

[0175] ■Groups of formula (A): or a group of formula (A'): wherein n is an integer between 1 and 12; and

[0176] ■Groups of formula (B): in:

[0177] ○ L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group;

[0178] ○ L2 is tert-butyloxycarbonyl or a protein-binding linker, and

[0179] ○m is an integer equal to 0 or 1;

[0180] or a pharmaceutically acceptable salt thereof.

[0181] Indole derivatives, etc. ("payload")

[0182] In a specific embodiment, the indole derivatives and the compound of formula (I) are (Z)-isomers. In a further specific embodiment, the indole derivatives and the compound of formula (I) are (E)-isomers.

[0183] According to the present invention, R1 and R1' independently represent hydrogen, halogen, (C1-C6) alkoxy or -SO2-CH3 group, provided that if R1 or R1' is (C1-C6) alkoxy, then R2 is not halogen. In a particular embodiment, one of R1 and R1' is H, and the other represents halogen, (C1-C6) alkoxy or -SO2-CH3 group, provided that if R1 or R1' is (C1-C6) alkoxy, then R2 is not halogen. Preferably, one of R1 and R1' is H, and the other represents halogen, typically bromine, chlorine or fluorine, advantageously bromine or chlorine, more specifically bromine.

[0184] According to the present invention, R2 represents:

[0185] a (C1-C6)alkoxy, (C3-C6)cycloalkoxy, (C3-C6)heterocycloalkoxy, aryloxy, heteroaryloxy, (C1-C6)alkyl-aryloxy, (C1-C6)alkyl-heteroaryloxy group, which is optionally substituted by at least one halogen, or a thio-(C1-C6)alkyl, thio-aryl, thio-heteroaryl, thio-(C1-C6)alkyl-aryl or thio-(C1-C6)alkyl-heteroaryl group, which is optionally substituted by at least one halogen or (C1-C6)alkoxy group,

[0186] ■-NR4R5 unit, O-(C1-C6)alkyl-NR4R5 unit or S-(C1-C6)alkyl-NR4R5 unit, wherein R4 and R5 represent H, (C1-C6)alkyl, or R4 and R5 together form a 3- to 7-membered ring, which is optionally interrupted by one or more heteroatoms, provided that at least one of R4 and R5 is not H,

[0187] ■NHCOR6 unit, wherein R6 represents (C1-C6) alkyl,

[0188] ■ aryl or heteroaryl, which is optionally substituted by at least one halogen, trifluoromethyl or (C1-C3)alkoxy, or

[0189] ■Halogen.

[0190] In particular, R2 represents:

[0191] ■ a (C1-C6)alkoxy, phenoxy or oxetanoxy group, which is optionally substituted by at least one halogen, preferably bromine, chlorine or fluorine, more preferably fluorine, such as trifluoromethyl;

[0192] ■ halogen, preferably bromine, chlorine or fluorine, more preferably bromine or chlorine;

[0193] ■R4-N-R5 units or S-(C1-C6)alkyl-NR4R5 units, wherein R4 and R5 represent H or (C1-C6)alkyl, provided that at least one of R4 and R5 is not H,

[0194] ■NHCOR6 units, wherein R6 represents (C1-C6) alkyl, advantageously methyl, ethyl or tert-butyl; or

[0195] ■ aryl optionally substituted with at least one halogen or trifluoromethyl; or

[0196] ■ Heteroaryl, advantageously furan, triazole, pyridine, thiazole, pyran, pyrrole, imidazole, tetrazole, benzofuran, triazinyl, pyrazinyl, pyridazine or tetrazole.

[0197] Preferably, R2 represents:

[0198] ■ a (C1-C6)alkoxy group selected from methoxy, ethoxy, isopropoxy, preferably selected from methoxy and isopropoxy, or a phenoxy group optionally substituted by fluorine, such as trifluoromethyl, or an oxetanoxy group;

[0199] ■ a halogen selected from fluorine and chlorine,

[0200] ■R4-N-R5 unit or S-(C1-C6)alkyl-NR4R5 unit, wherein R4 and R5 represent methyl or ethyl:

[0201] ■ a group selected from thiomethyl, thioethyl, thiobenzyl, thiopyridyl and thiophenyl, which is optionally substituted by at least one fluorine or trifluoromethyl group;

[0202] ■ phenyl optionally substituted with at least one bromo or trifluoromethyl group; or

[0203] ■ A heteroaryl group selected from furan or triazole.

[0204] More preferably, R2 represents:

[0205] ■ (C1-C6) alkoxy group, preferably methoxy,

[0206] ■ (C3-C6) heterocycloalkoxy group, preferably oxetanyloxy, or

[0207] ■ Heteroaryl, preferably furyl or triazolyl.

[0208] According to the present invention, R3 represents hydrogen, (C1-C3) alkyl, preferably methyl, ethyl or isopropyl, (C1-C3) alkoxy, preferably methoxy, ethoxy or isopropoxy, or halogen, advantageously fluorine. Preferably, R3 is hydrogen, methoxy or fluorine. More preferably, R3 is hydrogen.

[0209] In a preferred embodiment:

[0210] -X represents C-CN unit,

[0211] -R1 represents halogen, preferably bromine,

[0212] -R1' represents hydrogen,

[0213] - R2 represents a (C1-C6) alkoxy group, preferably a methoxy group, and

[0214] -R3 represents hydrogen.

[0215] In a further preferred embodiment:

[0216] -X represents C-CN unit,

[0217] -R1 represents hydrogen,

[0218] - R1' represents a (C1-C6) alkoxy group, preferably a methoxy group,

[0219] - R2 represents a (C1-C6) alkoxy group, preferably a methoxy group, and

[0220] -R3 represents hydrogen.

[0221] In a particular aspect, the indole derivative corresponds to the indole derivatives disclosed in WO 2014 / 086964, more specifically the indole derivative corresponds to the indole derivatives within the scope of the claims of EP 2 928 864 B1.

[0222] In a preferred embodiment, the indole derivatives are selected from the following compounds:

[0223] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-chloropyridin-3-yl)-acrylonitrile;

[0224] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-chloropyridin-3-yl)-acrylonitrile;

[0225] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)-acrylonitrile;

[0226] -(E)-2-(5-bromo-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)-acrylonitrile;

[0227] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-(dimethylamino)pyridin-3-yl)-acrylonitrile;

[0228] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-dimethylamino)pyridin-3-yl)-acrylonitrile, hydrochloride;

[0229] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(dimethylamino)pyridin-3-yl)-acrylonitrile;

[0230] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)-acrylonitrile;

[0231] -(E)-2-(5-chloro-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)-acrylonitrile;

[0232] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-phenoxypyridin-3-yl)-acrylonitrile;

[0233] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-phenoxypyridin-3-yl)-acrylonitrile;

[0234] -(Z)-2-(5-methoxy-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)-acrylonitrile;

[0235] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-ethoxypyridin-3-yl)-acrylonitrile;

[0236] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-isopropoxypyridin-3-yl)-acrylonitrile;

[0237] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(methylthio)pyridin-3-yl)-acrylonitrile;

[0238] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(ethylthio)pyridin-3-yl)-acrylonitrile;

[0239] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(3-bromophenyl)pyridin-3-yl)-acrylonitrile;

[0240] -(Z)-3-(4-(3-bromophenyl)pyridin-3-yl)-2-(5-chloro-1H-indol-3-yl)-acrylonitrile;

[0241] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(phenylthio)pyridin-3-yl)-acrylonitrile;

[0242] -(Z)-3-(4-(Benzylthio)pyridin-3-yl)-2-(5-bromo-1H-indol-3-yl)-acrylonitrile;

[0243] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(3,4-dimethoxy)thio)pyridin-3-yl)-acrylonitrile;

[0244] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(4-fluorophenoxy)pyridin-3-yl)-acrylonitrile;

[0245] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-(4-fluorophenoxy)pyridin-3-yl)-acrylonitrile;

[0246] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(diethylamino)pyridin-3-yl)-acrylonitrile;

[0247] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(4-(trifluoromethyl)phenyl)pyridin-3-yl)-acrylonitrile;

[0248] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-(4-(trifluoromethyl)phenyl)pyridin-3-yl)-acrylonitrile;

[0249] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-((4-fluorophenyl)thio)pyridin-3-yl)-acrylonitrile;

[0250] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-((4-fluorophenyl)thio)pyridin-3-yl)-acrylonitrile;

[0251] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-(furan-3-yl)pyridin-3-yl)-acrylonitrile;

[0252] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-(pyridin-2-ylthio)pyridin-3-yl)-acrylonitrile;

[0253] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(pyridin-2-ylthio)pyridin-3-yl)-acrylonitrile;

[0254] -(Z)-3-(4-(1H-1,2,4-triazol-1-yl)pyridin-3-yl)-2-(5-bromo-1H-indol-3-yl)-acrylonitrile;

[0255] -(Z)-3-(4-(1H-1,2,4-triazol-1-yl)pyridin-3-yl)-2-(5-chloro-1H-indol-3-yl)-acrylonitrile;

[0256] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(furan-3-yl)pyridin-3-yl)-acrylonitrile;

[0257] -(E)-2-(5-bromo-1H-indol-3-yl)-3-(4-(furan-3-yl)pyridin-3-yl)-acrylonitrile;

[0258] -(Z)-2-(5-chloro-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)-acrylonitrile;

[0259] -(Z)-2-(5-bromo-1H-indol-3-yl)-3-(4-(2-(dimethylamino)ethylthio)pyridin-3-yl)-acrylonitrile;

[0260] -(Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-(4-fluorophenoxy)benzonitrile;

[0261] -(Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile;

[0262] -(E)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile;

[0263] -(Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-(dimethylamino)benzonitrile;

[0264] -(Z)-3-(2-(5-chloro-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile;

[0265] -(Z)-3-(2-(5-chloro-1H-indol-3-yl)-2-cyanovinyl)-4-(dimethylamino)benzonitrile;

[0266] -(Z)-3-(2-(5-chloro-1H-indol-3-yl)-2-cyanovinyl)-4-(ethylthio)benzonitrile;

[0267] -(Z)-2-(6-bromo-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)acrylonitrile;

[0268] -(Z)-2-(6-fluoro-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)acrylonitrile;

[0269] -(Z)-2-(6-chloro-1H-indol-3-yl)-3-(4-methoxypyridin-3-yl)acrylonitrile;

[0270] -(Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-(furan-3-yl)pyridine-1-oxide;

[0271] -(Z)-3-(2-(5-chloro-1H-indol-3-yl)-2-cyanovinyl)-4-methoxypyridine-1-oxide;

[0272] -(Z)-3-(2-(5-chloro-1H-indol-3-yl)-2-cyanovinyl)-4-(trifluoromethoxy)benzonitrile;

[0273] -(Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-(trifluoromethoxy)benzonitrile;

[0274] and pharmaceutically acceptable salts thereof.

[0275] In a more preferred embodiment, the indole derivative is Example 38 of WO 2014 / 086964: (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile, which has the following formula:

[0276]

[0277] Phosphonic acid series

[0278] The present invention provides compounds of formula (I) as "phosphonic acid prodrugs" of indole derivatives as defined above.

[0279] Thus, in a particular embodiment, Ra is a group of formula (A): or a group of formula (A'): wherein n is an integer between 1 and 12. In a preferred embodiment, n is an integer between 2 and 10, more preferably an integer between 2 and 8, even more preferably an integer between 2 and 6. Advantageously, n is 2, 3, 4, 5 or 6.

[0280] In a specific embodiment, "phosphonic acid prodrugs" include their pharmaceutically acceptable salts. Preferably, the pharmaceutically acceptable salt is selected from sodium salt, disodium salt, lysine salt, dilysine salt, arginine salt or diarginine salt. More preferably, the pharmaceutically acceptable salt is selected from sodium salt or disodium salt, and even more preferably sodium salt.

[0281] In a preferred embodiment, the compound of formula (I) is selected from the following compounds:

[0282] - Example 1: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid;

[0283] - Example 2: (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid;

[0284] - Example 3: (Z)-7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonic acid;

[0285] - Example 4: (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonic acid;

[0286] - Example 5: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid;

[0287] - Example 6: (Z)-sodium hydrogen 5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonate;

[0288] - Example 7: (Z)-sodium hydrogen 3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonate;

[0289] - Example 8: (Z)-sodium hydrogen 4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonate;

[0290] - Example 9: (Z)-sodium hydrogen 6-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxohexylphosphonate;

[0291] - Example 10: (Z)-sodium hydrogen 7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonate;

[0292] - Example 11: Sodium hydrogen (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonate;

[0293] - Example 12: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid sodium hydrogen;

[0294] - Example 13: (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid disodium salt;

[0295] - Example 14: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid disodium salt;

[0296] - Example 15: (S)-2,6-diaminohexanoic acid compound and (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid (2:1);

[0297] - Example 16: (S)-2,6-diaminohexanoic acid compound and (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid (2:1);

[0298] - Example 17: 5-bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[hydroxy(dioxo)-λ6-phosphino]propyl ester; and

[0299] - Example 18: 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[hydroxy(dioxo)-λ6-phosphino]butyl ester.

[0300] L1 Series

[0301] The present invention further provides compounds of formula (I) as "L1 prodrugs" of indole derivatives as defined above. "L1 prodrugs" as used herein correspond to prodrugs comprising a cleavable group L1.

[0302] Thus, in a particular embodiment, Ra is a group of formula (B): in:

[0303] ○ L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group;

[0304] ○ L2 is tert-butyloxycarbonyl or a protein-binding linker, and

[0305] ○m is an integer equal to 0 or 1.

[0306] According to the present invention, L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group.

[0307] In a particular embodiment, L1 is a pH sensitive group. As used herein, a "pH sensitive" group is sensitive to hydrolysis and may be cleaved under certain pH values ​​(particularly under acidic conditions where the pH range is 4 to 6). Therefore, this pH sensitive group can be an acid-induced cleavable group or an acid-labile group, which is relatively stable in the neutral environment (pH 7.3-7.5) of blood and can be hydrolyzed in slightly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). As examples of hydrolyzable pH sensitive groups, hydrazine, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide, orthoester, carbonate, acetal, ketal, borate, etc. can be cited but are not limited to. In a preferred embodiment, the pH sensitive group as defined above is an acid-induced cleavable group.

[0308] In further specific embodiments, L1 is a photo-induced cleavable group.

