CN114213395A - Pyrimidone acyl piperazine compound and preparation method and application thereof - Google Patents

Abstract

The invention provides a pyrimidone acyl piperazine compound and a preparation method and application thereof. The compound has a formula IThe structure of (1). The invention also relates to a preparation method of the compound containing the structure of the formula I, a pharmaceutical composition and application of the compound in preparing anti-SARS-CoV-2M^proApplication in medicine.

Description

Pyrimidone acyl piperazine compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of organic compound synthesis and medical application, and particularly relates to a pyrimidinone acyl piperazine compound for inhibiting protease such as novel coronavirus main protease, a composition containing the compound, a method for preparing the compound and a method for treating diseases or disease symptoms caused by coronavirus by using the compound.

Background

The Acute Respiratory Syndrome Coronavirus type 2 (Severe acid Respiratory Syndrome Virus Coronavir 2, SARS-CoV-2) seriously threatens the life and health of people. SARS-CoV-2 is a single-strand positive-strand RNA virus belonging to the genus beta coronavirus, and its replication cycle includes the links of adsorption, fusion, transcription, translation, assembly and release. Compared with the existing coronavirus, SARS-CoV-2 has stronger transmission, longer latent period and more asymptomatic infectors, which greatly increases the risk of spreading the epidemic situation and hinders the effective control of the epidemic situation. New coronary pneumonia or infectious disease that will become a long-term epidemic in the global or local area. However, the existing medicines lack pertinence to treatment of novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) and have poor curative effect. Although many new corona vaccines have been successfully developed, there is still a risk of infection after vaccination, and in addition, the emergence of new variant viruses leads to increased viral transmission and a continuous worsening of the local epidemic, which presents a greater challenge to the control of the epidemic. Therefore, the development of anti-SARS-CoV-2 drugs with clinical application value is not slow.

Proteases play an important role in the critical stages of transcription and translation of the viral replication cycle. SARS-CoV-2 Main protease (M)^pro) Is a protease which is composed of homodimer and takes cysteine as an active center, and the active catalytic site of the protease is composed of His41 and Cys145 (shown in figure 1). Main eggThe white enzyme is used as a common protease with extremely high similarity among coronary virus species, and the catalytic active site is highly conserved. At the same time, there is no M in the human body^proThe homologous protease is specific to the virus, so the target inhibitor has good selectivity and small toxic and side effects, which ensures that M is prepared by the homologous protease^proBecomes an ideal anti-SARS-CoV-2 medicine target.

After the new crown epidemic outbreak, SARS-CoV-2 main protease becomes an important target for drug design, most of the existing main protease inhibitors are peptide-like covalent inhibitors, but the covalent inhibitors have the problem of poor selectivity, not only inhibit the activity of virus main protease, but also inhibit human cysteine cathepsin L and B, and have potential safety hazard. The non-covalent inhibitor has higher selectivity and is an important direction for developing anti-new crown drugs, but the activity of the inhibitor is weaker, so that the finding of the high-activity SARS-CoV-2 main protease non-covalent inhibitor is a current research hotspot.

The pyrimidone acyl piperazine compound 7 (Mculte-5948770040) is a SARS-CoV-2 main protease Inhibitor (IC) with a new framework type discovered by high-throughput screening₅₀4.2 μ M), the co-crystal structure shows that compound 7 binds to the host protease non-covalently to inhibit M^proActivity (PDB ID:7 LTJ). However, the further development of the compounds is severely restricted due to lower activity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a pyrimidone acyl piperazine compound for inhibiting a main protease, a composition containing the compound and a preparation method thereof, and also provides the compound as SARS-CoV-2M^proThe screening result of the activity of the inhibitor and the application thereof.

The technical scheme of the invention is as follows:

1. pyrimidone acyl piperazine compounds

A pyrimidone acyl piperazine compound or a pharmaceutically acceptable salt, ester or prodrug thereof has a structure shown in a general formula I:

wherein the content of the first and second substances,

R₁comprises the following steps: various substituted aliphatic amines, aromatic amines, six-membered heterocyclic methylamines or five-membered heterocyclic methylamines; the substituent is selected from methyl, halogenated methyl, halogen or tert-butyloxycarbonyl.

According to a preferred aspect of the invention, R₁Is substituted aniline, substituted benzylamine, substituted phenethylamine, indolylmethylamine, indolylethylamine, biphenylmethylamine, substituted cyclic methylamine, substituted cyclic ethylamine, spiromethylamine, substituted piperidine ring, substituted furanmethylamine, substituted thiophenemethylamine, pyrrolemethylamine, pyridylmethylamine, pyrazolemethylamine, imidazolemethylamine, oxazolemethylamine, isoxazolemethylamine, thiazolemethylamine; the substituent is selected from methyl, halogenated methyl, halogen or tert-butyloxycarbonyl; the compound shown in the formula I is in a racemate configuration, an R configuration or an S configuration.

According to a further preferred embodiment of the invention, R₁Is any one of the following substituents I (a), I (b) or I (c):

n is an integer selected from 0, 1 or 2;

m is an integer selected from 0, 1 or 2, R is independently at each occurrence C₁-C₂Alkyl, phenyl, halogen, hydroxy, cyano, nitro, halomethyl, halophenyl of (a);

m and n are integers selected from 0, 1 or 2;

R₂、R₃is H, C₁-C₂Alkyl, halogen, halomethyl, or cycloalkyl.

n is an integer selected from 0, 1 and 2, X and Y are each-S-, -O-, ═ NH-, or ═ CH-, and at most one is ═ CH-; ar is H, a benzene ring or a 5-6-membered aromatic heterocycle; r₄Is H, methyl or halogen.

According to a further preferred embodiment of the invention, the pyrimidinoacylpiperazine compound is one of the following compounds:

as used herein, "pharmaceutically acceptable salts" means salts of the compounds which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue toxicity, irritation, and allergic response and the like, are commensurate with a reasonable benefit-to-risk ratio, are generally water or oil soluble or dispersible, and are effective for their intended use. Including pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are contemplated herein and are compatible with the chemical nature of the compound of formula I, II. A list of suitable salts is found on pages 1-19 of s.m. berge et al, j.pharm.sci.,1977, 66.

The term "prodrug" as used herein refers to pharmaceutically acceptable derivatives such that the resulting biotransformation product of these derivatives is the active drug as defined for the compound of formula I.

2. Preparation method of pyrimidone acyl piperazine compound

The preparation method of the pyrimidone acyl piperazine compound comprises the following steps: taking 3, 4-dichlorophenylboronic acid 1 and Boc-piperazine formate 2 as starting materials, taking dichloromethane as a reaction solvent, and obtaining an intermediate 3 through a Chan-Lam coupling reaction under the catalysis of copper acetate; then dissolving the intermediate 3 in a proper amount of dichloromethane, and deprotecting with trifluoroacetic acid to obtain an intermediate 4; then condensing the intermediate 4 with orotic acid under the action of a condensing agent HATU to obtain a key intermediate 5; and hydrolyzing the methyl ester of the intermediate 5 by using lithium hydroxide to obtain an intermediate 6, and then carrying out amide condensation on the intermediate 6 and various amines in dichloromethane under the action of HATU to obtain each target product.

The synthetic route is as follows:

reagents and conditions: (i) acetic acid ketone, oxygen, pyridine, dichloromethane, room temperature; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) orotic acid, HATU, N, -diisopropylethylamine, dichloromethane, room temperature; (iv) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (v) various amine compounds, HATU, N, -diisopropylethylamine, dichloromethane, room temperature.

Wherein R is₁As described above in formula I; the room temperature of the invention is 20-30 ℃.

