CN114539162B - Substituted aryl urea imino diaryl pyrimidine derivative and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a diaryl pyrimidine derivative containing substituted aryl ureido imino, a preparation method and application thereof. The structure of the compound is shown as a general formula ZFI, and the compound also comprises pharmaceutically acceptable salts, hydrates and solvates thereof, polycrystal or eutectic crystals thereof, and precursors and derivatives with the same biological functions. The in vitro enzyme level anti-PARP-1 activity evaluation experimental result shows that the small molecules have different structural characteristics combined with PARP-1, and analysis is carried out on the protein crystal structure and drug interaction characteristics, thereby illustrating the basis of the molecular structural characteristics. The compounds have the biological activity of potentially treating and regulating diseases (such as tumors) related to abnormal PARP-1 expression, have lower cytotoxicity, and can be used for preparing medicines for treating diseases such as tumors related to abnormal PARP-1 expression.

Description

Substituted aryl urea imino diaryl pyrimidine derivative and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a diaryl pyrimidine derivative containing substituted aryl ureido imino, a preparation method and application thereof.

Background

Poly (ADP-ribose polymerase, PARP) is widely found in eukaryotic cells. There are at least 17 subtypes, of which PARP-1 and PARP-2 have a DNA binding domain. PARP-1 is the earliest discovered and most studied subtype, functioning about 85% of the PARP family. PARP-1 contains 1014 amino acids and has a relative molecular mass of 116kDa and comprises three domains: an N-terminal DNA binding domain, a C-terminal catalytic domain and an intermediate self-modifying domain. PARP-1 is a ribozyme activated by DNA cleavage, and mediates BER repair of DNA damage by catalyzing formation of polymer (Poly (ADP) ribosylation) at the site of DNA damage, and has close relation with occurrence and development of various malignant tumors ¹ . Studies have shown that PARP-1 expression in tumor cells is significantly elevated in untreated patients compared to normal cellsHigh, whereas the tumor cells of the patients undergoing chemotherapy have a more pronounced tendency to rise. Thus, inhibition of PARP-1 enzyme activity may block DNA repair and increase sensitivity to chemotherapeutic agents. The PARP-1inhibitor and the cytotoxic chemotherapy drug are combined to be applied, thereby providing a new thought for tumor treatment and being a new target point for anti-tumor treatment. Studies have shown that PARP is involved in HIV infection of cells. An important element in the life cycle of HIV-1 is the entry of viral DNA into the host cell genome by the action of integrase. This process requires a DNA double strand gap and thus may lead to subsequent activation of poly a-di-phosphoribosyl polymerase (PARP) activity. PARP is a potential target for inhibition of HIV-1.

The chemical structure types of PARP-1inhibitors (PARPi) are diverse and there are many drugs in clinical trial phase, with representative drugs including benzimidazole carboxamides (1), phthalazinones (2), tricyclic indololactams (3), indazole carboxamides (4), carbazole diimides (5) and nicotinamide analogs (6) according to basic structural features. Currently, six PARP inhibitors have been marketed in china as Olaparib (Olaparib AZD2281, KU0059436, 2), ruaparib (ruaparib, 3), nilaparib (nirapparib, 4), talazoparib (7), fluzoparib (Fluzoparib, 8) and pamipril (Pamiparib, 9), respectively (fig. 1). Parp-1inhibitors of olapari (2), which were first approved by the FDA as marketed in the 2014 end, contain a basic structural parent nucleus with phthalazinones and a cyclopropane side chain with a certain tonicity, mainly by participating in the DNA defect repair pathway to kill tumor cells ² . The compounds have inhibitory activity (IC) on PARP-1 and PARP-2 ₅₀ ) Is 5 nM and 1nM, can be orally taken, is an autophagy (autophagy) and mitochondrial autophagy (mitophagy) activator, has poor structural stability, and is easy to be metabolically opened in vivo. Nilaparil was approved by the U.S. FDA for maintenance therapy in adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer in month 3 2017 (6), the first PARP inhibitor approved by the U.S. FDA for treatment without BRCA mutation or other biomarker detection, which acts by inhibiting apoptosis of PARP1 and PARP2 mediated cancer cells, and is an orally administered, highly bioavailable and highly selective PARP inhibitor. Fluoxazopali(8) Is approved to be marketed by the national drug administration in 12 months and 14 days of 2020, is the first domestic PARP inhibitor, and induces cell cycle arrest by inhibiting the DNA repair process in BRCA1/2 dysfunctional cells, thereby inhibiting tumor cell proliferation. The trifluoromethyl structure of the fluxapyroxad seals the metabolic part, has small influence on the activity of the metabolic enzymes such as CYP enzyme and the like, is not easy to generate interaction when being combined with other medicines, and ensures stable medicine effect and safety. Parmipril (9), obtained in 5 months 2021, is a potent, selective inhibitor of PARP-1 and PARP-2, which induces tumor cell death by inhibiting repair of single-stranded lesions of tumor cell DNA and homologous recombination repair defects, and is particularly highly sensitive to tumor cells harboring DNA repair defects with BRCA gene mutations.

