A polysubstituted piperazine compound with a chiral center and its preparation method and application

Abstract

A polysubstituted piperazine compound with a chiral center and a preparation method and application thereof, relating to the field of medical technology; the structure of the compound is general formula (I): (I) A polysubstituted piperazine compound with a chiral center of the present invention can be used for targeting SARS-CoV-2 Mpro non-peptide non-covalent inhibitors, has a good inhibitory effect on SARS-CoV-2 and its mutants Omicron BA.2 and XBB.1, etc., has high activity and low cytotoxicity, and is suitable for preparing anti-SARS-CoV-2 virus drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a polysubstituted piperazine compound with a chiral center, a preparation method and an application thereof. Background Art

[0002] Mpro is a cysteine ​​protease that is responsible for cleaving the polyprotein PP1a / b together with PLpro to produce 16 non-structural proteins responsible for viral replication, of which Mpro is responsible for cleaving and producing 12 of them, playing an indispensable role in the replication of the virus. Mpro functions in the form of a homodimer, and its structure consists of monomers A and B. After the dimer is activated, it performs catalytic functions in a specific manner. Each of its monomers consists of three domains, namely domain I (amino acids 8-101), domain II (amino acids 102-184), and domain III (amino acids 201-303). Previous studies have revealed that the main proteases of almost all coronaviruses share substrate binding regions, indicating that Mpro is highly conserved. In addition, the protein's cleavage of the substrate involves the Leu-Gln (Ser, Ala, Gly) sequence, which is a common and unique characteristic sequence of coronaviruses, and there is no protease in the human body that cleaves this substrate. Therefore, it is possible to develop highly selective and broad-spectrum anti-COVID-19 drugs targeting this target, so Mpro has always been a potential target for the development of small molecule drugs against the new coronavirus.

[0003] For inhibitors of SARS-CoV-2 Mpro, most of the drugs on the market are peptidomimetic covalent inhibitors, such as namatevir and sennotervir. These inhibitors need to be used in combination with CYP inhibitors (ritonavir) to improve pharmacokinetic parameters, which poses a risk of drug-drug interaction and limits the population of applications. In addition, as the virus mutates, in vitro passage experiments have shown that namatevir has developed drug resistance. Among non-covalent inhibitors, only enstevir has been approved for emergency marketing in Japan, but its effectiveness in reducing severe illness and mortality has not yet been announced. Currently in the clinical research stage, such as FB2001 and PF-00835231, they are peptidomimetic covalent inhibitors with a single structure and severe homogeneity. Non-covalent inhibitors usually have better drugability and do not need to be used in combination with CYP inhibitors. In view of the recurrence of new coronavirus disease, potential drug resistance, and technical reserves for future coronaviruses, it is of great significance to develop new non-peptide-like non-covalent anti-new coronavirus drugs.

[0004]

[0005] Pyridoylpiperazine compounds GC-14 (IC 50 = 0.4 μM, EC 50=1.1μM) is a new type of SARS-CoV-2 non-peptide main protease inhibitor. After further optimization, the substituted nicotinyl piperazine compound GC-78 (IC50=0.22 μM, EC50=0.68 μM) was obtained. The structures are as follows: It has been improved in terms of activity and pharmacokinetic properties, but still needs further optimization (J Med Chem. 2022;65(19):13343-13364; J Med Chem. 2023;66(23):16426-16440.) in order to obtain non-peptide mimetic and non-covalent anti-COVID-19 drugs with the potential to become candidate drugs. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a polysubstituted piperazine compound having a chiral center, the structure of which is shown in the general formula (I), which can be used as an inhibitor of SARS-CoV-2 Mpro and has the characteristics of high activity and low cytotoxicity.

[0007] A second object of the present invention is to provide a method for preparing the above compound.

[0008] A third object of the present invention is to provide a pharmaceutical composition.

