CN116199682A - Phenyl thiazolyl benzamide compound and application thereof - Google Patents

Abstract

The invention provides a phenylthiazolyl benzamide compound and application thereof, wherein the phenylthiazolyl benzamide compound has a structure shown in the following formula I: wherein R is ₁ Selected from the group consisting of

R _a 、R _b Independently selected from-Me, -CD ₃ 、

Or (b)

Any one of them; the R is _f Selected from the group consisting of

The R is _g Selected from hydrogen, -Me, -n-Pr, -n-Bu,

Or (b)

Any one of them; r is R ₂ Selected from hydrogen or halogen; r is R ₃ Any one selected from hydrogen and substituted or unsubstituted C6-C12 aryl;

Description

Phenyl thiazolyl benzamide compound and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a phenylthiazolyl benzamide compound and application thereof, in particular to a phenylthiazolyl benzamide compound with high activity and application thereof.

Background

Nuclear receptors (nuclear receptors) are a family of ligand-dependent transcription factors homologous to steroid hormone receptors that are involved in regulating the growth and cellular differentiation of the body, thereby affecting a variety of physiological and metabolic processes in the body. Chicken ovalbumin upstream promoter transcription factor 2 (chicken ovalbumin upstream promoter transcription factor 2, coup-TFII) is one of the important members of the nuclear receptor family. The COUP-TF family includes two highly homologous subtypes, COUP-TFI and COUP-TFII, located on chromosomes 5 and 15, respectively, also referred to as nuclear receptor 2 families 1 and 2 (NR 2F1 and NR2F 2). COUP-TF is currently considered to be an orphan nuclear receptor because no ligand has been identified for its specific binding.

Current studies indicate that COUP-TFI plays an important role in neural development and COUP-TFII plays an important role in organ development, respectively. Further research shows that COUP-TFII plays an important role in various aspects of the physiological growth and development process of the organism, can regulate various signal paths, and participates in controlling tumor growth, angiogenesis, regeneration of organism tissues or cells and the like.

It is worth mentioning that the expression of COUP-TFII is abnormally up-regulated in the states of embryo development stage, tissue regeneration, diseases and the like; whereas in normal tissue or body maturation stage, COUP-TFII expression is maintained at a lower level. The current research result also shows that the COUP-TFII gene is knocked out from adult mice, and the mice do not show abnormal physiological phenomena such as phenotype change and the like. This demonstrates to some extent that inhibition of COUP-TFII does not produce toxic side effects. Therefore, small molecule drugs which target binding and inhibit COUP-TFII activity are developed, i.e. have practical and potential feasibility.

COUP-TFII is a necessary condition for angiogenesis during tumor growth, and its abnormal expression can lead to the occurrence and metastasis of cancer. Therefore, COUP-TFII is an ideal completely new target for potential cancer treatment.

NRF2 (nuclear factor E rythroid 2related factor, nuclear factor E2 related factor) was cloned in 1994 from Moi et al as a factor binding to the nuclear factor E2 repeat of the beta globin gene promoter. Belongs to the CNC family of transcription factors, and contains a leucine zipper structure.

The NRF2/Keap1 (Kelch-like ECH-associated protein 1, kelch-like epichlorohydrin-related protein 1) -ARE (antioxidant response element ) signaling pathway is an endogenous antioxidant signaling pathway of the organism discovered in recent years.

Activation of the NRF2/Keap 1-ARE pathway is one of the important mechanisms for anti-tumorigenesis. Activation of NRF2 enables cancer cells to adapt to adverse microenvironments and high endogenous ROS levels during early stages of tumorigenesis; whereas in rapidly proliferating cells, NRF2 can support intermediary metabolism by promoting nucleotide and amino acid biosynthesis. Studies have shown that low activity NRF2 promotes tumorigenesis, while sustained high activity NRF2 can accelerate cancer progression and tolerance to treatment.

Therefore, the development of the compound which can inhibit COUP-TFII and NRF2 has very important practical value and wide application prospect in tumor treatment.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a phenylthiazolyl benzamide compound and application thereof, in particular to a phenylthiazolyl benzamide compound with high activity and application thereof. The phenylthiazolyl benzamide compound provided by the invention can effectively inhibit the activities of COUP-TFII and NRF2, and has an excellent inhibition effect on the proliferation of prostate cancer cells.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a phenylthiazolyl benzamide compound having a structure according to formula I:

wherein R is ₁ Selected from the group consisting of

R _a 、R _b Independently selected from-Me, -CD ₃ 、

Any one of the following.

The R is _f Selected from the group consisting of

The R is _g Selected from hydrogen, -Me, -n-Pr, -n-Bu,

Any one of the following.

R ₂ Selected from hydrogen or halogen.

R ₃ Selected from any one of hydrogen and substituted or unsubstituted C6-C12 aryl.

Representing the attachment site of the group.

The compound with the specific structure can effectively inhibit the activity of COUP-TFII and NRF2, and has excellent inhibition effect on the proliferation of prostate cancer cells.

Preferably, the substituents of the C6-C12 aryl are selected from the group consisting of C1-C10 straight-chain or branched alkyl, C1-C10 haloalkyl or-COOR _c Any one of them; the R is _c Selected from hydrogen or C1-C10 straight chain alkyl.

Preferably, said R ₂ Selected from hydrogen or-F.

Preferably, said R ₃ Selected from hydrogen or

The R is _i Selected from-CF ₃ -COOH or-COOEt.