Some representative PARP-1inhibitors have the following structure:

because the PARP-1 and the PARP-2 catalytic domains have similar structures, the PARP-1inhibitor is developed at present, has poor selectivity and has inhibition effect on both PARP-1 and PARP-2. On the other hand, the normal repair function of DNA is also maintained for normal cells to maintain viability, so that the activity of normal cells needs to be maintained and activated while antitumor drugs PARPi are used to make tumor cells synthetic lethal. Therefore, the design and research of PARP-1inhibitors (PARP-1 inhibitors) and agonists (PARP-1 agonists) with high selectivity have important research significance and clinical application value.

Disclosure of Invention

The invention aims to provide a diaryl pyrimidine derivative containing substituted aryl ureido imino, a preparation method and application thereof.

In order to achieve the above purpose, the invention retains the basic structural parent nucleus part of common pyridine formamide or benzamide in the traditional PARPi structure, reverses the arrangement direction of substituent groups of the basic structural unit of amide, designs the basic structural unit of amide as an analogue of urea structure, uses pyrimidine ring to replace nitrogen-containing heterocycle in most of original molecular structures, ensures that a certain number of nitrogen atoms are in the molecular structure, so that hydrogen bond interaction is formed between the molecule and amino acid residues on the inner wall of an active pocket of a target protein, and uses the type and the substitution site of the substituent groups on the benzene ring to regulate the affinity of the compound to the target protein, and designs the basic idea of the molecular structure of the target compound as follows:

the substituted aryl urea imino diaryl pyrimidine derivative provided by the invention is ZFI and has the following structural formula:

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

The invention also comprises the medicinal salt of the substituted aryl urea imino diaryl pyrimidine derivative, the hydrate and solvate thereof, polycrystal and eutectic thereof, and precursors and derivatives with the same biological functions.

In the invention, the medicinal salt of the substituted aryl urea imino diaryl pyrimidine derivative comprises hydrochloride, hydrobromide, sulfate, phosphate, acetate, methanesulfonate, p-toluenesulfonate, tartrate, citrate, fumarate or malate.

The invention also provides a preparation method of the diaryl pyrimidine derivative containing the substituted aryl urea imino, and the specific synthetic route is as follows

The specific steps of the synthesis are as follows:

(1) 2- (4-cyanoanilino) -4-chloro-pyrimidine (10,1.0-1.1 equiv.) and 2-halogeno-benzyl cyanide (11,1.0-1.1 equiv) are subjected to nucleophilic substitution reaction in N, N-dimethylformamide under the action of sodium hydride (60%), the temperature is controlled to be between minus 10 ℃ and room temperature, and an intermediate Cyan-CH is obtained under the anhydrous and anaerobic condition ₂ -DAPYs(12)；

(2) The intermediate Cyan-CH ₂ The DAPYs (12) is unstable, and is put into air, and after the reaction is carried out for 40 to 48 hours at room temperature, the key intermediate oxo methylene substituted diaryl pyrimidine compound (13) can be obtained by slow oxidation;