[0009] A fourth object of the present invention is to provide the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of an Mpro enzyme inhibitor.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable salt in the preparation of anti-coronavirus drugs

[0011] The purpose of the present invention is achieved by the following technical solutions:

[0012] The present invention provides a compound of general formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, solvate, hydrate, polymorph or isotopic variant thereof, and a mixture thereof:

[0013] (I) wherein R is selected from a benzylamine group, a C3-C8 fatty amine group, or a 3-7 membered heterocyclic amine group which is optionally substituted by R*;

[0014] The R* is selected from aryl, C1-C 10 Alkyl, X, -R'OR'', -R'OOR'', and -R'C(O)NR'';

[0015] Said X is selected from F and Cl;

[0016] Said R' is selected from C1-C6 alkyl;

[0017] The R'' is selected from H, C1-C8 alkyl, 3-8 membered heterocyclic group and 3-8 membered halogenated heterocyclic group.

[0018] In some embodiments, the compound of formula (I), or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, solvate, hydrate, polymorph or isotopic variant, and mixtures thereof, is characterized in that the structure of R is ; Wherein, R1 is selected from H, C1-C6 alkyl;

[0019] R2 is selected from H, C1-C8 alkyl, -R2'OR2'', -R2'OOR2'' and -R2'C(O)NR2'';

[0020] Said R2' is selected from C1-C6 alkyl;

[0021] The R2'' is selected from H, OH, C1-C6 alkyl, 3-8 membered heterocyclic group and 3-8 membered halogenated heterocyclic group;

[0022] R3 is selected from C1-C8 alkyl, phenyl and its derivatives, 3-8 membered heterocyclic group containing N and / or S atoms and its derivatives; the derivatives are wherein one or more hydrogen atoms are replaced by fluorine atoms, chlorine atoms, aromatic groups, hydroxyl groups, amino groups, -OR3'; and R3' is selected from C1-C6 alkyl.

[0023] Among them, R1 and R2, R2 and R3, or R1 and R3 can be connected via a saturated or unsaturated CC bond.

[0024] In some embodiments, the compound of formula (I), or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, solvate, hydrate, polymorph or isotopic variant, and mixtures thereof, is characterized in that R is selected from:

[0025] .

[0026] In some embodiments, the compound comprises the general formula (II): (II) wherein R1* is selected from H, C1-C4 chain alkyl, C3-C6 cycloalkyl, -R 1-1 OR 1-2 and -R 1-1 OOR 1-2 ; said R 1-1 is selected from C1-C3 alkyl; said R 1-2 Selected from H, C1-C4 alkyl, 3-8 membered haloalkyl.

[0027] In some embodiments, the compound comprises the general formula (III): (III) Among them, R 2’ , R 2’’ R is independently selected from H, C1-C4 alkyl and C3-C6 cycloalkyl; or 2’ and R 2’’ The C3-C6 cycloalkyl group is formed by connection.

[0028] In some embodiments, the compound comprises the general formula (IV): Wherein, R3* is selected from C1-C5 alkyl or C1-C5 alkyl in which one or more hydrogen atoms are replaced by hydroxyl, chlorine or fluorine atoms.

[0029] In some embodiments, the compound comprises the general formula (V-1) or (V-2): (Ⅴ-1) (V-2) wherein R4* and R5* are independently selected from H, C1-C5 alkyl;

[0030] R6* is selected from C1-C5 alkyl, or C1-C5 alkyl in which one or more hydrogen atoms are replaced by hydroxyl group, chlorine atom or fluorine atom.

[0031] In some embodiments, the compound comprises the general formula (VI): (VI) wherein R7 is selected from C1-C5 alkyl, phenyl and its derivatives, 3-8 membered heterocyclic group containing N and / or S atoms and its derivatives and ; The derivative is one in which one or more hydrogen atoms are replaced by fluorine atoms, chlorine atoms, aromatic groups, hydroxyl groups, amino groups, -OR3'; and R3' and **R are independently selected from C1-C4 alkyl groups.

