TWI857798B - Indoline derivatives for treatment and/or prevention of tumor or cell proliferative and fibrosis diseases - Google Patents

Abstract

The invention relates to indoline derivatives and uses thereof for treating and/or preventing an inflammatory condition or fibrosis diseases, and tumor or cell proliferative diseases.

Description

Indoline derivatives for the treatment and/or prevention of tumors or cell proliferation and fibrosis diseases

The present invention relates to indoline derivatives and their use in the treatment and/or prevention of inflammatory conditions or fibrotic diseases and tumors or cell proliferative diseases.

Cyclin-dependent protein kinase 8 (CDK8) is a serotonin-threonine protein kinase that regulates several transcriptional factors and pathways, such as Wnt/β-catenin, TGF-β/Smad, and STAT. Among them, TGF-β and its involvement in the inflammatory process are considered to be crucial in inducing pulmonary fibrosis. Therefore, CDK8 plays an important role not only in regulating fibrogenic growth factor and inflammatory signaling pathways, but also in transcriptional regulation.

Recently, there has been an increase in the study of small molecule inhibitors targeting CDK8, and many studies have shown that CDK8 is promising as a target for the treatment of various cancers (such as colorectal cancer, metastatic prostate cancer, acute myeloid leukemia, and advanced ER(+) HER2(-) metastatic breast cancer) as well as anti-inflammatory and fibrotic therapies.

However, it has been observed that these CDK8 inhibitors exhibit modern metabolic instability and non-specific cytotoxicity, which may lead to adverse events in treated patients, highlighting the urgent need to discover new therapeutic targets to improve existing treatments and unmet medical needs.

The present invention provides a compound of formula (I): (I), or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate or solvate thereof, wherein: R ¹ is hydrogen, C _{1 - 6} alkyl, C _{1 - 6} halogenalkyl, C _{1 - 6} alkoxy, C _{1 - 6} halogenalkoxy or phenyl; R ^2a and R ^2b are each independently C _{1 - 6} alkyl, C _{1 - 6} alkenyl or C _{1 - 6} alkynyl, which may be substituted with a halogen, or R ^2a and R ^2b together with the carbon atom to which it is attached forms ring C, wherein ring C is a 4- to 6-membered cycloalkyl or a 4- to 6-membered heterocycloalkyl, wherein the 4- to 6-membered cycloalkyl or the 4- to 6-membered heterocycloalkyl is optionally substituted with a phenyl or benzyl group, wherein the phenyl or benzyl group is optionally substituted with a halogen or a hydroxyl group; each of R ³ , R ⁵ and R ⁶ is independently H, C _{1 - 6} alkyl, C _{2 - 6} alkenyl, C _{2 - 6} alkynyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 3- to 8-membered cycloalkyl, 3- to 8-membered cycloalkenyl, 3- to 8-membered heterocycloalkyl, 3- to 8-membered heterocycloalkenyl, halogen, cyano, nitro, OR _a , SR _a , S(O)R _a , SO ₂ R _a R a , _{R b} , NHC(O)-CH=CH-C(O)R _a , NHC(O)-CH=CH-C(O)NR _b R _c , SO ₂ NR _b R _c , OC(O)R _a , C(O)NR _b R _c , NR _b R c , NHC(O)R _a , NHC(O)NR _b R _c or NHC(S)R _b , wherein each of R _a , R _b and R _c is independently H, hydroxyl, C _{1 - 6} alkoxy, 6 to 10 membered aryloxy, 5 to 10 membered heteroaryloxy, C _{1 - 6} alkyl, C _{2 - 6} alkenyl, C _{2 - R4} is hydrogen, a C1-10 aliphatic group, a phenyl group or a 5-6 membered heteroaryl group, which is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, nitro _, cyano, amino, phenyl, _5-6 membered heteroaryl, _C1-6 alkoxy and _{C1-6 halogen} alkoxy; Ring ^A is phenyl, oxadiazole, thiadiazole, thiazobenzophenone or benzopiperidinium, which is optionally substituted by one or more substituents selected from the group consisting of oxo, _halogen , _C1-6 oxadiazole, thiazobenzophenone or _{benzopiperidinium .} wherein the substituents _C1-6 alkyl, phenyl and _oxadiazole are optionally further substituted by halogen, _{C1-3 alkyl} or any combination _thereof ; _-L- is -O-, ^-NRa- , -C(= _O )-, -NRa- _C (=O)- or _-C (=O) ^-NRa- , wherein R ^a has the meaning defined above; and ring B _is phenyl, oxadiazole or pyridine, optionally substituted by one _or more substituents selected from the group consisting of nitro, halogen, alkyl, pyridine and _{C1-3 alkylaminocarbonyl} ; or ^-LB is absent.

In various embodiments, in the compound of formula (I), R ¹ is hydrogen and R ^2a and R ^2b are each independently methyl. In various embodiments, Ring A is substituted with halogen.

In one embodiment, in the compound of formula (I), ring A is phenyl, oxadiazole, thiadiazole, thiazolylbenzophenone or benzopiperidinium, which is optionally substituted with one or more substituents selected from the group consisting of: oxo, halogen, C _1-6 alkyl, phenyl, oxadiazole and _{C 1-3 alkylaminocarbonyl} , wherein the substituents C _1-6 alkyl, phenyl and oxadiazole are optionally further substituted with halogen, C _1-3 alkyl or any combination _thereof ; _-L- is -O-, _-NR ^a - or -C(═O) _{- ; and} ring B is phenyl, _oxadiazole or pyridine, which is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkyl and C _{1-3 alkylaminocarbonyl} ; or -LB is absent.

In one embodiment, in the compound of formula (I), ring A _is phenyl optionally substituted with one or more substituents selected from the group consisting of halogen and C _{1-6 alkyl} ; L is -O-, -NH-, -C(=O)- or -C( ₌ O ₎ -NR ^a -, wherein ring A is connected to the N atom; and ring B is phenyl or pyridine optionally substituted with one or more substituents selected from the group consisting of nitro, halogen, _{C 1-6} alkyl and _{C 1-3} alkylaminocarbonyl.

In one embodiment, in the compound of formula (I), ring A is phenyl, oxadiazole or thiadiazole, which is substituted with one or more substituents selected from the group consisting _of phenyl, oxadiazole or C _1-3 alkylaminocarbonyl, and the substituents phenyl and _oxadiazole are optionally further substituted with _{C 1-3 alkyl} ; and -LB is absent.

In one embodiment, in the compound of formula (I), ring A is thiazolylacene or benzopiperidinium, which is optionally substituted with one or more substituents selected from the group consisting of pendoxy, halogen and alkyl; and -L-B is absent.

In one embodiment, in the compound of formula (I), Ring A is phenyl, which is optionally substituted with one or more substituents selected from the group consisting of halogen _and alkyl; -L- is -NH-; and Ring B is pyridine, which is optionally substituted with one or more substituents selected from the group consisting of halogen and C _{1-6 alkyl} .

In one embodiment, in the compound of formula (I), ring A is phenyl substituted by a halogen; -L- _is -O-; and ring B is pyridine substituted by a C _{1-3 alkylaminocarbonyl} .

In one embodiment, the compound of formula (I) has formula (II): , where R is selected from the group consisting of: .

In one embodiment, in the compound of formula (I), ring C is cyclobutane, cyclohexane or piperidine, and ring C is optionally substituted with benzyl or phenyl, wherein the benzyl or phenyl is optionally substituted with hydroxyl.

In one embodiment, the compound of formula (I) has formula (III): , wherein R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , Ring A, Ring B and L are as defined above, and Ring C is selected from the group consisting of: , , and .

The present invention also provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate and solvate thereof, and a pharmaceutically acceptable carrier.

The present invention also provides a method for inhibiting CDK8 in an individual, comprising administering to the individual a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate or solvate thereof.

The present invention also provides a method for preventing or treating inflammatory conditions and/or fibrotic diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a compound as disclosed herein or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate and solvate thereof.

The present invention also provides a method for preventing or treating tumors and/or cell proliferative diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate or solvate thereof.

The present invention is based, at least in part, on the discovery of indoline derivatives and their effective dosages for preventing and/or treating inflammatory conditions or fibrotic diseases. The compounds are effective for preventing and/or treating inflammatory conditions or fibrotic diseases without being cytotoxic or genotoxic.

As used herein, unless the context requires otherwise, the term "comprise" and variations of the term such as "comprising", "comprises" and "comprised" are not intended to exclude other additives, components, integers or steps.

As used herein, unless the context requires otherwise, disclosed method steps are not intended to be limiting or to indicate that each step is essential to the method or that the steps must be performed in the order disclosed.

As used herein, the use of "or" means "and/or" unless otherwise specified. In the case of multiple dependent clauses, the use of "or" only refers to more than one of the preceding independent or dependent clauses in an alternative manner.

As used herein, all numbers are approximate and may vary to account for measurement error and rounding of significant figures. The use of "about" before certain measurements includes variations due to sample impurity, measurement error, human error and statistical variations, and rounding of significant figures.

As used herein, the term "about" refers to an acceptable deviation from a given value as measured by one of ordinary skill in the art, depending in part on how the value is measured or determined.

As used herein, the term "aliphatic" or "aliphatic group" may include saturated or unsaturated straight chain, branched chain or cyclic groups, such as (cyclo)alkane, (cyclo)alkene, (cyclo)alkyne, etc. In some cases, the aliphatic group may contain a heteroatom in the chain or as a ring member atom.

As used herein, the term "alkyl" refers to a saturated straight chain or branched chain alkyl group, preferably containing 1 to 20 carbon atoms, and more preferably 1 to 6 carbon atoms, or a specified number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, dibutyl, isobutyl, tertiary butyl, 2-ethylbutyl, n-pentyl, isopentyl, 1-methylpentyl, 1,3-dimethylbutyl, n-hexyl, 1-methylhexyl, n-heptyl, isoheptyl, 1,1,3,3-tetramethylbutyl, 1-methylheptyl, 3-methylheptyl, n-octyl, 2-ethylhexyl, 1,1,3-trimethylhexyl, 1,1,3,3-tetramethylpentyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, 1,1,3,3,5,5-hexamethylhexyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, or the like.

As used herein, the term "alkoxyl" or "alkoxy" refers to a group having the formula "-O-alkyl", wherein the definition of "alkyl" in the formula has the meaning of "alkyl" as set forth above.

As used herein, the term "cycloalkyl" as used herein means a saturated or partially unsaturated cyclic carbon group containing 3 to 10 ring carbon atoms and more preferably 3 to 8 ring carbon atoms and optionally containing alkyl substituents on the ring. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclopropenyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-cyclohexen-1-yl and the like.

As used herein, the term "aromatic", "aromatic group" or "aryl" refers to a group having aromatic properties, which may have 6 to 12 ring members and may have a monocyclic or polycyclic (fused) ring. Examples of aromatic groups include, but are not limited to, phenyl, benzyl, biphenyl, naphthalene, fluorene, etc.

As used herein, the term "heteroaryl" refers to a group having aromaticity, which may have 5 to 12 ring members and at least one heteroatom preferably selected from nitrogen, oxygen and sulfur as a ring member and may have a monocyclic or polycyclic (fused) ring. Examples of aromatic groups include, but are not limited to, pyrrole, pyridine, thiophene, oxazole, furan, pyrimidine, diazole, triazole, thiadiazole, thiazolylacene, benzopiperidinium, etc.

As used herein, the term "halogen" or "halogen" means fluoro, chloro, bromo or iodo.

As used herein, the term "amino" as used herein refers to a functional group of the formula -NR'R", wherein R' and R" each independently represent hydrogen, an aliphatic group as defined above, or an aromatic group.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of an amine group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactic acid Salts of the invention include but are not limited to: succinate, malonic acid, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, bis(hydroxynaphthoate), pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartaric acid, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts and the like. Where appropriate, additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

The term "solvent" refers to a form of a compound that is associated with a solvent, usually by a solvent decomposition reaction. This physical/chemical association may include hydrogen bonding. Conventional solvents include water (i.e., forming hydrates), methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form and may be solvated. Suitable solvents include pharmaceutically acceptable solvents and further include both stoichiometric solvents and non-stoichiometric solvents. In certain circumstances, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvent will be able to separate. "Solvate" encompasses both solution phases and separable solvents. Representative solvent complexes include hydrates, ethanolates, and methanolates.

The term "prodrug" refers to a compound having a cleavable group and becoming a compound described herein that is pharmaceutically active in vivo by solvent decomposition or under physiological conditions, including derivatives of the compounds described herein. Such examples include, but are not limited to, choline ester derivatives and their analogs, N-alkyl phosphonoesters and their analogs. The acid and acid derivative forms of other derivatives of the compounds of the present invention are active, but the acid-sensitive form generally provides advantages in solubility, tissue compatibility or delayed release in mammalian organisms. Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reacting the parent acid with a suitable alcohol, or amides prepared by reacting the parent acid compound with a substituted or unsubstituted amine, or anhydrides or mixed anhydrides.

As used herein, the terms "administer", "administering" or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing a compound of the present invention or a pharmaceutical composition thereof into or onto the body of a subject.