[0309] In another specific embodiment, L1 is a bioreducible cleavable group. As used herein, a "bioreducible cleavable" group is cleavable under bioreducing conditions. For example, the group is cleavable in the presence of a reducing agent (such as glutathione or dithiothreitol). Therefore, the glutathione sensitive group can be cleaved by a disulfide exchange reaction with the glutathione substance in the cell. Preferably, L1 is a glutathione sensitive group or a group having a disulfide bond, or a group having a sulfonamide. A variety of disulfide bond groups are known in the art, including, for example, those disulfide bond groups that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene). As used herein, a sulfonamide group is a sulfonyl group attached to an amine group, wherein the sulfur-nitrogen bond can be cleaved.

[0310] In another specific embodiment, L1 is an enzymatically cleavable group. As used herein, an "enzymatically cleavable" group is a group that is cleaved by an enzyme (e.g., a protease, a peptidase, an esterase, a β-glucuronidase, a glycosidase, a phosphodiesterase, a phosphatase, a pyrophosphatase, a tubulin-tyrosine ligase, or a lipase).

[0311] In one embodiment, the enzymatically cleavable group L1 is cleaved by a protease or a peptidase.

[0312] Examples of proteases include, but are not limited to, protease B, VAGP tetrapeptide, etc. Preferably, the enzymatically cleavable group L1 is cleaved by a peptidase. In this embodiment, the enzymatically cleavable group L1 comprises a peptide, which is a cleavage site of a linker, so as to facilitate the release of the drug when exposed to intracellular proteases (e.g., lysosomal enzymes). Peptides can be designed and optimized to be enzymatically cleaved by specific enzymes (e.g., tumor-associated proteases, proteases B, C, and D, or plasmin proteases). Examples of peptides with two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), valine-citrulline (vc or val-cit), valine-arginine (val-arg), valine-lysine (val-lys), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides with three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine-alanine-asparagine (ala-ala-asn), alanine-phenylalanine-lysine (ala-phe-lys), glutamic acid-cysteine-glycine (glu-cys-gly), glycine-glycine-phenylalanine (gly-gly-phe), and glycine-glycine-glycine (gly-gly-gly). Examples of peptides with four amino acids include, but are not limited to, glycine-glycine-valine-citrulline (gly-gly-val-cit), glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly), and glutamic acid-glycine-glutamic acid-glutamic acid (glu-gly-glu-glu). The above amino acid combinations may also be present in the reverse order (i.e., cit-val).

[0313] Examples of peptides are specifically provided in Table A.

[0314] Table A

[0315] sequence Serial Number 1 Gly Gly Val X 2 X Val Gly Gly 3 Gly Gly Phe Gly Serial Number: 1 4 Gly Phe Gly Gly Serial Number: 2 5 Glu Gly Glu Glu Serial Number: 3 6 Glu Glu Gly Glu Serial Number: 4

[0316] X = cit or citrulline

[0317] The peptides disclosed herein may comprise L- or D-isomers of amino acid residues. The term "naturally occurring amino acids" refers to Ala, Asp, Asx, Cit, Cys, Glu, Phe, Glx, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp and Tyr. "D-" means an amino acid with a "D" (dextrorotatory) configuration, which is opposite to the configuration of naturally occurring ("L-") amino acids. The amino acids described herein are commercially available or can be synthesized using methods known in the art.

[0318] Preferably, L1 is p-aminobenzyloxycarbonyl-AA1 w -AA2 x -AA3 y -AA4 z A group wherein AA1, AA2, AA3 and AA4 independently represent amino acids as defined above, and w, x, y and z independently represent integers equal to 0 or 1. The group "p-aminobenzyloxycarbonyl" as used herein is also referred to as PABC. More preferably, L1 is p-aminobenzyloxycarbonyl-AA1 w -AA2 x -AA3 y -AA4 z A group wherein AA1, AA2, AA3 and AA4 independently represent an amino acid selected from alanine, arginine, valine, citrulline, phenylalanine, lysine, glycine, aspartic acid, asparagine, glutamic acid and derivatives thereof, preferably citrulline, valine, cysteic acid, glycine and glutamic acid, more preferably citrulline, valine and cysteic acid, and w, x, y and z are independently integers equal to 0 or 1. Even more preferably, L1 is a group selected from -PABC-Cit-Val and -PABC-Cit-Val-cysteic acid.

[0319] In one embodiment, the enzymatically cleavable group L1 is cleaved by a pyrophosphatase. In this embodiment, L1 contains a pyrophosphate group.

[0320] In one embodiment, the enzymatically cleavable group L1 is cleaved by beta-glucuronidase.In a preferred embodiment, L1 is a -PABC-glucuronide group.

[0321] In one aspect, a "L1 prodrug" is one in which L1 is as defined above and L2 is absent. In this regard, a compound of formula (I) is one in which Ra is a group of formula (B): wherein L1 is as defined above and m is an integer equal to 0.

[0322] According to this aspect, preferred compounds are those selected from the group consisting of:

[0323] - Example 19: (2S,3S,4S,5R,6S)-6-[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid;

[0324] - Example 21: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropionyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester hydrochloride; and

[0325] -Example 25: 2-Amino-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propane-1-sulfonic acid.

[0326] In one aspect, a "L1 prodrug" is one in which L1 is as defined above, m is an integer equal to 1, and L2 is a tert-butyloxycarbonyl (Boc) group. In this aspect, a compound of formula (I) is one in which Ra is a group of formula (B): wherein L1 is as defined above, and m is an integer equal to 0.

[0327] According to this aspect, preferred compounds are those selected from the group consisting of:

[0328] - Example 20: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester; and

[0329] -Example 24: 3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-(tert-butoxycarbonylamino)-3-oxo-propane-1-sulfonic acid.

[0330] In one aspect, a "L1 prodrug" is one wherein L1 is as defined above, m is an integer equal to 1, and L2 is a binding protein linker.

[0331] As used herein, a "binding protein linker" is a group that can react or bind or connect to a peptide, protein or antibody. More specifically, a "binding protein linker" includes any chemical part or group that can connect a peptide, protein or antibody through a stable covalent bond. For example, a "binding protein linker" has a functional group that can react with a free cysteine ​​present on a peptide, protein or antibody.

[0332] Examples of "protein-binding linkers" include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) groups, succinimidyl thioether groups. Additional examples of "protein-binding linkers" include groups having the following molecular formulas:

[0333]

[0334] wherein r is an integer between 1 and 36, preferably an integer between 1 and 24, more preferably equal to 2, 4, 8, 12, 16, 20 or 24.

[0335] According to this aspect, preferred compounds are those selected from the group consisting of:

[0336] - Example 22: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy](peg4)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester; and

[0337] -Example 26: (2R)-3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy]propionylamino]-3-oxo-propane-1-sulfonic acid.

[0338] Conjugate

[0339] The present invention also provides a conjugate of formula (II):

[0340]

[0341] in:

[0342] -X, R1, R 1' , R2, R3 and L1 are as defined herein;

[0343] -L2 is the binding protein linker;

[0344] - P is a peptide or protein, preferably an antibody, antibody fragment or antigen-binding fragment, which is capable of binding to a target of interest; and

[0345] -v is an integer from 1 to 10.

[0346] According to the invention, the conjugate of formula (II) is, for example:

[0347] -X, R1, R 1' , R2, R3 are defined above in the "Indole Derivatives" or "Payload" section (including all specific and preferred embodiments);

[0348] - L1 is a cleavable group as defined above in the "L1 series" section (including all specific and preferred embodiments); and

[0349] - L2 is a binding protein linker as defined above in the section "L1 series" (including all specific and preferred embodiments).

[0350] According to the present invention, the peptide or protein "P" can be coupled to more than one or v compounds of formula (I), wherein m is 1 and L2 is a protein-binding linker. For example, the peptide or protein "P" can be coupled to 1 to 10, 1 to 9, 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compounds of formula (I), wherein m is 1 and L2 is a protein-binding linker. In a preferred embodiment, v is an integer from 1 to 8, 2 to 8. In a particularly preferred embodiment, v is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8.

[0351] According to the present invention, P is a peptide or protein capable of binding to a target of interest. As used herein, "peptide or protein capable of binding to a target of interest" refers to any peptide and / or protein that recognizes and binds to a cell surface molecule (particularly a cell surface marker, antigen, epitope or receptor). In a particular aspect, protein P binds to another protein, not limited to a polypeptide portion. Protein P targeting a specific cell, tissue or location may also have certain therapeutic effects, such as anti-proliferation (cytostatic and / or cytotoxic) activity against a target cell or pathway. In a particular aspect, protein P may contain or may be designed to contain at least one chemically reactive group, such as a carboxylic acid, an amine, a thiol or a chemically reactive amino acid portion or side chain. In a particular aspect, protein P may contain a targeting portion that may bind to or complex with a cell surface molecule (such as a cell surface receptor or antigen) of a given target cell population. After specific binding or complexation with a receptor, the cell may absorb the targeting conjugate and then internalize it into the cell.

[0352] In a particular embodiment, P is a protein, preferably albumin. These albumin conjugates of formula (II) can increase the circulation half-life of the payload and facilitate intracellular delivery to tumors.

[0353] In preferred embodiments, P is an antibody, antibody fragment, or antigen-binding fragment.

[0354] Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens usually have multiple binding sites, also called epitopes or antigenic determinants, which are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to different epitopes has a different structure. Therefore, there may be multiple corresponding antibodies for an antigen. The term "antibody" as used herein has the broadest meaning, specifically covering monoclonal antibodies, single domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as it exhibits the desired biological activity. The antibody can be a mouse antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antibody derived from other species.

[0355] Monoclonal antibodies are homogeneous antibody groups directed against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) directed against target antigens can be prepared using any technique known in the art, which can produce antibody molecules through a continuous cell line cultured. These include, but are not limited to, human B cell hybridoma technology and EBV hybridoma technology. Such antibodies can be any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAb used in the present disclosure can be cultured in vitro or in vivo.

[0356] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be prepared by any of a variety of techniques known in the art.

[0357] In certain aspects, the antibody can be a bispecific antibody (BsAb), i.e., a protein (usually an artificial protein) that can simultaneously bind to two different types of antigens or two different epitopes on the same antigen. BsAb can be a bispecific monoclonal antibody (BsMAb). Methods for preparing bispecific antibodies are known in the art. The traditional preparation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities. The main types of preparation methods are quadromas, chemical coupling, and genetic recombination, each of which produces a unique form. BsAbs are usually prepared using quadromas or hybridoma methods.

[0358] In the quadroma process, due to the random assortment of immunoglobulin heavy and light chains, a mixture of 10 different antibody molecules may be produced, of which only one has the correct bispecific structure. The correct molecule is usually purified using an affinity chromatography step, which is rather cumbersome and produces low product yields.

[0359] According to different approaches, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion can be to an immunoglobulin heavy chain constant domain, which contains at least part of the hinge region, C H 2 and C H 3. The first heavy chain constant region (C H 1) may contain a site required for light chain binding, which site is present in at least one of the fusions. Nucleic acids with sequences encoding immunoglobulin heavy chain fusions and (if desired) immunoglobulin light chains are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility for adjusting the mutual ratios of the three polypeptide fragments when the three polypeptide chains used for the construction are in unequal proportions, which can provide the best yield. However, when the expression of at least two polypeptide chains in equal proportions can produce high yields or the proportions have no special significance, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

[0360] Bispecific antibodies may contain mixed immunoglobulin heavy chains with a first binding specificity on one arm and mixed immunoglobulin heavy chain-light chain pairs (providing a second binding specificity) on the other arm. This asymmetric structure facilitates separation of the desired bispecific compound from the unwanted immunoglobulin chain combination, since only half of the bispecific molecule has an immunoglobulin light chain, which provides a convenient separation method. Using such techniques, bispecific antibodies can be prepared for the treatment or prevention of diseases defined herein.

[0361] Hybrid or bifunctional antibodies can be obtained biologically (ie, by cell fusion techniques) or chemically (particularly using cross-linking agents or disulfide bond forming agents), and may include intact antibodies or fragments thereof.

[0362] The antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen or a microbial antigen, or other antibodies that bind to a tumor cell or matrix. In this regard, "functional activity" refers to that a fragment, derivative or analog can elicit an anti-anti-idiotype antibody that recognizes the same antigen as the antibody derived from the fragment, derivative or analog. Specifically, in exemplary aspects, the antigenicity of the idiotype of an immunoglobulin molecule can be enhanced by deleting the framework sequence and the CDR sequence at the C-terminus of the CDR sequence that specifically recognizes the antigen. In order to determine which CDR sequences bind to the antigen, a synthetic peptide containing the CDR sequence can be used to bind to the antigen by any binding assay method known in the art.

[0363] Other useful antibodies include antibody fragments such as, but not limited to, F(ab')2 fragments (which comprise the variable region, the light chain constant region, and the heavy chain C H 1 domain), and Fab fragments that can be generated by reducing the disulfide bonds of F(ab')2 fragments. Other useful antibodies are antibody heavy and light chain dimers, or any minimal fragment thereof, such as Fv or single chain antibodies (SCA), or any other molecule with the same specificity as an antibody. As used herein, antibody fragments are antibody fragments as defined herein.

[0364] In addition, recombinant antibodies (e.g., chimeric and humanized monoclonal antibodies) comprising human and non-human parts that can be prepared using standard recombinant DNA techniques are useful antibodies. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those molecules with variable regions derived from mouse monoclonals and human immunoglobulin constant regions. Humanized antibodies are antibody molecules from non-human species that have one or more complementary determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0365] Fully human antibodies can be produced using transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes, but can express human heavy and light chain genes. The transgenic mice are immunized with a selected antigen (e.g., all or part of the polypeptides of the present disclosure) in a normal manner. Monoclonal antibodies against antigens can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation, followed by class switching and somatic mutation. Therefore, using this technology, IgG, IgA, IgM, and IgE antibodies with therapeutic uses can be produced. For an overview of the technology used to generate human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0366] A technique called "guided selection" can be used to generate fully human antibodies that can recognize a selected epitope. In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to guide the selection of fully human antibodies that recognize the same epitope. Human antibodies can also be generated using various techniques known in the art, including phage display libraries.