[0032] In some embodiments, the compound comprises the general formula (VII-1) or (VII-2): (VII-1) (VII-2) wherein R8 and R9 are independently selected from H, C1-C5 alkyl and -R8'OR8'';

[0033] The R8' is selected from C1-C3 alkyl; the R8'' is selected from H, C1-C4 alkyl, 3-8 membered haloalkyl;

[0034] Among them, R 10 Selected from C1-C5 alkyl, C1-C5 haloalkyl, 3-6 membered heterocyclic group containing O, S or N.

[0035] In some embodiments, the compound comprises the general formula (VIII-1) or (VIII-2): (VIII-1) (VIII-2) In the general formula (VIII-1), R 11 Selected from F and Cl, R 12 is selected from H, F and Cl; or R 11 and R 12 Connect to form C3-C6 cycloalkyl, C3-C6 halogenated cycloalkyl; and the C3-C6 cycloalkyl, C3-C6 halogenated cycloalkyl can be selected from saturated or unsaturated structures;

[0036] R 13 Selected from H, C1-C4 alkyl, C1-C4 haloalkyl;

[0037] In the general formula (VIII-2), R 11 is selected from F and Cl; R 11 ' and R 11 '' is independently selected from H, C1-C4 alkyl, C1-C4 haloalkyl.

[0038] In some embodiments, the compound of formula (I) is selected from:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] The present invention provides a method for preparing a compound, and the synthetic route thereof comprises:

[0049]

[0050] Preferably, when the target product is A1-A6, B1-B3, C1-C4, D1-D4, E2-E3, E5-E24, the preparation method of the compound, see synthetic route A, comprises the following steps:

[0051] Using 3,4-dichlorophenylboronic acid 1 and Boc-piperazinecarboxylic acid methyl ester 2 as starting materials and dichloromethane as the reaction solvent, an intermediate 3 is obtained by a Chan-Lam coupling reaction under the catalysis of copper acetate; then the Boc protecting group is removed by trifluoroacetic acid to obtain an intermediate 4; under the action of HATU, intermediate 4 undergoes an amide condensation reaction with 4-methoxynicotinic acid in DMF to obtain an intermediate 5; intermediate 5 is hydrolyzed with lithium hydroxide to obtain an intermediate 6; under the action of HATU, intermediate 6 undergoes an amide condensation reaction with various amine compounds in DMF to obtain the target products A1-A6, B1-B3, C1-C4, D1-D4, E2-E3, and E5-E24.

[0052] Synthesis route A includes:

[0053] Reagents and conditions: (i) copper acetate, oxygen, pyridine, dichloromethane, room temperature; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) 4-methoxynicotinic acid, HATU, DIEA, DMF, room temperature; (iv) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (v) various amine compounds, HATU, DIEA, DMF, room temperature.

[0054] In some embodiments, the preparation method of the compound, see synthesis route B, comprises the following steps: using compound A6 synthesized in route A as a raw material, hydrolyzing with lithium hydroxide to obtain intermediate 7; intermediate 7 is reacted with various amine compounds in DMF under the action of HATU through amide condensation reaction to obtain target products F1-F10.

[0055] Synthesis route B:

[0056] Among them, R2 * Selected from -NR2 ** ; said R2 ** Selected from H, OH, methoxy, C1-C6 alkyl, 3-8 membered heterocyclic group and 3-8 membered halogenated heterocyclic group.

[0057] The present invention provides a method for preparing a compound, and its synthetic route comprises:

[0058] Preferably, the preparation method of the compound, as shown in Synthesis Route C, comprises the following steps: 3,4-dichlorophenylboronic acid 1 and (S)-Boc-piperazinecarboxylic acid 8 are used as starting materials, dichloromethane is used as the reaction solvent, and an intermediate 9 is obtained by a Chan-Lam coupling reaction under the catalysis of copper acetate; intermediate 9 is reacted with various amine derivatives (containing R G Amine compounds of the structure, R is RG When the target compound is selected from compound G1-G7), intermediates 10a-10h are obtained by amide condensation reaction in DMF; intermediates 10a-10h are deprotected by trifluoroacetic acid to obtain intermediates 11a-11h; intermediates 11a-11h undergo amide condensation reaction with 4-methoxynicotinic acid in DMF under the action of HATU to obtain final products G1-G7.