As used herein, the terms "condition," "disease," and "disorder" are used interchangeably.

An "effective amount" of a compound described herein is an amount sufficient to elicit the desired biological response, i.e., to treat a condition. As will be appreciated by one of ordinary skill in the art, an effective amount of a compound described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the individual. An effective amount encompasses both therapeutic and preventive treatments.

A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of a therapeutic agent that, alone or in combination with other therapies, provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can encompass an amount that improves the overall therapy, reduces or avoids symptoms or causes of a condition, and/or enhances the therapeutic efficacy of another therapeutic agent.

As used herein, the term "pharmaceutically acceptable carrier" refers to a solid, semisolid or liquid filler, diluent, encapsulating material, formulation aid or vehicle known in the art for administration to a subject together with a therapeutic agent. A pharmaceutically acceptable carrier is nontoxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is determined in part by the specific composition being administered and the specific method used to administer the composition.

As used herein, the term "subject" is defined herein to include animals, such as mammals, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In certain embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein with respect to, for example, a mammalian subject (e.g., a human).

As used herein, the terms "treat," "treating," and "treatment" refer to the eradication or amelioration of a disease or disorder or one or more symptoms associated with a disease or disorder. In certain embodiments, the terms refer to minimizing the spread or worsening of a disease or disorder resulting from the administration of one or more preventive or therapeutic agents to an individual suffering from such a disease or disorder. In some embodiments, the terms refer to administration of a compound or dosage form provided herein, with or without one or more additional active agents, following diagnosis or onset of symptoms of a particular disease.

As used herein, the terms "prevent," "preventing," and "prevention" refer to preventing the onset, recurrence, or spread of a disease or disorder or one or more symptoms thereof. In certain embodiments, the terms refer to treatment or administration of a compound or antibody or dosage form provided herein, with or without one or more other additional active agents, prior to the onset of symptoms, particularly to a patient at risk for a disease or disorder provided herein. The terms encompass the inhibition or reduction of symptoms of a particular disease.

As used herein, unless otherwise indicated, the terms "co-administered" and "in combination with" include simultaneous, concurrent, separate or sequential administration of two or more therapeutic agents without specific time limits. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms.

The inventors have found that specific indoline derivative compounds can exhibit excellent inhibitory effects on CDK8 and the potential to treat CDK8-related diseases. Therefore, in one embodiment, the present invention relates to a compound of formula (I):

,

or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate or solvate thereof, wherein Ring A, Ring B, Linker L, R ¹ , R ^2a , R ^2b , R ³ and R ⁴ are as defined above.

In various embodiments, the groups are as defined above.

In one embodiment, the compound of formula (I) is selected from the group consisting of: N-(4-(4-nitrophenoxy)phenyl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-oxo-N-(4-(anilino)phenyl)-3-(propan-2-ylidene)indoline-5-sulfonamide, N-(4-((2-oxo-3-(propan-2-ylidene)indoline)-5-sulfonamido)phenyl)benzamide, N-(4-benzylphenyl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-oxo-N-(5-phenyl-1H-pyrazol-3-yl)-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-oxo-3-(propan-2-ylidene)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)indoline-5-sulfonamide, 2-oxo-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-3-(propan-2-ylidene)indoline-5-sulfonamide, N-(benzo[d]thiazol-2-yl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, N-(4-(1H-imidazol-1-yl)phenyl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, N-methyl-4-(4-((2-oxo-3-(propan-2-ylidene)indoline)-5-sulfonamido)phenoxy)pyridinecarboxamide, 4-(3-fluoro-4-((2-oxo-3-(propan-2-ylidene)indoline)-5-sulfonamido)phenoxy)-N-methylpyridinecarboxamide, 2-fluoro-N-methyl-4-((2-oxo-3-(propan-2-ylidene)indoline)-5-sulfonamido)benzamide, N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, and N-(1,4-dioxo-1,2,3,4-tetrahydropyridine-5-yl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, or their pharmaceutically acceptable salts, stereoisomers, mirror isomers, prodrugs, hydrates or solvates.

The compound of formula (I) can be prepared according to various chemical synthesis methods. In one embodiment, the compound can be prepared by the following process 1: , wherein R ¹ , R ^2a , R ^2b , R ³ , R ⁴ , R ⁵ , R ⁶ , Ring A, Ring B and L are as defined above.

The compounds described herein can be prepared into pharmaceutical compositions by conventional methods using excipients commonly used in the art (i.e., pharmaceutical excipients, pharmaceutical carriers, or the like).

As solid compositions for oral administration, tablets, powders, granules and the like are used. In such solid compositions, one or two or more active ingredients are mixed with at least one inert excipient. The composition may contain inert additives such as lubricants, disintegrants, stabilizers, solubilizers and the like by conventional methods.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs and the like, and include commonly used inert diluents, such as purified water or ethanol. In addition to inert diluents, liquid compositions may contain adjuvants such as solubilizers, wetting agents and suspensions, sweeteners, flavors, fragrances or preservatives.

The solid composition or liquid composition may also be encapsulated to form capsules, including but not limited to soft capsules or hard capsules.

The pharmaceutical composition can also be used by injection into an individual. Parenteral injections include sterile aqueous or non-aqueous preparations, suspensions or emulsions. As aqueous solvents, for example, distilled water for injection or physiological saline are included. As non-aqueous solvents, for example, alcohols such as ethanol are included. Such compositions may further include tonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers or solubilizers. These are sterilized, for example, by filtering through a bacteria-retaining filter, mixing with a sterilizing agent or irradiating. In addition, these can also be used by preparing sterile solid compositions and dissolving or suspending them in sterile water or a sterile solvent for injection before use.

Transmucosal preparations (such as nasal preparations and the like) are used in solid, liquid or semisolid forms and can be prepared according to methods known in the relevant art. For example, known excipients, pH adjusters, preservatives, surfactants, lubricants, stabilizers, thickeners and the like can be appropriately added. In administration, appropriate devices for inhalation or insufflation can be used.

In another aspect, the present invention relates to a method for preventing or treating inflammatory conditions or fibrotic diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a compound as defined herein or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate or solvate thereof.

Exemplary inflammatory conditions or fibrotic diseases that can be treated using CDK8 inhibitors include, but are not limited to, type 1 diabetes graft-versus-host disease, inflammatory bowel disease, psoriasis, psoriasis arthritis, Hashimoto's thyroiditis, food allergies, HCV vasculitis, alopecia areata, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis; skin fibrosis, lung fibrosis, kidney fibrosis, liver fibrosis, intestinal fibrosis, cystic fibrosis, myocardial fibrosis, uterine leiomyoma, and adenomyosis.

In certain embodiments, the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).

In another aspect, the present invention relates to a method for preventing or treating a tumor or a cell proliferative disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, stereoisomer, mirror image isomer, prodrug, hydrate or solvate thereof.

In certain embodiments, the tumor or cell proliferative disorder or cancer metastasis is selected from at least one of the group consisting of epithelial cancer, epidermoid carcinoma, Merkel cell carcinoma, liver cancer, cervical cancer, anal cancer, penile cancer, vulvar cancer, vaginal cancer, breast cancer, ovarian cancer, uterine cancer, skin cancer, melanoma, oral cancer, colon cancer, cervical cancer, renal cancer, kidney cancer, liver cancer, colorectal cancer, esophageal cancer, thyroid cancer, uveal cancer, acute myelogenous leukemia, acute myeloid leukemia, In some embodiments, the cancer is identified as overexpressing CDK8. In certain embodiments, the cancer is colorectal cancer, breast cancer, and prostate cancer (CRC).

Having now described the present invention with the aid of written description, one skilled in the art will recognize that the present invention can be implemented in a variety of embodiments, and the foregoing description and the following examples are intended to illustrate rather than limit the scope of the following patent application .

Materials and Methods

Material

Primary antibodies against N-calcified protein, Snail, p-Smad2 (Ser465/467), Smad3, p-RNA Pol II (Ser2/5), Akt, p-Akt (Ser473), p-GSK3β (Ser9), active form of β-catenin, histone 3 and secondary antibody labeled with anti-rabbit IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against STAT1 and p-STAT1 (Ser727) were from Abcam (Cambridge, MA, USA), and primary antibodies against E-calcified protein, vimentin, p-Smad3 (Thr179), and β-actin were from ABclonal (Woburn, MA, USA). CDK8, β-SMA, GSK3β-tubulin primary antibodies and anti-rabbit IgG secondary antibody DyLight 594 were purchased from GeneTex Inc. (Hsinchu, Taiwan). pcDNA3 CDK8 HA (P#634) was a gift from Matija Peterlin (Addgene plasmid #14649; http://n2t. net/addgene:14649; RRID:Addgene_14649); 7TFP CDH1 reporter was a gift from Bob Weinberg (Addgene plasmid #91704; http://n2t. net/addgene:91704; RRID:Addgene_91704). TurboFect transfection reagent was from Thermo Fisher Scientific (Waltham, MA, USA).

Kinase inhibition assay

Compounds were tested for kinase inhibitory activity using the Thermo Fisher SelectScreen service (www.thermofisher.com/selectscreen). For CDK8, the LanthaScreen Eu binding assay was used. Briefly, the assay used utilizes a fluorescent tracer for a given kinase. This is used to detect the addition of an anti-tag antibody. When the tracer and antibody bind to the targeted kinase, the Forster resonance energy transfer is measured. When a kinase inhibitor is added to the assay, the tracer/kinase binding will be disrupted, resulting in low FRET detection and indicating kinase inhibitory activity. Assessment of kinase activity for other kinase targets utilizes the LanthaScreen binding assay or the Z'-Lyte activity assay provided by the Thermo Fisher SelectScreen service. IC ₅₀ values are determined using a continuous dose, and inhibition curves are provided by Thermo Fisher Scientific.

Cell culture

Human A549 alveolar epithelial cells, human prostate cancer cell lines PC-3 and DU145 were obtained from the Bioresource Collection and Research Center (Hsinchu City, Taiwan). Human A549 alveolar epithelial cells were maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen Life Technologies, USA) supplemented with 10% fetal bovine serum (v/v) (Invitrogen Life Technologies, USA), penicillin (100 units/mL), and streptomycin (100 μg/mL) (Biological Industries, Israel). Human prostate cancer cell lines PC-3 and DU145 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium or minimum essential medium Eagle supplemented with 10% fetal bovine serum (v/v), penicillin (100 units/mL) and streptomycin (100 μg/mL). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO ₂ .

Cytotoxicity assay

Human A549 alveolar epithelial cells

Cytotoxicity was measured by colorimetric MTT assay. Cells (10 ⁴ ) in 1 mL of medium in 96-well plates were incubated with vehicle (control) or vehicle containing test compounds for 48 hours. After each treatment, 1 mg/mL MTT was added and the plates were incubated for an additional 2 hours at 37°C. Cells were then pelleted and lysed with 10% sodium dodecyl sulfate (SDS) containing 0.01 M HCl, and the absorbance at 570 nm was measured on a microplate reader.

Human prostate cancer cell lines PC-3 and DU145

Cytotoxicity was assessed using the MTT assay. Cells in 100 μL of medium in a 96-well plate were treated with vehicle or test compounds for 24 hours. Subsequently, 0.5 mg/mL MTT was added, and the plate was incubated for another 2 hours at 37°C. Cells were then pelleted and dissolved in 100 μL of dimethyl sulfoxide, and the absorbance at 550 nm was measured using a microplate reader.

RNA Extraction and real-time polymerase chain reaction ( PCR )

Human A549 alveolar epithelial cells

Total RNA was isolated using TRIzol and Direct-zol™ RNA MiniPrep Kit (ZYMO Research, USA) following the manufacturer's instructions. Reverse transcription into complementary (c)DNA was performed using random primers and M-MLRT. Briefly, first-strand cDNA was synthesized using 1 μg of messenger (m)RNA by incubation with random primers at 65°C for 5 minutes and then with M-MLRT at 37°C for 1 hour. For real-time PCR, cDNA was amplified in SYBR Green PCR Master Mix (Life Technologies, USA) and detected with Applied Biosystems StepOnePlus™ Q-PCR Detection System (Thermo Fisher Scientific, USA). Relative gene expression was normalized to GAPDH and calculated by using ^{the 2(-ΔΔCT )} method.

