Carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor as well as preparation method and application thereof

Abstract

The invention relates to a carbonyl-substituted pyrimidine derivative as well as a preparation method and application thereof, and belongs to the technical field of organic compound synthesis and medical application. The carbonyl-substituted pyrimidine derivative has a structure as shown in a formula I. The carbonyl-substituted pyrimidine derivative disclosed by the invention is novel in structure and shows good anti-HIV-1 activity as an HIV-1 inhibitor, and the EC50 value of the carbonyl-substituted pyrimidine derivative ranges from 4.28 nM to 274 nM and is obviously superior to that of a drug nevirapine (EC50 = 281 + / -38.7 nM) on the market.

Description

Technical Field

[0001] This invention relates to a carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor, its preparation method, and its application, belonging to the field of organic compound synthesis and pharmaceutical application technology. Background Technology

[0002] AIDS (Acquired Immunodeficiency Syndrome) is a major infectious disease primarily caused by infection with human immunodeficiency virus type 1 (HIV-1). HIV-1 reverse transcriptase (RT) is an asymmetric heterodimer composed of the p66 and p51 subunits, formed by the transcription and translation of the pol gene in the HIV-1 genome. Its core function is to catalyze the synthesis of double-stranded viral DNA using single-stranded viral RNA as a template, a crucial step in the HIV-1 replication cycle. As a key enzyme in the HIV-1 replication cycle, reverse transcriptase has become a preferred target for anti-AIDS drug development due to its significant advantages, such as its clearly defined three-dimensional crystal structure and the absence of its homologous enzyme in the human body.

[0003] Non-nucleoside reverse transcriptase inhibitors (NNRTIs) targeting this target have the advantages of high efficacy, low toxicity, and high selectivity. These inhibitors do not require intracellular phosphorylation to obtain their active form, therefore their activity is unaffected by phosphorylation conversion rate, conversion speed, or post-conversion stability. Furthermore, these inhibitors can bind to extracellular (e.g., in plasma) reverse transcriptases, reducing the activity of free viruses and thus weakening viral infectivity. In addition, NNRTIs possess structural diversity and can synergize with other drugs, making them an indispensable component of antiretroviral therapy.

[0004] Currently, six non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection, and three other NNRTIs have been approved for marketing in other countries and regions. However, after several years of clinical use, many of these drugs have gradually revealed problems such as drug resistance, toxic side effects, and poor pharmacokinetic properties.

[0005] Therefore, the development of a new generation of NNRTIs with high drug resistance and good safety remains a long and arduous task. The early development of new, highly effective, and low-toxicity anti-AIDS drugs with independent intellectual property rights in my country is of great significance for clinical treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention relates to a carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor, its preparation method, and its application.

[0007] This invention is achieved through the following technical solution: The first objective of this invention is to provide a carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

[0008] A carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof, said carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor having the structure shown in Formula I:

[0009]

[0010] in,

[0011] R1 can be H, CH3, OH, OCH3, OCH2CH3, NH2, NHNH2, NHCH3, NHOH, or NHOCH3;

[0012] R2 is a halogen atom, H, CH3, OH, CN, NH2, NO2, NHSO2NH2, NHSO2CH3, COOCH3, COOH, SO2NH2, CONH2, SO2CH3, or B(OH)2; the substituent is ortho, meta, para monosubstituted or polysubstituted;

[0013] R3 can be CN, CH3, CH=CHCN, or p-C4H6-CN.

[0014] According to a preferred embodiment of the present invention, the carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof is selected from one of the following:

[0015] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester (E1);

[0016] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid ethyl ester (E2);

[0017] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester (E3);

[0018] (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(ethoxycarbonyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E4);

[0019] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid methyl ester (E5);

[0020] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid methyl ester (E6);

[0021] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid methyl ester (E7);

[0022] (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(methoxycarbonyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E8);

[0023] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid (E9);

[0024] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid (E10);

[0025] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid (E11);

[0026] 2-((1-(4-boronbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid (E12);

[0027] 4-(4-cyano-2,6-dimethylphenoxy)-N-methoxy-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxamide (E13);

[0028] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)-N-methoxypyrimidine-5-carboxamide (E14);

[0029] 4-(4-cyano-2,6-dimethylphenoxy)-N-methoxy-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxamide (E15);

[0030] (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(methoxycarbamoyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E16).

[0031] A second objective of this invention is to provide a method for preparing the above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitors or pharmaceutically acceptable salts thereof.

[0032] The preparation method of the above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitors or pharmaceutically acceptable salts thereof includes the following steps:

[0033] 1) Using ethyl 2,4-dichloropyrimidine-5-carboxylate A1 or methyl 2,4-dichloropyrimidine-5-carboxylate A2 as starting materials, a nucleophilic substitution reaction is carried out with 4-hydroxy-3,5-dimethylbenzonitrile in N,N-dimethylformamide solution to obtain intermediate B1 or B2.

[0034] 2) Intermediate B1 or B2 reacts with tert-butyl 4-aminopiperidine-1-carboxylate via a nucleophilic substitution reaction to obtain intermediate C1 or C2, and the Boc protecting group is removed by trifluoroacetic acid to generate key intermediate D1 or D2.

[0035] 3) Intermediate D1 or D2 reacts with substituted benzyl bromo or benzyl chloride to give target compounds E1-E4 or E5-E8, respectively; compounds E1-E4 undergo ester hydrolysis in a mixed solution of methanol and water under the action of lithium hydroxide hydrate to give target compounds E9-E12; simultaneously, compounds E9-E12 undergo amide condensation with O-methylhydroxylamine hydrochloride in the action of condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate to give target compounds E13-E16;

[0036] The synthesis route is as follows:

[0037]

[0038] Reagents and conditions: (i) 4-hydroxy-3,5-dimethylbenzonitrile, potassium carbonate, N,N-dimethylformamide, room temperature; (ii) tert-butyl 4-aminopiperidine-1-carboxylate, potassium carbonate, N,N-dimethylformamide, 120 °C; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) substituted benzyl bromo or benzyl chloride, potassium carbonate, N,N-dimethylformamide, room temperature;

[0039]

[0040] Reagents and conditions: (i) Lithium hydroxide hydrate, methanol:water = 1:1 V / V, room temperature; (ii) O-methylhydroxylamine hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, 0 °C to room temperature.

