Aza-alicyclic ring substituted pyrimidine derivative as well as preparation method and application thereof

Abstract

The invention relates to an aza-alicyclic ring substituted pyrimidine derivative and a preparation method thereof, the aza-alicyclic ring substituted pyrimidine derivative has a structure as shown in a formula I. The aza-alicyclic ring substituted pyrimidine derivative provided by the invention is novel in structure, most of the compounds show relatively strong anti-HIV-1 activity, and the EC50 value is between 9.40 nM and 238 nM. Wherein the activity of compounds WP-10a (EC50 is equal to 12.6 + / -0.68 nM), WP-10b (EC50 is equal to 9.40 + / -0.96 nM), WP-10c (EC50 is equal to 18.3 + / -1.6 nM) and WP-10d (EC50 is equal to 26.2 + / -6.0 nM) of which the X site is substituted by amino azetidine is particularly prominent, the WP-10b with the optimal activity has the antiviral effect of single-digit nanomole, the activity is improved by about 30 times compared with that of a drug nevirapine (EC50 is equal to 281 + / -38.7 nM) on the market, and the activity is equivalent to that of efavirenz (EC50 is equal to 5.20 + / -0.90 nM)

Description

Technical Field

[0001] This invention relates to an aza-aliphatic substituted pyrimidine derivative, its preparation method and application, belonging to the field of organic compound synthesis and pharmaceutical application technology. Background Technology

[0002] AIDS is a major infectious disease primarily caused by infection with human immunodeficiency virus type 1 (HIV-1). Reverse transcriptase (RT) in the HIV-1 life cycle, due to its well-defined mechanism of action and rich structural biological information, has become a preferred target for anti-AIDS drug development. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) targeting this target have become a research hotspot in the field of anti-AIDS drug development in recent years due to their advantages such as high efficacy and low toxicity. Although nine NNRTIs have been approved for marketing in the United States, China, Europe, and Russia, many of these drugs have gradually revealed problems such as drug resistance, toxic side effects, and undesirable pharmacokinetic properties after several years of clinical use.

[0003] Therefore, the development of a new generation of NNRTIs with high anti-drug resistance activity and good safety remains a long and arduous task; it is of great significance to develop new, highly effective, and low-toxicity anti-AIDS drugs with independent intellectual property rights in my country as soon as possible. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an aza-aliphatic ring-substituted pyrimidine derivative and its preparation method.

[0005] The technical solution of the present invention is as follows:

[0006] The first objective of this invention is to provide an aza-alicyclic substituted pyrimidine derivative.

[0007] A nitrogen-containing aliphatic ring-substituted pyrimidine derivative has the structure shown in Formula I:

[0008]

[0009] in,

[0010] X is an aza-aliphatic ring, including piperazine ring, aminopiperidine ring, aminoazacyclobutane, aminoazabicyclooctane, azaspiro ring or high piperazine ring;

[0011] R1 is Boc, H, CH3, SO2NH2, SO2CH3, SO2CH2CH3, SO2N(CH3)2, SO2CH(CH3)2, CONH2, COCH3, COCH2CH3, CON(CH3)2 or COCH(CH3)2;

[0012] R2 is CN, CH3, CH=CHCN, or a substituted phenyl group.

[0013] According to a preferred embodiment of the present invention, the aza-alicyclic substituted pyrimidine derivative is selected from one of the following:

[0014] 4-((2-((4-cyanophenyl)amino)-6-(piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4a);

[0015] 4-((2-((4-cyanophenyl)amino)-6-(4-(methanesulfonyl)piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4b);

[0016] 4-((2-((4-cyanophenyl)amino)-6-(4-(ethylsulfonyl)piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4c);

[0017] 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethylpiperazine-1-sulfonamide (WP-4d);

[0018] 4-((6-(4-aminopiperidin-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-6a);

[0019] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)methanesulfonamide (WP-6b);

[0020] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)ethanesulfonamide (WP-6c);

[0021] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)-N,N-dimethylsulfonamide (WP-6d);

[0022] 4-((2-((4-cyanophenyl)amino)-6-(piperidin-4-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8a);

[0023] 4-((2-((4-cyanophenyl)amino)-6-((1-(methanesulfonyl)piperidin-4-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8b);

[0024] 4-((2-((4-cyanophenyl)amino)-6-((1-(ethylsulfonyl)piperidin-4-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8c);

[0025] 4-((6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylpiperidine-1-sulfonamide (WP-8d);

[0026] 4-((6-(3-aminoazacyclobutane-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-10a);

[0027] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)methanesulfonamide (WP-10b);

[0028] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)ethanesulfonamide (WP-10c);

[0029] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)-N,N-dimethylsulfonamide (WP-10d);

[0030] 4-((6-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14a);

[0031] 4-((2-((4-cyanophenyl)amino)-6-((1R,5S)-8-(methanesulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14b);

[0032] 4-((2-((4-cyanophenyl)amino)-6-((1R,5S)-8-(ethylsulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14c);

[0033] (1R,5S)-3-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethyl-3,8-diazabicyclo[3.2.1]octane-8-sulfonamide (WP-14d);

[0034] 4-((2-((4-cyanophenyl)amino)-6-(1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16a);

[0035] 4-((2-((4-cyanophenyl)amino)-6-(4-(methanesulfonyl)-1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16b);

[0036] 4-((2-((4-cyanophenyl)amino)-6-(4-(ethylsulfonyl)-1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16c);

[0037] 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethyl-1,4-diazacycloheptane-1-sulfonamide (WP-16d).

[0038] A second objective of this invention is to provide a method for preparing the above-mentioned aza-aliphatic ring substituted pyrimidine derivatives.