Human prostate cancer cell lines PC-3 and DU145

Total RNA was isolated from cells using TRIzol reagent (Invitrogen). Single-stranded cDNA as a polymerase chain reaction template was synthesized from 5 μg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase (Promega). Quantitative PCR was performed using TaqMan One-Step RT-PCR Master Mix (ABI) in a total reaction volume of 20 μL per reaction, consisting of 10 μL SYBR Green PCR Master Mix (Applied Biosystems), 5 pmol of each forward/reverse primer, and 2 μL cDNA. Oligonucleotide primers used for amplification were as follows: for E-calcineurin, 5'-AAAGGCCCATTTCCTAAAAACCT-3' (forward) and 5'-TGCGTTCTCTATCCAGAGGCT-3'(reverse); Snail, 5'-GAGGACAGTGGGAAAGGCTC-3' (forward) and 5'-TGGCTTCGGATGTGCATCTT-3'(reverse); α-SMA, 5'-AAAAGACAGCTACGTGGGTGA-3' (forward) and 5'-GCCATGTTCTATCGGGTACTTC-3'(reverse); GAPDH, 5'-CCATCACCATCTTCCAGGAGCG-3' (forward) and 5'-AGAGATGATGACCCTTTTGGC-3' (reverse). GAPDH served as an endogenous control to normalize the variation of total RNA levels in each sample. The critical cycle (Ct) in the exponential phase of amplification was detected, the relative mRNA expression was determined by calculating the ΔΔCt value, and the fold change was expressed as 2 ^{- ΔΔCt} . The value of each control sample was set to 1 and used to calculate the fold change of target gene expression.

Immunoblot and immunoprecipitation analysis

Human A549 alveolar epithelial cells

Cells ( ¹⁰⁶ ) were incubated in lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM β-glycerophosphate, 0.1% Triton X-100, 10% glycerol, 1 mM DTT, 1 μg/mL leupeptin, 5 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) at 4°C for 10 min, scraped, incubated on ice for another 10 min, and centrifuged at 17,000 g for 30 min at 4°C. Protein samples (20 μg) were then electrophoresed on SDS-polyacrylamide gel electrophoresis (PAGE) gels and transferred to nitrocellulose membranes, followed by blocking by incubation with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBST) containing 0.1% Tween-20 for 30 minutes at room temperature. Immunoblotting was performed as follows: incubation with primary antibodies in TBST at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Bound antibodies were measured using enhanced chemiluminescence (ECL) reagents (GE Healthcare, UK) and exposed to photographic film.

Human prostate cancer cell lines PC-3 and DU145

Cells were incubated in lysis buffer for 10 min at 4°C, scraped, incubated on ice for another 10 min and subsequently centrifuged at 17,000 g for 30 min at 4°C. Protein samples were electrophoresed on SDS-PAGE gels, transferred to PVDF membranes and blocked with 5% skimmed milk in PBS for 30 min at room temperature. Immunoblotting was performed as follows: primary antibody in PBS overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody for 1 hour at room temperature. Bound antibodies were measured using ECL reagents and exposed to photographic film. In immunoprecipitation analysis, cell lysates were immunoprecipitated with 1 μg of antibody and A/G agarose beads overnight at 4°C. After washing the precipitated beads three times with 1 mL of ice-cold lysis buffer, the bound immune complexes were separated by 10% SDS-PAGE and analyzed by immunoblotting.

Immunofluorescence staining

Human prostate cancer cell lines PC-3 and DU145

Cells were seeded in 24-well plates, treated with drugs for 12 hours, and fixed with 8% paraformaldehyde in PBS for 15 minutes. After washing twice with PBS (10 minutes each wash), cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Cells were rinsed twice with PBS for 10 minutes each, blocked with 5% BSA in PBS for 1 hour, and then incubated with primary antibodies overnight, followed by FITC-conjugated anti-rabbit IgG antibodies for 2 hours. Mounting agent containing DAPI stain was dropped onto the slide, and the cover slip was re-covered on the slide. Images were detected by ZEISS ApoTome.2 microscope.

Wound Healing Analysis

Human A549 alveolar epithelial cells

Cells were seeded in six-well plates and cultured to 90% confluence, at which point the cell monolayer was wounded with a sterile 10 μL pipette tip and washed with medium to remove detached cells. Cells were incubated for 24 hours in the presence or absence of test compounds and photographed. The percentage of wound closure was then determined using ImageJ software (National Institutes of Health, USA).

Cell dissection methods

Human A549 alveolar epithelial cells

The nuclear/cytosol fractionation kit (Biovision, USA) was used to separate the cytosol and nuclear fractions. Briefly, cells were collected and centrifuged at 600 g for 5 min, the supernatant was removed, and the lysate was resuspended in cytosol extraction buffer-A, vortexed vigorously for 15 sec, and placed on ice for 10 min. Cytosol extraction buffer-B was then added to the mixture, vortexed for 5 sec, incubated on ice for 1 min, and centrifuged at 14,500 rpm to obtain the cytosol fraction. The remaining precipitate was resuspended in nuclear extraction buffer, vortexed for 15 seconds, and then frozen for 10 minutes. After repeating this procedure four times, the sample was centrifuged at 14,500 rpm to obtain nuclear extracts.

Estimation of oxidative stress

Human A549 alveolar epithelial cells

Malondialdehyde (MDA), the final product of lipid peroxidation, was measured spectrophotometrically at a wavelength of 532 nm using malondialdehyde bis(dimethylacetal) as a standard according to the analytical protocol (Cell Biolabs, USA).

Immunohistochemical analysis

Human prostate cancer cell lines PC-3 and DU145

Tissue sections were dewaxed and rehydrated. Antigen retrieval was performed by autoclaving the slides in Trilogy solution (Cell Marque, Hot Springs, AR) at 121°C for 10 min. Slides were blocked with 3% _H2O2 and 5% fetal bovine _serum , incubated with primary antibodies overnight at 4°C, and subsequently treated with polymer-HRP reagent (Dako Cytomation, Glostrup, Denmark). Peroxidase activity was observed using diaminobenzidine tetrahydrochloride solution (DAKO), and sections were counterstained with hematoxylin. Dark brown nuclear staining was defined as positive, and no staining as negative.

Data analysis and statistical analysis

Data are presented as mean ± standard error of the mean (SEM) and analyzed using one-way analysis of variance (ANOVA). When ANOVA showed significant differences between groups, Tukey's post hoc test was used to identify pairs of groups showing statistically significant differences. Parameters with p < 0.05 were considered statistically significant.

For differential expression analysis of individual genes, two online database repositories, cBioPortal (www.cbioportal.org) and Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/), were used to access clinical transcriptome datasets. The datasets (TCGA, n=494; GSE74685, n=171) were downloaded and the mRNA levels of the query genes across different prostate cancer patient groups were analyzed. Calculation of statistical significance and construction of box plots were performed using SigmaPlot software. Karben-Meier plots were generated using the KM Plotter online tool (www.kmplot.com). Data sets obtained from cBioPortal and GEO databases containing information on time until cancer progression (TCGA, n=492) and biochemical recurrence (GSE70769, n=92) were transferred to the site for curve plotting. Gene expression cutoffs defining low and high expression groups were determined by Cox proportional hazards regression analysis, and the optimal cutoff was selected for final analysis. Expression patterns of entire signaling pathways or protein complexes were evaluated by Gene Set Enrichment Analysis (GSEA) software version 4.2.2. Patient samples from the datasets obtained from cBioPortal (Abida et al.) and GEO databases (GSE68882, GSE16560) were first manually sorted into two groups (based on Gleason score or metastatic events) and then imported into the program for gene sequencing.

Synthesis Procedure

Used to synthesize compounds a - n General Procedure

Nuclear magnetic resonance (NMR) spectra (1H and 13C NMR) were obtained using Bruker Fourier 300 MHz, JEOL 400 MHz, AVIII 500 MHz, and Agilent 600 MHz spectrometers with standard add-ons. Chemical shifts are presented in parts per million (ppm, δ ) with TMS as internal standard. Mass spectrometry (MS) data were measured on a Finnigan Mat TSQ-7000 mass spectrometer (HRESIMS). HPLC was performed using a C18 column (250×4.6 mm, Waters) and a L-2130 (Hitachi, Japan)/LC-20AT (Shimadzu, Japan) pump. Column chromatography was performed on silica gel (70-230 mesh, Merck, Germany). Thin layer chromatography (TLC) analysis was performed on silica gel plates (KG60-F254, Merck). Unless otherwise mentioned, all chemicals and materials were used as received from commercial suppliers without purification. Anhydrous dichloromethane was distilled from calcium hydride under _N2 .

Examples 1 : Synthetic compound library ( Compounds a to n )

An exemplary general-purpose path for Example 1 is shown below: .

Preparation of 3- ( propan - 2 - ylidene ) indolin - 2 - one (Compound 2)

To a stirred solution of compound 1 (100 mg, 0.75 mmol) in CH ₃ H ₆ O (3 mL, 0.25 M) was added iodine (65 mg, 0.75 mmol). The solution was stirred at reflux temperature for 22 hours and then concentrated in vacuo. The residue was diluted with distilled H ₂ O. The solid was collected by filtration and washed with distilled H ₂ O to give compound 2 (154 mg, 89%) as a solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.22 (s,1H), 7.52 (d, J = 7.6 Hz, 1H), 7.19 (m, 1H), 7.01 (m, 1H), 6.86 (d, J = 7.8 Hz, 1H), 2.62 (s, 3H), 2.38 (s, 3H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ 169.9, 155.7, 139.5, 127.7, 124.5, 123.8, 123.2, 121.7, 109.5, 25.4, 23.3

Preparation of 2 - oxo - 3- ( prop - 2 - ylidene ) indoline - 5 - sulfonyl chloride (Compound 3)

Chlorosulfonic acid (269 mg, 2.31 mmol) was slowly added dropwise to compound 2 (100 mg, 0.58 mmol). The solution was stirred for 2 h in an ice bath. Following the procedure described for compound 2, 3 (153 mg, 98%) was obtained as a solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.33 (s, 1H), 8.14 (d, J = 1.3 Hz, 1H), 7.93 (dd, J = 1.9, 8.4 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 2.68 (s, 3H), 2.50 (s, 3H). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.8, 161.2, 147.4, 137.2, 128.9, 125.7, 122.7, 121.9, 110.4, 25.5, 23.2

The compounds described herein, such as the following exemplary compounds, can be synthesized in analogy to the procedures described above using commercially available and appropriate starting materials or intermediates prepared as described herein.

Preparation N -( 4 -( 4 - Nitrophenoxy ) Phenyl )- 2 - Pendant - 3 -( C - 2 - Yaki ) Indoline - 5 - Sulfonamide ( Compounds a )

To a solution of compound 3 (100 mg, 0.37 mmol) in THF-H ₂ O (5:3, 16 mL) were added K ₂ CO ₃ (102 mg, 0.74 mmol) and 4-(4-nitrophenoxy)aniline (85 mg, 0.37 mmol). The resulting solution was stirred at room temperature (RT) for 5 hours and then concentrated in vacuo. The residue was diluted with distilled H ₂ O (10 mL), neutralized with 1 N HCl _{( aq )} to pH 7, and extracted with EtOAc (30 mL×3). The organic layer was dried over Na ₂ SO ₄ and then removed in vacuo after filtration. The residue was purified by silica gel chromatography (MeOH/CH ₂ Cl ₂ =1:99) to obtain compound a (146 mg, 40%) as a solid. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 10.90 (s, 1 H, H-1), 10.19 (s, 1 H, H-9), 8.15 (m, 2 H, H-19, 21), 7.82 (s, 1 H, H-4), 7.58 (dd, J = 1.3, 8.3 Hz, 1 H, H-6), 7.17 (d, J = 8.9 Hz, 2 H, H-11, 15), 7.08 (d, J = 8.9 Hz, 2 H, H-12, H-14), 7.01 (m, 2 H, H-18, 22), 6.93 (d, J = 8.2 Hz, 1 H, H-7), 2.53 (s, 3H, H-1'), 2.30 (s, 3 H, H- 3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 165.0 (C-20), 162.9 (C-2'), 158.0 (C-13), 143.9 (C-7a), 142.1 (C-17), 135.3 (C-10), 133.7 (C- 5), 127.2 (C-6), 126.1 (C-19, 21), 123.7 (C-4a), 122.6 (C-11, 15), 121.5 (C-3, 4), 121.4 (C-12, 14), 117.0 (C-18, 22), 108.8 (C-7), 24.9 (C-3'), 22.6 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. for C ₂₃ H ₂₀ O ₆ N ₃ S 466.1067, found 466.1070.

Preparation 2 - Pendant - N -( 4 -( Aniline ) Phenyl )- 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds b )

To a stirred solution of N 1-phenylbenzene-1,4-diamine (68 mg, 0.37 mmol) in anhydrous CH ₂ Cl ₂ (10 mL) were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under N ₂ for 3 h and then concentrated in vacuo. Following the procedure described for compound a , b (159 mg, 99%) was obtained as a solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 10.84 (s, 1 H, H-1), 9.67 (s, 1 H, H-9), 8.03 (s, 1 H, H-16), 7.72 (m, 1 H, H-4), 7.54 (dd, J = 1.8, 8.2 Hz, 1 H, H-6), 7.18 (m, 2 H, H-19, 21), 6.95 (dd, J = 1.1, 8.6 Hz, 2 H, H-18, 22), 6.93 (m, 4 H, H-11, 12, 14, 15), 6.90 (d, J = 8.2 Hz, 1 H, H-7), 6.77 (m, 1 H, H-20), 2.51 (s, 3 H, H-1'), 2.26 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 157.6 (C-2'), 143.6 (C-7a), 143.5 (C-17), 140.5 (C-10), 131.6 (C-5), 129.7 (C-13), 129.1 (C-1 4, 19, 21), 127.1 (C-4a, 6), 123.5 (C-11, 15), 121.6 (C-3), 121.5 (C-4), 119.4(C-20), 117.5 (C-12), 116.2 (C-18, 22), 108.7 (C-7), 24.9 (C-3'), 22.5 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. for C ₂₃ H ₂₂ O ₃ N ₃ S 420.1376, found 420.1377.