According to the invention, the preferable preparation method of the pyrimidone acyl piperazine compound comprises the following specific steps:

(1) adding 1-tert-butyl-3-methylpiperazine-1, 3 dicarboxylic acid (1) and 3, 4-dichlorophenylboronic acid (2) into dichloromethane, and dissolving the dichloromethane in a stirring manner to obtain a solution which is nearly clear; adding anhydrous copper acetate and 2 equivalents of pyridine into the solution at one time; after the suspension is uniform, reacting for 24 hours under an oxygen atmosphere, and detecting by TLC that the reaction EA/PE is 1:2, v/v; after the reaction is completed, adding water into the system to quench the reaction; separating liquid, repeatedly washing with distilled water until the organic phase is blue-free; washing an organic phase by a saturated sodium chloride solution, separating the organic phase, drying by anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain an intermediate 3 which is a colorless oily liquid;

(2) dissolving the intermediate 3 in dichloromethane in an ice-water bath, and dropwise adding a mixed solution of trifluoroacetic acid and dichloromethane; after the dropwise addition is finished, the reaction is carried out at room temperature; after about 6 hours, concentrating the reaction solution under reduced pressure to obtain a mauve oily substance; after ethyl acetate is added, a large amount of white solid is separated out; filtering, washing the solid with ethyl acetate/petroleum ether, and drying to obtain the trifluoroacetate of the target product intermediate 4;

(3) under ice bath, adding orotic acid and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) into dichloromethane, activating for 20min, adding N, N, -diisopropylethylamine and the intermediate 4 trifluoroacetate, reacting for 20 hours at room temperature, changing the solution to dark yellow, precipitating a solid, and monitoring by TLC; filtering the reaction solution, washing the obtained solid with DCM to be offwhite, and drying to obtain an intermediate 5;

(4) dissolving the intermediate 5 in a mixed solution of methanol and tetrahydrofuran, dripping a lithium hydroxide aqueous solution in an ice bath, reacting at room temperature, stirring for 4 hours, and concentrating part of the organic solvent under reduced pressure; 1M dilute hydrochloric acid solution was added dropwise to the remaining aqueous solution in an ice bath to adjust the pH to 3 or less, and a large amount of milky white solid was precipitated. Filtering, washing and drying to obtain an intermediate 6 which is yellowish powdery solid;

(5) adding the intermediate 6 and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) into dichloromethane, activating for 20min, adding N, N, -diisopropylethylamine and various substituted amines, reacting at room temperature for 12h, and monitoring by TLC; the reaction solution was filtered and the solid was washed with dichloromethane to obtain the final GA series.

3. Target activity and application of pyrimidone acyl piperazine compounds

The invention discloses an activity screening result of pyrimidone acyl piperazine compounds and the first application of the compounds as main protease inhibitors. Experiments prove that the pyrimidone acyl piperazine compound can be used as a main protease inhibitor for preparing anti-coronavirus medicines. The invention also provides application of the compound in preparing anti-coronavirus medicines.

anti-SARS-CoV-2 major protease activity assay of target compound

SARS-CoV-2 main protease target activity test is carried out on a class of pyrimidone acyl piperazine compounds synthesized according to the method, the anti-main protease activity data of the compounds are shown in figure 2, and main protease inhibitor compound 7 and ML188 reported in the literature are taken as positive controls.

The most of the newly synthesized pyrimidone acyl piperazine compounds of the invention show significant main protease inhibitory activity. For example, compounds GA-13, GA-13S, GA-17S, GA-19, GA-25, GA-27, and GA-33, in which GA-17S activity is particularly prominent (IC)₅₀0.38 ± 0.07 μ M), which has approximately 20-fold higher activity of inhibiting the main protease than lead compound 7; in addition, the series of compounds have S configuration activity superior to R configuration as can be seen from GA-13S and GA-28. Therefore, the pyrimidone acyl piperazine compound has further research and development value and can be used as a lead compound for resisting SARS-CoV-2.

The pyrimidone acyl piperazine compound can be used as SARS-CoV-2 main protease inhibitor, in particular to SARS-CoV-2 inhibitor used for preparing anti-new coronavirus drugs.

An anti-neocoronaviruse pharmaceutical composition comprising the pyrimidone acyl piperazine compound and one or more pharmaceutically acceptable carriers or excipients.

The invention provides a pyrimidone acyl piperazine compound with a brand-new structure and a preparation method thereof, and also provides a screening result of the SARS-CoV-2 major protease resistance activity of the compound and the first application of the compound in the field of antivirus. The pyrimidone acyl piperazine compound can be used as SARS-CoV-2 main protease inhibitor and has high application value. Specifically, the invention discovers a main protease inhibitor with higher activity and novel structure through structure optimization, and the main protease inhibitor can be used as a SARS-CoV-2 main protease inhibitor for preparing anti-new coronavirus medicaments.

Drawings

FIG. 1 is M^proThree-dimensional structures and their active center maps;

FIG. 2 shows the inhibition of the host protease by the compounds at a concentration of 10. mu.M;

FIG. 3 shows SARS-CoV-2M^proIs a drug screening schematic diagram of a target point.

Detailed Description

The invention will be understood by the following examples, which are given by way of illustration and are not intended to limit the scope of the invention.

Example 1: preparation of GA-1 to GA-34

1-tert-butyl-3-methylpiperazine-1, 3 dicarboxylic acid (1, 5.0g, 20.5mmol, 1.0eq.) and 3, 4-dichlorophenylboronic acid (2, 7.8g, 41mmol, 2.0eq.) were added to 100mL of dichloromethane and dissolved with stirring to near clarity. To the solution was added anhydrous copper acetate (3.71g, 20.5mmol, 1.0eq.) and pyridine (3.20g, 41mmol, 2.0eq.) in one portion. After the suspension is uniformly suspended, the mixture is reacted for 24 hours under an oxygen atmosphere, and the reaction EA/PE is detected by TLC to be 1:2, v/v. After the reaction is completed for 16h, water is added into the system to quench the reaction. And separating, and repeatedly washing with distilled water until the water phase is blue-free. Washing an organic phase by a saturated sodium chloride solution, separating the organic phase, drying by anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography (EA/PE gradient elution) to obtain an intermediate 3 which is 4.0g of colorless oily liquid with the yield of 51%. ESI-MS of M/z 389.2[ M + H ]]⁺.C₁₇H₂₂Cl₂N₂O₄(388.1).

(2) Intermediate 3(4.0g, 10.3mmol, 1.0eq.) was dissolved in 100mL of dichloromethane under an ice-water bath, and a mixed solution of 5mL of trifluoroacetic acid and 25mL of dichloromethane was added dropwise. After the addition, the reaction mixture was cooled to room temperature. After about 6h, TLC detection was performed, and the reaction solution was concentrated under reduced pressure to obtain a reddish-purple oil. After addition of 50mL of ethyl acetate, a large amount of white solid precipitated. Filtration and washing of the solid with 30mL ethyl acetate 2 times and drying in vacuo. To obtain the target productIntermediate 4 trifluoroacetic acid salt 3.9g, yield 94.2%. ESI-MS of M/z 289.2[ M + H ]]⁺.C₁₂H₁₄Cl₂N₂O₂(288.1).

(3) Under an ice-water bath, adding orotic acid (1.67g, 10.67mmol, 1.1eq.) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 5.5g, 14.55mmol, 1.5eq.) into 100mL dichloromethane, activating for 20min, adding diisopropylethylamine (DIPEA, 3.75g, 29.1mmol, 3.0eq.) and trifluoroacetate salt (3.9g, 9.7mmol, 1.0eq.) of intermediate 4, reacting for 20 hours at room temperature until the solution turns dark yellow, a solid precipitates, and monitoring the reaction completion by TLC; the solid in the reaction solution was filtered by suction, washed with dichloromethane, and dried under vacuum to give 53.2 g of an off-white intermediate in 78.5% yield. ESI-MS of M/z 427.2[ M + H ]]⁺.C₁₇H₁₆Cl₂N₄O₅(426.1).

(4) Intermediate 5(3.2g, 7.5mmol, 1.0eq.) was dissolved in a mixture of 50mL of methanol and 50mL of tetrahydrofuran, and an aqueous solution of lithium hydroxide (1.8g, 75mmol, 10eq.) was added dropwise in an ice bath to react at room temperature, followed by stirring for 4 hours and then concentrating a portion of the organic solvent under reduced pressure. Adding 1M dilute hydrochloric acid solution dropwise into the rest water solution under ice bath to adjust pH to<3, a large amount of milky white solid was precipitated. Filtering, washing with 100mL water for multiple times, and vacuum drying to obtain intermediate 6 about 2.8g with yield of 90.6%, which is yellowish powdery solid with melting point 275-,¹H NMR(400MHz,DMSO-d₆)δ13.16(s,1H),11.27(d,J＝29.1Hz,2H),7.42(d,J＝9.0Hz,1H),7.14(d,J＝2.9Hz,1H),6.91(d,J＝11.9Hz,1H),5.52(s,1H),4.80(d,J＝20.8Hz,1H),4.24(dd,J＝62.0,12.5Hz,1H),3.95–3.46(m,3H),3.02(d,J＝13.4Hz,1H).ESI-MS:m/z 411.1[M-H]^-.C₁₆H₁₄Cl₂N₄O₅(412.03).