[0041] A third objective of this invention is to provide the use of the above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitors or pharmaceutically acceptable salts thereof.

[0042] The above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitors or their pharmaceutically acceptable salts are used as HIV-1 inhibitors.

[0043] The above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitors or their pharmaceutically acceptable salts are used to prepare anti-AIDS drugs.

[0044] The above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitors or their pharmaceutically acceptable salts are used as non-nucleoside NNRTIs in the preparation of anti-HIV drugs.

[0045] An anti-HIV-1 pharmaceutical composition comprising the above-mentioned carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof and pharmaceutical excipients.

[0046] Technical features and advantages of the present invention:

[0047] 1. The carbonyl-substituted pyrimidine derivatives of this invention have novel structures and exhibit good anti-HIV-1 activity as HIV-1 inhibitors, with their EC... 50 The values ​​ranged from 4.28 nM to 274 nM, significantly superior to the marketed drug nevirapine (EC). 50 =281 ± 38.7 nM).

[0048] 2. The structure-activity relationship study results of the carbonyl-substituted pyrimidine derivatives provided by this invention show that compound E5 (EC) with methyl formate substitution is... 50 = 5.82 ± 0.85 nM), E6 (EC 50 = 4.28 ± 0.47 nM), E7 (EC 50 = 6.01 ± 0.76 nM) and E8 (EC 50 The anti-HIV-1 activity of compound E6 (7.18 ± 0.66 nM) was particularly outstanding, with compound E6 (EC) exhibiting the best activity. 50 = 4.28 ± 0.47 nM) compared to nevirapine (EC) 50 = 281 ± 38.7 nM) activity increased nearly 65 times, superior to zidovudine (EC) 50 = 7.50 ± 1.8 nM) and efavirenz (EC 50 = 5.20 ± 0.90 nM), and exhibits low cytotoxicity and a high selectivity index (CC). 50= 35.4 ± 3.3 µM, SI = 8273).

[0049] 3. The carbonyl-substituted pyrimidine derivatives provided by this invention, compound E6, exhibits the strongest antiviral activity at the cellular level and demonstrates significant HIV-1 reverse transcriptase inhibitory activity (IC50). 50 = 0.065 ± 0.019 µM), far superior to ectovirine (IC50). 50 = 0.232 ± 0.036 µM) and rilpivirine (IC50) 50 = 0.118 ± 0.027 µM), indicating that the target of this type of compound is reverse transcriptase, belonging to the typical non-nucleoside reverse transcriptase inhibitors. Detailed Implementation

[0050] The following examples help to understand the present invention, but they are not intended to limit the scope of the invention.

[0051] Example 1: Preparation of ethyl 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate (B1)

[0052] The starting materials ethyl 2,4-dichloropyrimidin-5-carboxylate A1 (0.22 g, 1.0 mmol) and 4-hydroxy-3,5-dimethylbenzonitrile (0.17 g, 1.1 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Potassium carbonate (0.28 g, 2.0 mmol) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was complete as monitored by TLC, 50 mL of water was slowly added to the reaction solution, and stirring was continued for 30 min. The precipitate in the suspension was collected by filtration, washed with ice water (3 × 5 mL), and dried to give intermediate ethyl 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidin-5-carboxylate B1. It was a white solid with a yield of 51%. 1 H NMR(400 MHz, DMSO-d6) δ 9.04 (s, 1H), 7.57 (s, 1H), 7.53 (s, 1H), 4.36 (q, J =7.1 Hz, 2H), 2.01 (s, 3H), 1.95 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). ESI-MS: m / z 332.47 [M+H] + . C 16 H 14 ClN3O3 (331.07).

[0053] Example 2: Preparation of methyl 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate (B2)

[0054] The preparation method is the same as in Example 1, except that:

[0055] The starting material, ethyl 2,4-dichloropyrimidin-5-carboxylate A1 (0.22 g, 1.0 mmol), was replaced with methyl 2,4-dichloropyrimidin-5-carboxylate A2 (0.21 g, 1.0 mmol). After drying, the intermediate methyl 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidin-5-carboxylate B2 was obtained. It was a white solid with a yield of 56%. 1 H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 7.57 (s, 1H), 7.53 (s, 1H), 3.89 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H). ESI-MS: m / z 362.61 [M+COOH] - . C 15 H 12 ClN3O3 (317.06).

[0056] Example 3: Preparation of ethyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate (C1)

[0057] The intermediates 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate B1 (0.33 g, 1.0 mmol) and tert-butyl 4-aminopiperidine-1-carboxylate (0.24 g, 1.2 mmol) prepared in Example 1 were dissolved in 10 mL of N,N-dimethylformamide, and potassium carbonate (0.28 g, 2.0 mmol) was added. The mixture was then heated at 120 °C for 8 h. After the reaction was completed as monitored by TLC, 50 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated sodium chloride solution (3 × 5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The solution was purified by silica gel column chromatography using ethyl acetate:petroleum ether (1:10) as the eluent. Finally, recrystallization from ethyl acetate and petroleum ether yielded intermediate ethyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid ester C1. It was a white solid with a yield of 43%. 1H NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H), 8.16 (d, J = 7.1 Hz,1H), 7.69 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 3.82 (d, J = 13.2 Hz, 2H), 3.75–3.66 (m, 1H), 2.69 (d, J = 4.2 Hz, 2H), 2.09 (s, 6H), 1.82–1.65 (m, 2H), 1.40 (s, 9H), 1.35 (d, J = 3.7 Hz, 1H), 1.32 (d, J = 3.7 Hz, 1H), 1.28 (t, J = 7.1Hz, 3H). ESI-MS: m / z 496.18 [M+H] + . C 26 H 33 N5O5 (495.25).