[0367] The antibody can be a fusion protein of an antibody or a functionally active fragment thereof and an amino acid sequence of another protein (or a portion thereof, such as at least 10, 20 or 50 amino acid portions of a protein) other than an antibody, for example, the antibody is fused to the N-terminus or C-terminus of the amino acid sequence by a covalent bond (such as a peptide bond). For example, the antibody or its fragment can be covalently linked to another protein at the N-terminus of the constant domain.

[0368] Antibodies include analogs and derivatives that have been modified, i.e., by covalent attachment of any type of molecule, as long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, derivatives and analogs of antibodies include those that have been further modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to cellular antibody units or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, the analogs or derivatives may contain one or more unnatural amino acids.

[0369] Antibody conjugates can include antibodies with modifications (e.g., substitutions, deletions, or additions) in the amino acid residues that interact with the Fc receptors. Specifically, antibodies include antibodies with modifications of amino acid residues identified as participating in the interaction between the anti-Fc domain and the FcRn receptor. Immunospecific antibodies directed against cancer cell antigens can be obtained commercially, for example, from Genentech (San Francisco, Calif.), or generated by any method known to those skilled in the art (e.g., chemical synthesis or recombinant expression techniques). Nucleotide sequences encoding immunospecific antibodies directed against cancer cell antigens can be obtained from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing.

[0370] In some aspects, the antibody conjugate can be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody or a humanized antibody. In some aspects, the antibody can be an antibody fragment, such as a Fab fragment.

[0371] Known antibodies that can be coupled for treating or preventing cancer are described herein. Immunospecific antibodies directed against cancer cell antigens can be obtained commercially or prepared by any method known to those skilled in the art (e.g., recombinant expression techniques). Nucleotide sequences encoding immunospecific antibodies directed against cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing. Examples of antibodies that can be used to treat cancer include, but are not limited to, humanized anti-HER2 monoclonal antibodies for treating patients with metastatic breast cancer; (rituximab; Genentech), which is a chimeric anti-CD20 monoclonal antibody used to treat patients with non-Hodgkin's lymphoma; OvaRex (orgovoumab; AltaRex Corporation, MA), which is a murine antibody used to treat ovarian cancer; Panorex (edrecolomab, Glaxo Wellcome, NC), which is a murine IgG2a antibody used to treat colorectal cancer; CetuximabErbitux (cetuximab, Imclone Systems Inc., NY), which is an anti-EGFR IgG chimeric antibody used to treat epidermal growth factor-positive cancers such as head and neck cancer; Vitaxin (etaracizumab, MedImmune, Inc., MD), which is a humanized antibody used to treat sarcomas; Campath (etaracizumab, MedImmune, Inc., MD), which is a humanized antibody used to treat sarcomas; I / H (alemtuzumab, Leukosite, MA), a humanized IgG1 antibody used to treat chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA), a humanized anti-CD33 IgG antibody used to treat acute myeloid leukemia (AML); LymphoCide (epratuzumab, Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody used to treat non-Hodgkin's lymphoma; Smart ID 10 (Protein Design Labs, Inc., Labs, Inc., CA), which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA), which is a radiolabeled murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA), which is a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (bevacizumab, Genentech, Inc., CA), which is an anti-VEGF humanized antibody for the treatment of lung and colorectal cancer; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA), which is an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ), which is a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0372] Other useful antibodies include, but are not limited to, brentuximab (anti-CD30), polatuzumab (anti-CD79b), enfortumab (anti-Nectin 4), tisotumad (anti-tissue factor), loncastuximab (anti-CD19), rituximab (anti-CD20), trastuzumab (anti-HER2), inotuzumab (anti-CD22), gemtuzumab (anti-CD33), pertuzumab (anti-HER2), obinutuzumab (anti-CD20), ofatumumab (anti-CD20), olaratumab (anti-PDGFR-α), ishazab (anti-CD20), selegiline ... isatuximab (anti-CD38), sacituzumab (anti-TROP2), U3-1784 (anti-FGFR4), daratumumab (anti-CD38), STI-6129 (anti-CD38), lintuzumab (anti-CD33), huMy9-6 (anti-CD33), belantamab (anti-BCMA), indatuximab (anti-CD138), cetuximab (anti-EGFR), dinutuximab (anti-GD2), anti-CD38 A2 antibody, HuAT13 / 5 antibody, alemtuzumab (anti-CD52), ibritumomab (anti-CD20), tositumomab (anti-CD20 conjugated to I231), bevacizumab (anti-VEGF), panitumumab (anti-EGFR), tremelimumab (formerly known as ticilimumab-anti-CTA-4), catumaxomab (anti-CD3 and EpCAM), oregovomab (anti-CA125), h 1959 antibody (anti-Gal-3BP), and veltuzumab (anti-CD20).

[0373] Other useful antibodies include, but are not limited to, antibodies against the following antigens: CA125 (ovary), CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA

[00136] The present invention relates to the expression of proteins involved in the present invention in the context of cancer, leukemia, prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (cancer), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE-4 (cancer), anti-transferrin receptor (cancer), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (cancer), CD38 (multiple myeloma), CD40 (lymphoma), mucin (cancer), P21 (cancer), MPG (melanoma), and Neu oncogene products (cancer). Some specific, useful antibodies include, but are not limited to, BR96 mAb (Trail, PA, et al. Science (1993) 261, 212-215), BR64 (Trail, PA, et al. Cancer Research (1997) 57, 100-105), mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, JA, et al. Cancer Res. (2000) 60: 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb, and mAbs against the CD30 antigen, such as AC10. Many other internalizing antibodies that bind to tumor-associated antigens can be used and have been reviewed.

[0374] Other antigens that the conjugates of the invention can bind to include, but are not limited to, 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axinl, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD4 0, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, aggregation factor, cKit, Claudin 3. Claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Cripto protein, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosylgalactose-3BP, GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMF24, HMWMAA, HPVE6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1IRa, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrin (including α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, αIIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIRl, LAMP-1, LCK, Legumain, LewisY, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, Mel an A / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostaglandins, Pseudomonas aeruginosa, rabies, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, respiratory syncytial virus, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRb, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-b2, TNF-a, TGS5, Tie 2, TIM-1, TnAg, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and / or XAGE1.

[0375] Antibodies that bind to antigens associated with antigen-presenting cells (e.g., CD40, OX40L, endoglin, DEC-205, 4-1BBL, CD36, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLECIOA, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, BDCA-2, XCR-1, and CCR2) can also be coupled.

[0376] Antibody conjugates can be combined with receptors or receptor complexes expressed on activated lymphocytes. Receptors or receptor complexes can include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins or complement control proteins. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1 and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103 and CD 104. Non-limiting examples of suitable lectins are C-type, S-type and I-type lectins.

[0377] Preferably, the antibodies include 3F8, 8H9, abagovomab, abciximab Abizumab, abrezekimab, abrilumab, actoxumab, adalimumab Adekalimumab, aducanumab, afasevikumab, afelimomab, atezolizumab, alacizumab, ALD518, alemtuzumab Alirocumab Atumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrulitumab, anti-TnC-A1-SIP (F16), apolizumab, aprotumab, acitumomab (CEA- ), ascrinvacumab, aseluzumab, atidortoxumab, atlizumab (tocilizumab, ), atezolizumab atinumab, atumumab, avelumab (Bavencio), azintuxizumab, balantamab, bapinezumab, basiliximab Baveximab, BCD-100, betumomab begelomab, belantamab, belimumab bemarituzumab, benralizumab bermekimab, bersanlimab, bermekimab, besozumab Bevacizumab Bezlotoxumab Biximab bimagrumab, bimekizumab, birtamimab, bivacizumab, bleselumab, rantumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab, brentuximab, brodalumab (SILIQ TM ), brolucizumab Brontictuzumab, burosumab cabiralizumab, caplacizumab Camidanlumab, camrelizumab, canakinumab cantuzumab, carotuximab, carlumab, catuzumab cBR96, CC49, cedizumab, cemiplimab cergutuzumab, certrelimab, certolizumab, cetuximab cibisatamab, cirmtuzumab, citatuzumab, citumumab, clazakizumab, cleximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab crotedumab, cusatuzumab, daclizumab, daclizumab dalotuzumab, dapirolizumab, daratumumab dectrekumab, demcizumab, denintuzumab, denosumab ( depatuxizumab, derlotuximab, demuximab, dezamizumab, dinutuximab diridavumab, domagrozumab, dostarlimab, dorlimomab, dorlixizumab, drozitumab, DS-8201, duligotuzumab, dupilumab Durvalumab dusigitumab, emeticlizumab, eculizumab Ebalizumab, Edratumomab Efalizumab Efficacy eldelumab, elezanumab, elgemtumab, elozumab Elsilimomab, emactuzumab, emapalumab emibetuzumab, emicizumab enapotamab, enavatuzumab, enfortumab Enmotumomab, enoblituzumab, enokizumab, enoticumab, ensituximab, ipilimumab, eptinezumab Epratuzumab, erenumab erlizumab, ermaquine (REXOMUN"), edarizumab (ABEGRIN"), etigilimab, etrolizumab, evinacumab, evolocumab Avelumab, fanolesomab faricimab, faricimab, farizumab, fasinumab, FBTA05, famizumab, fizanumab, fibatuzumab, ficlatuzumab, fentolimumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontuzumab foralumab, flavavir, fremanezumab Fusutuximab, frovocimab, fmnevetmab, fullanumab, futuximab, galcanezumab galiximab, gancotamab, ganitumab, ganitumomab, gavitumomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, gereximab, glembatumumab, golimumab gomiliximab, guselkumab huMy9-6, OR000213, ianalumab, ibalizumab IBI308, ibritumomab, icrucumab, idarucizumab ifabotuzumab, igovolumab (INDIMACIS-125), iladatuzumab, IMAB362, imalumab, imaprelimab, incitabine imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, infliximab inotuzumab, inotuzumab, iomab-B, ipilimumab, itumumab, isatuximab iscalimab, istiratumab, itolizumab, ixekizumab Keliximab, labetuzumab (CEA-CIDE TM ), lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab landogrozumab, laprituximab, larcaviximab, levitrazumab, lemasomab, lendalizumab, lenvervimab, lenzilumab, leronlimab, lesofavumab, letolizumab, lexatumumab, livremab, lifastuzumab, ligelizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, loncastuximab, lorvotuzumab, losatuxizumab, lucamumab, lulizumab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, masitumomab, matuzumab, mavrilimumab, mepolizumab metizumab, milatuzumab, mirelimumab, mirikizumab, mirvetuximab, mitumomab, modotuximab, molalizumab, mogamulizumab Muromab, mosunetuzumab, motavizumab moxetumomab muromonab-CD3 (ORTHOCLONE ), nacolomab, namilumab, naptumomab, naratuximab, namatumab, natalizumab navicixizumab, navivumab, naxitamab, nebacumab, nexitumomab nemolizumab, NEODOOl, nerimumab, nesvacumab, netakimab, nimotuzumab nirsevimab, nivolumab, novofilimab, obiltoxaximab Atezolizumab, ocaratuzumab, ocrelizumab Odomumab, Ofatumumab Olaratumab oleclumab, olendalizumab, olokizumab, omalizumab omburtamab, OMS721, onartuzumab, ontecizumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, ovarex, orticumab, oxetimumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ogamicin, ozoralizumab, pavelizumab, palivizumab Pamrevlumab, panitumumab pankomab, panokumab, parsatuzumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab perakizumab, pertuzumab pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, polatuzumab (Polivy), prezalumab, plozalizumab, pogalizumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, pritoxaximab, plitoxaximab, PRO140, quilizumab, ranevetmab, radretumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab ravagalimab, ravulizumab Raxicumab, refanezumab, regavir, REGN-EB3, renatlimab, remtolumab, reslizumab Ritumumab, rinucumab, risankizumab Rituximab rivabazumab, rmab, rolotumumab, roledumab, romilkimab, romosozumab Rolizumab, rosmantuzumab, rovalvuzumab, rovalpituzumab rozanolixizumab, rulizumab (ANTOVA), SA237, sacituzumab, samalizumab, samrotamab, sarilumab satralizumab, satumomab pendetide, seculumab selicrelumab, seribantumab, setoxaximab, setmsumab, sevimmab, SGN-CD19A, SHP647, sirolimumab, sifalimumab, setuximab, simtuzumab, silizumab, sirtratumab, sirukumab, sofituzumab, solanizumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, setuximab, STI-6129, thiosomab suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab (AFP- ), talacotuzumab, tavolimab, tamtuvetmab, tanizumab, patamumab, tarextumab, tavolimab, tefizumab telimomab, telisotuzumab, tesidolumab, tetraxetan, tetulomab, tenetuzumab, tenetuzumab, tetitumomab Tilizumab, tezepelumab, TGN1412, tibulizumab, ticilimumab Tilgizumab, timigutuzumab, timolumab, tiragolumab, tiragotumab, tislelizumab, tisotumab, tiuxetan, tildrakizumab TNX-650, atlizumab, ), tomozuotuximab, tolizumab, tosatoxumab, tositumomab tovetumab, tralokinumab, trastuzumab TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirizumab, ustekinumab ublituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vanalimab, vandortuzumab, vantictumab, vanucizumab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepatumomab, vesencumab, vesizumab vobarilizumab, voriximab vonlerolizumab, vopratelimab, vorsetuzumab, vortumumab, vunakizumab, xentuzumab, XMAB-5574, zalumab (HuMEX-EGFr), zalumab (HuMAX-CD4), zatuximab, zenocutuzumab, zilamumab, zolbetuximab, or zolimomab.

[0378] An antibody that "binds" a target of interest (eg, a molecular target or target antigen) is one that is capable of binding the antigen with sufficient affinity such that the antibody can be used to target cells expressing the antigen.

[0379] In another specific embodiment, P is a peptide, preferably with specific targeting ability to a protein receptor overexpressed in tumor tissue. These peptides are generally precedented in the literature to have strong binding affinity to the target site, which can be measured in the nanomolar range by different techniques, including, for example, surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC). More specifically, the peptide is the tripeptide RGD (arg-gly-asp) targeting integrin (α5β1, α8β1 and αIIbβ3), or GnRH (gonadotropin-releasing hormone) targeting GnRH-R (receptor version of the hormone), or SST (somatostatin) targeting SSTR1-5 (somatostatin receptor), or EGF (epidermal growth factor) targeting EGFR: HER1, HER2, HER3, HER4, or Angiopep-2 targeting LRP-1 (low-density lipoprotein receptor-related protein-1), or bradykinin potentiating peptide (BPP) targeting ACE (angiotensin converting enzyme receptor).

