CN101899008B - N-substituted pyrimidinesulfonyl-substituted benzamide compounds and their use in preparing medicines - Google Patents

Abstract

The N-substituted pyrimidine sulfonyl-substituted benzamide small molecule inhibitor of Bcl-2 protein family anti-apoptosis member and application thereof, the present invention relates to inhibiting Bcl-2 protein family anti-apoptotic member (such as Bcl- 2. N-substituted benzoyl-pyrimidinesulfonamide compounds of formula I acting on Bcl-x _L , Mcl-1, etc.): For the definition of each group, please refer to the description. These compounds (and their stereoisomers if present), their pharmaceutically acceptable salts, their hydrates, their solvates, and their pharmaceutical compositions are useful in the treatment of The use in diseases or symptoms, or in the treatment of tumors, or in combination with other anti-tumor drugs as a synergist in the treatment of tumors. It also relates to a process for the preparation of compounds of formula I and pharmaceutically acceptable salts thereof.

Description

N-substituted pyrimidinesulfonyl-substituted benzamide compounds and their use in preparing medicines

The present invention relates to N-substituted pyrimidine sulfonyl-substituted benzamide compounds, to their preparation method, to their anti-apoptosis members as Bcl-2 protein family (such as Bcl-2, Bcl- _xL , Mcl-1, etc. ) inhibitors, for treating diseases or symptoms related to the high expression of anti-apoptotic members of the Bcl-2 protein family, for treating tumors, and as a synergist with other anti-tumor drugs Use in combined treatment of tumors.

Recent studies have found that cell apoptosis is closely related to tumorigenesis and drug resistance. The Bcl-2 protein family plays an important regulatory role in the apoptosis pathway. It can be divided into anti-apoptotic members (such as Bcl-2, Bcl-x _L , Mcl-1, etc.) and pro-apoptotic members. Studies have shown that anti-apoptotic members interact with the conserved region (BH) 3 of the Bcl-2 protein family through the hydrophobic groove on its surface to regulate the normal physiological apoptosis of cells.

Anti-apoptotic members of the Bcl-2 protein family are found to be overexpressed in many tumors, which is one of the important reasons for tumor generation and drug resistance. By inhibiting the anti-apoptotic effect of overexpressed anti-apoptotic members in tumor cells, restoring its normal apoptotic pathway and increasing its sensitivity to chemotherapy and radiotherapy is a new strategy for treating tumors. The antisense nucleotide drug (Genasense) against Bcl-2 has entered Phase III clinical research, and its clinical data have confirmed the good selectivity of this treatment strategy and its ability to improve the sensitivity of chemotherapy and radiotherapy drugs. However, due to the inherent defects of antisense nucleotide drugs, small molecule inhibitors will be more suitable for clinical application. the

From the molecular mechanism of the Bcl-2 protein family, it can be seen that small molecule inhibitors can interfere with the binding of the BH3 region of the pro-apoptotic member to promote cell apoptosis by binding to the hydrophobic groove on the surface of the anti-apoptotic member. At present, the research on small molecule inhibitors of anti-tumor Bcl-2 protein has become a hot spot, and some small molecule inhibitors with different structure types have been reported successively. The related compounds of the present invention are used as Bcl-2 protein family anti-apoptotic member inhibitors, and their application in anti-tumor has not been reported. the

The object of the present invention is to provide anti-apoptotic members (such as Bcl-2, Bcl- _xL , Mcl-1 etc.) that can selectively inhibit Bcl-2 protein family, restore the normal apoptosis pathway of tumor cells and increase its resistance to chemotherapy. Radiation Sensitive Compounds.

More specifically, the first aspect of the present invention relates to N-substituted benzoyl-pyrimidinesulfonamide compounds of formula I, their stereoisomers, their pharmaceutically acceptable salts, their hydrates or their solvates,

in

R ₁ can be substituted at the 2- or 3-position, hydrogen atom, hydroxyl, amino, halogen, C ₁ -C ₅ straight or branched chain alkyl, C ₁ -C ₅ alkanoyl, C ₁ -C ₅ alkoxy Group, C ₁ ~C ₅ alkanoyl amido.

The R ₂ group is a hydrogen atom, C ₁ ~ C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six membered aromatic monocyclic or condensed ring aromatic hydrocarbon groups (preferably naphthyl, indolyl), C ₃ ~ C ₆ cycloalkane, or XR ₅ (II), or NR ₅ R ₆ (III). Wherein, X is O, S, carbonyl, sulfone group or sulfoxide group; R ₅ , R ₆ are each independently a hydrogen atom, C ₁ ~ C ₇ straight chain or branched chain alkyl, unsubstituted or substituted aromatic hydrocarbon group (preferably Five-six-membered aromatic single ring, naphthyl, indolyl), C ₃ -C ₆ cycloalkane, or groups represented by formula IV:

(CH2) _n YR ₇ (IV)

Among them, n=1-7; Y is O, S, NH, carbonyl, sulfone or sulfoxide; R ₇ is a hydrogen atom, C ₁ ~ C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five ~ Six-membered aromatic monocyclic or condensed aromatic hydrocarbon group (preferably naphthyl, indolyl).