[0058] Example 4: Preparation of methyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate (C2)

[0059] The preparation method is the same as in Example 3, except that:

[0060] Ethyl 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate B1 (0.33 g, 1.0 mmol) was replaced with methyl 2-chloro-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate B2 (0.31 g, 1.0 mmol) obtained in Example 2. Recrystallization from ethyl acetate and petroleum ether yielded intermediate methyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate C2. White solid, yield 38%. 1H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.17 (d, J = 6.7 Hz, 1H), 7.70(s, 1H), 7.68 (s, 1H), 3.81 (s, 3H), 3.00 (d, J = 6.8 Hz, 1H), 2.90 (d, J =4.2 Hz, 2H), 2.74 (d, J = 4.2 Hz, 2H), 2.09 (s, 6H), 1.74 (d, J = 12.3 Hz,1H), 1.48 (d, J = 12.1 Hz, 1H), 1.38 (s, 9H), 1.33–1.21 (m, 1H), 1.12 (qd, J= 11.9, 4.2 Hz, 1H). ESI-MS: m / z 482.44 [M+H] + . C 25 H 31 N5O5 (481.23).

[0061] Example 5: Preparation of ethyl 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylate (D1)

[0062] The intermediate 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate C1 (0.50 g, 1.0 mmol) obtained in Example 3 was dissolved in 10 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added. The reaction was carried out at room temperature for 6 h. After the reaction was completed by TLC monitoring, the pH of the reaction solution was adjusted to about 9 with saturated sodium bicarbonate solution, extracted with dichloromethane (3 × 10 mL), and the organic phases were combined. The mixture was washed with saturated sodium chloride solution (3 × 5 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and finally purified by silica gel column chromatography using methanol:dichloromethane (1:20) as the eluent to obtain intermediate 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylate D1. It was a white solid with a yield of 53%. 1H NMR (400 MHz, DMSO-d6): δ 8.66 (s, 1H), 8.20 (d, J =6.9 Hz, 1H), 7.69 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 3.88 (d, J = 6.5 Hz,1H), 3.31–3.19 (m, 3H), 2.87 (td, J = 12.5, 3.0 Hz, 2H), 2.09 (s, 6H), 1.98 (d, J = 14.0 Hz, 2H), 1.74–1.54 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H). ESI-MS: m / z 396.26 [M+H] + . C 21 H 25 N5O3 (395.20).

[0063] Example 6: Preparation of methyl 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylate (D2)

[0064] The preparation method is the same as in Example 5, except that:

[0065] Ethyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate C1 (0.50 g, 1.0 mmol) was replaced with methyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylate C2 (0.48 g, 1.0 mmol) obtained in Example 4 by silica gel column chromatography to obtain methyl 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylate D2, a white solid in 50% yield. 1 H NMR (400 MHz, DMSO-d6): δ 8.85–8.57 (m, 1H), 8.03 (dd, J = 145.0, 7.1 Hz, 1H), 7.69 (s, 2H), 3.80 (d, J = 4.6 Hz, 3H), 3.11–2.58 (m, 4H), 2.21 (t, J = 12.1 Hz, 1H), 2.09 (s, 6H), 1.86–1.71 (m,1H), 1.57–1.06 (m, 4H). ESI-MS: m / z 382.56 [M+H] + . C20 H 23 N5O3 (381.18).

[0066] Example 7: General method for preparing target compounds E1-E4

[0067] The intermediate 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylic acid ethyl ester D1 (0.40 g, 1.0 mmol) prepared in Example 5, along with substituted benzyl bromide or benzyl chloride (4-(bromomethyl)benzenesulfonamide, 4-(chloromethyl)benzamide, 4-methanesulfonyl benzyl bromide or 4-(bromomethyl)phenylboronic acid) (1.1 mmol), was dissolved in 10 mL of N,N-dimethylformamide. After adding potassium carbonate (0.28 g, 2.0 mmol), the mixture was reacted at room temperature for 4 h. After the reaction was monitored by TLC, 50 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated sodium chloride solution (3 × 5 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography using methanol:dichloromethane (1:20) as the eluent. Finally, the target compounds E1-E4 were obtained by recrystallization from ethyl acetate and petroleum ether.

[0068] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester (E1)

[0069] White solid, yield 55%, mp: 187-189 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.58 (s,1H), 8.08 (d, J = 7.0 Hz, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.61 (s, 2H), 7.42(d, J = 7.9 Hz, 2H), 7.26 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 3.43 (s, 3H), 2.59 (d, J = 11.4 Hz, 2H), 2.01 (s, 6H), 1.87 (d, J = 11.8 Hz, 2H), 1.65 (d,J = 12.0 Hz, 2H), 1.38 (q, J = 10.7 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 166.04, 165.08, 162.36, 162.13, 153.91, 143.20, 132.81,132.78, 129.49, 126.08, 119.09, 108.58, 101.57, 61.83, 61.28, 51.91, 31.14,16.12, 14.52. ESI-MS: m / z 565.26 [M+H] + . C 28 H 32 N6O5S (564.22).

[0070] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid ethyl ester (E2)

[0071] White solid, yield 59%, mp: 155-157 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.57 (s,1H), 8.08 (d, J = 6.9 Hz, 1H), 7.87 (s, 1H), 7.77 (d, J = 8.1 Hz, 2H), 7.60(s, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.25 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.41 (s, 3H), 2.67–2.52 (m, 2H), 2.01 (s, 6H), 1.84 (s, 2H), 1.65 (d, J =13.5 Hz, 2H), 1.38 (q, J = 17.4, 14.2 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13 CNMR (100 MHz, DMSO-d6): δ 168.22, 166.03, 165.08, 162.36, 162.12, 153.91,133.47, 132.81, 132.77, 128.92, 127.90, 119.08, 108.58, 101.56, 62.07, 61.27,51.90, 48.31, 31.14, 16.12, 14.51. ESI-MS: m / z 529.36 [M+H] + . C 29 H 32N6O4(528.25).