[0039] The preparation method of aza-alicyclic substituted pyrimidine derivatives includes the following steps:

[0040] 1) Starting from 2,4,6-trichloropyrimidine 1, intermediate 2 was prepared by nucleophilic substitution reaction with 4-hydroxy-3,5-dimethylbenzonitrile in a 1,4-dioxane solution;

[0041] 2) Intermediate 2 reacts with p-aminobenzonitrile under alkaline conditions in potassium tert-butoxide to obtain intermediate 3; using N,N-dimethylformamide as solvent, intermediate 3 reacts with piperazine-1-carboxylate tert-butyl ester, piperidine-4-carboxylate tert-butyl ester, 4-aminopiperidine-1-carboxylate tert-butyl ester, azacyclobutane-3-carboxylate tert-butyl ester, (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate tert-butyl ester, or 1,4-diazacycloheptane-1-carboxylate tert-butyl ester under high-temperature alkaline conditions via nucleophilic substitution to obtain key intermediates 4a-f; subsequently, intermediate 4a-f undergoes deprotection of the Boc protecting group in dichloromethane solution via trifluoroacetic acid to generate intermediate 5a-f;

[0042] 3) Intermediates 5a-f undergo acylation reactions with various substituted sulfonyl chlorides or acyl chlorides under basic conditions of triethylamine to obtain target compound I;

[0043] The synthesis route is as follows:

[0044]

[0045] Reagents and conditions: (i) 4-hydroxy-3,5-dimethylbenzonitrile, N,N-diisopropylethylamine, 1,4-dioxane, 65°C; (ii) p-aminobenzonitrile, potassium tert-butoxide, N-methylpyrrolidone, 0°C to room temperature; (iii) aminoazalicyclic, potassium carbonate, N,N-dimethylformamide, 120°C; (iv) trifluoroacetic acid, dichloromethane, room temperature; (v) substituted sulfonyl chloride or acyl chloride, triethylamine, dichloromethane, 0°C to room temperature.

[0046] The compounds 5a, 5b, 5c, 5d, 5e, and 5f obtained in this invention are the target compounds WP-4a, WP-6a, WP-8a, WP-10a, WP-14a, and WP-16a.

[0047] A third objective of this invention is to provide applications of the above-mentioned aza-alicyclic substituted pyrimidine derivatives.

[0048] The above-mentioned aza-alicyclic substituted pyrimidine derivatives are used as HIV-1 inhibitors.

[0049] The above-mentioned aza-alicyclic substituted pyrimidine derivatives are used in the preparation of anti-AIDS drugs.

[0050] The above-mentioned aza-alicyclic substituted pyrimidine derivatives are used as non-nucleoside NNRTIs in the preparation of anti-HIV drugs.

[0051] An anti-HIV-1 pharmaceutical composition comprising the above-mentioned azafatty ring substituted pyrimidine derivatives and their pharmaceutically acceptable salts and pharmaceutical excipients.

[0052] Technical features and advantages of the present invention:

[0053] 1. This invention discloses the results of antiviral activity, cytotoxicity, and enzyme-level inhibitory activity of aza-alicyclic substituted pyrimidine derivatives at the cellular level, as well as their first application as an anti-AIDS drug.

[0054] 2. The aza-aliphatic ring-substituted pyrimidine compounds provided by this invention have novel structures, and most of them exhibit strong anti-HIV-1 activity, EC. 50 The values ​​range from 9.40 nM to 238 nM. Among them, the compound WP-10a (EC) has an amino-aziridine substitution at the X position. 50= 12.6 ± 0.68 nM), WP-10b (EC 50 = 9.40 ± 0.96 nM), WP-10c (EC 50 = 18.3 ± 1.6 nM) and WP-10d (EC 50 = 26.2 ± 6.0 nM) with particularly outstanding activity; the most active WP-10b exhibits single-digit nanomolar antiviral efficacy, compared to the marketed drug nevirapine (EC). 50 = 281 ± 38.7 nM) activity increased nearly 30 times, compared with efavirenz (EC) 50 = 5.20 ± 0.90 nM) and zidovudine (EC) 50 = 7.50 ± 1.8 nM) with comparable activity. Furthermore, WP-10b did not exhibit significant cytotoxicity (CC). 50 > 40.9 µM), and therefore also has a high selectivity index (SI > 4348).

[0055] 3. Both compounds WP-10a and WP-10b exhibited good reverse transcriptase inhibitory activity, IC50... 50 The values ​​were 0.593 ± 0.051 µM and 0.543 ± 0.064 µM, respectively, demonstrating that the target of the azafatty ring-substituted pyrimidine compound is reverse transcriptase, which is a typical non-nucleoside reverse transcriptase inhibitor.

[0056] 4. Experiments have shown that the aza-aliphatic ring-substituted pyrimidine compounds of the present invention can be used as HIV-1 non-nucleoside reverse transcriptase inhibitors and have high application value. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0058] Example 1: Preparation of 4-((2,6-dichloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (2)

[0059] The starting materials 2,4,6-trichloropyrimidine 1 (1.0 g, 5.45 mmol), 4-hydroxy-3,5-dimethylbenzonitrile (0.8 g, 5.45 mmol), and N,N-diisopropylethylamine (1.8 ml, 10.9 mmol) were dissolved in 20 ml of 1,4-dioxane and reacted at 65 °C for 2 h with stirring. After the reaction was complete as detected by TLC, the reaction solution was cooled to room temperature, and then 50 mL of ice water was slowly added. The mixture was stirred for another 30 min. The precipitate in the suspension was collected by filtration, washed with ice water (3 × 5 mL), and dried to give intermediate 4-((2,6-dichloropyrimidine-4-yl)oxy)-3,5-dimethylbenzonitrile 2. It was a white solid, 89% yield, mp: 206–208 °C. 1 H NMR (400 MHz, DMSO-d6) δ 7.74 (s, 2H), 7.62 (s, 1H), 2.12 (s, 6H). ESI-MS: m / z 294.28 [M+H] + . C 13 H9Cl2N3O (293.01).