[0059] Synthesis route C includes:

[0060] The present invention provides a pharmaceutical composition, which contains the compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0061] The present invention provides an application of the compound or a pharmaceutically acceptable salt thereof in preparing an Mpro enzyme inhibitor.

[0062] The present invention provides an application of the compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-coronavirus drug.

[0063] Preferably, the coronavirus is a new coronavirus (SARS-CoV-2).

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The polysubstituted piperazine compound with a chiral center of the present invention can be used for targeting SARS-CoV-2Mpro non-peptide non-covalent inhibitors, has a good inhibitory effect on SARS-CoV-2 and its mutants Omicron BA.2 and XBB.1, has high activity and low cytotoxicity, and is suitable for preparing anti-SARS-CoV-2 virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is the high resolution mass spectrum (HRMS) of compound G1 in Example 3.

[0067] Figure 2 This is the high performance liquid chromatography (HPLC) chart of compound G1 in Example 3.

[0068] Figure 3 The figure is a curve diagram of the enzyme level inhibitory activity (IC50 nM) of various compounds in Example 4.

[0069] Figure 4 is the in vitro antiviral activity (EC 50 μM) graph.

[0070] Figure 5 The figure shows the cytotoxicity (CC50 μM) of various compounds in Example 5 on Vero E6. DETAILED DESCRIPTION

[0071] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0072] The "pharmaceutically acceptable salt" described in the present invention refers to a salt of a compound that is suitable for contact with human or lower animal tissues without undue toxicity, irritation, and allergic reaction within a reliable pharmaceutical evaluation range, has a fairly reasonable benefit-risk ratio, is usually water- or oil-soluble or dispersible, and can be effectively used for its intended purpose. It includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which can be used for the intended purpose and are compatible with the chemical properties of the compound of formula I. For a list of suitable salts, see SM Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.

[0073] More preferred salt forms include inorganic acid salts such as hydrochloride, sulfate or phosphate, and organic acid salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate or acetate.

[0074] The term "hydrate" refers to a compound that is combined with water. Generally, the ratio of the number of water molecules contained in the hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Therefore, the hydrate of a compound can be represented by, for example, the general formula R·xH2O, where R is the compound, and x is a number greater than 0. A given compound can form more than one type of hydrate, including, for example, a monohydrate (x is 1), a lower hydrate (x is a number greater than 0 and less than 1, for example, a hemihydrate (R·0.5H2O)) and a polyhydrate (x is a number greater than 1, for example, a dihydrate (R·2H2O) and a hexahydrate (R·6H2O)).

[0075] The compounds of the present invention can be in amorphous or crystalline form (polymorph). In addition, the compounds of the present invention can exist in one or more crystalline forms. Therefore, the present invention includes all amorphous or crystalline forms of the compounds of the present invention within its scope. The term "polymorph" refers to the crystalline form (or its salt, hydrate or solvate) of a compound with a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, photoelectric properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures and other factors may lead to a crystalline form dominating. The various polymorphs of a compound can be prepared by crystallization under different conditions.

[0076] The pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the composition of the present invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water or electrolytes. Example

[0077] A method for preparing a compound comprises the following steps:

[0078]

[0079] (1) 3,4-Dichlorophenylboronic acid 1 (5.87 g, 30.75 mmol, 1.5 eq), Boc-piperazinecarboxylic acid methyl ester 2 (5.00 g, 20.5 mmol, 1.0 eq), and copper acetate (3.71 g, 20.5 mmol, 1.0 eq) were added to a dry reaction bottle, and 100 mL of dichloromethane was injected as a solvent. Then, pyridine (3.20 g, 41 mmol, 2.0 eq) was added. The mixture was stirred at room temperature for 24 hours under oxygen-filled conditions. The reaction was monitored by TLC. After the reaction was complete, water was injected into the reaction solution to quench the reaction. Then, dichloromethane was used for extraction. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The organic phase was separated and purified by column chromatography to obtain intermediate 3. The elution phase was PE:EA=10:1 to 1:1, and the intermediate 3 was a colorless oily liquid of 3.1 g with a yield of 39.0%. The structural characterization is shown in Table 1.

