CN104926829A - Thieno miazines derivatives and preparation method and application thereof - Google Patents

Abstract

The invention discloses a thienopyrimidine derivative and its preparation method and application. The thienopyrimidine derivative is a compound having a structure shown in general formula I and a pharmaceutically acceptable salt, ester or prodrug thereof. The invention also provides the preparation method of the compound, and the application of the composition containing one or more such compounds in the preparation of medicines for treating and preventing human immunodeficiency virus (HIV) infection.

Description

A kind of thienopyrimidine derivatives and its preparation method and application

technical field

The invention belongs to the technical field of organic compound synthesis and medical application, and specifically relates to a thienopyrimidine derivative, a preparation method thereof and an application as an HIV-1 inhibitor.

Background technique

AIDS (Acquired Immune Deficiency Syndrome, AIDS) has become a major infectious disease that endangers human life and health, and its main pathogen is Human Immunodeficiency Virus Type 1 (HIV-1). Although the implementation of Highly Active Antiretroviral Therapy (HAART) significantly prolongs the survival time of patients, the problems of drug resistance, drug side effects, and the cost of long-term medication have forced researchers to develop new types of HIV with high efficiency and low toxicity. Inhibitors. HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important part of HAART therapy. This type of drug has the advantages of high efficiency, low toxicity, and strong specificity. However, the defect of easy drug resistance makes this type of drug quickly lose Therefore, the research and development of new, high-efficiency, low-toxicity, and broad-spectrum anti-drug resistance NNRTIs is one of the hot spots in the research of anti-HIV drugs.

Diarylpyrimidine (DAPY) is a typical class of HIV-1 NNRTIs, which has strong anti-HIV activity and also has a good inhibitory effect on drug-resistant mutant strains. Etravirine (Etravirine) and Rilpivirine (Rilpivirine) in this class of drugs have been listed, but the water solubility of this class of compounds is poor, and the oral bioavailability is low, so the chemical structure of this class is further modified. New anti-HIV drugs with broad-spectrum high-efficiency, good bioavailability and independent intellectual property rights are of great significance.

Contents of the invention

Aiming at the deficiencies of the prior art, the invention provides a thienopyrimidine derivative and a preparation method thereof, and also provides the application of the thienopyrimidine derivative as an HIV-1 inhibitor.

Technical scheme of the present invention is as follows:

1. Thienopyrimidine derivatives

A thienopyrimidine derivative, or a pharmaceutically acceptable salt, ester or prodrug thereof, has the structure shown in general formula I:

Wherein, the dotted line represents a double bond between A and B, a double bond between B and D, a single bond between A and B, or a single bond between B and D;

A is: S or C(U);

B is: S or C(V);

D is: S or C(W);

And A, B and D have and only one is S;

Where U, V and W are each independently: H, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C _2- C ₆ alkenyl, C ₃ -C ₆ cycloalkyl, O C ₃ -C ₆ cycloalkyl, phenyl, benzyl, trifluoromethyl, amino, hydroxyl, various substituted six-membered heterocycles, various substituted five-membered heterocycles;

Z is: F, F ₂ , OH, COOH, CONH ₂ , COOC ₂ H ₅ ;

X is one of O, NH or S;

R ₁ , R ₂ , and R ₃ are each independently: H, halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C _2- C ₆ alkenyl, trifluoromethyl, Amino, hydroxycyanovinyl;

Ar is: substituted benzene ring, substituted naphthalene ring, various substituted six-membered heterocyclic rings, various substituted five-membered heterocyclic rings, various substituted six-membered and five-membered heterocyclic rings, various substituted six-membered and six-membered heterocyclic rings Heterocycles, various substituted five-membered heterocycles, various substituted benzo five-membered heterocycles, or various substituted benzo six-membered heterocycles.

Preferably, Ar is a substituted benzene ring with general formula (a) or general formula (b):

Wherein, R ₄ is H, CH ₃ , COCH ₃ , COOCH ₃ , COOC ₂ H ₅ , COOH, SO ₂ CH ₃ or SO ₂ CF ₃ ; R ₅ is Cl, Br, Me, NHCH ₃ , NHCOCH ₃ , NHSO _{2 CH3 or NHSO2CF3} .