[0060] Example 2: Preparation of 4-((6-chloro-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (3)

[0061] Intermediate 4-((2,6-dichloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile 2 (1.0 g, 3.4 mmol) and p-aminobenzonitrile (0.4 g, 3.4 mmol) prepared in Example 1 were dissolved in 5 mL of N-methylpyrrolidone. Potassium tert-butoxide (0.75 g, 6.8 mmol) was added in portions at 0 °C. After the addition was complete, the mixture was stirred at room temperature for 2 h. After the reaction was complete as detected by TLC, the reaction solution was slowly added dropwise to 50 mL of ice water. The mixture was filtered, washed with ethyl acetate (3 × 5 mL), and dried to obtain intermediate 4-((6-chloro-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile 3. It was a white solid with a yield of 48%, mp: 275-278 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.40 (s,1H), 7.82 (d, J = 8.9 Hz, 2H), 7.78–7.76 (m, 2H), 7.75 (s, 2H), 5.76 (s, 1H),2.14 (s, 6H). ESI-MS: m / z 376.50 [M+H] + . C 20 H14 Cl2N5O (375.09).

[0062] Example 3: Preparation of target compounds 5a-f (WP-4a, WP-6a, WP-8a, WP-10a, WP-14a and WP-16a)

[0063] The intermediate 4-((6-chloro-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile 3 (0.5 g, 1.3 mmol), potassium carbonate (0.37 g, 2.7 mmol), and 1.6 mmol of piperazine-1-carboxylate, or piperidine-4-carboxylate, or 4-aminopiperidine-1-carboxylate, or aziridine-3-carboxylate, or (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate, or 1,4-diazabicycloheptane-1-carboxylate were dissolved in 10 mL of N,N-dimethylformamide and reacted at 120 °C for 8 h. After the reaction was detected by TLC, the reaction solution was cooled to room temperature, 50 mL of water was added, and stirring was continued for 30 min. The precipitate was collected by filtration, washed with ice water (3 × 5 mL), and dried to obtain white intermediates 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester 4a, 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester 4b, 4-((6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)amino)piperazine-1-carboxylic acid tert-butyl ester 4c, 1- ... (-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)tert-butyl carbamate 4d, (1R,5S)-3-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate tert-butyl ester 4e or 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-1,4-diazacycloheptane-1-carboxylate tert-butyl ester 4f, can be directly added to the next step without purification.

[0064] The obtained crude intermediate 4a-f (0.5 g) 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, the pH of the reaction solution was adjusted to about 9 with saturated sodium bicarbonate solution, and then extracted with dichloromethane (3 × 10⁻⁶). The organic phases were combined, 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 eluent to obtain the target compounds 4-((2-(((4-cyanophenyl)amino)-6-(piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-4a, 4-((6-(4-aminopiperidin-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-6a, and 4-((2-((4-cyanophenyl)amino)-6-(piperidin-4-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-6a. WP-8a, WP-10a, WP-10a, WP-10a, WP-14a, and WP-14a, and WP-16a, respectively.

[0065] 4-((2-((4-cyanophenyl)amino)-6-(piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4a)

[0066] White solid, yield 65%, mp: 219-221 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.70 (d,J = 75.6 Hz, 1H), 7.78 (d, J = 8.9 Hz, 1H), 7.73 (s, 2H), 7.71 (s, 1H), 7.54–7.43 (m, 2H), 6.00 (s, 1H), 3.62–3.44 (m, 4H), 2.89–2.69 (m, 4H), 2.12 (s,6H), 1.23 (d, J = 5.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 169.51, 168.83,164.93, 162.78, 161.43, 158.50, 154.51, 153.76, 145.54, 145.14, 133.63,133.35, 133.24, 132.98, 132.95, 132.92, 119.99, 119.89, 119.23, 118.47,108.60, 108.41, 103.06, 102.29, 78.92, 78.57, 45.43, 45.10, 44.31, 16.39,16.26. ESI-MS: m / z 426.32 [M+H] + . C 24 H 23 N7O (425.20).

[0067] 4-((6-(4-aminopiperidin-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-6a)

[0068] White solid, yield 72%, mp: 209-211 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.59 (s,1H), 7.87–7.65 (m, 3H), 7.58–7.37 (m, 3H), 6.05 (s, 1H), 4.32 (s, 3H), 3.24–2.85 (m, 3H), 2.13 (s, 6H), 1.99–1.79 (m, 3H), 1.38 (qd, J = 12.1, 11.4, 2.9Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 169.67, 164.60, 158.63, 154.51, 145.54,133.63, 133.35, 133.24, 132.94, 132.91, 119.97, 119.19, 118.48, 108.43,102.35, 78.66, 48.28, 42.97, 31.83, 16.37, 16.24. ESI-MS: m / z 440.33 [M+H] + .C 25 H 25 N7O (439.21).

[0069] 4-((2-((4-cyanophenyl)amino)-6-(piperidin-4-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8a)

[0070] White solid, yield 65%, mp: 245-247 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.62 (s,1H), 7.72 (s, 3H), 7.66 (d, J = 19.5 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 5.52(s, 1H), 4.03 (s, 1H), 3.09–2.89 (m, 2H), 2.12 (s, 6H), 2.05 (s, 1H), 1.82–1.64 (m, 2H), 1.23 (td, J = 7.2, 2.3 Hz, 4H). 13 C NMR (100 MHz, DMSO-d6): δ164.79, 154.26, 145.67, 133.36, 133.04, 133.00, 120.06, 119.15, 118.58,108.46, 102.24, 58.24, 52.46, 42.45, 40.42, 28.82, 16.32. ESI-MS: m / z 440.24[M+H] + . C 25 H 25 N7O (439.21).

[0071] 4-((6-(3-aminoazacyclobutane-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-10a)

[0072] White solid, yield 54%, mp: 167-169 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.67 (s,1H), 7.73 (s, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 5.54(s, 1H), 5.35 (s, 2H), 4.22 (t, J = 8.0 Hz, 2H), 3.95–3.85 (m, 1H), 3.75 (d,J = 7.5 Hz, 2H), 2.11 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 168.42, 165.90,158.84, 154.47, 145.63, 133.36, 132.93, 132.86, 120.00, 119.18, 118.45,108.45, 102.28, 77.51, 63.08, 59.43, 52.50, 43.34, 16.31, 7.68. ESI-MS: m / z412.43 [M+H] + . C 23 H 21 N7O (411.18).