(5) adding key intermediate 6(0.1g, 0.24mmol, 1.0eq.) and HATU (0.137g, 0.36mmol, 1.5eq.) into 10mL of dichloromethane, activating for 20min, adding diisopropylethylamine (DIPEA, 0.093g, 0.72mmol, 3.0eq.) and cyclohexylmethylamine (0.03g, 0.264mmol, 1.1eq.), reacting for 12h at room temperature, monitored by TLC; the reaction solution was filtered, and the solid was washed with dichloromethane to obtain the objective compound N- (cyclohexylmethyl) -1- (3, 4-dichlorophenyl) -4- (2, 6-dioxo-1, 2,3, 6-tetrahydropyrimidine-4-formyl) piperazine-2-carboxamide (GA-1). Or dichloromethane (80mL) was dissolved, washed with water (20 mL. times.3), washed with a saturated sodium chloride solution (20mL), dried over anhydrous sodium sulfate, filtered, and subjected to silica gel column chromatography to obtain the objective compound.

The product was a pale yellow solid in 55% yield, mp 211-213 ℃.

¹H NMR(600MHz,DMSO-d₆)δ11.20(s,2H),8.09(dt,J＝28.9,5.9Hz,1H),7.41(d,J＝9.0Hz,1H),7.00(d,J＝18.2Hz,1H),6.82(d,J＝9.1Hz,1H),5.42(d,J＝16.2Hz,1H),4.35(d,J＝39.6Hz,1H),4.06(dd,J＝44.4,13.5Hz,1H),3.86-3.67(m,1H),3.66-3.42(m,3H),3.28-3.15(m,1H),3.01-2.71(m,2H),1.64-1.49(m,5H),1.36-0.98(m,6H),0.77(d,J＝8.5Hz,2H).¹³CNMR(150MHz,DMSO-d₆)δ169.88,164.16,161.64,151.48,149.86,147.17,131.98,130.91,120.02,115.86,114.88,99.40,58.07,47.52,45.29,43.18,41.55,37.77,30.78,26.41,25.86.ESI-MS:m/z 507.3[M-H]^-.C₂₃H₂₇Cl₂N₅O₄(508.40).

The procedure was as above, except that 2-phenylethylamine was used.

The product was a white solid, yield: 72%, melting point 177-.

¹H NMR(600MHz,DMSO-d₆)δ11.37–11.11(m,2H),8.20(d,J＝16.7Hz,1H),7.40(d,J＝9.1Hz,1H),7.27(t,J＝7.4Hz,2H),7.22–7.11(m,3H),7.02(d,J＝2.9Hz,1H),6.76(td,J＝9.7,9.1,2.9Hz,1H),5.52–5.42(m,1H),4.37(d,J＝36.6Hz,1H),4.10(d,J＝13.4Hz,1H),3.74–3.55(m,2H),3.47(d,J＝39.0Hz,2H),3.32(d,J＝10.2Hz,1H),3.18(s,1H),2.66(qt,J＝14.0,7.4Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.7,164.2,161.7,151.5,149.9,147.2,139.8,132.0,130.9,129.1,128.7,126.5,119.9,115.8,114.7,99.4,58.0,47.5,43.1,41.4,40.8,35.3.ESI-MS:m/z 516.1[M+H]⁺.C₂₄H₂₃Cl₂N₅O₄(515.11).

The procedure is as above, except that indole-3-methylamine is used.

The product was an off-white solid, yield: 59%, melting point 242-.

¹H NMR(600MHz,DMSO-d₆)δ11.22(d,J＝37.6Hz,2H),10.90(s,1H),8.37(d,J＝32.0Hz,1H),7.46–7.30(m,3H),7.19(d,J＝28.9Hz,1H),7.07(t,J＝7.0Hz,1H),7.00(dd,J＝11.9,3.0Hz,1H),6.92(t,J＝7.4Hz,1H),6.84–6.73(m,1H),5.47(d,J＝11.4Hz,1H),4.56(dd,J＝14.6,6.4Hz,1H),4.50–4.29(m,2H),4.23–4.04(m,2H),3.71–3.58(m,2H),3.52–3.41(m,1H),3.27–3.11(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ173.00,170.03,164.22,152.62,149.92,146.95,137.76,136.86,132.04,130.88,126.84,124.23,122.08,121.65,119.04,116.69,115.26,114.87,111.89,100.06,47.75,43.24,41.39,34.89.ESI-MS:m/z 563.3[M+Na]⁺.C₂₅H₂₂Cl₂N₆O₄(540.11).

The procedure is as above, except that (4-fluorophenyl) methylamine is used.

The product was a white solid, yield: 66%, melting point 244-.

¹H NMR(600MHz,DMSO-d₆)δ11.22(s,2H),8.69(d,J＝23.7Hz,1H),7.42(d,J＝9.1Hz,1H),7.18(dd,J＝8.4,5.5Hz,2H),7.13–7.00(m,3H),6.83(d,J＝9.2Hz,1H),5.42(d,J＝14.2Hz,1H),4.51–4.31(m,2H),4.09(d,J＝15.6Hz,1H),3.91–3.70(m,1H),3.66–3.45(m,3H),3.29–3.16(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ170.51,169.99,164.28,162.46,161.83,160.85,151.64,149.85,147.38,135.63,132.03,130.95,129.66,129.60,129.51,120.00,116.24,115.85,115.51,115.37,114.83,99.37,58.26,47.36,43.10,42.14,41.50.ESI-MS:m/z 520.2[M+H]⁺.C₂₃H₂₀Cl₂FN₅O₄(519.09).

The procedure was as above, except that 4, 4-difluorocyclohexyl-1-amine was used.

The product was a white solid, yield: 71%, melting point 162-.

¹H NMR(600MHz,DMSO-d₆)δ11.40–11.07(m,2H),8.11(dd,J＝37.5,7.7Hz,1H),7.41(d,J＝9.0Hz,1H),7.01(s,1H),6.80(d,J＝9.0Hz,1H),5.45(s,1H),4.54–4.11(m,2H),3.90–3.67(m,2H),3.67–3.38(m,3H),3.16(t,J＝9.4Hz,1H),1.96(s,2H),1.86(d,J＝15.9Hz,2H),1.70(d,J＝13.4Hz,2H),1.42(d,J＝11.7Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.62,163.34,161.60,151.50,149.91,147.08,132.00,130.95,119.92,115.67,114.65,99.91,57.60,57.10,47.68,45.62,43.68,41.44,31.77,31.61,28.32,27.94.ESI-MS:m/z 530.1[M+H]⁺.C₂₂H₂₃Cl₂F₂N₅O₄(529.11).

The procedure was as above, except that 3, 5-dimethylaniline was used.

The product was a white solid, yield: 73%, melting point 196 ℃ and 198 ℃.

¹H NMR(600MHz,DMSO-d₆)δ11.24(s,1H),11.21(s,1H),9.97(d,J＝126.1Hz,1H),7.43(d,J＝8.9Hz,1H),7.16–7.07(m,3H),6.88(dd,J＝9.0,2.9Hz,1H),6.69(s,1H),5.40(d,J＝73.7Hz,1H),4.67–4.56(m,1H),4.22(dd,J＝81.3,13.6Hz,1H),3.95–3.73(m,1H),3.68(d,J＝12.7Hz,1H),3.61–3.41(m,2H),3.23–3.17(m,1H),2.21(s,6H).¹³C NMR(150MHz,DMSO-d₆)δ161.76,151.53,149.97,147.30,138.08,133.59,130.16,127.41,126.35,118.53,117.91,116.03,114.89,99.49,98.97,58.51,57.42,49.07,47.50,44.32,43.54,43.19,41.43,21.44.ESI-MS:m/z 538.2[M+Na]⁺.C₂₄H₂₃Cl₂N₅O₄(515.11).

The procedure was as above, except that tryptamine was used.

The product was a pale yellow solid, yield: 68%, melting point 143-.

