阅读说明：本技术 含有苯并噻唑的苯丙氨酸衍生物及其制备方法与应用 (Benzothiazole-containing phenylalanine derivative and preparation method and application thereof ) 是由 展鹏 徐淑静 刘新泳 孙林 黄天广 张续杰 屈直言 于 2021-06-04 设计创作，主要内容包括：本发明提供了一种含有苯并噻唑的苯丙氨酸衍生物及其制备方法和应用。所述衍生物具有如下通式I所示的结构。本发明还涉及该类衍生物的制备方法及其作为HIV-1/HIV-2抑制剂在制备抗艾滋病药物中的应用,以及代表性化合物在人肝微粒体和血浆中的代谢稳定性结果。(The invention provides a benzothiazole-containing phenylalanine derivative and a preparation method and application thereof. The derivative has a structure shown in the following general formula I. The invention also relates to a preparation method of the derivative, application of the derivative as an HIV-1/HIV-2 inhibitor in preparing anti-AIDS drugs, and metabolic stability results of representative compounds in human liver microsomes and blood plasma.)

1. The phenylalanine derivative containing benzothiazole or pharmaceutically acceptable salt and ester thereof has a structure shown in a general formula I:

wherein the content of the first and second substances,

r is: c₁-C₆Alkyl, OC₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₃-C₆Cycloalkyl, OC₃-C₆Cycloalkyl, propargyl, pyridyl, phenyl, substituted benzyl, substituted naphthalene rings, various substituted six-membered heterocycles or various substituted five-membered heterocycles; the substituent is selected from halogen, methyl, methoxy, trifluoromethyl, acetyl, trifluoromethoxy, hydroxyl, cyano, nitro, amino and N-tert-butyloxycarbonyl.

2. The benzothiazole-containing phenylalanine derivative according to claim 1, wherein R is: OC₁-C₆Alkyl, propargyl, pyridyl, benzeneA group, a substituted phenyl group, various substituted six-membered heterocyclic rings; the substituent is selected from halogen, methyl, methoxy, trifluoromethyl, acetyl, trifluoromethoxy, hydroxyl, cyano, nitro, amino and N-tert-butyloxycarbonyl.

3. A benzothiazole-containing phenylalanine derivative according to claim 1 or 2, characterized in that the compound is one of the following:

4. the method for preparing a benzothiazole-containing phenylalanine derivative according to claim 3, comprising the steps of:

1, 3-benzothiazole-5-amine (1) is used as a starting material to react with paraformaldehyde, sodium methoxide and sodium borohydride to obtain an intermediate 2; carrying out amide condensation reaction on the intermediate 2 and N-tert-butyloxycarbonyl-L-phenylalanine to obtain an intermediate 3; removing Boc group from the intermediate 3 under the action of trifluoroacetic acid to obtain an intermediate 4; then the intermediate 4 and the monomethyl isophthalate are subjected to amide condensation reaction to obtain an intermediate 5; hydrolyzing the intermediate 5 by lithium hydroxide to remove ester groups to obtain an intermediate 6, and then performing amide condensation reaction with various substituted aromatic amines or aliphatic amines to obtain a target compound (7 a-t); further removing Boc group from the compound (7r-t) under the action of trifluoroacetic acid to obtain a target compound (7 u-w);

the synthetic route is as follows:

reagents and conditions: (i) paraformaldehyde, sodium methoxide, methanol, 60 ℃, 16h, sodium borohydride, room temperature, and 16 h; (ii) N-tert-butoxycarbonyl-L-phenylalanine, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane, and the temperature is changed to room temperature at 0 ℃; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) the monomethyl isophthalate, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are turned to room temperature at 0 ℃; (v) lithium hydroxide, tetrahydrofuran, water, room temperature; (vi) correspondingly substituted aromatic amine or aliphatic amine, O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are rotated to room temperature at 0 ℃; (vii) trifluoroacetic acid, dichloromethane, room temperature;

wherein R is phenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, 3-fluorophenyl, 2-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 4-acetylphenyl, 4-trifluoromethoxyphenyl, 4-cyanophenyl, propargyl, 2-methoxyethyl, 4-pyridyl, N-t-butoxycarbonyl-piperazinyl, N-t-butoxycarbonyl-piperidinyl, N-t-butoxycarbonyl-4-aminophenyl, N-t-butoxycarbonyl-3-aminophenyl, N-t-butoxycarbonyl-2-aminophenyl;

the substituted aromatic amine or aliphatic amine is aniline, 4-fluoroaniline, 4-bromoaniline, 4-chloroaniline, 3-fluoroaniline, 2-fluoroaniline, p-toluidine, p-methoxyaniline, p-trifluoromethylaniline, p-aminoacetophenone, p-trifluoromethoxyaniline, p-aminobenzonitrile, propargylamine, 2-methoxyethylamine, 4-aminopyridine, N-tert-butoxycarbonyl-piperazine, N-tert-butoxycarbonyl-4-aminopiperidine, N-tert-butoxycarbonyl-p-phenylenediamine, N-tert-butoxycarbonyl-m-phenylenediamine, N-tert-butoxycarbonyl-1, 2-phenylenediamine.

5. The method for preparing a benzothiazole-containing phenylalanine derivative according to claim 4, comprising the following steps:

(1) adding 1, 3-benzothiazole-5-amine (1) into methanol, then slowly adding paraformaldehyde, and slowly adding a methanol solution of sodium methoxide; reacting at 60 ℃ for 16h, then gradually cooling to room temperature, and then adding NaBH₄And continuously reacting for 16h at room temperature; after TLC monitoring reaction, filtering reaction liquid, evaporating under reduced pressure to remove a solvent, adding a saturated sodium chloride solution, extracting with ethyl acetate, combining organic phases, washing twice with the saturated sodium chloride solution, drying the organic phase with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 2;

(2) adding N-tert-butoxycarbonyl-L-phenylalanine and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate into dichloromethane to dissolve, stirring for 30min under an ice bath condition, dissolving the intermediate 2 into dichloromethane, and dropwise adding the dichloromethane into a reaction system by using a constant-pressure dropping funnel; then adding N, N-diisopropylethylamine into the reaction system, and returning to room temperature for reaction for 3 hours; TLC monitoring reaction, decompressing and distilling to remove solvent, adding saturated sodium chloride solution, and extracting with dichloromethane; combining organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 3;

(3) adding the intermediate 3 into dichloromethane to dissolve, then adding trifluoroacetic acid into the reaction system, and reacting for 4h at room temperature; after TLC monitoring reaction, decompressing and distilling off the solvent, then adding saturated sodium bicarbonate solution to adjust the pH of the reaction solution to 7, adding water, and then adding dichloromethane for extraction; combining organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, and evaporating the solvent to dryness under reduced pressure to obtain an intermediate 4;

(4) adding monomethyl isophthalate and O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate into dichloromethane to dissolve, then stirring for 30min under an ice bath condition, dissolving the intermediate 4 into dichloromethane, and dropwise adding the dichloromethane into a reaction system by using a constant-pressure dropping funnel; then, adding N, N-diisopropylethylamine into the reaction system, and returning to room temperature for reaction for 6 hours; TLC monitoring reaction, decompressing and distilling to remove solvent, adding saturated sodium chloride solution, and extracting with dichloromethane; combining organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 5;

(5) adding the intermediate 5 into tetrahydrofuran and water to dissolve, then adding lithium hydroxide into a reaction system, and reacting for 4 hours at room temperature; after TLC monitoring reaction, decompressing and distilling off tetrahydrofuran, then adjusting pH to about 3 by using 2mol/L hydrochloric acid, separating out white solid, and performing suction filtration and drying to obtain an intermediate 6;

(6) adding the intermediate 6 and O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate into dichloromethane for dissolving, then stirring for 30min under an ice bath condition, dissolving the amino derivatives with different substitutions into dichloromethane, and dropwise adding the amino derivatives into a reaction system by using a constant pressure dropping funnel; then, adding N, N-diisopropylethylamine into the reaction system, and returning to room temperature for reaction for 6 hours; after the TLC monitoring reaction, the solvent is evaporated under reduced pressure, and a saturated sodium chloride solution is added, and then dichloromethane is used for extraction twice; mixing organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain a pure product (7a-7t) of the target compound;

(7) adding the target compound (7r-t) into dichloromethane to dissolve the target compound, then adding trifluoroacetic acid into the reaction system, and reacting for 4h at room temperature; after TLC monitoring reaction, decompressing and distilling off the solvent, then adding saturated sodium bicarbonate solution to adjust the pH of the reaction solution to 7, adding water, and then adding dichloromethane for extraction; and combining organic phases, washing twice by using a saturated sodium chloride solution, drying the organic phases by using anhydrous magnesium sulfate, filtering, decompressing and evaporating a solvent to obtain a crude product of the target compound, and purifying by using a silica gel preparation plate to obtain a pure product (7u-7w) of the target compound.

6. Use of a benzothiazole-containing phenylalanine derivative according to any of claims 1-3 in the preparation of a medicament for the treatment and prevention of aids.

7. An anti-HIV pharmaceutical composition comprising a benzothiazole-containing phenylalanine derivative of any one of claims 1-3 or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers or excipients.

Technical Field

The invention belongs to the technical field of organic compound synthesis and medical application, and particularly relates to a benzothiazole-containing phenylalanine derivative and a preparation method and application thereof.