[0073] 4-((6-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14a)

[0074] White solid, 70% yield, mp: 247-249 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.55 (s,1H), 7.71 (s, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 5.85(s, 1H), 3.89 (s, 2H), 3.38 (dd, J = 8.1, 6.6 Hz, 1H), 3.01 (d, J = 12.1 Hz, 2H), 2.12 (s, 6H), 1.80–1.12 (m, 6H). 13 C NMR (100 MHz, DMSO-d6): δ 169.20,166.32, 162.43, 158.27, 154.47, 145.58, 145.24, 133.61, 133.37, 133.24,132.95, 132.91, 119.98, 119.19, 118.50, 108.42, 102.29, 78.47, 53.51, 52.48,51.64, 28.74, 16.40, 16.24, 7.65. ESI-MS: m / z 452.46 [M+H] + . C 26 H 25 N7O(451.21).

[0075] 4-((2-((4-cyanophenyl)amino)-6-(1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16a)

[0076] White solid, yield 67%, mp: 141-143 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.53 (s,1H), 7.71 (s, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 5.82(s, 1H), 3.65 (s, 4H), 2.91 (dd, J = 6.4, 4.2 Hz, 2H), 2.75 (t, J = 5.7 Hz, 2H), 2.13 (s, 6H), 2.02–1.71 (m, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 169.23,164.43, 158.52, 154.55, 145.70, 133.54, 133.39, 133.29, 132.92, 132.89,132.72, 120.01, 119.19, 119.03, 118.44, 108.38, 102.21, 77.92, 52.49, 48.27,47.90, 16.39, 16.23. ESI-MS: m / z 440.32 [M+H] + . C 25 H 25 N7O (439.21).

[0077] Example 4: Preparation of target compounds WP-4b-d, WP-6b-d, WP-8b-d, WP-10b-d, WP-14b-d and WP-16b-d

[0078] The compound 4-((2-((4-cyanophenyl)amino)-6-(piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-4a (0.42 g, 1.0 mmol) and triethylamine (0.30 g, 3.0 mmol) prepared in Example 3 were dissolved in dichloromethane (5 mL). A substituted sulfonyl chloride or acyl chloride (methylsulfonyl chloride, ethylsulfonyl chloride, or N,N-dimethylaminosulfonyl chloride) (1.1 mmol) was slowly added dropwise to the reaction system at 0 °C. After the addition was completed, the reaction was moved to room temperature for 4 h. The reaction was monitored by TLC until it was complete. 20 mL of water was added to the reaction solution to quench the reaction, and the mixture was extracted with dichloromethane (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 (1:50) as the eluent. The target compound WP-4b-d was obtained by recrystallization from ethyl acetate and petroleum ether.

[0079] 4-((2-((4-cyanophenyl)amino)-6-(4-(methanesulfonyl)piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4b)

[0080] White solid, yield 59%, mp: > 270 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.70 (s,1H), 7.75 (q, J = 8.1, 7.3 Hz, 3H), 7.48 (q, J = 8.8 Hz, 3H), 6.10 (s, 1H),3.95–3.66 (m, 4H), 3.29–3.08 (m, 4H), 2.93 (s, 3H), 2.12 (s, 6H). 13 C NMR (100MHz, DMSO-d6): δ 169.67, 164.72, 158.56, 154.46, 145.43, 133.67, 133.33,133.21, 132.95, 119.95, 119.37, 119.18, 118.55, 108.50, 102.46, 78.98, 45.51,43.94, 34.53, 34.43, 16.38, 16.24. ESI-MS: m / z 504.30 [M+H] + . C 25 H 25 N7O3S(503.17).

[0081] 4-((2-((4-cyanophenyl)amino)-6-(4-(ethylsulfonyl)piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4c)

[0082] White solid, yield 74%, mp: 221-223 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.60 (s,1H), 7.82–7.55 (m, 3H), 7.52–7.27 (m, 3H), 6.01 (s, 1H), 3.81–3.55 (m, 4H), 3.18 (dt, J = 29.1, 5.0 Hz, 4H), 3.08–2.95 (m, 2H), 2.05 (s, 6H), 1.22–1.13 (m, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 169.66, 164.79, 161.31, 158.55, 154.46,153.66, 145.43, 145.01, 133.66, 133.34, 133.22, 133.04, 132.94, 119.94,119.39, 119.17, 118.56, 108.50, 103.25, 102.47, 79.22, 79.00, 45.36, 44.32,44.00, 43.08, 16.36, 16.23, 7.92.m / z 518.37 [M+H] + . C 26 H 27 N7O3S (517.19).

[0083] 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethylpiperazine-1-sulfonamide (WP-4d)

[0084] White solid, yield 59%, mp: 255-257 ℃. 1H NMR (400 MHz, DMSO-d6): δ 9.68 (s,1H), 7.73 (s, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 6.06(s, 1H), 3.80–3.57 (m, 4H), 3.24 (dt, J = 28.7, 5.0 Hz, 4H), 2.81 (s, 6H), 2.12 (s, 6H). 13 C NMR (100 MHz, DMSO-d6): δ 169.63, 168.92, 164.82, 158.54,154.44, 153.65, 145.43, 145.00, 133.66, 133.33, 133.21, 133.05, 132.97,132.94, 119.96, 119.38, 119.18, 118.57, 108.72, 108.49, 102.45, 79.22, 78.96,46.21, 44.14, 43.84, 38.37, 38.34, 21.23, 16.37, 16.23. m / z 533.28 [M+H] + .C 26 H 28 N8O3S (532.20).

[0085] The synthesis of the target compound WP-6b-d was the same as that of compound WP-4b-d, except that compound WP-4a was replaced with 4-((6-(4-aminopiperidin-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-6a (0.44 g, 1.0 mmol).

