CN114539163A - HIV-1 reverse transcriptase inhibitor and synthetic method thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an HIV-1 reverse transcriptase inhibitor and a synthesis method thereof. The HIV-1 reverse transcriptase inhibitor is a substituted aryl uretonimine diaryl pyrimidine derivative, and also comprises a medicinal salt, a hydrate and a solvate thereof, polycrystal or eutectic thereof, and a precursor and a derivative thereof with the same biological function. The results of in vitro cell level anti-HIV-1 activity experiments show that the compounds have stronger anti-HIV-1 bioactivity, can obviously inhibit the virus replication in MT-4 cells infected by HIV-1 virus, and have lower cytotoxicity and better selectivity. The invention also comprises the application of the compound composition in the preparation of medicaments for treating AIDS and the like.

Description

HIV-1 reverse transcriptase inhibitor and synthetic method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an HIV-1 reverse transcriptase inhibitor and a synthesis method thereof.

Background

The Reverse transcription process (Reverse transcription) is the first link after the HIV-1 virus invades host cells and plays a key role in the HIV-1 life cycle. In this link, Reverse Transcriptase (RT) is responsible for Reverse transcription of viral RNA into DNA-RNA hybrids and degradation of RNA in the hybrids to form single-stranded viral DNA, which is then integrated into the host cell by integrase. Therefore, RT can be used as an important selective target for anti-HIV-1 virus drug design¹。

Among the existing targeted HIV-1 reverse transcriptase compounds, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have stronger antiviral activity and lower cytotoxicity²Thus, it is the first choice for highly effective antiretroviral therapy (HAART) cocktail therapy for anti-HIV-1 therapy³. There are five anti-HIV reverse transcriptase inhibitors that are approved for marketing by the FDA in the united states: nevirapine (Nevirapine), Delavirdine (Delavirdine), Efavirenz (Efavirenz), Etravirine (Etravirine), Rilpivirine (Rilpivirine). In addition, RDEA806, IDX899, UK-453061 are in clinical research⁴. However, amino acid mutations in reverse transcriptase inactivate otherwise potent drugs, i.e., create drug-resistant HIV strains⁵. Therefore, the development of novel high-efficiency non-nucleoside reverse transcriptase inhibitors with broad-spectrum drug resistance becomes one of the hot spots of the research of medicinal chemists⁶。

The invention aims to optimize the structure of etravirine and rilpivirine, designs and synthesizes a series of derivatives containing substituted aryl urea imino diaryl pyrimidine by carrying out structural modification on a linking group of a left wing aromatic ring and a middle pyrimidine ring of the compounds, introduces various substituents on the aromatic ring, enhances the interaction between the compounds and conserved aromatic amino acid on the inner wall of a binding pocket of a non-nucleoside drug of reverse transcriptase, and can improve the biological activity of the series of compounds against drug-resistant virus strains.

Disclosure of Invention

The invention aims to provide an HIV-1 non-nucleoside reverse transcriptase inhibitor with excellent performance and a synthesis method thereof.

The invention provides an HIV-1 reverse transcriptase inhibitor, which is a diaryl pyrimidine derivative containing substituted aryl ureide group, and has the structural formula shown in the following formula (I):

wherein R is¹Independently selected from hydrogen, methyl, cyano, nitro, methoxy, ethoxy, hydroxyl and halogen, and the substitution position can be ortho-position, para-position or meta-position; x is halogen.

The HIV-1 reverse transcriptase inhibitor also comprises medicinal salts of the diaryl pyrimidine derivatives containing the substituted aryl ureidino, hydrates and solvates thereof, polycrystals and eutectic crystals thereof, and precursors and derivatives with the same biological functions.

The medicinal salt specifically comprises hydrochloride, hydrobromide, sulfate, phosphate, acetate, methanesulfonate, p-toluenesulfonate, tartrate, citrate, fumarate or malate.

The invention also provides a synthetic method of the HIV-1 reverse transcriptase inhibitor-substituted aryl uretidioimido diaryl pyrimidine derivative, and the synthetic route is as follows:

the specific steps of the synthesis are as follows;

the method comprises the following steps of (I) taking cheap and easily-obtained thiouracil (5, 1.5-1.6 equiv.) as a starting material, taking iodomethane (6, 1.0-1.1 equiv.) as a methylating agent, reacting for 20-24 h at room temperature under the action of sodium hydroxide (the molar concentration is about 1.25M and the molar concentration is 1.0-1.1 equiv.), and carrying out S-alkylation reaction to obtain a high-purity white solid, namely 2-methylthiopyrimidine-4-ketone (7), wherein the yield is 89.7%⁷；

(II) 2-methylthio pyrimidine-4-ketone (7, 1.0-1.1 equiv.) and excess 4-cyanoaniline (8, 2.5-3.0 equiv.) are reacted in a molten state at 180-185 ℃ for 10-18 h under the solvent-free condition, and the reaction solution is subjected toAfter dissolving in acetonitrile, appropriate workup gave 2- (4-cyanoanilino) pyrimidin-4-one (9) as a yellow solid in 67.6% yield⁸；

Dissolving and refluxing 2- (4-cyanoanilino) pyrimidine-4-one (9, 1.0-1.1 equiv.) in a large excess of phosphorus oxychloride (10.0-11.0 equiv.), carrying out chlorination reaction on hydroxyl at the C-4 position of a pyrimidine heterocyclic ring, carrying out post-treatment, dissolving in a proper amount of cold water, neutralizing to neutrality by using sodium hydroxide (20%) to obtain a yellow precipitate, filtering and drying to obtain a yellow solid 2- (4-cyanoanilino) -4-chloro-pyrimidine (10), wherein the yield is 84.0%, and the compound is an important intermediate for synthesizing a target compound;

performing nucleophilic substitution reaction on (tetra) 2- (4-cyanoanilino) -4-chloro-pyrimidine (10, 1.0-1.1 equiv.) and 2-halogenophenylacetonitrile (11a,11b, 1.5-1.6 equiv.) in dried N, N-dimethylformamide (2.5-3.0 equiv.mL) under the action of sodium hydride (60 percent, 2.0-2.2 equiv.), and obtaining an unstable intermediate Cyan-CH under anhydrous and anaerobic conditions₂-DAPYs(12a,12b)。；

(V) because of the intermediate Cyan-CH₂the-DAPYs (12a,12b) are unstable, can remove the nitrogen protection after the reaction, are placed in the air to react for 48 to 72 hours at room temperature, can be slowly oxidized to obtain a key intermediate Oxo-CH₂2-halophenyl 2- (4-cyanophenylamino) -pyrimidone pure products (13a,13b) can be obtained by post-treatment and column chromatography separation of the-DAPYs;

and (VI) simultaneously carrying out the steps (I) - (V), and carrying out the preparation of various substituted semicarbazides (18a-18t) in parallel by using various substituted anilines as starting materials through two-step reaction. The specific method comprises dissolving various substituted anilines (14a-14t, 1.0-1.1 equiv.) in tetrahydrofuran, and simultaneously dissolving NaHCO₃(1.2-1.4 equiv.) is dissolved in water, and NaHCO is added₃Mixing the aqueous solution with a tetrahydrofuran solution of substituted aniline, placing the mixture in an ice bath, adding phenyl chloroformate (15, 1.2-1.4 equiv.) after the temperature is stabilized to be 0-5 ℃. The reaction speed is very fast, and the reaction can be finished after reactants are added. Because the substituted phenyl carbamate intermediate is unstable in the reaction liquid, the temperature is controlled to be 0-5 ℃ in the reaction process so as to prevent the intermediate from decomposing and affectingYield and purity. The post-treatment process is simple, and various substituted phenyl carbamate intermediates stable at room temperature can be obtained by rotary evaporation after extraction with ethyl acetate⁹. Dissolving the substituted phenyl carbamate intermediate (16a-16t, 1.0-1.1 equiv.) in acetonitrile, adding 80% hydrazine hydrate (17, 2.5-3.0 equiv.), and carrying out ultrasonic room-temperature reaction for 1-3 h to obtain the corresponding substituted semicarbazide (18a-18 t). (ii) a

(VII) finally, the intermediate Oxo-CH₂Heating, refluxing and dehydrating DAPYs (13a,13b, 1.0-1.1 equiv.) and various substituted semicarbazides (18a-18t, 1.0-1.1 equiv.) in ethanol for 4-5 h under the condition of taking hydrochloric acid as a catalyst to obtain corresponding target compounds (1a, 1b, …, 1z, 1aa).