The R ₃ group is a hydrogen atom, a C ₁ to C ₇ straight chain or branched chain alkyl group, an unsubstituted or substituted five to six membered aromatic monocyclic or condensed ring aromatic hydrocarbon group (preferably naphthyl, indolyl), a heterocycle ( Preferably piperidine, piperazine, tetrahydropyrrole, morpholine), C ₃ -C ₆ cycloalkane, or ZR ₈ (V), or NR ₈ R ₉ (VI). Among them, Z is O, S, carbonyl, sulfone or sulfoxide; R ₈ and R ₉ are each independently a hydrogen atom, C ₁ to C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six members Aromatic monocyclic or fused-ring aromatic hydrocarbon groups (preferably naphthyl, indolyl), C ₃ -C ₆ cycloalkane groups, or groups represented by formula VII:

WR ₁₀ (VII)

Wherein, W is carbonyl, sulfone or sulfoxide. the

The R ₁₀ group is a C ₁ to C ₇ straight chain or branched chain alkyl group, a C ₃ to C ₆ cycloalkane group, an unsubstituted or substituted five to six membered aromatic monocyclic or condensed ring aromatic hydrocarbon group (preferably naphthyl, indole group), wherein the substituents can be mono-substituted or multi-substituted, halogen, unsubstituted or halogenated C ₁ ~C ₅ straight chain or branched chain alkyl, C ₁ ~C ₅ alkoxy group, nitro , trifluoromethyl group, cyano group, sulfonic acid group, carboxyl group, C ₁ ~C ₅ alkanoyl group, C ₁ ~C ₅ alkanoyl group, hydroxyl group, amino group.

The R ₁₀ group can also be CHR ₁₁ R ₁₂ (Ⅷ), wherein, R ₁₁ and R ₁₂ are independently hydrogen, C ₁ to C ₇ straight chain or branched chain alkyl, thio C ₁ to C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five- to six-membered aromatic single-ring or condensed-ring aromatic hydrocarbon group, heterocycle, C ₃ -C ₆ cycloalkane group, unsubstituted or substituted amino (substituents are preferably acetyl, methoxy formyl, formyl, ethoxyacetyl, methyl, ethyl), or (CH ₂ ) _n R ₁₃ (IX). Wherein, n=1-3, R ₁₃ is an unsubstituted or substituted five- to six-membered aromatic single-ring or condensed-ring aromatic hydrocarbon group, a heterocycle, and a C ₃ -C ₆ cycloalkane group.

The second part of the present invention relates to a pharmaceutical composition, including at least one compound of general formula I or its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate, and a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier. the

The third part of the present invention relates to any compound of general formula I, its stereoisomer, its pharmaceutically acceptable salt, its hydrate, its solvate, and their pharmaceutical composition, as the anti-apoptotic member of Bcl-2 protein family (such as Bcl-2, Bcl-x _L , Mcl-1, etc.) the use of inhibitors for the treatment of diseases or symptoms related to the high expression of anti-apoptotic members of the Bcl-2 protein family, for the treatment of tumors The use in the treatment of tumors, as well as the use as a synergist in combination with other anti-tumor drugs.

According to the present invention, R ₁ , R ₂ , R ₃ are particularly preferably the following groups, but these preferred groups do not imply any limitation to the present invention.

According to the present invention, among the groups containing substituents, unspecified substituents can be halogen, unsubstituted or halogenated C ₁ -C ₅ linear or branched chain alkyl, C ₁ -C ₅ alkoxy groups, C ₁ -C ₅ alkylthio group, nitro group, trifluoromethyl group, cyano group, sulfonic acid group, carboxyl group, C ₁ -C 5 alkanoyl group, C ₁ -C _{5 alkanoyl} group, hydroxyl group, amino group, etc.

According to the invention, the term "halogen" means fluorine, chlorine, bromine or iodine. the

According to the present invention, the substitution position of _R1 can be at the 2-position or the 3-position, preferably a hydrogen atom, a hydroxyl group, or an amino group.

The R ₂ group is preferably NR ₅ R ₆ (III). Wherein, R ₅ and R ₆ are each independently a hydrogen atom, C ₁ to C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six membered aromatic single ring, naphthyl, indolyl, or the formula IV Show group:

(CH ₂ ) _n YR ₇ (Ⅳ)

Among them, n=1-7; Y is S; R ₇ is a hydrogen atom, C ₁ ~C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six membered aromatic single ring, naphthyl, indolyl.

R ₃ group is a hydrogen atom, C ₁ ~ C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six membered aromatic single ring, naphthyl, indolyl, piperidine, piperazine, tetrahydropyrrole, Morpholine, or NR ₈ R ₉ (VI). Wherein, R ₈ and R ₉ are each independently a hydrogen atom, C ₁ to C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six membered aromatic single ring, naphthyl, indolyl, or the formula VII Show group:

WR ₁₀ (VII)

Wherein, W is carbonyl, sulfone group. the

The R ₁₀ group is C ₁ ~ C ₇ straight chain or branched chain alkyl, unsubstituted or substituted five to six membered aromatic single ring, naphthyl, indolyl, wherein the substituents can be mono-substituted or multi-substituted , is halogen, unsubstituted or halogenated C ₁ ~C ₅ linear or branched chain alkyl, C ₁ ~C ₅ alkoxy group, nitro, trifluoromethyl, cyano, sulfonic acid, carboxyl, acetyl group, acetamido group, hydroxyl group, amino group.

The R ₁₀ group can also be CHR ₁₁ R ₁₂ (Ⅷ), wherein, R ₁₁ and R ₁₂ are independently hydrogen, C ₁ to C ₇ straight chain or branched chain alkyl, thio C ₁ to C ₇ straight chain Or branched chain alkyl, unsubstituted or substituted five- to six-membered aromatic single-ring or condensed-ring aromatic hydrocarbon group, heterocycle, C ₃ -C ₆ cycloalkane group, unsubstituted or substituted amino (substituent is acetyl, methoxymethyl acyl, formyl, ethoxyacetyl, methyl, ethyl), or (CH ₂ ) _n R ₁₃ (IX). Wherein, n=1-3, R ₁₃ is an unsubstituted or substituted five- to six-membered aromatic single-ring or condensed-ring aromatic hydrocarbon group, a heterocycle, and a C ₃ -C ₆ cycloalkane group.