Background

Acquired Immune Deficiency Syndrome (AIDS), a chronic infectious disease caused by Human Immunodeficiency Virus (HIV). HIV belongs to the group of RNA retroviruses and is divided into two subtypes, HIV-1 and HIV-2, of which HIV-1 is currently the predominant pathogen. HIV-2 is mainly epidemic in Western Africa, but with the continuous development of globalization and the increasingly frequent exchange of people, the risk of HIV-2 infection is increasing, and the cases of HIV-2 infection are found in countries and regions such as the United states, Europe, south Africa, India and China, and sufficient attention should be paid. Currently, anti-HIV drugs remain effective weapons for the prevention and treatment of aids. To date, more than 30 chemical entities have been approved by the U.S. FDA for marketing against HIV-1 life cycle critical elements, targeting reverse transcriptase, protease, integrase, CCR5 coreceptors and membrane fusion processes, respectively. The high-efficiency Antiretroviral Therapy (HAART) prolongs the survival time of patients to a great extent and improves the life quality of the patients, but the problems of drug resistance, drug toxic and side effects, latent infection, high cost of long-term taking drugs and the like greatly reduce the efficacy of the Therapy, limit the application of the Therapy and further force researchers to develop new targets, new mechanisms and new structures of anti-AIDS drugs.

The HIV-1 capsid is assembled by cutting Gag precursor protein to obtain capsid protein monomer. During the conversion of immature viral particles into mature viral particles, the capsid proteins assemble into mature capsids, encapsulating viral RNA and nuclear-associated proteins (reverse transcriptase, protease, integrase, etc.) to form mature HIV-1 viral particles. The mature virus particle is infectious and the next round of replication of the virus can take place. In recent years, with the researchers' deep knowledge of capsid protein structure, their crystal structures have been reported in succession. Therefore, the capsid protein of HIV-1 can be used as the action target of a novel anti-HIV-1 medicament.

The Pfizer company obtains a compound PF74 which can obviously inhibit the replication of HIV-1 through high-throughput screening of a compound library, and the structure-activity relationship and mechanism research of the compound shows that the compound interferes with the uncoating process of viruses and the formation process of infectious particles by binding HIV-1 capsid protein. Although PF74 has novel structure, unique mechanism and definite target, PF74 has lower curative effect, poorer drug-like property and is easy to induce drug resistance compared with the anti-HIV-1 drugs on the market at present. Therefore, the development of capsid protein inhibitors that are more efficient and have good drug-like properties and prevent the development of drug resistance has become an attractive direction in the field of development of anti-aids drugs in recent years.

According to the crystal structure characteristics of the binding site of PF74 and HIV-1 capsid protein, the invention discovers the benzothiazole-containing phenylalanine HIV-1 capsid protein inhibitor with a brand-new structure through reasonable drug design, chemical synthesis and biological activity evaluation, and is expected to improve the problems of low curative effect, poor drug property and drug resistance of the existing HIV-1 capsid protein inhibitor. The inhibitor with the structure has good to excellent activity on HIV-2 and can be further modified as a lead compound.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a benzothiazole-containing phenylalanine derivative and a preparation method thereof, and also provides an activity screening result of the compound as an HIV-1/2 inhibitor and application thereof, and a metabolic stability result of a representative compound in human liver microsomes and plasma.

The technical scheme of the invention is as follows:

1. phenylalanine derivatives containing benzothiazole

The phenylalanine derivative containing benzothiazole or pharmaceutically acceptable salt and ester thereof has a structure shown in a general formula I:

wherein the content of the first and second substances,

r is: c₁-C₆Alkyl, OC₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₃-C₆Cycloalkyl, OC₃-C₆Cycloalkyl, propargyl, pyridyl, phenyl, substituted benzyl, substituted naphthalene rings, various substituted six-membered heterocycles or various substituted five-membered heterocycles; the substituent is selected from halogen, methyl, methoxy, trifluoromethyl, acetyl, trifluoromethoxy, hydroxyl, cyano, nitro, amino and N-tert-butyloxycarbonyl.

In accordance with a preferred aspect of the present invention,

r is: OC₁-C₆Alkyl, propargyl, pyridyl, phenyl, substituted phenyl, various substituted six-membered heterocycles; the substituent is selected from halogen, methyl, methoxy, trifluoromethyl, acetyl, trifluoromethoxy, hydroxyl, cyano, nitro, amino and N-tert-butyloxycarbonyl.

According to a further preferred embodiment of the invention, the benzothiazole-containing phenylalanine derivative is one of the following compounds:

as used herein, "pharmaceutically acceptable salts" means salts of the compounds which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue toxicity, irritation, and allergic response and the like, are commensurate with a reasonable benefit-to-risk ratio, are generally water or oil soluble or dispersible, and are effective for their intended use. Including pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are contemplated herein and are compatible with the chemical nature of the compounds of formula I. A list of suitable salts is found on pages 1-19 of s.m. berge et al, j.pharm.sci.,1977, 66.

2. Preparation method of benzothiazole-containing phenylalanine derivative

A method for preparing a benzothiazole-containing phenylalanine derivative, comprising the steps of: 1, 3-benzothiazole-5-amine (1) is used as a starting material to react with paraformaldehyde, sodium methoxide and sodium borohydride to obtain an intermediate 2; carrying out amide condensation reaction on the intermediate 2 and N-tert-butyloxycarbonyl-L-phenylalanine to obtain an intermediate 3; removing Boc group from the intermediate 3 under the action of trifluoroacetic acid to obtain an intermediate 4; then the intermediate 4 and the monomethyl isophthalate are subjected to amide condensation reaction to obtain an intermediate 5; hydrolyzing the intermediate 5 by lithium hydroxide to remove ester groups to obtain an intermediate 6, and then performing amide condensation reaction with various substituted aromatic amines or aliphatic amines to obtain a target compound (7 a-t); the Boc group of the compound (7r-t) is further removed under the action of trifluoroacetic acid to obtain a target compound (7 u-w).

The synthetic route is as follows:

reagents and conditions: (i) paraformaldehyde, sodium methoxide, methanol, 60 ℃, 16h, sodium borohydride, room temperature, and 16 h; (ii) N-tert-butoxycarbonyl-L-phenylalanine, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane, and the temperature is changed to room temperature at 0 ℃; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) the monomethyl isophthalate, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are turned to room temperature at 0 ℃; (v) lithium hydroxide, tetrahydrofuran, water, room temperature; (vi) correspondingly substituted aromatic amine or aliphatic amine, O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are rotated to room temperature at 0 ℃; (vii) trifluoroacetic acid, dichloromethane, room temperature.

Wherein R is phenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl, 3-fluorophenyl, 2-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 4-acetylphenyl, 4-trifluoromethoxyphenyl, 4-cyanophenyl, propargyl, 2-methoxyethyl, 4-pyridyl, N-tert-butoxycarbonyl-piperazinyl, N-tert-butoxycarbonyl-piperidinyl, N-tert-butoxycarbonyl-4-aminophenyl, N-tert-butoxycarbonyl-3-aminophenyl, N-tert-butoxycarbonyl-2-aminophenyl.

The substituted aromatic amine or aliphatic amine is aniline, 4-fluoroaniline, 4-bromoaniline, 4-chloroaniline, 3-fluoroaniline, 2-fluoroaniline, p-toluidine, p-methoxyaniline, p-trifluoromethylaniline, p-aminoacetophenone, p-trifluoromethoxyaniline, p-aminobenzonitrile, propargylamine, 2-methoxyethylamine, 4-aminopyridine, N-tert-butoxycarbonyl-piperazine, N-tert-butoxycarbonyl-4-aminopiperidine, N-tert-butoxycarbonyl-p-phenylenediamine, N-tert-butoxycarbonyl-m-phenylenediamine, N-tert-butoxycarbonyl-1, 2-phenylenediamine.

The room temperature of the invention is 20-30 ℃.

According to the preferred preparation method of the benzothiazole-containing phenylalanine derivative, the specific steps are as follows:

(1) 1, 3-benzothiazol-5-amine (1) was added to methanol, then paraformaldehyde was slowly added, and then sodium methoxide in methanol was slowly added. Reacting at 60 ℃ for 16h, then gradually cooling to room temperature, and then adding NaBH₄And the reaction was continued at room temperature for 16 h. After TLC monitoring reaction, filtering reaction liquid, evaporating under reduced pressure to remove a solvent, adding a saturated sodium chloride solution, extracting with ethyl acetate, combining organic phases, washing twice with the saturated sodium chloride solution, drying the organic phase with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 2;

(2) adding N-tert-butoxycarbonyl-L-phenylalanine and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate into dichloromethane to dissolve, stirring for 30min under an ice bath condition, dissolving the intermediate 2 into dichloromethane, and dropwise adding the dichloromethane into a reaction system by using a constant-pressure dropping funnel; then adding N, N-diisopropylethylamine into the reaction system, and returning to room temperature for reaction for 3 h. After the completion of the TLC monitoring reaction, the solvent was distilled off under reduced pressure, and a saturated sodium chloride solution was added to conduct extraction with methylene chloride. Combining organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 3;

(3) the intermediate 3 was dissolved by adding it to methylene chloride, and then trifluoroacetic acid was added to the reaction system to react at room temperature for 4 hours. After the completion of the TLC monitoring reaction, the solvent was distilled off under reduced pressure, and then a saturated sodium bicarbonate solution was added to adjust the pH of the reaction solution to 7, water was added, and dichloromethane was added for extraction. Combining organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, and evaporating the solvent to dryness under reduced pressure to obtain an intermediate 4;