The "pharmaceutically acceptable salt" mentioned in the present invention means that within the scope of reliable medical evaluation, the salt of the compound is suitable for contacting with human or lower animal tissues without undue toxicity, irritation and allergy reactions, etc., have a fairly reasonable ratio of benefit to risk, are usually water or oil soluble or dispersible, and are effective for their intended use. Included are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are acceptable for the intended use and chemically compatible with the compounds of formula I herein. For a list of suitable salts see S.M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.

The "prodrugs" mentioned in the present invention refer to pharmaceutically acceptable derivatives, so that the biotransformation products obtained from these derivatives are active drugs as defined by the compound of formula I.

In more detail of the present invention, the structural formula of the thienopyrimidine compound is one of the following:

Wherein, R, U, V, W, Z and Ar are as described in general formula I above.

Further preferably, compounds IA-1, IA-2, IA-3, IA-4, IA-5, IB-1, IB-2, IB-3, IB-4, IB-5 and IC-1, IC -2, IC-3, IC-4, IC-5, Ar is the general formula (a) or general formula (b).

In more detail of the present invention, the thienopyrimidine derivatives are one of the following specific compounds:

2. Preparation method of thienopyrimidine derivatives

The preparation method of thienopyrimidine derivatives, the steps are as follows: using 2,4-dichloro-substituted thienopyrimidine 1 as an initial raw material, first in N,N-dimethylformamide solution with substituted phenol, benzenethiol or Aniline undergoes nucleophilic substitution to generate intermediate 2; intermediate 2 is then reacted with N-Boc-4-aminopiperidine in dimethyl sulfoxide and subsequently deprotected from the Boc group under trifluoroacetic acid conditions to generate key intermediate 4 ; or intermediate 2 undergoes Buchwald-Hartwig (Buchwald-Hartwig) coupling reaction to generate intermediate 3 and then removes BOC protection to obtain intermediate 4; finally this key intermediate 4 is in N,N-dimethyl Under the condition of formamide solution and potassium carbonate as alkali, react with various substituted benzyl chloride or benzyl bromide to generate the target product; the synthetic route is as follows:

Reagents and conditions: (i) substituted phenol, aniline or phenylthiol, dimethylformamide, potassium carbonate, room temperature; (ii) N-Boc-4-aminopiperidine, dimethyl sulfoxide, potassium carbonate, 120°C; (iii) N-Boc-4-aminopiperidine, palladium acetate, 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene, cesium carbonate, 90°C, dioxygen Hexacyclic; (iv) dichloromethane, trifluoroacetic acid, room temperature; (v) substituted benzyl chloride or bromide, dimethylformamide, potassium carbonate, room temperature.

The substituted phenol, benzenethiol or aniline are s-trimethylphenol, 2,6-dimethyl-4-cyanophenol, 2,6-dimethyl-4-(E)-cyanovinyl Phenol, mes-trimethylphenylthiol, 2,6-dimethyl-4-cyanophenylthiol, 2,6-dimethyl-4-(E)-cyanovinylphenylthiol , Trimethylaniline, 2,6-dimethyl-4-cyanoaniline, 2,6-dimethyl-4-(E)-cyanovinylaniline;

Substituted chlorobenzyl or bromobenzyl is o-chlorobenzyl, m-chlorobenzyl, p-chlorobenzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl, o-fluorochlorobenzyl, m-fluorochlorobenzyl, p-fluorochlorobenzyl Benzyl, 2,4-difluorobenzyl bromide, 3,4-difluorobenzyl bromide, o-cyanobenzyl chloride, m-cyanobenzyl chloride, p-cyanobenzyl chloride, o-nitrobenzyl chloride, m-nitrobenzyl chloride, p-nitrobenzyl chloride, o-methoxybenzyl chloride, m-methoxybenzyl chloride, p-methoxybenzyl chloride, p-methylsulfonyl benzyl bromide, p-sulfonyl benzyl bromide, p-formamidobenzyl bromide, 4- Methyl (bromomethyl)benzoate.

The room temperature described in the present invention is 20-30°C.

3. Anti-HIV-1 wild strain and mutant strain activity and application of thienopyrimidine derivatives

Part of the thienopyrimidine derivatives synthesized according to the above method were tested against HIV-1 (III _B ) at the cellular level, single-drug-resistant mutants K103N, Y181C, Y188L and double-drug-resistant mutants RES056 (K103N/Y181C). For activity screening, nevirapine (NVP), efavirenz (EFV), etravirine (ETV) and zidovudine (AZT) were used as positive controls. Their anti-HIV-1 activity and toxicity data are listed in Table 1 and Table 2, respectively.