[0380] The conjugate of formula (II) defined above can also be represented by the following formula III:

[0381]

[0382] In this regard, X, R1, R 1' , R2 and R3 are as defined above in the "Payload" section (including all specific and preferred embodiments). P and v are as defined above.

[0383] In this regard, L3 represents a linker that allows the peptide or protein P to be conjugated to a "payload" (molecule or indole derivative). As used herein, the term "linker" refers to any chemical moiety capable of connecting a peptide or protein "P" to a "molecule".

[0384] In one embodiment, the linker L3 may comprise a heterobifunctional group. As used herein, the term "heterobifunctional group" refers to a chemical portion that connects the linker to which it belongs and the peptide or protein P. The heterobifunctional group is characterized by having different reactive groups at either end of the chemical portion. The connection to "P" can be achieved by chemical or enzymatic coupling or a combination of the two. Chemical coupling involves a controlled reaction of accessible amino acid residues on the surface of protein P with a reactive handle on the heterobifunctional group. Examples of chemical coupling include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling of non-natural amino acids introduced by genetic engineering, wherein non-natural amino acid residues with the desired reactive handle are installed on "P". In enzymatic coupling, an enzyme mediates the coupling of a linker to accessible amino residues on a peptide or protein P. Examples of enzymatic coupling include, but are not limited to, transpeptidation reactions using sortase, transpeptidation reactions using microbial transglutaminase, and N-glycan engineering. Chemical coupling and enzymatic coupling can also be used sequentially. For example, enzymatic coupling can also be used to install unique reactive handles on "P" for subsequent chemical coupling. In particular, the heterobifunctional group is selected from:

[0385]

[0386] in is the point of attachment to the remainder of the linker L3, and It is the connection point with "P".

[0387] In one embodiment, the linker L3 is a group that can be cleaved by a protease (e.g., protease B, VGAP tetrapeptide, etc.). Specifically, the linker L3 is a linker that can be cleaved by a protease selected from the following:

[0388]

[0389] wherein q is an integer from 2 to 10; Z 1 , Z 2 , Z 3 and Z 4 Each independently does not exist or is in the L- or D-configuration of a naturally occurring amino acid residue, provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of them are amino acid residues; is the point of attachment to the molecule; and is the point of connection with "P". Preferably, Z 1 , Z 2 , Z 3 and Z 4is independently absent or selected from L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of them are amino acid residues. More preferably, Z 1 Not present or glycine; Z 2 is absent or is selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine and glycine; Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine. Even more preferably, L3 is where q is 5, and is the point of attachment to the molecule; and It is the connection point with "P".

[0390] In one embodiment, the linker L3 is a pyrophosphatase cleavable linker, preferably a pyrophosphatase cleavable linker having the formula: wherein q is an integer from 2 to 10, and is the point of attachment to the molecule; and It is the connection point with "P".

[0391] In one embodiment, the linker L3 is a β-glucuronidase cleavable linker, preferably selected from

[0392] Where q is an integer from 2 to 10; ---- does not exist or is a bond; and It is the point of connection to the molecule; It is the connection point with "P".

[0393] In one embodiment, the linker L3 is bioreducible. The bioreducible linker utilizes the difference in reduction potential between the intracellular compartment and the plasma. The content of reduced glutathione in the cytoplasm of tumor cells is 1000 times higher than that in the cytoplasm of normal cells, and tumor cells also contain enzymes that can lead to a decrease in reduced glutathione in the intracellular compartment. The linker keeps the conjugate intact during systemic circulation and is selectively cleaved by high concentrations of glutathione in the cell, thereby releasing the active drug at the tumor site from the non-toxic prodrug. Preferably, L3 is a bioreducible linker selected from the following:

[0394]

[0395] wherein q is an integer from 2 to 10; R, R', R" and R'" are each independently selected from hydrogen, C1-C6 alkoxy, C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or the two geminal R groups together with the carbon atoms to which they are attached may form a cyclobutyl or cyclopropyl ring; and is the point of attachment to the molecule; and It is the connection point with "P".

[0396] In one embodiment, the linker L3 is acid cleavable. Preferably, L3 is an acid cleavable linker selected from

[0397] wherein q is an integer from 2 to 10; and It is the point of connection to the molecule; It is the connection point with "P".

[0398] In one embodiment, L3 is a click-release linker, wherein the release of the molecule is chemically triggered by tetrazine or related compounds. Preferably, L3 is a click-release linker selected from:

[0399] wherein q is an integer from 2 to 10; and It is the point of connection to the molecule; It is the connection point with "P".

[0400] In one embodiment, L3 is a linker L1-L2, wherein L1 is a cleavable group as defined above in the "L1 series" section (including all specific and preferred embodiments); and L2 is a binding protein linker as defined above in the "L1 series" section (including all specific and preferred embodiments).

[0401] The present invention also provides a "payload" or "drug" as defined above and a compound of formula (I) as defined herein coupled to a nanoparticle (particularly a silica nanoparticle). In a particular aspect, the "payload" can be substituted with a linker to form a linker-payload conjugate, which is attached to the silica nanoparticle to form a nanoparticle-drug conjugate. Examples of such nanoparticle-drug conjugates (NDCs) are described, for example, in international patent applications WO2022 / 093800 and WO2022 / 093794.

[0402] A preferred aspect is a nanoparticle-drug conjugate comprising:

[0403] (a) a nanoparticle comprising a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) is covalently bound to the surface of the nanoparticle, and optionally a fluorescent compound is covalently encapsulated within the core of the nanoparticle;

[0404] (b) a targeting ligand that binds to a folate receptor, wherein the targeting ligand is selected from the group consisting of folic acid, dihydrofolate, tetrahydrofolate, and a folate receptor binding derivative of any of the foregoing, wherein the targeting ligand is directly or indirectly attached to the nanoparticle via a spacer; and

[0405] (c) a compound of formula (I) as defined herein, wherein Ra is of formula (B) A group, wherein L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group, and m is equal to 0; wherein the compound of formula (I) is attached to the nanoparticle directly or indirectly through a spacer.

[0406] Therapeutic Uses

[0407] The present invention relates to:

[0408] - a pharmaceutical composition comprising any compound of formula (I), (II), (III) as defined above (including any disclosed embodiment); and / or

[0409] - a pharmaceutical composition comprising any compound of formula (I), (II) or (III) as defined above (including any disclosed embodiment) and a pharmaceutically acceptable excipient; and / or

[0410] a pharmaceutical composition comprising (a) any compound of formula (I), (II) or (III) as defined above or including any of the disclosed embodiments, and (b) an additional active ingredient, preferably an additional anti-tumor drug; and / or

[0411] - a pharmaceutical composition as defined above or any compound of formula (I), (II) or (III) as defined above (including any disclosed embodiment) for use as a medicament; and / or

[0412] - a pharmaceutical composition as defined above or any compound of formula (I), (II) or (III) as defined above for use in the treatment of cancer; and / or

[0413] - a product or kit comprising (a) any compound of formula (I), (II) or (III) as disclosed above (including any of the disclosed embodiments), and (b) an additional active ingredient, preferably an additional anti-tumor drug, as a combined preparation for simultaneous, separate or sequential use, in particular for the treatment of cancer; and / or

[0414] - a combined preparation comprising (a) any compound of formula (I), (II) or (III) as disclosed above (including any of the disclosed embodiments), and (b) an additional active ingredient, preferably an additional anti-tumor drug, for simultaneous, separate or sequential use, in particular for the treatment of cancer; and / or

[0415] - a pharmaceutical composition as defined above or any compound of formula (I), (II) or (III) as defined above (including any disclosed embodiment) for use in the treatment of cancer in combination with radiation therapy, surgery (e.g., tumor resection), hyperthermia and / or other anti-tumor therapy or before, simultaneously with or after surgery (e.g., tumor resection); and / or

[0416] - use of a pharmaceutical composition as defined above or any compound of formula (I), (II) or (III) as defined above (including any disclosed embodiment) for the preparation of a medicament for the treatment of cancer; and / or

[0417] - use of a pharmaceutical composition as defined above or any compound of formula (I), (II) or (III) as defined above (including any disclosed embodiment) and (b) an additional active ingredient (preferably an additional anti-tumor drug) for the preparation of a medicament for the treatment of cancer; and / or

[0418] - a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition as defined above or a compound of any of formula (I), (II) or (III) as defined above (including any disclosed embodiment); and / or

[0419] - a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition as defined above or a compound of any of formula (I), (II) or (III) as defined above (including any disclosed embodiment), and (b) an additional active ingredient (preferably an additional anti-tumor drug); and / or

[0420] - A method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition as defined above or a compound of any of formula (I), (II) or (III) as defined above (including any disclosed embodiment), in combination with radiation therapy, surgery (e.g., tumor resection), hyperthermia and / or other anti-tumor therapies.

[0421] As used herein, the term "cancer" refers to the presence of cells with typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer can be a solid tumor or a hematopoietic tumor. Examples of cancer include, for example, leukemias, lymphomas, blastomas, carcinomas, and sarcomas. More specific examples of such cancers include chronic myeloid leukemia, acute lymphocytic leukemia, Philadelphia chromosome positive acute lymphocytic leukemia (Ph+ALL), squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, osteosarcoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, esophageal cancer, colon cancer, head and neck cancer, gastric cancer, germ cell tumors, childhood sarcoma, sinus natural killer cells, multiple myeloma, acute myeloid leukemia (AML), chronic lymphocytic leukemia, mastocytosis and any symptoms associated with mastocytosis. Preferably, the cancer is leukemia, acute myeloid leukemia, lymphoma, breast cancer, pancreatic cancer, lung cancer or colon cancer. More preferably, the cancer is acute myeloid leukemia or pancreatic cancer. Optionally, the cancer is associated with dysregulation of MKlp2 or its pathway. Specifically, the cancer is associated with overexpression of MKlp2.

[0422] The terms "treatment", "treat" or "treating" as used herein refer to any action intended to improve the health status of a patient, such as treatment, prevention, prophylaxis, and delay of disease. In certain embodiments, the term refers to the improvement or eradication of a disease or symptoms associated with a disease. In other embodiments, the term refers to minimizing the spread or worsening of a disease caused by administering one or more therapeutic agents to a subject suffering from such a disease.

[0423] "Effective amount" refers to the amount of the pharmaceutical composition of the present invention that prevents, eliminates or reduces the harmful effects of the treated disease in mammals (including humans). It should be understood that those skilled in the art can adjust the dosage according to the patient, pathology, mode of administration, etc. For example, the compound of the present invention can be used at a dosage of 0.01 to 500 mg / kg body weight / day. In a specific embodiment, the pharmaceutical composition of the present invention comprises 0.01 to 500 mg / kg of the compound of the present invention. It should be understood that those skilled in the art can adjust the dosage according to the patient, pathology, mode of administration, etc.

[0424] The route of administration can be topical, transdermal, oral, rectal, sublingual, intranasal, intrathecal, intratumoral or parenteral (including subcutaneous, intramuscular, intravenous and / or intradermal). Preferably, the route of administration is parenteral, oral or topical. The pharmaceutical composition is suitable for one or more of the above-mentioned routes. The pharmaceutical composition, kit, product or combined preparation is preferably administered by injection or intravenous infusion or suitable sterile solution, or is administered by the digestive tract in the form of a liquid or solid dosage.

[0425] The pharmaceutical composition can be formulated as a solution in a pharmaceutically compatible solvent, or as an emulsion, suspension or dispersion in a suitable pharmaceutical solvent or carrier, or as a pill, tablet or capsule containing a solid carrier in a manner known in the art. The preparation of the present invention suitable for oral administration can be in the form of discrete units such as capsules, sachets, tablets or lozenges, each unit containing a predetermined amount of active ingredient; can be in the form of powder or granules; can be in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or can be in the form of a water-in-oil emulsion or an oil-in-water emulsion. Preparations for rectal administration can be in the form of suppositories, in which the active ingredient and a carrier such as cocoa butter are incorporated, or can be in the form of enemas. Preparations suitable for parenteral administration conveniently contain a sterile oily or aqueous preparation of the active ingredient (which is preferably isotonic with the blood of the recipient). Each such preparation may also contain other pharmaceutically compatible and non-toxic adjuvants, such as stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances. The preparation of the present invention comprises an active ingredient and a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients. The carrier must be "acceptable", i.e., compatible with the other ingredients of the formulation and not deleterious to the recipient. The pharmaceutical composition is advantageously administered by injection or intravenous infusion of a suitable sterile solution or orally via the digestive tract. Safe and effective methods of administering most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in standard literature.

[0426] Additional antineoplastic agents can be selected from a non-exhaustive list of antineoplastic agents consisting of inhibitors of topoisomerase I or II, antimitotic agents, DNA alkylating agents, antimetabolites, targeted agents (e.g., kinase inhibitors), therapeutic antibodies and / or antibody-drug conjugates designed to mediate cytotoxicity against cancer cells or to modulate one of their key biological functions.

[0427] Antimitotic agents include, but are not limited to, paclitaxel, docetaxel, and their analogs, such as larotaxel (also known as XRP9881; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol-Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), ortataxel (also known as IDN 5109, BAY 59-8862, or SB-T-101131; Bristol-Meyer-Squibb), RPR109881A (Bristol-Meyer-Squibb), RPR 116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), PG-paclitaxel (also known as CT-2103, PPX, polyglutamic acid paclitaxel, polyglutamic acid paclitaxel, or Xyotax TM ), (also known as Nab-Paclitaxel; ABRAXIS BIOSCIENCE), Tesetaxel (also known as DJ-927), IDN 5390 (INDENA), Taxoprexin (also known as docosahexaenoic acid-paclitaxel; PROTARGA), DHA-paclitaxel (also known as ) and MAC-321(WYETH).

[0428] Inhibitors of topoisomerase I and / or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin, and mitoxantrone. Inhibitors of topoisomerase I and II include, but are not limited to, intoplicin.

[0429] DNA alkylating agents include, but are not limited to, cisplatin, carboplatin, and oxaliplatin. In a preferred embodiment, the DNA alkylating agent is cisplatin.