¹H NMR(600MHz,DMSO-d₆)δ11.19(d,J＝11.7Hz,1H),10.79(d,J＝6.8Hz,1H),8.24(q,J＝6.3,5.7Hz,1H),7.52(d,J＝7.9Hz,1H),7.36(d,J＝8.8Hz,1H),7.33(d,J＝8.1Hz,1H),7.12(d,J＝33.6Hz,1H),7.06(t,J＝7.5Hz,1H),7.02(d,J＝3.0Hz,1H),6.96(t,J＝7.4Hz,1H),6.75(dd,J＝18.7,7.7Hz,1H),5.47(d,J＝12.6Hz,1H),4.37(d,J＝39.9Hz,1H),4.16–4.04(m,1H),3.72–3.56(m,2H),3.51(d,J＝9.1Hz,1H),3.39(q,J＝6.9Hz,2H),3.27–3.15(m,2H),2.76(dd,J＝15.9,9.2Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.69,164.22,162.53,151.32,149.88,147.83,136.78,132.00,130.94,127.66,123.11,121.37,119.78,118.69,115.66,114.52,112.12,111.83,99.93,58.20,47.39,42.92,41.55,38.72,25.50.ESI-MS:m/z 553.0[M-H]^-.C₂₆H₂₄Cl₂N₆O₄(554.12).

The procedure was as above, except that 2-cyclohexylethylamine was used.

The product was a white solid, yield: 59%, melting point 222-.

¹H NMR(600MHz,DMSO-d₆)δ11.19(s,2H),8.09–7.98(m,1H),7.41(d,J＝9.1Hz,1H),7.00(d,J＝10.4Hz,1H),6.81(t,J＝9.5Hz,1H),5.43(d,J＝25.6Hz,1H),4.33(d,J＝32.7Hz,1H),4.08–4.02(m,1H),3.85–3.64(m,1H),3.64–3.54(m,2H),3.46(d,J＝12.6Hz,1H),3.26–3.11(m,1H),3.05(tp,J＝13.5,6.7Hz,1H),2.92(dq,J＝12.8,6.5Hz,1H),1.60(t,J＝10.3Hz,5H),1.22(q,J＝7.0Hz,2H),1.08(t,J＝8.5Hz,4H),0.80(t,J＝12.8Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.60,164.15,161.66,151.47,149.89,147.54,147.51,147.16,131.98,130.91,120.31,119.98,116.21,115.84,115.12,114.79,99.40,98.90,58.15,47.47,43.13,41.48,36.82,34.89,33.14,32.85,26.57,26.17,26.10.ESI-MS:m/z 521.9[M+H]⁺.C₂₄H₂₉Cl₂N₅O₄(521.16).

The procedure is as above, except that cyclohexylamine is used.

The product was a white solid, yield: 63%, melting point 196 ℃ and 198 ℃.

¹H NMR(600MHz,DMSO-d₆)δ11.23(s,1H),11.20(s,1H),7.97(dd,J＝29.0,7.9Hz,1H),7.41(d,J＝9.0Hz,1H),7.00(d,J＝9.4Hz,1H),6.79(d,J＝9.2Hz,1H),5.44(d,J＝1.9Hz,1H),4.45–4.12(m,2H),4.08–3.97(m,1H),3.89–3.68(m,1H),3.67–3.53(m,2H),3.52–3.38(m,2H),1.62(d,J＝10.4Hz,4H),1.51(d,J＝14.7Hz,1H),1.26–1.18(m,2H),1.10(q,J＝11.3,10.6Hz,3H).¹³C NMR(150MHz,DMSO-d₆)δ169.15,164.08,161.60,151.46,149.91,147.04,131.98,130.93,119.86,115.64,114.63,99.53,57.61,48.16,47.75,43.20,41.48,32.81,32.34,25.63,24.84.ESI-MS:m/z 494.2[M+H]⁺.C₂₂H₂₅Cl₂N₅O₄(493.13)

The procedure was as above, except that 2- (4-fluorophenyl) ethylamine was used.

The product was an off-white solid, yield: 79%, melting point 184-.

¹H NMR(600MHz,DMSO-d₆)δ11.24(q,J＝13.4,12.1Hz,2H),8.23–8.10(m,1H),7.39(d,J＝9.1Hz,1H),7.23–7.15(m,2H),7.07(t,J＝8.9Hz,2H),7.00(d,J＝3.0Hz,1H),6.74(d,J＝9.1Hz,1H),5.46(d,J＝16.3Hz,1H),4.36(d,J＝29.3Hz,1H),4.10(d,J＝12.6Hz,1H),3.71–3.54(m,2H),3.53–3.38(m,2H),3.32–3.26(m,1H),3.25–3.10(m,2H),2.70–2.59(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.71,164.22,162.12,161.67,160.51,151.52,149.87,147.17,135.89,132.00,130.88,119.87,115.44,115.31,114.67,99.41,98.92,57.97,47.54,43.06,41.36,40.78,34.40.ESI-MS:m/z 534.1[M+H]⁺.C₂₄H₂₂Cl₂FN₅O₄(533.10).

The procedure was as above, except that 2- (4-chlorophenyl) ethylamine was used.

The product was a white solid, yield: 77%, melting point 214-.

¹H NMR(600MHz,DMSO-d₆)δ11.26(s,1H),11.22(d,J＝10.5Hz,1H),8.17(q,J＝5.7Hz,1H),7.39(d,J＝9.0Hz,1H),7.30(d,J＝8.5Hz,2H),7.18(t,J＝9.6Hz,2H),7.00(d,J＝2.9Hz,1H),6.73(t,J＝10.7Hz,1H),5.46(d,J＝20.1Hz,1H),4.35(d,J＝27.1Hz,1H),4.10(d,J＝13.6Hz,1H),3.70–3.54(m,2H),3.54–3.38(m,2H),3.30(d,J＝6.8Hz,1H),3.20–3.09(m,1H),2.72–2.57(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.72,164.24,161.70,151.58,149.86,147.26,138.82,132.00,131.24,130.98,130.90,128.63,119.86,115.77,114.64,99.38,57.95,47.54,43.05,41.35,40.54,34.54.ESI-MS:m/z 550.1[M+H]⁺.C₂₄H₂₂Cl₃N₅O₄(549.07).

The procedure was as above except that 2- (3, 5-dichlorophenyl) ethylamine was used.

The product was a white solid, yield: 70%, melting point 212-.

¹H NMR(600MHz,DMSO-d₆)δ11.23(t,J＝15.0Hz,2H),8.20(dt,J＝20.8,5.8Hz,1H),7.45–7.32(m,2H),7.28(d,J＝29.8Hz,2H),6.99(s,1H),6.72(t,J＝7.0Hz,1H),5.48(d,J＝17.2Hz,1H),4.47–4.29(m,1H),4.10(d,J＝12.0Hz,1H),3.70–3.55(m,2H),3.53–3.46(m,1H),3.43(d,J＝17.9Hz,1H),3.27–3.10(m,2H),2.70(tt,J＝14.6,6.7Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.77,164.25,161.67,151.54,149.84,147.18,144.21,134.28,132.03,130.86,128.00,126.32,119.88,115.74,114.55,99.40,57.96,57.11,47.53,43.04,41.32,34.46.ESI-MS:m/z 584.3[M+H]⁺.C₂₄H₂₁Cl₄N₅O₄(583.03).

The procedure is as above, except that furan-2-methanamine is used.

The product was an off-white solid, yield: 63%, melting point 216-.

¹H NMR(600MHz,DMSO-d₆)δ9.56(d,J＝47.6Hz,2H),7.52(d,J＝25.0Hz,1H),7.38(t,J＝9.7Hz,1H),6.97(d,J＝2.9Hz,1H),6.79(dd,J＝9.1,2.9Hz,1H),6.34(d,J＝15.1Hz,1H),6.14(dd,J＝56.3,3.1Hz,1H),5.07(d,J＝40.8Hz,1H),4.56–4.34(m,2H),4.34–4.14(m,2H),4.10(d,J＝13.1Hz,1H),3.66–3.57(m,1H),3.57–3.50(m,1H),3.47(d,J＝12.0Hz,1H),3.17–2.98(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ170.23,168.36,167.13,159.18,153.00,150.42,142.19,131.86,130.16,119.28,115.55,114.58,110.36,106.75,95.23,93.85,58.45,46.92,43.14,41.18,36.24.ESI-MS:m/z 492.2[M+H]⁺.C₂₁H₁₉Cl₂N₅O₅(491.08).