(3) Simultaneously with the preparation of intermediate 13, intermediate 18 is prepared starting from various substituted anilines; the method comprises the following specific steps: substituted anilines (14 a-14t,1.0 to 1.1 equiv.) are dissolved in tetrahydrofuran while NaHCO is added ₃ (1.2-1.4 equiv.) dissolving in water, and mixing NaHCO with water ₃ Mixing the aqueous solution with tetrahydrofuran solution of substituted aniline, placing in ice bath, adding phenyl chloroformate (15,1.2-1.4 equiv.) after the temperature is stabilized at 0 ℃, extracting with ethyl acetate, and steaming to obtain various substituted phenyl carbamate intermediates (16 a-16 t) which exist stably at room temperature ³ The method comprises the steps of carrying out a first treatment on the surface of the Dissolving a substituted phenyl carbamate intermediate (16 a-16t, 1.0-1.1 equiv.) in acetonitrile, adding 80% hydrazine hydrate (17,2.5-3.0 equiv.), and reacting at ultrasonic room temperature for 1-3 h to obtain a corresponding substituted semicarbazide (18 a-18 t);

(4) And finally, heating, refluxing and dehydrating an equivalent amount of an oxo methylene substituted diaryl pyrimidine compound intermediate (13,1.0-1.1 equiv.) and a substituted semicarbazide intermediate (18 a-18t, 1.0-1.1 equiv.) in ethanol under the condition of taking hydrochloric acid as a catalyst to obtain 22 corresponding target compounds (ZFIa-ZFIv).

Substituted anilines (14 a-14 t), substituted phenylcarbamates (16 a-16 t), substituted semicarbazides (18 a-18 t) in step (3), and the target compound (ZFIa-ZFIv) in step (4), corresponding R, X, are specifically as follows:

wherein the numbers (a-v) of the substituent enantiomers respectively map respective series of intermediates (14, 16, 18) and respective series of target compounds (ZFIa-ZFIv), the intermediate numbers and the substituent type numbers are combined with each other to represent respective numbers of the respective compounds, such as intermediates (14 a-v, 16 a-v, 18 a-v) and target compounds (ZFIa-ZFIv), respectively.

The target compound can be used as a selective regulator for abnormal PARP-1 expression, has finer adjustable activity, can finely adjust the biological enzyme activity of PARP-1 by changing the type and the substitution position of partial substituent groups in the structure of the compound, and can inhibit or properly activate; the cytotoxicity was evaluated at the cellular level to be small. In particular, substitution of the aromatic ring with the idle-hydroxy group inhibits PARP-1 activity, while substitution with the ortho-methoxy group suitably activates PARP-1 activity, as analyzed below. The modulation of other substituents can be seen in FIG. 1 (as inhibition modulation when ΔTm is positive and as activation modulation when ΔTm is negative).

The invention also relates to a pharmaceutical composition containing an effective dose of the compound and a related medicinal carrier, and application of the compound or the composition in preparing medicines for preventing and treating AIDS.

Drawings

FIG. 1 is a graph showing the ΔTm change of PARP-1 interacting with a target compound.

FIG. 2 shows the chemical structure comparison of compound ZFIm and compound ZFIt.

FIG. 3 is a pattern of interaction of compound ZFIm (left) and compound ZFIt (right) with PARP-1 (3 GOY) binding pocket amino acid residues: wherein, (a) is a side view of the compound showing the interaction pattern of ZFIm with the PARP-1 binding pocket amino acid residue, (b) is a side view of the compound showing the interaction pattern of zfil with the PARP-1 binding pocket amino acid residue, (c) is a side view of the compound showing the interaction pattern of ZFIm with the PARP-1 binding pocket amino acid residue, and (d) is a side view of the compound showing the interaction pattern of zfil with the PARP-1 binding pocket amino acid residue.

Detailed Description

The present invention will be better understood by the following examples of embodiments, but is not limited thereto.

Example 1: preparation of 2-halophenyl 2- (4-cyanophenylamino) -pyrimidinone (13)