(4) adding monomethyl isophthalate and O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate into dichloromethane to dissolve, then stirring for 30min under an ice bath condition, dissolving the intermediate 4 into dichloromethane, and dropwise adding the dichloromethane into a reaction system by using a constant-pressure dropping funnel; then, N-diisopropylethylamine was added to the reaction system, and the reaction was returned to room temperature for 6 hours. After the completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and a saturated sodium chloride solution was added and extracted with dichloromethane. Combining organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 5;

(5) the intermediate 5 was dissolved by adding it to tetrahydrofuran and water, and then lithium hydroxide was added to the reaction system to react at room temperature for 4 hours. After TLC monitoring reaction, decompressing and distilling off tetrahydrofuran, then adjusting pH to about 3 by using 2mol/L hydrochloric acid, separating out white solid, and performing suction filtration and drying to obtain an intermediate 6;

(6) adding the intermediate 6 and O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate into dichloromethane for dissolving, then stirring for 30min under an ice bath condition, dissolving the amino derivatives with different substitutions into dichloromethane, and dropwise adding the amino derivatives into a reaction system by using a constant pressure dropping funnel; then, N-diisopropylethylamine was added to the reaction system, and the reaction was returned to room temperature for 6 hours. After the completion of the TLC monitoring reaction, the solvent was distilled off under reduced pressure, and a saturated sodium chloride solution was added, followed by extraction with dichloromethane twice. Mixing organic phases, washing twice with saturated sodium chloride solution, drying the organic phases with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain a pure product (7a-7t) of the target compound;

(7) the objective compound (7r-t) was dissolved by adding it to methylene chloride, and then trifluoroacetic acid was added to the reaction system and reacted at room temperature for 4 hours. After the completion of the TLC monitoring reaction, the solvent was distilled off under reduced pressure, and then a saturated sodium bicarbonate solution was added to adjust the pH of the reaction solution to 7, water was added, and dichloromethane was added for extraction. And combining organic phases, washing twice by using a saturated sodium chloride solution, drying the organic phases by using anhydrous magnesium sulfate, filtering, decompressing and evaporating a solvent to obtain a crude product of the target compound, and purifying by using a silica gel preparation plate to obtain a pure product (7u-7w) of the target compound.

3. Application of benzothiazole-containing phenylalanine derivative

The invention discloses a screening result of anti-HIV-1/2 activity of a benzothiazole-containing phenylalanine derivative and application of the phenylalanine derivative as an HIV-1/2 inhibitor for the first time. Experiments prove that the benzothiazole-containing phenylalanine derivative can be used as an HIV-1/2 inhibitor for preparing anti-AIDS medicaments. The invention also provides application of the compound in preparing anti-HIV-1/2 medicaments.

anti-HIV-1/2 Activity and toxicity test of the target Compound

A class of benzothiazole-containing phenylalanine derivatives synthesized according to the above method were tested for anti-HIV-1/2 activity and toxicity at cellular level, and their anti-HIV-1 and anti-HIV-2 activity and toxicity data are shown in Table 1, with the capsid protein inhibitor PF74 reported in literature as a positive control.

The newly synthesized benzothiazole-containing phenylalanine derivativeExhibit certain anti-HIV-1 activity, wherein the compound 7u (EC)₅₀＝3.57±0.15μM,CC₅₀57.36 ± 9.82 μ M, SI 16) and 7M (EC)₅₀＝5.02±1.00μM,CC₅₀133.61 ± 4.8 μ M, SI ═ 27) exhibited single digit micromolar levels of anti-HIV-1 activity. Notably, 16 compounds had anti-HIV-2 activity in the 0.85-3.85 μ M range, which was superior to the positive control PF74 (EC)₅₀＝4.16±0.86μM,CC₅₀32.27 ± 1.25 μ M, SI ═ 8), where the anti-HIV-2 activity and selectivity (EC) of the target compound 7M was₅₀＝0.85±0.24μM,CC₅₀133.61 ± 4.8 μ M, SI 157) is particularly prominent and of further interest. Meanwhile, the metabolic stability of the representative compounds 7u and 7m in human liver microsomes and plasma is superior to that of the lead PF 74. Particularly, 7u (t) is_1/254.6min) has a half-life in human liver microsomes that is greater than that of PF74 (t)_1/20.5min) is increased by 109 times, and can be further modified as a lead compound to obtain the anti-AIDS drug with greatly improved activity and metabolic stability.

The benzothiazole-containing phenylalanine derivatives can be used as HIV-1/2 inhibitors. In particular to the application of the compound as an HIV-1/2 inhibitor in preparing anti-AIDS drugs.

An anti-HIV-1/2 pharmaceutical composition comprising a benzothiazole-containing phenylalanine derivative of the present invention and one or more pharmaceutically acceptable carriers or excipients.

The invention provides a benzothiazole-containing phenylalanine derivative and a preparation method thereof, and also provides a screening result of anti-HIV-1/2 activity of partial compounds and the first application thereof in the field of antivirus. Tests prove that the benzothiazole-containing phenylalanine derivatives can be used as HIV-1/2 inhibitors and have high application value. In particular to the application of the compound as an HIV-1/2 inhibitor in preparing anti-AIDS drugs.

Detailed Description

The invention will be understood by the following examples, which are given by way of illustration and are not intended to limit the scope of the invention.

Example 1: preparation of intermediate N-methylbenzo [ d ] thiazol-5-amine (2)

1, 3-benzothiazol-5-amine (0.5g, 3.33mmol) was added to a 100mL round bottom flask, and 20mL of methanol was added, followed by the slow addition of paraformaldehyde (0.22g, 6.66mmol), and the slow addition of sodium methoxide (0.90g, 16.64mmol) in methanol. Reacting at 60 ℃ for 16h, then gradually cooling to room temperature, and then adding NaBH₄(0.31g, 8.32mmol) and reaction continued at room temperature for 16 h. After completion of TLC monitoring, the reaction mixture was filtered, the solvent was evaporated under reduced pressure, 30mL of water was added, extraction was performed with ethyl acetate (30 mL. times.3), the organic phases were combined, washed with a saturated sodium chloride solution (20 mL. times.2), the organic phase was dried over anhydrous magnesium sulfate, filtration was performed, the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by silica gel column chromatography (eluent EA: PE ═ 1:8+ 1% TEA) to give N-methylbenzo [ d ] as an intermediate]0.43g of crude thiazol-5-amine (2) was obtained in 78.6% yield as yellow oil.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ9.20(s,1H),7.77(d,J＝8.7Hz,1H),7.08(d,J＝2.2Hz,1H),6.83(dd,J＝8.7,2.2Hz,1H),5.92(d,J＝5.2Hz,1H),2.75(d,J＝5.1Hz,3H).

C₈H₈N₂S[M+H]⁺(165.04),ESI-MS m/z:165.00(M+1).

example 2: preparation of intermediate tert-butyl (S) - (1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamate (3)

After N-t-butoxycarbonyl-L-phenylalanine (1.94g, 7.31mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (4.75g, 9.13mmol) and 30mL of dichloromethane were added to a 100mL round-bottom flask and dissolved, followed by stirring under ice bath conditions for 30min, intermediate 2(1.0g, 6.09mmol) was dissolved in 10mL of dichloromethane and added dropwise to the reaction system using a constant pressure dropping funnel; n, N-diisopropylethylamine (2.83g, 21.87mmol) was then added to the reaction and the reaction was allowed to return to room temperature for 3 h. After completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and 30mL of a saturated sodium chloride solution was added to conduct extraction with methylene chloride (30 mL. times.3). The organic phases were combined, washed with a saturated sodium chloride solution (20mL × 2), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by silica gel column chromatography (eluent EA: PE ═ 1:4) to give 1.9g of a crude product of intermediate t-butyl (S) - (1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamate (3) as a yellow solid in a yield of 76.0%.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ9.50(s,1H),8.27(d,J＝8.6Hz,1H),7.99(s,1H),7.38(d,J＝8.6Hz,1H),7.17(d,J＝8.1Hz,1H),7.09(d,J＝7.2Hz,3H),6.73(d,J＝7.0Hz,2H),4.18(q,J＝7.3Hz,1H),3.24(s,3H),2.84(dd,J＝13.7,4.5Hz,1H),2.65(dd,J＝13.6,9.7Hz,1H),1.31(s,9H).

C₂₂H₂₅N₃O₃S[M+H]⁺(412.16),ESI-MS m/z:411.87[M+H]⁺；433.99[M+Na]⁺

example 3: preparation of intermediate (S) -2-amino-N- (benzothiazol-5-yl) -N-methyl-3-phenylpropylamine (4)

Intermediate 3(1.0g, 2.43mmol) was charged into a 100mL round-bottomed flask and dissolved by adding 20mL of dichloromethane, followed by adding 4mL of trifluoroacetic acid to the reaction system and reacting at room temperature for 4 hours. After completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and then the reaction solution was adjusted to pH 7 with saturated sodium bicarbonate solution, and 30mL of water was added and extracted with dichloromethane (30 mL. times.3). The organic phases were combined, washed with a saturated sodium chloride solution (20 mL. times.2), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give 0.6g of crude intermediate (S) -2-amino-N- (benzothiazol-5-yl) -N-methyl-3-phenylpropylamine (4) as a yellow oil in 79.3% yield.