In the sixth step, substituted anilines (14a-14t), substituted phenyl carbamates intermediates (16a-16t), substituted semicarbazides (18a-18t), and the 27 targets (1a, 1b, …, 1z, 1aa) obtained in the seventh step, wherein the corresponding X and R are listed as follows:

the numbers (a to v) corresponding to the substituents in the table correspond to the respective series of intermediates (14,16,18) and the corresponding series of target compounds (1a to v), and the intermediate numbers and the substituent type numbers in combination represent the respective numbers of the corresponding compounds, such as the intermediates (14a to v,16a to v,18a to v) and the target compounds (1a to v), respectively.

The compound (I) is an HIV-1 non-nucleoside reverse transcriptase inhibitor, has stronger biological activity, higher selection coefficient and lower cytotoxicity.

The invention also relates to a pharmaceutical composition which contains the compound (I) and related medicinal carriers with effective dose, and application of the compound or the composition in preparing medicaments for preventing and treating AIDS.

In conclusion, the invention designs and synthesizes a series of diaryl pyrimidine compounds with semicarbazone structures by keeping main pharmacophores according to the structural characteristics of marketed drugs, namely etravirine and rilpivirine and according to the structure-activity relationship of CH-DAPYs and introducing the semicarbazone structures with antiviral activity to a methylene connecting group of a left wing aromatic ring of the DAPY compounds by using a split principle. The molecular simulation shows that: the compound is in a classical U-shaped conformation when being combined with reverse transcriptase, the left wing aromatic ring enters a hydrophobic region and forms pi-pi stacking effect with Y181 and Y188, and NH connecting the right wing aromatic ring and the pyrimidine ring can form hydrogen bond with K101. Thereby attempting to increase the interaction of the compound with the target in an attempt to increase the biological activity of the compound of interest against drug-resistant HIV strains. The invention takes thiouracil and methyl iodide as initial raw materials, and a target compound is obtained by S-methylation, nucleophilic substitution, chlorination, oxidation, secondary nucleophilic substitution and condensation reaction in sequence. All new compounds are prepared by¹H NMR and¹³and (4) identifying by a spectrum analysis method such as C NMR and the like, and verifying part of compounds by ESI-MS. Screening experiments for anti-HIV-1 activity were performed at the extracellular and enzymatic levels of the target compound. Cell level activity test results show that the target compound has low micromolar inhibition activity on HIV-1 wild strains, and partial compounds have lower inhibition activity on double-mutation HIV-1 strains RES056 and HIV-2 ROD. Among them, the target compound has the best 1h activity (EC)₅₀Value of 0.0329 μ M, SI value of 3712), activity stronger than that of the reference drugs NEV and DEV, and low cytotoxicity and high selectivity. The test result of the enzyme level on the activity of the HIV-1RT shows that the target compound has stronger inhibitory activity on the HIV-1 RT.

Drawings

FIG. 1 shows the design strategy from marketed drugs etravirine and rilpivirine to the target compound of the present invention.

FIG. 2 is a graph showing the binding pattern of a target to the HIV-1RT non-nucleoside inhibitor binding pocket. Wherein, (a) the target 1q, (b) the target 1p, (c) the target 1k, and (d) the target 1 d.

Detailed Description

The invention will be better understood by the following examples of implementation, but is not intended to limit the scope of the invention.

Example 1: preparation of intermediates

(1) Preparation of 2- (4-cyanoanilino) -4-chlorouracil (10)

Sodium hydroxide (50.4g,1.26mol) was made into an aqueous solution (1.0L), 2-thiouracil (5,212.9g,1.50mol) was added to the sodium hydroxide solution, stirred until completely dissolved, allowed to stand, and cooled to room temperature. Methyl iodide (6,153.6g,1.2mol) was added and stirring continued for 24h, TLC showed complete conversion of the starting material. Adjusting the pH value to be neutral, separating out a white solid, filtering, washing with water, and drying to obtain a white solid (153g, 89.7%), namely the 2-methylthio pyrimidine-4-ketone (7).

2-methylthio-4-pyrimidyl-ketone (7,42.7g,0.30mol) and p-aminobenzonitrile (8,88.6g,0.75mol) were mixed well, and the temperature was slowly raised to 180 ℃ for 10 hours. Thin layer chromatography showed that a small amount of raw material was not converted at 10h, the reaction time was prolonged to 18h, the conversion of raw material was complete, but the product was impure and the yield decreased. After the reaction was completed, it was slowly cooled to room temperature, and the reaction solution solidified to form a pale yellow solid stuck to the bottom of the flask, which was then slurried with acetonitrile to obtain a crude product of 2- (4-cyanoanilino) pyrimidin-4-one (9,43.0g, 67.6%).

The 2- (4-cyanoanilino) pyrimidin-4-one (9,32.0g,0.165mol) obtained above was dissolved in phosphorus oxychloride (150mL, 1.64mol), which was now available as both the chlorinated reagent and the solvent. The reaction was completed by heating and refluxing for 30 min. Cooling to room temperature, slowly pouring into ice water under vigorous stirring, controlling the temperature, wherein the speed is not too fast, otherwise, the product is easy to cause bumping and the purity of the product is influenced. A pale yellow solid precipitated, cooled, filtered, and the solid suspended in cold water (200mL) and neutralized to neutrality with 20% sodium hydroxide. Filtration, washing with water and drying gave 2- (4-cyanoanilino) -4-chlorouracil as a pale yellow solid (10,32.0g, 84.0%).

(2) Preparation of 2-halophenyl 2- (4-cyanophenylamino) -pyrimidones (13a,13b)

2- (4-Cyanoanilino) -4-chlorouracil (10,2.77g,12.0mmol) and o-halophenylacetonitrile (11a,11b,2.73g/3.53g,18.0mmol) in anhydrous DMF (30mL) in the presence of NaH (0.96g,24.0mmol, 60%) as solvent undergo an aromatic nucleophilic substitution reaction to yield 2- (p-Cyanoanilino) -4- (2-halophenylcyano) methylenepyrimidine intermediate (12a,12 b). The reaction is sensitive to air, nitrogen protection is required, anhydrous conditions are high, and DMF is used after being dried overnight by using an activated molecular sieve. The 2- (p-cyanophenylamino) -4- (2-chlorophenylcyano) methylene pyrimidine (12a,12b) is unstable, nitrogen protection is removed, and the 2- (p-cyanophenylamino) -4-aroyl pyrimidine (13a,13b) is obtained by oxidation in the air. The reaction in the step is the rate-limiting reaction in the whole synthesis, the time consumption is long, side reactions are more, and the reaction is not easy to control. After 48-72h of reaction, TLC showed the reaction was complete. Pouring the reaction liquid into water, neutralizing the reaction liquid to be neutral by using diluted hydrochloric acid, extracting the reaction liquid by using ethyl acetate, combining ethyl acetate layers, drying the ethyl acetate layers by using anhydrous sodium sulfate, removing the ethyl acetate layers by rotary evaporation, and performing rapid reduced pressure column chromatography (PE: EA ═ 5:1) on the obtained crude product to obtain a pure product 2-halogenated phenyl 2- (4-cyanophenylamino) -pyrimidone (13a,13 b).

(3) Synthesis of various substituted semicarbazides

The substituted semicarbazide is obtained by using various substituted anilines as starting materials through two steps. The method has the advantages of simple and easy route, mild conditions, economy, high efficiency, high yield and high purity.

Various substituted anilines (14a-t, 1.86-3.44 g,20.0mmol) were dissolved in tetrahydrofuran, while NaHCO was added₃(2.0g,24.0mmol) was dissolved in water and NaHCO was added₃The aqueous solution was mixed with a tetrahydrofuran solution of substituted aniline and placed in an ice bath, after the temperature stabilized at 0 deg.C phenyl chloroformate (15,3.8g,24.0mmol) was added. The reaction speed is very fast, and the reaction can be finished after reactants are added. Because the substituted phenyl carbamate intermediate is unstable in the reaction liquid, the temperature is controlled to be 0 ℃ in the reaction process, so that the intermediate is prevented from decomposing and affecting the yield and the purity. The post-treatment process is simple, various substituted phenyl carbamate intermediates are obtained by rotary evaporation after extraction with ethyl acetate, and the intermediates are stable at room temperature⁹。

Dissolving the substituted phenyl carbamate intermediate (16a-t, 2.13-2.92 g,10.0mmol) in acetonitrile, adding 80% hydrazine hydrate (17,1.6g,25.0mmol), and carrying out ultrasonic room temperature reaction for 1-3 h to obtain the corresponding substituted semicarbazide (18a-t), wherein the specific experimental number is shown in Table 1.