It can be seen from Table 1 that the thienopyrimidine derivatives of the present invention are a series of non-nucleoside HIV-1 inhibitors with novel structures, and exhibit extremely strong anti-HIV-1 wild strain and mutant strain activity. The EC ₅₀ values of most of the compounds for inhibiting wild strains and mutant strains reached the nanomolar level. Among them, the activity of compound IA-1-2 was particularly prominent, and its EC ₅₀ value for HIV-1 wild strains was the first generation of anti-AIDS Drug nevirapine (NVP) more than 150 times, is more than 2 times of the latest generation drug etravirine (ETV). Compound IA-1-2 also exhibited extremely high safety, and its selectivity index to HIV-1 wild strain was greater than 100,000, much higher than that of marketed drugs. For single mutant strains K103N and Y181C, compound IA-1-2 exhibited inhibitory activity equivalent to that of etravirine, and for single mutant strain Y188L, its inhibitory activity was much higher than that of etravirine, which was the key to its inhibitory activity. more than 3 times. In addition, the cytotoxicity of compound IA-1-2 was much less than that of the positive drug (Table 2), resulting in a very high selectivity of this compound. Therefore, the thienopyrimidine compound has the value of further research and development, and can be used as a lead compound against HIV.

The thienopyrimidine derivatives of the invention can be used as non-nucleoside HIV-1 inhibitors. Specifically, it is used as an HIV-1 inhibitor for the preparation of anti-AIDS drugs.

An anti-HIV-1 pharmaceutical composition, comprising the thienopyrimidine derivative of the present invention and one or more pharmaceutically acceptable carriers or excipients.

The invention provides a thienopyrimidine derivative with a new structure, its preparation method, its anti-HIV-1 activity screening result and its first application in the field of anti-virus. Tests have proved that the thienopyrimidine derivatives of the invention can be used as HIV-1 inhibitors and have high application value. Specifically, it is used as an HIV-1 inhibitor for the preparation of anti-AIDS drugs.

detailed description

The following examples help to understand the present invention, but the content of the present invention cannot be limited.

The synthetic route involved in the embodiment is as follows:

Synthetic route one:

Synthetic route two:

Example 1: 4-((2-(piperidin-4-amine)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10) preparation

Weigh 4-hydroxy-3,5-dimethylbenzonitrile (0.15g, 1mmol) and potassium carbonate (0.17g, 1.2mmol) in 5mL of N,N-dimethylformamide (DMF) solution, room temperature Stir for 15 minutes, then add 2,4-dichlorothieno[3,2-d]pyrimidine (0.21g, 1mmol) and continue to stir at room temperature for 2h (TLC detects that the reaction is complete). At this time, a large amount of white solid was generated, and 25mL of ice water was slowly added thereto, filtered, and dried in a vacuum oven to obtain a white solid which was the compound 4-((2-chlorothieno[3,2-d]pyrimidine- 4-yl)oxy)-3,5-dimethylbenzonitrile, yield 93.8%, melting point 258-260°C.

Weigh the compound 4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (0.32g, 1.0mmol), N-Boc- 4-Aminopiperidine (0.24g, 1.2mmol) and potassium carbonate (0.28g, 2mmol) were dissolved in 5mL of dimethyl sulfoxide, then heated to reflux for 12 hours. After the reaction was cooled to room temperature, the reaction solution was slowly added dropwise to 20 mL of aqueous solution, stirred, and a large amount of yellow solid was formed. Filter and dry to obtain crude product 4-((2-(1-Boc-piperidin-4-amine)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzene Nitrile. The crude product (0.60g, 1.21mmol) was weighed and dissolved in 4mL of dichloromethane, and then trifluoroacetic acid (0.74mL, 10mmol) was slowly added thereto, and stirred at room temperature for 6 hours (TLC detected that the reaction was complete). Add 10 mL of water to the reaction solution, adjust the pH to 9 with saturated aqueous sodium bicarbonate, extract with dichloromethane (3×5 mL), wash with saturated brine, separate the organic layer, and dry over anhydrous sodium sulfate. Then perform flash column chromatography to obtain a white solid that is the compound 4-((2-(piperidin-4-amine)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5- Dimethylbenzonitrile. Yield 84.2%, melting point 114-116°C.