The R is _j Selected from-F or-Cl;

representing the attachment site of the group. />

Preferably, the phenylthiazolyl benzamide compound is selected from any one of the following formulas 1 to 20:

the compounds of formula I can be prepared by the following methods:

starting from para-aminoacetylbenzene, the compound III is obtained by substitution, then the compound III is further substituted and the compound III is combined with a ring to obtain an intermediate M (R) ₁ Selected from the group consisting of

From intermediate M (R ₁ Having the same defined ranges as above), to obtain compounds 1-2 by substitution, and to obtain compounds represented by formula I by substitution of bromine atoms.

In a second aspect, the present invention provides stereoisomers of the phenylthiazolylbenzamide compounds described above, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable pharmaceutical excipients.

In a third aspect, the present invention also provides the use of a phenylthiazolylbenzamide compound as described above or a stereoisomer of a phenylthiazolylbenzamide compound as described above, a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment and/or prophylaxis of a disease associated with excessive activity of COUP-TFII or overexpression of COUP-TFII.

Preferably, the disease associated with excessive COUP-TFII activity or COUP-TFII overexpression comprises prostate cancer.

In a fourth aspect, the present invention also provides the use of a phenylthiazolylbenzamide compound as described above or a stereoisomer of a phenylthiazolylbenzamide compound as described above, a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment and/or prevention of a disease associated with NRF2 (nuclear factor erythroid 2-related factor 2) being too active or with NRF2 being overexpressed.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a phenylthiazolyl benzamide compound with a specific structure, which can effectively inhibit the activities of COUP-TFII and NRF2, has excellent inhibition effect on the proliferation of prostate cancer cells, and is expected to become a novel tumor prevention and treatment drug.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The term "pharmaceutically acceptable salt" means that the compound can be converted by conventional means into the corresponding salt, which is chemically or physically compatible with the other ingredients comprising the pharmaceutical dosage form, and physiologically compatible with the recipient. The salts may be acid and/or base salts of the compounds with inorganic and/or organic acids and/or with inorganic and/or organic bases, and also include zwitterionic salts (inner salts) and also include quaternary ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. Or by appropriately mixing the compound of the present invention or a stereoisomer or solvate thereof with a certain amount of an acid or a base. These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by reaction in an aqueous medium and then cooled and dried. In particular, the salt is preferably a water-soluble pharmaceutically acceptable non-toxic acid addition salt, examples being salts of amino groups 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 using other methods conventional in the art (e.g. ion exchange methods).

The term "alkyl" refers to a radical containing primary (normal) carbon atomsSaturated hydrocarbons of secondary or tertiary or quaternary carbon atoms, or combinations thereof. The phrase containing the term, for example, "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, which may be, for each occurrence, independently of one another, C1 alkyl, C2 alkyl, C3 alkyl. Suitable examples include, but are not limited to: methyl (Me, -CH) ₃ ) Ethyl (Et, -CH) ₂ CH ₃ ) 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ) 2-propyl (i-Pr, i-propyl, -CH (CH) ₃ ) ₂ )。

The term "heterocycloalkyl" refers to a non-aromatic cyclic group in which one or more of the atoms making up the ring is a heteroatom and the remainder is carbon, including but not limited to nitrogen, oxygen, sulfur, and the like. Preferred heterocycloalkyl groups are 3-10 membered saturated heterocycloalkyl groups. Unless specifically indicated otherwise in this specification, heterocycloalkyl groups may be monocyclic ("monocyclic heterocycloalkyl"), or bicyclic, tricyclic or more ring systems which may include a fused, bridged or spiro ring system (e.g., a bicyclic system ("bicyclic heterocycloalkyl"). Heterocycloalkyl bicyclic ring system may include one or more heteroatoms in one or both rings, and saturated.

The term "alkoxy" refers to a group having an-O-alkyl group, i.e. an alkyl group as defined above, attached to the parent core structure via an oxygen atom. The phrase containing the term, for example, "C1-C3 alkoxy" means that the alkyl moiety contains from 1 to 3 carbon atoms.

The term "aryl" refers to an aromatic hydrocarbon radical derived from the removal of one hydrogen atom on the basis of an aromatic ring compound, which may be a monocyclic aryl radical, or a fused ring aryl radical, or a polycyclic aryl radical, at least one of which is an aromatic ring system for a polycyclic species. Preferred aryl groups are 6-10 membered aryl groups, which may be selected from phenyl and naphthyl, as examples.

The terms "halo", "halogen" refer to halogen atoms or are substituted with halogen atoms including fluorine, chlorine, bromine or iodine atoms.

In the various parts of the invention, linking substituents are described. When the structure clearly requires a linking group, the markush variables recited for that group are understood to be linking groups. For example, if the structure requires a linking group and the markush group definition for that variable enumerates an "alkyl" or "aryl" group, it will be understood that the "alkyl" or "aryl" represents a linked alkylene group or arylene group, respectively. In some specific structures, when an alkyl group is explicitly represented as a linking group, then the alkyl group represents a linked alkylene group, e.g., the alkyl in the group "-C1-C3 haloalkyl" is to be understood as alkylene.

Furthermore, the term "comprising" is an open-ended limitation and does not exclude other aspects, i.e. it includes the content indicated by the invention.

Unless otherwise indicated, the present invention employs conventional methods of mass spectrometry, nuclear magnetism, and the like to identify compounds, and the procedures and conditions may be referred to procedures and conditions conventional in the art.