Preparation N -( 4 -(( 2 - Pendant - 3 -( C - 2 - Aki ) Indoline )- 5 - Sulfonamide ) Phenyl ) Benzamide ( Compounds c )

To a stirred solution of N- (4-aminophenyl)benzamide (78 mg, 0.37 mmol) in anhydrous _CH2Cl2 (10 mL ₎ were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 5 h and then concentrated in vacuo. Following the procedure described for compound a , c (169 mg, 99%) was obtained as a solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 10.85 (s, 1 H, H-1), 10.16 (s, 1 H, H-16), 10.00 (s, 1 H, H-9), 7.90 (m, 2 H, H-19, 23), 7.82 (m, 1 H, H-4), 7.61 (d , J = 8.8 Hz, 2 H, H-11, 15), 7.57 (m, 2 H, H- 6, 21), 7.50 (t, J = 7.5 Hz, 2 H, H-20, 22), 7.07 (m, 2 H, H-12, 14), 6.90 (d, J = 8.2 Hz, 1 H, H-7) , 2.52 (s, 3 H, H-1'), 2.30 (s, 3 H, H-3') ^{. 13} C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 165.3 (C-17), 157.8 (C-2'), 143.7 (C-7a), 135.7 (C-18), 134.8 (C-10), 133.4 (C-5), 131.5 (C-13 ), 131.4 (C-21), 128.3 (C-20, 22), 127.5 (C-19, 23), 127.2 (C-6), 123.6 (C-4a), 121.5 (C-3), 121.4 (C-4), 121.2 (C-11, 15), 121.1 (C -12, 14), 108.8 (C-7), 24.9 (C-3'), 22.5 (C-1'). HRMS-ESI: m/z [M+H]+ calcd. for C ₂₄ H ₂₂ O ₄ N ₃ S 448.1326, found 448.1325.

Preparation N -( 4 - Benzylphenyl )- 2 - Pendant - 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds d )

To a stirred solution of (4-aminophenyl)(phenyl)methanone (58 mg, 0.29 mmol) in anhydrous _CH2Cl2 (10 mL) were added pyridine (47 mg, 0.59 mmol) and compound 3 (100 _mg , 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 6 h and then concentrated in vacuo. Following the procedure described for compound a , d (137 mg, 86%) was obtained as a solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 10.90 (s, 1 H, H-1), 10.76 (s, 1 H, H-9), 7.91 (m, 1 H, H-4), 7.71 (dd, J = 1.8, 8.2 Hz, 1 H, H-6), 7.65 (d, J = 8.7 Hz, 2 H, H-12, 14), 7.64 (dt, J = 1.8, 3.2 Hz, 1 H, H-20), 7.63 (q, J = 1.8 Hz, 2 H, H-18, 22), 7.52 (m, 2 H, H-19, 21), 7.28 (m, 2 H, H-11, 15), 6 .95(d, J = 8.2 Hz, 1 H, H-7), 2.52 (s, 3 H, H-1'), 2.32 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 194.4 (C-16), 168.3 (C-2), 158.3 (C-2'), 144.1 (C-7a), 142.3 (C-10), 137.3 (C-17), 132.3 (C-13), 131.6 (C-5 ), 131.4 (C-12, 14), 131.3 (C-20), 129.3 (C-19, 21), 128.4 (C-18, 22), 127.3 (C-6), 123.8 (C-4a), 121.4 (C-3), 121.3 (C-4), 118.0 (C -14, 15), 109.0 (C-7), 24.9 (C-3'), 22.6 (C-1'). HRMS-ESI: m/z [M+H]+ calcd. for C ₂₄ H ₂₁ O ₄ N ₂ S 433.1217, found 433.1212.

Preparation 2 - Pendant - N -( 5 - Phenyl - 1H - Pyrazole - 3 - base )- 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds e )

To a stirred solution of 5-phenyl- 1H -pyrazol-3-amine (59 mg, 0.37 mmol) in anhydrous CH ₂ Cl ₂ (10 mL) were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) in sequence. The solution was stirred at RT under N ₂ for 4 hours and then concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH/CH ₂ Cl ₂ =1:33) following the procedure described for compound a to give e (112 mg, 30%) as a solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 12.81 (s, 1 H, H-12), 10.84 (s, 1 H, H-1), 10.32 (s, 1 H, H-9), 7.91 (s, 1 H, H-4), 7.67 (t, J = 1.5 Hz, 2 H, H-16, 20 ), 7.66 (m, 2 H, H-17, 19), 7.42 (t, J = 7.5 Hz, 1 H, H-6), 7.34 (d, J = 7.3 Hz, 1 H, H-18), 6.92 (d, J = 8.2 Hz, 1 H, H-7), 6.44 (s, 1 H, H-14), 2. 51 (s, 3 H, H-1'), 2.27 (s, 3 H, H- 3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.4 (C-2), 157.5 (C-2'), 147.0 (C-10), 143.7 (C-7a), 142.5 (C-13), 132.3 (C-5), 128.9 (C-18), 128.3 (C-1 5), 127.1 (C-6, 17, 19), 124.9 (C-16, 20), 123.5 (C-4a), 121.7 (C-21), 121.5 (C-3), 108.7 (C-7), 94.6 (C-14), 24.8 (C-3'), 22.5 (C-1' ). HRMS-ESI: m/z [M+H]+ calcd. for C ₂₀ H ₁₉ O ₃ N ₄ S: 395.1172, found: 395.1171.

Preparation 2 - Pendant - 3 -( C - 2 - Yaki )- N -( 5 -( p-Tolyl )- 1H - Pyrazole - 3 - base ) Indoline - 5 - Sulfonamide ( Compounds f )

To a stirred solution of 5-(p-tolyl) 1H -pyrazol-3-amine (64 mg, 0.37 mmol) in anhydrous _CH2Cl2 (10 mL ₎ was added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 6 h and then concentrated in vacuo. The solid was collected by filtration and washed _{with CH2Cl2} to give compound f (91 mg, 61%) as a solid. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.72 (s, 1 H, H-12), 10.85 (s, 1 H, H-1), 10.31 (s, 1 H, H-9), 7.90 (s, 1 H, H-4), 7.65 (dd, J = 1.6, 8.2 Hz, 1 H, H-6 ), 7.54 (d, J = 8.1 Hz, 2 H, H-16, 20), 7.22 (d, J = 8.0 Hz, 2 H, H-17, 19), 6.92 (d, J = 8.2 Hz, 1 H, H-7), 6.36 (s, 1 H, H-14), 2.49 (s, 3 H, H-1' ), 2.31 (s, 3 H, H-21), 2.27 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.4 (C-2), 157.5 (C-2'), 147.0 (C-10), 143.7 (C-7a), 142.7 (C-13), 137.7 (C-18), 132.3 (C-5), 129.5 (C-1 5, 17, 19), 127.1 (C-6), 124.9 (C-16, 20), 123.5 (C-4a), 121.7 (C-3), 121.5 (C-4), 108.7 (C-7), 94.2 (C-14), 24.8 (C-3'), 22.5 (C-1' ), 20.8 (C-21). HRMS-ESI: m/z [M+H]+ calcd. for C ₂₁ H ₂₁ O ₃ N ₄ S 409.1329, found 409.1325.

Preparation 2 - Pendant - N -( 5 - Phenyl - 1 , 3 , 4 - Thiadiazole - 2 - base )- 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds g )

To a stirred solution of 5-phenyl-1,3,4-thiadiazol-2-amine (65 mg, 0.37 mmol) in anhydrous CH ₂ Cl ₂ (10 mL) were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under N 2 for 6 h and then concentrated in vacuo. Following the procedure described for compound f , g (95 mg, 26%) was obtained as a solid. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 10.88 (s, 1 H, H-1), 10.48 (s, 1 H, H-9), 7.91 (d, J = 1.4 Hz, 1 H, H-4), 7.82 (m, 1 H, H- 17), 7.70 (dd, J = 1.7, 8.3 Hz, 1 H, H-6), 7.55 (m, 2 H, H-18, 19), 7.45 (dd, J = 1.4, 8.0 Hz, 1 H, H-16), 6.97 (d, J = 8.3 Hz, 1 H, H-7), 6.75 (d, J = 8.0 Hz, 1 H, H-20), 52 (s, 3H, H-1'), 2.32 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.7 (C-2), 168.4 (C-10), 157.8 (C-2'), 155.2 (C-15), 143.7 (C-7a), 141.5 (C-13), 134.1 (C-5), 131.4 (C-18) , 129.4 (C-17), 126.3 (C-19), 126.2 (C-6), 125.4 (C-16), 123.6 (C-4a), 121.5 (C-3), 120.6 (C-4), 109.1 (C-7), 107.8 (C-20), 24.7 (C-3 '), 22.2 (C-1'). HRMS-ESI: m/z [M ₊ H]+ calcd. _{for C19H15O3N4S2 :} 411.0580, _{found :} 411.0590.

Preparation N -( Benzo [ d ] Thiazole - 2 - base )- 2 - Pendant - 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds h )

To a stirred solution of benzo[ d ]thiazol-2-amine (55 mg, 0.37 mmol) in anhydrous _CH2Cl2 (10 mL ₎ were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 24 h and then concentrated in vacuo. Following the procedure described for compound f , h (84 mg, 59%) was obtained as a solid. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 10.48 (s, 1 H, H-1), 7.87 (d, J = 7.9 Hz, 1 H, H-4), 7.78 (s, 1 H, H-6), 7.46 (m, 2 H, H-13, 14), 7.42 (m, 1 H, H-15), 7.29 (t, J = 7.5 Hz, 1 H, H-12), 6.75 (d, J = 7.9 Hz, 1 H, H-7), 2.50 (s, 3 H, H-1'), 2.31 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.9 (C-2), 168.7 (C-10), 154.4 (C-2'), 140.4 (C-7a), 134.2 (C-5), 127.4 (C-15), 125.3 (C-13), 124.0 (C-12 ), 123.5 (C-4a), 122.9 (C- 4), 122.6 (C-3, 12a), 122.5 (C-15a), 120.9 (C-6), 114.5 (C-14), 107.9 (C- 7), 24.7 (C-3'), 22.2 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. 384.0471 for C18H14O3N3S2, found 384.0482.

Preparation N -( 4 -( 1H - Imidazole - 1 - base ) Phenyl )- 2 - Pendant - 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds i )

To a stirred solution of 4-( 1H -imidazol-1-yl)aniline (41 mg, 0.26 mmol) in anhydrous _CH2Cl2 (10 mL) were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 _mg , 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 20 h and then concentrated in vacuo. Following the procedure described for compound f , i (100 mg, 68%) was obtained as a solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 10.90 (s, 1 H, H-1), 10.51 (s, 1 H, H-9), 8.91 (s, 1 H, H-17), 7.90 (d, J = 10.6 Hz, 2 H, H-19, 20), 7.64 (dd, J = 1.6, 8.2 Hz, 1 H, H-6), 7.58 (m, 2 H, H-12, 14), 7.50 (s, 1 H, H-4), 7.27 (dd, J = 2.5, 9.4 Hz, 2 H, H-11, 15), 6.93 (d, J = 8.2 Hz, 1 H, H-7), 2.52 (s , 3H, H-1'), 2.32 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 158.2 (C-2'), 144.0 (C-7a), 138.0 (C-10), 134.8 (C-17), 131.2 (C-5), 127.3 (C-6), 124.8 (C-13) , 123.8 (C- 4a), 122.4 (C-12, 14), 121.4 (C-19), 121.3 (C-3), 121.0 (C-4), 120.7 (C- 11, 15), 119.6 (C-20), 108.9 (C-7), 25.0 (C-3'), 22.6 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. for C ₂₀ H ₁₉ O ₃ N ₄ S: 395.1172, found: 395.1169.