[0086] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)methanesulfonamide (WP-6b)

[0087] White solid, yield 54%, mp: 265-267 ℃. 1H NMR (400 MHz, DMSO-d6): δ 9.58 (s,1H), 7.81–7.69 (m, 3H), 7.56–7.37 (m, 3H), 7.11 (dd, J = 26.6, 7.4 Hz, 1H), 6.05 (s, 1H), 4.25 (s, 2H), 3.49 (d, J = 9.4 Hz, 1H), 3.10 (t, J = 12.4 Hz, 2H), 2.97 (s, 3H), 2.12 (s, 6H), 1.99–1.78 (m, 2H), 1.56–1.18 (m, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 169.66, 164.51, 161.21, 158.62, 154.55, 145.56, 145.17,133.62, 133.37, 133.25, 132.91, 119.96, 119.19, 118.46, 108.42, 103.12,102.33, 78.57, 50.46, 43.23, 41.59, 32.88, 16.38, 16.24. m / z 518.36 [M+H] + .C 26 H 27 N7O3S (517.19).

[0088] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)ethanesulfonamide (WP-6c)

[0089] White solid, yield 74%, mp: 255-257 ℃. 1H NMR (400 MHz, DMSO-d6): δ 9.53 (s,1H), 7.69 (d, J = 3.0 Hz, 1H), 7.66 (s, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.37(d, J = 8.7 Hz, 2H), 5.97 1.27 (qd,J = 11.5, 4.0 Hz, 2H), 0.93 (t, J = 7.5 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ172.57, 169.65, 164.61, 158.62, 154.55, 145.57, 133.64, 133.36, 133.25,132.91, 119.97, 119.20, 118.42, 108.41, 102.29, 78.59, 46.10, 43.44, 31.57,29.02, 16.37, 16.24, 10.42. m / z 530.13 [MH] - . C 27 H 29 N7O3S (531.21).

[0090] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)-N,N-dimethylsulfonamide (WP-6d)

[0091] White solid, yield 71%, mp: 139-141 ℃. 1H NMR (400 MHz, DMSO-d6): δ 9.53 (s,1H), 7.66 (s, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.9 Hz, 2H), 7.21(d, J = 7.9 Hz, 1H), 5.97 (s, 1H), 4.20 (s, 2H), 3.01 (t, J = 12.5 Hz, 2H), 2.60 (s, 6H), 2.05 (s, 6H), 1.92–1.74 (m, 2H), 1.47–1.14 (m, 3H). 13 C NMR (100MHz, DMSO-d6): δ 169.65, 164.46, 158.61, 154.54, 145.55, 133.36, 132.94,132.91, 119.99, 119.21, 118.43, 108.40, 102.29, 78.52, 51.01, 43.22, 38.17,32.69, 16.39. m / z 547.23 [M+H] + . C 27 H 30 N8O3S (546.22).

[0092] The synthesis of the target compound WP-8b-d was the same as that of compound WP-4b-d, except that compound WP-4a was replaced with 4-((2-((4-cyanophenyl)amino)-6-(piperidin-4-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-8a (0.44 g, 1.0 mmol).

[0093] 4-((2-((4-cyanophenyl)amino)-6-((1-(methanesulfonyl)piperidin-4-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8b)

[0094] White solid, yield 54%, mp: 150-152 ℃. 1H NMR (400 MHz, DMSO-d6): δ 9.60 (s,1H), 7.71 (d, J = 6.4 Hz, 4H), 7.56 (d, J = 8.8 Hz, 2H), 7.40 (s, 1H), 5.45(s, 1H), 3.95 (s, 1H), 3.56 (dt, J = 11.9, 4.0 Hz, 2H), 2.92 (s, 3H), 2.88(dd, J = 12.0, 2.8 Hz, 2H), 2.13 (s, 6H), 2.02 (td, J = 11.1, 10.0, 5.3 Hz, 2H), 1.62–1.41 (m, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 164.84, 159.22, 154.25,145.86, 145.71, 133.37, 133.07, 133.00, 120.07, 119.13, 118.50, 108.49,102.28, 44.97, 34.75, 31.45, 16.31. m / z 518.27 [M+H] + . C 26 H 27 N7O3S (517.19).

[0095] 4-((2-((4-cyanophenyl)amino)-6-((1-(ethylsulfonyl)piperidin-4-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8c)

[0096] White solid, yield 69%, mp: 241-243 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.57 (s,1H), 7.71 (s, 2H), 7.68 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.37(s, 1H), 5.45 (s, 1H), 4.08–3.86 (m, 1H), 3.61 (d, J = 12.4 Hz, 2H), 3.09 (q,J = 7.3 Hz, 2H), 3.03–2.93 (m, 2H), 2.13 (s, 6H), 2.02–1.96 (m, 2H), 1.48 (d,J = 11.8 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H).13 C NMR (100 MHz, DMSO-d6): δ164.82, 154.26, 145.70, 133.38, 133.04, 132.99, 120.06, 119.13, 118.53,108.49, 102.28, 44.70, 43.25, 31.84, 16.30, 8.03. m / z 532.28 [M+H] + .C 27 H 29 N7O3S (531.21).

[0097] 4-((6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylpiperidine-1-sulfonamide (WP-8d)

[0098] White solid, 75% yield, mp: 159-161 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.59 (s,1H), 7.72 (s, 2H), 7.68 (d, J = 7.0 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.38(s, 1H), 5.46 (s, 1H), 3.57 (d, J = 12.3 Hz, 2H), 3.11–2.94 (m, 2H), 2.77 (s,6H), 2.12 (s, 6H), 2.02–1.92 (m, 2H), 1.48 (d, J = 11.4 Hz, 2H), 1.33–1.19(m, 1H). 13 C NMR (100 MHz, DMSO-d6): δ 164.79, 154.28, 145.69, 133.37, 133.01,120.07, 119.14, 119.07, 118.53, 108.47, 102.24, 45.69, 45.42, 39.85, 38.35,31.69, 31.43, 22.54, 16.32, 14.44. m / z 547.24 [M+H] + . C 27 H 30 N8O3S (546.22).