Those skilled in the art will appreciate that, in accordance with convention used in the art, the present application describes the structural formula of a group as used in

Meaning that the corresponding group is linked to other fragments, groups in the compound through this site. The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

Intermediate Ma:

the synthetic route is as follows:

compound Ma-1 (300 mg,2.22 mmol), triethylamine (449 mg,4.44 mmol) were added to dichloromethane (10 mL), compound Ma-2 (694 mg,2.44 mmol) was slowly added under ice-bath conditions, then stirred at 20℃for 2 hours, after completion of the LCMS and TLC detection reactions, water (100 mL) was added to quench the reaction, after extraction with dichloromethane, the organic phase was concentrated by drying and purified by column chromatography to give compound Ma-3 (530 mg, yield 62.3%) as a yellow solid.

Compound Ma-3 (530 mg,1.38 mmol), potassium carbonate (284 mg,2.08 mmol) was added to N, N-dimethylformamide (10 mL), and compound Ma-4 (241 mg,1.66 mmol) was slowly added under ice-bath, then stirred at 20℃for 2 hours, after completion of LCMS and TLC detection, quenched with water (100 mL), extracted with ethyl acetate, and the organic phase was concentrated by drying and purified by column chromatography to give compound Ma-5 (520 mg, yield 94.2%) as a yellow solid. LCMS [ M+H ]] ⁺ ＝400.1。

Compound Ma-5 (520 mg,1.30 mmol), compound Ma-6 (198 mg,2.61 mmol) and elemental iodine (331 mg,1.30 mmol) were added to ethanol (10 mL), stirred overnight at 90℃and after completion of the LCMS and TLC detection reaction, 1M sodium hydroxide solution was added to adjust the pH of the solution to between 12 and 13, and after extraction with ethyl acetate, the organic phase was dried and concentrated and purified by column chromatography to give Compound Ma-7 (300 mg, yield 64.9%) as a gray solid. LCMS [ M+H ]] ⁺ ＝356.1。

Compound Ma-7 (300 mg,0.85 mmol), potassium carbonate (233 mg,1.69 mmol) were added to N, N-dimethylformamide (10 mL), di-tert-butyl dicarbonate (221 mg,1.0 mmol) was slowly added under ice-bath, then stirred at 20℃for 2 hours, after completion of the LCMS and TLC detection reaction, water (50 mL) was added, the organic phase was concentrated by drying with ethyl acetate, and then purified by column chromatography to give compound Ma as a yellow solid(270 mg, yield 70.1%). LCMS [ M+H ]] ⁺ ＝456.1。

Example 1:

the synthetic route is as follows:

referring to the synthesis of intermediate Ma, the compound Ma-2 was replaced with an equivalent amount of N-t-butoxycarbonyl-4-acetylchloropiperidine to afford intermediate Mb.

Compound Mb (7.73 mmol), pyridine (6.1 g,77.3 mmol) and 4-dimethylaminopyridine (189 mg,1.55 mmol) were added to dichloromethane (40 mL), a solution of parabromobenzoyl chloride 1-1 (2.0 g,9.28 mol) in dichloromethane (10 mL) was slowly added under ice-bath conditions, then stirred overnight at 20℃and after completion of the LCMS and TLC detection reactions, concentrated and purified by column chromatography to give solid compound 1-2.

Compounds 1-2 (5.3 mmol), compounds 1-3 (6.4 mmol), [1,1' -bis (diphenylphosphine) ferrocene]Palladium dichloride dichloromethane complex (0.53 mmol) and sodium carbonate (10.6 mmol) were added to a mixed solution of 1, 4-dioxane (40 mL) and water (10 mL), stirred overnight at 100℃and after completion of LCMS and TLC detection, compound 1-4 was obtained by concentration and purification by column chromatography followed by deprotection to give compound 1 (17.9 mg, yield 29.2%) as a yellow solid. LCMS [ M+H] ⁺ ＝568.1。

¹ H NMR(400MHz,DMSO-d6)δ12.88(s,1H),9.01(s,1H),8.80(s,1H),8.25(d,J＝8.4Hz,2H),8.04-7.99(m,4H),7.93(d,J＝8.0Hz,2H),7.85(d,J＝8.0Hz,2H),7.80(s,1H),7.41(d,J＝7.2Hz,2H),3.17-3.11(m,1H),2.69-2.53(m,4H),1.86-1.68(m,4H)。

Example 2:

the synthetic route is as follows:

referring to example 1, compound 1-3 was replaced with an equivalent amount of compound 2-2, and compound 2 (67.8 mg, yield 72.4%) was synthesized as a yellow solid. LCMS [ M+H] ⁺ ＝572.1。

¹ H NMR(400MHz,DMSO-d6)δ12.90(s,1H),8.79(s,1H),8.56(s,1H),8.26(d,J＝8.4Hz,2H),8.08-8.04(m,4H),7.94(d,J＝8.0Hz,4H),7.81(s,1H),7.43(d,J＝6.8Hz,2H),4.34(q,J＝7.2Hz,2H),3.36(s,1H),3.15(d,J＝8.8Hz,2H),2.70-2.64(m,2H),1.81-1.75(m,4H),1.34(t,J＝7.2Hz,3H)。

Example 3:

the synthetic route is as follows:

referring to example 1, compound Mb was replaced with an equivalent of Mf (i.e., ma) and compound 1-3 was replaced with an equivalent of 3-3, to synthesize yellow solid compound 3 (140.5 mg, yield 86.1%). LCMS [ M+H] ⁺ ＝608.1。