Preparation N - methyl - 4 -( 4 -(( 2 - Pendant - 3 -( C - 2 - Aki ) Indoline )- 5 - Sulfonamide ) Phenoxy ) Pyridinecarboxamide ( Compounds j )

To a stirred solution of 4-(4-aminophenoxy) -N -methylpicolinamide (90 mg, 0.37 mmol) in anhydrous _CH2Cl2 (10 mL ₎ were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under N2 for 2 h and then concentrated in vacuo. Following the procedure described for compound a , j (174 mg, 97%) was obtained as a solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 10.88 (s, 1 H, H-1), 10.21 (s, 1 H, H-9), 8.73 (m, 1 H, H-24), 8.47 (d, J = 5.7 Hz, 1 H, H-21), 7.84 (s, 1 H, H-4), 7. 58 (dd, J = 1.7, 8.1 Hz, 1 H, H-6), 7.31 (d, J = 2.3 Hz, 1 H, H-18), 7.20 (m, 2 H, H-11, 15), 7.12 (m, 2 H, H-12, 14), 7.04 (dd, J = 5.7, 2.6 Hz, 1 H , H-22), 6.94 (d, J = 8.1 Hz, 1 H, H-7), 2.78 (d, J = 4.8 Hz, 3 H, H-25), 2.53 (s, 3 H, H-1'), 2.31 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 165.5 (C-19), 163.7 (C-23), 158.0 (C-2'), 152.5 (C-17), 150.4 (C-21), 149.6 (C- 13), 143.9 (C- 7a), 135.6 (C-10), 131.3 (C-5), 127.2 (C-6), 123.7 (C-4a), 122.6 (C-11, 15), 121.8 (C-12, 14), 121.4 (C-3, 4), 114.0 (C-22), 108.9 (C -7), 108.8 (C-18), 26.0 (C-25), 24.9 (C-3'), 22.5 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. 479.1384 for C ₂₄ H ₂₃ O ₅ N ₄ S, found 479.1382.

Preparation 4 -( 3 - fluorine - 4 -(( 2 - Pendant - 3 -( C - 2 - Aki ) Indoline )- 5 - Sulfonamide ) Phenoxy )- N - Pyridine formamide ( Compounds k )

To a stirred solution of 4-(4-amino-3-fluorophenoxy) -N -methylpicolinamide (96 mg, 0.37 mmol) in anhydrous _CH2Cl2 (10 mL ₎ were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 6 h and then concentrated in vacuo. Following the procedure described for compound a , k (178 mg, 97%) was obtained as a solid. ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 10.92 (s, 1 H, H-1), 10.10 (s, 1 H, H-9), 8.78 (q, J = 4.7 Hz, 1 H, H-24), 8.52 (d, J = 5.5 Hz, 1 H, H-21), 7.84 (d, J = 1.3 Hz, 1 H, H-4), 7.54 (dd, J = 1.7, 8.2 Hz, 1 H, H-6), 7.38 (d, J = 2.5 Hz, 1 H, H-18), 7.34 (t, J = 8.8 Hz, 1 H, H-15), 7.22 (dd, J = 2.7, 10.9 Hz, 1H, H-12), 7.13 (dd, J = 2.7, 5.5 Hz, 1 H, H-22), 7.04 (m, 1 H, H-14), 6.95 (d, J = 8.2 Hz, 1 H, H-7), 2.79 (d, J = 4.9 Hz, 3 H, H-25), 2.54 (s, 3 H, H- 1'), 2.31 (s, 3 H, H-3'). 13C NMR (125 MHz, DMSO- d ₆ ) δ 168.4 (C-2), 164.9 (C-19), 163.6 (C-23), 158.0 (C-2'), 157.3 (C-10), 155.3 (C-11), 152.5 (C- 17), 151.6 (C- 13), 150.5 (C-21), 143.9 (C-7a), 131.7 (C-6), 128.4 (C-15), 127.1 (C-7), 123.7 (C-5), 122.3 (C-12), 121.5 (C-3), 121.4 (C-4), 117.1 (C -14), 114.4 (C-22), 109.3 (C-18), 108.8 (C-7), 26.0 (C-25), 24.9 (C-3'), 22.5 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. 497.1289 for C ₂₄ H ₂₂ O ₅ N ₄ FS, found 497.1290.

Preparation 2 - fluorine - N - methyl - 4 -(( 2 - Pendant - 3 -( C - 2 - Aki ) Indoline )- 5 - Sulfonamide ) Benzamide ( Compounds l )

To a stirred solution of 4-amino-2-fluoro- N -methylbenzamide (62 mg, 0.37 mmol) in anhydrous _CH2Cl2 (10 mL ₎ were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under _N2 for 4 h and then concentrated in vacuo. Following the procedure described for compound a , l (120 mg, 81%) was obtained as a solid. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 10.91 (s, 1 H, H-1), 10.75 (s, 1 H, H-9), 8.01 (m, 1 H, H-17), 7.89 (s, 1 H, H-4), 7.68 (dd, J = 1.7, 8.2 Hz, 1 H, H-6) , 7.51 (t, J = 8.5 Hz, 1 H, H-12), 6.97 (m, 2 H, H-11, 15), 6.92 (m, 1 H, H-7), 2.71 (d, J = 4.6 Hz, 3 H, H-18), 2.52 (s, 3 H, H-1'), 2.33 (s, 3 H, H-3'). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 163.3 (C-16), 160.4 (C-13), 158.4 (C-2'), 144.2 (C-7a), 141.7 (C-14), 133.2 (C-5), 131.3 (C-10 ), 130.9 (C-12), 127.3 (C-6), 123.9 (C-4a), 121.4 (C-3), 121.3 (C-4), 114.4 (C-11), 109.0 (C-7), 105.7 (C-15), 26.2 (C-18), 25.0 (C-3 '), 22.6 (C-1'). HRMS-ESI: m/z [M+H] ⁺ calcd. for C ₁₉ H ₁₉ O ₄ N ₃ FS: 404.1075, found: 404.1074.

Preparation N -( 4 - methyl - 3 -(( 4 -( Pyridine - 3 - base ) Pyrimidine - 2 - base ) Amine ) Phenyl )- 2 - Pendant - 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds m )

To a stirred solution of 6-methyl- N 1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine (102 mg, 0.37 mmol) in anhydrous CH ₂ Cl ₂ (10 mL) were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) sequentially. The solution was stirred at RT under N 2 for 3 h and then concentrated in vacuo. Following the procedure described for compound f , m (64 mg, 34%) was obtained as a solid. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 10.80 (s, 1 H, H-1), 10.05 (s, 1 H, H-9), 9.24 (d, J = 1.5 Hz, 1 H, H-25), 8.87 (s, 1 H, H-17), 8.72 (dd, J = 1.5, 4.9 Hz, 1 H, H-22), 8.49 (m, 1 H, H-29), 8.46 (m, 1 H, H-27), 7.80 (s, 1 H, H-4), 7.60 (m, 2 H, H-6, 28), 7.44 (m, 2 H, H-11, 21), 7.07 (d, J = 8.4 Hz , 1 H, H- 14), 6.88 (d, J = 8.2 Hz, 1 H, H-7), 6.81 (dd, J = 2.3, 8.1 Hz, 1 H, H-15), 2.45 (s, 3 H, H-1'), 2.17 (s, 3 H, H-3'), 2.12 (s, 3 H, H-16). ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 168.3 (C-2), 160.9 (C-18), 160.8 (C-20), 159.5 (C-27), 157.6 (C-2'), 149.7 (C-22), 146.6 (C-25), 143.7 (C-7 a), 138.1 (C-12), 136.1 (C-29), 135.9 (C-10), 132.7 (C-24), 131.6 (C-5), 130.6 (C-14), 127.6 (C-13), 127.1 (C-6), 124.5 (C-28), 123. 5 (C-4a), 121.6 (C-4), 121.4 (C-3), 116.7 (C-11), 116.4 (C-15), 108.9 (C-7), 107.8 (C-21), 24.7 (C-3'), 22.5 (C-1'), 17.4 (C-16). HRMS-ESI: m/z [M+H] ⁺ calcd. 513.1703 for C ₂₇ H ₂₅ O ₃ N ₆ S, found 513.1703.

Preparation N -( 1 , 4 - Dioxy - 1 , 2 , 3 , 4 - Tetrahydrogen Tie 𠯤 - 5 - base )- 2 - Pendant - 3 -( C - 2 - Aki ) Indoline - 5 - Sulfonamide ( Compounds n )

To a stirred solution of 5-amino-2,3-dihydro-1,4-piperidin-1-one (73 mg, 0.37 mmol) in anhydrous CH ₂ Cl ₂ (10 mL) were added pyridine (58 mg, 0.74 mmol) and compound 3 (100 mg, 0.37 mmol) in sequence. The solution was stirred at RT under N ₂ for 5 h and then concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH/CH ₂ Cl ₂ =1:20-1:5) following the procedure described for compound a to give n (25 mg, 17%) as a solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 13.06 (s, 1 H, H-9), 11.79 (s, 2 H, H-15, 16), 10.83 (s, 1 H, H-1), 7.84 (d, J = 1.6 Hz, 1 H, H-4), 7.77 (m, 2 H, H-1 1, 12), 7.68 (dd, J = 1.6, 8.0 Hz, 1 H, H-6), 7.54 (dd, J = 2.8, 6.4 Hz, 1 H, H-13), 6.92 (d, J = 8.0 Hz, 1 H, H-7), 2.49 (s, 3 H, H-1'), 2.28 (s, 3 H, H-3'). ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 168.0 (C-2), 160.3 (C-17), 158.0, (C-2'), 151.8 (C-14), 144.1 (C-7a), 140.2 (C-10), 134.3 (C-12), 130.9 (C- 5), 127.2 (C-6), 126.3 (C-14a), 123.7 (C-4a), 121.2 (C-3, 4), 119.2 (C-11), 117.9 (C-13), 114.8 (C-17a), 109.0 (C-7), 24.6 (C-3'), 22 .4 (C-1'). HRMS-ESI: m/z [M+H]+ calcd. 413.0914 for C19H17O5N4S, found 413.0911.

Examples 2 . Evaluation of synthetic compounds

In this study, a fragment-based drug design strategy was used to optimize the reference CDK8 inhibitor E966-0539 , N- (3- acetylphenyl )-2-oxo-3-( propan -2-ylidene)-indoline-5-sulfonamide, which was previously discovered by structure-based virtual screening (SBVS) activity. The 50% inhibition concentration ( IC50 ) value of E966-0530 in the enzyme assay was 1,684 nM. The E966-0530 structure contains an indolin-2-one backbone with a sulfonamide group. Sulfonamides are key functional groups in organic chemistry with high hydrolytic stability and amino acid interaction ability (A. Ovung et al., 2021 Biophys. Rev. 13 (2) 259-272). Indolin-2-one is one of the proprietary core skeletons that continues to appear in natural products and pharmacologically active compounds (YM Khetmalis et al., 2021 Biomed . Pharmacother. 141, 111842). The above experiments and analyses indicate that E966-0530 is a promising starting point for further optimization. The optimization includes the design and synthesis of a series of 2-hydroxy- 3- (propan-2-ylidene)indoline-5-sulfonamide derivatives.

The fragment-based drug design strategy in this study produced optimized CDK8 inhibitors with promising results. The CDK8 inhibitory activity of compounds ( a - n ) designed by this fragment-based drug design strategy was tested using SelectScreen Kinase Service from ThermoFisher Scientific. The inhibitory effects of 14 compounds and reference compounds were tested at a concentration of 10 μM. The results are shown in Table 1 below:

Table 1 Compound No. The structure of R Percentage inhibition of CDK8 at 10 μM CDK8 Inhibition IC ₅₀ (nM ) a 48% Undetermined b 76% 1567 c 33% Undetermined d 99% 380 e 82% 1639 f 89% 798 g 99% 338 h 91% 870 i 92% 945 j 85% 1235 k 102% 129 l 92% 652 m 46% Undetermined n 74% 3004 Reference compounds 87% 1684

As can be seen from Table 1 , the results showed that 11 compounds exhibited ≥50% inhibition. Further testing showed that the compounds exhibited IC50 values between 129 and 3,004 nM. Seven compounds exhibited IC50 values in the range of hundreds of nM. Notably, three compounds inhibited CDK8 with IC50 values of <400 nM. Those compounds were d , g , and k , with IC50 values of 380, 338, and 129 nM, respectively. Compared to the original compound E966-0530 , compound k exhibited a 13-fold increase in CDK8 inhibitory activity. The experimental results showed that compound k was the most potent of the synthesized compounds.

Examples 3 . Compounds a - n Docking and SAR analyze

Figure 1 shows the 2D interaction pose of compound k.

To investigate interactions that promote CDK8 inhibition, compound k molecules were docked into the CDK8 binding site ( FIG1 ).

The structure of compound k is divided into three groups (G1, G2 and G3). The docking results show that the hydroxyindole ring of the G1 group occupies the adenine position and forms hydrogen bonds with the residues D98 and A100 of the hinge residue. These interactions are generated by the nitrogen and oxygen atoms located on the hydroxyindole ring and are identified as binding interactions. Additional π-π T-shaped interactions with the residue F97 were also observed in the G1 group. The G2 group is a sulfonamide structure. The residue K52 forms a hydrogen bond with the oxygen atom and forms an attractive charge with the amine. Finally, the G3 group is 4-(4-amino-3-fluorophenoxy) -N -methylpicolylamide. π-alkyl interactions occur between residue M174 and the aromatic ring of phenoxypyridine. Additional halogen interactions of the fluorine atom with residue N156 were also observed. Overall, these interactions suggest that compound k exhibits favorable interactions against CDK8.

In addition, SAR analysis was performed using compounds a - n . In summary, the analysis revealed favorable functional groups and interactions that may be important for potent CDK8 inhibition.