2- (4-cyanoanilino) -4-chlorouracil (10, 2.77g,12.0 mmol) was aromatically nucleophilic substituted with o-halophenylacetonitrile (11 a,11b,2.73g/3.53g,18.0 mmol) in the presence of NaH (0.96 g,24.0mmol, 60%) in anhydrous DMF (30 mL) to give 2- (p-cyanoanilino) -4- (2-halophenylcyano) methylenepyrimidine intermediate (12 a,12 b). The reaction is sensitive to air, requires nitrogen protection, and requires high anhydrous conditions, and DMF is used after drying overnight with activated molecular sieves. 2- (p-cyano-phenylamino) -4- (2-chlorphenyl cyano) methylene pyrimidine (12 a,12 b) is unstable, nitrogen protection is removed, and 2- (p-cyano-phenylamino) -4-aroyl pyrimidine (13 a,13 b) is obtained by oxidation in air. The reaction is a speed-limiting reaction in the whole synthesis, the time consumption is long, the side reaction is more, and the reaction is not easy to control. After 48-72h of reaction, TLC showed complete reaction. The reaction solution was poured into water, neutralized to neutrality with dilute hydrochloric acid, extracted with ethyl acetate, the ethyl acetate layers were combined and dried over anhydrous sodium sulfate and removed by rotary evaporation, and the crude product obtained was subjected to flash column chromatography (PE: ea=5:1) to obtain pure 2-halophenyl 2- (4-cyanophenylamino) -pyrimidinone (13 a,13 b).

Synthesis of various substituted semicarbazides

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

Various substituted anilines (14 a-14t, 1.86-3.44 g,20.0 mmol) were dissolved in tetrahydrofuran while NaHCO was added ₃ (2.0 g,24.0 mmol) was dissolved in water and NaHCO was added ₃ Aqueous solution and substituted anilineAfter being mixed and placed in an ice bath at a temperature of 0℃phenyl chloroformate (15, 3.8g,24.0 mmol) was added. The reaction speed is extremely high, and the reaction can be completed after the reactants are added. Because the substituted phenyl carbamate intermediate is unstable in the reaction liquid, the temperature in the reaction process is controlled at 0 ℃ so as to prevent the intermediate from being decomposed and influencing the yield and purity. The post-treatment process is simple, ethyl acetate is used for extraction and then rotary evaporation is carried out to obtain various substituted phenyl carbamate intermediates, and the intermediates are stable at room temperature ³ 。

The substituted phenyl carbamate intermediate (16 a-16t, 2.13-2.92 g,10.0 mmol) was dissolved in acetonitrile, 80% hydrazine hydrate (17, 1.6g,25.0 mmol) was added and reacted at room temperature with ultrasound for 1-3 h to give the corresponding substituted semicarbazide (18 a-18 t), the specific experimental numbers are shown in Table 1.

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

Example 2: synthesis of PAARi target ZFIa-ZFIv

Substituted semicarbazide (18 a-t, 0.23-0.35 g,1.5 mmol) and 2-chlorophenyl 2- (4-cyanophenylamino) -pyrimidinone (13 a,13b,0.50g/0.57g,1.5 mmol) are dissolved in absolute ethanol (10.0 mL), concentrated hydrochloric acid (2-3 drops) is added as a catalyst, and the mixture is heated and refluxed for 4-5.5 hours. Light yellow solid is separated out in the reaction process, and the thin layer chromatography shows that the reaction is complete and is filtered. The product has poor solubility, and the target compound ZFI with higher purity can be obtained by washing with ethyl acetate. The reaction times, yields and physical properties of the target compounds ZFIa to ZFIv are shown in table 2.

TABLE 2 reaction time, yield and physical Properties for the preparation of the target Compounds ZFIa to ZFIv