¹ H NMR(400MHz,DMSO-d6)δ8.24(d,J＝8.0Hz,2H),8.06(d,J＝8.4Hz,2H),7.96-7.91(m,6H),7.86(d,J＝8.0Hz,2H),7.71(s,1H),7.50(d,J＝8.8Hz,2H),4.33(q,J＝7.2Hz,2H),3.07(d,J＝4.4Hz,3H),2.60-2.54(m,2H),1.93(d,J＝12.0Hz,2H),1.63-1.54(m,2H),1.33(t,J＝7.2Hz,3H)。

Example 4:

the synthetic route is as follows:

compound 3 (50 mg,0.08 mmol) was added to a mixture of tetrahydrofuran (1 mL) and ethanol (1 mL), then an aqueous solution of sodium hydroxide (3.6 mg,0.09 mmol) was added (1 mL), stirred overnight at 45℃and after completion of the reaction, LCMS and TLC detection, concentrated and purified by column chromatography to give Compound 4 (12.8 mg, yield 27%) as a yellow solid, LCMS: [ M+H] ⁺ ＝580.1。

¹ H NMR(400MHz,DMSO-d6)δ8.13(d,J＝8.0Hz,2H),7.94(d,J＝8.4Hz,2H),7.86-7.81(m,6H),7.73(d,J＝8.0Hz,2H),7.59(s,1H),7.37(d,J＝8.8Hz,2H),3.02(d,J＝4.4Hz,3H),2.49-2.43(m,2H),1.86(d,J＝12.0Hz,2H),1.54-1.45(m,2H)。

Example 5:

the synthetic route is as follows:

referring to example 1, compound 1-2 was replaced with an equivalent amount of 3-2, and thus, compound 5 (2.21 g, yield 46.8%) was synthesized as a yellow solid. LCMS [ M+H] ⁺ ＝604.1。

¹ H NMR(400MHz,DMSO-d6)δ8.25(d,J＝8.0Hz,2H),8.00(d,J＝8.0Hz,2H),7.94(t,J＝9.2Hz,4H),7.86(d,J＝8.0Hz,2H),7.72(s,1H),7.50(d,J＝8.4Hz,2H),3.05(d,J＝12.8Hz,2H),2.59-2.52(m,2H),2.43-2.38(m,1H),1.94-1.88(m,2H),1.61-1.52(m,2H)。

Example 6:

the synthetic route is as follows:

compound 5 (100 mg,0.166 mmol) and potassium carbonate (30 mg,0.22 mmol) were added to N, N-dimethylformamide (5 mL), then methyl iodide (25.6 mg,0.18 mmol) was added, stirred overnight at 20℃after completion of the LCMS and TLC detection reaction, water (20 mL) was added, the organic phase was dried after extraction with ethyl acetate and concentrated, and then purified by column chromatography to give compound 6 (80.8 mg, yield 78.9%) as a white solid. LCMS [ M+H ]] ⁺ ＝618.1。

¹ H NMR(400MHz,DMSO-d6)δ12.88(s,1H),8.25(d,J＝8.4Hz,2H),8.01-7.92(m,6H),7.86(d,J＝8.4Hz,2H),7.73(s,1H),7.51(d,J＝8.8Hz,2H),2.65-2.62(m,1H),2.41-2.36(m,2H),2.33-2.29(m,3H),2.02-1.99(m,3H),1.75-1.67(m,3H)。

Example 7:

the synthetic route is as follows:

referring to example 6, the yellow solid compound 7 (84.2 mg, yield 78.6%) was synthesized by substituting an equivalent amount of ethyl iodide for methyl iodide. LCMS [ M+H] ⁺ ＝646.1。 ¹ H NMR(400MHz,DMSO-d6)δ12.88(s,1H),8.27(d,J＝7.6Hz,2H),8.04–7.84(m,8H),7.73(s,1H),7.51(d,J＝8.0Hz,2H),3.29–3.21(m,1H),2.95–2.85(m,2H),2.28–2.13(m,2H),2.01–1.84(m,4H),1.70–1.56(m,2H),1.47–1.34(m,2H),0.82(t,J＝7.2Hz,3H)。

Example 8:

the synthetic route is as follows:

referring to example 6, the yellow solid compound 8 (56.2 mg, yield 62.2%) was synthesized by substituting methyl iodide with an equivalent amount of 1-bromopropane. LCMS [ M+H] ⁺ ＝660.1。

¹ H NMR(400MHz,DMSO-d6)δ12.88(s,1H),8.26(d,J＝7.6Hz,2H),8.00(d,J＝7.6Hz,2H),7.94(t,J＝7.6Hz,4H),7.86(d,J＝8.0Hz,2H),7.72(s,1H),7.50(d,J＝7.6Hz,2H),3.23-3.19(m,1H),2.88(d,J＝10.8Hz,2H),2.21(t,J＝7.2Hz,2H),1.93-1.84(m,4H),1.66 -1.57(m,2H),1.39 -1.32(m,2H),1.28 -1.19(m,2H),0.84(t,J＝7.2Hz,3H)。

Example 9:

the synthetic route is as follows:

referring to example 6, the yellow solid compound 9 (82.3 mg, yield 50.8%) was synthesized by substituting methyl iodide with an equivalent amount of 1-fluoro-2-ethyl iodide. LCMS [ M+H] ⁺ ＝647.1。