According to the invention, the compounds of the formula I in Tables 1, 2 and 3 are particularly preferred, but these compounds do not imply any limitation of the invention. the

Particularly preferred compound structures in the formula I of table 1

Particularly preferred compound structures in Table 2 Formula I (R ₃ is N R ₈ R ₉ , R ₈ is H, R ₉ is WR ₁₀ )

Particularly preferred compound structures in formula I in Table 3 (R ₃ is N R ₈ R ₉ , R ₈ is H, R ₉ is WR ₁₀ , R ₁₀ is CHR ₁₁ R ₁₂ )

The compounds with acidic groups according to the present invention I can form pharmaceutically acceptable salts of alkali metals, such as sodium salts, potassium salts, magnesium salts or calcium salts. the

According to the present invention, the term "stereoisomer" in the present invention refers to various stereoisomer forms of the compound of general formula I, such as racemic isomers and optical isomers. the

According to the present invention, the pharmaceutical combination in the present invention can be prepared according to methods known in the art, such as combining the compound in formula I, its stereoisomer, its pharmaceutically acceptable salt, or their hydrate or solvate with a pharmaceutically acceptable carrier or Excipients are mixed. the

The present invention relates to providing the results of the protein binding activity test of the compound in formula I with Bcl-2 protein (and its homologous proteins Bcl-xL, Mcl-1), the test results of anti-tumor activity, and the results of the synergistic test Test Results. the

According to the present invention, the compounds of the present invention can be administered alone or in combination, and the route of administration can be oral, parenteral or topical. The pharmaceutical composition can be formulated into various suitable dosage forms according to the route of administration. the

A method for preparing a compound of formula I, its stereoisomer, its pharmaceutically acceptable salt, its hydrate, and its solvate, which includes (1) the compound of formula X

After adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP), react with the compound of formula XI under normal temperature conditions

wherein R ₂ , R ₁ , R ₃ are as defined above, or

(2) If necessary, form a pharmaceutically acceptable salt of the compound of formula I with the compound of formula I obtained in step (1) with a pharmaceutically acceptable base, or

(3) If necessary, the compound of formula I obtained in step (1) or (2) is resolved into pure optical isomers of the compound of formula I by stereochemical separation methods such as fractionation, recrystallization, chromatography, etc. the

According to this invention, the compound of formula (I) can exist in the form of stereoisomers, and there is an asymmetric center in the compound part of formula (I), which can have S configuration or R configuration. The present invention includes all possible stereoisomers such as enantiomers or diastereomers, as well as mixtures of two or more stereoisomers, such as any desired ratio of enantiomers and/or diastereomers mixture. Accordingly, the present invention contemplates enantiomers, eg the le- and dex-enantiomers in enantiomerically pure form, and mixtures or racemates of the two enantiomers in different proportions. the

According to the present invention, the compound of formula (I) can interfere with the binding of the BH3 region of the pro-apoptotic member to promote cell apoptosis by binding to the hydrophobic groove on the surface of the anti-apoptotic member. Therefore, it can be used as an anti-tumor disease or symptom drug for animals, preferably for mammals, especially for humans. the

The present invention therefore also relates to pharmaceutical compositions comprising as active ingredient an effective dose of at least one compound of formula (I) and/or a stereoisomer thereof together with customary pharmaceutical excipients or adjuvants. Generally, the pharmaceutical compositions according to the invention contain 0.1-90% by weight of the compound of formula (I) and/or its physiologically acceptable salts. Pharmaceutical compositions can be prepared according to methods known in the art. When used for this purpose, if necessary, the compound of formula (I) and/or stereoisomers can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare as human suitable administration form or dosage form. the

The compound of formula (1) of the present invention or the pharmaceutical composition containing it can be administered in unit dose form, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal cavity, oral mucosa, skin, peritoneal or rectum etc. Dosage forms such as tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, lipid agents, transdermal agents, buccal tablets, suppositories, lyophilized powder for injection wait. It can be common preparations, sustained release preparations, controlled release preparations and various granule drug delivery systems. For making a unit dosage form into tablets, various carriers known in the art can be widely used, and examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose , calcium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, paste Essence, syrup, honey, glucose solution, acacia mucilage, gelatin pulp, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate , agar powder, algae starch, sodium bicarbonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary Ammonium salts, sodium lauryl sulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, Evergreen-coated tablets, or double-layer tablets and multi-layer tablets. In order to formulate a dosage unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents. Such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc. ; Disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc. In order to formulate the administration unit into a suppository, various carriers known in the art can be widely used. As examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, lipids of higher alcohols, gelatin, semi-synthetic glycerides and the like. In order to form a dosage unit into a capsule, the active ingredient compound of formula (1) or its stereoisomer is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard capsule or a soft capsule. The active ingredient formula (1) compound or its stereoisomers can also be made into microcapsules, suspended in an aqueous medium to form a suspension, such as solutions, emulsions, freeze-dried powder injections and suspensions, which can be used All diluents commonly used in the field, e.g. water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters wait. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin, and conventional solubilizers, buffers, pH regulators, etc. can also be added to the preparation for injection. the