TABLE 1 physical Properties and yields of various intermediate substituted semicarbazides 18a-t

Example 2: synthesis of DAPY-based target 1a-aa

Dissolving substituted semicarbazide (18a-t, 0.23-0.35 g,1.5mmol) and 2-chlorophenyl 2- (4-cyanophenylamino) -pyrimidone (13a,13b,0.50g/0.57g,1.5mmol) in absolute ethanol (10.0mL), adding concentrated hydrochloric acid (2-3 drops) as a catalyst, and heating and refluxing for 4-5.5 h. Light yellow solid is separated out in the reaction process, the thin layer chromatography shows that the reaction is complete, and the filtration is carried out. The product has poor solubility, and the target product with higher purity can be obtained by ethyl acetate washing.

TABLE 2 reaction time, yield and physical Properties for the preparation of target Compounds 1a-1aa

Characterization of target Compounds

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-ketone-N-phenyl semicarbazone (1a).¹H NMR(400MHz,DMSO-d₆) δ 10.15(s,1H, Ph-NH-pyrimidine ring), 9.83(s,1H, ═ N-NH-CO),9.24(s,1H, CO-NH-Ph "), 8.61(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.87(d, J ═ 5.1Hz,1H, pyrimidine ring CH₅),7.78–7.67(m,2H,Ph’H),7.59(dd,J＝10.4,4.2Hz,1H,Ph’H),7.54(d,J＝7.9Hz,2H,Ph”H_2,6),7.44(t,J＝7.0Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6),7.31(t,J＝7.9Hz,2H,Ph”H_3,5),7.03(t,J＝7.4Hz,1H,Ph”H₄)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.74,159.48,159.04,152.02,145.26,143.96,139.15,133.15,132.95,131.81,131.57,130.27,129.20,128.45,123.35,120.10,119.99,118.29,109.27,102.48ppm；MS(ESI+)490(M+Na)⁺；HPLC:t_R＝17.60min,98.5％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-methylphenyl) semicarbazone (1b).¹H NMR(400MHz,DMSO-d₆) δ 10.15(s,1H, Ph-NH-pyrimidine ring), 9.78(s,1H, ═ N-NH-CO),9.16(s,1H, CO-NH-Ph "), 8.61(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.88(d, J-5.1 Hz,1H, pyrimidine ring CH₅),7.74–7.68(m,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.43(t,J＝9.0Hz,5H,PhH_3,5+Ph’H+Ph”H_2,6),7.37(d,J＝8.8Hz,2H,PhH_2,6),7.11(d,J＝8.3Hz,2H,Ph”H_3,5),1.98(s,3H,CH₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.76,159.47,159.03,152.06,145.27,143.57,136.55,133.15,132.95,132.29,131.79,131.59,130.27,129.59,128.44,120.22,120.00,118.29,109.27,102.46,20.88ppm；MS(ESI+)504(M+Na)⁺；HRMS(ESI+):m/z＝482.1495,484.1470[M+H]⁺；calcd.482.1496,484.1467for C₂₆H₂₁ClN₇O+H；HPLC:t_R＝18.60min,99.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-cyanophenyl) semicarbazone (1c).¹H NMR(400MHz,DMSO-d₆) δ:10.17(s,1H, Ph-NH-pyrimidine ring), 10.08(s,1H, ═ N-NH-CO),9.67(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.86(d, J ═ 4.6Hz,1H, pyrimidine ring CH₅),7.74–7.68(m,6H,Ph’H,Ph”H_2,3,5,6),7.60(t,J＝6.7Hz,1H,Ph’H),7.43(t,J＝7.0Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.55,159.48,159.18,151.85,145.21,145.05,143.72,133.68,132.96,131.90,131.55,131.47,130.28,128.44,119.98,119.73,119.65,118.30,109.38,104.83,102.53ppm。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-ketone-N- (4-nitrophenyl) semicarbazone (1d).1H NMR (400MHz, DMSO-d)₆) δ 10.23(s,1H, Ph-NH-pyrimidine ring), 10.20(s,1H, ═ N-NH-CO),10.13(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆),8.21(d,J＝9.1Hz,2H,Ph”H_2,6) 7.82(d, J ═ 8.9Hz,3H, pyrimidine ring CH5, Ph "H_3,5),7.71(q,J＝7.9Hz,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.44(t,J＝8.6Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.64,159.38,159.06,151.80,145.93,145.32,145.16,142.19,133.04,132.96,131.92,131.54,131.40,130.29,128.45,125.45,119.97,119.06,118.34,109.36,102.57,56.49,19.03ppm；HRMS(ESI+):m/z＝513.1191,515.1165[M+H]⁺；calcd.513.1190,515.1161for C₂₅H₁₈ClN₈O₃+H。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-methoxyphenyl) semicarbazone (1e).¹H NMR(400MHz,DMSO-d₆) δ:10.14(s,1H, Ph-NH-pyrimidine ring), 9.78(s,1H, ═ N-NH-CO),9.12(s,1H, CO-NH-Ph "), 8.60(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 5.0Hz,1H, pyrimidine ring CH₅),7.77–7.64(m,2H,Ph’H),7.59(t,J＝7.4Hz,1H,Ph’H),7.43(dt,J＝7.6,5.1Hz,5H,PhH_3,5+Ph’H+Ph”H_2,6),7.37(d,J＝8.9Hz,2H,PhH_2,6),6.89(d,J＝8.9Hz,2H,Ph”H_3,5),3.35(s,3H,CH₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.77,159.47,158.98,155.71,152.32,145.28,143.56,133.17,132.95,132.03,131.76,131.61,130.26,128.42,122.23,120.01,118.29,114.34,109.29,102.45,55.66ppm；MS(ESI+)520.5(M+Na)⁺；HPLC:t_R＝17.35min,99.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-ethoxyphenyl) semicarbazone (1f).¹H NMR(400MHz,DMSO-d₆) δ:10.14(s,1H, Ph-NH-pyrimidine ring), 9.76(s,1H, ═ N-NH-CO),9.11(s,1H, CO-NH-Ph "), 8.60(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 5.1Hz,1H, pyrimidine ringCH₅),7.77–7.65(m,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.43(t,J＝9.8Hz,5H,PhH_3,5,Ph’H+Ph”H_2,6),7.37(d,J＝8.8Hz,2H,PhH_2,6),6.88(d,J＝8.9Hz,2H,Ph”H_3,5),3.98(q,J＝7.0Hz,2H,CH₂),1.30(t,J＝7.0Hz,3H,CH₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.78,159.47,158.98,154.98,152.31,145.28,143.53,133.17,132.95,131.93,131.76,131.61,130.26,128.48,122.23,120.00,118.28,114.87,109.29,102.45,63.58,15.19ppm；MS(ESI+)534.5(M+Na)⁺；HRMS(ESI+)m/z:＝512.1602,514.1572[M+H]⁺；calcd.512.1602,514.1572for C₂₇H₂₃ClN₇O₂+H；HPLC:t_R＝18.15min,98.5％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-hydroxyphenyl) semicarbazone (1g).¹H NMR(400MHz,DMSO-d₆) δ 10.13(s,1H, Ph-NH-pyrimidine ring), 9.69(s,1H, ═ N-NH-CO),9.19(s,1H, OH),9.02(s,1H, CO-NH-Ph "), 8.60(d, J ═ 5.2Hz,1H, pyrimidine ring CH)₆) 7.91(d, J ═ 5.2Hz,1H, pyrimidine ring CH₅),7.76–7.64(m,2H,Ph’H),7.58(t,J＝7.3Hz,1H,Ph’H),7.42(dd,J＝12.1,8.3Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6),7.28(d,J＝8.8Hz,2H,Ph”H_2,6),6.72(d,J＝8.8Hz,2H,Ph”H_3,5)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.81,159.46,158.95,153.89,152.36,145.28,143.33,133.17,132.95,131.61,131.58,130.41,130.25,128.48,122.65,120.00,118.28,115.58,109.27,102.43,56.51,19.03ppm；MS(ESI+)506(M+Na)⁺；HRMS(ESI+)m/z:＝484.1283,486.1256[M+H]⁺；calcd.484.1289,486.1259for C₂₅H₁₉ClN₇O₂+H；HPLC:t_R＝15.25min,98.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-fluorophenyl) semicarbazone (1h).¹H NMR(400MHz,DMSO-d₆) δ 10.15(s,1H, Ph-NH-pyrimidine ring), 9.87(s,1H, ═ N-NH-CO),9.29(s,1H, CO-NH-Ph "), 8.62(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 4.8Hz,1H, pyrimidine ring CH₅),7.71(dt,J＝15.3,7.8Hz,2H,Ph’H),7.57(dt,J＝8.9,6.3Hz,3H,Ph’H+Ph”H_2,6),7.43(t,J＝8.3Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.6Hz,2H,PhH_2,6),7.15(d,J＝8.8Hz,2H,Ph”H_3,5)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.70,159.47,159.04,157.30,145.26,143.99,135.45,133.15,132.95,131.79,131.58,130.26,128.41,122.27,120.00,118.29,115.82,115.60 109.33,102.47ppm；MS(ESI+)486.5(M+Na)⁺；HPLC:t_R＝17.52min,98.5％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-chlorophenyl) semicarbazone (1i).¹H NMR(400MHz,DMSO-d₆) δ:10.16(s,1H, Ph-NH-pyrimidine ring), 9.93(s,1H, ═ N-NH-CO),9.43(s,1H, CO-NH-Ph "), 8.62(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.88(d, J ═ 4.8Hz,1H, pyrimidine ring CH₅),7.76-7.65(m,2H,Ph’H),7.58(d,J＝8.9Hz,3H,Ph’H+Ph”H_2,6),7.42(t,J＝9.1Hz,3H,PhH_3,5+Ph’H),7.36(dd,J＝8.8,3.8Hz,4H,PhH_2,6+Ph”H_3,5)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.69,159.42,159.03,152.09,145.22,144.29,138.19,133.11,132.95,131.52,130.26,129.05,128.43,126.96,121.63,119.99,118.31,109.33,102.50ppm；MS(ESI+)502.5(M+Na)⁺；HRMS(ESI+)m/z:＝502.0957,504.0933[M+H]⁺；calcd.502.0950,504.0920for C₂₅H₁₈Cl₂N₇O₂+H；HPLC:t_R＝14.84min,98.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (4-bromophenyl) semicarbazone (1j).