¹ H NMR (400MHz, DMSO-d6, ppm) δ: 8.20 (d, 1H, J = 5.4Hz,), 7.72 (s, 1H), 7.26 (s, 1H), 6.92 (s, br, 1H), 3.78(s,br,1H),2.89(s,br,2H),2.12(s.6H),1.74(m,2H),1.23(m,4H).ESI-MS: m/z 380.5(M+ 1) C ₂₀ H ₂₁ N ₅ OS(379.15).

Example 2: Preparation of 2-(piperidin-4-amine)-4-(2,4,6-trimethylphenoxy)thieno[3,2-d]pyrimidine (13)

The operation steps are the same as in Example 1, except that the starting material is 2,4,6-trimethylphenol.

The product is a white solid. Yield 87.5%, 178-180°C.

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.73 (d, 1H, J = 5.4 Hz, C ₆ -thienopyrimidine-H), 7.19 (d, 1H, J = 5.3 Hz, C ₇ -thienopyrimidine- H), 6.90 (s, 2H, C ₃ , C ₅ -Ph”-H), 4.89 (d, 1H, J=7.4Hz, NH), 3.77 (s, 1H, NH), 3.20 (d, 2H, J=12.6Hz), 2.74-2.76(m,2H), 2.32(s,3H,C ₄ -Ph”-CH ₃ ), 2.04-2.09(m,9H), 1.46-1.54(m,2H).ESI - MS: m/z 369.5 (M+1) C ₂₀ H ₂₄ N ₄ OS (368.17)

Embodiment 3: the preparation of compound IA-1

Weigh compound 4-((2-(piperidin-4-amine)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10) (0.5 mmol) in 10mL DMF, stirred and dissolved at room temperature, then added anhydrous potassium carbonate (0.14g, 1.0mmol) and substituted benzyl chloride or benzyl bromide (0.6mmol), stirred at room temperature for 12h (reaction completed by TLC). The solvent was distilled off under reduced pressure, then 30ml of ethyl acetate was added to the residual substrate, washed with saturated saline solution 3 times, 10mL each time, the organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The target compound was separated by flash column chromatography, and then recrystallized in ethyl acetate-petroleum ether system to obtain the target compound IA-1.

With different substituted benzyl and 4-((2-(piperidin-4-amine)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile ( 10) The target products of compounds IA-1-1～IA-1-12 were obtained by the above method, and some results are as follows:

The operation is the same as above, except that 4-thiamphenicol benzyl bromide is used.

The product is a white solid, yield: 52.4%, melting point>300°C.

¹ H NMR (400MHz, DMSO-d6, ppm) δ: 8.18 (d, 1H, J = 5.3Hz), 7.86 (d, 2H, J = 8.2Hz), 7.71 (s, 2H), 7.54 (d, 2H ,J=8.2Hz),7.24(s,1H),6.88(s,1H),4.02(d,1H,J=7.2Hz),3.17-3.19(m,7H),2.71(s,2H),2.10 (s,6H),1.38-1.44(m,2H),1.14-1.19(m,2H).ESI-MS:m/z 548.4(M+1),570.5(M+Na).C ₂₈ H ₂₉ N ₅ O ₃ S ₂ (547.69).

The operation is the same as above, except that p-bromomethylbenzenesulfonamide is used.

The product is a white solid, yield: 50.3%, melting point 223-225°C.

¹ H NMR (400MHz, DMSO-d6, ppm) δ: 8.18 (d, 1H, J = 5.3Hz), 7.76 (d, 2H, J = 8.3Hz), 7.72 (s, 2H), 7.45 (d, 2H ,J=8.2Hz),7.29(s,2H),7.25(s,1H),6.87(s,1H),4.02(d,1H,J=7.2Hz),3.48(s,2H),2.72(s ,2H),2.10(s,6H),1.69-1.79(m,2H),1.39-1.44(m,2H),1.16-1.19(m,2H).ESI-MS: m/z 549.5(M+1 ), C ₂₇ H ₂₈ N ₆ O ₃ S ₂ (548.50).

The operation is the same as above, except that p-fluorobenzyl bromide is used.

The product is a white solid, yield: 61.8%, melting point>300°C.

¹ H NMR (400MHz, DMSO-d6, ppm) δ: 8.34 (d, 1H, J = 5.3Hz), 8.20 (d, 2H, J = 8.3Hz), 7.72 (s, 1H), 7.35 (d, 2H ,J=8.2Hz),7.28(s,2H),4.03(d,1H,J=7.2Hz),3.47(s,2H),2.72(s,2H),2.12(s,6H),1.72-1.76 (m, 2H), 1.39-1.43 (m, 2H), 1.15-1.18 (m, 2H). ESI-MS: m/z 488.5 (M+ ₁ ), _C27H26FN5OS ( _487.59 ).