[0072] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester (E3)

[0073] White solid, yield 49%, mp: 150-152 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.65 (s,1H), 8.15 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.9 Hz, 2H), 7.67 (s, 2H), 7.57(d, J = 7.9 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 3.55 (s, 2H), 3.21 (s, 3H), 2.66 (d, J = 11.2 Hz, 2H), 2.08 (s, 6H), 1.99–1.88 (m, 2H), 1.73 (d, J = 12.2Hz, 2H), 1.55–1.37 (m, 3H), 1.29 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 166.04, 165.09, 162.37, 162.15, 153.92, 145.24, 139.90, 132.81,132.78, 129.81, 127.41, 119.09, 108.59, 101.59, 61.74, 61.28, 51.92, 48.22,44.06, 31.16, 16.12, 14.52. ESI-MS: m / z 564.30 [M+H] + . C 29 H 33 N5O5S (563.22).

[0074] (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(ethoxycarbonyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E4)

[0075] White solid, yield 44%, mp: 148-150 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.57 (s,1H), 8.08 (d, J = 6.9 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 7.6 Hz,2H), 7.60 (s, 2H), 7.19 (d, J = 7.4 Hz, 2H), 4.21 (q, J = 7.1 Hz, 2H), 3.43(s, 1H), 3.37 (s, 2H), 2.73–2.52 (m, 2H), 2.01 (s, 6H), 1.84 (s, 2H), 1.71–1.59 (m, 2H), 1.49–1.30 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 166.03, 165.08, 162.36, 162.12, 153.91, 134.53, 134.42, 132.80,132.77, 128.35, 125.82, 119.07, 108.59, 101.56, 63.37, 62.56, 61.27, 51.90,31.11, 16.12, 14.52. C 28 H 32 BN5O5 (529.25).

[0076] Example 8: General method for preparing target compounds E5-E8

[0077] The preparation method is the same as in Example 7, except that:

[0078] The starting material ethyl 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylate D1 (0.40 g, 1.0 mmol) was replaced with methyl 4-(4-cyano-2,6-dimethylphenoxy)-2-(piperidin-4-ylamino)pyrimidine-5-carboxylate D2 (0.38 g, 1.0 mmol) prepared in Example 6; finally, the target compounds E5-E8 were obtained by recrystallization from ethyl acetate and petroleum ether.

[0079] Methyl 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylate (E5)

[0080] White solid, yield 67%, mp: 141-143 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.66 (d,J = 15.8 Hz, 1H), 8.11 (d, J = 6.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.61(s, 2H), 7.40–7.33 (m, 2H), 7.24 (d, J = 6.9 Hz, 2H), 3.72 (d, J = 6.6 Hz, 3H), 3.39 (d, J = 28.5 Hz, 2H), 2.84–2.50 (m, 3H), 1.99 (s, 6H), 1.75–1.08 (m, 6H). 13 C NMR (100 MHz, DMSO-d6): δ 166.76, 166.36, 163.88, 162.58, 154.13,143.17, 132.97, 132.92, 132.86, 132.52, 129.49, 129.40, 126.06, 119.12,108.65, 98.83, 61.93, 52.61, 52.42, 52.00, 49.79, 31.55, 31.05, 16.35, 16.10.ESI-MS: m / z 551.22 [M+H] + . C 27 H 30 N6O5S (550.20).

[0081] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid methyl ester (E6)

[0082] White solid, 70% yield, mp: 165-167 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.66 (d,J = 16.3 Hz, 1H), 8.10 (d, J = 6.6 Hz, 1H), 7.85 (s, 1H), 7.75 (d, J = 8.2Hz, 2H), 7.61 (s, 2H), 7.26 (dd, J = 8.6, 3.0 Hz, 3H), 3.72 (d, J = 6.5 Hz,3H), 3.36 (d, J = 29.0 Hz, 2H), 2.91–2.50 (m, 3H), 2.00 (s, 6H), 1.73–1.12(m, 6H).13 C NMR (100 MHz, DMSO-d6): δ 168.23, 166.76, 163.87, 162.57, 162.45,154.12, 153.74, 142.39, 133.44, 132.97, 132.91, 132.86, 132.52, 128.91,128.84, 127.89, 119.09, 108.74, 108.66, 99.88, 98.81, 62.17, 52.62, 52.43,52.00, 49.83, 31.56, 31.06, 16.35, 16.10. ESI-MS: m / z 515.26 [M+H] + . C 28 H 30 N6O4(514.23).

[0083] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid methyl ester (E7)

[0084] White solid, 75% yield, mp: 180-182 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.74 (d,J = 15.7 Hz, 1H), 8.18 (d, J = 6.6 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.69(s, 2H), 7.54 (d, J = 8.2 Hz, 2H), 3.81 (s, 3H), 3.51 (d, J = 25.9 Hz, 2H), 3.21 (s, 3H), 2.98–2.59 (m, 3H), 2.07 (s, 6H), 1.89–1.16 (m, 6H). 13C NMR (100MHz, DMSO-d6): δ 166.76, 163.88, 163.81, 162.58, 162.46, 154.12, 153.74,145.29, 139.84, 132.98, 132.91, 132.86, 132.52, 129.83, 129.74, 127.41,127.28, 119.12, 99.92, 98.84, 61.82, 52.65, 52.41, 52.00, 49.71, 44.05,31.55, 31.07, 16.35, 16.10. ESI-MS: m / z 550.25 [M+H] + . C 28 H 31 N5O5S (549.20).