¹H NMR(400MHz,DMSO-d₆) δ:10.16(s,1H, Ph-NH-pyrimidine ring), 9.93(s,1H, ═ N-NH-CO),9.36(s,1H, CO-NH-Ph "), 8.62(d, J ═ 5.1Hz,1H, pyrimidine ring CH₆) 7.89(d, J ═ 4.2Hz,1H, pyrimidine ring CH₅),7.71(q,J＝7.8Hz,2H,Ph’H),7.63-7.52(m,3H,Ph’H+Ph”H_2,6),7.49(d,J＝8.6Hz,2H,Ph”H_3,5),7.43(t,J＝7.4Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.5Hz,2H,PhH_2,6)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.65,159.48,159.09,152.06,145.25,144.30,138.62,133.13,132.95,131.95,131.83,131.55,130.26,128.43,122.04,119.99,118.29,114.95,109.35,102.49ppm；MS(ESI+)568.5(M+Na)⁺；HRMS(ESI+)m/z:＝546.0439,548.0419[M+H]+；calcd.546.0445,548.0424for C₂₅H₁₈BrClN₇O₂+H；HPLC:t_R＝19.58min,99.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (3-methylphenyl) semicarbazone (1k) 48.2%; light yellow solid; mp is 258.4-259.1 ℃;¹H NMR(400MHz,DMSO-d₆) δ:10.16(s,1H, Ph-NH-pyrimidine ring), 9.83(s,1H, ═ N-NH-CO),9.21(s,1H, CO-NH-Ph "), 8.61(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.86(d, J ═ 5.0Hz,1H, pyrimidine ring CH₅),7.77–7.66(m,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.43(t,J＝7.9Hz,3H,PhH_3,5+Ph’H),7.36(t,J＝11Hz,4H,PhH_2,6+Ph”H_2,6),7.18(t,J＝7.7Hz,1H,Ph”H₅),6.85(d,J＝7.4Hz,1H,Ph”H₄),2.28(s,3H,CH₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.76,159.44,159.02,151.99,145.25,143.88,139.06,138.40,132.96,131.81,131.56,130.27,129.05,128.45,124.07,120.56,120.00,118.30,117.22,109.25,102.47,21.66ppm；MS(ESI+)582(M+Na)⁺；HRMS(ESI+)m/z:＝482.1493,484.1474[M+H]⁺；calcd.482.1496,484.1467for C₂₆H₂₁ClN₇O+H；HPLC:t_R＝14.55min,99.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-nitrophenyl) semicarbazone (1l).¹H NMR(400MHz,DMSO-d₆) δ:10.17(s,1H, Ph-NH-pyrimidine ring), 10.13(s,1H, ═ N-NH-CO),9.78(s,1H, CO-NH-Ph), 8.62(s,1H, Ph "H", Ph₂) 8.65(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆),7.94(s,2H,Ph”H_4,6) 7.89(d, J ═ 9.8Hz,1H, pyrimidine ring CH₅),7.73(d,J＝7.4Hz,2H,Ph’H),7.60(t,J＝8.3Hz,2H,Ph’H+Ph”H₅),7.44(t,J＝8.8Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.57,159.48,159.14,152.34,148.49,145.23,144.84,140.59,133.12,132.94,131.84,131.54,130.47,130.26,128.40,119.98,118.30,117.75,114.13,109.46,102.50ppm；MS(ESI+)513.5(M+H)⁺；HRMS(ESI+)m/z:＝513.1191,515.1165[M+H]+；calcd.513.1191,515.1164for C₂₅H₁₈ClN₈O₃+H；HPLC:t_R＝14.05min,98.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-hydroxyphenyl) semicarbazone (1m).¹H NMR(400MHz,DMSO-d₆) δ 10.19(s,1H, Ph-NH-pyrimidine ring), 9.82(s,1H, ═ N-NH-CO),9.24(s,1H, CO-NH-Ph "), 8.60(d, J ═ 5.3Hz,1H, pyrimidine ring CH₆) 7.81(d, J ═ 5.1Hz,1H, pyrimidine ring CH₅),7.77–7.63(m,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.43(t,J＝8.8Hz,3H,PhH_3,5+Ph’H),7.37(t,J＝8.8Hz,2H,PhH_2,6),7.15-6.99(m,2H,Ph”H_2,5),6.89(d,J＝8.3Hz,1H,Ph”H₂),6.44(d,J＝9.5Hz,1H,Ph”H₄)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.92,159.29,158.81,158.19,151.73,145.18,143.82,140.22,133.11,132.96,131.83,131.60,131.50,130.28,129.85,128.47,119.98,118.35,110.43,106.87,102.55,56.50,19.03ppm。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-methoxyphenyl) semicarbazone (1N).¹H NMR(400MHz,DMSO-d₆) δ:10.19(s,1H, Ph-NH-pyrimidine ring), 9.86(s,1H, ═ N-NH-CO),9.36(s,1H, CO-NH-Ph "), 8.61(d, J ═ 5.3Hz,1H, pyrimidine ring CH₆) 7.82(d, J ═ 5.0Hz,1H, pyrimidine ring CH₅),7.78–7.65(m,2H,Ph’H),7.59(t,J＝7.4Hz,1H,Ph’H),7.43(t,J＝9.0Hz,3H,PhH_3,5+Ph’H),7.38(t,J＝9.5Hz,2H,PhH_2,6),7.20(dd,J＝11.2,4.9Hz,2H,Ph”H_2,5),7.07(d,J＝8.0Hz,1H,Ph”H₆),6.61(d,J＝8.2Hz,1H,Ph”H₄),2.49(s,3H,CH₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.87,160.07,159.31,158.86,151.86,145.18,144.02,133.10,132.96,131.84,131.58,131.49,130.28,129.99,128.47,119.97,118.36,112.13,109.21,108.67,105.68,102.56,55.47ppm。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-fluorophenyl) semicarbazone (1o).¹H NMR(400MHz,DMSO-d₆) δ:10.16(s,1H, Ph-NH-pyrimidine ring), 9.95(s,1H, ═ N-NH-CO),9.42(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.88(d, J ═ 4.9Hz,1H, pyrimidine ring CH₅),7.72(dt,J＝15.4,8.1Hz,2H,Ph’H),7.62-7.51(m,2H,Ph’H+Ph”H₂),7.47-7.40(m,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6),7.34-7.28(m,2H,Ph”H_4,6),6.85(t,J＝7.7Hz,1H,Ph”H₅)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:163.19,162.62,161.52,159.48,159.12,152.01,145.24,144.48,141.10,140.98,133.12,132.96,131.85,131.53,130.71,130.27,128.44,119.99,118.29,115.71,109.34,106.55,102.49ppm；MS(ESI+)508(M+H)⁺；HPLC:t_R＝16.05min,98.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-chlorophenyl) semicarbazone (1p).¹H NMR(400MHz,DMSO-d₆) δ 10.16(s,1H, Ph-NH-pyrimidine ring), 9.97(s,1H, ═ N-NH-CO),9.40(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.90(d, J ═ 5.0Hz,1H, pyrimidine ring CH₅),7.77–7.68(m,3H,Ph’H+Ph”H₂),7.70-7.41(m,4H,PhH_3,5+Ph’H+Ph”H₂),7.59(t,J＝7.3Hz,1H,Ph’H),7.38-7.31(m,3H,PhH_2,6+Ph”H_5,6),7.09(d,J＝7.8Hz,1H,Ph”H₄)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.61,159.48,159.12,152.10,145.24,144.48,140.74,133.53,133.13,132.96,131.84,131.55,130.81,130.26,128.42,122.99,119.99,119.50,118.52,118.29,102.49ppm；MS(ESI+)524.5(M+Na)⁺；HRMS(ESI+):m/z:＝502.0939,504.0914[M+H]+；calcd.502.0950,504.0920for C₂₅H₁₈Cl₂N₇O₂+H；HPLC:t_R＝19.20min,97.9％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (3-bromophenyl) semicarbazone (1q).¹H NMR(400MHz,DMSO-d₆) δ 10.15(s,1H, Ph-NH-pyrimidine ring), 9.98(s,1H, ═ N-NH-CO),9.41(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 5.0Hz,2H, pyrimidine ring CH₅+Ph”H₂),7.78–7.66(m,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.50(d,J＝8.0Hz,1H,Ph”H₆),7.43(t,J＝4.6Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.7Hz,2H,PhH_2,6),7.27(t,J＝8.0Hz,1H,Ph”H₅),7.21(d,J＝8.0Hz,1H,Ph”H₄)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.61,159.47,159.11,152.10,145.24,144.48,140.48,133.13,132.95,131.83,131.54,131.11,130.26,128.42,125.89,122.34,122.01,119.99,118.90,118.30,109.39,102.50ppm；MS(ESI+)568.5(M+Na)⁺；HRMS(ESI+)m/z:＝546.0434,548.0412[M+H]+；calcd.546.0445,548.0424for C₂₅H₁₈BrClN₇O₂+H；HPLC:t_R＝16.18min,98.3％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (2-methylphenyl) depsipeptide (1r).¹H NMR(400MHz,DMSO-d₆) δ:10.17(s,1H, Ph-NH-pyrimidine ring), 9.85(s,1H, ═ N-NH-CO),9.27(s,1H, CO-NH-Ph "), 8.61(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.85(d, J-5.1 Hz,1H, pyrimidine ring CH)₅),7.77–7.66(m,2H,Ph’H),7.59(t,J＝7.2Hz,1H,Ph’H),7.43(t,J＝9.5Hz,3H,PhH_3,5+Ph’H),7.38-7.33(m,4H,PhH_2,6+Ph”H_5,6),7.18(t,J＝7.7Hz,1H,Ph”H₄),6.84(d,J＝7.4Hz,1H,Ph”H₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.85,159.49,159.35,158.89,151.98,145.21,143.84,139.09,138.38,133.13,132.95,131.81,131.59,131.54,130.27,129.04,128.45,124.04,120.53,119.98,118.34,117.18,109.24,102.52,21.66ppm；MS(ESI+)m/z 482(M+H)⁺；HPLC:t_R＝14.56min,98.6％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (2-hydroxyphenyl) semicarbazone (1s).¹H NMR(400MHz,DMSO-d₆) δ 10.35(s,1H, Ph-NH-pyrimidine ring), 10.21(s,1H, ═ N-NH-CO),8.94(s,1H, CO-NH-Ph "), 8.60(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 8.00(d, J ═ 7.9Hz,1H, pyrimidine ring CH)₅),7.74–7.67(m,2H,Ph’H),7.59(dd,J＝13.7,6.3Hz,2H,Ph’H+Ph”H₆),7.43(t,J＝7.6Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6),6.90(d,J＝7.8Hz,1H,Ph”H₃),6.84(t,J＝7.5Hz,1H,Ph”H₄),6.76(t,J＝7.6Hz,1H,Ph”H₅)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:163.10,159.34,158.80,151.87,146.97,145.15,143.52,133.14,132.97,131.78,131.72,130.18,128.36,127.16,123.32,119.98,119.87,119.58,118.38,115.16,108.57,102.59ppm。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (2-methoxyphenyl) semicarbazone (1t). delta.10.35 (s,1H, Ph-NH-pyrimidine ring), 10.21(s,1H,. gtn-NH-CO), 8.94(s,1H, CO-NH-Ph "), 8.61(d, J ═ 5.2Hz,1H, pyrimidine ring CH)₆) 7.83(d, J ═ 4.9Hz,1H, pyrimidine ring CH₅),7.75–7.68(m,2H,Ph’H),7.59(t,J＝7.2Hz,1H,Ph’H),7.43(t,J＝9.2Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.6Hz,2H,PhH_2,6),7.21-7.18(m,2H,Ph”H_4,5),7.07(d,J＝8.0Hz,1H,Ph”H₆),6.60(d,J＝8.0Hz,1H,Ph”H₃),2.49(s,3H,CH₃)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.88,160.06,159.30,158.83,151.85,145.17,144.00,140.42,133.11,132.96,131.84,131.59,131.50,130.28,129.99,128.47,119.98,118.36,112.13,109.20,108.66,105.67,102.56,55.47ppm。