[0099] The synthesis of the target compound WP-10b-d was the same as that of compound WP-4b-d, except that compound WP-4a was replaced with 4-((6-(3-aminoazacyclobutane-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-10a (0.41 g, 1.0 mmol).

[0100] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)methanesulfonamide (WP-10b)

[0101] White solid, yield 53%, mp: > 270 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.73 (s,1H), 7.92 (d, J = 6.4 Hz, 1H), 7.74 (s, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.44(d, J = 8.6 Hz, 2H), 5.58 (s, 1H), 4.37 (d, J = 5.3 Hz, 3H), 3.93 (d, J = 4.9Hz, 2H), 2.96 (s, 3H), 2.11 (s, 6H). 13 C NMR (100 MHz, DMSO-d6): δ 168.52,165.84, 158.83, 154.39, 145.51, 133.32, 132.96, 132.90, 119.97, 119.17,118.49, 108.52, 102.41, 77.72, 58.01, 43.49, 40.81, 16.31. m / z 490.30 [M+H] + .C 24 H 23 N7O3S (489.16).

[0102] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)ethanesulfonamide (WP-10c)

[0103] White solid, yield 64%, mp: 218-220 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.65 (s,1H), 7.87 (d, J = 5.5 Hz, 1H), 7.66 (s, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.36(d, J = 8.6 Hz, 2H), 5.52 (s, 1H), 4.28 (d, J = 5.3 Hz, 3H), 3.85 (s, 2H), 2.97 (q, J = 7.3 Hz, 2H), 2.04 (s, 6H), 1.15 (t, J = 7.3 Hz, 3H). 13 C NMR (100MHz, DMSO-d6): δ 168.52, 165.85, 158.83, 154.40, 145.51, 133.32, 132.96,132.89, 119.96, 119.16, 118.49, 108.52, 102.41, 77.76, 58.14, 46.69, 43.44,16.31, 8.47. m / z 504.27 [M+H] + . C 25 H 25 N7O3S (503.17).

[0104] N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)-N,N-dimethylsulfonamide (WP-10d)

[0105] White solid, yield 63%, mp: 159-160 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.72 (s,1H), 7.99 (d, J = 7.9 Hz, 1H), 7.74 (s, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.44(d, J = 8.6 Hz, 2H), 5.61 (s, 1H), 4.37–4.26 (m, 3H), 3.91 (s, 2H), 2.69 (s,6H), 2.12 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 168.51, 165.84, 158.82,154.41, 145.51, 133.32, 132.96, 132.90, 119.97, 119.17, 118.48, 108.51,102.40, 77.78, 58.03, 43.94, 39.47, 38.04, 16.31. m / z 519.17 [M+H] + .C 25 H 26 N8O3S (518.18).

[0106] The synthesis of the target compound WP-14b-d is the same as that of compound WP-4b-d, except that compound WP-4a is replaced with 4-((6-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-14a (0.45 g, 1.0 mmol).

[0107] 4-((2-((4-cyanophenyl)amino)-6-((1R,5S)-8-(methanesulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14b)

[0108] White solid, yield 65%, mp: 259-261 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.64 (s,1H), 7.72 (s, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 5.96(s, 1H), 4.60–3.84 (m, 4H), 3.19–2.95 (m, 5H), 2.13 (s, 6H), 2.04–1.87 (m, 2H), 1.69 (q, J = 6.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 169.38, 166.28,158.29, 154.41, 145.45, 133.65, 133.35, 133.21, 132.97, 132.93, 119.96,119.36, 119.17, 118.59, 108.49, 102.45, 78.93, 55.95, 51.77, 27.72, 22.53,16.40, 16.23, 14.43. m / z 530.41 [M+H] + . C 27 H 27 N7O3S (529.19).

[0109] 4-((2-((4-cyanophenyl)amino)-6-((1R,5S)-8-(ethylsulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14c)

[0110] White solid, 78% yield, mp: 225-227 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.63 (s,1H), 7.72 (s, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 5.94(s, 1H), 4.45–3.91 (m, 4H), 3.20 (q, J = 7.4, 6.9 Hz, 2H), 3.11 (d, J = 12.5Hz, 2H), 2.12 (s, 6H), 1.99–1.80 (m, 2H), 1.70 (d, J = 7.7 Hz, 2H), 1.26 (t,J = 7.2 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 169.38, 166.31, 158.31, 154.40,145.45, 133.35, 133.21, 132.97, 132.93, 119.95, 119.36, 119.16, 118.61,108.50, 102.45, 79.34, 78.94, 55.96, 51.61, 47.14, 28.27, 16.38, 16.22, 8.63.m / z 544.34 [M+H] + . C28 H 29 N7O3S (543.21).

[0111] (1R,5S)-3-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethyl-3,8-diazabicyclo[3.2.1]octane-8-sulfonamide (WP-14d)

[0112] White solid, yield 57%, mp: 245-247 ℃. 1 H NMR (400 MHz, DMSO-d6): δ 9.56 (s,1H), 7.65 (s, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 5.85(s, 1H), 4.09 (d, J = 4.3 Hz, 4H), 3.10 (d, J = 12.1 Hz, 2H), 2.70 (s, 6H), 2.05 (s, 6H), 1.90–1.79 (m, 2H), 1.63 (t, J = 6.9 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 169.36, 166.36, 158.32, 154.37, 145.44, 133.34, 132.99, 132.95,119.95, 119.16, 118.64, 108.51, 102.46, 78.92, 57.40, 51.23, 40.48, 38.40,27.95, 16.38. m / z 559.26 [M+H] + . C 28 H 30 N8O3S (558.22).

[0113] The synthesis of the target compound WP-16b-d was the same as that of compound WP-4b-d, except that compound WP-4a was replaced with 4-((2-((4-cyanophenyl)amino)-6-(1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile WP-16a (0.44 g, 1.0 mmol).