The procedure was as above, except that 4-phenylbenzylamine was used.

The product was an off-white solid, yield: 68% melting point 266-.

¹H NMR(600MHz,DMSO-d₆)δ11.24(d,J＝24.7Hz,2H),8.76(d,J＝47.1Hz,1H),7.63(d,J＝7.6Hz,2H),7.60–7.51(m,2H),7.45(q,J＝9.4,8.6Hz,3H),7.35(t,J＝7.4Hz,1H),7.23(d,J＝8.0Hz,2H),7.06(dd,J＝16.0,3.0Hz,1H),6.87(t,J＝9.7Hz,1H),5.45(s,1H),4.49(d,J＝19.7Hz,1H),4.37–4.21(m,1H),4.19–4.08(m,2H),3.64(t,J＝12.7Hz,1H),3.58(p,J＝6.7Hz,2H),3.49(d,J＝8.4Hz,1H),3.25(t,J＝9.2Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ170.08,164.25,161.57,151.54,149.89,147.24,140.46,139.31,138.67,132.06,130.99,129.40,128.57,128.25,128.08,127.80,127.05,115.88,114.87,99.45,58.20,53.76,47.50,42.03,12.71.ESI-MS:m/z 578.2[M+H]⁺.C₂₉H₂₅Cl₂N₅O₄(577.13).

The procedure is as above, except that 2-tert-butoxycarbonyl-2-azaspiro [3.3] heptan-6-amine is used.

The product was a white solid, yield: 78%, melting point 268-.

¹H NMR(600MHz,DMSO-d₆)δ11.20(d,J＝39.4Hz,2H),8.39(dd,J＝36.3,7.1Hz,1H),7.40(d,J＝8.8Hz,1H),7.01(d,J＝3.0Hz,1H),6.79(dd,J＝9.0,3.1Hz,1H),5.41(s,1H),4.52–4.24(m,1H),4.11(t,J＝13.3Hz,1H),3.95(hept,J＝7.8Hz,1H),3.77(d,J＝64.6Hz,4H),3.68–3.62(m,1H),3.56(d,J＝9.6Hz,1H),3.46(t,J＝9.3Hz,2H),3.12(t,J＝9.9Hz,1H),2.43–2.30(m,2H),2.11–1.92(m,2H),1.35(s,9H).¹³C NMR(150MHz,DMSO-d₆)δ169.08,164.25,161.83,155.86,151.75,150.01,131.96,130.92,119.83,115.81,114.78,99.43,78.86,57.77,57.08,47.58,45.51,43.44,43.13,41.34,40.76,31.78,28.54.ESI-MS:m/z 607.2[M+H]⁺.C₂₇H₃₂Cl₂N₆O₆(606.18).

The procedure is as above except that 4, 4-difluoropiperidine hydrochloride is used.

The product was a white solid, yield: 78%, melting point 218-.

¹H NMR(600MHz,DMSO-d₆)δ11.29(s,1H),11.22(d,J＝10.9Hz,1H),7.48–7.35(m,1H),7.10(s,1H),6.88(d,J＝9.0Hz,1H),5.37(d,J＝70.1Hz,1H),5.21(d,J＝60.1Hz,1H),4.40(dd,J＝115.4,13.1Hz,1H),3.97–3.85(m,1H),3.81–3.55(m,4H),3.45(d,J＝10.3Hz,1H),3.06(t,J＝14.5Hz,1H),1.90(q,J＝57.1,52.9Hz,4H).¹³C NMR(150MHz,DMSO-d₆)δ169.29,164.08,161.75,151.45,149.69,147.24,132.11,131.18,120.39,116.24,115.10,98.88,53.92,53.50,47.21,45.80,44.35,43.72,43.17,42.11,40.94.ESI-MS:m/z 516.1[M+H]⁺.C₂₁H₂₁Cl₂F₂N₅O₄(515.09).

The procedure is as above, except that thiophene-2-methylamine is used.

The product was a white solid, yield: 78%, melting point 176-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(d,J＝16.6Hz,2H),8.85–8.70(m,1H),7.40(d,J＝9.0Hz,1H),7.36(d,J＝4.8Hz,1H),7.00(d,J＝3.0Hz,1H),6.94–6.91(m,1H),6.89(s,1H),6.83–6.78(m,1H),5.42(d,J＝15.6Hz,1H),4.54(dd,J＝15.4,6.6Hz,1H),4.46–4.36(m,2H),4.26(dd,J＝15.4,5.1Hz,1H),4.15–4.05(m,1H),3.82–3.59(m,2H),3.60–3.44(m,2H).¹³CNMR(150MHz,DMSO-d₆)δ169.32,164.23,161.71,151.08,149.34,147.14,142.56,132.84,130.91,128.30,125.68,125.45,119.91,115.75,114.29,99.91,58.24,47.30,43.07,41.47,37.94.ESI-MS:m/z 508.2[M+H]⁺.C₂₁H₁₉Cl₂N₅O₄S(507.05).

The procedure was as above except that 3, 5-difluoroaniline was used.

The product was a white solid, yield: 69%, melting point 174-.

¹H NMR(600MHz,DMSO-d₆)δ11.17(d,J＝44.7Hz,2H),10.55(d,J＝179.4Hz,1H),7.43(d,J＝8.8Hz,1H),7.26(d,J＝9.1Hz,2H),7.15(d,J＝12.1Hz,1H),6.90(dq,J＝18.4,10.0,9.3Hz,2H),5.36(d,J＝126.9Hz,1H),4.68(t,J＝25.8Hz,1H),4.28(dd,J＝80.4,13.6Hz,1H),3.96–3.74(m,1H),3.73–3.54(m,2H),3.54–3.40(m,2H),3.13(dd,J＝16.0,9.5Hz,1H).¹³CNMR(150MHz,DMSO-d₆)δ170.06,169.26,164.31,163.89,163.67,163.30,162.05,161.66,151.47,150.31,147.38,147.10,132.09,131.02,120.77,119.89,116.23,115.05,103.21,103.04,99.34,99.29,58.53,53.90,47.52,43.36.ESI-MS:m/z 524.4[M+H]⁺.C₂₂H₁₇Cl₂F₂N₅O₄(523.06)

The procedure is as above, except that 4-trifluoromethylaniline is used.

The product was a pale yellow solid, yield: 78%, melting point 220-.

¹H NMR(600MHz,DMSO-d₆)δ11.27(d,J＝53.2Hz,1H),11.17(d,J＝52.1Hz,1H),10.47(d,J＝137.1Hz,1H),7.73(d,J＝8.4Hz,2H),7.64(dd,J＝35.4,8.5Hz,2H),7.44(dd,J＝9.3,4.0Hz,1H),7.15(d,J＝9.2Hz,1H),6.91(s,1H),5.39(d,J＝101.8Hz,1H),4.67(s,1H),4.27(dd,J＝86.9,13.8Hz,1H),3.86(dd,J＝72.4,11.5Hz,1H),3.69(dd,J＝38.8,12.0Hz,1H),3.62–3.44(m,2H),3.17(td,J＝17.6,15.8,9.0Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ169.25,164.17,163.93,161.77,151.52,149.91,146.75,142.30,132.09,131.03,126.33,124.39,120.38,116.21,115.03,99.40,58.59,47.43,43.36,41.29.ESI-MS:m/z 554.1[M-H]^-.C₂₃H₁₈Cl₂F₃N₅O₄(555.07).

The procedure was as above, except that (5-methylthiophene) -2-methylamine was used.

The product was a pale yellow solid, yield: 68%, melting point 208-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(d,J＝17.2Hz,2H),8.71(d,J＝22.0Hz,1H),7.40(d,J＝9.0Hz,1H),6.99(d,J＝8.4Hz,1H),6.81(d,J＝9.1Hz,1H),6.65(d,J＝3.3Hz,1H),6.57(s,1H),5.41(d,J＝15.3Hz,1H),4.44–4.33(m,2H),4.21(ddd,J＝60.6,15.3,5.3Hz,1H),4.08(t,J＝12.5Hz,1H),3.81–3.69(m,1H),3.64(d,J＝12.6Hz,1H),3.56–3.42(m,2H),3.28–3.17(m,1H),2.36(s,3H).¹³C NMR(150MHz,DMSO-d₆)δ170.20,169.11,164.23,161.69,151.52,149.79,147.16,140.09,138.09,132.03,130.91,125.52,125.08,115.82,114.76,99.45,58.59,47.35,42.63,41.88,38.08,15.37.ESI-MS:m/z 522.2[M+H]⁺.C₂₂H₂₁Cl₂N₅O₄S(521.07).