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (4-bromophenyl) semicarbazone (1u).¹H NMR(400MHz,DMSO-d₆) δ 10.17(s,1H, Ph-NH-pyrimidine ring), 9.96(s,1H, ═ N-NH-CO),9.51(s,1H, CO-NH-Ph "), 8.64(d, J ═ 4.8Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 4.2Hz,3H, pyrimidine ring CH₅+Ph”H_2,6),7.63(s,2H,Ph’H),7.51(d,J＝7.3Hz,1H,Ph’H),7.45(d,J＝8.0Hz,3H,PhH_3,5+Ph’H),7.38(d,J＝7.9Hz,2H,PhH_2,6),7.28(d,J＝7.8Hz,2H,Ph”H_3,5)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.53,159.41,159.06,145.67,145.23,145.23,140.89,133.68,133.46,133.26,132.92,131.92,131.62,131.10,128.84,123.08,122.00,119.97,119.05,118.30,109.42,102.46ppm。

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (4-chlorophenyl) semicarbazone (1v).¹H NMR(400MHz,DMSO-d₆) Delta 10.18(s,1H, Ph-NH-pyrimidine ring), 9.93(s,1H, ═ N-NH-CO),9.52(s,1H, CO-NH-Ph "), 8.62(d, J ═ 5.2Hz,1H, pyrimidine ring CH —)₆) 7.88(d, J ═ 4.8Hz,1H, pyrimidine ring CH)₅),7.76-7.65(m,2H,Ph’H),7.58(d,J＝8.9Hz,3H,Ph’H+Ph”H_2,6),7.42(t,J＝9.1Hz,3H,PhH_3,5+Ph’H),7.36(dd,J＝8.8,3.8Hz,4H,PhH_2,6+Ph”H_3,5)ppm；¹³C NMR(100MHz,DMSO-d₆)δ:162.59,159.32,158.95,152.02,145.41,145.21,138.21,133.68,133.32,132.92,131.90,131.63,129.03,128.91,126.85,123.02,121.45,119.85,118.32,102.49ppm。