The operation is the same as above, except that 4-chloromethylpyridine hydrochloride is used.

The product is a white solid, yield: 51.3%, melting point 154-156°C.

¹ H NMR (400MHz, DMSO-d6, ppm) δ: 8.48(d, 1H, J=5.3Hz), 8.20(d, 2H, J=8.3Hz), 7.72(s, 1H), 7.57(d, 2H ,J=8.2Hz),7.28(s,2H),4.02(d,1H,J=7.2Hz),3.48(s,2H),2.73(s,2H),2.12(s,6H),1.70-1.76 (m, 2H), 1.37-1.43 (m, 2H), 1.17-1.18 (m, 2H). ESI-MS: m/z _471.5 (M+ ₁ ), _C26H26N6OS (470.59).

The operation is the same as above, except that 2,4-difluorobenzyl bromide is used.

The product is a white solid, yield: 55.7%, melting point>300°C.

¹ HNMR (400MHz, DMSO-d6, ppm) δ: 8.20 (d, 1H, J = 5.4Hz), 7.67-7.70 (m, 2H), 7.39 (d, 1H, J = 6.4Hz), 7.24 (m, 1H), 7.14(td, 1H, J=10.2, 2.5Hz), 7.02(td, 1H, J=8.4, 2.1Hz), 6.89(s, 1H), 4.13-4.15(m, 1H), 3.45(s ,2H),2.74(s,2H),2.11(s,6H),1.33-1.84(m,2H),1.35-1.42(m,4H).ESI-MS: m/z 506.3(M+1), _{C27H25F2N5OS ( 505.17 )} .

The operation is the same as above, except that p-methylsulfonyl benzyl chloride is used.

The product is a white solid, yield: 57.4%, melting point 92-95°C.

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.87 (d, 2H, J = 8.3Hz, C ₃ , C ₅ -Ph'-H), 7.70 (d, 1H, J = 5.4Hz, C ₆ -thienopyrimidine-H), 7.53 (d, 2H, J=8.1Hz, C ₂ , C ₆ -Ph'-H), 7.16 (d, 1H, J=5.4Hz, C ₇ -thienopyrimidine-H), 6.90 (s,2H,C ₃ ,C ₅ -Ph”-H),5.02(s,1H,NH),3.58(s,2H,N-CH ₂ ),3.06(s,3H,SO ₂ -CH ₃ ) ,2.74-2.76(m,2H),2.32(s,3H,C ₄ -Ph”-CH ₃ ),1.93-2.09(m,11H),1.42-1.51(m,2H).ESI-MS:m/ z 537.6 (M _{+ 1} ), _559.5 (M ₊ Na), _C28H32N4O3S2 (536.19).

The operation is the same as above, except that methyl p-bromomethylbenzoate is used.

The product is a white solid, yield: 52.7%, melting point 97-100°C.

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.97 (d, 2H, J = 8.2Hz, C ₃ , C ₅ -Ph'-H), 7.70 (d, 1H, J = 5.4Hz, C ₆ -thienopyrimidine-H), 7.39 (d, 2H, J＝8.1Hz, C ₂ , C ₆ -Ph'-H), 7.17 (d, 1H, J＝5.6Hz, C ₇ -thienopyrimidine-H), 6.90 (s,2H,C ₃ ,C ₅ -Ph”-H),5.10(s,1H,NH),3.53(s,2H,N-CH ₂ ),2.73-2.76(m,2H),2.31(s ,3H,CO ₂ -CH ₃ ),1.91-2.09(m,14H),1.41-1.49(m,2H).ESI-MS: m/z517.6(M+1),533.5(M+Na), C ₂₉ H ₃₂ N ₄ O ₃ S (516.22)

The operation is the same as above, except that 4-fluorobenzyl bromide is used.