1. The spectroscopic data for the target compounds are characterized as follows:

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-ketone-N-phenylsemicarbazone (ZFIa). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.87 (d, J=5.1 Hz,1H, pyrimidine ring CH ₅ ),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”H ₄ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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+):m/z＝490(M+Na) ⁺ ；HPLC:t _R ＝17.60min,98.5％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-methylphenyl) semicarbazone (ZFIb). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.88 (d, J=5.1 Hz,1H, pyrimidine ring CH ₅ ),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,CH ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₆ H ₂₁ ClN ₇ O+H；HPLC:t _R ＝18.60min,99.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-cyanophenyl) semicarbazone (ZFIc). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.86 (d, j=4.6 hz,1h, pyrimidine ring CH ₅ ),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； ¹³ C NMR(100MHz,DMSO-d ₆ )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-one-N- (4-nitrophenyl) semicarbazone (ZFId) 1H NMR (400 MHz, DMSO-d) ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ),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； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₅ H ₁₈ ClN ₈ O ₃ +H。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-methoxyphenyl) semicarbazone (ZFIe). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.91 (d, J=5.0 Hz,1H, pyrimidine ring CH ₅ ),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,CH ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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+):m/z＝520.5(M+Na) ⁺ ；HPLC:t _R ＝17.35min,99.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-ethoxyphenyl) semicarbazone (ZFIf). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.91 (d, J=5.1 Hz,1H, pyrimidine ring CH ₅ ),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,CH ₂ ),1.30(t,J＝7.0Hz,3H,CH ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₇ H ₂₃ ClN ₇ O ₂ +H；HPLC:t _R ＝18.15min,98.5％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-ketone-N- (4-hydroxyphenyl) semicarbazone (ZFIG). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta: 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=5.2 hz,1H, pyrimidine ring CH) ₆ ) 7.91 (d, J=5.2 Hz,1H, pyrimidine ring CH ₅ ),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； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₅ H ₁₉ ClN ₇ O ₂ +H；HPLC:t _R ＝15.25min,98.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-fluorophenyl) semicarbazone (ZFIh). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.91 (d, J=4.8 Hz,1H, pyrimidine ring CH ₅ ),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； ¹³ C NMR(100MHz,DMSO-d ₆ )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+):m/z＝486.5(M+Na) ⁺ ；HPLC:t _R ＝17.52min,98.5％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-chlorophenyl) semicarbazone (ZFIi). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 hz,1H, pyrimidine ring CH) ₆ ) 7.88 (d, J=4.8 Hz,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； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₅ H ₁₈ Cl ₂ N ₇ O ₂ +H；HPLC:t _R ＝14.84min,98.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (4-bromophenyl) semicarbazone (ZFIj). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.1 hz,1H, pyrimidine ring CH) ₆ ) 7.89 (d, J=4.2 Hz,1H, pyrimidine ring CH ₅ ),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； ¹³ C NMR(100MHz,DMSO-d ₆ )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+):m/z＝568.5(M+Na) ⁺ ；HRMS(ESI+)m/z:＝546.0439,548.0419[M+H]+；calcd.546.0445,548.0424for C ₂₅ H ₁₈ BrClN ₇ O ₂ +H；HPLC:t _R ＝19.58min,99.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-ketone-N- (3-methylphenyl) semicarbazone (ZFIk). YIeld:48.2%; light yellow solid; mp 258.4-259.1 ℃; ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 hz,1H, pyrimidine ring CH) ₆ ) 7.86 (d, J=5.0 Hz,1H, pyrimidine ring CH ₅ ),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”H ₅ ),6.85(d,J＝7.4Hz,1H,Ph”H ₄ ),2.28(s,3H,CH ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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+):m/z＝582(M+Na) ⁺ ；HRMS(ESI+)m/z:＝482.1493,484.1474[M+H] ⁺ ；calcd.482.1496,484.1467for C ₂₆ H ₂₁ ClN ₇ O+H；HPLC:t _R ＝14.55min,99.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-nitrophenyl) semicarbazone (ZFIl). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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) ₂ ) 8.65 (d, J=5.2 Hz,1H, pyrimidine ring CH ₆ ),7.94(s,2H,Ph”H _4,6 ) 7.89 (d, J=9.8 Hz,1H, pyrimidine ring CH ₅ ),7.73(d,J＝7.4Hz,2H,Ph’H),7.60(t,J＝8.3Hz,2H,Ph’H+Ph”H ₅ ),7.44(t,J＝8.8Hz,3H,PhH _3,5 +Ph’H),7.37(d,J＝8.8Hz,2H,PhH _2,6 )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₅ H ₁₈ ClN ₈ O ₃ +H；HPLC:t _R ＝14.05min,98.1％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-hydroxyphenyl) semicarbazone (ZFIm). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.3 Hz,1H, pyrimidine ring CH) ₆ ) 7.81 (d, J=5.1 Hz,1H, pyrimidine ring CH ₅ ),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”H ₂ ),6.44(d,J＝9.5Hz,1H,Ph”H ₄ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 (ZFIn). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.3 Hz,1H, pyrimidine ring CH) ₆ ) 7.82 (d, j=5.0 hz,1h, pyrimidine ring CH ₅ ),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”H ₆ ),6.61(d,J＝8.2Hz,1H,Ph”H ₄ ),2.49(s,3H,CH ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 (ZFIo). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 hz,1H, pyrimidine ring CH) ₆ ) 7.88 (d, J=4.9 Hz,1H, pyrimidine ring CH ₅ ),7.72(dt,J＝15.4,8.1Hz,2H,Ph’H),7.62-7.51(m,2H,Ph’H+Ph”H ₂ ),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”H ₅ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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+):m/z＝508(M+H) ⁺ ；HPLC:t _R ＝16.05min,98.2％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-chlorophenyl) semicarbazone (ZFIp). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 hz,1H, pyrimidine ring CH) ₆ ) 7.90 (d, j=5.0 hz,1h, pyrimidine ring CH ₅ ),7.77–7.68(m,3H,Ph’H+Ph”H ₂ ),7.70-7.41(m,4H,PhH _3,5 +Ph’H+Ph”H ₂ ),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”H ₄ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₅ H ₁₈ Cl ₂ N ₇ O ₂ +H；HPLC:t _R ＝19.20min,97.9％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (3-bromophenyl) semicarbazone (ZFIq). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.91 (d, J=5.0 Hz,2H, pyrimidine ring CH ₅ +Ph”H ₂ ),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”H ₆ ),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”H ₅ ),7.21(d,J＝8.0Hz,1H,Ph”H ₄ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 C ₂₅ H ₁₈ BrClN ₇ O ₂ +H；HPLC:t _R ＝16.18min,98.3％。