In addition, colorants, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations, if necessary. the

The dosage of the compound of formula (1) of the present invention or its stereoisomer depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight and individual response of the patient or animal, the specific used Compound, route of administration and frequency of administration, etc. The above dose may be administered in a single dose divided into several, for example two, three or four doses. the

Example

The present invention is illustrated by the following examples, but these examples are not meant to limit the invention in any way. the

Embodiment 1: Preparation of 2-bromoethylbenzenesulfane

Add 7.2ml (0.076mol) of phosphorus tribromide dropwise to 30ml (0.22mol) of 2-phenylthioethanol. After the reaction, add 50ml of water and 100ml of ether to separate the two phases. Add magnesium sulfate to the organic phase to dry. Filter and concentrate to obtain 33 g of colorless transparent oil, yield: 98%. the

Embodiment 2: Preparation of 2-(phenylthio)ethylamine

Add 8.68g (0.040mol) of (2-bromoethyl)thiobenzenesulfane and 9.11g (0.040mol) of potassium salt of 4-nitrophthalimide to 70ml of anhydrous DMF, and react at 35°C for 1h. Add 100ml of dichloromethane and 250ml of water, separate the two phases, and extract the aqueous phase with dichloromethane (80ml×2). The organic phases were combined and washed successively with 80 ml of 0.2 mol/L sodium hydroxide aqueous solution and 80 ml of water. Dry over anhydrous magnesium sulfate, filter, and concentrate the filtrate to obtain a yellow solid, which is ground by adding a small amount of ether, and allowed to stand to precipitate a solid. After filtration, the filter cake was recrystallized with ethyl acetate to obtain 4-nitro-2-[2-(phenylthio)ethyl]isoindoline-1,3-dione as yellow crystals. the

The yellow crystals obtained above (11.4 g, 0.035 mol) and 85% hydrazine hydrate (6.3 ml, 0.108 mol) were added to 200 ml of methanol. Heated to 65°C for 1h. Filter to remove filter cake. Add 6% hydrochloric acid (about 60ml) to the filtrate to adjust the pH to 4, and leave it in the refrigerator to precipitate a yellow solid. Filter to remove filter cake. The filtrate was concentrated to dryness to obtain a light yellow solid, which was added with 250 ml of water and washed with diethyl ether (100 ml x 2 times). The aqueous layer was adjusted to pH>12 by adding saturated aqueous sodium hydroxide solution (about 30 ml), and extracted with diethyl ether (100 ml×3). The ether layers were combined, and the filtrate was concentrated to obtain 4.65 g of 2-(phenylthio)ethylamine, yield: 73.45%, mp 65-68°C. the

Example 3: Synthesis of 2-aminopyrimidine-5-sulfonic acid

After slowly adding 8.00 g (0.0840 mol) of 2-aminopyrimidine to 50 mL of chlorosulfonic acid, the reaction was refluxed at 150° C. for 8 hours. Part of the chlorosulfonic acid was distilled off after the reaction, and the residue was cooled and poured into 50 g of ice water to precipitate a solid. After filtration and recrystallization from water, 8.60 g of white crystals were obtained with a yield of 58.2%. the

Example 4: Synthesis of 2-hydroxypyrimidine-5-sulfonic acid

8.60g (0.049mol) of 2-aminopyrimidine-5-sulfonic acid, 50mL of sulfuric acid and 2mL of water were placed in a 250mL round-bottomed three-neck flask, and refluxed at 180°C for 5 hours. The reaction solution was cooled and poured into about 200 g of ice water to precipitate a solid. After filtration and recrystallization from water, 4.66 g of white crystals were obtained with a yield of 57.8%. the

Example 5: Synthesis of 2-chloropyrimidine-5-sulfonyl chloride

2.64g (0.0150mol) of 2-hydroxypyrimidine-5-sulfonic acid and 10.9g (0.0525mol) of phosphorus pentachloride were refluxed at 180°C for 8 hours. 60 mL of toluene was added to the reaction solution, filtered, and the solvent was evaporated to dryness under reduced pressure. The remaining yellow solid was placed in a Soxhlet extractor and continuously extracted with 100 mL of petroleum ether for 16 hours. Half of the extract was evaporated, the solid was precipitated by cooling, and filtered to obtain 2.40 g of white crystals with a yield of 75.1%. the

Embodiment 6: the synthesis of 2-chloropyrimidine-5-sulfonamide

32.9 g (0.150 mol) of 2-chloropyrimidine-5-sulfonyl chloride was dissolved in 150 mL of anhydrous tetrahydrofuran, and dry ammonia gas was introduced under an ice bath. Reaction 1.5 hours, TCL monitors and stops reaction immediately until raw material reaction completes. After the reaction, adjust the pH to <6, filter, evaporate the solvent to dryness under reduced pressure, add 150 mL of dichloromethane to the residue, filter, evaporate the solvent to dryness under reduced pressure, and purify the residue by silica gel column chromatography, eluent: dichloromethane : Ethyl acetate=50:3, 16.1g of white solid was obtained, yield 54.0%

Example 7: Synthesis of 2-[2-(phenylthio)ethylamine]pyrimidine-5-sulfonamide

5.80g (0.0300mol) of 2-chloropyrimidine-5-sulfonamide and 4.60g (0.03mol) of 2-(phenylthio)ethylamine, N,N-diisopropylethylamine (DIEA), in the form of dimethyl Sulfone was used as a solvent to react at room temperature for 5 hours. After the reaction was completed, 80ml of 1M hydrochloric acid solution was added, and a white solid was precipitated, which was then refrigerated. After filtering, the solid was washed three times with water, and recrystallized from ethyl acetate to obtain 7.7 g of white crystals. Yield 82.7%, mp: 172-176°C. the