¹ H NMR(400MHz,DMSO-d6)δ12.89(s,1H),8.26(d,J＝8.4Hz,2H),8.00(d,J＝8.0Hz,2H),7.94(t,J＝8.8Hz,4H),7.86(d,J＝8.0Hz,2H),7.73(s,1H),7.51(d,J＝8.0Hz,2H),4.54(t,J＝4.8Hz,1H),4.42(t,J＝4.8Hz,1H),3.32-3.26(m,4H),2.93(d,J＝11.6Hz,2H),2.62(t,J＝4.8Hz,1H),2.55(t,J＝4.8Hz,1H),2.04(t,J＝12.0Hz,2H),1.93(d,J＝12.0Hz,2H),1.67-1.59(m,2H)。

Example 10:

the synthetic route is as follows:

referring to example 6, the substitution of methyl iodide for an equivalent amount of 1-fluoro-3-iodopropane afforded compound 10 (45.3 mg, 53.2% yield) as a yellow solid. LCMS [ M+H] ⁺ ＝664.1。

¹ H NMR(400MHz,DMSO-d6)δ12.88(s,1H),8.26(d,J＝8.0Hz,2H),8.00(d,J＝8.0Hz,2H),7.95(t,J＝8.4Hz,4H),7.86(d,J＝8.4Hz,2H),7.72(s,1H),7.51(d,J＝8.0Hz,2H),4.50(t,J＝6.0Hz,1H),4.38(t,J＝6.0Hz,1H),3.28–3.21(m,1H),2.90(d,J＝11.2Hz,2H),2.33(t,J＝6.8Hz,2H),1.99–1.85(m,4H),1.82–1.75(m,1H),1.75–1.70(m,1H),1.68–1.55(m,2H)。

Example 11:

the synthetic route is as follows:

referring to example 6, the yellow solid compound 11 (13.9 mg, yield 16.1%) was synthesized by substituting methyl iodide with an equivalent amount of acetyl chloride. LCMS [ M+H] ⁺ ＝646.1。

¹ H NMR(400MHz,DMSO-d6)δ12.89(s,1H),8.27(d,J＝8.0Hz,2H),8.02(d,J＝8.0Hz,2H),7.96(t,J＝9.2Hz,4H),7.87(d,J＝8.4Hz,2H),7.75(s,1H),7.53(d,J＝8.0Hz,2H),4.43(d,J＝13.2Hz,1H),3.87(d,J＝12.8Hz,1H),3.62(t,J＝11.2Hz,1H),3.07(t,J＝12.4Hz,1H),2.63–2.55(m,1H),2.06–1.93(m,6H),1.71–1.51(m,1H),1.51–1.36(m,1H)。

Example 12:

the synthetic route is as follows:

referring to example 6, the yellow solid compound 12 (22.7 mg, yield 19.8%) was synthesized by substituting an equivalent amount of acryloyl chloride with methyl iodide. LCMS [ M+H] ⁺ ＝658.1。

¹ H NMR(400MHz,DMSO-d6)δ12.89(s,1H),8.27(d,J＝8.4Hz,2H),8.03–7.92(m,6H),7.86(d,J＝8.0Hz,2H),7.74(s,1H),7.53(d,J＝8.4Hz,2H),6.79(dd,J＝16.8,10.4Hz,1H),6.09(dd,J＝16.8,2.0Hz,1H),5.66(dd,J＝10.4,2.0Hz,1H),4.48(d,J＝11.2Hz,1H),4.12(d,J＝10.4Hz,1H),3.66(t,J＝12.0Hz,1H),3.10(t,J＝12.8Hz,1H),2.75–2.64(m,1H),2.09–1.98(m,2H),1.59–1.41(m,2H)。

Example 13:

the synthetic route is as follows:

referring to example 1, the compound Mb was replaced with an equivalent amount of Mg (i.e., ma) to give the compound 13-5, and then referring to example 6, the methyl iodide was replaced with an equivalent amount of 1-fluoro-2-iodoethane, to thereby obtain the compound 13 as a yellow solid (82.3 Mg, yield 50.8%). LCMS [ M+H] ⁺ ＝647.1。

¹ H NMR(400MHz,DMSO-d6)δ12.89(s,1H),8.26(d,J＝8.4Hz,2H),8.00(d,J＝8.0Hz,2H),7.94(t,J＝8.8Hz,4H),7.86(d,J＝8.0Hz,2H),7.73(s,1H),7.51(d,J＝8.0Hz,2H),4.54(t,J＝4.8Hz,1H),4.42(t,J＝4.8Hz,1H),3.32-3.26(m,4H),2.93(d,J＝11.6Hz,2H),2.62(t,J＝4.8Hz,1H),2.55(t,J＝4.8Hz,1H),2.04(t,J＝12.0Hz,2H),1.93(d,J＝12.0Hz,2H),1.67-1.59(m,2H)。

Example 14:

the synthetic route is as follows:

referring to example 9, compound 5 was replaced with an equivalent amount of compound 3, and thus, compound 14 (82.3 mg, yield 50.8%) was synthesized as a yellow solid. LCMS [ M+H] ⁺ ＝647.1。

¹ H NMR(400MHz,DMSO-d6)δ12.89(s,1H),8.26(d,J＝8.0Hz,2H),8.07(d,J＝8.0Hz,2H),7.95(t,J＝8.8Hz,6H),7.73(s,1H),7.51(d,J＝8.8Hz,2H),4.54(t,J＝4.8Hz,1H),4.42(t,J＝4.8Hz,1H),4.34(q,J＝7.2Hz,2H),3.28–3.21(m,1H),2.93(d,J＝11.2Hz,2H),2.75–2.49(m,2H),2.16–1.87(m,4H),1.74–1.55(m,2H),1.34(t,J＝7.2Hz,3H)。