First, compound j, which has a single moiety difference compared to compound k, was then selected for analysis. Based on the inhibitory activity, compound k was found to have an inhibitory potency that was almost 1.5 times higher due to the substitution of the halogen atom. In addition, the effects of compounds e - i whose amine groups are at C5-C6 or C6-C5 were explored. The composition of C5 includes pyrazole, thiadiazole and thiazole. Among them, compound g composed of thiadiazole showed good inhibitory activity and was also the second most effective in this series of compounds. Docking analysis revealed that compound g formed π-anionic interactions with residue K52 through its sulfonamide and thiadiazole, which may play an important role in CDK8 inhibition.

Compounds a - d were obtained by connecting diphenyl structures with different bonding atoms to sulfonamide. Among them, compound d showed inhibitory activity with an IC50 value of 380 nM. According to structural analysis, it was found that the benzophenone of compound d formed additional hydrophobic interactions with residues V27, V35, Y99 and W105, and the compound had better inhibitory activity.

Examples 4 . With significant selective inhibition CDK8 active

Figures 2A-2D show the evaluation of CDK8 inhibition by compound k.

The kinase selectivity of compound k was evaluated. A group of 54 kinases from different families were selected, and the kinase inhibition of compound k was screened at 150 nM. As shown in Figure 2A , among all kinases, only CDK8 was inhibited by compound k , with an inhibition percentage of >60% (64%). The inhibition of compound k on CDK19 (a paralog of CDK8) was slightly weaker than the inhibition on CDK8 (40%), and the compound had no significant effect on other CDKs and kinases. The higher selectivity of the compound can reduce the risk of adverse side effects, and the selectivity of compound k can enhance its safety and avoid adverse side effects. The cytotoxicity of compound k was then evaluated in DU145 and PC-3 cells (human prostate cancer cell lines isolated from patients with brain metastases and bone metastases, respectively). As shown in Figure 2B , the IC50 of compound k in both cell lines was greater than 30 μM for 24 hours and maintained for 72 hours. These results indicate that compound k administered at concentrations below 30 μM from 24 hours to 72 hours did not cause significant cytotoxicity in either cell line. To further examine whether compound k still inhibits CDK8/19 inhibition in cells, we evaluated whether compound treatment in DU145/PC-3 androgen-independent prostate cancer cells could inhibit STAT1-S727 phosphorylation. Since STAT1 is a substrate for CDK8/19, STAT1-S727 phosphorylation is often used to measure CDK8/19 kinase activity (J. Bancerek et al., 2013 Immunity 38 (2) 250-262). As shown, compound k treatment inhibited the phosphorylation of STAT1, a known CDK8 substrate, at the Ser727 residue in a concentration-dependent manner ( Figure 2C to Figure 2D ). To further confirm the CDK8 inhibitory effect of the compound, cells were transfected with pcDNA3 CDK8 HA plasmid to overexpress CDK8. In both cell lines, compound k significantly inhibited the phosphorylation of STAT1 at Ser727 in a concentration-dependent manner without affecting CDK8 expression ( Figure 2C to Figure 2D ). These results indicate that compound k can inhibit CDK8/19 activity in cells.

Examples 5 . During prostate cancer progression CDK8 Transformation growth factor β ( TGF - β ) Signal increase

Figures 3A to 3H show that CDK8 expression and TGF-β signaling are increased in metastatic prostate cancer and correlate with poor patient survival.

To examine the relationship between CDK8 and prostate cancer progression, public datasets of clinical samples were accessed through the GEO database and cBioPortal. As shown, higher CDK8 RNA expression was found in metastatic prostate cancer samples than in primary prostate cancer samples ( Figure 3A ), and similar trends were noted in an independent series when samples were sorted according to the presence of lymph node metastases ( Figure 3B ). Expression array data collected from 94 radical prostatectomy patient samples were used to analyze the time until relapse, and high CDK8 RNA levels were found to be significantly associated with earlier biochemical relapse over a 100-month period ( Figure 3C ). Given the affiliation of CDK8 with the Mediator complex, gene set enrichment analysis (GSEA) was performed in an exploratory attempt to analyze the expression of the multiprotein complex as a whole (GSE68882). The analysis revealed that components of the Mediator complex, including CDK8/19, MED12, and MED13, were also upregulated in metastatic prostate cancer ( Figure 3D ). These observed trends echo the findings of previous studies and prompted us to explore causal models that could explain such associations (IB Roninson et al., 2019 Cells 8 (8) 821; J. Bragelmann et al., 2017 Clin. Cancer Res 23 (7) 1829-1840). In addition, TGF-β is an important inducing factor for cancer metastasis, and previous studies have shown that overproduction of TGF-β is associated with metastasis, androgen receptor inhibition resistance, and adverse clinical outcomes in prostate cancer (T. Trivedi et al., 2021 Biomolecules 11 (11) 1643). In addition, the Gleason score is a grading system used to determine the aggressiveness of prostate cancer. Scores above 7 indicate cancers that may spread more rapidly. Using a Gleason score of 7 (intermediate grade) as a cutoff, changes in TGF-β/Smad signaling were analyzed. Database enrichment analysis showed that TGF-β/Smad signaling was enhanced in samples with high pathological Gleason scores ( Figure 3E , Figure 3F ). High TGF-β/Smad signaling was associated with poor 10-year progression-free survival ( Figure 3G ), and high TGFBR1 transcript levels were observed in metastatic prostate cancer samples ( Figure 3H ). These results suggest that the TGF-β/Smad pathway plays a key role in prostate cancer metastasis and that the CDK8/Mediator complex may be an important regulator of this signaling.

Example 6. Inhibition of Cell Migration and EMT Protein Expression FIG. 4A to FIG. 4B show the inhibition of epithelial-mesenchymal transition protein expression in human alveolar epithelial cells by a CDK8 inhibitor.

FIG. 5A and FIG. 5B show that compound k inhibits the migration of A549 cells.

FIG. 6A to FIG. 6D show that cell migration and EMT characteristics of prostate cancer cells were significantly inhibited by compound k .

FIG. 7A to FIG. 7F show that compound k significantly inhibited the expression of EMT proteins and genes induced by TGF-β1.

Previous studies have shown that EMT in pulmonary fibrosis is a process in which alveolar epithelial cells lose contact and adhesion, change their shape, transform into myofibroblasts, and exhibit characteristics of invasion, migration, and ECM production (L. Richeldi et al., 2017 Lancet 389 (10082 1941-1952)).

TGF-β1 is one of the most potent fibrogenic interleukins. Previous studies have identified that TGF-β1 can trigger Smad-dependent signaling to increase EMT protein expression and epithelial migration, ultimately leading to lung fibrosis (NG Frangogiannis et al., 2020 J. Exp. Med. 217 (3), e20190103). In this study, CDK8 inhibitors were investigated to determine how they would regulate TGF-β1 signaling. We found that TGF-β1 treatment significantly increased the expression of myofibroblast markers such as collagen I, α-smooth muscle actin (α-SMA), and Snail in human alveolar epithelial A549 cells; at the same time, treatment with TGF-β1 also downregulated the level of epithelial cell marker E-calcification ( Figure 4A ). These results indicate that TGF-β1 treatment triggers EMT progression in our model. Cynicin A (CDK8 inhibitor) and pirfenidone (clinical drug for the treatment of idiopathic pulmonary fibrosis) were used as reference compounds, both of which showed slight inhibition of EMT protein levels. Interestingly, when A549 epithelial cells were treated with compound k , a significant inhibition of TGF-β1 triggering EMT protein expression was observed. In contrast, compound b showed a slight decrease in A549 cells, while compound a showed no significant decrease in EMT protein content ( Figure 4A ).

Among the compounds tested, compound k showed the greatest efficacy compared to other test compounds and reference reagents. This seems to be consistent with its CDK8 inhibitory activity. In addition, the effect of compound k on the cell viability of A549 cells was also evaluated. As shown, when treated with compound k for 12, 24 and 48 hours, the cell viability of treated cells did not change significantly ( Figure 4B ). This was also observed when the compound concentration was increased to 10 μM. This indicates that the inhibition of EMT protein expression by compound k is not due to cytotoxicity.

In addition, cell migration was assessed when treated with CDK8 inhibitors. A549 alveolar epithelial cells treated with TGF-β1 showed a clearer appearance and cell migration at 24 hours; after 48 hours of treatment, more significant cell migration was observed ( Figure 5A to Figure 5B ). Compound k showed a potent inhibition of cell migration; in contrast, mild ( a ) and moderate ( b ) CDK8 inhibitors and two reference reagents showed less inhibition of cell migration. This indicates that compound k is a more potent regulator of EMT in this lung fibrosis model. ( Figure 5A to Figure 5B )

In addition, TGF-β is also an important inducing factor of cancer metastasis. Overexpression of TGF-β is significantly associated with poor clinical outcomes in prostate cancer metastasis (T. Trivedi et al., 2021 Biomolecules 11 (11) 1643). Therefore, functional analysis of prostate cancer cells was performed to explore the potential effects of CDK8 inhibition on tumor cell metastasis. In response to TGF-β1 (10 ng/mL) stimulation for 24 hours, a transformation from self-aggregated pebble-like shape to dispersed elongated morphological characteristics was observed in DU145 cells ( Figure 6A ). Compound k treatment appeared to reverse these effects. Next, we examined whether compound k inhibited the migration of prostate cancer cells. The results showed that in response to TGF-β1 treatment for 48 hours, wound closure was significantly enhanced, and compound k significantly inhibited TGF-β1-induced migration in prostate cancer cells ( Figure 6B to Figure 6C ). In addition, a transwell assay was performed to confirm the inhibitory effect of the compound on migration. The results showed that compound k treatment significantly inhibited TGF-β1-triggered cell migration in both prostate cancer cells. We further studied the mechanism by which the compound inhibited cell migration. As shown, prostate cancer cells exposed to TGF-β1 increased several epithelial-mesenchymal transition (EMT) proteins, such as N-calcification, Snail and vimentin, and reduced the content of the epithelial marker E-calcification ( Figure 6D and Figure 7A to Figure 7D ). Compound k treatment attenuated the effect of TGF-β1 by upregulating the epithelial marker E-calcification and downregulating the mesenchymal markers N-calcification, Snail, and vimentin ( Figure 6D and Figure 7A to Figure 7D ). To determine whether such results were the result of transcriptional inhibition, real-time PCR experiments were also performed. Changes in mRNA content reflect protein changes ( Figure 7E to Figure 7F ). The commercial CDK8 inhibitor senexin A (IC50=280 nM) was used as a reference compound. Compound k exhibited a more potent EMT protein inhibitory effect than senexin A ( Figure 6D and Figure 7A to Figure 7F ). These results indicate that compound k significantly inhibits TGF-β1-induced EMT gene expression and migration in prostate cancer cells.

Examples 7 . Compounds k Through inhibition RNA Polymerase II and Smad Phosphorylation of the linker inhibits TGF - β 1 / Smad Signal transduction

FIG8A and FIG8B show the inhibitory effect of compound k on TGF-β1-induced cellular distribution of p-Smad3 T179, RNA polymerase II pS2/S5, and Smad3.

Figures 9A to 9G show that compound k significantly inhibits TGF-β/Smad/RNA polymerase II signaling.

It is known that TGF-β1 mainly induces the Smad pathway to enhance EMT proteins and redox imbalance, both of which contribute to the pathogenesis of pulmonary fibrosis (Y. Park et al., 2021 Sci. Rep. 11 (1) 4318). After TGF-β1 binds to its receptor, Smad3 binds to Smad4, then translocates to the cell nucleus and binds to DNA. CDK8 phosphorylates Smad3 at T179, leading to the activation of RNA polymerase II and the regulation of ECM protein transcription (J. Massague et al., 2012 Nat. Rev. Mol. Cell Biol. 13 (10) 616-630; E. Aragon et al., 2011 Genes Dev. 25 (12) 1275-1288). When treated with CDK8 inhibitors, the effects of Smad3 phosphorylation and RNA polymerase II activation were evaluated. After treatment with various compounds, cells were fractionated into cytoplasmic and nuclear fractions, and α-tubulin and histone H3 were used as their markers, respectively. As expected, total Smad3 was mainly distributed in the cytoplasmic fraction under resting conditions; only a small amount of Smad3 protein was observed in the nuclear fraction ( Figure 8A to Figure 8B ). In addition, a small amount of phosphorylated Smad3 T179 was detected in both the cytoplasmic and nuclear fractions. In contrast, when compared with the control group, TGF-β1 treatment not only significantly increased the total Smad3 content in the nucleus, but also significantly enhanced the expression of p-Smad3 T179 and RNA polymerase II pSer2/5 in the nucleus. These results indicate that in alveolar epithelial A549 cells, after TGF-β treatment, Smad3 protein translocated to the nucleus, phosphorylated at Thr179, and subsequently activated RNA polymerase II. Compound k treatment significantly reversed the phosphorylation of p-Smad3 T179 and RNA Pol II pSer2/5; in contrast, the two reference reagents, senexin A and pirfenidone, produced weak inhibition of the phosphorylation of p-Smad3 and RNA Pol II ( Figure 8A to Figure 8B ).