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (4-fluorophenyl) semicarbazone (1w).¹H NMR(400MHz,DMSO-d₆) δ:10.16(s,1H, Ph-NH-pyrimidine ring), 9.85(s,1H, ═ N-NH-CO),9.36(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 3.5Hz,2H, pyrimidine ring CH₅+Ph’H),7.65–7.62(m,2H,Ph’H),7.57(dd,J＝8.8,4.9Hz,2H,Ph’H),7.45(d,J＝8.8Hz,2H,PhH_3,5),7.38(d,J＝8.8Hz,3H,PhH_2,6+Ph’H),7.19-7.10(m,2H,Ph”_3,5)ppm。

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (3-nitrophenyl) semicarbazone (1X).¹H NMR(400MHz,DMSO-d₆) δ 10.19(s,1H, Ph-NH-pyrimidine ring), 10.11(s,1H, ═ N-NH-CO),9.90(s,1H, CO-NH-Ph "), 8.66(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆),8.63(s,1H,Ph”H₂),7.95(d,J＝6.6Hz,2H,Ph”H_4,6) 7.92-7.87(m,2H, pyrimidine ring CH)₅+Ph’H),7.64-7.59(m,3H,Ph’H),7.45(t,J＝8.8Hz,2H,PhH_3,5),7.40(dd,J＝13.7,5.8Hz,3H,PhH_2,6+Ph’H)ppm。

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (4-cyanophenyl) semicarbazone (1y).¹H NMR(400MHz,DMSO-d₆) δ:10.16(s,1H, Ph-NH-pyrimidine ring), 9.85(s,1H, ═ N-NH-CO),9.36(s,1H, CO-NH-Ph "), 8.63(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.86(d, J ═ 4.6Hz,1H, pyrimidine ring CH₅),7.74–7.68(m,6H,Ph’H,Ph”H_2,3,5,6),7.60(t,J＝6.7Hz,1H,Ph’H),7.43(t,J＝7.0Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.8Hz,2H,PhH_2,6)ppm。

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-ylketone-N- (3-bromophenyl) semicarbazone (1z).¹H NMR(400MHz,DMSO-d₆) δ 10.17(s,1H, Ph-NH-pyrimidine ring), 9.95(s,1H, ═ N-NH-CO),9.46(s,1H, CO-NH-Ph "), 8.64(d, J ═ 5.2Hz,1H, pyrimidine ring CH₆) 7.91(d, J ═ 5.0Hz,2H, pyrimidine ring CH₅+Ph”H₂),7.78–7.66(m,2H,Ph’H),7.59(t,J＝7.3Hz,1H,Ph’H),7.50(d,J＝8.0Hz,1H,Ph”H₆),7.43(t,J＝4.6Hz,3H,PhH_3,5+Ph’H),7.37(d,J＝8.7Hz,2H,PhH_2,6),7.27(t,J＝8.0Hz,1H,Ph”H₅),7.21(d,J＝8.0Hz,1H,Ph”H₄)ppm.

2-bromophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-ketone-N- (3-methoxyphenyl) semicarbazone (1aa).¹H NMR(400MHz,DMSO-d₆) δ 10.17(s,1H, Ph-NH-pyrimidine ring), 9.95(s,1H, ═ N-NH-CO),9.46(s,1H, CO-NH-Ph "), 8.64(d, J ═ 5.3Hz,1H, pyrimidine ring CH₆) 7.90(d, J ═ 5.0Hz,1H, pyrimidine ring CH₅),7.78–7.65(m,2H,Ph’H),7.59(t,J＝7.4Hz,1H,Ph’H),7.43(t,J＝9.0Hz,3H,PhH_3,5+Ph’H),7.38(t,J＝9.5Hz,2H,PhH_2,6),7.20(dd,J＝11.2,4.9Hz,2H,Ph”H_2,5),7.07(d,J＝8.0Hz,1H,Ph”H₆),6.61(d,J＝8.2Hz,1H,Ph”H₄),2.49(s,3H,OCH₃)ppm。

Example 3: anti-HIV biological Activity assay

The in vitro cell level anti-HIV virus activity test mainly comprises the following steps: inhibiting activity and cytotoxicity on HIV-infected MT-4 cells. The method comprises the following steps: the protective effect of the drug on HIV-induced cytopathic effects was determined by MTT method in HIV-infected MT-4 cells at different times of HIV infection, and the half-effective concentration EC required to protect 50% of the cells from HIV-induced cytopathic effects was calculated₅₀Toxicity assay was performed in parallel with anti-HIV activity assay, and the concentration (CC) at which 50% of uninfected cells were cytopathic was measured by MTT method in MT-4 cell culture₅₀) And calculating the selectivity index SI ═ CC₅₀/EC₅₀。

The material and the method are as follows:

the anti-HIV activity of each compound was monitored by the efficiency of the drug's inhibition of the cytopathic effects of HIV in cells. MT-4 cells were used for cell culture. The viral strains used were: HIV-1 strain IIIB and HIV-2 strain ROD.

The specific operation is as follows: dissolving the compound in DMSO or water, diluting in phosphate buffered saline solution, and mixing 3 × 10⁵MT-4 cells were pre-incubated with 100. mu.L of each compound in different concentrations in this solution at 37 ℃ for 1h, and then 100. mu.L of an appropriate viral diluent was added to the compound and the cells were incubated at 37 ℃ for 1 h. After three washes, the cells were resuspended in culture medium with or without compound, respectively. Cells were then incubated at 5% CO₂Incubate at 37 ℃ for 7 more days in an atmosphere and replace the supplemented medium with culture medium with or without compound on the third day post infection. The procedure was repeated twice for each broth condition. Cytopathic effects on the virus were monitored daily by reverse light microscopy. Typically, the viral dilutions used in this experiment often lead to cytopathic effects the fifth day after viral infection. The inhibitory concentration of the drug is such that the drug produces a 50% inhibition of the viral cytopathic effect while having no direct toxicity to the cells (CC)₅₀) And (4) showing. It is emphasized that, when the compound is poorly water-soluble and DMSO is required for dissolution, the specific concentration of DMSO is generally less than 10% relative to water (the final concentration of DMSO in the MT-4 cell culture medium is less than 2%). Since DMSO can affect the antiviral activity of the test compound, the antiviral activity of solutions containing the same concentration of DMSO should also be performed in parallel to the control blank. In addition, the final concentration of DMSO (1/1000) was much lower than the concentration required for HIV-1 replication in T cells.

According to the invention, marketed drugs of Nevirapine (NVP), Efavirenz (EFV) and Etravirine (ETravirine, ETV) are used as reference substances, and the HIV inhibition activity results of part of target compounds are summarized in tables 3-4. Experimental results show that the compounds contained in the chemical general formula generally have stronger anti-HIV-1 virus activity, smaller cytotoxicity and higher selectivity index. The results of the active cytotoxicity of DAPY-SC compounds against HIV-1IIIB, the variant strain RES056 and the HIV-2 strain ROD are shown in Table 3.

TABLE 3 anti-HIV Activity and cytotoxicity of the target Compounds

As shown in Table 3, the EC of 20 target compounds against wild-type HIV-1₅₀The value is within the range of 0.0329-1.1538 mu M, the inhibitor has low micromolar level inhibitory activity on HIV-1 wild strains, and the SI value is within the range of 22-3712, so that the inhibitor has lower inhibitory activity compared with a lead compound. Among them, the target compound 1h has the best activity (R ═ 4-F, EC)₅₀A value of 0.0329. mu.M, SI value of 3712), which is stronger than the reference drug NEV (EC)₅₀Value of 0.114. mu.M, SI value of 132), DEV (EC)₅₀Value 0.0366 μ M, SI value 1200) and ddI (EC)₅₀The value was 19.96. mu.M, and the SI value was 11). In addition, 1a (H, EC)₅₀A value of 0.0449. mu.M, an SI value of 1575),1c (4-CN, EC₅₀Value 0.0650. mu.M, SI value 519),1n (3-OCH)₃,EC₅₀Value 0.0402. mu.M, SI value 213),1p (3-Cl, EC₅₀A value of 0.0737. mu.M, a SI value of 1729) and 1s (2-OH, EC)₅₀Value 0.0372 μ M, SI value 135) was also stronger, superior to the reference drugs NEV and ddI. And EC of intermediate 13a₅₀The values were 0.0111. mu.M, SI value 5548, stronger than the reference drugs NEV, DEV and ddI, and more active than 20 target compounds.