[0080] Table 1

[0081] (2) Intermediate 3 (1 g, 2.58 mmol, 1 eq) was dissolved in 10 mL of dichloromethane, and 2.5 mL of trifluoroacetic acid was added dropwise to the reaction solution. The reaction was stirred at room temperature for 6 hours and monitored by TLC. After the reaction was completed, the crude oily liquid was obtained by concentration under reduced pressure. Subsequently, 5 mL of ethyl acetate was added to precipitate a solid. The solid was washed with petroleum ether and filtered to obtain intermediate 4 as a white solid (0.664 g, with a yield of 81%). The solid was directly used in the next step without further purification. The structural characterization is shown in Table 2.

[0082] Table 2

[0083]

[0084]

[0085] (3) Intermediate 4 (1 g, 3.47 mmol, 1 eq), 4-methoxynicotinic acid (0.58 g, 3.82 mmol, 1.1 eq), and HATU (1.98 g, 5.20 mmol, 1.5 eq) were added to 15 mL of DMF, and then DIEA (1.12 g, 8.68 mmol, 2.5 eq) was added thereto. The reaction was allowed to react at room temperature for 6 h. The reaction was monitored by TLC. After the reaction was completed, 15 mL of water was added to the reaction solution to quench the reaction. The reaction was extracted with ethyl acetate (20 mL×3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. The organic phase was separated and purified by column chromatography to obtain intermediate 5 as a white solid, 1.17 g, with a yield of 80%. The elution phase was EA:methanol = 1:0-10:1. The structural characterization is shown in Table 3.

[0086] Table 3

[0087]

[0088] (4) Intermediate 5 (1 g, 2.36 mmol, 1 eq) was placed in a reaction flask, and 10 mL of a 1:1 mixed solvent of tetrahydrofuran:methanol was added for dissolution. Subsequently, lithium hydroxide (0.227 g, 9.44 mmol, 4 eq) was added and stirred at room temperature for 3 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was neutralized with 1 M HCl solution to pH 7, extracted with EA (20 mL×5), and the organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was concentrated by vacuum distillation to obtain intermediate 6 as a white solid, 0.868 g, with a yield of 90%; the structural characterization is shown in Table 4.

[0089] Table 4

[0090]

[0091] (5) Intermediate 6 (0.1 g, 0.244 mmol, 1 eq), (S)-α-phenylethylamine (0.036 g, 0.293 mmol, 1.2 eq), and HATU (0.139 g, 0.366 mmol, 1.5 eq) were placed in a reaction flask, 1 mL of DMF was added to dissolve, and DIEA (0.079 g, 0.61 mmol, 2.5 eq) was subsequently added. The reaction was allowed to react at room temperature for 6 h and monitored by TLC. After the reaction was completed, 10 mL of water was added to the reaction solution for quenching, and the mixture was extracted with ethyl acetate (15 mL×3). The organic phase was collected and washed with saturated brine. The organic phase was concentrated by vacuum distillation and purified by column chromatography to obtain the target product A1 as a white solid, 0.1 g, with a yield of 80%.

[0092] The (S)-α-phenylethylamine in step (5) was replaced with various amine compounds, and the compounds synthesized by the route of Example 1 were shown in Table 5:

[0093] Table 5

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] Example 2

[0113] A method for preparing a compound comprises the following steps:

[0114] (1) Compound A6 (1 g, 1.75 mmol, 1 eq) prepared in Example 1 was placed in a reaction flask, and 10 mL of a 1:1 mixed solvent of tetrahydrofuran:methanol was added for dissolution. Subsequently, lithium hydroxide (0.168 g, 7.00 mmol, 4 eq) was added and stirred at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was neutralized with 1 M HCl solution to a pH of 7, extracted with EA (15 mL×5), and the organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was concentrated by distillation under reduced pressure to obtain intermediate 7, 0.865 g, with a yield of 90%, as a white solid, which was directly subjected to the next step of reaction without purification.