Embodiment 8: the preparation of ethyl benzamidobenzoate

1.22g (0.01mol) of benzoic acid and 1.65g (0.01mol) of ethyl p-aminobenzoate, 2.3g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (0.012mol), a small amount of 4-dimethylaminopyridine (DMAP), with dichloromethane as the solvent for normal temperature reaction for 3h, after the reaction, add 100ml of 0.5M hydrochloric acid, solids are precipitated, filtered out, and the dichloromethane phase is reused Wash with sodium bicarbonate, wash with saturated sodium chloride, dry over anhydrous magnesium sulfate, and concentrate. The concentrated product was combined with the filtered solid to obtain 2.5 g of crude product, which was directly used in the next reaction. the

Other substituted benzamido ethyl benzoate is prepared by reacting different substituted benzoic acid with ethyl p-aminobenzoate, and repeating the steps of 8. the

Other 4-(2 substituted-3 substituted) ethyl benzoates were prepared by reacting different N-protected hydrophobic amino acids with ethyl p-aminobenzoate, and repeating the steps of 8. the

Embodiment 9: the preparation of benzamidobenzoic acid

2.5 g of ethyl benzamidobenzoate obtained in Example 8 was dissolved in 60 ml of tetrahydrofuran: methanol=1: 1 solution, after dissolving completely, aqueous sodium hydroxide solution was added (1 g of sodium hydroxide was dissolved in 10 ml of water), After the reaction, the organic solvent was evaporated to dryness, and 30ml of 1M hydrochloric acid solution was added to form a white precipitate. The precipitate was filtered, dried, and the filtrate was extracted with 50ml of ethyl acetate, dried over anhydrous magnesium sulfate, and concentrated. The concentrated product was combined with the above filtered solid and recrystallized from THF. 2 g of white crystals were obtained. Yield 90%, mp: 280-284°C. the

Other substituted benzamidobenzoic acids are obtained by alkali hydrolysis with different substituted ethyl benzamidobenzoates, and the steps of 9 are repeated. the

Other 4-(2-substituted-3-substituted) benzoic acids were prepared by repeating the steps of 9 by using ethyl 4-(2-substituted-3-substituted) benzoates with different substitutions through alkaline hydrolysis. the

Other benzoic acids, p-toluic acid, 4-fluorophenylbenzoic acid are commercially available. the

Embodiment 10: Preparation of target compound

0.14g (0.57mmol) of benzamidobenzoic acid and 0.17g (0.57mmol) of 2-[2-(phenylthio)ethylamine]pyrimidine-5-sulfonamide, 1-ethyl-3-(3-di Ethylaminopropyl) carbodiimide hydrochloride (EDCI) 0.24g (1.14mmol), a small amount of 4-dimethylaminopyridine, react with methylene chloride at room temperature for 3 hours, after the reaction, add 0.5M hydrochloric acid 20ml , a white precipitate was precipitated, the precipitate was filtered, dried, and the filtrate was extracted with 50 ml of ethyl acetate, dried over anhydrous magnesium sulfate, and concentrated. The concentrated product was combined with the above filtered solid and passed through a gel column. (Developer: dichloromethane:methanol=1:4) Yield: 88%, mp: 246-247. the

Other target compounds were prepared by reacting the compound obtained in Example 9 with the 2-[2-(phenylthio)ethylamine]pyrimidine-5-sulfonamide obtained in Example 7, and repeating the steps in Example 10. the

The melting points, yields and spectral data of some preferred compounds synthesized in the present invention are shown in Table 4. the

Melting point, productive rate and spectral data of preferred compound of table 4 part