[0085] (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(methoxycarbonyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E8)

[0086] White solid, yield 51%, mp: 163-165 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.86–8.55(m, 1H), 8.08 (d, J = 6.6 Hz, 1H), 7.89 (s, 2H), 7.67 (d, J = 7.7 Hz, 2H),7.61 (s, 2H), 7.15 (d, J = 7.5 Hz, 2H), 3.73 (s, 3H), 3.35 (s, 2H), 2.82–2.56(m, 3H), 2.00 (s, 6H), 1.88–1.11 (m, 6H). 13 C NMR (100 MHz, DMSO-d6): δ166.77, 163.87, 163.81, 162.58, 162.47, 154.12, 153.74, 134.52, 132.96,132.91, 132.87, 132.51, 128.23, 119.07, 108.69, 98.83, 52.45, 51.99, 31.09,16.35, 16.10. C 27 H 30 BN5O5 (515.23).

[0087] Example 9: General method for preparing target compounds E9-E12

[0088] Compounds E1-E4 (1.0 mmol) were dissolved in 10 mL of methanol, and then a solution of hydrated lithium hydroxide (0.21 g, 5.0 mmol) dissolved in 10 mL of water was added. The reaction was carried out at room temperature for 4 h. After the reaction was completed by TLC monitoring, the methanol solvent in the reaction solution was concentrated under reduced pressure. 1 mol / L dilute hydrochloric acid solution was slowly added dropwise to the reaction solution until no more white solid precipitated. The mixture was stirred for 30 min, and the precipitate in the suspension was collected by filtration. The precipitate was washed with ice water (3 × 5 mL) and dried to obtain the target compounds E9-E12.

[0089] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid (E9)

[0090] White solid, yield 67%, mp: 218-220 °C. 1 H NMR (400 MHz, DMSO-d6): δ 12.60 (s,1H), 8.71 (d, J = 18.7 Hz, 1H), 8.08 (d, J = 6.4 Hz, 1H), 7.79 (d, J = 7.9Hz, 2H), 7.67 (s, 2H), 7.48 (s, 2H), 7.38–7.25 (m, 2H), 3.43 (s, 2H), 3.10–2.55 (m, 3H), 2.07 (s, 6H), 1.86–1.11 (m, 6H). 13 C NMR (100 MHz, DMSO-d6): δ166.98, 164.91, 164.85, 164.19, 162.60, 154.28, 153.88, 132.92, 132.84,132.49, 126.10, 108.63, 108.53, 16.35, 16.10. ESI-MS: m / z 547.23 [M+H] + .C 26 H 28 N6O5S (536.18).

[0091] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid (E10)

[0092] White solid, yield 56%, mp: 187-189 ℃.1 H NMR (400 MHz, DMSO-d6): δ 8.93 (s,1H), 8.53 (s, 1H), 7.99 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.66 (s, 2H), 7.53(d, J = 7.9 Hz, 2H), 7.40 (s, 1H), 3.88 (s, 2H), 3.70 (s, 1H), 2.92 (s, 2H), 2.41 (s, 2H), 2.08 (s, 6H), 1.91 (d, J = 13.7 Hz, 3H), 1.55 (q, J = 11.0 Hz,2H). 13 C NMR (100 MHz, DMSO-d6): δ 168.60, 168.03, 164.62, 162.88, 161.60,154.09, 134.36, 132.87, 132.77, 130.13, 128.08, 119.16, 108.36, 51.12, 46.72,30.12, 16.20. ESI-MS: m / z 501.27 [M+H] + . C 27 H 28 N6O4 (500.22).

[0093] 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid (E11)

[0094] White solid, yield 60%, mp: 210-212 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.58 (s,1H), 8.54 (d, J = 6.9 Hz, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.69 (d, J = 8.7 Hz,2H), 7.66 (s, 2H), 3.85 (s, 2H), 3.66 (s, 1H), 3.23 (s, 3H), 2.87 (d, J =11.6 Hz, 2H), 2.33 (d, J = 11.9 Hz, 2H), 2.08 (s, 6H), 2.01–1.81 (m, 3H),1.58 (q, J = 11.7, 11.1 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 168.21, 164.93,162.73, 162.20, 153.99, 140.62, 132.84, 132.79, 130.77, 127.52, 119.11,108.48, 102.86, 51.38, 47.21, 43.97, 39.70, 30.24, 16.17. ESI-MS: m / z 536.21[M+H] + . C 27 H 29 N5O5S (535.19).

[0095] 2-((1-(4-boronbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid (E12)

[0096] White solid, yield 63%, mp: 222-224 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 8.71 (d,J = 18.7 Hz, 1H), 8.08 (s, 1H), 8.01 (d, J = 6.6 Hz, 1H), 7.74 (d, J = 7.5Hz, 2H), 7.67 (s, 2H), 7.23 (d, J = 7.1 Hz, 2H), 3.78 (s, 2H), 3.47 (s, 2H), 2.82 (dt, J = 25.8, 8.5 Hz, 2H), 2.66 (d, J = 11.2 Hz, 1H), 2.07 (s, 6H), 1.83–1.19 (m, 6H). 13 C NMR (100 MHz, DMSO-d6): δ 166.97, 166.62, 165.09,164.16, 162.55, 154.32, 134.51, 132.92, 132.45, 128.32, 119.11, 108.55,62.61, 52.39, 49.74, 39.64, 31.42, 31.01, 16.35, 16.10. C 26 H 28 BN5O5 (501.22).

[0097] Example 10: General method for preparing target compounds E13-E16

[0098] Compounds E9-E12 (1.0 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.14 g, 3.0 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Then, N,N-diisopropylethylamine (0.26 g, 2.0 mmol) was added at 0 °C, and the mixture was stirred for 15 min. O-methylhydroxylamine hydrochloride (0.09 g, 1.1 mmol) was then added, and the reaction was continued at room temperature for 8 h. After the reaction was completed by TLC monitoring, 50 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated sodium chloride solution (3 × 5 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography using methanol:dichloromethane (1:20) as the eluent. Finally, the target compounds E13-E16 were obtained by recrystallization from ethyl acetate and petroleum ether.