[0430] Antimetabolites block the enzymes responsible for nucleic acid synthesis or are incorporated into DNA, thereby generating an erroneous genetic code and leading to apoptosis. Non-exhaustive examples include, but are not limited to, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, more specifically methotrexate, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, 5-fluorouracil, gemcitabine, and capecitabine.

[0431] The anti-tumor agent may be an alkylating agent, including but not limited to nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes. Non-exhaustive examples include uracil nitrogen mustard, chlormethylamine, cyclophosphamide (CYTOXAN (R)), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, cisplatin, carboplatin, oxaliplatin, thiotepa, streptozocin, dacarbazine and temozolomide.

[0432] Antitumor agents can also be targeting agents, particularly kinase inhibitors. Kinases can be selected from intracellular tyrosine or serine / threonine kinases, receptor tyrosine or serine / threonine kinases. For example, these drugs may have the ability to inhibit angiogenesis based on the inhibitory activity of VEGFR and PDGFR kinases. Specifically, targeted drugs can be selected from a variety of approved kinase inhibitor drugs: Gleevec that inhibits Abl, and Iressa and Tarceva that both inhibit EGFR, sorafenib (Nexavar, BAY 43-9006) that inhibits Raf, dasatinib (BMS-354825) and nilotinib (AMN-107, Tasigna) that also inhibit Abl, lapatinib that also inhibits EGFR, temsirolimus (Torisel, CCI-779) that targets the mTOR pathway, sunitinib (Stuten, SU11248) that inhibits multiple targets including VEGFR, and specific antibodies that inactivate kinase receptors: Herceptin and Avastin.

[0433] Antibody-drug conjugates include, but are not limited to, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, Sacituzumab govitecan belantamab mafodotin trastuzumab duocarmazine, BAT8001, mirvetuximab soravtansine, SAR408701, loncastuximab tesirine, camidanlumab tesirine, vadastuximab talirine, trastuzumab duocarmazine, BAT8001, mirvetuximab soravtansine, SAR408701, loncastuximab tesirine, camidanlumab tesirine, vadastuximab talirine, rovalpituzumab tesirine, or depatuxizumab mafodotin.

[0434] The term "treatment" as used herein refers to any type of cancer treatment (ie, anti-tumor therapy), including adjuvant therapy and neoadjuvant therapy. Treatment includes radiation therapy and therapy, preferably systemic therapy, such as hormone therapy, chemotherapy, immunotherapy, and monoclonal antibody therapy.

[0435] As used herein, the term "adjuvant therapy" refers to any type of cancer treatment, usually after surgical removal of the primary tumor, as an additional treatment for patients with cancer who are at risk of metastasis and / or who may relapse. The purpose of such adjuvant therapy is to improve prognosis. Adjuvant therapy includes radiation therapy and therapy, preferably systemic therapy, such as hormone therapy, chemotherapy, immunotherapy, and monoclonal antibody therapy.

[0436] "Hormone therapy" or "hormone treatment" refers to cancer treatments that are designed to block, add, or remove hormones. For example, in breast cancer, the female hormones estrogen and progesterone can promote the growth of some breast cancer cells. Therefore, for these patients, hormone therapy is needed to block estrogen. Commonly used drugs include: tamoxifen, toremifene, anastrozole, exemestane, letrozole, goserelin / leuprolide, megestrol acetate, and fluoxymesterone.

[0437] As used herein, the term "chemotherapeutic treatment" or "chemotherapy" refers to the treatment of cancer with chemical or biological substances, particularly with one or more anti-neoplastic drugs.

[0438] The terms "radiation therapy" or "radiotherapy" are commonly used in the art and refer to various types of radiation therapy, including internal and external radiation therapy or radioimmunotherapy, and the use of various types of radiation, including x-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiation.

[0439] Further aspects and advantages of the present invention will be described in the following examples which are to be regarded as illustrative rather than limiting. Example

[0440] I. "Phosphonic acid prodrugs"

[0441] General Synthesis Scheme I:

[0442]

[0443] Dibenzyl phosphite is condensed with bromofatty acid esters (bromalkanoates) to form phosphonates, which are hydrolyzed by alkaline to form phosphonic acid. The acid is then coupled to the NH-indole of the drug by a peptide coupling agent to obtain a benzyl phosphonate prodrug, which is hydrolyzed in an acidic medium to form a prodrug phosphonic acid. Treatment with an alkali can yield a phosphonic acid prodrug in the form of a salt.

[0444] Preparation method of phosphonic acid prodrug

[0445] Phosphonoalkanoic acid was synthesized according to the method described by C. Zingle et al. in Bioorganic & Medicinal Chemistry letters, 22(21), 6563-6567 (2012).

[0446] A. Phosphonoalkanoate (Step 1)

[0447] 1.5-(Bis(benzyloxy)phosphoryl)valerate ethyl ester

[0448] Under argon atmosphere, at 0°C, a solution of dibenzyl phosphite (7mL, 28.5mmol) in DMF (20mL) was slowly added to a suspension of sodium hydride (1.141g, 28.5mmol) in DMF (40mL). The reaction mixture was stirred at room temperature for 15 minutes, cooled to 0°C again, and ethyl 5-bromopentanoate (4.51mL, 28.5mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, poured into 150mL of water and extracted with 150mL of EtOAc. The organic layer was washed twice with water (75mL). The combined aqueous layer was extracted again with 75mL of EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to obtain ethyl 5-(bis(benzyloxy)phosphoryl)pentanoate (11.41g) as a colorless oil. Yield = 87%

[0449] LC-MS: [M+H]=391

[0450] 1 H NMR (300MHz, DMSO) δppm: 7.45–7.20 (m, 10H), 5.10–4.86 (m, 4H), 4.02 (q, J = 7.3Hz, 2H), 2.29 (dt, J=14.3,7.4Hz,2H),1.81(ddd,J=10.7,7.9,5.9Hz,2H),1.70–1.35(m,4H),1.15(t,J=7.1Hz,3H)

[0451] The following compounds were prepared using the same method as above:

[0452] 2. Ethyl 3-(bis(benzyloxy)phosphoryl)propionate

[0453] Yield = 25%

[0454] LC-MS: [M+H]=363

[0455] 1H NMR (300MHz, DMSO) δppm: 7.37 (s, 10H), 4.99 (qd, J = 12.1, 8.1Hz, 4H), 4.03 (q, J=7.1Hz,2H),2.37(t,J=7.3Hz,2H),2.25-2.05(m,2H),1.15(t,J=7.1Hz,3H)

[0456] 3.7-(Bis(benzyloxy)phosphoryl)heptanoic acid ethyl ester

[0457] Yield = 99%

[0458] LC-MS: [M+H] = 419

[0459] 1H NMR (300MHz, DMSO) δppm: 7.43–7.24 (m, 10H), 4.97 (qd, J=12.1, 8.3Hz, 4H), 4.03 (q, J=7.1, 2H), 2.24 (dt,J=12.2,7.3Hz,2H),1.88–1.67(m,2H),1.57–1.31(m,6H),1.34–1.18(m,2H),1.16(t,J=7.1,3H)

[0460] B. Phosphonoalkanoic acid (step 2)

[0461] 1.5-(Bis(benzyloxy)phosphoryl)pentanoic acid

[0462] 1N sodium hydroxide (58.5 mL, 58.5 mmol) was added to a solution of ethyl 5-(bis(benzyloxy)phosphoryl)pentanoate (11.41 g, 29.2 mmol) in ethanol (100 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with 200 mL of water and washed with ethyl acetate (3 x 100 mL). The combined organic layers were washed with water. The aqueous layer was acidified to pH=3-4 with 1N HCl (60 mL) and then extracted three times with ethyl acetate (100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to dryness to give 5-(bis(benzyloxy)phosphoryl)pentanoic acid (7.93 g) as a colorless oil. Yield=74%

[0463] LC-MS: [M+H]=363

[0464] 1 H NMR (300MHz, DMSO) δppm: 7.46–7.23 (m, 10H), 5.10–4.85 (m, 4H), 2.18 (t, J = 7.0Hz, 2H), 1.88–1.71 (m, 2H), 1.61–1.38 (m, 4H)

[0465] The following compounds were prepared using the same method:

[0466] 2.3-(Bis(benzyloxy)phosphoryl)propionic acid

[0467] Yield = 87%

[0468] LC-MS: [M+H]=335

[0469] 1H NMR (300MHz, DMSO) δppm: 12.35 (s, 1H), 7.43-7.27 (m, 10H), 5.11-4.88 (m, 4H), 2.45-2.34 (m, 2H), 2.13-1.97 (m, 2H)

[0470] 3.7-(Bis(benzyloxy)phosphoryl)heptanoic acid

[0471] Yield = 83%

[0472] LC-MS: [M+H]=391

[0473] 1 H NMR (300MHz, DMSO) δppm: 7.45–7.24 (m, 10H), 4.96 (qd, J=12.1, 8.2Hz, 4H), 2.17 (dt, J=10 .8,7.3Hz,2H),1.86–1.67(m,2H),1.53–1.33(m,4H),1.23(ddt,J=32.4,13.4,6.6Hz,4H)

[0474] C. Coupling of prodrug moiety (step 3)

[0475] 1. (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid dibenzyl ester

[0476] To a solution of (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile (2.10 g, 5.55 mmol), 5-(bis(benzyloxy)phosphoryl)pentanoic acid (2.40 g, 6.62 mmol) and PyBOP (4.31 g, 8.28 mmol) in DMF (30 mL) was added TEA (1.539 mL, 11.04 mmol). The reaction mixture was stirred at room temperature overnight and then poured into 200 mL of water. The resulting suspension was stirred at room temperature for 30 minutes, filtered, washed twice with water (50 mL) and dried under vacuum to give (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid dibenzyl ester (4.10 g) as a pale yellow solid. Yield = 51%

[0477] LC-MS: [M+H] = 722

[0478] 1H NMR (300MHz, DMSO) δppm: 8.39 (d, J = 8.5 Hz, 2H), 8.29 (d, J = 1.7 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 8.01 (dd, J = 8.7, 2.1 Hz, 1H), 7.94 (s, 1H), 7.62 (dd, J = 8. 9,1.9Hz,1H),7.40–7.30(m,11H),5.08–4.90(m,4H),3.97(s,3H),3.15(t, J=7.1Hz,2H),2.01–1.85(m,2H),1.75(dd,J=14.2,7.0Hz,2H),1.62(m,2H)

[0479] The following compounds were prepared using the same method:

[0480] 2. (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid dibenzyl ester

[0481] Yield = 74%

[0482] LC-MS: [M+H]=694

[0483] 1H NMR (300MHz, DMSO) δppm: 8.35 (d, J = 7.6 Hz, 2H), 8.30 (d, J = 1.8 Hz, 1H), 8.15 (d, J = 1.8 Hz, 1H), 8.01 (dd, J = 8.7, 2.1 Hz, 1H), 7.97 (s, 1 H),7.62(dd,J=8.9,1.9Hz,1H),7.43-7.23(m,11H),5.14-4.94(m,4H),3.98(s,3H),3.43-3.35(m,2H),2.29(dt,J=8.3,7.3Hz,2H)

[0484] 3. (Z)-7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonic acid dibenzyl ester

[0485] Yield = 52%

[0486] LC-MS: [M+H]=750

[0487] 1H NMR (300MHz, CDCl3) δppm: 8.38 (d, J = 8.9 Hz, 1H), 8.21 (d, J = 1.8 Hz, 1H), 7.93 (d,J=1.8Hz,1H),7.79(s,1H),7.72(s,1H),7.66(dd,J=8.7,2.0Hz,1H),7.4 7(dd,J=8.9,1.9Hz,1H),7.26(s,10H),6.97(d,J=8.7Hz,1H),4.93(ddd,J=3 0.1,11.8,8.5Hz,4H),3.93(s,3H),2.83(t,J=7.3Hz,2H),1.86-1.34(m,10H)

[0488] 4. (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonic acid dibenzyl ester

[0489] Yield = 52%

[0490] LC-MS: [M+H]=674

[0491] 1 H NMR (300MHz, DMSO) δppm: 8.28(d,J=1.8Hz,1H),8.15(s,1H),8.05(d,J=2.4Hz,1H ),8.02-7.97(m,1H),7.93(s,1H),7.88(d,J=8.8Hz,1H),7.44-7.25(m,11H),7.06 (dd,J=8.8,2.4Hz,1H),4.99(p,J=12.1Hz,4H),3.97(s,3H),3.83(s,3H),3.13(t ,J=6.9Hz,2H),2.02-1.85(m,2H),1.75(dd,J=13.9,7.0Hz,2H),1.69-1.53(m,2H)

[0492] 5. (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid dibenzyl ester

[0493] Yield = 46%

[0494] LC-MS: [M+H]=702

[0495] 1H NMR (300MHz, DMSO) δppm: 8.27 (d, J = 1.9 Hz, 1H), 8.14 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.9 9(dd,J=8.7,2.1Hz,1H),7.94(s,1H),7.88(d,J=8.8Hz,1H),7.40-7.30(m,11H),7.06(d d,J=8.8,2.4Hz,1H),4.98(qd,J=12.1,8.2Hz,4H),3.98(d,J=5.1Hz,3H),3.09(t,J=7. 1Hz,2H),1.88-1.75(m,2H),1.71-1.59(m,2H),1.47(d,J=7.7Hz,2H),1.41-1.32(m,4H)

[0496] D. Phosphonic acid prodrug (step 4)

[0497] Example 1: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid

[0498]

[0499] (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid dibenzyl ester (1.531 g, 2.119 mmol) was dissolved in 4M HCl in 1,4-dioxane (47.7 ml, 190.8 mmol) to give a pale yellow solution. The reaction mixture was heated at 90°C for 2 hours. After cooling, the precipitate was filtered and washed twice with dioxane (3 ml) and diisopropyl ether (20 ml) to give (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid (1.003 g) as a light grey solid. Yield = 86%

[0500] LC-MS: [M+H] = 541.9

[0501] 1H NMR (300MHz, DMSO) δppm: 8.48-8.35 (m, 2H), 8.30 (d, J = 1.9Hz, 1H), 8.16 (d, J = 1.8Hz, 1H), 8.02 (dd, J = 8.7, 2.1Hz, 1H), 7.96 (s,1H),7.62(dd,J=8.9,1.9Hz,1H),7.38(d,J=8.8Hz,1H),3.99(s,3H),3.18(t,J=7.1Hz,2H),1.69(dd,J=49.3,8.9Hz,6H)