The product is a white solid, yield: 65.3%, melting point 133-136°C

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.71 (d, 1H, J = 5.4Hz, C ₆ -thienopyrimidine-H), 7.29 (m, 2H), 7.17 (d, 1H, J = 5.4 Hz, C ₇ -thienopyrimidine-H), 7.02 (t, 2H, J=8.6Hz), 6.90 (s, 2H, C ₃ , C ₅ -Ph”-H), 4.86 (s, 1H, NH), 3.50 (s,2H,N-CH ₂ ),2.73-2.78(m,2H),2.32(s,3H,C ₄ -Ph”-CH ₃ ),1.94-2.09(m,11H),1.40-1.51(m , 2H). ESI-MS: m/z 477.5 (M+1), C ₂₇ H ₂₉ FN ₄ OS (476.20).

The operation is the same as above, except that 3-fluorobenzyl bromide is used.

The product is a white solid, yield: 63.1%, melting point 140-142°C.

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.68 (d, 1H, J=5.4Hz, C ₆ -thienopyrimidine-H), 7.37(t, 1H, J=6.1Hz, C ₅ -Ph' -H), 7.22(t, 1H, J=6.0Hz, C ₄ -Ph'-H), 7.17(d, 1H, J=5.3Hz, C ₇ -thienopyrimidine-H), 7.10(t, 1H,J = 7.4Hz, C ₆ -Ph'-H), 7.03 (t, 1H, J = 9.5Hz, C ₂ -Ph'-H), 6.88 (s, 2H, C ₃ , C ₅ -Ph"-H) ,5.05(s,1H,NH),3.57(s,2H,N-CH ₂ ),2.78-2.80(m,2H),2.31(s,3H,C ₄ -Ph”-CH ₃ ),1.92-2.08 (m, 11H), 1.41-1.48 (m, 2H). ESI-MS: m/z 477.5 (M+ ₁ ), _C27H29FN4OS ( _476.20 ).

The operation is the same as above, except that 3-cyanobenzyl bromide is used.

The product is a white solid, yield: 63.1%, melting point 99-102°C

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.72 (d, 1H, J=5.4Hz, C ₆ -thienopyrimidine-H), 7.64 (s, 1H, C ₂ -Ph'-H), 7.58 (t, 2H, J=9.0Hz, C ₄ -Ph'-H), 7.44(t, 1H, J=7.7Hz, C ₅ -Ph'-H), 7.17(d, 1H, J=5.3Hz, C ₇ -thienopyrimidine-H), 6.90(s,2H,C ₃ ,C ₅ -Ph”-H), 5.14(s,1H,NH), 3.51(s,2H,N-CH ₂ ), 2.72-2.75 (m,2H),2.32(s,3H,C ₄ -Ph”-CH ₃ ),2.09(s,9H),2.93(s,2H),1.42-1.50(m,2H).ESI-MS:m /z 484.6 (M+ ₁ ), 506.5 (M+Na), _C28H29N5OS ( _483.21 ).

The operation is the same as above, except that 4-chloromethylpyridine hydrochloride is used.

The product is a white solid, yield: 50.7%, melting point 135-137°C

¹ H NMR (400MHz, CDCl ₃ -d6, ppm) δ: 8.54 (d, 2H, J=5.9Hz, C ₂ , C ₆ -pyridine-H), 7.71 (d, 1H, J=5.4Hz, C ₆ -thienopyrimidine-H),7.28(d,2H,J＝6.6Hz,C ₃ ,C ₅ -pyridine-H),7.18(d,1H,J＝5.4Hz,C ₇ -thienopyrimidine-H),6.90(s ,2H,C ₃ ,C ₅ -Ph”-H),4.86(s,1H,NH),3.50(s,2H,N-CH ₂ ),2.74-2.77(m,2H),2.32(s,3H ,C ₄ -Ph”-CH ₃ ), 1.95-2.09(m,11H), 1.44-1.52(m,2H).ESI-MS: m/z 460.6(M+1), 482.6(M+Na), _{C26H29N5OS ( 459.21} ).

The operation is the same as above, except that 2,4-difluorobenzyl bromide is used.

The product is a white solid, yield: 59.6%, melting point 110-112°C.

¹ HNMR (400MHz, CDCl ₃ -d6, ppm) δ: 7.70 (d, 1H, J = 5.4Hz, C ₆ -thienopyrimidine-H), 7.36 (dd, 1H, J ₁ = 15.1Hz, J ₂ = 8.3Hz _{( _ _ _} m,2H),4.89(d,1H,J＝7.3Hz,NH),3.52(s,2H,N-CH ₂ ),2.75-2.78(m,2H),2.32(s,3H,C ₄ -Ph "-CH ₃ ), 2.09-2.17(m,9H), 1.93-1.95(m,2H), 1.39-1.47(m,2H).ESI-MS: m/z 495.5(M+1), 517.6(M ₊ Na), _{C27H28F2N4OS ( 494.20} ).