[0099] 4-(4-cyano-2,6-dimethylphenoxy)-N-methoxy-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxamide (E13)

[0100] White solid, yield 72%, mp: 166-168 °C. 1 H NMR (400 MHz, DMSO-d6): δ 11.78 (s,1H), 8.56 (d, J = 6.9 Hz, 1H), 8.35 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.67(s, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.32 (s, 2H), 3.69 (s, 3H), 3.52 (s, 2H), 2.99–2.57 (m, 3H), 2.07 (s, 6H), 1.95 (t, J = 15.3 Hz, 2H), 1.73 (d, J = 12.4Hz, 2H), 1.50–1.35 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 164.43, 164.25,162.04, 157.69, 154.02, 151.76, 143.21, 143.16, 132.87, 132.74, 129.50,129.09, 126.08, 119.12, 108.45, 102.67, 63.95, 61.83, 51.88, 31.22, 16.14.ESI-MS: m / z 566.21 [M+H] + . C 27 H 31 N7O5S (565.21).

[0101] 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)-N-methoxypyrimidine-5-carboxamide (E14)

[0102] White solid, 77% yield, mp: 147-149 °C. 1 H NMR (400 MHz, DMSO-d6): δ 11.78 (s,1H), 8.55 (d, J = 6.9 Hz, 1H), 8.34 (s, 1H), 7.93 (s, 1H), 7.84 (d, J = 7.8Hz, 2H), 7.66 (s, 2H), 7.38 (d, J = 7.6 Hz, 2H), 7.32 (s, 1H), 3.69 (s, 3H), 3.51 (s, 2H), 2.82–2.58 (m, 2H), 2.07 (s, 6H), 1.73 (s, 2H), 1.39 (d, J =32.7 Hz, 2H), 1.24 (d, J = 6.9 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 168.19,164.24, 162.03, 157.67, 154.01, 152.86, 132.86, 132.74, 130.85, 128.92,127.93, 119.11, 108.46, 102.70, 63.93, 51.84, 29.49, 16.14. ESI-MS: m / z530.42 [M+H] + . C 28 H 31 N7O4 (529.24).

[0103] 4-(4-cyano-2,6-dimethylphenoxy)-N-methoxy-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxamide (E15)

[0104] White solid, yield 65%, mp: 133-135 °C. 1 H NMR (400 MHz, DMSO-d6): δ 11.80 (s,1H), 8.57 (d, J = 6.8 Hz, 1H), 8.34 (s, 1H), 7.99–7.76 (m, 2H), 7.67 (s, 2H),7.62–7.53 (m, 2H), 3.68 (s, 3H), 3.53 (d, J = 17.1 Hz, 2H), 3.22 (s, 3H), 2.67 (d, J = 4.9 Hz, 2H), 2.07 (s, 6H), 1.97 (d, J = 14.5 Hz, 2H), 1.73 (s,2H), 1.40 (t, J = 11.9 Hz, 2H), 1.25 (q, J = 5.0, 4.4 Hz, 1H). 13 C NMR (100MHz, DMSO-d6): δ 164.40, 164.24, 162.06, 157.68, 153.99, 132.85, 132.76,129.85, 127.47, 119.13, 108.44, 102.69, 63.97, 61.77, 51.87, 44.01, 31.10,16.15. ESI-MS: m / z 565.22 [M+H] + . C 28 H 32 N6O5S (564.22).

[0105] (4-((4-((4-cyano-2,6-dimethylphenoxy)-5-(methoxycarbamoyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E16)

[0106] White solid, yield 43%, mp: 216-218 ℃. 1H NMR (400 MHz, DMSO-d6): δ 11.13 (d,J = 13.7 Hz, 1H), 8.62 (s, 1H), 8.02 (s, 2H), 7.95 (d, J = 6.4 Hz, 1H), 7.74(d, J = 7.6 Hz, 2H), 7.68 (s, 2H), 7.24 (t, J = 7.2 Hz, 2H), 3.70 (s, 3H), 2.82 (s, 2H), 2.70–2.62 (m, 1H), 2.09 (s, 6H), 1.99 (s, 1H), 1.74 (d, J =12.1 Hz, 1H), 1.47 (d, J = 11.5 Hz, 3H), 1.24 (d, J = 9.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 170.83, 164.98, 164.84, 162.31, 153.82, 153.50, 134.54,133.21, 132.86, 132.42, 128.43, 119.09, 108.72, 63.84, 60.24, 52.37, 21.24,16.46, 16.20, 14.55. C 27 H 31 BN6O5 (530.24).

[0107] Experimental Example: In vitro anti-HIV-1 activity experiment

[0108] Experimental Example 1: Anti-HIV-1 activity and cytotoxicity of the target compound at the cellular level

[0109] Experimental Principle

[0110] TZM-bl cells carry a luciferase (Luc) reporter gene, which is regulated by the tat element of HIV-1 and can express luciferase under the action of the Tat protein. When TZM-bl cells are infected with HIV-1, the Tat protein produced by the virus upregulates the luciferase reporter gene in the cells, resulting in high luciferase expression. This can then be detected using specific luciferase detection reagents to assess viral infection. When a target compound or positive control drug is added, the viral replication cycle is blocked, and the amount of Tat protein produced decreases, leading to a reduction in the expression level of luciferase in TZM-bl cells, thus reflecting the inhibitory effect of the inhibitor on the virus.

[0111] Experimental materials

[0112] The target compounds, positive control drugs nevirapine (NVP), efavirenz (EFV), ectavirine (ETR), and zidovudine (AZT), TZM-bl cells, 2,3-di-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxaniline (XTT), and HIV-1 NL4-3 wild-type strain.