Example 15:

the synthetic route is as follows:

referring to example 3, 15-4 was synthesized by substituting methyl iodide for Ma-4 and 15-1 for 3-2 and 15-2 for 3-3, and then referring to example 6, 15-9 mg of yellow solid compound 15 (yield 18.6%) was synthesized by substituting 1-fluoro-2-iodoethane for methyl iodide. LCMS [ M+H] ⁺ ＝665.1。

¹ H NMR(400MHz,DMSO-d6)δ12.90(s,1H),8.26(d,J＝8.0Hz,2H),8.15(s,1H),8.00-7.94(m,6H),7.73(s,1H),7.51(d,J＝8.4Hz,2H),4.54(t,J＝4.8Hz,1H),4.42(t,J＝4.8Hz,1H),3.29-3.21(m,4H),2.93(d,J＝10.8Hz,2H),2.75-2.49(m,2H),2.12-1.87(m,4H),1.67 -1.59(m,2H)。

Example 16:

the synthetic route is as follows:

referring to example 15, compound 15-2 was replaced with an equivalent amount of 16-2, and thus, compound 16 (5.0 mg, yield 6%) was synthesized as a yellow solid. LCMS [ M+H] ⁺ ＝665.1。

¹ H NMR(400MHz,DMSO-d ₆ )δ12.88(s,1H),8.24-8.22(m,2H),8.02(s,1H),7.96-7.94(m,2H),7.84-7.82(m,2H),7.72-7.71(m,2H),7.67-7.65(m,2H),7.51-7.49(m,2H),4.56(s,1H),4.44(s,1H),3.33(s,3H),2.95-2.87(m,1H),2.33-2.31(m,1H),1.96-1.93(m,2H),1.67-1.64(m,4H)。

Example 17:

the synthetic route is as follows:

referring to example 15, compound 15-2 was replaced with an equivalent amount of 17-2, and thus, compound 17 (14.7 mg, yield 17.9%) was synthesized as a yellow solid. LCMS [ M+H] ⁺ ＝681.1。

¹ H NMR(400MHz,DMSO-d6)δ12.90(s,1H),8.26(d,J＝7.6Hz,2H),8.00-7.94(m,5H),7.92-7.83(m,2H),7.73(s,1H),7.51(d,J＝8.0Hz,2H),4.54(t,J＝4.8Hz,1H),4.42(t,J＝4.8Hz,1H),3.31-3.24(m,4H),2.93(d,J＝10.8Hz,2H),2.72-2.49(m,2H),2.12-1.87(m,4H),1.68-1.58(m,2H)。

Example 18:

the synthetic route is as follows:

referring to example 3, compound 3-1 was replaced with an equivalent amount of 18-1 to give compound 18-2, which was then deprotected to synthesize compound 18 as a yellow solid (25 mg, yield 94.3%). LCMS [ M+H] ⁺ ＝478.1。

¹ H NMR(400MHz,DMSO-d6)δ7.97-7.93(m,4H),7.65(s,1H),7.61-7.55(m,1H),7.49-7.44(m,3H),3.15(s,1H),3.00(d,J＝12.0Hz,2H),2.47-2.45(m,2H),1.88(d,J＝11.6Hz,2H),1.56-1.46(m,2H)。

Example 19:

the synthetic route is as follows:

referring to example 9, compound 5 was replaced with an equivalent amount of compound 18, and thus, compound 19 (11.8 mg, yield 15.4%) was synthesized as a yellow solid. LCMS [ M+H] ⁺ ＝524.1。

¹ H NMR(400MHz,DMSO-d6)δ12.89(s,1H),7.98-7.93(m,4H),7.74(s,1H),7.63-7.58(m,1H),7.51-7.48(m,3H),4.54(t,J＝4.4Hz,1H),4.42(t,J＝4.4Hz,1H),3.27-3.26(m,1H),2.93(d,J＝11.2Hz,2H),2.71-2.49(m,2H),2.12-1.84(m,4H),1.67-1.58(m,2H)。

Example 20:

the synthetic route is as follows:

referring to example 12, compound 5 was replaced with an equivalent amount of compound 18, and a yellow solid compound 20 (11.6 mg, yield 20.8%) was obtained by synthesis. LCMS [ M+H] ⁺ ＝532.1。

¹ H NMR(400MHz,DMSO-d6)δ7.98-7.93(m,4H),7.72(s,1H),7.63-7.57(m,1H),7.52-7.46(m,3H),6.81-6.75(m,1H),6.17-5.58(m,2H),4.56–4.03(m,2H),3.68-3.62(m,1H),3.12-3.06(m,1H),2.70-2.66(m,1H),2.06-1.99(m,2H),1.51-1.44(m,2H)。

Effect of COUP inhibitors on proliferation Activity of prostate cancer cells

1. Method of

Prostate cancer cells PC-3, DU-145 were grown at 1.5X10 ⁴ Density of/well in 96 well plate, 5% CO ₂ Incubated overnight at 37 ℃. The next day, the compounds were diluted at a maximum concentration of 100 μm in a 3-fold gradient, 10 total concentrations were added to the cell culture medium for a further treatment for 24h, 2 total multiplex wells.

After 24h of treatment of the cells with the compound, they were treated according to CellTiter

AQueous One Solution Cell Proliferation Assay kit (Promega, G3580) protocol for determining cell viability is outlined below: mu.L MTS reagent was added to each well, and after mixing, the mixture was incubated in an incubator at 37℃for 1 hour, and absorbance was measured using an MD i3X multifunctional microplate reader at 490nM.