The procedure was as above except that cyclobutylmethylamine was used.

The product was a white solid, yield: 55%, melting point 222-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(d,J＝9.7Hz,2H),8.10(s,1H),7.41(s,1H),6.98(s,1H),6.80(s,1H),5.42(s,1H),4.34(d,J＝46.5Hz,1H),4.06(dd,J＝23.3,13.4Hz,1H),3.90–3.67(m,1H),3.66–3.39(m,3H),3.28–2.86(m,3H),2.34(p,J＝7.5Hz,1H),1.86(qt,J＝7.7,3.3Hz,2H),1.82–1.65(m,2H),1.61–1.48(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ169.98,163.54,161.64,150.89,148.70,146.04,131.99,130.92,119.89,115.66,114.64,99.42,58.11,47.48,44.05,43.05,41.52,35.04,25.43,18.14.ESI-MS:m/z 502.1[M+Na]⁺.C₂₁H₂₃Cl₂N₅O₄(479.11).

The procedure is as above except that cyclobutylamine is used.

The product was a white solid, yield: 64%, melting point 188-.

¹H NMR(600MHz,DMSO-d₆)δ10.86(s,2H),8.37(dd,J＝41.2,7.5Hz,1H),7.36(dd,J＝58.1,9.0Hz,1H),7.01(d,J＝2.9Hz,1H),6.79(dd,J＝9.1,2.9Hz,1H),5.41(s,1H),4.37–4.25(m,1H),4.19–4.05(m,2H),3.83–3.57(m,2H),3.43(d,J＝71.3Hz,2H),3.12(t,J＝11.8Hz,1H),2.10(t,J＝8.2Hz,2H),1.91–1.74(m,2H),1.69–1.49(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ168.74,164.17,161.65,151.55,150.02,147.20,131.97,130.93,119.83,115.80,114.77,99.51,57.65,47.74,44.53,43.16,41.36,30.13,27.86,15.13.ESI-MS:m/z 466.3[M+H]⁺.C₂₀H₂₁Cl₂N₅O₄(465.10).

The procedure was as above, except that cyclopentylamine was used.

The product was a white solid, yield: 71%, melting point 176-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(s,2H),8.05(dd,J＝28.8,7.2Hz,1H),7.41(d,J＝9.0Hz,1H),7.10–6.91(m,1H),6.80(t,J＝11.3Hz,1H),5.44(s,1H),4.49–4.13(m,2H),3.92(q,J＝6.8Hz,1H),3.87–3.67(m,1H),3.63(d,J＝12.3Hz,1H),3.59–3.38(m,2H),1.72(dp,J＝13.1,7.1,6.3Hz,2H),1.58(d,J＝16.8Hz,2H),1.54–1.39(m,3H),1.39–1.23(m,2H).¹³CNMR(150MHz,DMSO-d₆)δ169.42,164.10,161.58,151.49,150.01,147.09,131.98,130.93,115.71,114.70,100.68,99.53,57.49,50.90,43.24,41.46,33.03,32.32,23.90.ESI-MS:m/z 480.1[M+H]⁺.C₂₁H₂₃Cl₂N₅O₄(479.11).

The procedure is as above, except that (5-methylfuran) -2-methanamine is used.

The product was a white solid, yield: 73%, melting point 188-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(d,J＝9.3Hz,2H),8.58(d,J＝30.2Hz,1H),7.41(d,J＝9.0Hz,1H),7.00(t,J＝3.8Hz,1H),6.81(dt,J＝8.9,4.2Hz,1H),6.01(d,J＝3.0Hz,1H),5.94(s,1H),5.42(s,1H),4.43(d,J＝34.5Hz,1H),4.31(dd,J＝15.7,6.2Hz,1H),4.15–3.96(m,2H),3.85–3.67(m,1H),3.67–3.40(m,3H),3.27–3.04(m,1H),2.20(s,3H).¹³C NMR(150MHz,DMSO-d₆)δ169.81,164.24,161.68,151.53,151.19,150.43,149.86,147.17,131.99,130.91,119.90,115.74,114.70,108.00,106.76,99.39,58.09,47.48,43.06,41.45,36.25,13.66.ESI-MS:m/z 506.1[M+H]⁺.C₂₂H₂₁Cl₂N₅O₅(505.09).

The procedure is as above, except that pyrrole-2-methylamine is used.

The product was a pale yellow solid, yield: 73%, melting point 152-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(d,J＝16.0Hz,1H),10.48(d,J＝36.5Hz,1H),8.44(d,J＝37.8Hz,1H),7.40(t,J＝8.0Hz,1H),7.03(d,J＝3.1Hz,1H),6.82(dd,J＝9.2,2.9Hz,1H),6.62(d,J＝6.5Hz,1H),5.94–5.86(m,1H),5.82(d,J＝11.4Hz,1H),5.46(s,1H),4.43(d,J＝29.7Hz,1H),4.33–4.15(m,2H),4.15–4.04(m,2H),3.72–3.46(m,4H).¹³C NMR(150MHz,DMSO-d₆)δ169.65,164.35,161.79,151.63,149.88,147.39,132.59,130.91,127.21,118.68,117.58,115.84,114.02,107.72,106.42,99.37,57.94,47.50,43.10,41.80,41.36.ESI-MS:m/z489.0[M-H]^-.C₂₁H₂₀Cl₂N₆O₄(490.09).

The procedure was as above, except that aniline was used.

The product was a pale yellow solid, yield: 83%, melting point 179-.

¹H NMR(600MHz,DMSO-d₆)δ11.28(d,J＝42.4Hz,1H),11.19(d,J＝36.5Hz,1H),10.10(d,J＝117.8Hz,1H),7.48(d,J＝8.0Hz,2H),7.44(dd,J＝9.2,3.2Hz,1H),7.28(dt,J＝20.6,7.7Hz,2H),7.13(d,J＝7.8Hz,1H),7.06(t,J＝7.4Hz,1H),6.89(d,J＝9.1Hz,1H),5.42(d,J＝71.3Hz,1H),4.62(d,J＝32.5Hz,1H),4.22(dd,J＝85.8,13.5Hz,1H),3.97–3.76(m,1H),3.69(t,J＝13.7Hz,1H),3.62–3.43(m,2H),3.22(t,J＝9.6Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ168.64,164.57,161.77,152.01,150.35,148.24,146.12,137.63,131.02,129.29,129.05,125.17,124.34,120.76,120.32,116.72,114.94,101.33,58.63,47.84,45.66,41.37.ESI-MS:m/z488.0[M+H]⁺.C₂₂H₁₉Cl₂N₅O₄(487.08).

The procedure is as above, except that (4-trifluoromethylcyclohexyl) amine is used.

The product was a white solid, yield: 78%, melting point 168-.

¹H NMR(600MHz,DMSO-d₆)δ11.21(d,J＝12.4Hz,2H),8.09(dd,J＝59.5,6.9Hz,1H),7.41(d,J＝9.1Hz,1H),7.07–6.96(m,1H),6.89–6.80(m,1H),5.43(d,J＝24.0Hz,1H),4.48(d,J＝63.0Hz,1H),4.16(dd,J＝137.5,13.8Hz,2H),3.93–3.69(m,2H),3.69–3.55(m,2H),3.45(dd,J＝11.8,6.6Hz,1H),3.30(s,1H),3.20–3.04(m,1H),1.60(s,4H),1.54–1.36(m,4H).¹³CNMR(150MHz,DMSO-d₆)δ169.89,164.03,161.41,151.47,149.94,147.09,132.04,131.37,115.58,114.66,99.37,98.80,56.60,55.35,48.24,43.95,43.40,41.44,28.30,28.06,19.89.ESI-MS:m/z 562.2[M+H]⁺.C₂₃H₂₄Cl₂F₃N₅O₄(561.12).

The procedure is as above, except that (1H-pyrazol-5-yl) methylamine is used.

The product was a white solid, yield: 58%, melting point 172-.