[0502] The following compounds were prepared using the same method:

[0503] Example 2: (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid

[0504]

[0505] Yield = 96%

[0506] LC-MS: [M+H] = 513.9

[0507] 1 H NMR (300MHz, DMSO) δppm: 8.42-8.31(m,2H),8.28(d,J=1.7Hz,1H),8.17(d,J=1.8Hz,1H),8.04-7.94(m,2H ),7.63(dd,J=8.9,1.9Hz,1H),7.37(d,J=8.8Hz,1H),3.99(s,3H),3.33(d,J=6.8Hz,2H),2.04-1.90(m,2H)

[0508] Example 3: (Z)-7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonic acid

[0509]

[0510] Yield = 98%

[0511] LC-MS: [M+H]=570

[0512] 1H NMR (300MHz, DMSO) δppm: 8.40 (d, J = 7.7Hz, 2H), 8.28 (s, 1H), 8.14 (s, 1H), 8.00 (d, J = 8.6Hz, 1H), 7.94 (s, 1H), 7.6 0(d,J=8.9Hz,1H),7.37(d,J=8.8Hz,1H),3.97(s,3H),3.15(t,J=7.1Hz,2H),1.69(s,2H),1.44(d,J=25.9Hz,8H)

[0513] Example 4: (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonic acid

[0514]

[0515] Yield = 69%

[0516] LC-MS: [M+H]=494

[0517] 1 H NMR (300MHz, DMSO) δppm: 8.25(s,1H),8.16(s,1H),8.04(s,1H),7.98(d,1H),7.91(s,1H),7.86(d,1H),7 .35(d,1H),7.03(dd,1H),3.97(s,3H),3.83(s,3H),3.15(t,J=7Hz,2H),1.77(t,J=7Hz,2H),1.61(m,4H)

[0518] Example 5: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid

[0519]

[0520] Yield = 73%

[0521] LC-MS: [M+H] = 522

[0522] E. Phosphonic acid sodium salt prodrug (step 5)

[0523] Example 6: Sodium (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonate

[0524]

[0525] (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid (700 mg, 1.291 mmol) was suspended in water (25 mL) and then 1N sodium hydroxide (1.291 mL, 1.291 mmol) was added at +5°C. The reaction mixture was stirred at room temperature for 0.5 hours to obtain a clear solution. The solution was lyophilized to give sodium hydrogen (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonate (0.72 g) as a pale yellow solid.

[0526] Yield = 98%

[0527] LC-MS: [M+H] = 562

[0528] 1 H NMR (300MHz, DMSO) δppm: 12.12(brs,1H),8.5 -7.0(m,8H),3.96(brs,3H),3.53(m,6H),1.69(m,2H)

[0529] Using the same protocol, the following compounds were obtained:

[0530] Example 7: Sodium (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonate

[0531]

[0532] Yield = 97%

[0533] LC-MS: [MH] = 511.9

[0534] 1 H NMR (300MHz, DMSO) δppm: 12.04(brs,1H),8.21(s,1H),8.08(s,1H),7.90(m,2H),7.70(s,1H),7.48(d,1H),7.35(m,2H),3.97(s,3H),3.41(brs,4H)

[0535] Example 8: Sodium (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonate

[0536]

[0537] LC-MS: [M+H]=528

[0538] Example 9: Sodium (Z)-6-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxohexylphosphonate

[0539]

[0540] LC-MS: [M+H]=556

[0541] Example 10: Sodium (Z)-7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonate

[0542]

[0543] Yield = 98%

[0544] LC-MS: [MH] = 568

[0545] 1 H NMR (300MHz, DMSO) δppm: 12.11(brs,1H),8.25-7.35(m,8H),3.97(s,3H),3.59(brs,8H),1.37(brs,4H)

[0546] Example 11: Sodium (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonate

[0547]

[0548] Yield quantification

[0549] LC-MS: [M+H]=494

[0550] 1 H NMR (300MHz, D2O) δppm: 7.5-6.5 (m, 8H), 3.59 (s, 6H), 3.25 (brs, 2H), 1.57 (m, 6H)

[0551] Example 12: Sodium (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonate

[0552]

[0553] Yield = 96%

[0554] LC-MS: [M+H] = 522

[0555] Example 13: (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid disodium salt

[0556]

[0557] (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid (74.2 mg, 0.14 mmol) and sodium bicarbonate (23.6 mg, 0.281 mmol) were suspended in water (10 mL) and heated at 40° C. for 10 minutes to obtain a clear solution. The solution was lyophilized to obtain (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid disodium salt (0.72 g) as a pale yellow solid.

[0558] LC-MS: [M+H]=528

[0559] 1 H NMR (500MHz, D2O) δppm: 7.9-6.7(m,10H),3.78(s,3H),2.9(brs,2H),1.9(d,2H),1.5(d,2H)

[0560] Example 14: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid disodium salt

[0561]

[0562] (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid (229 mg, 0.422 mmol) was suspended in water (10 mL), followed by the addition of 1N sodium hydroxide (0.845 mL, 0.845 mmol). Acetone (50 mL) was slowly added, and the precipitate was filtered and washed twice with acetone (5 mL) to obtain (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid disodium salt (0.214 g) as a pale yellow solid.

[0563] LC-MS: [M+H] = 542

[0564] F. Phosphonate Lysine Salt Prodrug (Step 5)

[0565] Example 15: (S)-2,6-diaminohexanoic acid compound and (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid (2:1)

[0566]

[0567] Sodium (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonate (0.399 g, 0.776 mmol) was suspended in water (10 mL). L-lysine (0.227 g, 1.552 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes until completely dissolved. Acetone (50 mL) was then slowly added and the precipitate was filtered, washed with acetone (2 x 5 mL) and dried to give a yellow solid (0.52 g). Yield = 78%

[0568] LC-MS: [MH] = 511.9

[0569] Example 16: (S)-2,6-diaminohexanoic acid compound and (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid (2:1)

[0570]

[0571] LC-MS: [MH] = 525.8

[0572] General Synthetic Scheme II

[0573]

[0574] Dibenzyl phosphite is condensed with a THP-protected bromoalkanol to give a phosphono-THP-protected alcohol, which is then hydrolyzed in an acidic manner to give a phosphono alcohol. Then, the NH-indole in the drug is treated with triphosgene to give the corresponding chloroformate, which is reacted with a phosphono alcohol to give a benzyl-phosphonate prodrug. These latter compounds are hydrolyzed in an acidic medium to give the prodrug phosphonic acid.

[0575] Example 17: 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[hydroxy(dioxo)-λ6-phosphino]propyl ester

[0576]

[0577] A) Dibenzyl (3-hydroxypropyl)phosphonate

[0578] Dibenzyl phosphite (4.4 mL, 20 mmol) was added to a stirred suspension of HNa (1 g, 25 mmol) in dry DMF at 0°C under nitrogen. After 15 minutes at room temperature, the solution was cooled to 0°C, then 2-(3-bromopropoxy)tetrahydro-2H-pyran (3.4 mL, 20 mmol) was added and the reaction mixture was stirred at room temperature for 5 hours. The solution was diluted with water (100 mL) and extracted with ether (2 x 150 mL). The combined extracts were washed with aqueous NH4Cl (4 x 30 mL), brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography on a SiO2 column (eluent: 0 to 100% EtOAc in petroleum ether) to give 5.41 g of a colorless liquid. Yield = 67%

[0579] PPTS (0.025 g, 0.1 mmol) was added to a stirred solution of the previous compound (0.404 g, 1 mmol) in EtOH (10 mL), and the solution was heated at 55° C. for 3 hours. The solution was concentrated in vacuo and the residue was purified by flash chromatography on a SiO2 column (eluent: 0 to 100% EtOAc in petroleum ether, then 0 to 10% MeOH in EtOAc) to give 0.264 g of a colorless oil. Yield = 82%

[0580] LC-MS: [M+H] = 321

[0581] 1 H NMR (300MHz, CDCl3) δppm: 7.35 (s, 10H), 5.07 (dd, J = 11.7, 9Hz, 2H), 4.97 (dd, J = 11.7, 5.7Hz, 2H), 3.65 (t, J = 5.7Hz, 2H), 1.96-1.74 (m, 4H)

[0582] B) 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[dibenzyloxy(dioxo)-λ6-phosphino]propyl ester

[0583] Dibenzyl (3-hydroxypropyl)phosphonate (0.042 g, 0.132 mmol) was dissolved in DCM (1 mL) under N2 and the solution was cooled to 0°C. Triphosgene (14 mg, 0.049 mmol) was added followed by DIPEA (0.023 mL, 0.132 mmol). The solution was stirred for 1 hour and then at room temperature for 1 hour. In a separate flask, (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile (0.05 g, 0.132 mmol) was dissolved in DMF (1 mL) under N2 and cooled to 0°C. HNa (0.063 g, 0.158 mmol) was added and the solution was stirred at room temperature for 1 hour. The chloroformate solution was added dropwise to the indole solution at room temperature. The reaction mixture was stirred at room temperature for 2 hours, then quenched by adding saturated NH4Cl aqueous solution (5mL), and extracted with EtOAc (20mL). The organic layer was washed with saturated NH4Cl aqueous solution (3x 5mL), dried and concentrated in vacuo with Na2SO4. The residue was purified by flash chromatography (eluent: 0 to 100% EtOAc petroleum ether solution) of SiO2 column to obtain 0.041g of light yellow solid. Yield = 43%

[0584] 1 H NMR (300MHz, DMSO) δppm: 8.27(d,J=1.8Hz,1H),8.17(d,J=1.8Hz,1H),8.14(s,1H),8.11(s,1H),8.0(dd,J=1.8,8.7Hz,1H),7.92 (s,1H),7.61(dd,J=1.8,9.0Hz,1H),7.44-7.25(m,11H),5.11-4.93(m,4H),4.45(t,J=5.7Hz,2H),3.97(s,3H)2.15-1.90(m,4H)

[0585] C) 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[hydroxy(dioxo)-λ6-phosphino]propyl ester

[0586] 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[dibenzyloxy(dioxo)-λ6-phosphino]propyl ester (0.348 g, 0.48 mmol) was dissolved in 4M HCl in dioxane (9.5 mL) and the solution was stirred at 90°C for 2 hours. The solution was concentrated in vacuo and the residue was triturated in DCM to precipitate the product. The solid was filtered and dried in vacuo to give 0.21 g of the desired compound as a white solid. Yield = 80%

[0587] LC-MS: [MH] = 542

[0588] 1 H NMR (400MHz, DMSO) δppm: 8.26 (s, 1H), 8.14 (m, 3H), 7.99 (d, J = 8.4Hz, 1H), 7.92 (s, 1H), 7.64 (d, J = 9.0 Hz,1H),7.36(d,J=8.7Hz,1H),4.49(t,J=6.3Hz,2H),3.98(s,3H),2.01(m,2H),1.70(q,J=8.2Hz,2H)

[0589] 31 P NMR (162MHz, DMSO d6, δppm: 25.44

[0590] Example 18: 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[hydroxy(dioxo)-λ6-phosphino]butyl ester

[0591]

[0592] A) 2-(4-bromobutoxy)tetrahydro-2H-pyran

[0593] To an ice-cold solution of 4-bromobutanol (0.153 g, 1 mmol) in DCM (2 mL) was added DHP (0.091 mL, 1.1 mmol) and PTSA (0.002 g, 0.01 mmol) in sequence. The solution was stirred at room temperature for 2 hours, diluted with DCM (15 mL), washed with saturated NaHCO3 (10 mL), brine (10 mL), dried and concentrated to dryness. The residue was purified by flash chromatography on a SiO2 column (eluent 0 to 10% EtOAc in petroleum ether) to give 0.145 g of a clear liquid. Yield = 61%

[0594] 1H NMR (300MHz, CDCl3) δppm: 4.68-4.54(m,1H), 3.96-3.66(m,2H), 3.64-3.32(m,4H), 2.12-1.44(m,10H)

[0595] B) Dibenzyl (4-(tetrahydropyran-2-yloxy)butyl)phosphonate

[0596] Under nitrogen, dibenzyl phosphite (1.56 mL, 7 mmol) was added to a stirred suspension of HNa (0.35 g, 8.75 mmol) in anhydrous DMF (10.5 mL) at 0 ° C. After 15 minutes at room temperature, the solution was cooled to 0 ° C, then a solution of 2-(4-bromobutoxy)tetrahydro-2H-pyran (1.67 g, 7 mmol) in anhydrous DMF (4 mL) was added, and the reaction mixture was stirred at room temperature for 3 hours. The solution was diluted with water (20 mL) and extracted with ether (2x70 mL). The combined extracts were washed with NH4Cl aqueous solution (4x20 mL), dried with Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography on a SiO2 column (eluent: 10 to 100% EtOAc in petroleum ether) to give 1.71 g of a colorless liquid. Yield = 58%.