Spectral data:

C₁₇H₁₇N₃OS[M+H]⁺(312.11),ESI-MS m/z:312.02[M+H]⁺

example 4: preparation of intermediate methyl (S) -3- ((1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamoyl) benzoate (5)

To a 100mL round-bottom flask was added monomethyl isophthalate (0.48g, 2.67mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (1.27g, 3.33mmol), and dissolved by addition of 30mL dichloromethane, followed by stirring under ice bath conditions for 30min, after which intermediate 4(0.69g, 2.22mmol) was dissolved in 10mL dichloromethane and added dropwise to the reaction using a constant pressure dropping funnel; after that, N-diisopropylethylamine (0.57g, 4.44mmol) was added to the reaction system, and the reaction was returned to room temperature for 6 hours. After the completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and 30mL of a saturated sodium chloride solution was added and extracted with methylene chloride (30 mL. times.3). The organic phases were combined, washed with a saturated sodium chloride solution (20mL × 2), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by silica gel column chromatography (eluent EA: PE ═ 1:3) to give 0.64g of intermediate (S) -methyl 3- ((1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamoyl) benzoate (5) as a yellow solid in a yield of 60.8%.

Spectral data:

C₂₆H₂₃N₃O₄S[M+H]⁺(474.14),ESI-MS m/z:474.02[M+H]⁺

example 5: preparation of intermediate (S) -3- ((1- (benzo [ d ] thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamoyl) benzoic acid (6)

Intermediate 5(0.63g, 1.33mmol) was charged into a 100mL round-bottomed flask, and 10mL of tetrahydrofuran and 10mL of water were added to dissolve it, followed by adding lithium hydroxide (63.7mg, 2.66mmol) to the reaction system and reacting at room temperature for 4 hours. After the reaction was monitored by TLC, the solvent was evaporated under reduced pressure, then pH was adjusted to about 3 with 2mol/L hydrochloric acid to precipitate a white solid, which was filtered and dried to obtain 0.47g of intermediate (S) -3- ((1- (benzo [ d ] thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamoyl) benzoic acid (6) as a white solid in a yield of 76.9%.

Spectral data:

C₂₅H₂₁N₃O₄S[M+H]⁺(460.13),ESI-MS m/z:460.05[M+H]⁺

example 6: preparation of target Compound (7a-t)

Intermediate 6(0.1g, 0.2176mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (0.1241g, 0.3264mmol) were added to a 50mL round bottom flask and dissolved by the addition of 15mL of dichloromethane, followed by stirring under ice bath conditions for 30min, after which the variously substituted amino derivative (0.2611mmol) was dissolved in 10mL of dichloromethane and added dropwise to the reaction using an isopiestic dropping funnel; then, N-diisopropylethylamine (0.0844g, 0.6529mmol) was added to the reaction system, and the reaction was returned to room temperature for 6 hours. After the completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and 30mL of a saturated sodium chloride solution was added, followed by extraction with methylene chloride (30 mL. times.3). And combining organic phases, washing with a saturated sodium chloride solution (20mL multiplied by 2), drying with anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure, and performing silica gel column chromatography to obtain a pure target compound (7 a-w).

The corresponding substituted aromatic amine is prepared by reacting aniline with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³Phenylalanine (7a), white powdery solid, yield 81%. Melting point 102-103 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.39(s,1H),9.52(s,1H),8.96(d,J＝7.6Hz,1H),8.41(s,1H),8.30(d,J＝8.5Hz,1H),8.19–8.05(m,2H),8.02(d,J＝7.8Hz,1H),7.80(d,J＝8.0Hz,2H),7.62(t,J＝7.7Hz,1H),7.49(d,J＝8.4Hz,1H),7.41–7.34(m,2H),7.12(q,J＝6.2,5.1Hz,4H),6.86(d,J＝4.9Hz,2H),4.68(d,J＝8.2Hz,1H),3.34(d,J＝1.5Hz,3H),3.02(qd,J＝13.7,7.0Hz,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.67,166.31,165.62,158.58,154.21,141.81,139.56,138.36,135.65,134.37,133.57,130.95,130.82,129.21,129.14,128.85,128.61,127.45,126.91,125.69,124.26,123.84,122.62,120.83,53.24,38.05,37.00.

C₃₁H₂₆N₄O₃S[M+H]⁺(535.17),ESI-MS m/z:535.03[M+H]⁺

the corresponding substituted aromatic amine is 4-fluoroaniline and intermediate 6, O- (7-azabenzotriazole-1-yl) -NReacting N, N ', N' -tetramethylurea hexafluorophosphate with N, N-diisopropylethylamine to obtain (S) -N¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (4-fluorophenyl) isophthalamide (7b), white powdery solid, yield 80%. Melting point 111-112 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.45(s,1H),9.52(s,1H),8.96(d,J＝7.6Hz,1H),8.41(s,1H),8.29(d,J＝8.5Hz,1H),8.16–8.05(m,2H),8.02(d,J＝7.8Hz,1H),7.82(dd,J＝8.7,5.0Hz,2H),7.62(t,J＝7.7Hz,1H),7.49(d,J＝8.4Hz,1H),7.22(t,J＝8.7Hz,2H),7.11(d,J＝5.2Hz,3H),6.97–6.76(m,2H),4.69(s,1H),3.28(s,3H),3.02(qd,J＝13.8,7.1Hz,2H).

¹³C-NMR(100MHz,DMSO-d₆):δ171.66,166.30,165.53,160.05,158.49,157.66,154.20,141.81,138.31,135.91,135.88,135.47,134.43,130.90,130.85,129.21,128.86,128.60,127.41,126.91,125.65,123.80,122.75,122.67,53.20,38.06,37.09.

C₃₁H₂₅FN₄O₃S[M+H]⁺(553.16),ESI-MS m/z:553.03[M+H]⁺

the corresponding substituted aromatic amine is prepared by reacting 4-bromoaniline with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylprop-2-yl) -N³- (4-bromophenyl) isophthalamide (7c), white powdery solid, yield 76%. Melting point 103-105 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.52(s,1H),9.52(s,1H),8.96(d,J＝7.6Hz,1H),8.41(s,1H),8.29(d,J＝8.5Hz,1H),8.18–7.96(m,3H),7.83–7.76(m,2H),7.63(t,J＝7.8Hz,1H),7.59–7.54(m,2H),7.49(d,J＝8.3Hz,1H),7.10(d,J＝5.4Hz,3H),6.85(d,J＝6.3Hz,2H),4.69(s,1H),3.28(s,3H),3.03(td,J＝16.4,15.2,7.4Hz,2H).

¹³C-NMR(100MHz,DMSO-d₆):δ171.66,166.28,165.73,158.51,154.18,141.79,138.93,138.30,135.36,134.42,133.56,131.96,130.97,129.21,128.90,128.60,127.46,126.91,125.65,123.81,122.74,122.59,115.98,53.21,38.06,37.06.

C₃₁H₂₅BrN₄O₃S[M+H]⁺(613.08),ESI-MS m/z:613.01[M+H]⁺

the corresponding substituted aromatic amine is prepared by reacting 4-chloroaniline with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (4-chlorophenyl) isophthalamide (7d), white powdery solid, yield 75%. Melting point 134-137 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.52(s,1H),9.52(s,1H),8.96(d,J＝7.6Hz,1H),8.41(s,1H),8.29(d,J＝8.5Hz,1H),8.05(dd,J＝20.4,8.0Hz,3H),7.85(d,J＝8.4Hz,2H),7.63(t,J＝7.8Hz,1H),7.44(d,J＝8.3Hz,3H),7.11(s,3H),6.85(d,J＝6.3Hz,2H),4.68(q,J＝7.3Hz,1H),3.28(s,3H),3.15–2.90(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.66,166.27,165.71,158.55,154.20,141.80,138.54,138.34,135.37,134.41,133.57,130.97,129.21,129.06,128.90,128.61,127.87,127.47,126.91,125.67,123.83,122.61,122.34,53.23,38.05,37.02.

C₃₁H₂₅ClN₄O₃S[M+H]⁺(568.13),ESI-MS m/z:568.05[M+H]⁺

the corresponding substituted aromatic amine is prepared by reacting 3-fluoroaniline with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (3-fluorophenyl) isophthalamide (7e), white powdery solid, yield 65%. Melting point 104-105 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.58(s,1H),9.52(s,1H),8.97(d,J＝7.6Hz,1H),8.42(s,1H),8.30(d,J＝8.5Hz,1H),8.07(dt,J＝17.9,7.8Hz,3H),7.78(dd,J＝11.8,2.2Hz,1H),7.62(dd,J＝19.7,8.1Hz,2H),7.42(q,J＝7.8Hz,2H),7.11(s,3H),6.96(t,J＝8.5Hz,1H),6.85(d,J＝6.3Hz,2H),4.76–4.60(m,1H),3.28(s,3H),3.02(qd,J＝13.7,7.2Hz,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.66,166.26,165.89,158.55,154.20,141.81,141.39,141.28,138.35,135.30,134.43,133.57,131.03,130.82,130.72,129.21,128.92,128.61,127.50,126.91,125.67,123.83,122.61,116.46,110.81,110.60,107.59,107.32,53.24,38.06,37.01.