[0115] (2) Intermediate 7 (0.1 g, 0.180 mmol, 1 eq), methylamine hydrochloride (CH3NH2·HCl) (0.015 g, 0.216 mmol, 1.2 eq), and HATU (0.103 g, 0.27 mmol, 1.5 eq) were added to a reaction flask, and 1 mL of DMF was added for dissolution. Subsequently, DIEA (0.058 g, 0.45 mmol, 2.5 eq) was added and stirred at room temperature for 6 hours. The reaction was monitored by TLC. After the reaction was completed, water was added to the reaction solution to quench the reaction. The reaction was extracted with ethyl acetate (10 mL×3). The organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was concentrated by distillation under reduced pressure and separated and purified by column chromatography to obtain the target product F1, 0.075 g, with a yield of 74%, as a white solid. The elution phase was EA:methanol = 1:0-10:1.

[0116] The methylamine hydrochloride in step (2) is replaced by various amine compounds, and the compounds synthesized by the route of Example 2 are shown in Table 6:

[0117] Table 6

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Example 3

[0125] A method for preparing a compound comprises the following steps:

[0126] (1) 3,4-Dichlorophenylboronic acid 1 (2.5 g, 13.04 mmol, 1.5 eq), (S)-Boc-piperazinecarboxylic acid 8 (2 g, 8.70 mmol, 1 eq), and copper acetate (3.2 g, 17.4 mmol, 2 eq) were added to a dry reaction bottle, and 30 mL of dichloromethane was injected as a solvent. Then, pyridine (1.4 g, 17.4 mmol, 2 eq) was added. The reaction was stirred at room temperature for 24 hours under the condition of full oxygen. The reaction was monitored by TLC. After the reaction was complete, water was injected into the reaction solution to quench the reaction. Then, dichloromethane (30 mL×3) was used for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. Column chromatography was performed to separate and purify the intermediate 9, which was a colorless oily liquid, 0.488 g, with a yield of 15%. The elution phase was PE:EA=1:1 to 1:3.

[0127] (2) Intermediate 9 (0.1 g, 0.267 mmol, 1 eq), (S)-1-(2-fluorophenyl)ethylamine (0.045 g, 0.321 mmol, 1.2 eq) and HATU (0.152 g, 0.401 mmol, 1.5 eq) were placed in a reaction flask, 1 mL of DMF solvent was added for dissolution, and DIEA (0.086 g, 0.668 mmol, 2.5 eq) was subsequently added. The mixture was stirred at room temperature for 6 h and the reaction was monitored by TLC. After the reaction was completed, water was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (10 mL×3), the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. The intermediate 10a was separated and purified by column chromatography to obtain 0.11 g of white solid with a yield of 83%. The elution phase was PE:EA=1:2-1:3. The structural characterization is shown in Table 7.

[0128] Table 7

[0129]

[0130] (3) Intermediate 10a (0.1 g, 0.202 mmol, 1 eq) was placed in a reaction bottle, 5 mL of dichloromethane was added, and then 1 mL of trifluoroacetic acid was added. The reaction was stirred at room temperature for 6 hours and monitored by TLC. After the reaction was completed, the crude product was concentrated under reduced pressure and washed with petroleum ether to obtain intermediate 11a, 0.076 g, yield 95%, as a white solid. The structural characterization is shown in Table 8. The product was directly used in the next step without further purification.