Embodiment 4: In vitro anti-HIV activity test of target compound

Test Principle

The anti-HIV activity of compounds was screened in vitro by MTT method. The full name of MTT is bromide-3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium nitrogen (trade name: thiazolium blue), which can be used to detect the survival and growth of cells. The detection principle is: MTT can combine with living intracellular succinate dehydrogenase to reduce to water-insoluble blue-purple crystalline formazan and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide can dissolve formazan in cells, and its absorbance (A) value at 590 nm can indirectly reflect the number of living cells by using a microplate reader. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number.

Because the MT-4 cell that HIV infects can take place lesion within a certain period of time (5-7 days), therefore add the compound solution to be detected of appropriate concentration in the MT-4 cell suspension liquid that HIV infects, after a period of time (5-7 days) After 7 days) of culture, the viability of MT-4 cells was measured by MTT assay, and the drug concentration (EC ₅₀ ) that protected 50% of the cells from cytopathic changes was obtained to obtain the anti-HIV activity of the target compound. At the same time, the concentration (CC ₅₀ ) at which the target compound causes 50% of HIV-uninfected cells to develop pathological changes was obtained, and the selectivity index (SI=CC ₅₀ /EC ₅₀ ) was calculated.

Test Materials and Methods

(1) HIV-1 (III _B ), HIV-2 (ROD) strains, and various HIV-1 drug-resistant strains: provided by Rega Institute, Faculty of Medicine, University of Leuven, Belgium.

(2) MT-4 cells: provided by the Rega Research Institute, Faculty of Medicine, University of Leuven, Belgium.

(3) MTT: purchased from Sigma, USA.

(4) Sample treatment: Before use, the sample was dissolved in DMSO to make an appropriate concentration, and diluted 5 times with double distilled water, each with 5 dilutions.

(5) Positive control drugs: nevirapine (NVP), efavirenz (EFV), etravirine (TMC125), zidovudine (AZT).

(6) Test method: the sample is diluted and added to the suspension of HIV-infected MT-4 cells. After a period of time, the cell viability is measured by MTT colorimetry, and the absorbance (A) value at 590nm is recorded in a microplate reader. , Calculate EC ₅₀ , CC ₅₀ and SI.

(7) MTT colorimetric method: After adding the sample solution and incubating for a period of time, add 20 μL of MTT solution (5 mg/ML) to each well, continue to cultivate for several hours, discard the staining solution, and add 150 μL DMSO to each well, mix thoroughly, The absorbance (A) value at 590 nm was measured in a microplate reader.

experimental method

On a 96-well cell culture plate, add 50 μL of MT-4 cell culture medium containing 1×10 ⁴ , and then add 20 μL of MT-4 cell suspension infected with HIV-1 (III _B or RES056) or HIV-2 (ROD) solution (containing 100 times CCID ₅₀ per ml) or blank culture medium (toxicity assay), and then add different concentrations of test compound solutions or positive control drugs, and design 3 replicate wells for each concentration. Then the cells were cultured at 37°C for 5 days in a 5% CO ₂ atmosphere, and 20 μL (5 mg/mL) MTT solution was added to each well, and the culture was continued for 2 hours, then DMSO was added, and the reaction solution was measured at 540 nm using a microplate reader. Calculate the cell proliferation rate P% at different concentrations of the compound. At the same time, a blank control group, a drug control group and a positive drug control group were set up to calculate the concentration (EC ₅₀ ) of the compound required to protect 50% of the cells from HIV-induced cytopathy. Calculation of selection index: SI=CC ₅₀ /EC ₅₀ .

According to the above-mentioned experimental method, the synthetic partial thienopyrimidine derivatives were screened against HIV-1 (III _B ) at the cell level, single mutants K103N, Y181C, Y188L and double mutants RES056 (K103N/Y181C), The activity results are shown in Table 1.

Table 1 Part IA series compounds and reference drug structures and their anti-HIV-1 activity (MT-4 cells)

Note: ^a EC ₅₀ : the compound concentration that protects 50% of the MT-4 cells infected with HIV-1 from cytopathic changes; A represents the compound EC ₅₀ <10nM, B represents the compound EC ₅₀ value of 10-100nM, and C represents the compound EC ₅₀ >100nM; NVP, EFV, ETV, and AZT represent marketed drugs nevirapine, efavirenz, etravirine, and zidovudine, respectively.