C₃₁H₂₅FN₄O₃S[M+H]⁺(553.16),ESI-MS m/z:578.17[M+Na]⁺

the corresponding substituted aromatic amine is prepared by reacting 2-fluoroaniline with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (2-fluorophenyl) isophthalamide (7f), white powdery solid, yield 82%. Melting point 105-107 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.29(s,1H),9.55(s,1H),8.99(d,J＝7.6Hz,1H),8.48(s,1H),8.33(d,J＝8.5Hz,1H),8.14(d,J＝7.4Hz,2H),8.06(d,J＝7.8Hz,1H),7.68(q,J＝8.4Hz,2H),7.52(d,J＝8.4Hz,1H),7.38–7.27(m,3H),7.15(d,J＝5.1Hz,3H),6.90(d,J＝6.2Hz,2H),4.73(d,J＝7.2Hz,1H),3.32(s,3H),3.14–2.96(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.64,166.27,165.51,154.99,154.20,141.79,138.32,134.58,134.45,133.57,131.11,131.04,129.22,128.94,128.61,127.57,127.49,126.92,126.18,126.06,125.66,124.83,124.80,123.83,122.60,116.44,116.24,53.18,38.05,37.09.

C₃₁H₂₅FN₄O₃S[M+H]⁺(553.16),ESI-MS m/z:553.04[M+H]⁺

the corresponding substituted aromatic amine is prepared by the reaction of p-toluidine with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (p-tolyl) isophthalamide (7g), white powdery solid, yield 81%. Melting point 132-.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.30(s,1H),9.51(s,1H),8.94(d,J＝7.5Hz,1H),8.40(s,1H),8.29(d,J＝8.5Hz,1H),8.18–8.04(m,2H),8.00(d,J＝7.8Hz,1H),7.68(d,J＝8.0Hz,2H),7.61(t,J＝7.8Hz,1H),7.55–7.42(m,1H),7.18(d,J＝8.0Hz,2H),7.11(d,J＝5.4Hz,3H),6.85(d,J＝6.3Hz,2H),4.69(s,1H),3.28(s,3H),3.12–2.93(m,2H),2.30(s,3H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.66,166.33,165.40,158.55,154.20,141.80,138.35,137.03,135.72,134.36,133.56,133.22,130.89,130.73,129.52,129.21,128.82,128.60,127.39,126.91,125.68,123.83,122.61,120.85,53.21,38.05,37.03,20.98.

C₃₂H₂₈N₄O₃S[M+H]⁺(549.19),ESI-MS m/z:549.07[M+H]⁺

the corresponding substituted aromatic amine is prepared by the reaction of p-anisidine, an intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (4-methoxyphenyl) isophthalamide (7h), white powdery solid, yield 80%. Melting point 143-144 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.27(s,1H),9.52(s,1H),8.95(d,J＝7.6Hz,1H),8.40(s,1H),8.30(d,J＝8.5Hz,1H),8.17–7.93(m,3H),7.79–7.67(m,2H),7.61(t,J＝7.7Hz,1H),7.55–7.42(m,1H),7.11(d,J＝5.2Hz,3H),7.04–6.92(m,2H),6.85(d,J＝4.5Hz,2H),4.69(t,J＝7.6Hz,1H),3.76(d,J＝1.5Hz,3H),3.28(s,3H),3.01(qd,J＝14.6,14.0,7.5Hz,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.67,166.35,165.16,158.51,156.13,154.20,138.33,135.72,134.38,133.55,132.61,130.82,130.65,129.21,128.80,128.60,127.33,126.90,125.66,123.80,122.59,122.46,114.28,55.67,53.20,38.06,37.07.

C₃₂H₂₈N₄O₄S[M+H]⁺(565.18),ESI-MS m/z:588.20[M+Na]⁺

the corresponding substituted aromatic amine is prepared by the reaction of p-trifluoromethylaniline, an intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (4- (trifluoromethyl) phenyl) isophthalamide (7i), white powdery solid, yield 79%. Melting point 134-136 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.74(s,1H),9.52(s,1H),8.98(d,J＝7.6Hz,1H),8.43(s,1H),8.30(d,J＝8.6Hz,1H),8.07(dd,J＝23.9,8.2Hz,5H),7.76(d,J＝8.4Hz,2H),7.64(t,J＝7.8Hz,1H),7.49(s,1H),7.11(s,3H),6.85(d,J＝6.3Hz,2H),4.68(s,1H),3.28(s,3H),3.11–2.89(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.65,166.23,166.11,165.07,158.50,154.20,143.21,141.81,138.32,135.17,134.48,133.55,131.14,131.08,129.22,128.93,128.60,127.58,126.90,126.45,126.41,126.21,125.65,124.40,124.09,123.80,123.52,122.60,120.65,53.22,38.06,37.08.

C₃₂H₂₅F₃N₄O₃S[M+H]⁺(603.16),ESI-MS m/z:603.03[M+H]⁺

the corresponding substituted aromatic amine is prepared by the reaction of p-aminoacetophenone, intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (4-acetylphenyl) -N³- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxy-3-ph phenylpropanol-2-yl) isopropanol (7j), white powdery solid, yield 74%. Melting point 103-104 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.71(s,1H),9.51(s,1H),8.97(d,J＝7.6Hz,1H),8.43(s,1H),8.30(d,J＝8.5Hz,1H),8.10(d,J＝7.5Hz,2H),7.99(q,J＝8.6Hz,5H),7.64(t,J＝7.8Hz,1H),7.49(d,J＝8.5Hz,1H),7.11(d,J＝5.2Hz,3H),6.85(d,J＝6.2Hz,2H),4.69(d,J＝7.0Hz,1H),3.28(s,3H),3.14–2.93(m,2H),2.57(s,3H).

¹³C-NMR(100Hz,DMSO-d₆):δ197.11,171.65,166.24,166.06,158.59,154.20,143.97,141.78,138.34,135.24,134.40,133.57,132.60,131.13,129.83,129.20,128.95,128.62,127.60,126.92,125.67,123.85,122.62,119.95,53.26,38.05,36.97,26.97.

C₃₃H₂₈N₄O₄S[M+H]⁺(576.18),ESI-MS m/z:576.01[M+H]⁺

the corresponding substituted aromatic amine is prepared by the reaction of p-trifluoro-methoxyaniline, intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (4- (trifluoromethoxy) phenyl) isophthalamide (7k), white powdery solid, yield 71%. Melting point 112-113 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.58(s,1H),9.52(s,1H),8.97(d,J＝7.6Hz,1H),8.42(s,1H),8.29(d,J＝8.5Hz,1H),8.09(d,J＝8.3Hz,2H),8.04(d,J＝7.8Hz,1H),7.98–7.86(m,2H),7.63(t,J＝7.9Hz,1H),7.49(d,J＝8.4Hz,1H),7.40(d,J＝8.5Hz,2H),7.11(d,J＝5.3Hz,3H),6.85(d,J＝6.3Hz,2H),4.69(d,J＝7.4Hz,1H),3.28(s,3H),3.14–2.90(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.66,166.27,165.76,158.55,154.20,144.44,141.80,138.77,138.35,135.31,134.43,133.57,131.00,129.21,128.90,128.61,127.50,126.91,125.67,123.83,122.61,122.18,121.99,119.37,53.25,38.05,37.01.

C₃₂H₂₅F₃N₄O₄S[M+H]⁺(618.15),ESI-MS m/z:618.04[M+H]⁺

the corresponding substituted aromatic amine is prepared by the reaction of p-aminobenzonitrile, an intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (4-cyanophenyl) isophthalamide (7l), white powdery solid, yield 68%. Melting point 171-172 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.78(s,1H),9.50(s,1H),8.96(d,J＝7.5Hz,1H),8.42(s,1H),8.28(d,J＝8.6Hz,1H),8.09(d,J＝7.8Hz,2H),8.01(dd,J＝8.8,1.7Hz,3H),7.86–7.82(m,2H),7.64(t,J＝7.8Hz,1H),7.47(d,J＝8.5Hz,1H),7.10(d,J＝5.5Hz,3H),6.85(d,J＝6.3Hz,2H),4.69(t,J＝8.4Hz,1H),3.27(s,3H),3.11–2.93(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.64,166.24,166.19,158.54,154.20,143.88,141.80,138.33,135.04,134.47,133.65,131.26,131.14,129.21,128.98,128.61,127.63,126.91,125.65,123.83,122.61,120.72,119.52,105.99,53.25,38.06,37.02.

C₃₂H₂₅N₅O₃S[M+H]⁺(560.17),ESI-MS m/z:594.02[M+Cl]^-

the corresponding substituted aromatic amine is prepared by the reaction of propargylamine, an intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (Prop-2-yl-1-yl) isophthalamide (7m), white powdery solid, yield 76%. Melting point 134-135 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ9.51(s,1H),9.04(t,J＝5.6Hz,1H),8.89(d,J＝7.6Hz,1H),8.42–8.22(m,2H),8.08(s,1H),7.97(t,J＝7.6Hz,2H),7.56(t,J＝7.7Hz,1H),7.47(d,J＝8.1Hz,1H),7.11(d,J＝4.9Hz,3H),6.97–6.72(m,2H),4.67(d,J＝7.0Hz,1H),4.09(dd,J＝5.7,2.4Hz,2H),3.27(s,3H),3.16(s,1H),3.08–2.91(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.62,166.28,166.01,158.54,154.19,141.78,138.30,134.44,134.39,133.55,130.73,130.50,129.21,128.84,128.61,127.10,126.91,125.66,123.82,122.58,81.67,73.43,53.13,38.05,37.10,29.05.