2-chlorophenyl-2- (4-cyanophenylamino) -pyrimidin-4-yl-one-N- (2-methylphenyl) amino-ketal (ZFIr). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 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”H ₄ ),6.84(d,J＝7.4Hz,1H,Ph”H ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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 (ZFIs). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 8.00 (d, J=7.9 Hz,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”H ₆ ),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”H ₃ ),6.84(t,J＝7.5Hz,1H,Ph”H ₄ ),6.76(t,J＝7.6Hz,1H,Ph”H ₅ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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-ketone-N- (2-methoxyphenyl) semicarbazone (zfil). Delta. 10.35 (s, 1H, ph-NH-pyrimidine ring), 10.21 (s, 1H, =N-NH-CO), 8.94 (s, 1H, CO-NH-Ph "), 8.61 (d, J=5.2 Hz,1H, pyrimidine ring CH) ₆ ) 7.83 (d, j=4.9hz, 1h, pyrimidine ring CH) ₅ ),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”H ₆ ),6.60(d,J＝8.0Hz,1H,Ph”H ₃ ),2.49(s,3H,CH ₃ )ppm； ¹³ C NMR(100MHz,DMSO-d ₆ )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-yl-ketone-N- (4-bromophenyl) semicarbazone (ZFIu). ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 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=4.8 Hz,1H, pyrimidine ring CH) ₆ ) 7.91 (d, j=4.2 hz,3h, pyrimidine ring CH ₅ +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； ¹³ C NMR(100MHz,DMSO-d ₆ )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-yl-ketone-N- (4-chlorophenyl) semicarbazone (ZFIv). ¹ H NMR(400MHz,DMSO-d ₆ ) 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=5.2 hz,1H, pyrimidine ring CH) ₆ ) 7.88 (d, J=4.8 Hz,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； ¹³ C NMR(100MHz,DMSO-d ₆ )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. PARP-1 Activity test of target Compounds

Binding assays for the target poly (adenosine diphosphate) ribose polymerase-1 (PARP-1) enzyme levels were performed on 22 semicarbazone-substituted bisaryl pyrimidine target compounds using classical thermal drift assay (Thermal shift analysis, TSA) methods, the potential biological activity of which was primarily reviewed, and specific test methods for TSA are described below, with the primary evaluation results summarized in Table 3 and FIG. 1.

2.1 Experimental materials

(1) Blank (Control) sample: fluorescent dye + DMSO + buffer, control (Reference) sample: protein + fluorescent dye + DMSO + buffer, experimental (Sample) Sample: protein + fluorescent dye + inhibitor + buffer;

(2) Instrument: real-time quantitative fluorescent PCR;

(3) Data analysis software: protein hot melting software.