[0114] 4-((2-((4-cyanophenyl)amino)-6-(4-(methanesulfonyl)-1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16b)

[0115] White solid, yield 65%, mp: 127-129 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.59 (s,1H), 7.72 (s, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 5.94(s, 1H), 4.04–3.61 (m, 4H), 3.46 (t, J = 5.6 Hz, 2H), 3.30 (t, J = 5.7 Hz, 2H), 2.88 (s, 3H), 2.13 (s, 6H), 1.86 (q, J = 6.9, 6.3 Hz, 2H). 13 C NMR (100MHz, DMSO-d6): δ 169.40, 164.26, 158.61, 154.51, 145.57, 133.61, 133.38,133.28, 132.96, 132.92, 119.98, 119.19, 118.50, 108.43, 102.34, 99.98, 78.25,48.67, 47.31, 46.46, 37.45, 26.81, 16.40, 16.20. m / z 518.41 [M+H] + . C 26 H 27 N7O3S(517.19).

[0116] 4-((2-((4-cyanophenyl)amino)-6-(4-(ethylsulfonyl)-1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16c)

[0117] White solid, yield 73%, mp: 127-129 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.52 (s,1H), 7.65 (s, 2H), 7.50 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 5.86(s, 1H), 3.90–3.54 (m, 4H), 3.43 (t, J = 5.4 Hz, 2H), 3.28 (s, 2H), 2.99 (q,J = 7.3 Hz, 2H), 2.06 (s, 6H), 1.78 (q, J = 6.0 Hz, 2H), 1.08 (t, J = 7.3 Hz,3H). 13 C NMR (100 MHz, DMSO-d6): δ 169.38, 164.24, 158.60, 154.51, 145.58,133.60, 133.38, 132.96, 132.92, 119.98, 119.19, 118.49, 108.42, 102.33,78.26, 49.14, 47.36, 46.55, 45.05, 16.38, 16.19, 8.23, 8.20. m / z 532.33 [M+H] + . C 27 H 29 N7O3S (531.21).

[0118] 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethyl-1,4-diazacycloheptane-1-sulfonamide (WP-16d)

[0119] White solid, yield 65%, mp: 167-169 °C. 1 H NMR (400 MHz, DMSO-d6): δ 9.60 (s,1H), 7.72 (s, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 5.94(s, 1H), 3.73 (d, J = 60.5 Hz, 4H), 3.49 (t, J = 5.5 Hz, 2H), 3.37 (d, J =9.3 Hz, 2H), 2.63 (s, 6H), 2.13 (s, 6H), 1.84 (q, J = 6.0 Hz, 2H). 13C NMR(100 MHz, DMSO-d6): δ 169.36, 164.19, 158.60, 154.51, 145.58, 133.62, 133.37,132.97, 132.93, 120.00, 119.20, 118.46, 108.41, 103.00, 102.31, 78.28, 60.23,48.59, 47.85, 38.00, 37.93, 21.24, 16.37, 16.18, 14.55. m / z 547.30 [M+H] + .C 27 H 30 N8O3S (546.22).

[0120] Experimental example: Anti-HIV-1 activity and cytotoxicity of the target compound at the cellular level.

[0121] Experimental Example 1: In vitro anti-HIV-1 activity experiment

[0122] Experimental Principle

[0123] 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.

[0124] Experimental materials

[0125] The target compounds in the examples are the 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-carboxanilide (XTT), and the HIV-1 NL4-3 wild-type strain.

[0126] Experimental methods

[0127] Cell viability assay method (EC) 50TCID 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.

[0128] 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.

[0129] The synthesized partially aza-alicyclic substituted pyrimidine derivatives were tested for antiviral activity and cytotoxicity at the cellular level using the experimental methods described above. Cell viability (ECG) was measured. 50 ), cytotoxicity (CC) 50 The results of the tests for the Selectivity Index (SI) are shown in Table 1.

[0130] Table 1. Antiviral activity, cytotoxicity, and selectivity indices of azafatty ring substituted pyrimidine derivatives and positive control drugs

[0131]

[0132] Note: a EC 50 The concentration of compounds that protect 50% of HIV-infected cells from cytopathic effects;

[0133] b CC 50 The concentration at which the target compound causes 50% of uninfected HIV cells to become diseased;

[0134] c SI:CC 50 / EC 50 , Selectivity index.

[0135] As shown in Table 1, the aza-alicyclic substituted pyrimidine compounds provided by this invention are a series of novel HIV-1 inhibitors, and most of them exhibit strong anti-HIV-1 activity. 50 The values ​​range from 9.40 nM to 238 nM. Among them, the compound WP-10a (EC) has an amino-aziridine substitution at the X position. 50 = 12.6 ± 0.68 nM), WP-10b (EC 50 = 9.40 ± 0.96 nM), WP-10c (EC 50 = 18.3 ± 1.6 nM) and WP-10d (EC 50 = 26.2 ± 6.0 nM) with particularly outstanding activity; the most active WP-10b exhibits single-digit nanomolar antiviral efficacy, compared to the marketed drug nevirapine (EC). 50 = 281 ± 38.7 nM) activity increased nearly 30 times, compared with efavirenz (EC) 50 = 5.20 ± 0.90 nM) and zidovudine (EC) 50 = 7.50 ± 1.8 nM) with comparable activity. Furthermore, WP-10b did not exhibit significant cytotoxicity (CC). 50 > 40.9 µM), and therefore also has a high selectivity index (SI > 4348).

[0136] Experiment Example 2: HIV-1 Reverse Transcriptase Inhibitory Activity Experiment

[0137] Experimental Principle

[0138] 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.

[0139] Experimental materials

[0140] 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.

[0141] Experimental methods

[0142] 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).

[0143] Inhibition rate calculation formula: Inhibition rate % × 100%

[0144] 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.

[0145] Enzyme inhibitory activity (IC50) 50 The results are shown in Table 2.