C₂₈H₂₄N₄O₃S[M+H]⁺(497.16),ESI-MS m/z:497.06[M+H]⁺

The corresponding substituted fatty amine is prepared by reacting 2-methoxy ethylamine with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (2-methoxyethyl) isophthalamide (7n), white powdery solid, yield 80%. Melting point 105-106 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ9.50(s,1H),8.85(d,J＝7.6Hz,1H),8.60(d,J＝5.7Hz,1H),8.28(d,J＝14.2Hz,2H),8.07(s,1H),7.95(dd,J＝13.1,7.7Hz,2H),7.54(t,J＝7.7Hz,1H),7.46(d,J＝8.3Hz,1H),7.10(d,J＝5.2Hz,3H),6.84(d,J＝6.2Hz,2H),4.67(d,J＝7.4Hz,1H),3.47(t,J＝5.1Hz,4H),3.31–3.21(m,6H),3.09–2.91(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.65,166.38,166.30,158.52,154.19,141.80,138.32,135.02,134.31,133.55,130.49,130.43,129.21,128.71,128.60,126.97,126.90,125.65,123.81,122.59,70.93,58.41,53.13,39.54,38.05,37.11.

C₂₈H₂₈N₄O₄S[M+H]⁺(517.18),ESI-MS m/z:517.04[M+H]⁺

The corresponding substituted aromatic amine is prepared by reacting 4-aminopyridine with intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine¹- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) -N³- (pyridin-4-yl) isophthalamide (7o), white powdery solid, yield 80%. Melting point 143-145 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ10.75(s,1H),9.51(s,1H),8.97(d,J＝7.5Hz,1H),8.51(d,J＝5.4Hz,2H),8.43(s,1H),8.29(d,J＝8.5Hz,1H),8.08(dd,J＝16.3,7.5Hz,3H),7.82(d,J＝5.5Hz,2H),7.65(t,J＝7.8Hz,1H),7.48(d,J＝8.5Hz,1H),7.11(d,J＝5.3Hz,3H),6.86(d,J＝6.2Hz,2H),4.70(t,J＝7.6Hz,1H),3.28(s,3H),3.02(dq,J＝23.0,12.8,10.9Hz,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.64,166.55,166.19,158.54,154.20,150.79,146.37,141.80,138.33,134.90,134.48,133.57,131.33,131.15,129.21,128.99,128.61,127.64,126.92,125.66,123.82,122.60,114.53,53.25,38.06,37.04.

C₃₀H₂₅N₅O₃S[M+H]⁺(536.17),ESI-MS m/z:536.05[M+H]⁺

The corresponding substituted aliphatic amine is N-tert-butyloxycarbonyl-piperazine, and the intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine react to prepare tert-butyl (S) -4- (3- (((1- (benzo [ d ] thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamoyl) benzoyl) piperazine-1-carboxylate (7p) which is white powdery solid with the yield of 77% and the melting point of 152-.

¹H-NMR(400MHz,DMSO-d₆):δ9.51(s,1H),8.89(d,J＝7.7Hz,1H),8.30(d,J＝8.5Hz,1H),8.11(s,1H),7.98–7.81(m,2H),7.66–7.42(m,3H),7.09(d,J＝5.3Hz,3H),6.82(d,J＝6.3Hz,2H),4.65(d,J＝9.4Hz,1H),3.62(s,8H),3.27(s,3H),3.01(td,J＝16.9,15.4,7.5Hz,2H),1.42(s,9H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.62,169.08,166.05,158.49,154.33,138.37,136.17,134.27,130.27,129.17,129.01,128.56,126.86,126.42,122.58,79.73,53.17,38.71,38.06,36.98,28.50.

C₃₄H₃₇N₅O₅S[M+H]⁺(628.25),ESI-MS m/z:628.13[M+H]⁺

The corresponding substituted aliphatic amine is N-tert-butyloxycarbonyl-4-aminopiperidine, an intermediate 6, O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate and N, N-diisopropylethylamine are used for reaction to prepare tert-butyl (S) -4- (3- ((1- (benzo [ d ] thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamoyl) benzamido) piperidine-1-carboxylate (7q), which is white powdery solid with the yield of 73%. Melting point 138 ℃ and 140 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ9.52(s,1H),8.90(d,J＝7.6Hz,1H),8.42(d,J＝7.8Hz,1H),8.35–8.20(m,2H),8.11(s,1H),7.95(t,J＝7.9Hz,2H),7.52(dt,J＝15.8,8.1Hz,2H),7.10(d,J＝5.3Hz,3H),6.97–6.79(m,2H),4.65(q,J＝7.2,6.1Hz,1H),3.98(dd,J＝27.4,9.6Hz,3H),3.27(s,3H),3.05–2.79(m,4H),2.00(s,1H),1.81(dd,J＝13.1,3.9Hz,2H),1.42(s,9H),1.18(t,J＝7.1Hz,1H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.69,166.50,165.61,158.59,154.41,154.19,141.80,138.38,135.25,134.27,133.57,130.62,130.37,129.19,128.60,127.15,126.90,125.67,123.84,122.61,79.12,53.25,47.05,38.03,36.93,31.76,28.56.

C₃₅H₃₉N₅O₅S[M+H]⁺(642.27),ESI-MS m/z:642.13[M+H]⁺

N-tert-butyloxycarbonyl-p-phenylenediamine is selected as a correspondingly substituted aromatic amine to react with an intermediate 6, O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine to prepare tert-butyl (S) - (4- (3- ((1- (benzothiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-N-yl) carbamoyl) benzamido) phenyl) carbamate (7r), wherein the yield is 76%. Melting point 162-163 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.29(s,1H),9.52(s,1H),9.32(s,1H),8.94(d,J＝7.6Hz,1H),8.40(s,1H),8.30(d,J＝8.5Hz,1H),8.17–8.03(m,2H),8.00(d,J＝7.8Hz,1H),7.67(d,J＝8.5Hz,2H),7.60(t,J＝7.8Hz,1H),7.44(d,J＝8.6Hz,3H),7.10(d,J＝5.2Hz,3H),6.85(d,J＝6.4Hz,2H),4.68(d,J＝6.8Hz,1H),3.28(s,3H),3.01(qd,J＝14.0,7.3Hz,2H),1.49(s,9H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.65,166.34,165.21,158.48,154.20,153.32,141.82,138.32,135.99,135.69,134.38,133.95,130.83,130.68,129.22,128.79,128.60,127.34,126.90,125.65,123.80,122.60,121.42,118.84,79.38,53.18,38.71,38.06,28.64.

C₃₆H₃₅N₅O₅S[M+H]⁺(650.24),ESI-MS m/z:[M+H]⁺

n-tert-butyloxycarbonyl-m-phenylenediamine is selected as a correspondingly substituted aromatic amine to react with an intermediate 6, O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine to prepare tert-butyl (S) - (3- (3- ((1- (benzo [ d ] thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-N-2-yl) carbamoyl) benzamide) phenyl carbamate (7S) which is white powdery solid with the yield of 72 percent and the melting point of 151-152 ℃.

Spectral data:

¹H-NMR(400MHz,DMSO-d₆):δ10.36(s,1H),9.52(s,1H),9.41(s,1H),8.94(d,J＝7.6Hz,1H),8.39(s,1H),8.30(d,J＝8.5Hz,1H),8.17–8.05(m,2H),8.06–7.94(m,2H),7.60(t,J＝7.8Hz,1H),7.47(dd,J＝18.2,8.3Hz,2H),7.23(t,J＝8.1Hz,1H),7.12(t,J＝8.4Hz,4H),6.97–6.77(m,2H),4.68(d,J＝7.2Hz,1H),3.28(s,3H),3.15–2.92(m,2H),1.49(s,9H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.66,166.33,165.52,158.55,154.20,153.23,141.80,140.28,139.81,138.34,135.63,134.31,133.56,130.97,130.79,129.22,129.07,128.78,128.61,127.47,126.91,125.68,123.83,122.61,114.93,114.46,111.09,79.44,53.20,38.05,37.05,28.63.

C₃₆H₃₅N₅O₅S[M+H]⁺(650.24),ESI-MS m/z:650.06[M+H]⁺

n-tert-butoxycarbonyl-1, 2-phenylenediamine is selected to react with an intermediate 6, O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine to prepare tert-butyl (S) - (2- (3- ((1- (benzo [ d ] thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropane N-2-yl) carbamoyl) benzamide) phenyl carbamate (7t) which is white powdery solid with the yield of 69 percent, the melting point of 104-.

¹H-NMR(400MHz,DMSO-d₆):δ9.93(s,1H),9.51(s,1H),8.98(d,J＝7.6Hz,1H),8.69(s,1H),8.45(s,1H),8.29(d,J＝8.5Hz,1H),8.06(dd,J＝20.3,7.6Hz,3H),7.67–7.44(m,4H),7.26–7.08(m,5H),6.86(d,J＝6.2Hz,2H),4.69(d,J＝6.8Hz,1H),3.28(s,3H),3.10–2.92(m,2H),1.44(s,9H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.64,166.24,165.48,158.55,154.21,153.82,141.79,138.34,135.01,134.47,132.50,131.01,130.91,129.93,129.22,128.92,128.61,127.47,126.91,126.71,126.22,125.65,124.44,124.19,123.82,80.07,53.22,38.05,37.05,28.51.