In addition, CDK8 promotes ligand-induced transcriptional activity through phosphorylation of transcription factors (I. Menzl et al., 2019 Pharmaceuticals 12 (2) 92). To better understand whether the inhibition of migration by the compounds involves inhibition of CDK8 transcriptional regulation, we further examined the effects of the compounds on the TGF-β/Smad canonical pathway. After TGF-β1 activates the receptor, the Smad family signal transducers Smad2 and Smad3 are phosphorylated at the C-terminal domain (e.g., Ser465/467), and these tail-phosphorylated Smads subsequently complex with the co-mediator Smad4 that translocates to the nucleus. In the nucleus, the CDK8/Mediator complex phosphorylates Smad3 at Thr179, which creates a high-affinity binding site for the coactivator Pin1 and other transcriptional partners, which then phosphorylates active RNA polymerase II S2/S5 to achieve peak transcription and promote EMT and metastasis (C. Alarcon et al., 2009 Cell 139 (4) 757-769). Through interactions with different transcription factors, cofactors and RNA polymerase II (2013 Cell 153 (6) 1327-13). As shown, the levels of p-Smad2 (Ser465/467) and the mesenchymal markers N-calcified and Snail increased in response to 24-hour postintervention by TGF-β1; Compound K treatment significantly downregulated the expression of mesenchymal markers ( FIG. 9A - 9B ).

To confirm that CDK8 is involved in this interaction, CDK8 was overexpressed to counteract the inhibitory effect of compound k . Of interest, the inhibitory effect of the compound was restored in DU145 cells overexpressing CDK8 ( Figures 9A to 9B ), indicating the inhibitory effect of the compound on CDK8. Next, downstream signal changes after compound treatment were examined. A nuclear extraction protocol was performed to separate the cytosol and nuclear fractions, and α-tubulin and histone H3 were used as cytoplasmic and nuclear markers, respectively ( Figures 9C to 9D ). TGF-β1 treatment significantly increased the levels of p-Smad3 T179 and p-RNA polymerase II S2/5 in the nuclear fraction, and compound k significantly inhibited the phosphorylation of Smad3 and RNA polymerase II ( Figures 9C to 9D ). We also reconfirmed the interaction between CDK8, transcription factors, RNA polymerase II and Pin1 coactivator in response to compound k treatment by immunoprecipitation.

TGF-β1 treatment significantly increased the binding of CDK8 to RNA polymerase II (Ser2/5), Smad3, and Pin1, and these interactions were inhibited by compound k treatment ( Figure 9E to Figure 9F ). In addition, to identify the inhibitory effect of the compound on EMT, we transfected DU145 cells with a CDH1 reporter encoding the key epithelial protein marker E-calcified mucin and evaluated CDH1-driven luciferase expression. As expected, TGF-β1 treatment significantly reduced CDH1-driven luciferase expression, and compound k reversed this effect to the control amount ( Figure 9G ). These results indicate that compound k inhibits EMT signaling through CDK8 inhibition.

Examples 8 . Compound reduction TGF - β 1 Induced oxidative stress

Figures 10A and 10B show that CDK8 inhibition reduces TGF-β1-induced oxidative stress.

Increasing evidence also indicates that TGF-β1 increases reactive oxygen species (ROS) production by impairing mitochondrial function through the Smad pathway that causes redox imbalance and inducing NADPH oxidase (NOX) (mainly NOX4). Redox imbalance also induces TGF-β1 expression and promotes fibrosis, forming a vicious cycle (RM Liu et al., 2015 Redox Biol. (6) 565-577). As shown in Figures 10A to 10B , TGF-β1 treatment significantly increased oxidative stress, characterized by increased malondialdehyde (MDA) and NOX4 levels in alveolar epithelial A549 cells, and compound k significantly reduced TGF-β1-induced oxidative stress. In contrast, the identified inhibitor compounds b and a produced weaker inhibition of MDA and Nox4 expression ( Figure 10A to Figure 10B ). Taken together, these results indicate that compound k is a potent CDK8 inhibitor and can potentially modulate signaling pathways associated with fibroblast response.

Examples 9 . Compounds k Obstruction TGF - β 1 Induce β - Catenin signaling

Figures 11A to 11L show that compound k blocks TGF-β1-induced β-catenin signaling.

In addition, the TGF-β/Akt/glycogen synthase kinase 3β (GSK3β)/β-catenin atypical pathway also plays a role in metastasis of various cancers, and recent studies have demonstrated that CDK8 kinase activity is required for β-catenin-driven transcription (R. Firestein et al., 2008 Nature 455 (7212) 547-551; A. Hamidi et al., 2017 Sci. Signal. 10 (486), eaal4186). Therefore, we investigated whether β -catenin signaling also plays a role in the treatment. Phosphorylation of Akt at Ser473, GSK3β at Ser9 (inactive form), and active form of β -catenin were significantly increased in DU145 prostate cancer cells ( Figure 11A to Figure 11B ). In immunoprecipitation analysis, the level of interaction between the active form of β -catenin and CDK8 increased after TGF- β treatment and was significantly inhibited by compound treatment ( Figure 11C to Figure 11D ), indicating that CDK8 also plays a role in the regulation of β -catenin signaling. Interestingly, CDK8 and β-catenin gene expression also showed a positive correlation in 498 prostate cancer patients in the TCGA database ( Figure 11E ). Since LiCl can significantly increase the amount of phosphorylation of GSK3β at Ser9 (inactive form) ( Figure 11F to Figure 11G ), we used LiCl to inactivate GSK3β and evaluate the changes in β-catenin signaling and downstream proteins in response to compound treatment. As shown, compared with control cells, DU145 cells treated with LiCl showed a significant increase in the active form of β -catenin and the content of Snail downstream proteins; CDK8 inhibition (using compound k and using cynicin A) reduced the content of these factors, and compound k showed better efficacy than cynicin A ( Figure 11H to Figure 11I ). In addition, significant p-Akt (Ser473)/p-GSK3β (Ser9) expression was observed under basal conditions in PC-3 cells ( Figure 11J ), indicating that activator treatment is not required in PC-3 cells to stimulate this signal. Compound k treatment significantly inhibited Snail expression in PC-3 cells ( Figure 11K to Figure 11L ). These results support that the compound inhibits β -catenin signaling and downstream proteins through CDK8 inhibition.

Examples 10 . In vivo anti-metastatic properties

FIG. 12A to FIG. 12G show that compound k exhibits anti-invasive effects in vivo.

The in vivo efficacy of compound k in the PC-3 SCID mouse model was further evaluated. The results showed that oral administration of the compound (50 mg/kg, qd) did not induce significant weight changes ( Figure 12A ). In addition, the invasion of the edges of the harvested tumors into the surrounding tissues was examined to identify metastasis. In the H&E staining results, the control group showed continuous tumor edges, indicating that tumor cells infiltrated the surrounding tissues ( Figure 12B ); however, in the compound k -treated group, a clear boundary was observed between tumor and normal cells ( Figure 12B ). It is noteworthy that the integrity of the tumor capsule was preserved in the compound-treated group, and in clinical studies, tumor capsule invasion is associated with poor prognosis and a higher risk of recurrence (A. Buhmeida et al., 2006 Diagn. Pathol. 1:4). According to IHC staining results, key EMT proteins vimentin, N-calcified mucin, and Snail were overexpressed in the control group, while the levels of these proteins were significantly reduced in the compound k- treated group ( Figure 12C ). In addition, immunoblots of tumor samples showed that compound treatment significantly inhibited the expression of EMT proteins (e.g., vimentin), myofibroblast markers α-SMA, p-Smad3 (Thr179), active forms of β-catenin, CDK8 active markers (e.g., p-STAT1 Ser727), and increased the expression of epithelial cell markers (e.g., E-calcification) ( Figure 12D to Figure 12E ). In addition, immunoprecipitation analysis was performed to investigate whether the compounds inhibited the cooperation of CDK8 with the mediator complex to regulate the transfer signal. As shown, a significant interaction between CDK8, MED12, and p-Smad3 (Thr179) was observed in the untreated group ( Figure 12F to Figure 12G ). Compound k treatment significantly inhibited the binding of CDK8 to p-Smad3 (Thr179), but did not affect the interaction between CDK8 and MED12. Overall, these results support that compound k inhibits prostate cancer metastasis via CDK8 inhibition.

In view of the above disclosure, the compounds described herein may have great potential in medicine and medical applications, especially in the treatment of CDK8-mediated diseases.

It should be understood that the foregoing examples are merely illustrative of the present invention. Certain modifications may be made to the objects and/or methods employed and still achieve the objectives of the present invention. Such modifications are encompassed within the scope of the claimed invention.

Figure 1 shows the 2D interaction poses of compound k . 2D interaction poses of compound k. Red dashed lines represent hydrogen bonds. Hydrophobic interactions are represented by green lines. 2D interactions were generated in LeadIT.

Figures 2A to 2D show the evaluation of CDK8 inhibition of compound k . Figure 2A shows the inhibitory efficiency of compound k against a panel of 54 kinases from different kinase families. Figure 2B shows DU145 and PC-3 cells incubated with or without different concentrations (0.1, 0.3, 1, 3, 10, 30 μM) of compound k for 24 and 72 hours. Cell viability was measured by MTT assay, and 50% inhibitory concentration (IC50) values were calculated by sigmoidal dose-response equation. Figures 2C and 2D show DU145 and PC-3 cells transfected with or without pcDNA3 CDK8-HA plasmid (1 μg) for 24 hours, and then treated with the indicated concentrations of compound k for another 24 hours. Western blots were performed on whole cell lysates with the indicated antibodies. The results are shown as the mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001 relative to the control group; # p < 0.05 relative to the relevant control).

Figures 3A to 3H show that CDK8 expression and TGF-β signaling are increased in metastatic prostate cancer and are associated with poor patient survival. Figure 3A shows a box plot comparing CDK8 mRNA expression in primary and metastatic prostate cancer samples from the GSE74685 dataset. Figure 3B shows a comparison of CDK8 mRNA transcript levels in lymph node-positive and lymph node-negative prostate cancer samples from the Cancer Genome Atlas (TCGA) database. Figure 3C shows a Kaplan-Meier curve that depicts the time until biochemical recurrence in radical prostatectomy samples with high and low CDK8 expression based on data from the GSE70769 dataset. Figure 3D shows gene set enrichment analysis (GSEA) of mediator complex components in metastatic and primary prostate cancer samples from the GSE68882 dataset. Figures 3E and 3F show GSEA of TGF-β/Smad signaling pathway genes in prostate cancer patients grouped according to the Gleason score cutoff value 7 (intermediate grade) using patient samples provided by Abida et al. and the GSE16560 dataset. Figure 3G shows the Karben-Meier curve, which shows the 10-year progression-free survival rate of prostate cancer patients with high and low TGFB1 expression based on the TCGA dataset. Figure 3H shows a box plot comparing TGFBR1 mRNA expression in primary and metastatic prostate cancer patients using samples from the GSE74685 dataset.

Figures 4A-4B show the inhibition of epithelial-mesenchymal transition protein expression in human alveolar epithelial cells by CDK8 inhibitors. Figure 4A shows A549 cells treated with test compound (2 μM), senexin A (5 μM) or pirfenidone (1 mM) for 1 hour and stimulated with TGF-β1 (10 ng/mL) for another 1 hour. Cell lysates were analyzed by Western blot using the indicated antibodies. Results are shown as mean ± SEM from three independent experiments. * p <0.05 and *** p <0.001 compared with the control group; # p <0.05, ## p <0.01, and ### p <0.001 compared with the TGF-β1-treated group. Figure 4B shows cells incubated with or without the indicated concentrations of compound k for 12, 24, or 48 hours. Cell viability was determined by MTT assay. Results are shown as the mean ± SEM of three independent experiments.

Figures 5A to 5B show that compound k inhibits the migration of A549 cells. A549 cells were cultured in a six-well dish until 90% confluency was reached, scraped with a pipette tip, and photographed immediately (0 hours). Cells were cultured for 1 hour with or without compound k (2 μM), cernicin A (5 μM), or pirfenidone (1 mM), then treated with TGF-β1 (10 ng/mL) and allowed to migrate to the wound area for 24 or 48 hours, and photographed. Cell migration to the wound was quantified using ImageJ software; the solid line indicates time zero. Quantitative assessment of the average number of cells in the clean band is expressed as the mean ± SEM from three independent experiments. ** p < 0.01 compared with the control group; ## p < 0.01 and ### p < 0.001 compared with the TGF-β1-treated group.

Figures 6A to 6D show that cell migration and EMT characteristics of prostate cancer cells are significantly inhibited by compound k . Figure 6A shows that DU145 cells were treated with or without the specified concentration of compound k for 24 hours in the presence of TGF-β1 (10 ng/mL). Images of cell morphology were taken at ×100 magnification. Figure 6B shows that DU145 cells were cultured in a 6-well dish until 90% confluence was reached, scraped with a pipette tip, and immediately photographed (0 hours). Cells were cultured with or without TGF-β1 (10 ng/mL) and compound k (2.5 and 10 μM), allowed to migrate to the wound area for 48 hours and photographed. Cell migration into the wound was quantified using ImageJ software; the solid white line indicates time zero. Quantitative assessment of the average number of cells in the scraped area is presented as the mean ± SEM from at least three independent experiments (*** p < 0.001 vs. control group; ## p < 0.01 and ### p < 0.001 vs. TGF-β1-treated group). (C) PC-3 cells were incubated with 10 μM compound k/synesin A for 1 hour, followed by treatment with TGF-β1 (10 ng/mL) for 24 hours and incubated with the indicated antibodies and DAPI. Immunofluorescence images were captured by a ZEISS ApoTome.2 microscope. Images were captured at 600× magnification.