2.2 test principle and method

Proteins are sensitive to external temperatures and heating can cause denaturation of the proteins. The thermal stability of a protein, which refers to the ability to maintain biological activity under the influence of temperature elevation and other factors, is an important indicator of protein stability. TSA is one method of detecting protein thermostability. In TSA, proteins gradually increase in temperature with an increase in external temperature, and when the temperature is higher than a critical temperature, proteins are denatured, and the structure is stretched and unfolded, and the denaturation temperature is the denaturation temperature (Tm) or melting temperature. The higher the Tm value, the better the thermal stability. The denatured protein structure stretches exposing hydrophobic areas for binding to the fluorochromes in solution. Since the adopted fluorescent dye is weak in fluorescence when being contacted with water, and can be excited to generate a fluorescent signal when being contacted with a hydrophobic environment, the change of the fluorescent intensity of the fluorescent dye reflects the denaturation condition of the protein. After the compound binds to the protein, the protein is stabilized, and the Tm value is increased. At Tm ₂ (addition of Compound) minus Tm ₁ (no compound is added) to give a Δtm value, the higher the Δtm value, the stronger the binding of the protein to the compound. The method utilizes a fluorescence real-time quantitative PCR instrument to run a melting curve to screen small molecule ligands. Each protein of interest has a relatively constant melting temperature (Tm) under certain conditions (buffer, pH, salt ion strength).

2.3 Experimental procedure

The protein is dissolved in buffer and the small molecule ligand is dissolved in DMSO. The protein is mixed with small molecule ligand and fluorescent dye to obtain experiment sample, and blank and control sample are set for 4 times. The molar ratio of the protein to the small molecule ligand is 1:3-1:5. The samples are respectively added into a 96-well plate, the temperature rising strategy is 25-95 ℃, the temperature rising speed is 1 ℃/min, and Tm and delta Tm values are measured.

2.4 experimental results and discussion

The Δtm values are shown in table 3. It is noted that the interaction with PARP-1 is positively correlated with the stability of the protein for some compounds like ZFIt and negatively correlated with the stability of the protein for some compounds like ZFIm.

TABLE 3 DeltaTm change profile of PARP-1 interaction with target Compounds (ZFIa to ZFIv)

In order to investigate the phenomenon that the Tm value of PARP-1 increases and the Tm value of a part of the compound decreases after the addition of a part of the compound. Selecting a composite crystal with a PDB code of 3GOY ⁴ Molecular docking studies were performed on DAPY-SC and PARP-1 using Surflex-Dock molecular modeling software of the Sybyl software package. The Dock scoring values are shown in Table 1. Comparative analyses were performed using ZFIm and zfilt as examples, and as shown in figures 3-a, b, c, d, ZFIm and zfilt interacted with PARP-1in different conformations and spatial orientations, ZFIm assumed a butterfly bipfin conformation with the diaryl pyrimidine three rings of zfilt irregularly opened. This may be responsible for the opposite effects of both on PARP-1 stability modulation. The site of action comprises a plurality of aromatic amino acids, such as Tyr1633, tyr1640 and Tyr1646, which form pi-pi stacking and hydrophobic interactions with ZFIm and ZFIt. ZFI simultaneously has hydrogen bonding interactions with two amino acid residues (Tyr 1633 and Tyr 1640), respectively. Zfil only interacts with hydrogen bonds between one amino acid residue (Gly 1602) (fig. 3).

Analysis of the structural features and differences of the two compounds revealed that: the subtle change of the meta-3-hydroxy group on the ZFIm semicarbazone linked benzene ring to the ZFIt ortho-2-methoxy group can change the direction of the binding strength between the compound and the target PARP-1 molecule, which indicates that the subtle adjustment of substituent groups on the compound with similar structure type can possibly change the PARP-1 inhibition activity or the activation activity. This is itself a very interesting phenomenon, and the binding of the molecular docking is also different (fig. 3).

In summary, the experimental results of PARP-1 enzyme levels show that the compounds contained in the chemical general formula generally have variable regulation functions on PARP-1, and are expected to become auxiliary therapeutic drugs for PARPi and diseases related to tumor cell proliferation.

The invention is not limited to the examples described above.