¹H NMR(600MHz,DMSO-d₆)δ12.56(s,1H),11.27(d,J＝20.7Hz,2H),8.49(s,1H),7.41(d,J＝13.8Hz,1H),7.01(s,1H),6.83(d,J＝9.4Hz,1H),6.01(d,J＝24.9Hz,1H),5.46(s,1H),4.54–4.32(m,2H),4.10(q,J＝13.3,12.4Hz,2H),3.88–3.42(m,4H),3.20(d,J＝9.5Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ171.06,166.06,162.62,152.44,152.31,149.78,142.05,132.00,130.91,129.30,119.69,115.44,114.69,104.21,99.31,56.25,47.39,45.45,43.08,41.13.ESI-MS:m/z 492.3[M+H]⁺.C₂₀H₁₉Cl₂N₇O₄(491.09).

The procedure is as above, except that thiazole-5-methylamine is used.

The product was a white solid, yield: 68%, melting point 218-.

¹H NMR(600MHz,DMSO-d₆)δ10.99(s,2H),9.00(s,1H),8.87(s,1H),7.63(s,1H),7.32(d,J＝9.0Hz,1H),6.92(d,J＝10.4Hz,1H),6.72(d,J＝9.0Hz,1H),5.34(s,1H),4.52(dd,J＝15.3,6.2Hz,1H),4.38(d,J＝25.6Hz,2H),4.26(dd,J＝15.3,5.1Hz,1H),4.11–3.95(m,1H),3.68–3.49(m,2H),3.38(dd,J＝12.4,8.8Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ170.04,165.42,161.86,154.91,148.91,146.42,141.63,137.73,132.62,130.30,125.98,118.51,116.32,114.65,99.46,58.64,47.12,43.01,41.44,35.33.ESI-MS:m/z 509.3[M+H]⁺.C₂₀H₁₈Cl₂N₆O₄S(508.05).

The procedure is as above, except that thiazole-2-methylamine is used.

The product was a white solid, yield: 68%, melting point 161-.

¹H NMR(600MHz,DMSO-d₆)δ11.31–11.19(m,2H),9.08(t,J＝5.7Hz,1H),7.70(d,J＝3.3Hz,1H),7.61(d,J＝3.2Hz,1H),7.43(d,J＝9.0Hz,1H),7.03(d,J＝3.0Hz,1H),6.84(dd,J＝9.1,3.0Hz,1H),5.46(s,1H),4.69(dd,J＝16.2,6.6Hz,1H),4.62–4.45(m,2H),4.37–4.14(m,1H),3.90–3.64(m,2H),3.60–3.45(m,2H),3.30–3.22(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ170.43,169.44,164.25,161.72,152.28,149.74,147.13,143.26,132.04,130.91,120.44,119.53,115.75,114.67,100.62,58.38,49.07,47.22,43.01,41.02.ESI-MS:m/z 509.1[M+H]⁺.C₂₀H₁₈Cl₂N₆O₄S(508.05).

The procedure is as above, except that oxazole-5-methylamine is used.

The product was a pale yellow solid, yield: 73%, melting point 188-.

¹H NMR(600MHz,DMSO-d₆)δ11.16(s,2H),8.85–8.66(m,1H),8.16(s,1H),7.34(d,J＝9.0Hz,1H),6.94(s,1H),6.83(d,J＝6.6Hz,1H),6.73(d,J＝5.8Hz,1H),5.33(d,J＝9.1Hz,1H),4.45–4.31(m,2H),4.15–3.97(m,2H),3.78–3.54(m,2H),3.52–3.34(m,3H),3.12(t,J＝11.5Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ169.31,165.26,162.29,152.00,151.53,149.88,149.56,133.55,130.53,123.64,123.05,119.86,115.72,114.34,99.03,58.19,47.30,43.04,41.41,34.65.ESI-MS:m/z 493.2[M+H⁺].C₂₀H₁₈Cl₂N₆O₅(492.07).

Example 2: preparation of (S) -1- (3, 4-dichlorophenyl) -4- (2, 6-dioxy-1, 2,3, 6-tetrahydropyrimidine-4-carbonyl) -N- (furan-2-ylmethyl) piperazine-2-carboxamide (GA-13S)

The synthetic route is as follows:

reagents and conditions: (i) acetic acid ketone, pyridine, oxygen, dichloromethane, room temperature; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) orotic acid, HATU, diisopropylethylamine, dichloromethane, room temperature; (iv) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (v) furanmethanamine or thiophenemethylamine, HATU, diisopropylethylamine, dichloromethane, room temperature.

The procedure is as in example 1, except that in step 1 (S) -1-tert-butyl-3-methylpiperazine-1, 3-dicarboxylic acid is used; in the step 5, furan methylamine is used;

the product was a white solid, yield: 67%, melting point 216-.

¹H NMR(600MHz,DMSO-d₆)δ9.74–9.36(m,2H),7.52(d,J＝26.3Hz,1H),7.37(dd,J＝11.5,9.0Hz,1H),6.97(d,J＝2.9Hz,1H),6.79(dd,J＝9.1,3.1Hz,1H),6.43–6.28(m,1H),6.14(dd,J＝57.7,3.2Hz,1H),5.06(d,J＝44.5Hz,1H),4.55–4.36(m,2H),4.33–4.04(m,3H),3.66–3.48(m,2H),3.49–3.38(m,1H),3.15–3.02(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ171.29,170.20,167.12,153.04,152.56,150.46,142.38,142.17,131.85,130.04,120.10,119.26,115.16,114.59,110.89,106.88,106.73,95.20,58.43,47.73,43.14,41.19,36.24.ESI-MS:m/z515.1[M+Na]⁺.C₂₁H₁₉Cl₂N₅O₅(491.08)

Example 3: preparation of (S) -1- (3, 4-dichlorophenyl) -4- (2, 6-dioxy-1, 2,3, 6-tetrahydropyrimidine-4-carbonyl) -N- (thien-2-ylmethyl) piperazine-2-carboxamide (GA-17S)

The procedure is as above, except that thienylmethylamine is used.

The product was a pale yellow solid, yield: 69%, melting point 176-.

¹H NMR(600MHz,DMSO-d₆)δ11.29–11.16(m,2H),8.78(dt,J＝19.5,5.8Hz,1H),7.43–7.28(m,2H),7.00(t,J＝3.8Hz,1H),6.96–6.87(m,2H),6.81(dd,J＝9.0,3.3Hz,1H),5.42(d,J＝15.3Hz,1H),4.54(dd,J＝15.4,6.6Hz,1H),4.47–4.37(m,2H),4.26(dd,J＝15.4,5.1Hz,1H),4.10(t,J＝13.8Hz,1H),3.81–3.42(m,4H),3.27–3.17(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ168.94,164.24,161.70,152.35,149.77,147.14,142.56,132.03,130.22,127.11,125.68,124.38,119.28,115.74,114.00,100.84,58.22,48.07,43.06,41.47,37.86.ESI-MS:m/z508.1[M+H]⁺.C₂₁H₁₉Cl₂N₅O₄S(507.05)

Example 4: preparation of (R) -1- (3, 4-dichlorophenyl) -4- (2, 6-dioxy-1, 2,3, 6-tetrahydropyrimidine-4-carbonyl) -N- (furan-2-ylmethyl) piperazine-2-carboxamide (GA-28)

The procedure is as in example 1, except that in step 1, (R) -1-tert-butyl-3-methylpiperazine-1, 3-dicarboxylic acid is used; in the step 5, furan methylamine is used;

the product was a white solid, yield: 78%, melting point 248-.