[0113] Experimental methods

[0114] Cell viability assay method (EC) 50 TCID values: The anti-HIV-1 activity of the test samples at the cellular level was reflected by the degree of reduction in luciferase reporter gene expression after infection of TZM-bl cells with different viruses (wild-type or mutant strains). First, 200-fold TCID values ​​were applied. 50 HIV-1 at various concentrations, test samples at various concentrations (maximum concentration 10 μg / mL, serially diluted 8 times in 4-fold increments), and TZM-bl cells (10,000 cells) were mixed and incubated in a 96-well black cell culture plate (Corning-Costar) in a total volume of 150 μL of growth medium. Then, one set of eight wells was incubated with both cells and virus (virus control group), while another set of eight wells was incubated with only cells (blank control group). After 48 hours of incubation, the medium was removed from each well, and 100 μL of Promega Bright-Glo luciferase assay reagent was added to the cells. After incubation at room temperature for 2 minutes, luciferase activity in the test wells was measured using a Perkin-Elmer Victor X-ray spectrophotometer. 50% inhibitory concentration (EC50) was used. 50 The RLU value is defined as the concentration of a test sample that results in a 50% reduction in the RLU value compared to the virus control well after subtracting the RLU value of the blank control well.

[0115] Cytotoxicity assay method (CC) 50 The cytotoxicity of the test samples was determined by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxyaniline (XTT) colorimetric method. First, equal volumes of TZM-bl cells (5 × 10⁻⁶ cells per mL) were seeded. 5 100 μL of test samples at different concentration gradients were added to 96-well plates containing 100 cells. After incubation at 37 °C for 4 days, 50 μL of 0.02 μM methyl phenazine sulfate XTT solution was added to the 96-well plates, and after another 4 hours of incubation, the absorbance at 450 nm was measured. 50% cytotoxic concentration (CC) 50 The value is defined as the concentration of the test sample that causes a 50% reduction in cell viability.

[0116] The synthesized partially carbonyl-substituted pyrimidine derivatives were subjected to cellular-level antiviral activity and cytotoxicity tests using the experimental methods described above. Cellular activity (ECG) was measured. 50 ), cytotoxicity (CC) 50 The selectivity index (SI) and the selectivity index are shown in Table 1.

[0117] Table 1. Antiviral activity, cytotoxicity, and selectivity index of carbonyl-substituted pyrimidine derivatives and positive control drugs

[0118]

[0119] Note: a EC 50 The concentration of compounds that protect 50% of HIV-infected cells from cytopathic effects;

[0120] b CC 50 The concentration at which the target compound causes 50% of uninfected HIV cells to become diseased;

[0121] c SI:CC 50 / EC 50 , Selectivity index.

[0122] As shown in Table 1, the carbonyl-substituted pyrimidine compounds provided by this invention are a novel class of HIV-1 inhibitors, exhibiting good anti-HIV-1 activity, and their EC50 values ​​are [not specified]. 50 The values ​​ranged from 4.28 nM to 274 nM, significantly superior to the marketed drug nevirapine (EC). 50 = 281 ± 38.7 nM). Structure-activity relationship studies indicate that compound E5 (EC) with methyl formate substituted group... 50 = 5.82 ± 0.85 nM), E6 (EC 50 = 4.28 ± 0.47 nM), E7 (EC 50 = 6.01 ± 0.76 nM) and E8 (EC 50 The anti-HIV-1 activity of compound E6 (7.18 ± 0.66 nM) was particularly outstanding, with compound E6 (EC) exhibiting the best activity. 50 =4.28 ± 0.47 nM) compared to nevirapine (EC) 50 = 281 ± 38.7 nM) activity increased nearly 65 times, superior to zidovudine (EC) 50 = 7.50 ± 1.8 nM) and efavirenz (EC 50 = 5.20 ± 0.90 nM), and exhibits low cytotoxicity and a high selectivity index (CC). 50= 35.4 ± 3.3 µM, SI = 8273).

[0123] Experiment Example 2: HIV-1 Reverse Transcriptase Inhibitory Activity Experiment

[0124] Experimental Principle

[0125] ELISA (Enzyme-Linked Immunosorbent Assay) is a method used to test the inhibitory activity (IC50) of a target compound against HIV-1 reverse transcriptase (RT). 50 (Value). During reverse transcription, RT uses Poly(A) as a template and utilizes dNTPs simultaneously labeled with biotin and digoxigenin as raw materials to introduce primer oligo(dT). 15 DNA strand elongation was performed. After the synthesis of the DNA:RNA hybrid strand was completed, the hybrid strand containing biotin and digoxigenin-labeled substrates could firmly bind to a microplate coated with streptavidin. Subsequently, horseradish peroxidase-conjugated digoxigenin antibody was added, which bound to the hybrid strand attached to the microplate. Finally, a colorimetric reaction was generated by adding the horseradish peroxidase substrate ABTS, and the absorbance value of the color product was detected using a microplate reader. The inhibition rate of reverse transcriptase at each concentration of the test sample was calculated, and the IC50 of its enzyme-inhibiting activity was thus determined. 50 value.

[0126] Experimental materials

[0127] Target compound, positive control drugs nevirapine (NVP), efavirenz (EFV), ectavirine (ETR), and rilpivirine (RPV), HIV-1 reverse transcriptase kit (Roche, Switzerland), micropipette, and analytical grade DMSO.

[0128] Experimental methods

[0129] The HIV-1 RT inhibitory activity was determined using a reverse transcriptase kit. First, the stock solution of the test compound was serially diluted with lysis buffer to prepare five sample solutions of different concentrations. Then, the reverse transcriptase was prepared using triple-distilled water and lysis buffer. Next, 20 μL of each concentration of sample solution and 20 μL of the reverse transcriptase solution were added to 20 μL of a reaction mixture containing primers, template, and substrate, and incubated together at 37 °C for 1 hour. Then, 60 μL of the incubated sample solution was transferred to a 96-well plate coated with streptavidin and incubated at 37 °C for another hour to ensure strong binding of the biotin- and digoxigenin-labeled hybrid strands to streptavidin. Subsequently, the reaction wells were rinsed with washing buffer (5 × 250 μL) to thoroughly remove unbound substrate. Then, 200 μL of horseradish peroxidase solution conjugated with digoxigenin antibody was added, and the mixture was incubated at 37 °C for 1 hour to ensure strong binding of the digoxigenin antibody to the digoxigenin-labeled hybrid strand. The wells were then rinsed again with washing buffer (5 × 250 μL) to thoroughly remove excess horseradish peroxidase. Finally, 200 μL of the horseradish peroxidase substrate ABTS was added to the wells, and the absorbance of the color product was measured using a microplate reader at a measurement wavelength of 405 nm and a reference wavelength of 490 nm. Similarly, in addition to the test sample group, parallel measurements were performed on the negative control group (no test sample, with reverse transcriptase) and the blank control group (no test sample, no reverse transcriptase).