C₃₆H₃₅N₅O₅S[M+H]⁺(650.24),ESI-MS m/z:650.06[M+H]⁺

Example 7: preparation of target Compound (7u-w)

A50 mL round-bottomed flask was charged with the aimed compound 7r (0.1g, 0.1539mmol) and dissolved by adding 15mL of methylene chloride, followed by adding 4mL of trifluoroacetic acid to the reaction system and reacting at room temperature for 4 hours. After completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and then a saturated sodium bicarbonate solution was added to adjust the pH of the reaction solution to 7, and 30mL of water was added and extracted with dichloromethane (30 mL. times.3). Mixing organic phases, washing with saturated sodium chloride solution (20mL × 2), drying with anhydrous magnesium sulfate, filtering, evaporating solvent under reduced pressure to obtain crude product of target compound, and purifying with silica gel preparation plate to obtain pure product (S) -N of target compound¹- (4-aminophenyl) -N³- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phen-amin-2-yl) isophthalamide (7u) as a white powdery solid in 77% yield. Melting point 131-132 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ9.98(s,1H),9.50(s,1H),8.88(d,J＝7.6Hz,1H),8.36(s,1H),8.28(d,J＝8.5Hz,1H),8.14–7.91(m,3H),7.57(t,J＝7.8Hz,1H),7.47(d,J＝8.5Hz,1H),7.39(d,J＝8.2Hz,2H),7.10(s,3H),6.85(s,2H),6.67–6.50(m,2H),4.94(s,2H),4.68(s,1H),3.27(s,3H),3.00(dt,J＝23.5,13.5Hz,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.67,166.39,164.71,158.54,154.21,145.79,141.81,138.34,136.01,134.31,133.40,130.70,130.40,129.22,128.73,128.60,128.50,127.21,126.91,125.68,123.82,122.66,114.18,53.17,38.06,37.07.

C₃₁H₂₇N₅O₃S[M+H]⁺(550.18),ESI-MS m/z:550.04[M+H]⁺

A50 mL round-bottomed flask was charged with the objective compound 7s (0.1g, 0.1539mmol) and dissolved by adding 15mL of dichloromethane, followed by adding 4mL of trifluoroacetic acid to the reaction system and reacting at room temperature for 4 hours. After completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and then a saturated sodium bicarbonate solution was added to adjust the pH of the reaction solution to 7, and 30mL of water was added and extracted with dichloromethane (30 mL. times.3). Mixing organic phases, washing with saturated sodium chloride solution (20mL × 2), drying with anhydrous magnesium sulfate, filtering, evaporating solvent under reduced pressure to obtain crude product of target compound, and purifying with silica gel preparation plate to obtain pure product (S) -N of target compound¹- (3-aminophenyl) -N³- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phen-amin-2-yl) isophthalamide (7v) as white powdery solid in 76% yield. Melting point 129-130 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ10.05(s,1H),9.50(s,1H),8.89(d,J＝7.6Hz,1H),8.35(s,1H),8.28(d,J＝8.5Hz,1H),8.13–7.93(m,3H),7.58(t,J＝7.8Hz,1H),7.47(d,J＝8.4Hz,1H),7.23–7.06(m,4H),6.98(t,J＝7.9Hz,1H),6.87(t,J＝8.9Hz,3H),6.34(d,J＝7.9Hz,1H),5.09(s,2H),4.68(d,J＝7.0Hz,1H),3.27(s,3H),3.13–2.91(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.67,166.35,165.36,158.52,154.20,149.45,141.82,140.12,138.33,135.99,134.31,133.56,130.88,130.60,129.31,129.22,128.76,128.61,127.37,126.91,125.67,123.82,122.61,110.37,108.83,106.57,53.18,38.06,37.09.

C₃₁H₂₇N₅O₃S[M+H]⁺(550.18),ESI-MS m/z:584.18[M+Cl]^-

In a 50mL round-bottomed flask, 7t (0.1g, 0.1539mmol) of the objective compound was charged and dissolved by adding 15mL of methylene chloride, followed by adding 4mL of trifluoroacetic acid to the reaction system and reacting at room temperature for 4 hours. After completion of the TLC monitoring, the solvent was distilled off under reduced pressure, and then a saturated sodium bicarbonate solution was added to adjust the pH of the reaction solution to 7, and 30mL of water was added and extracted with dichloromethane (30 mL. times.3). Mixing organic phases, washing with saturated sodium chloride solution (20mL × 2), drying with anhydrous magnesium sulfate, filtering, evaporating solvent under reduced pressure to obtain crude product of target compound, and purifying with silica gel preparation plate to obtain pure product (S) -N of target compound¹- (2-aminophenyl) -N³- (1- (benzo [ d ]]Thiazol-5-yl (methyl) amino) -1-oxo-3-phenylpropan-2-yl) isophthalamide (7w) as a white powdery solid in 77% yield. Melting point 169-171 ℃.

¹H-NMR(400MHz,DMSO-d₆):δ9.76(s,1H),9.52(s,1H),8.92(d,J＝7.6Hz,1H),8.45(s,1H),8.29(d,J＝8.6Hz,1H),8.11(d,J＝9.4Hz,2H),8.00(d,J＝7.8Hz,1H),7.60(t,J＝7.8Hz,1H),7.48(d,J＝8.5Hz,1H),7.20(d,J＝7.9Hz,1H),7.11(d,J＝5.1Hz,3H),7.00(t,J＝7.7Hz,1H),6.86(d,J＝6.2Hz,2H),6.81(d,J＝8.0Hz,1H),6.62(t,J＝7.6Hz,1H),4.96(s,2H),4.69(d,J＝7.2Hz,1H),3.28(s,3H),3.13–2.90(m,2H).

¹³C-NMR(100Hz,DMSO-d₆):δ171.66,166.32,165.53,158.50,154.19,142.51,141.80,138.28,135.25,134.29,131.05,130.74,129.23,128.77,128.62,127.51,127.19,127.11,126.92,125.65,124.19,123.80,122.58,117.61,117.17,53.12,38.07,37.18.

C₃₁H₂₇N₅O₃S[M+H]⁺(550.18),ESI-MS m/z:550.05[M+H]⁺

Example 8 in vitro anti-HIV Activity test (MT-4 cells) of the Compounds of interest

Interpretation of terms: MT-4 cells: human acute lymphoblastic leukemia cells; MTT assay: MTT is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide, and the trade name is thiazole blue; DMSO, DMSO: dimethyl sulfoxide (DMSO).

Principle of testing

Because the HIV-infected MT-4 cells can be diseased within a certain period of time (5-7 days), a solution of a compound to be detected with a proper concentration is added into the suspension of the HIV-infected MT-4 cells, and after a period of culture (5-7 days), the activity of the MT-4 cells is measured by an MTT (methyl thiazolyl tetrazolium) analysis method, so that the concentration of a drug (EC) for protecting 50% of the cells from cytopathic diseases is obtained (the concentration of the drug is the same as that of the drug (EC)₅₀) The anti-HIV activity of the target compound can be obtained. Simultaneously obtaining the concentration (CC) of the target compound which can cause 50 percent of cells not infected by HIV to generate pathological changes₅₀) Calculating the selection coefficient (SI ═ CC)₅₀/EC₅₀)。

Principle of MTT assay: MTT, i.e., bromo-3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium nitrogen, binds to live intracellular succinate dehydrogenase and does not react with dead cells. At present, the MTT method is an enzyme analysis method for rapidly reflecting the cell activity.

Test materials and methods

(1)HIV-1(III_B) HIV-2(ROD) strain: provided by the institute of microbiology and immunology, Rega institute of university of Leuven, belgium;

(2) MT-4 cells: provided by the institute of microbiology and immunology, Rega institute of university of Leuven, belgium;

(3) MTT: purchased from Sigma, usa;

(4) sample treatment: dissolving a sample in DMSO to prepare a proper concentration before use, and diluting by 5 times with double distilled water, wherein each dilution is 5 dilutions;

(5) positive control: PF 74;

(6) the test method comprises the following steps: diluting the sample, adding into suspension of HIV-infected MT-4 cell, measuring cell activity by MTT colorimetric method after a period of time, recording absorbance (A) value at 590nm in enzyme labeling instrument, and calculating EC₅₀、CC₅₀And a SI;

(7) MTT staining method: after adding the sample for incubation for a period of time, 20. mu.L of MTT solution (5mg/mL) was added to each well, incubation was continued for several hours, the staining solution was discarded, 150. mu.L of DMSO was added to each well, mixed well, and absorbance was recorded at 590nm in a microplate reader.

The specific operation is as follows: dissolving the compound in DMSO or water, diluting with phosphate buffer solution, and mixing 3 × 10 solutions⁵MT-4 cells were preincubated with 100. mu.L of compound solutions at various concentrations for 1h at 37 ℃. Then, 100. mu.L of a virus dilution of appropriate concentration was added to the mixture, and the cells were incubated at 37 ℃ for 1 h. After three washes, the cells were resuspended in culture medium with or without compound, respectively. Cells were then incubated at 5% CO₂Incubate at 37 ℃ for 7 more days in the environment and supplement the stock culture with culture medium with or without compound on the third day after infection. The procedure was repeated twice for each culture condition. Cytopathic effects on the virus were monitored daily with a reverse optical microscope. Generally, the virus dilutions used in this experiment often developed cytopathic effects the fifth day after viral infection. The inhibitory concentration of the drug is that concentration at which the drug produces 50% inhibition of viral cytopathic effects while not being directly toxic to cells (EC)₅₀) And (4) showing. It is to be emphasized that, when the compound is poorly water soluble and requires DMSO to dissolve, the volume-specific concentration of DMSO relative to water is typically less than 10% (final DMSO concentration in MT-4 cell culture medium is less than 2%). Since DMSO can affect the antiviral activity of the test compound, the antiviral activity of a solution containing the same concentration of DMSO should also be run in parallel to the control blank. In addition, the final DMSO concentration (1/1000) was much lower than that required to affect HIV replication in MT-4 cells.