Reference is made to:

1.Gilliams-Francis,K.L.；Quaye,A.A.；Naegele,J.R.,PARP cleavage,DNA fragmentation,and pyknosis during excitotoxin-induced neuronal death.Exp.Neurol.2003,184(1),359-372.

2.Curtin,N.,PARP inhibitors for anticancer therapy.Biochem.Soc.Trans.2014,42(1),82-88.

3.R.Hron,B.S.J.,Preparation of substituted semicarbazides from corresponding amines and hydrazines via phenyl carbamates.Tetrahedron Letters 2014,55(9),1540-1543.

4.Wahlberg,E.；Karlberg,T.；Kouznetsova,E.；Markova,N.；Macchiarulo,A.；Thorsell,A.-G.；Pol,E.；Frostell,

Ekblad,T.；Oncu,D.；Kull,B.；Robertson,G.M.；Pellicciari,R.；Schuler,H.；Weigelt,J.,Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.Nat Biotechnol 2012,30(3),283-388。/>

Claims (2)

1. a preparation method of a diaryl pyrimidine derivative containing substituted aryl urea imino is characterized in that the derivative has the following structural formula:

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

the specific synthetic route is as follows:

(1) 2- (4-cyanoanilino) -4-chloro-pyrimidine (10) and 2-halogeno benzyl cyanide (11) are subjected to nucleophilic substitution reaction in N, N-dimethylformamide under the action of sodium hydride, the temperature is controlled between minus 10 ℃ and minus 5 ℃ to room temperature, and an intermediate Cyan-CH is obtained under the anhydrous and anaerobic condition ₂ -DAPYs (12); wherein, 2- (4-cyanoanilino) -4-chloro-pyrimidine (10) is 1.0 to 1.1equiv, and 2-halogenated benzyl cyanide (11) is 1.0 to 1.1equiv;

(2) The intermediate Cyan-CH ₂ The DAPYs (12) is unstable, and is put into air, and after reaction for 40 to 48 hours at room temperature, the key intermediate oxo methylene substituted diaryl pyrimidine compound (13) is obtained by slow oxidation;

(3) Simultaneously with the preparation of intermediate 13, intermediate 18 is prepared starting from various substituted anilines; the method comprises the following specific steps: substituted anilines (14 a-14 t) were dissolved in tetrahydrofuran while NaHCO was added ₃ Dissolving NaHCO in water ₃ Mixing the aqueous solution with tetrahydrofuran solution of substituted aniline, placing in ice bath, adding phenyl chloroformate (15) after stabilizing the temperature to 0 ℃, completing the reaction quickly, extracting and steaming with ethyl acetate, and obtaining various substituted phenyl carbamate intermediates (16 a-16 t) which exist stably at room temperature; dissolving the substituted phenyl carbamate intermediate (16 a-16 t) in acetonitrile, adding 80% hydrazine hydrate (17), and carrying out ultrasonic room temperature reaction for 1-3 h to obtain corresponding substituted semicarbazide (18 a-18 t); wherein, the substituted aniline (14 a-14 t) is 1.0 to 1.1equiv., naHCO ₃ 1.20 to 1.4equiv., phenyl chloroformate (15) 1.2 to 1.4equiv., substituted phenyl carbamate intermediate (16 a-16 t) 1.0 to 1.1equiv., hydrazine hydrate (17) 2.5 to 3.0equiv.;

(4) Heating, refluxing and dehydrating equivalent oxo methylene substituted diaryl pyrimidine compound intermediate (13) and substituted semicarbazide intermediate (18 a-18 t) in ethanol under the condition of taking hydrochloric acid as a catalyst to obtain a corresponding target compound: ZFIa-ZFIv, 22 total; wherein the intermediate (13) is 1.0-1.1 equiv., and the substituted semicarbazide intermediate (18 a-18 t) is 1.0-1.1 equiv.;

substituted anilines (14 a-14 t), substituted phenylcarbamates (16 a-16 t), substituted semicarbazides (18 a-18 t) in step (3), and the target compound in step (4): ZFIa-ZFIv, corresponding R, X, is specifically as follows:

2. the use of the diaryl pyrimidine derivative containing substituted aryl ureido, which is prepared by the preparation method of claim 1, in preparing medicines for preventing and treating diseases related to abnormal PARP1 expression.

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