Figures 7A to 7F show that compound k significantly inhibited TGF-β1-induced EMT protein and gene expression. In the presence or absence of TGF-β1 (10 ng/mL), cells were exposed to the specified concentrations of compound k or senexin A (10 μM) for 24 hours, and the protein and mRNA levels of E-calcification, N-calcification, Snail, vimentin, and α-SMA were determined by Western blot (A) or real-time PCR (B). The results are shown as the mean ± SEM from at least three independent experiments (relative to the control group, ** p <0.01, *** p <0.001; relative to the TGF-β1-treated group, # p <0.05, ## p <0.01, and ### p <0.001).

Figures 8A-8B show the inhibitory effect of compound k treatment on the cellular distribution of TGF-β1-induced p-Smad3 T179, RNA polymerase II pS2/S5, and Smad3. Human alveolar epithelial A549 cells were incubated with compound k (5 μM), ceranicin A (5 μM), or pirfenidone (1 mM) for 1 hour and then stimulated with TGF-β1 (10 ng/mL) for 3 hours. Western blot analysis of protein expression in cytoplasmic and nuclear fractions was performed using the indicated antibodies. α-Tubulin and histone H3 served as loading controls for the cytoplasmic and nuclear fractions, respectively. Results are shown as the mean ± SEM of three independent experiments. ** p < 0.01 and *** p < 0.001 compared with the control group; # p < 0.05 and ## p < 0.01 compared with the TGF-β1-treated group.

Figures 9A to 9G show that compound k significantly inhibits TGF-β/Smad/RNA polymerase II signaling. Figures 9A to 9B show that human prostate cancer DU145 cells were transfected with pcDNA3 CDK8-HA plasmid (1 μg) for 24 hours, treated with compound k (10 μM) for 1 hour and then incubated with TGF-β1 (10 ng/mL) for another 24 hours. Western blotting was performed on whole cell lysates with the indicated antibodies. Figures 9C to 9D show that DU145 cells were treated with TGF-β1 (10 ng/mL) for 1 hour in the presence or absence of compound k (10 μM) and then subjected to nuclear-cytosolic fractionation. Proteins in the cytosol and nucleus were detected by Western blot analysis, and protein expression in the nucleus was quantified. Figures 9E to 9F show that DU145 cells were incubated with compound k (10 μM) in the presence or absence of TGF-β1 (10 ng/mL) for 1 hour, and total cell lysates were immunoprecipitated with antibodies and subjected to immunoblotting. Figure 9G shows that DU145 cells were transfected with 7TFP CDH1 reporter plasmids (1 μg) for 24 hours, followed by subsequent treatment with TGF-β1 (10 ng/mL) with or without compound k and senisin A (10 μM) for 24 hours, and luciferase expression was subsequently determined. The results are shown as the mean ± SEM of at least three independent experiments (vs. control group, * p < 0.05, ** p < 0.01, and *** p <0.001; vs. TGF-β1-treated group, # p < 0.05, ## p < 0.01, and ### p <0.001; vs. indicated groups, § p < 0.05, §§ p < 0.01).

Figures 10A to 10B show that CDK8 inhibition reduces TGF-β1-induced oxidative stress. A549 cells were incubated with compounds k , b , a (2 μM), ceranicin A (5 μM), or pirfenidone (1 mM) for 1 hour and then treated with 10 ng/mL TGF-β1 for another 24 hours. Malondialdehyde (MDA) ( Figure 10A ) and Nox4 ( Figure 10B ) levels were detected. *** p <0.001 compared with the control group; #p <0.05, ## p <0.01, and ### p <0.001 compared with the TGF-β1-treated group.

Figures 11A to 11L show that compound k blocks TGF-β1-induced β-catenin signaling. Figures 11A to 11B show that DU145 cells were cultured with TGF-β1 (10 ng/mL) for 24 hours, and whole cell lysates were collected and used for protein concentration assessment, as indicated. Figures 11C to 11D show that cells were cultured with compound k (10 μM) for 12 hours, and with or without TGF-β1 (10 ng/mL) for another 2 hours, and nuclear lysates were immunoprecipitated with antibodies and subjected to immunoblotting. Figure 11E shows a comparison of gene expression between CDK8 and β-catenin in prostate cancer patients analyzed by the TCGA database (n=498). Figures 11F to 11G show DU145 cells incubated with 20 mM LiCl for 6 hours. Inhibitory phosphorylation of GSK3β was assessed by Western blotting. Figures 11H to 11I show DU145 cells treated with indicated concentrations of compound k and senisin A (10 μM) for 20 hours and then incubated with or without LiCl (20 mM) for another 6 hours. Western blotting was performed on whole cell lysates with indicated antibodies. Figures 11J to 11K show a comparison of basal activity of the Akt/GSK3β axis between PC-3 and DU145 cells. Figure 11L shows concentration-dependent inhibition of β-catenin target protein in PC-3 cells by compound k after 24 hours of treatment with compound k . The results are shown as the mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 relative to the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 relative to the relevant control).

Figures 12A to 12G show that compound k exhibits anti-invasive effects in vivo. PC-3 cells (1×107 cells) were injected subcutaneously into the flank of 7-week-old male SCID mice and treated with vehicle or compound k (50 mg/kg) qd for 31 days. Figure 12A shows that weight changes were recorded during compound treatment (n=7, mean ± SEM). After 31 days of treatment, tumors were excised from each mouse. Figures 12B to 12C show paraffin sections of tumors stained with hematoxylin and eosin and the indicated antibodies. Length is represented by the indicated scale bar. Figures 12D to 12G show immunoblots and immunoprecipitation analysis of tumor samples analyzed with the indicated antibodies.

Claims (20)

A compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof,

wherein: R ¹ is hydrogen, C _1-6 alkyl, C _1-6 halogenalkyl, C _1-6 alkoxy or C _1-6 halogenalkoxy; R ^2a and R ^2b are each independently C _1-6 alkyl, C _2-6 alkenyl or C _2-6 alkynyl, which is optionally substituted with a halogen; R ³ , R ⁵ and R ⁶ are each independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, halogen, cyano, nitro, OR _a , SR _a , S(O)R _a , SO ₂ R _a , SO ₂ NR _b R _c , OC(O)R _a , C(O)NR _b R _c , NR _b R c , NHC(O)R _a or NHC(S)R _b , wherein R _a , R _b and R _c are each independently H, hydroxyl, C 2-6 wherein: ( ₁ ) Ring ^{A is phenyl which is optionally substituted by one or more substituents selected from the group consisting of halogen and C 1-6 alkyl; L} _{is -O-, -NH- , -C} (=O)- or -C(=O ₎ -NR a -, wherein when L is -C(=O)-NR ^a -, Ring ^A is connected to the N atom of L; and Ring B is phenyl or pyridine which is optionally substituted by one _{or more} substituents selected from the group consisting of nitro, halogen, C _1-6 alkyl and C 1-6 alkyl; (2) Ring A is phenyl, oxadiazole or thiadiazole, which is substituted by one or more substituents selected from the group consisting of phenyl or oxadiazole, and the substituents phenyl and oxadiazole are further substituted by C _1-3 alkyl as appropriate; and -LB is absent; (3) Ring _A is thiazolylbenzophenone or benzopiperidinone

, which is optionally substituted by one or more substituents selected from the group consisting of pendooxy, halogen and C _1-6 alkyl; and -LB is absent; or (4) Ring A is phenyl, which is optionally substituted by one or more substituents selected from the group consisting of halogen and C _1-6 alkyl; -L- is -NH-; and Ring B is pyridine, which is optionally substituted by one or more substituents selected from the group consisting of halogen and C _1-6 alkyl. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein R ¹ is hydrogen, and R ^2a and R ^2b are each independently methyl. The compound of claim 1 or 2 or its pharmaceutically acceptable salt, stereoisomer or hydrate, wherein ring A is substituted with a halogen. The compound of claim 1 or 2 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein ring A is a phenyl group substituted by a halogen; -L- is -O-; and ring B is a pyridine group substituted by a C _1-3 alkylaminocarbonyl group. A compound having formula (II) or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein formula (II) is:

Where R is selected from the group consisting of:

A compound or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof: N-(4-(4-nitrophenoxy)phenyl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-oxo-N-(4-(anilino)phenyl)-3-(propan-2-ylidene)indoline-5-sulfonamide, N-(4-((2-oxo-3-(propan-2-ylidene)indoline)-5-sulfonamido)phenyl)benzamide, N-(4-benzoylphenyl) -2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-oxo-N-(5-phenyl-1H-pyrazol-3-yl)-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-oxo-3-(propan-2-ylidene)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)indoline-5-sulfonamide, 2-oxo-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-3-(propan-2-ylidene)indoline-5-sulfonamide, N-(Benzo[d]thiazol-2-yl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, N-(4-(1H-imidazol-1-yl)phenyl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide, 2-(N-methylformamide)-4-(4-((2-oxo-3-(propan-2-ylidene)indoline)-5-sulfonamido)phenoxy)-pyridine, 2-(N-methylformamide)-4-(3-fluoro-4-((2 -((2-oxo-3-(propan-2-ylidene)indolin)-5-sulfonamido)phenoxy)-pyridine, 2-fluoro-N-methyl-4-((2-oxo-3-(propan-2-ylidene)indolin)-5-sulfonamido)benzamide, N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-2-oxo-3-(propan-2-ylidene)indolin-5-sulfonamide, and N-(1,4-dioxo-1,2,3,4-tetrahydronaphthalene

-5-yl)-2-oxo-3-(propan-2-ylidene)indoline-5-sulfonamide. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 7 further comprises another therapeutic agent. A pharmaceutical composition as claimed in claim 8, wherein the additional therapeutic agent is selected from the group consisting of: antifibrotic agents; hormones or hormone antagonists; kinase inhibitors; targeted signal transduction agents; biological response regulators; anticancer agents; and any combination thereof. A pharmaceutical composition as claimed in claim 8, wherein the additional therapeutic agent is selected from the group consisting of: alkylating agents; antibiotics; anti-metabolites; anti-angiogenic agents; and any combination thereof. A pharmaceutical composition as claimed in claim 8, wherein the additional therapeutic agent is selected from the group consisting of: antibody therapeutic agents; chemotherapeutic agents; and any combination thereof. The pharmaceutical composition of claim 8, wherein the additional therapeutic agent is selected from the group consisting of: taxanes; retinoids; alkaloids; topoisomerase inhibitors; Hsp90 inhibitors; farnesyl transferase inhibitors; aromatase inhibitors; indoleamine 2,3-dioxygenase (IDO) inhibitors; histone acetyl transferase (HAT) inhibitors; histone deacetylase (HDAC) inhibitors; sirtuin (SIRT) inhibitors; bromodomain and extra-terminal motif (BET) inhibitors; and any combination thereof. A use of a compound as claimed in any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof for the manufacture of a drug for inhibiting CDK8 in an individual. A use of a compound as claimed in any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof for the manufacture of a medicament for preventing or treating inflammatory conditions or fibrotic diseases. The use of claim 14, wherein the inflammatory condition or fibrotic disease is selected from the group consisting of: type 1 diabetes, graft-versus-host disease, inflammatory bowel disease, psoriasis, psoriasis arthritis, Hashimoto's thyroiditis, food allergies, HCV vasculitis, alopecia areata, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, skin fibrosis, lung fibrosis, kidney fibrosis, liver fibrosis, intestinal fibrosis, cystic fibrosis, myocardial fibrosis, uterine leiomyoma, adenomyosis, and any combination thereof. For use as claimed in claim 15, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis. A use of a compound as claimed in any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof for the manufacture of a medicament for preventing or treating a tumor or a cell proliferative disease. The use of claim 17, wherein the tumor or cell proliferation disease is selected from the group consisting of brain cancer, lung cancer, epidermoid carcinoma, squamous cell carcinoma, bladder cancer, gastric cancer, pancreatic cancer, breast cancer, head cancer, cervical cancer, renal cancer, liver cancer, ovarian cancer, prostate cancer, colorectal cancer, uterine cancer, esophageal cancer, testicular cancer, thyroid cancer, melanoma, multiple myeloma, myeloid leukemia, neuroglioma, Kaposi's sarcoma, and any combination thereof. The use of claim 17, wherein the tumor or cell proliferative disease is selected from the group consisting of: colon cancer, rectal cancer, uveal melanoma, acute myelogenous leukemia (AML), chronic myelogenous leukemia, human laryngeal squamous cell carcinoma, and any combination thereof. The use of claim 18, wherein the tumor or cell proliferative disease is prostate cancer.