In vitro anti-HIV-1 (III) of a target Compound_B) And HIV-2(ROD) Activity screening data were provided by the institute of microbiology and immunology, university of Leuven, Belgium, Rega, all activity data were obtained in at least two independent, parallel experiments, and the results are shown in Table 1.

TABLE 1 phenylalanine derivatives containing in part benzothiazole anti-HIV-1/2 Activity, toxicity and selection index (MT-4 cells)

^aEC₅₀: concentration of compound that protects 50% of HIV-1 infected cells from cytopathic effects;^bCC₅₀: (ii) concentration of compound that causes lesions in 50% of cells not infected with HIV-1;^cand (3) SI: coefficient of selectivity, CC₅₀/EC₅₀The ratio of (A) to (B);^dND is not determined; PF 74: a class of HIV-1 capsid inhibitors has been reported as positive controls.

And (4) experimental conclusion analysis: as shown in Table 1, the newly synthesized benzothiazole-containing phenylalanine derivative of the present invention exhibits certain anti-HIV-1 activity, wherein the compound 7u (EC)₅₀＝3.57±0.15μM,CC₅₀57.36 ± 9.82 μ M, SI 16) and 7M (EC)₅₀＝5.02±1.00μM,CC₅₀133.61 ± 4.8 μ M, SI ═ 27) exhibited single digit micromolar levels of anti-HIV-1 activity. Notably, 16 compounds had anti-HIV-2 activity in the 0.85-3.85 μ M range, which was superior to the positive control PF74 (EC)₅₀＝4.16±0.86μM,CC₅₀32.27 ± 1.25 μ M, SI ═ 8), where the anti-HIV-2 activity and selectivity (EC) of the target compound 7M was₅₀＝0.85±0.24μM,CC₅₀133.61 ± 4.8 μ M, SI 157) is particularly prominent and of further interest.

Example 9 Metabolic stability Studies of representative Compounds 7u and 7m in human liver microsomes

The most major and most important metabolic organ for drugs is the liver, and the metabolism of most drugs is mainly accomplished in the liver pharmacozymase system, so the metabolic stability of representative compounds 7u and 7m in Human Liver Microsomes (HLM) was first tested.

Experimental methods

1. Preparation of test compound and control working solution:

1.1 intermediate solution: mu.L of compound stock solution (10mM, DMSO as vehicle) was diluted to 100. mu.M (methanol content 99%) with 495. mu.L of methanol.

1.2 working solution: mu.L of the compound intermediate solution (100. mu.M) was diluted to 10. mu.M (methanol content 9.9%) with 450. mu.L of 100mM potassium phosphate buffer.

Preparation of NADPH cofactor:

2.1 materials:

NADPH powder: a reduced form of β -nicotinamide adenine dinucleotide phosphate, tetrasodium salt; NADPH.4 Na (supplier: Chem-Impex International, Cat. No. 00616)

2.2 preparation steps:

the appropriate amount of NADPH powder was weighed and added with 10mM MgCl₂The solution was diluted (working solution concentration: 10 units/mL; final concentration: 1 unit/mL)

3. Preparation of liver microsomes:

an appropriate concentration of microsomal working solution was prepared with 100mM potassium phosphate buffer.

4. Preparation of stop solution:

a cold (4 ℃) acetonitrile solution containing 100ng/mL of tosylbutamide and 100ng/mL of labetalol as Internal Standard (IS) was used as stop solution.

5. The determination step comprises:

5.1 Using an Apricot automated workstation, 10. mu.L/well of compound working solution was added to all 96-well reaction plates, excluding blanks (T0, T5, T10, T20, T30, T60 and NCF 60).

5.2 Add 80. mu.L/well of microsome solution to all reaction plates (blank, T0, T5, T10, T20, T30, T60 and NCF60) using an Apricot automated workstation.

5.3 all reaction plates containing the mixture of compounds and microsomes were preincubated at 37 ℃ for 10 min.

5.4 Add 10. mu.L/well of 100mM potassium phosphate buffer to the reaction plate NCF60 using the Apricot automated workstation.

5.5 the reaction plate NCF60 was incubated at 37 ℃ and timer 1 was started.

5.6 after the preincubation, 10. mu.L/well of NADPH regeneration system was added to each reaction plate except NCF60 (blank, T0, T5, T10, T20, T30 and T60) using an Apricot automated workstation to start the reaction.

5.7 incubate the reaction plate at 37 ℃ and start timer 2.

5.8 Add 300. mu.L/well of stop solution to each reaction plate at the appropriate end time point using an Apricot automated workstation to stop the reaction.

5.9 seal each plate and shake for 10 minutes.

5.10 after shaking, each plate was centrifuged at 4000rpm and 4 ℃ for 20 minutes.

5.11 during centrifugation, 300. mu.L/well of HPLC grade water was added to eight new 96-well plates using an Apricot automated workstation.

After 5.12 centrifugation, 100 μ Ι _ of supernatant was transferred from each reaction plate to its corresponding bioassay plate using the Apricot automation workstation.

5.13 seal each bioassay plate and shake for 10 minutes before LC-MS/MS analysis. The results are shown in Table 2.

TABLE 2 Metabolic stability results of representative compounds 7u and 7m in human liver microsomes

^aR²: correlation coefficients for linear regression used to determine kinetic constants;^bT_1/2: a half-life;^cCL_int(mic): intrinsic clearance;^dCL_int(liver): clearance in liver microsomes;^eNCF: no cofactor was present. During the 60 min incubation, no NADPH regenerating system was added to the NCF sample (replaced with buffer). If the residual amount is less than 60%, NADPH-independent reaction occurs.

And (4) experimental conclusion analysis: testosterone (testosterone), diclofenac (diclofenac) and propranolol (propranolol) with moderate metabolic stability in liver microsomes were selected as control drugs in this experiment. The results are shown in Table 2, where PF74 was rapidly metabolized in liver microsomes, leaving little after 60 minutes with a half-life of 0.5 minutes. While the half-lives of representative compounds 7u and 7m were 54.6 and 25.7 minutes, respectively, significantly better than the lead PF 74. Particularly, the half-life of 7u is improved by 109 times compared with PF74, and the 7u can be used as a lead compound to be further modified so as to obtain an anti-HIV inhibitor with greatly improved activity and metabolic stability.

EXAMPLE 10 Metabolic stability Studies of representative Compounds 7u and 7m in human plasma

The oral drug enters the systemic circulation after being metabolized by the liver to exert pharmacological activity, so the plasma stability of the compound is also a key factor for evaluating the druggability of the compound. Next, a test of human plasma stability will be performed on representative compounds 7u and 7 m.

Experimental methods

Before the experiment was started, the pooled frozen plasma was thawed in a water bath at 37 ℃. The plasma was centrifuged at 4000rpm for 5 minutes and if there were clots, it was removed. If necessary, the pH can be adjusted to 7.4. + -. 0.1. 10 μ L of stock solution was diluted with 90 μ L of DMSO to prepare 1mM intermediate solution; a1 mM positive control Propantheline bromide (Propantheline) intermediate solution was prepared by diluting 10. mu.L of the stock solution with 90. mu.L of ultrapure water. By using 180 u L45% MeOH/H₂O solution 20. mu.L of the intermediate solution (1mM) was diluted to prepare 100. mu.M addition solution. To 98. mu.L of blank plasma, 2. mu.L of loading solution (100. mu.M) was added as a standard to reach a final concentration of 2. mu.M (triplicate), and the sample was incubated in a water bath at 37 ℃. At each time point (0, 10, 30, 60 and 120 minutes), 400 μ L of stop solution (200ng/mL tosylbutamide and 200ng/mL labetalol in 50% acetonitrile/methanol) was added to precipitate the proteins and mixed well. The sample plate was centrifuged at 4000rpm for 10 minutes. An aliquot of the supernatant (50. mu.L) was transferred from each well and mixed with 100. mu.L of ultrapure water. The samples were centrifuged at 800rpm for about 10 minutes before LC-MS/MS analysis. The results are shown in Table 3.

TABLE 3 Metabolic stability results of representative compounds 7u and 7m in human plasma

^a% remaining: the remaining percentage was 100 × (PAR for the specified incubation time/PAR for time T0). PAR is the peak area ratio of the test compound to the internal standard. The accuracy should be within 80-120% of the indicated value.^bT_1/2: half-life.

And (4) experimental conclusion analysis: the results of the human plasma stability experiments for representative compounds 7u and 7m are shown in table 3. After 120 minutes of incubation, the residual amount of 7u technical material is 98.7%, 7m technical material can still be completely reserved (106.8%), and the residual amount of PF74 technical material is reduced to 85.2%. The results show that the metabolic stability of human plasma of 7u and 7m is slightly improved compared with PF74, and is far better than that of propathline bromide (0.1% of the original drug after 120 min).