[0597] 1 H NMR (300MHz, CDCl3) δppm: 7.35 (s, 10H), 5.06 (dd, J=11.7, 8.7Hz, 2H), 4.97 (dd, J=11.7, 8.1Hz, 2H), 4.54 (t, J=3.6Hz,1H),3.88-3.77(m,1H),3.75-3.65(m,1H),3.54-3.43(m,1H),3.39-3.28(m,1H),1.94-1.40(m,12H)

[0598] C) Dibenzyl (4-hydroxybutyl)phosphonate

[0599] PPTS (0.086 g, 0.34 mmol) was added to a stirred solution of the previous compound (1.44 g, 3.44 mmol) in EtOH (34 mL) and the solution was heated at 55°C for 4 hours. The solution was concentrated in vacuo and the residue was purified by flash chromatography on a SiO2 column (eluent: 0 to 10% MeOH in DCM) to give 1.1 g of a colorless liquid. Yield = 95%

[0600] D) 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[dibenzyloxy(dioxo)-λ6-phosphino]butyl ester

[0601] Dibenzyl (4-hydroxybutyl)phosphonate (0.265 g, 0.796 mmol) was dissolved in DCM (6 mL) under N2 and the solution was cooled to 0°C. Triphosgene (87 mg, 0.295 mmol) was added followed by DIPEA (0.139 mL, 0.796 mmol). The solution was stirred for 1 hour and then at room temperature for 1 hour. In a separate flask, (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile (0.3 g, 0.796 mmol) was dissolved in anhydrous DMF (6 mL) under N2 and cooled to 0°C. HNa (0.038 g, 0.955 mmol) was added and the solution was stirred at room temperature for 1 hour. The chloroformate solution was added dropwise to the indole solution at room temperature. The reaction mixture was stirred at room temperature overnight, then saturated NH4Cl aqueous solution (30mL) was added to quench the reaction and extracted with EtOAc (120mL). The organic layer was washed with saturated NH4Cl aqueous solution (3x30mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography on SiO2 column (eluent: 0 to 100% EtOAc in petroleum ether) to obtain 0.342g of light yellow solid. Yield = 43%

[0602] E) 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[hydroxy(dioxo)-λ6-phosphino]butyl ester

[0603] 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[dibenzyloxy(dioxo)-λ6-phosphino]butyl ester (0.334 g, 0.452 mmol) was dissolved in 4M HCl in dioxane (9.5 mL) and the solution was stirred at 90°C for 2 hours. The solution was concentrated in vacuo and the residue was triturated in DCM to precipitate the product. The solid was collected by filtration and dried under vacuum to give 0.222 g of the desired compound as a white solid. Yield = 88%

[0604] LC-MS: [MH] = 557.8

[0605] 1H NMR (300MHz, DMSO) δppm: 8.26 (s, 1H), 8.12 (m, 3H), 7.99 (d, J = 8.4Hz, 1H), 7.92 (s, 1H), 7.63 (d, J =8.7Hz,1H),7.36(d,J=9.0Hz,1H),4.46(t,J=5.4Hz,2H),3.98(s,3H),1.90(m,2H),1.63(m,4H)

[0606] 31 P NMR (162MHz, DMSO, δppm: 26.18

[0607] II. L1 Prodrug

[0608] Embodiment 19 :(2S,3S,4S,5R,6S)-6-[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid

[0609]

[0610] 1)(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylic acid methyl ester

[0611] The compound was synthesized in two steps according to Yu-Ling Leu et al. J. Med. Chem. 2008, 51, 1470-1746

[0612]

[0613] a) Methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formylphenoxy)tetrahydropyran-2-carboxylate

[0614] In a 100mL round-bottom flask equipped with magnetic stirring, acetyl bromide-α-D-glucuronic acid methyl acetate (2.00g, 5.04mmol) was dissolved in CH3CN (40mL), and then 4-hydroxybenzaldehyde (584mg, 4.78mmol) and Ag2O (1.11g, 4.78mmol) were slowly added. The mixture was stirred at room temperature for 18 hours. Insoluble matter was filtered off. The filtrate was evaporated under reduced pressure to obtain a dark brown oil. The crude oil was ground with EtOH (10mL) to obtain 1.64g of brown solid. The solid was dissolved in DCM (50 mL) and purified by chromatography on a SiO2 filter cake, eluting first with 100% DCM and then with DCM / MeOH (98 / 2) to give 1.10 g of (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formylphenoxy)tetrahydropyran-2-carboxylic acid methyl ester as an off-white solid (yield = 50%).

[0615] LCMS-ESI: [M+H+18] + =456

[0616] b) Methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylate

[0617] In a 250 mL round bottom flask, (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formylphenoxy)tetrahydropyran-2-carboxylic acid methyl ester (1.10 g, 2.52 mmol) was dissolved in CHCl3 (94 mL) and 2-propanol (16 mL). SiO2 (5.50 g) was added. Then NaBH4 (95 mg, 2.52 mmol) was slowly added at 5°C. The mixture was stirred at +5°C for 2 hours, hydrolyzed with 200 ml of H2O, filtered to remove SiO2, and extracted with DCM. The organic layer was dried over MgSO4 and evaporated under reduced pressure to give a colorless oil, which was triturated with ethanol (5 mL) to give 1.00 g of methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylate as a white solid (yield = 90%).

[0618] LCMS-ESI: [M+H+18] + =458

[0619] 2)(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]tetrahydropyran-2-carboxylic acid methyl ester

[0620]

[0621] In a 100 ml pear-shaped flask equipped with magnetic stirring, (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylic acid methyl ester (1.00 g, 2.27 mmol) was dissolved in anhydrous THF (30 mL) and DCM (15 mL). 4-Nitrophenyl chloroformate (687 mg, 3.41 mmol) was added slowly at room temperature. After 5 minutes, pyridine (286 μL, 3.41 mmol) was added and the reaction mixture was stirred at 20 ° C for 2 hours. Ethyl acetate (150 mL) and 10% aqueous citric acid solution (150 mL) were added to the reaction mixture. The organic layer was separated, washed with water and brine, dried over magnesium sulfate, and the solvent was removed in vacuo to give 1.6 g of a light yellow oil. The crude oil was triturated with EtOH (5 mL) to give 1.64 g of methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]tetrahydropyran-2-carboxylate as a light yellow oil (yield = 92%).

[0622] LCMS-ESI: [M+H+18] + =623

[0623] 3) 5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[(2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]oxyphenyl]methyl ester

[0624]

[0625] In a 15 ml reactor equipped with magnetic stirring, (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile (250 mg, 0.66 mmol) was added to THF (10 mL). HNa 60% (26 mg, 0.66 mmol) was slowly added at +5°C under nitrogen atmosphere. The reaction was stirred at +5°C for 10 minutes. In a separate 100 mL round bottom flask equipped with magnetic stirring, (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]tetrahydropyran-2-carboxylic acid methyl ester (400 mg, 0.66 mmol) was dissolved in THF (25 mL) under inert atmosphere. To this solution was slowly added the previous THF solution of indole sodium salt at 5°C. Then the reaction mixture was stirred at room temperature for 2 hours. Slowly add citric acid aqueous solution (150mL), stir the mixture for 18 hours, filter the solid of precipitation, wash with water, obtain 580mg yellow solid. The crude solid is directly passed through SiO2 column (24g) flash chromatography purification, gradient elution is carried out with heptane 100% to AcOEt 100%, obtain 377mg off-white solid 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl) vinyl] indole-1-carboxylic acid [4-[(2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl] oxyphenyl] methyl ester (yield = 67%).

[0626] LCMS-ESI: [M+H+18] + =863

[0627] 1 H NMR (400MHz, DMSO-d6) δppm: 8.25(s,1H),8.18-8.10(m,3H),7.99(d,1H),7.93(s,1H),7.63(d,1H),7.54(d,2H),7.34( d,1H),7.05(d,2H),5.70(d,1H),5.46(brs,3H),5.10-5.03(m,2H),4.71(d,1H),3.96(s,3H),3.62(s,3H),2.01(s,9H)

[0628] 4)(2S,3S,4S,5R,6S)-6-[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid

[0629]

[0630] In a 10 ml reactor equipped with magnetic stirring, 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[(2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]oxyphenyl]methyl ester (150 mg, 0.169 mmol) was added to acetonitrile (1.7 mL). Concentrated HCl (1.7 mL, 16.9 mmol) was added and the reaction mixture was heated at 40° C. for 2 days. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography on a SiO2 filter cake (eluted with DCM 100% to DCM / MeOH (95 / 5) and then with AcOEt / MeOH / AcCN / H2O (7 / 1 / 1 / 1)) to give a yellow solid which was purified again on a SiO2 filter cake eluting with a mixture AcOEt / MeOH / AcCN / H2O (7 / 1 / 1 / 1) to give 12 mg of (2S,3S,4S,5R,6S)-6-[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid as a yellow solid (yield = 10%).

[0631] LCMS-ESI: [M+H+18] + =723

[0632] Embodiment 22 : 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy](peg4)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester

[0633]

[0634] 1) Example 20: 5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester

[0635]

[0636] Under argon atmosphere, (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile (140 mg, 0.37 mmol) was dissolved in anhydrous THF (6 mL) in a 50 ml pear-shaped flask. HNa 60% (15.6 mg, 0.39 mmol) was added at 5°C to give a red-orange solution; the solution was then stirred at room temperature for 10 minutes. The solution was cooled to 5°C again, and Boc-Val-Cit-PABA-PNP (Iris Biotech, 240 mg, 0.37 mmol) dissolved in 15 mL THF was added. After 30 minutes, an orange gel appeared with a yellow precipitate underneath. After 3 hours, water was slowly added and the aqueous solution was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated to dryness to give 400 mg of crude product. The crude product was suspended in water and heated at 50°C for 15 minutes. 4-Nitro-phenol was filtered off and the filtrate was concentrated in vacuo. The resulting solid was suspended in MeOH (20 mL), heated at 50° C. for 15 minutes and filtered to give 230 mg of a pale yellow solid. The solvent of the filtrate was removed and the resulting solid residue was triturated with a small amount of MeOH to give a second crop of 50 mg of 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester as a pale yellow solid (overall yield = 85%).

[0637] LCMS-ESI: [M+H-Boc] + =783

[0638] 1 H NMR (400MHz, DMSO-d6) δppm: 10.19 (s, 1H), 8.27 (s, 1H), 8.20 (s, 1H), 8.14 (d, J = 12Hz, 2 H),8.04(t,J=12Hz,2H),8.0(s,1H),7.69(t,3H),7.52(d,2H),7.38(d,J=8Hz,1H),6.78 (d,J=12Hz,1H),5.99(brs,1H),5.45(d,J=12Hz,4H),4.46(brs,1H),3.99(s,3H),3.85( brs,1H),3.0(brd,2H),1.97(m,1H),1.65(m,2H),1.39(m,11H),0.85(dd,J=8,16Hz,6H)

[0639] 2) Example 21: 5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropionyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester hydrochloride

[0640]

[0641] Under argon atmosphere, to a solution of the previous compound (380 mg, 0.43 mmol) in 20 mL THF in a 50 ml pear-shaped flask was added 4N HCl in dioxane (3.23 mL, 12.92 mmol). The reaction mixture was stirred at 20° C. for 18 hours. The solvent was removed in vacuo to give a solid, which was triturated with THF (2 mL) and filtered to give 210 mg of a pale yellow powder of 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester hydrochloride (yield = 92%).

[0642] LCMS-ESI: [M+H] + =783

[0643] 1 H NMR (400MHz, DMSO-d6) δppm: 10.39 (s, 1H), 8.76 (d, J = 8Hz, 1H), 8.19–7.94 (m, 8H), 7.66 (t, 3H), 7.52 (d, 2H), 7.36 (d, J = 12Hz, 1H), 5.46 (s, 2H) ,4.52(brs,1H),4.11(brs,3H),3.97(s,3H),3.68(brs,1H),3.01(brd,2H),2.09(brs,1H),1.65(brs,2H),1.47(brs,2H),0.95(d,J=8Hz,6H)

[0644] 3) Example 22: 5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy](peg4)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester

[0645]

[0646] In a 15 ml reactor equipped with magnetic stirring, 19-maleimido-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanoic acid (122 mg, 0.29 mmol) and HBTU (185 mg, 0.49 mmol) were added to DMF (4 mL). At +5°C, DIPEA (148 μL, 0.85 mmol) was added. The solution was stirred at +5°C for 20 minutes and 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester hydrochloride (200 mg, 0.24 mmol) was added. The reaction was stirred at room temperature for 18 hours. The mixture was slowly poured into water (25 mL) under stirring, the precipitate was filtered and washed with water to give a brown solid. The crude solid was directly purified by flash chromatography on a SiO2 column (12 g), the solid was loaded and gradient eluted with DCM 100% to DCM / MeOH (8 / 2) to give 85 mg of 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy](peg4)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester as a yellow solid (yield = 29%).

[0647] LCMS-ESI: [M+H] + =1184

[0648] 1H NMR(400MHz,DMSO-d6)δ10.09(s,1H),8.18(s,1H),8.15-7.88(m,7H),7.66(t, 3H),7.50(d,2H),7.36(d,1H),7.00(s,2H),5.98(brs,1H),5.43(d,4H),4.37( brs,1H),4.22(t,1H),3.96(s,3H),3.58(t,4H),3.48(s,12H),3.14(m,2H),2. 97(dl,2H),2.32(m,2H),1.95(m,1H),1.60(dl,2H),1.43(dl,2H),0.84(dd,6H)

[0649] Embodiment 23:5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino](peg24)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester

[0650]

[0651] In a 15 ml reactor equipped with magnetic stirring, 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropionyl]amino]-5-urea-pentanoyl]amino]phenyl]methyl ester hydrochloride (140 mg, 0.17 mmol) and diisopropylethylamine (59 μL, 0.34 mmol) were added to DMF (2 mL). Mal-PEG(24)-NHS (Iris Biotech, 262 mg, 0.19 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was ground three times with DIPE (8 mL) to remove DMF, and the resulting oil was suspended in 0.2N HCl (20 mL) and extracted with DCM (4x15 mL). The organic phase was washed with water (15 mL), dried over Na2SO4 and concentrated under reduced pressure to give 350 mg of a yellow oil. The crude oil was directly purified twice by flash chromatography on a SiO2 column, solid loading, gradient elution from DCM 100% to DCM / MeOH 8 / 2 to give 100 mg of [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino](peg24)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino](peg24)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester (yield = 28%).

[0652] MS (MALDI-TOF): [M+Na] + =2083.91

[0653] Embodiment 26:(2R)-3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino][PEG4]propanoylamino]-3-oxo-propane-1-sulfonic acid

[0654]

[0655] 1) Example 24: 3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-(tert-butoxycarbonylamino)-3-oxo-propane-1-sulfonic acid

[0656]

[0657] In a 15 ml reactor equipped with magnetic stirring, BOC-cysteic acid, TEA (151 mg, 0.41 mmol) and HBTU (248 mg, 0.65 mmol) were added to DMF (1 mL). Diisopropylethylamine (198 μL, 1.14 mmol) was added. The solution was stirred at room temperature for 30 minutes. 5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropanoyl]amino]-5-urea-pentanoyl]amino]phenyl]methyl ester hydrochloride (268 mg, 0.33 mmol) was added at room temperature and the reaction was stirred at room temperature for 18 hours. The reaction mixture was poured into water, extracted with DCM, and the organic layer was washed with water, dried over Na2SO4 and concentrated under reduced pressure to give a brown oil. The crude oil was purified by flash chromatography on a SiO2 column (24 g) with gradient elution from DCM 100% to DCM / MeOH 3 / 1 to afford 190 mg of 3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-(tert-butoxycarbonylamino)-3-oxo-propane-1-sulfonic acid as a light yellow solid (yield = 56%).