The effect of compounds on cell proliferation was evaluated as cell viability and IC50 values were calculated using GraphPad prism8.0 software to fit dose-response curves with three parameters. Cell viability= (signal value of sample-signal value of medium control)/(signal value of DMSO control-signal value of medium control) ×100%. Inhibition ratio = (1-cell viability) ×100%.

2. Results

*：IC50>10μM；**：10μM≥IC50>1μM；***：1μM≥IC50>0.1μM；****：0.1μM≥IC50

30≥Max；/>

50≥Max>30；/>

70≥Max>50；/>

Max>70

Summarizing: the compound provided by the invention has better inhibition activity on the proliferation of prostate cancer cells.

Method for detecting inhibition activity of compound on COUP-TF II based on reporter gene activity detection

1. Method of

1.1 plasmid cotransfection of HEK293T cells and Compound treatment

HEK293T cells were plated at 1X 10 the day prior to plasmid transfection ⁴ Density/well was seeded in 96-well plates. According to transfection reagents

Cell transfection was performed in accordance with the instructions of HD (Promega, # E2311). The method mainly comprises the following steps: as an example of a single well, the plasmids pCR3.1-COUP-TFII and pXP2-NGFIA-Luc were added in a ratio of 20ng to 5ng to 10. Mu.L of Opti-MEM ^TM Mixing the above materials in medium I (Gibco, # 11058021); a further 0.2. Mu.L of +.>

HD, mixing, standing at 20deg.C for 5min; this 10. Mu.L mixture was then added to the cell well containing 100. Mu.L of culture medium. 6h after cell cotransfection, the compound is subjected to gradient dilution with 100 mu M as the highest concentration and half-logarithmic dilution multiple, 10 total concentrations are added into cell culture solution for treatment for 24h, and 2 total multiple wells are formed.

1.2One-Glo Luciferase assay

After 24h of treatment of the cells with the compounds, the cells were treated according to ONE-

Luciferase Assay System (Promega, # E6120) instructions. The method mainly comprises the following steps: mu.L of culture medium is pipetted out per well, and 50. Mu.L of ONE/Tiger are added>

Luciferase reagent, shake for 10min at 20 ℃; mu.L of the cleavage reaction solution was transferred to a white opaque optiPlate-96 well plate, and the luminescence signal value (Firefly-Luc) of Firefly luciferase (Firefly luciferase) was detected by an MD i3x multifunctional microplate reader. EC50 values were calculated using the Firefly-Luc signal values as inhibitory activity of the compounds on COUP-TF II, normalized to the ratio of solvent DMSO groups, and dose-response curves fitted with three parameters using GraphPad prism8.0 software. And the inhibition ratio was calculated as the following formula = (Luc of Luc/DMSO group of 1-compound treated samples) ×100%. VCT-8 was used as a positive control compound (WO 2019/222134).

2. Results

Experimental data are shown in the following table.

*：20μM≥EC50>5μM；**：5μM≥EC50>1μM；***：1μM≥EC50>0.1μM；

****：0.1μM≥EC50

20≥Max；/>

40≥Max>20；/>

70≥Max>40；/>

Max>70

Summarizing: the compound provided by the invention has better inhibition activity on COUP-TFII.

Method for detecting inhibition activity of compound on NRF2 based on reporter gene activity detection

1. Method of

1.1 plasmid cotransfection of HEK293T cells and Compound treatment

pGL4.37[luc2P/ARE](Promega, # E3641) and pRL-TK (Promega, # E2241) plasmids were purchased from Promega; the DH 5. Alpha. E.coli was transformed with the plasmid by CaCl2 method, and after further culture amplification, the corresponding plasmid DNA was obtained by purification using plasmid extraction kit (TIANGEN, #D107). HEK293T cells were plated at 1X 10 the day prior to plasmid transfection ⁴ Density/well was seeded in 96-well plates. According to transfection reagents

Cell transfection was performed in accordance with the instructions of HD (Promega, # E2311). The method mainly comprises the following steps: taking the single well as an example, plasmid pGL4.37[ luc2P/ARE]And pRL-TK at a ratio of 80ng to 8ng with 10. Mu.L of Opti-MEM ^TM Mixing the above materials in medium I (Gibco, # 11058021); a further 0.2. Mu.L of +.>

HD, mixing, standing at 20deg.C for 5min; this 10. Mu.L mixture was then added to the cell well containing 100. Mu.L of culture medium. 6h after cell cotransfection, the compound is subjected to gradient dilution with 100 mu M as the highest concentration and half-logarithmic dilution multiple, 10 total concentrations are added into cell culture solution for treatment for 24h, and 2 total multiple wells are formed.

1.2 Dual-Glo Luciferase assay

After 24h of treatment of the cells with the compounds, the cells are treated according to the Dual-

Luciferase Assay System (Promega, # E2940) instructions. The method mainly comprises the following steps: mu.L of culture medium is pipetted off per well, and then 50. Mu.L of Dual-/I are added>

Luciferase reagent, shake for 10min at 20 ℃; taking 80 mu L of the cleavage reaction solution to a white opaque optiPlate-96 well plate, and detecting a luminescence signal value (Firefly-Luc) of Firefly luciferase (Firefly luciferase) by using an MD i3x multifunctional enzyme-labeled instrument; a further 40. Mu.L of Dual-/is added>

Stop&/>

Reagent, oscillating for 10min at 20 ℃; the luminescence signal value (Renilla-Luc) of Renilla luciferase (Renilla luciferase) was detected by an MD i3x multifunctional microplate reader. The ratio of Firefly-Luc/Renilla-Luc is used as the inhibiting activity of the compound on NRF2, and the ratio of the solvent DMSO group is usedEC50 values were calculated using GraphPad prism8.0 software to fit dose-response curves with three parameters. And the inhibition ratio was calculated as = (ratio of 1-compound treated sample of Firefly-Luc/Renilla-Luc/ratio of DMSO group of Firefly-Luc/Renilla-Luc) ×100%.