No. melting point Yield MS(m/z) (M-H)- 1H-NMR(DMSO)/δ

1 183-185 8.1% 413.4 3.14-3.16(t, 2H, S-CH ₂ ), 3.53-3.57(t, 2H, N-CH ₂ ), 7.16-7.38(m, 5H, S-Ar-H), 7.46-7.88(m, 5H , COAr-H), 8.46(s, 1H, NH), 8.70-8.74(d, 2H, pyrimidine), 12.50(s, 1H, SO ₂ NHCO) 2 196-200 8.0% 427.74 2.34(s, 3H, Ar-CH ₃ ), 3.12-3.17(t, 2H, S-CH ₂ ), 3.52-3.56(t, 2H, N-CH ₂ ), 7.13-7.35(m, 5H, S- Ar-H), 7.37-7.88(m, 4H, COAr-H), 8.55(s, 1H, NH), 8.70-8.75(d, 2H, pyrimidine), 12.47(s, 1H, SO ₂ NHCO) 3 222-224 8.0% 507.5 3.15-3.10(t, 2H, S-CH ₂ ), 3.53-3.48(t, 2H, N-CH ₂ ), 7.14-7.17(t, 1H, S-Ar-H), 7.25-7.36(d, 2H , F-Ar-H and 4H, dd, S-Ar-H), 7.74-7.78 (dd, 4H, H-Ar-Ar-H), 7.92-7.95 (d, 2 H, COAr-H), 8.55 (s, 1H, NH), 8.69-8.75 (d, 2H, pyrimidine), 12.58 (s, 1H, SO ₂ NHCO) 4 246-247 7.3% 532.91 3.14-3.19 (t, 2H, J=6.3Hz, S-CH ₂ ), 3.52-3.57 (t, 2H, J=6.3Hz, N-CH ₂ ), 7.18-7.94 (m, 14H, Ar-H) , 8.54 (s, 1H, NH), 8.69-8.75 (d, 2H, pyrimidine), 10.54 (s, 1H, ArNHCO), 12.45 (s, 1H, SO ₂ NHCO) 5 248-250 6.6% 550.58 3.15-3.18(t, 2H, S-CH ₂ ), 3.53-3.56(t, 2H, N-CH ₂ ), 7.18-7.91(m, 13H, Ar-H), 8.71(s, 1H, NH), 8.72-8.76 (d, 2H, pyrimidine), 10.74 (s, 1H, ArNHCO), 12.42 (s, 1H, SO ₂ NHCO) 6 242-244 6.5% 550.95 3.14-3.17 (t, 2H, S-CH ₂ ), 3.54-3.56 (t, 2H, N-CH ₂ ), 7.18-7.92 (m, 13H, Ar-H), 8.71 (s, 1H, NH), 8.72-8.76 (d, 2H, pyrimidine), 10.57 (s, 1H, ArNHCO), 12.42 (s, 1H, SO ₂ NHCO) 7 251-252 6.4% 550.49 3.15-3.17(t, 2H, S-CH ₂ ), 3.54-3.57(t, 2H, N-CH ₂ ), 7.18-7.92(m, 13H, Ar-H), 8.71(s, 1H, NH), 8.72-8.76 (d, 2H, pyrimidine), 10.54 (s, 1H, ArNHCO), 12.40 (s, 1H, SO ₂ NHCO) 8 178-182 6.5% 566.33 3.12-3.17(t, 2H, S-CH ₂ ), 3.50-3.57(t, 2H, N-CH ₂ ), 7.14-7.91(m, 13H, Ar-H), 8.70(s, 1H, NH), 8.75-8.76 (d, 2H, pyrimidine), 10.84 (s, 1H, ArNHCO), 12.45 (s, 1H, SO ₂ NHCO) 9 228-231 6.5% 566.32 3.11-3.17 (t, 2H, S-CH ₂ ), 3.48-3.52 (t, 2H, N-CH ₂ ), 7.16-7.99 (m, 13H, Ar-H), 8.20 (s, 1H, NH), 8.66-8.69 (d, 2H, pyrimidine), 10.53 (s, 1H, ArNHCO), 12.48 (s, 1H, SO ₂ NHCO) 10 268-271 6.4% 566.37 3.12-3.17 (t, 2H, S-CH ₂ ), 3.52-3.54 (t, 2H, N-CH ₂ ), 7.14-7.99 (m, 13H, Ar-H), 8.71 (s, 1H, NH), 8.70-8.76 (d, 2H, pyrimidine), 10.53 (s, 1H, ArNHCO), 12.45 (s, 1H, SO ₂ NHCO) 11 192-196 5.5% 546.90 2.36-2.37(s, 3H, CH ₃ ), 3.15-3.16(t, 2H, S-CH ₂ ), 3.53-3.57(t, 2H, N-CH ₂ ), 7.18-7.90(m, 13H, Ar- H), 8.61(s, 1H, NH), 8.72-8.76(d, 2H, pyrimidine), 10.61(s, 1H, ArNHCO), 12.41(s, 1H, SO ₂ NHCO) 12 235-244 5.5% 546.62 2.40 (s, 3H, CH ₃ ), 3.15-3.18 (t, 2H, S-CH ₂ ), 3.55-3.57 (t, 2H, N-CH ₂ ), 7.16-7.90 (m, 13H, Ar-H) , 8.72 (s, 1H, NH), 8.72-8.78 (d, 2H, pyrimidine), 10.50 (s, 1H, ArNHCO), 12.40 (s, 1H, SO ₂ NHCO)