[0658] LCMS-ESI: [MH] - =1034

[0659] 1 H NMR (400MHz, DMSO) δppm: 9.49 (s, 1H), 8.25 (s, 1H), 8.16 (s, 1H), 8.12 (m, 2H), 7.98 (d, 1H) ,7.93(s,1H),7.79(d,2H),7.63(d,1H),7.47(d,2H),7.34(d,1H),6.94(d,1H),5.93(brs ,1H),5.46(s,2H),5.42(s,2H),4.37(brs,1H),4.16(brs,1H),3.97(brs,4H),2.98(brd, 4H),2.13(brs,1H),1.71(brs,2H),1.33-1.35(m,11H),1.22(brs,2H),0.89(d,J=8Hz,6H)

[0660] 2) Example 25: 2-Amino-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propane-1-sulfonic acid

[0661]

[0662] In a 15 ml reactor equipped with magnetic stirring, the previous compound (120 mg, 0.12 mmol) was added to THF (2 mL). A 4N HCl solution in dioxane (870 μL, 3.48 mmol) was added. The solution was heated to reflux for 5 minutes, cooled, and the precipitate was filtered and washed with THF to give 65 mg of 2-amino-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propane-1-sulfonic acid as a yellow solid (yield = 58%).

[0663] LCMS-ESI: [MH] - =934

[0664] 1H NMR (400MHz, DMSO) δppm: 9.93 (s, 1H), 8.77 (d J=8Hz,1H),8.25(s,2H),8.14(m,2H),7.98(d,2H),7.94(s,1H),7.70(d,2H),7.50(d,2H),7.34(d,1H),6.05(brs,1H),5.45(s,2H) ,4.32–4.15(m,3H),3.96(s,3H),3.56(brs,4H),2.98(m,4H),2.13(brs,1H),1.75(brs,2H),1.35-1.25(m,2H),0.87(d,J=8Hz,6H)

[0665] 3) Example 26: (2R)-3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy]propionylamino]-3-oxo-propane-1-sulfonic acid

[0666]

[0667] In a 15 ml reactor equipped with magnetic stirring, 19-maleimido-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanoic acid (77 mg, 0.19 mmol) and HBTU (117 mg, 0.31 mmol) were added to DMF (1.5 mL), followed by DIPEA (94 μL, 0.54 mmol). The solution was stirred at room temperature for 20 minutes, followed by the addition of 2-amino-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propane-1-sulfonic acid (150 mg, 0.15 mmol). The reaction mixture was stirred at room temperature for 18 hours. The solution was poured into 0.2N HCl, extracted with DCM, and the organic layer was washed with water, dried over Na2SO4 and concentrated under reduced pressure. The crude oil was directly purified by flash chromatography on a SiO2 column with gradient elution from DCM 100% to DCM / MeOH 8 / 2 to afford 90 mg of (2R)-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]propanoylamino]-3-oxo-propane-1-sulfonic acid as a yellow solid (yield = 44%).

[0668] LCMS-ESI: [M+H] + =1334

[0669] III. Solubility

[0670] A. Solution:

[0671] For each compound, three 500 μM solutions were prepared in 0.01N HCl (pH 1.0) and three 500 μM solutions were prepared in phosphate buffer (pH 7.4). Each compound was dissolved in an appropriate solvent in an opaque tube and stirred at room temperature for 24 hours to prepare the solution. After 24 hours, for each solution, a sample was centrifuged at a speed of 3500 rpm for 10 minutes to separate the insoluble part from the soluble part. The supernatant was taken and diluted in H2O (1 / 20) and then injected into LC-MSMS. After HPLC separation, the analyte was detected and quantified by mass spectrometry. A standard curve was drawn for each test substance. The concentration of each supernatant was calculated according to the standard curve to determine the solubility of each test substance.

[0672] B: Results

[0673] The solubility results are shown in Table 1 below:

[0674] Table 1:

[0675]

[0676]

[0677]

[0678] IV. In vitro evaluation: cathepsin B-mediated drug release

[0679] plan

[0680] Example 27: Synthesis of N-acetyl-S-(1-((R)-2-(((S)-1-(((S)-1-((4-(((5-bromo-3-((Z)-1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indole-1-carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)-4,20-dioxo-1-sulfo-7,10,13,16-tetraoxa-3,19-diazadocosan-22-yl)-2,5-dioxopyrrolidin-3-yl)-L-cysteine ​​(quenched mal prodrug)

[0681]

[0682] A solution of Example 26 (crude material from the previous step) (60 mg, 0.045 mmol) in 0.8 ml DMSO was treated with a solution of NAcCys (8 mg, 0.049 mmol) in 0.2 ml DMSO; DIPEA (16 μL, 0.09 mmol) was added to the mixture at room temperature. Stir for 1 hour: UPLC showed complete conversion to the title compound. HCOOH (0.1 mmol, 4 μL) was added and the pH was checked (4, orange to pH 1-12 on paper). The crude product was purified by preparative RP-HPLC. Eluent: acetonitrile (+0.1% HCOOH) and water (+0.1% HCOOH). Column: XBridge PROTEIN, 150x19 mm, 300A, 5um. Flow rate: 30 ml / min. Fractions containing the title compound were collected and freeze-dried. 16 mg of white solid was recovered. UPLC-MS (M3): Rt = 3.86 Observed peak m / z (ES+) = 1497.2 [MH] + consistent with the expected (MW: 1496.42); purity (peak area %) 93%

[0683] The drug release of (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile in Example 27 was performed using cathepsin B enzyme.

[0684] Cathepsin B (50 units / mL) was preincubated in activation buffer containing 25 mM sodium acetate (pH 5.5), 30 mM DTT, and 15 mM EDTA at 37°C for 15 min and then added at 20% (v / v) to start each reaction.

[0685] The final in vitro cathepsin B reaction mixture will contain 25 mM sodium acetate (pH 5.5), 10 units / mL activated cathepsin B, 30 mM DTT and 15 mM EDTA, and 0.5 μg / mL Example 27 (2.5% (v / v) incubation sample background).

[0686] Incubation was performed at 37°C for 2 hours (0-5-10-15-30-60-120 minutes).

[0687] The reaction was terminated by adding organic solvent and the supernatant was analyzed by LC-MS to quantify the released free drug.

[0688] Figure 1 The results showed effective drug release of (Z)-3-(2-(5-bromo-1H-indol-3-yl)-2-cyanovinyl)-4-methoxybenzonitrile.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: -X represents a nitrogen atom, a C-CN unit or a N+-O- unit; - R1 and R1' independently represent hydrogen, halogen, (C1-C6)alkoxy or a -SO2-CH3 group, provided that if R1 or R1' is (C1-C6)alkoxy, R2 is not halogen; -R2 stands for: a (C1-C6)alkoxy, (C3-C6)cycloalkyloxy, (C3-C6)heterocycloalkyloxy, aryloxy, heteroaryloxy, (C1-C6)alkyl-aryloxy, (C1-C6)alkyl-heteroaryloxy group, which is optionally substituted by at least one halogen, or represents a thio-(C1-C6)alkyl, thio-aryl, thio-heteroaryl, thio-(C1-C6)alkyl-aryl or thio-(C1-C6)alkyl-heteroaryl group, which is optionally substituted by at least one halogen or (C1-C6)alkoxy group, ■-NR4R5 unit, O-(C1-C6)alkyl-NR4R5 unit or S-(C1-C6)alkyl-NR4R5 unit, wherein R4 and R5 represent H, (C1-C6)alkyl, or R4 and R5 together form a 3- to 7-membered ring, which is optionally interrupted by one or more heteroatoms, provided that at least one of R4 and R5 is not H, ■NHCOR6 unit, wherein R6 represents (C1-C6) alkyl, ■ aryl or heteroaryl, which is optionally substituted by at least one halogen, trifluoromethyl or (C1-C3)alkoxy, or ■Halogen, - R3 represents hydrogen, (C1-C3)alkyl, (C1-C3)alkoxy or halogen; and -Ra is a group selected from the following: ■Groups of formula (A): or a group of formula (A'): wherein n is an integer between 1 and 12; and ■Groups of formula (B): in: oL1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group; oL2 is tert-butyloxycarbonyl or a protein-binding linker, and οm is an integer equal to 0 or 1.

2. A compound of formula (I), wherein R2 represents: ■ (C1-C6) alkoxy group, preferably methoxy, ■ (C3-C6) heterocycloalkoxy group, preferably oxetanyloxy, or ■ Heteroaryl, preferably furyl or triazolyl.

3. A compound of formula (I) according to claim 1 or 2, wherein: -X represents C-CN unit, -R1 represents halogen, preferably bromine, -R1' represents hydrogen, - R2 represents a (C1-C6) alkoxy group, preferably a methoxy group, and -R3 represents hydrogen.

4. A compound of formula (I) according to claim 1 or 2, wherein: -X represents C-CN unit, -R1 represents hydrogen, - R1' represents a (C1-C6) alkoxy group, preferably a methoxy group, - R2 represents a (C1-C6) alkoxy group, preferably a methoxy group, and -R3 represents hydrogen.

5. A compound of formula (I) according to any one of claims 1 to 4, wherein Ra is a group of formula (A): or a group of formula (A'): wherein n is an integer between 1 and 12, preferably an integer between 2 and 10, more preferably an integer between 2 and 8, an integer between 2 and 6, and even more preferably n is 2, 3, 4, 5 or 6.

6. A compound of formula (I) according to any one of claims 1 to 5, wherein the pharmaceutically acceptable salt is selected from sodium salt, disodium salt, lysine salt, dilysine salt, arginine salt or diarginine salt, preferably sodium salt or disodium salt, more preferably sodium salt.

7. The compound of formula (I) according to claim 1 or 5, wherein the compound is selected from: - Example 1: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid; - Example 2: (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid; - Example 3: (Z)-7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonic acid; - Example 4: (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonic acid; - Example 5: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid; - Example 6: (Z)-sodium hydrogen 5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonate; - Example 7: (Z)-sodium hydrogen 3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonate; - Example 8: (Z)-sodium hydrogen 4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonate; - Example 9: (Z)-sodium hydrogen 6-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxohexylphosphonate; - Example 10: (Z)-sodium hydrogen 7-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-7-oxoheptylphosphonate; - Example 11: Sodium hydrogen (Z)-5-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-5-oxopentylphosphonate; - Example 12: (Z)-7-(3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-6-methoxy-1H-indol-1-yl)-7-oxoheptylphosphonic acid sodium hydrogen; - Example 13: (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid disodium salt; - Example 14: (Z)-5-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-5-oxopentylphosphonic acid disodium salt; - Example 15: (S)-2,6-diaminohexanoic acid compound and (Z)-3-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-3-oxopropylphosphonic acid (2:1); - Example 16: (S)-2,6-diaminohexanoic acid compound and (Z)-4-(5-bromo-3-(1-cyano-2-(5-cyano-2-methoxyphenyl)vinyl)-1H-indol-1-yl)-4-oxobutylphosphonic acid (2:1); - Example 17: 5-bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 3-[hydroxy(dioxo)-λ6-phosphino]propyl ester; and - Example 18: 5-Bromo-3-[(Z)-1-cyano-2-(2-cyano-5-methoxy-phenyl)vinyl]indole-1-carboxylic acid 4-[hydroxy(dioxo)-λ6-phosphino]butyl ester.

8. A compound of formula (I) according to any one of claims 1 to 4, wherein Ra is a group of formula (B): in: o L1 is a cleavable group selected from a pH-sensitive group, a photo-induced cleavable group, a bioreducible cleavable group and an enzymatic cleavable group; o L2 is tert-butyloxycarbonyl or a protein-binding linker, and om is an integer equal to 0 or 1.

9. The compound of formula (I) according to claim 8, wherein L1 is an enzymatically cleavable group that is cleaved by a protease, peptidase, esterase, β-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, tubulin tyrosine ligase or lipase.

10. The compound of formula (I) according to claim 8 or 9, wherein L1 is p-aminobenzyloxycarbonyl-AA1 w -AA2 x -AA3 y -AA4 z A group wherein AA1, AA2, AA3 and AA4 independently represent an amino acid selected from alanine, valine, citrulline, phenylalanine, lysine, glycine, aspartic acid, asparagine, glutamic acid and derivatives thereof, preferably citrulline, valine, cysteic acid, glycine and glutamic acid, more preferably citrulline, valine and cysteic acid, and w, x, y and z independently represent an integer equal to 0 or 1.

11. A compound of formula (I) according to any one of claims 8 to 10, wherein m is 1, and L2 is a protein binding linker having the formula: wherein r is an integer between 1 and 36, preferably an integer between 1 and 24, more preferably equal to 2, 4, 8, 12, 16, 20 or 24.

12. The compound of formula (I) according to claim 1 or 8, wherein the compound is selected from: - Example 19: (2S,3S,4S,5R,6S)-6-[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid; - Example 20: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester; - Example 21: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-aminopropionyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester hydrochloride; - Example 22: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy](peg4)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester; - Example 23: 5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carboxylic acid [4-[[(2S)-2-[[(2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino](peg24)propionylamino]-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl ester; - Example 24: 3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-(tert-butoxycarbonylamino)-3-oxo-propane-1-sulfonic acid; - Example 25: 2-amino-3-[[(1S)-1-[[(1S)-1-[[4-[[5-bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propane-1-sulfonic acid; and -Example 26: (2R)-3-[[(1S)-1-[[(1S)-1-[[4-[[5-Bromo-3-[(Z)-1-cyano-2-(5-cyano-2-methoxy-phenyl)vinyl]indole-1-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy]propionylamino]-3-oxo-propane-1-sulfonic acid.

13. Conjugate of formula (II): in: -X, R1, R 1' , R2, R3 and L1 are as defined in any one of claims 1 to 4 and 8 to 10; -L2 is the binding protein linker; - P is a peptide or protein, preferably an antibody, antibody fragment or antigen-binding fragment, which is capable of binding to a target of interest; and -v is an integer from 1 to 10.

14. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 13 and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, for use in the treatment of cancer, preferably selected from leukemia, acute myeloid leukemia, lymphoma, breast cancer, pancreatic cancer, lung cancer or colon cancer.

Images