For the double mutant strain RES056, part of compounds show weaker inhibitory activity and comprise 1f (4-OC)₂H₅,EC₅₀Value 5.12. mu.M), 1g (4-OH, EC)₅₀Value 4.92. mu.M), 1M (3-OH, EC)₅₀Value 5.21. mu.M), 1n (3-OCH)₃,EC₅₀Value of 8.01. mu.M), 1s (2-OH, EC)₅₀Values of 5.31. mu.M) and 1t (2-OCH)₃,EC₅₀Value of 8.49 μ M), superior to the reference drug NEV (EC)₅₀Value of 15.04. mu.M) and DEV (EC)₅₀The value was 43.86. mu.M).

In addition, the first and second substrates are,some of the compounds showed some activity against HIV-2ROD, including 1f (4-OC)₂H₅,EC₅₀Value 6.84. mu.M), 1g (4-OH, EC)₅₀Value 5.14. mu.M), 1M (3-OH, EC)₅₀Value of 5.97. mu.M), 1n (3-OCH)₃,EC₅₀Value of 8.99. mu.M), 1s (2-OH, EC)₅₀Value 4.67. mu.M) and 1t (2-OCH)₃,EC₅₀The value was 9.94. mu.M). Notably, this is consistent with the inhibitory activity against the double mutant strain RES 056.

Example 4 testing of the Activity of the Compounds of interest against HIV-1RT at the enzyme level

To confirm that the target compound is HIV-1RT, the present patent performed enzyme level inhibition activity tests on the target compound. Test materials Reverse Transcriptase activity detection kit (EnzCheck Reverse Transcriptase Assay kit) was purchased from Invitrogen corporation. Positive controls Nevirapine (NEV) and Efavirenz (EFV).

(1) Test principle and method

Recombinant wild-type Reverse Transcriptase P66/P51 HIV-1RT was expressed and purified according to methods reported in the literature and tested for Reverse Transcriptase activity using a commercial Reverse Transcriptase activity test kit (EnzCheck Reverse Transcriptase Assay kit), all following strictly the instructions of the test kit. The detection box has the action principle that when the dye PicoGreen is combined with double-stranded DNA or RNA/DNA heterozygosis double-stranded, the fluorescent signal of the dye PicoGreen is obviously enhanced, so that the quantitative effect on the double-stranded nucleic acid is achieved. And even if very high dyes are present: in base-matched comparison, single-stranded nucleic acids all have only a slight fluorescent signal.

(2) Procedure of experiment

Using 350 bases Poly (rA) as template, oligo (dT)₁₆Annealing at room temperature for 60 minutes as primers in a molar ratio of 1: 1.2: mu.L of polymerization buffer (containing 60mM Tri-HCl,60mM KCl,8mM MgCl) containing 52ng of RNA/DNA mixture per well in a 96-well plate₂13mM DTT,100mM dTTP, pH 8.1); mu.L of RT enzyme solution was diluted to appropriate concentration with enzyme diluent (containing 50mM Tri-HCl, 20% glycerol,2mM DTT, pH 7.6); the reaction solution was incubated at 25 ℃ for 40 minutes, and the reaction was stopped by adding 15mM EDTA, followed by detection by adding PicoGreen dyeDetecting the hybrid double strand. The absorbance value at the emission wavelength of 523nm at the excitation wavelength of 490nm was determined by a microplate reader. To test the compounds for anti-retroviral activity, 1. mu.L of compound in DMSO was added to each well before adding the reverse transcriptase solution. The test samples were dissolved in DMSO to the appropriate concentration and then diluted 5-fold with DMSO, 8 dilutions each. While control wells were added 1 μ L DMSO without compound. The results are expressed as relative fluorescence values, i.e., fluorescence intensity of wells containing compound/fluorescence intensity of wells containing no compound.

(3) Results and discussion

The results of the inhibitory activity of the target compounds against wild-type HIV-RT are shown in Table 4.

TABLE 4 inhibitory Activity of the target Compounds on HIV-1RT

As shown in Table 4, 20 target compounds and 13a both have strong inhibitory activity against HIV-1RT, IC of the target compounds₅₀The value is 0.438 to 10.14. mu.M. Of these, 1h had the strongest inhibitory activity (IC)₅₀Value of 0.438 μ M), superior to the reference drug NEV (IC)₅₀The value was 0.971. mu.M). In addition, 1a (IC)₅₀Value of 0.522. mu.M), 1l (IC)₅₀Value of 0.970. mu.M), 1n (IC)₅₀Value 0.833. mu.M), 1q (IC)₅₀Values of 0.955. mu.M) and 1s (IC)₅₀Value of 0.594 μ M) is also stronger than the reference drug NEV. Indicating that the target compound is HIV-1 RT.

In conclusion, the experimental results of the cell level and the enzyme level show that the compounds contained in the chemical general formula generally have stronger anti-HIV-1 virus activity, smaller cytotoxicity and higher selectivity index.

Molecular Docking (Docking) analysis between compounds and target RT

In order to obtain the potential active conformation of the molecule, we select TMC125/HIV-1 complex 3MEC by means of Surflex-Dock module in Sybyl molecular simulation software package according to the research result of 2D-QSAR, and remove water and hydrogenate enzyme protein after extracting ligand. Carrying out conformation optimization by using a steepest energy gradient descent method under a Tripos force field, and then loading Gasteig-Huckel charge. And (3) taking the extracted TMC125 conformation as a reference, and performing simulated docking on the optimized molecule and the prepared receptor protein. Parameters of the docking results are in the appendix. When compared with Total Score (TS) values, 1ac is highest and TS values of 1q,1p,1j and 1k are also higher. The activity prediction of the target compound by using the QSAR model is consistent with the grading value.

The interaction of the ligand with the reverse transcriptase was observed, taking as an example the binding pattern of 1q,1p,1k, which is predicted to be more active, and 1d and 3MEC, which are predicted to be less effective, as shown in the analysis of fig. 2.

The conformations of 1q and 1p with stronger activity in the NNIBP of RT are similar and present a U-shaped conformation, the ligand enters a hydrophobic pocket formed by amino acids such as Tyr181, Tyr188 and Trp229 to form hydrophobic interaction, and a left wing aromatic ring and benzene rings of the Tyr181 and the Tyr188 form pi-pi stacking interaction in the same plane. At the same time, right wing aromatic ring linker NH forms a hydrogen bond with Lys 101. The NH on the semicarbazone of 1q can also form a hydrogen bond with Ile180, and the binding with the enzyme is tighter.

While the 1k and less active 1d predicted conformations in the hydrophobic binding pocket of the enzyme differed from the classical U-shaped conformation. The position of the left wing aromatic ring of 1k is exchanged with that of the aromatic ring of semicarbazone between 1q and 1p, and the benzene ring on the semicarbazone enters a hydrophobic pocket to form pi-pi stacking interaction. 1d is far away from a hydrophobic pocket formed by amino acids such as Tyr181, Tyr188 and Trp229, and cannot form pi-pi stacking interaction and hydrophobic interaction with enzyme, although 1d can also form hydrogen bond with Lys101, the affinity of the 1d with the enzyme is greatly reduced compared with 1p,1q and 1k, and the interaction with the hydrophobic pocket is very important for the combination of the ligand and HIV-1 RT.

The present invention is not limited to the above examples.

Reference documents:

1.De Clercq,E.,Fifty years in search of selective antiviral drugs.J.Med.Chem.2019,62,7322-7339.

2.Meng,G.；Chen,F.-E.；De Clercq,E.；Balzarini,J.；Pannecouque,C.,Nonnucleoside HIV-1reverse transcriptase inhibitors:part I.Synthesis and structure-activity relationship of1-alkoxymethyl-5-alkyl-6-naphthylmethyl uracils as HEPT analogues.Chemical and Pharmaceutical Bulletin 2003,51(7),779-789.

3.Jin,K.；Sang,Y.；De Clercq,E.；Pannecouque,C.；Meng,G.,Design and synthesis of a novel series of non-nucleoside HIV-1inhibitors bearing pyrimidine and N-substituted aromatic piperazine.Bioorganic&Medicinal Chemistry Letters 2018,28,3491–3495.