[0130] Inhibition rate calculation formula: Inhibition rate % × 100%

[0131] The inhibition rate of the test sample against RT at different concentrations can be calculated using this formula, and then the compound concentration corresponding to a 50% inhibition rate of RT can be obtained, which is its IC50 inhibitory activity. 50 Value. Enzyme inhibitory activity (IC50) 50 The results are shown in Table 2.

[0132] Table 2. Inhibitory activity of carbonyl-substituted pyrimidine derivatives against HIV-1 reverse transcriptase

[0133]

[0134] Note: a IC 50 : The concentration of the compound corresponding to a reverse transcriptase inhibition rate of 50%.

[0135] As shown in Table 2, compound E6, which exhibits the strongest antiviral activity at the cellular level, demonstrates significant HIV-1 reverse transcriptase inhibitory activity (IC50). 50 = 0.065 ± 0.019 µM), far superior to ectovirine (IC50).50 = 0.232 ± 0.036µM) and rilpivirine (IC50) 50 = 0.118 ± 0.027 µM), indicating that the target of this type of compound is reverse transcriptase, belonging to the typical non-nucleoside reverse transcriptase inhibitors.

Claims

1. A carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof, characterized in that... It has the structure shown in general formula I: ； in, R1 can be H, CH3, OH, OCH3, OCH2CH3, NH2, NHNH2, NHCH3, NHOH, or NHOCH3; R2 is a halogen atom, H, CH3, OH, CN, NH2, NO2, NHSO2NH2, NHSO2CH3, COOCH3, COOH, SO2NH2, CONH2, SO2CH3, or B(OH)2; the substituent is ortho, meta, para monosubstituted or polysubstituted; R3 can be CN, CH3, CH=CHCN, or p-C4H6-CN.

2. The carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor according to claim 1, characterized in that, The carbonyl-substituted pyrimidine derivative or its pharmaceutically acceptable salt is selected from one of the following: 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester (E1); 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid ethyl ester (E2); 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester (E3); (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(ethoxycarbonyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E4); 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid methyl ester (E5); 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid methyl ester (E6); 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid methyl ester (E7); (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(methoxycarbonyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E8); 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid (E9); 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid (E10); 4-(4-cyano-2,6-dimethylphenoxy)-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxylic acid (E11); 2-((1-(4-boronbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)pyrimidine-5-carboxylic acid (E12); 4-(4-cyano-2,6-dimethylphenoxy)-N-methoxy-2-((1-(4-sulfonamidobenzyl)piperidin-4-yl)amino)pyrimidine-5-carboxamide (E13); 2-((1-(4-carbamoylbenzyl)piperidin-4-yl)amino)-4-(4-cyano-2,6-dimethylphenoxy)-N-methoxypyrimidine-5-carboxamide (E14); 4-(4-cyano-2,6-dimethylphenoxy)-N-methoxy-2-((1-(4-(methanesulfonyl)benzyl)piperidin-4-yl)amino)pyrimidine-5-carboxamide (E15); (4-((4-((4-(4-cyano-2,6-dimethylphenoxy)-5-(methoxycarbamoyl)pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)boronic acid (E16).

3. A method for preparing the carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, comprising the following steps: 1) Using ethyl 2,4-dichloropyrimidine-5-carboxylate A1 or methyl 2,4-dichloropyrimidine-5-carboxylate A2 as starting materials, a nucleophilic substitution reaction is carried out with 4-hydroxy-3,5-dimethylbenzonitrile in N,N-dimethylformamide solution to obtain intermediate B1 or B2. 2) Intermediate B1 or B2 reacts with tert-butyl 4-aminopiperidine-1-carboxylate via a nucleophilic substitution reaction to obtain intermediate C1 or C2, and the Boc protecting group is removed by trifluoroacetic acid to generate key intermediate D1 or D2. 3) Intermediate D1 or D2 reacts with substituted benzyl bromo or benzyl chloride to give target compounds E1-E4 or E5-E8, respectively; compounds E1-E4 undergo ester hydrolysis in a mixed solution of methanol and water under the action of lithium hydroxide hydrate to give target compounds E9-E12; simultaneously, compounds E9-E12 undergo amide condensation with O-methylhydroxylamine hydrochloride in the action of condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate to give target compounds E13-E16; The synthesis route is as follows: ； Reagents and conditions: (i) 4-hydroxy-3,5-dimethylbenzonitrile, potassium carbonate, N,N-dimethylformamide, room temperature; (ii) tert-butyl 4-aminopiperidine-1-carboxylate, potassium carbonate, N,N-dimethylformamide, 120 °C; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) substituted benzyl bromo or benzyl chloride, potassium carbonate, N,N-dimethylformamide, room temperature; ； Reagents and conditions: (i) Lithium hydroxide hydrate, methanol:water = 1:1 V / V, room temperature; (ii) O-methylhydroxylamine hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, 0 °C to room temperature.

4. The use of the carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor of claim 1 or a pharmaceutically acceptable salt thereof as an HIV-1 inhibitor.

5. The use of the carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of an anti-AIDS drug.

6. The use of the carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor of claim 1 or a pharmaceutically acceptable salt thereof as a non-nucleoside NNRTI in the preparation of an anti-HIV drug.

7. An anti-HIV-1 pharmaceutical composition comprising the carbonyl-substituted pyrimidine HIV-1 reverse transcriptase inhibitor of claim 1 or a pharmaceutically acceptable salt thereof and pharmaceutical excipients.

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