Table 2 Cytotoxicity and selection index (MT-4 cells) of IA series compounds and reference drugs

Note: ^a CC ₅₀ : The drug concentration at which 50% of the MTT cells are affected; A represents the compound SI>10000, B represents the compound SI value is 1000-10000, and C represents the compound SI<1000; X1: SI≥1 or <1; NVP, EFV, ETV, and AZT represent marketed drugs nevirapine, efavirenz, etravirine, and zidovudine, respectively.

Claims (7)

1. A thienopyrimidine derivative, or a pharmaceutically acceptable salt, ester or prodrug thereof, is characterized in that it has a structure shown in general formula I: Wherein, the dotted line represents a double bond between A and B, a double bond between B and D, a single bond between A and B, or a single bond between B and D; A is: S or C(U); B is: S or C(V); D is: S or C(W); And A, B and D have and only one is S; Where U, V and W are each independently: H, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C _2- C ₆ alkenyl, C ₃ -C ₆ cycloalkyl, O C ₃ -C ₆ cycloalkyl, phenyl, benzyl, trifluoromethyl, amino, hydroxyl, various substituted six-membered heterocycles, various substituted five-membered heterocycles; Z is: F, F ₂ , OH, COOH, CONH ₂ , COOC ₂ H ₅ ; X is one of O, NH or S; R ₁ , R ₂ , and R ₃ are each independently: H, halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C _2- C ₆ alkenyl, trifluoromethyl, Amino, hydroxycyanovinyl; Ar is: substituted benzene ring, substituted naphthalene ring, various substituted six-membered heterocyclic rings, various substituted five-membered heterocyclic rings, various substituted six-membered and five-membered heterocyclic rings, various substituted six-membered and six-membered heterocyclic rings Heterocycles, various substituted five-membered heterocycles, various substituted benzo five-membered heterocycles, or various substituted benzo six-membered heterocycles. 2. The compound of claim 1, characterized in that Ar in the general formula I is a substituted benzene ring with the general formula (a) or the general formula (b): Wherein, R ₄ is H, CH ₃ , COCH ₃ , COOCH ₃ , COOC ₂ H ₅ , COOH, SO ₂ CH ₃ or SO ₂ CF ₃ ; R ₅ is Cl, Br, Me, NHCH ₃ , NHCOCH ₃ , NHSO _{2 CH3 or NHSO2CF3} . 3. The compound according to claim 1 or 2, characterized in that the structural formula is one of the following: Wherein, R, U, V, W, Z and Ar are as described in general formula I above. 4. The compound according to claim 3, characterized in that it is one of the following specific compounds: 5. The preparation method of the compound described in claim 1, the steps are as follows: using 2,4-dichloro-substituted thienopyrimidine 1 as the initial raw material, first in N,N-dimethylformamide solution with substituted phenol, Nucleophilic substitution of benzenethiol or aniline yields intermediate 2; intermediate 2 is then reacted with N-Boc-4-aminopiperidine in DMSO and subsequently de-Boc-protected under trifluoroacetic acid conditions to yield The key intermediate 4; or intermediate 2 undergoes Buchwald-Hartwig coupling reaction to generate intermediate 3 and then removes BOC protection to obtain intermediate 4; finally this key intermediate 4 is in N,N-dimethyl formaldehyde Under the condition of amide solution and potassium carbonate as alkali, it reacts with various substituted benzyl chloride or benzyl bromide to generate the target product; the synthetic route is as follows: Reagents and conditions: (i) substituted phenol, aniline or phenylthiol, dimethylformamide, potassium carbonate, room temperature; (ii) N-Boc-4-aminopiperidine, dimethyl sulfoxide, potassium carbonate, 120°C; (iii) N-Boc-4-aminopiperidine, palladium acetate, 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene, cesium carbonate, 90°C, dioxygen Hexacyclic; (iv) dichloromethane, trifluoroacetic acid, room temperature; (v) substituted benzyl chloride or bromide, dimethylformamide, potassium carbonate, room temperature. 6. A use of the compound according to any one of claims 1-4 in the preparation of medicines for treating and preventing human immunodeficiency virus. 7. A pharmaceutical composition comprising the compound of any one of claims 1-4 and one or more pharmaceutically acceptable carriers or excipients.