4.Jin,K.；Sang,Y.；Han,S.；Clercq,E.D.；Pannecouque,C.；Meng,G.；Chen,F.,Synthesis and biological evaluation of dihydroquinazoline-2-amines as potentnon-nucleoside reverse transcriptase inhibitors of wild-type and mutant HIV-1strains.European Journal of Medicinal Chemistry 2019,176,11-20.

5.Jin,K.；Liu,M.；Zhuang,C.；De Clercq,E.；Pannecouque,C.；Meng,G.；Chen,F.,Improving the positional adaptability:structure-based design of biphenyl-substituted diaryltriazines as novel non-nucleoside HIV-1reverse transcriptase inhibitors.Acta Pharm.Sin.B 2020,10(2),344-357.

6.Xiao,T.；Tang,J.-F.；Meng,G.；Pannecouque,C.；Zhu,Y.-Y.；Liu,G.-Y.；Xu,Z.-Q.；Wu,F.-S.；Gu,S.-X.；Chen,F.-E.,Indazolyl-substituted piperidin-4-yl-aminopyrimidines as HIV-1NNRTIs:Design,synthesis and biological activities.European Journal of Medicinal Chemistry 2020,186(2020)111864(186),111864.

7.Gu,S.X.；Yang,S.Q.；He,Q.Q.；Ma,X.D.；Chen,F.E.；Dai,H.F.；Clercq,E.D.；Balzarini,J.；Pannecouque,C.,Design,synthesis and biological evaluation of cycloalkyl arylpyrimidines(CAPYs)as HIV-1NNRTIs.Bioorg Med Chem 2011,19(23),7093-9.

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Claims (7)

1. an HIV-1 reverse transcriptase inhibitor, which is a diaryl pyrimidine derivative containing substituted aryl ureide group, and has the structural formula shown as the following formula (I):

wherein R is independently selected from hydrogen, methyl, cyano, nitro, methoxy, ethoxy, hydroxyl and halogen, and the substitution position can be ortho, para or meta; x is halogen.

2. The HIV-1 reverse transcriptase inhibitor of claim 1, wherein said substituted aryl ureido containing diarylpyrimidine derivatives are 27: 1a, 1b, …, 1z, 1aa, which corresponds to R, X as follows:

3. the HIV-1 reverse transcriptase inhibitor of claim 1, further comprising a pharmaceutically acceptable salt of a diarylpyrimidine derivative containing a substituted arylureeimide group, in particular hydrochloride, hydrobromide, sulfate, phosphate, acetate, methanesulfonate, p-toluenesulfonate, tartrate, citrate, fumarate or malate.

4. The method of synthesizing the HIV-1 reverse transcriptase inhibitor of claim 1, wherein the specific synthetic route is as follows:

the specific steps of the synthesis are as follows;

reacting thiouracil (5) serving as an initial raw material and methyl iodide (6) serving as a methylation reagent at room temperature for 20-24 hours under the action of sodium hydroxide to perform S-alkylation reaction to obtain a high-purity white solid, namely 2-methylthiopyrimidine-4-ketone (7); wherein, the amount of the thiouracil (5) is 1.5 to 1.6equiv., the amount of the methyl iodide (6) is 1.0 to 1.1equiv., the molar concentration of the sodium hydroxide is 1.20 to 1.35M, and the amount is 1.0 to 1.1 equiv.;

secondly, 2-methylthio pyrimidine-4-ketone (7) and excessive 4-cyano aniline (8) react for 10-18 h in a molten state at 180-185 ℃ under the solvent-free condition, and after acetonitrile is dissolved, post-treatment is carried out to obtain yellow solid 2- (4-cyano anilino) pyrimidine-4-ketone (9); wherein the amount of the 2-methylthio pyrimidine-4-ketone (7) is 1.0-1.1 equiv., and the amount of the 4-cyanoaniline (8) is 2.5-3.0 equiv.;

dissolving 2- (4-cyanoanilino) pyrimidine-4-ketone (9) in excessive phosphorus oxychloride for reflux, carrying out chlorination reaction on hydroxyl at the C-4 position of a pyrimidine heterocyclic ring, carrying out post-treatment, dissolving in a proper amount of cold water, neutralizing with sodium hydroxide to be neutral to obtain yellow precipitate, filtering and drying to obtain yellow solid 2- (4-cyanoanilino) -4-chloro-pyrimidine (10); wherein the amount of the 2- (4-cyanoanilino) pyrimidine-4-one (9) is 1.0-1.1 equiv., and the amount of the phosphorus oxychloride is 10.0-11.0 equiv.;

performing nucleophilic substitution reaction on (tetra) 2- (4-cyanoanilino) -4-chloro-pyrimidine (10) and 2-halogenated phenylacetonitrile (11a,11b) in dried N, N-dimethylformamide under the action of 60% sodium hydride under anhydrous and oxygen-free conditions to obtain unstable intermediate Cyan-CH₂-DAPYs (12a,12 b); wherein the amount of 2- (4-cyanoanilino) -4-chloro-pyrimidine (10) is 1.0 to 1.1equiv., the amount of 2-halophenylacetonitrile (11a,11b) is 1.5 to 1.6equiv., and the amount of sodium hydride is 2.0 to E2.4equiv., 2.5 equiv-3.0 mL of N, N-dimethylformamide;

(V) due to the intermediate Cyan-CH₂the-DAPYs (12a,12b) are unstable, the nitrogen protection is removed after the reaction, the reaction is carried out for 48 to 72 hours in the air at room temperature, and the slow oxidation is carried out to obtain an intermediate Oxo-CH₂-DAPYs, and obtaining pure 2-halophenyl 2- (4-cyanophenylamino) -pyrimidone (13a,13b) by post-treatment and column chromatography separation;

sixthly, simultaneously carrying out the steps (one) to (five), carrying out the preparation of various substituted semicarbazides (18a-18t) in parallel by using various substituted anilines as starting materials through two-step reaction; the method comprises dissolving various substituted anilines (14a-14t) in tetrahydrofuran, and adding NaHCO₃Dissolving in water, NaHCO₃Mixing the aqueous solution with a tetrahydrofuran solution of substituted aniline, placing the mixture in an ice bath, and adding phenyl chloroformate (15) after the temperature is stabilized to be 0-5 ℃; the reaction speed is very fast, and the reaction can be finished after reactants are added; extracting with ethyl acetate, and rotary steaming to obtain various substituted phenyl carbamate intermediates (16a-16t) stable at room temperature; dissolving the substituted phenyl carbamate intermediate (16a-16t) in acetonitrile, adding 80% hydrazine hydrate (17), and carrying out ultrasonic room temperature reaction for 1-3 h to obtain corresponding substituted semicarbazide (18a-18 t); wherein, the substituted aniline (14a-14t) is 1.0-1.1 equiv, NaHCO₃1.2 to 1.4equiv., and phenyl chloroformate (15) is 1.2 to 1.4 equiv.; 1.0-1.2 equiv. for the substituted phenyl carbamate intermediate (16a-16t), and 2.5-3.0 equiv. for the hydrazine hydrate (17);

(VII) finally, the intermediate Oxo-CH₂Heating, refluxing and dehydrating DAPYs (13a,13b) and various substituted semicarbazides (18a-18t) in ethanol for 4-5 hours under the condition of taking hydrochloric acid as a catalyst to obtain corresponding target compounds (1a, 1b, …, 1z, 1 aa); wherein, the intermediate Oxo-CH₂1.0-1.1 equiv. for DAPYs (13a,13b) and 1.0-1.1 equiv. for various substituted semicarbazides (18a-18 t);

in the sixth step, substituted aniline (14a-14t), substituted phenyl carbamate intermediate (16a-16t), substituted semicarbazide (18a-18t), and the target (1a, 1b, …, 1z, 1aa) obtained in the seventh step, wherein the corresponding X and R are listed as follows:

5. a pharmaceutical composition comprising an effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier.

6. The application of diaryl pyrimidine derivatives containing substituted aryl ureido imino in preparing HIV-1 reverse transcriptase inhibitors is disclosed, wherein the diaryl pyrimidine derivatives containing substituted aryl ureido have the structural formula shown in the formula (I):

wherein R is independently selected from hydrogen, methyl, cyano, nitro, methoxy, ethoxy, hydroxyl and halogen, and the substitution position can be ortho-position, para-position or meta-position; x is halogen.

7. The use according to claim 6, for the preparation of a medicament for the prevention and treatment of AIDS.

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