¹H NMR(600MHz,DMSO-d₆)δ11.22(d,J＝10.3Hz,1H),9.58(s,1H),8.71(dt,J＝54.8,5.7Hz,1H),7.53(s,1H),7.41(d,J＝9.0Hz,1H),7.00(t,J＝3.4Hz,1H),6.82(td,J＝9.0,8.2,2.9Hz,1H),6.38–6.34(m,1H),6.14(d,J＝3.1Hz,1H),5.42(s,1H),4.54–4.44(m,1H),4.36(dd,J＝15.6,6.3Hz,1H),4.24(qd,J＝15.8,5.7Hz,1H),4.15–4.02(m,2H),3.71(dd,J＝13.8,4.3Hz,1H),3.58(h,J＝6.9Hz,4H),3.54–3.41(m,2H),3.20(ddd,J＝13.5,9.7,4.1Hz,1H).¹³CNMR(150MHz,DMSO-d₆)δ169.96,164.24,161.68,151.51,149.86,147.19,142.58,132.01,130.92,119.89,115.72,114.69,110.86,107.17,99.38,98.63,53.72,41.99,18.44,17.22,12.59.ESI-MS:m/z 492.0[M+H]⁺.C₂₁H₁₉Cl₂N₅O₅(491.08)

Example 5 antipodal host protease (M) of the Compound of interest^pro) Inhibition experiment of

The experimental principle is as follows:

the Fluorescence Resonance Energy Transfer (FRET) method is adopted, and the substrate structure is as follows: MCA-AVLQSGFR-Lys (Dnp) -Lys-NH₂Wherein MCA is a fluorescence donor, Dnp is a fluorescence acceptor or a fluorescence quenching group, the complete sequence contains a fluorescent group and a fluorescence quenching group, and the fluorescent group does not generate fluorescence due to the inhibition effect of the quenching group because the two groups are close in space. When SARS-CoV-2 main protease M is added^proThereafter, since the main protease cleaves between amino acids Q and S so that the fluorescent group is away from the quencher group, fluorescence having a wavelength of 405nm is emitted at an excitation light of 320nm, and M is detected by measuring the fluorescence^proAnd thus the inhibitory activity of the indirectly reactive compound (see figure 3) (Dai Wenhao,et al.,Science.368(6497):1331-1335,2020.Qiao Jingxin,et al.,Science.371(6536):1374-1378,2021.)。

the experimental method comprises the following steps:

the inhibitory activity of the target compound against the main protease was tested using fluorescence resonance energy transfer method. Using MCA-AVLQSGFR-Lys (Dnp) -Lys-NH₂Is used as a reaction substrate. Under the condition of keeping out of the light, 1.5 mu M of SARS-CoV-2M was added^proAdding 500 mu M of substrate and 10 mu M of compound into a 96-well plate for primary screening, incubating for 10 minutes at 37 ℃, detecting the fluorescence intensity of each group by using a multifunctional microplate reader, wherein the excitation wavelength is 320nm, the emission wavelength is 405nm, and the fluorescence intensity is obtained by detecting every 10s for 10 min. First, the fluorescence intensity value is converted into the increase of fluorescence intensity per unit time from the standard curve. Taking the data of the first minute to obtain the rate, and expressing the inhibition degree of the inhibitor on the enzyme activity by the change of the initial reaction rate to study the enzyme inhibition, wherein the initial reaction rate of the blank control is V₀After addition of the inhibitor, it is V_iThen, the degree of inhibition of enzyme activity can be expressed by the following equation: i% ((1-V))_i)/V₀X 100%. The experiment was divided into a blank control group, a positive control group and an experimental group. Compound 7 was used as the positive control group for the experiment, and compounds with an inhibition rate exceeding that of the positive control at a concentration of 10. mu.M were rescreened. The results of the preliminary screening of the experiment are shown in the attached figure 2.

Re-screening: selecting 1.5. mu.M SARS-CoV-2M^proIC of compound tested at 500. mu.M substrate and four concentration gradients (0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M)₅₀. Each group is provided with 3 multiple holes, incubation is carried out for 10 minutes at 37 ℃, the fluorescence intensity of each group is detected by using a multifunctional microplate reader, the excitation wavelength is 320nm, the emission wavelength is 405nm, the fluorescence intensity is detected every 10s for 10 minutes, and the fluorescence intensity is obtained. Finally, IC is carried out by utilizing GraphPad Prism 5 according to the inhibition rate under different concentrations₅₀And (4) calculating. The results of the experiment are shown in table 1.

TABLE 1 rescreening results of inhibition of SARS-CoV-2 major protease by target compounds (pyrimidinone acyl piperazines)

^aIC₅₀(μ M) the concentration of the desired compound at which 50% inhibition of the enzyme is achieved, i.e.the half inhibitory concentration; ML 188: a SARS-CoV-2 major protease inhibitor has been reported as a positive control.

And (4) experimental conclusion analysis:

the most of the newly synthesized pyrimidone acyl piperazine compounds of the invention show significant main protease inhibitory activity. Compounds GA-13, GA-13S, GA-17S, GA-19, GA-25, GA-27, GA-33. The activity of the compound is better than that of lead compound 7 and another known SARS-CoV-2 main protease inhibitor ML188, wherein the activity of GA-17S is especially outstanding (IC)₅₀0.38 ± 0.07 μ M), which inhibits IC of the main protease₅₀The value is improved by nearly 20 times compared with that of the lead compound 7; in addition, the series of compounds have S configuration activity superior to R configuration as can be seen from GA-13S and GA-28. Therefore, the pyrimidone acyl piperazine compound has further development value.

Claims (8)

1. A pyrimidone acyl piperazine compound or a pharmaceutically acceptable salt, ester or prodrug thereof has a structure shown in a general formula I:

wherein the content of the first and second substances,

R₁comprises the following steps: various substituted aliphatic amines, aromatic amines, six-membered heterocyclic methylamines or five-membered heterocyclic methylamines; the substituent is selected from methyl, halogenated methyl, halogen or tert-butyloxycarbonyl.

2. Pyrimidinoacylpiperazines as claimed in claim 1, R₁Is any one of the following substituents:

R₁is substituted aniline, substituted benzylamine, substituted phenethylamine, indolylmethylamine, indolylethylamine, biphenylmethylamine, substituted cyclic methylamine, substituted cyclic ethylamine, spirocyclomethylamineAmine, substituted piperidine ring, substituted furanmethanamine, substituted thiophenemethylamine, pyrrolemethylamine, pyridylmethylamine, pyrazolylmethylamine, imidazolemethylamine, oxazolemethylamine, isoxazolemethylamine, thiazolemethylamine; the substituent is selected from methyl, halogenated methyl, halogen or tert-butyloxycarbonyl; the compound shown in the formula I is in a racemate configuration, an R configuration or an S configuration.

3. The pyrimidinone acyl piperazine compound according to claim 2, R₁Is any one of the following substituents:

R₁is any one of the following substituents I (a), I (b) or I (c):

n is an integer selected from 0, 1 or 2;

m is an integer selected from 0, 1 or 2, R is independently at each occurrence C₁-C₂Alkyl, phenyl, halogen, hydroxy, cyano, nitro, halomethyl, halophenyl of (a);

m and n are integers selected from 0, 1 or 2;

R₂、R₃is H, C₁-C₂Alkyl, halogen, halomethyl, or cycloalkyl of (a);

n is an integer selected from 0, 1 and 2, X and Y are each-S-, -O-, ═ NH-, or ═ CH-, and at most one is ═ CH-; ar is H, a benzene ring or a 5-6-membered aromatic heterocycle; r₄Is H, methyl or halogen.

4. A pyrimidinone acyl piperazine compound according to claim 3, which is one of the compounds having the following structure:

5. the process for preparing pyrimidinone acyl piperazines compound according to claim 1, comprising the steps of:

taking 3, 4-dichlorophenylboronic acid 1 and Boc-piperazine formate 2 as starting materials, taking dichloromethane as a reaction solvent, and obtaining an intermediate 3 through a Chan-Lam coupling reaction under the catalysis of copper acetate; then dissolving the intermediate 3 in a proper amount of dichloromethane, and deprotecting with trifluoroacetic acid to obtain an intermediate 4; then condensing the intermediate 4 with orotic acid under the action of a condensing agent HATU to obtain a key intermediate 5; hydrolyzing the intermediate 5 with lithium hydroxide to obtain an intermediate 6, and then carrying out amide condensation on the intermediate 6 and various amines in dichloromethane under the action of HATU to obtain each target product;

the synthetic route is as follows:

reagents and conditions: (i) acetic acid ketone, oxygen, pyridine, dichloromethane, room temperature; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) orotic acid, HATU, N, -diisopropylethylamine, dichloromethane, room temperature; (iv) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (v) various amine compounds, HATU, N, -diisopropylethylamine, dichloromethane, room temperature;

wherein R is₁As described in formula I in claim 1 above; the room temperature is 20-30 ℃.

6. Use of pyrimidone acyl piperazine compound of any one of claims 1 to 4 in preparation of anti-SARS-CoV-2M^proThe use of (1) in medicine.

7. An anti-coronavirus pharmaceutical composition comprising a pyrimidinone acyl piperazine compound according to any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers or excipients.

8. The coronavirus of claim 7 is SARS-CoV-2.

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