2. Results

Experimental data are shown in the following table.

*：EC50>20μM；**：20μM≥EC50>10μM；***：10μM≥EC50>1μM；

20≥Max；/>

40≥Max>20；/>

70≥Max>40；/>

Max>70

Summarizing: the compound provided by the invention also has better inhibition activity on NRF 2.

Pharmacokinetic evaluation

In mice, the bioavailability and pharmacokinetic behavior of the compounds were evaluated. 6 male ICR mice of similar body weight were selected, 3 of which were given 10mg/kg in a single intragastric administration and 3 of which were given intravenously in a single dose of 1 or 5 mg/kg. Blood samples were taken at time points of 5 minutes (intravenous), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 7 hours post-dose, plasma samples were analyzed for concentration by LC-MS/MS and the pharmacokinetic parameters of the compounds were analyzed using PKSolver free tools and non-compartmental model (NCA) software.

Experimental protocol:

experimental animals: each compound test group consisted of 6 healthy male ICR mice, 18-25g, purchased from Charles River, randomly divided into 2 groups of 3.

Preparation: a certain amount of the compound was weighed and added to 2% dmso+15% solutol+83% physiological saline to make a clear solution.

Dosage is as follows: ICR mice were fasted overnight and the compounds were administered at a gastric lavage dose of 10mg/kg or a dose of 1 or 5mg/kg for intravenous administration. The administration volumes for intragastric and intravenous administration were 10mL/kg and 1 or 5mL/kg, respectively. Unified feeding is performed 2 hours after administration.

Sample collection: about 30 μl of blood was collected through the great saphenous vein 5 minutes (intravenous), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 7 hours after administration. Blood is put into a container containing K ₂ In a commercially available tube of EDTA, the blood sample was then centrifuged at 4600rpm at 4℃for 5 minutes to obtain a plasma sample, and all plasma samples were then flash frozen on dry ice and kept at-70℃until LC-MS/MS analysis was performed.

Sample preparation: mu.L of plasma sample was aspirated, precipitated with 50nmol/L of alpha-naphthaleneflavone (internal standard) in methanol, the mixture was thoroughly mixed and centrifuged at 14000rpm for 5 minutes at 4℃and 75. Mu.L of supernatant was then mixed with 75. Mu.L of methanol for LC-MS/MS analysis.

The pharmacokinetic parameter results are shown in table 1.

TABLE 1

Conclusion: the compound has better absorption in mice, slow elimination, high exposure and higher bioavailability, and can be used for further research.

The applicant states that the phenylthiazolyl benzamide compounds of the present invention and their use are illustrated by the examples described above, but the present invention is not limited to, i.e. it is not meant that the present invention must be practiced in dependence upon the examples described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (10)

1. The phenylthiazolyl benzamide compound is characterized by having a structure shown in the following formula I:

wherein R is ₁ Selected from the group consisting of

R _a 、R _b Independently selected from-Me, -CD ₃ 、

Any one of them;

the R is _f Selected from the group consisting of

The R is _g Selected from hydrogen, -Me, -n-Pr, -n-Bu,

Any one of them;

R ₂ selected from hydrogen or halogen;

R ₃ any one selected from hydrogen and substituted or unsubstituted C6-C12 aryl;

representing the attachment site of the group.

2. The phenylthiazolylbenzamide compound according to claim 1, wherein the substituent of the C6-C12 aryl group is selected from the group consisting of a C1-C10 linear or branched alkyl group, a C1-C10 haloalkyl group, and a-COOR _c Any one of them; the R is _c Selected from hydrogen or C1-C10 straight chain alkyl.

3. The phenylthiazolyl benzamide compound according to claim 1 or 2, wherein R ₂ Selected from hydrogen or-F.

4. A phenylthiazolyl benzamide compound according to any of claims 1-3, wherein R ₃ Selected from hydrogen or

The R is _i Selected from-CF ₃ -COOH or-COOEt;

the R is _j Selected from-F or-Cl;

representing the attachment site of the group.

5. The phenylthiazolyl benzamide compound according to any one of claims 1 to 4, wherein the phenylthiazolyl benzamide compound is selected from any one of the following formulas 1 to 20:

6. a stereoisomer of a phenylthiazolyl benzamide compound according to any of claims 1 to 5, a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

7. The stereoisomer of the phenylthiazolylbenzamide compound of claim 6, the pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the same, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable pharmaceutical excipient.

8. Use of a phenylthiazolylbenzamide compound according to any of claims 1 to 5 or a stereoisomer of a phenylthiazolylbenzamide compound according to claim 6 or 7, a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment and/or prevention of a disease associated with excessive activity of COUP-TFII or with overexpression of COUP-TFII.

9. The use according to claim 8, wherein the disease associated with excessive activity of COUP-TFII or overexpression of COUP-TFII comprises prostate cancer.

10. Use of a phenylthiazolylbenzamide compound according to any of claims 1 to 5 or a stereoisomer of a phenylthiazolylbenzamide compound according to claim 6 or 7, a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment and/or prevention of a disease associated with NRF2 over-activity or with NRF2 overexpression.