13 257-260 5.4% 546.77 2.39 (s, 3H, CH ₃ ), 3.15-3.18 (t, 2H, S-CH ₂ ), 3.55-3.58 (t, 2H, N-CH ₂ ), 7.16-7.90 (m, 13H, Ar-H) , 8.72 (s, 1H, NH), 8.72-8.77 (d, 2H, pyrimidine), 10.45 (s, 1H, ArNHCO), 12.39 (s, 1H, SO ₂ NHCO) 14 218-220 7.3% 600.28 3.12-3.17 (t, 2H, J=6.3Hz, S-CH ₂ ), 3.50-3.57 (t, 2H, J=6.3Hz, N-CH ₂ ), 7.18-7.94 (m, 13H, Ar-H) , 8.48 (s, 1H, NH), 8.70-8.74 (d, 2H, pyrimidine), 10.72 (s, 1H, ArNHCO), 12.45 (s, 1H, SO ₂ NHCO) 15 180-182 6.1% 622.44 3.16-3.18 (t, 2H, J=6.5Hz, S-CH ₂ ), 3.54-3.56 (t, 2H, J=6.5Hz, N-CH ₂ ), 3.58-3.88 (m, 9H, OCH ₃ ), 6.93-7.90(m, 11H, Ar-H), 8.56(s, 1H, NH), 8.71-8.77(d, 2H, pyrimidine), 10.41(s, 1H, ArNHCO), 12.40(s, 1H, _SO2 NHCO) 16 250-252 6.1% 623.24 3.15-3.18 (t, 2H, J=6.5Hz, S-CH ₂ ), 3.51-3.55 (t, 2H, J=6.5Hz, N-CH ₂ ), 3.73-3.89 (m, 9H, OCH ₃ ), 6.97-7.89(m, 11H, Ar-H), 8.20(s, 1H, NH), 8.67-8.71(d, 2H, pyrimidine), 10.31(s, 1H, ArNHCO), 12.45(s, 1H, _SO2 NHCO) 17 292-296 6.8% 578.1 3.12-3.17 (t, 2H, J=6.3Hz, S-CH ₂ ), 3.50-3.56 (t, 2H, J=6.3Hz, N-CH ₂ ), 7.18-7.94 (m, 13H, Ar-H) , 8.48 (s, 1H, NH), 8.70-8.74 (d, 2H, pyrimidine), 10.72 (s, 1H, ArNHCO), 12.49 (s, 1H, SO ₂ NHCO) 18 240-244 5.2% 617.2 2.84-3.01 (dd, 2H, CH ₂ Ar), 3.13-3.16 (m, 2H, S-CH ₂ ), 3.57-3.54 (m, 2H, N-CH2), 4.61-4.65 (m, 1H, COC- HN), 7.16-7.38(m, 10H, Ar-H), 7.85(dd, 4H, COAr-HN), 7.65- 1.75(s, 1H, NHCOCH ₃ ), 8.34(d, 1H, NHCO), 8.50( s, 1H, NH), 8.69-8.74 (d, 2H, rimidine), 10.4 (s, 1H, ArNHCO), 12.37 (s, 1H, SO ₂ NHCO) 19 100-118 4.7% 583.9 0.84-0.90(m, 6H, C(CH ₃ ) ₂ ), 1.46-1.49(m, 2H, CCH ₂ C), 1.56-1.64(m, 1H, CCH(C)2), 1.83(s, 1H, NHCOCH ₃ ), 3.12-3.17 (2H, t, ArSCH ₂ ), 3.50-3.57 (m, 2H, N-CH ₂ ), 4.40 (s, 1H, COC-HN), 4.41-4.42 (m, 1H, COC -HN), 7.14-7.39(m, 5H, Ar-H), 7.68-7.86(dd, 4H, COAr-HN), 8.19(d, 1H, NHCO), 8.54(s, 1H, NH), 8.68- 8.74 (d, 2H, pyrimidine), 10.4 (s, 1H, ArNHCO), 12.42 (s, 1H, SO ₂ NHCO) 20 110-116 4.6% 601.5 1.86 (s, 1H, NHCOCH ₃ ), 1.89-1.98 (m, 2H, SCH ₂ ), 2.04 (s, 3H, SCH ₃ ), 2.45-2.50m, 2H, SCH2CH ₂ ), 3.16-3.14 (t, 2H , ArSCH ₂ ), 3.57-3.54 (m, 2H, N-CH ₂ ), 4.40 (m, 1H, COC-HN), 7.15-7.35 (m, 5H, S-Ar-H), 7.65-7.85 (dd , 4H, COAr-HN), 8.25 (d, 1H, NHCO), 8.50 (s, 1H, NH), 8.68-8.71 (d, 2H, pyrimidine), 10.34 (s, 1H, ArNHCO), 12.39 (s, 1H, SO ₂ NHCO)

Example 11: Affinity of the compounds of the present invention for Bcl-2, Bcl-xL, and Mcl-1 proteins

Bid BH3 domain peptide (sequence: EDIIRNIARHLAQVGDSMDR), N-terminally labeled with fluorescein isothiocyanate. 200nM of Bcl-xL, Bcl-2 or Mcl-1 protein and the same amount of 200nM fluorescein isothiocyanate-labeled BH3 peptide, and different concentrations of PBS (pH7.4), the compound of the present invention together at room temperature After incubation for 10 minutes, the fluorescence polarization was recorded at an excitation wavelength of 485 nm and an absorption wavelength of 520 nm, respectively. All experiments were repeated 3 times. _IC50 was obtained by dose-response curve. IC ₅₀ of compound 3 inhibiting Bcl-2 protein: 41.4 μM. The inhibitory rates of compound 18 to Bcl-2, Bcl-xL, and Mcl-1 proteins at 100 μM were 32%, 50%, and 34%, respectively.

Example 12: Inhibitory effect of compounds of the present invention on human tumor cell proliferation in vitro

After dissolving the compound in DMSO, add PBS(-) to make a 1000ug/mL solution or a uniform suspension, and then dilute it with DMSO-containing PBS(-) to form 5 concentration gradients. Gossypol was used as a control. Select five tumor cells: NCI-H446 (human non-small cell lung cancer cells), HCT116 (human colon cancer cells), PC-3M (human prostate cancer cells), MDA-MB-435 (human breast cancer cells), Raji (human Lymphoma cells) were all frozen and passaged by the pharmacology laboratory of Shanghai Pharmaceutical Industry Research Institute. Cultured in the culture medium of RPMI1640+15%NBS+double antibody (penicillin 100 units/mL, streptomycin 100ug/mL). 96-well plate Add 100 μl of cell suspension with a concentration of 4-5×104 cells/ml to each well, and place it in a 5% CO ₂ incubator at 37°C. After 24 hours, add sample solution, 10 μl/well, set up double wells, 37°C, 5% CO2 for 72 hours. Add 20 μl of 5 mg/ml MTT solution to each well, add solution after 4 hours of action, 100 μl/well, put it in an incubator, measure 570nm OD value with MK-2 automatic microplate reader after dissolution, pass Compared with the blank control to evaluate the inhibition of cell proliferation, calculate the IC _50. Table 5 shows the inhibitory effect of the compound of the present invention and positive control gossypol acetic acid (GA) on the proliferation of five human tumor cells in vitro.