[0146] Table 2. Inhibitory activity of azafatty ring substituted pyrimidine derivatives against HIV-1 reverse transcriptase

[0147]

[0148] Note: a IC 50 Table 2 shows the compound concentrations corresponding to a 50% reverse transcriptase inhibition rate. As can be seen from Table 2, both compounds WP-10a and WP-10b exhibited good reverse transcriptase inhibitory activity, with IC50 values ​​of [missing value]. 50 The values ​​were 0.593 ± 0.051 µM and 0.543 ± 0.064 µM, respectively, demonstrating that the target of the aza-adipic ring-substituted pyrimidine compound is reverse transcriptase, which is a typical non-nucleoside reverse transcriptase inhibitor.

Claims

1. A nitrogen-substituted aliphatic ring pyrimidine derivative having the structure shown in Formula I: ； in, X is an aza-aliphatic ring, including piperazine ring, aminopiperidine ring, aminoazacyclobutane, aminoazabicyclooctane, azaspiro ring or high piperazine ring; R1 is Boc, H, CH3, SO2NH2, SO2CH3, SO2CH2CH3, SO2N(CH3)2, SO2CH(CH3)2, CONH2, COCH3, COCH2CH3, CON(CH3)2 or COCH(CH3)2; R2 is CN, CH3, CH=CHCN, or a substituted phenyl group.

2. The aza-aliphatic substituted pyrimidine derivative according to claim 1, characterized in that: The aza-aliphatic substituted pyrimidine derivatives are selected from one of the following: 4-((2-((4-cyanophenyl)amino)-6-(piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4a); 4-((2-((4-cyanophenyl)amino)-6-(4-(methanesulfonyl)piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4b); 4-((2-((4-cyanophenyl)amino)-6-(4-(ethylsulfonyl)piperazin-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-4c); 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethylpiperazine-1-sulfonamide (WP-4d); 4-((6-(4-aminopiperidin-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-6a); N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)methanesulfonamide (WP-6b); N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)ethanesulfonamide (WP-6c); N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)piperidin-4-yl)-N,N-dimethylsulfonamide (WP-6d); 4-((2-((4-cyanophenyl)amino)-6-(piperidin-4-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8a); 4-((2-((4-cyanophenyl)amino)-6-((1-(methanesulfonyl)piperidin-4-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8b); 4-((2-((4-cyanophenyl)amino)-6-((1-(ethylsulfonyl)piperidin-4-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-8c); 4-((6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylpiperidine-1-sulfonamide (WP-8d); 4-((6-(3-aminoazacyclobutane-1-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-10a); N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)methanesulfonamide (WP-10b); N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)ethanesulfonamide (WP-10c); N-(1-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)azacyclobutane-3-yl)-N,N-dimethylsulfonamide (WP-10d); 4-((6-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14a); 4-((2-((4-cyanophenyl)amino)-6-((1R,5S)-8-(methanesulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14b); 4-((2-((4-cyanophenyl)amino)-6-((1R,5S)-8-(ethylsulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-14c); (1R,5S)-3-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethyl-3,8-diazabicyclo[3.2.1]octane-8-sulfonamide (WP-14d); 4-((2-((4-cyanophenyl)amino)-6-(1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16a); 4-((2-((4-cyanophenyl)amino)-6-(4-(methanesulfonyl)-1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16b); 4-((2-((4-cyanophenyl)amino)-6-(4-(ethylsulfonyl)-1,4-diazacycloheptane-1-yl)pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (WP-16c); 4-(6-(4-cyano-2,6-dimethylphenoxy)-2-((4-cyanophenyl)amino)pyrimidin-4-yl)-N,N-dimethyl-1,4-diazacycloheptane-1-sulfonamide (WP-16d).

3. The method for preparing the aza-aliphatic ring-substituted pyrimidine derivative according to claim 1, comprising the following steps: 1) Starting from 2,4,6-trichloropyrimidine 1, intermediate 2 was prepared by nucleophilic substitution reaction with 4-hydroxy-3,5-dimethylbenzonitrile in a 1,4-dioxane solution; 2) Intermediate 2 reacts with p-aminobenzonitrile under alkaline conditions in potassium tert-butoxide to obtain intermediate 3; using N,N-dimethylformamide as solvent, intermediate 3 reacts with piperazine-1-carboxylate tert-butyl ester, piperidine-4-carboxylate tert-butyl ester, 4-aminopiperidine-1-carboxylate tert-butyl ester, azacyclobutane-3-carboxylate tert-butyl ester, (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate tert-butyl ester, or 1,4-diazacycloheptane-1-carboxylate tert-butyl ester under high-temperature alkaline conditions via nucleophilic substitution to obtain key intermediates 4a-f; subsequently, intermediate 4a-f undergoes deprotection of the Boc protecting group in dichloromethane solution via trifluoroacetic acid to generate intermediate 5a-f; 3) Intermediates 5a-f undergo acylation reactions with various substituted sulfonyl chlorides or acyl chlorides under basic conditions of triethylamine to obtain target compound I; The synthesis route is as follows: ； Reagents and conditions: (i) 4-hydroxy-3,5-dimethylbenzonitrile, N,N-diisopropylethylamine, 1,4-dioxane, 65°C; (ii) p-aminobenzonitrile, potassium tert-butoxide, N-methylpyrrolidone, 0°C to room temperature; (iii) aminoazalicyclic, potassium carbonate, N,N-dimethylformamide, 120°C; (iv) trifluoroacetic acid, dichloromethane, room temperature; (v) substituted sulfonyl chloride or acyl chloride, triethylamine, dichloromethane, 0°C to room temperature.

4. The use of the aza-alicyclic substituted pyrimidine derivative of claim 1 as an HIV-1 inhibitor.

5. The application of the aza-alicyclic substituted pyrimidine derivative of claim 1 in the preparation of anti-AIDS drugs.

6. The application of the aza-alicyclic substituted pyrimidine derivatives of claim 1 as non-nucleoside NNRTIs in the preparation of anti-HIV drugs.

7. An anti-HIV-1 pharmaceutical composition comprising the azafatty ring substituted pyrimidine derivative of claim 1 and its pharmaceutically acceptable salt and pharmaceutical excipients.

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