Example 13: The compound of the present invention has a synergistic effect on the cellular level of cytotoxic antineoplastic drugs

Prepare paclitaxel solutions with concentrations of 10 μg/ml, 1 μg/ml, 0.1 μg/ml, 0.01 μg/ml, 0.001 μg/ml, 0.0001 μg/ml, and 0.00001 μg/ml as reference solutions, and measure the IC ₅₀ at this time Value, then in each concentration of paclitaxel solution, add 100 μg/ml, 50 μg/ml of compound 18 respectively, and determine the IC ₅₀ value. The Q value is calculated by the golden formula (Q=ICa+b/((ICa+ICb)-ICa*ICb)). When Q>0.85, it is combined, and when Q>1.15, it is synergistic, which shows that for cytotoxic antineoplastic drugs There is a synergistic effect. When the concentration of paclitaxel solution was 0.01 μg/ml, the Q value of 100 μg/ml compound 18 combined with it was 1.277, and the Q value of 50 μg/ml compound 18 combined with it was 1.264. In addition, when the concentration of paclitaxel solution was 0.001 μg /ml, the Q value of 100 μg/ml of compound 18 combined with it is 1.275, indicating that it has a synergistic effect on cytotoxic antineoplastic drugs at this time.

Claims (7)

1. N-substituted pyrimidinesulfonyl-substituted benzamide compounds of formula I, their stereoisomers, and their pharmaceutically acceptable salts, in The _R group is a hydrogen atom, The R ₂ group is NR ₅ R ₆ (III), wherein R ₅ and R ₆ are each independently a hydrogen atom or a group shown in formula IV: (CH2) _n YR ₇ (IV) Among them, n=1-7; Y is S, R ₇ is a hydrogen atom, C ₁ ~ C ₇ straight chain or branched chain alkyl, unsubstituted five to six membered aromatic single ring, The R ₃ group is a hydrogen atom, a C ₁ to C ₇ straight chain or branched chain alkyl group, an unsubstituted or substituted five to six membered aromatic single ring, or NR ₈ R ₉ (VI), wherein R ₈ and R ₉ are independent is a hydrogen atom, or a group shown in formula VII: WR ₁₀ (VII) Wherein, W is carbonyl; The R ₁₀ group is an unsubstituted or substituted five- to six-membered aromatic monocyclic ring, wherein the substituents can be mono-substituted or multi-substituted, halogen, unsubstituted or halogenated C ₁ -C ₅ straight chain or branched chain alkane group, C ₁ ~C ₅ alkoxy group, nitro group; The R ₁₀ group can also be CHR ₁₁ R ₁₂ (VIII), wherein, R ₁₁ and R ₁₂ are each independently C ₁ to C ₇ straight chain or branched chain alkyl, and thio C ₁ to C ₇ straight chain or branched Alkanyl, amino substituted by acetyl, or (CH ₂ ) _n R ₁₃ (IX); wherein n=1-3, R ₁₃ is an unsubstituted five- to six-membered aromatic single ring, Among the groups containing substituents, the substituents are halogen, unsubstituted or halogenated C ₁ -C ₅ linear or branched alkyl, C ₁ -C ₅ alkoxy groups, nitro. 2. The compound of claim 1, wherein said compound of formula I is selected from the group consisting of: (1) N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (2) 4-methyl-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (3) 4-p-fluorophenyl-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (4) 4-(phenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (5) 4-(2-fluorophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (6) 4-(3-fluorophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (7) 4-(4-fluorophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (8) 4-(2-Chlorophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (9) 4-(3-Chlorophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (10) 4-(4-Chlorophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (11) 4-(2-methylphenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (12) 4-(3-methylphenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (13) 4-(4-methylphenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (14) 4-(4-trifluoromethylphenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (15) 4-(2,3,4-trimethoxyphenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (16) 4-(3,4,5-trimethoxyphenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (17) 4-(4-nitrophenylcarboxamido)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (18) 4-(2-Acetamido-3-phenylpropylamino)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (19) 4-(2-Acetamido-4-methylpentylamino)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide (20) 4-(2-Acetamido-4-(methylmercapto)butylamino)-N-(2-(2-(phenylmercapto)ethylamino)pyrimidine-5-sulfonyl)benzamide. 3. A pharmaceutical composition comprising at least one compound of formula I according to any one of claims 1-2 and a pharmaceutically acceptable carrier. 4. Use of the compound of formula I according to any one of claims 1 to 2 in the preparation of medicines for treating diseases or symptoms related to high expression of anti-apoptotic members of Bcl-2 protein family. 5. Use of the compound of formula I according to any one of claims 1 to 2 in the preparation of a drug for treating tumors. 6. Use of the compound of formula I according to any one of claims 1 to 2 in the preparation of a synergist, which is used in combination with other antitumor drugs to treat tumors. 7. prepare the described formula I compound of claim 1, the method for its stereoisomer and pharmaceutically acceptable salt thereof, it comprises (1) Compounds of formula X After adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP), react with the compound of formula XI under normal temperature conditions

wherein R ₂ , R ₁ , R ₃ are as defined in claim 1, or (2) If necessary, form a pharmaceutically acceptable salt of the compound of formula I with the compound of formula I obtained in step (1) with a pharmaceutically acceptable base, or (3) If necessary, the compound of formula I obtained in step (1) or (2) is resolved into pure optical isomers of the compound of formula I by a stereochemical separation method selected from fractionation, recrystallization or chromatography.