[0131] Table 8

[0132]

[0133] (4) Intermediate 11a (0.05 g, 0.127 mmol, 1 eq), 4-methoxynicotinic acid (0.023 g, 0.152 mmol, 1.2 eq), and HATU (0.072 g, 0.191 mmol, 1.5 eq) were placed in a reaction flask, 1 mL of DMF was added for dissolution, and DIEA (0.041 g, 0.318 mmol, 2.5 eq) was subsequently added. The mixture was stirred at room temperature for 6 hours and the reaction was monitored by TLC. After the reaction was completed, water was added to the reaction solution to quench the reaction, and ethyl acetate (10 mL×3) was used for extraction. The organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was concentrated by vacuum distillation and purified by column chromatography to obtain the target product G1, 0.056 g, with a yield of 83%, as a white solid. The elution phase was EA:methanol = 1:0-10:1. Its high resolution mass spectrum (HRMS) is shown below: Figure 1 As shown, the high performance liquid chromatography (HPLC) diagram is Figure 2 As shown, the purity is 96.79%.

[0134] The (S)-1-(2-fluorophenyl)ethylamine in step (2) was replaced with various amine compounds, and the compounds synthesized by the route of Example 3 were shown in Table 9:

[0135] Table 9

[0136]

[0137]

[0138]

[0139]

[0140] Example 4

[0141] Inhibition experiment of compounds on main protease (Mpro)

[0142] Experimental principle: The fluorescence resonance energy transfer (FRET) method is used. The substrate structure is: MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2, where MCA is a fluorescence donor and Dnp is a fluorescence acceptor or fluorescence quencher. The complete sequence contains a fluorescent group and a fluorescent quencher. If the two groups are close in space, the inhibitory effect of the quencher will prevent the fluorescent group from producing fluorescence. When the substrate is added to the SARS-CoV-2 main protease Mpro, the main protease can cut between the amino acid residues Q and S, making the fluorescent group away from the quencher, and emitting fluorescence with a wavelength of 405 nm under an excitation light of 320 nm. The activity of Mpro is detected by measuring the fluorescence, which reflects the inhibitory activity of the compound.

[0143] Experimental method: Recombinant SARS-CoV-2 Mpro (final concentration of 750 nm) was mixed with each compound of Examples 1-3 and GC-78 and serial dilutions of Nirmatrelvir in 25 μL of assay buffer (20 nMTris-HCl, pH 7.5, 150 mM NaCl, 1 nM EDTA, 2 mM DTT) and incubated for 10 minutes. The reaction was initiated by adding 25 μL of fluorescent substrate (MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2) at a final concentration of 20 μM, and the fluorescence signal at 320 nm (excitation) / 405 nm (emission) was measured using a microplate reader. The Vmax of the reaction with different concentrations of the compound and the Vmax of the reaction with DMSO were calculated and used to generate the IC 50 All experimental data were analyzed using GraphPad Prism software. 50 (nM): The concentration of compound required to inhibit the enzyme by 50%, i.e. half inhibitory concentration; the experimental results are shown in Table 10.

[0144] Table 10 Enzyme level activity of target compounds

[0145]

[0146]

[0147] The results show that the polysubstituted piperazine compound containing a chiral center synthesized by the present invention exhibits significant main protease inhibitory activity.

[0148] like Figure 3As shown, the activities of compounds G1, G2, G3, G4, G5, G6, and G7 are better than those of the lead compound GC-78, and the inhibitory activity against the main protease is 1-6 times higher than that of GC-78, among which the activities of G1, G2, G4, and G5 are comparable to those of the marketed drug Nirmatrelvir. Therefore, the polysubstituted piperazine compounds containing a chiral center of the present invention are worth further research and development.

[0149] Evaluation of anti-SARS-CoV-2 activity and cytotoxicity of compounds at the cellular level

[0150] Anti-SARS-CoV-2 activity detection experimental method: Vero E6 cells were plated at 1×10 4 The cells were plated in 96-well plates at 100 μl per well. After overnight attachment, the supernatant was removed and 100 μl of the compound diluted 3-fold in DMEM medium containing 2% fetal bovine serum (FBS) was added to the cell wells. Then, an equal volume of diluted SARS-CoV-2 virus-infected cells (Moi = 0.01) were added. After 4 days of continuous culture, the CPE of the cells was detected using Celltiter-Glo (Promega) reagent, and the cell survival rate was calculated. Graphpad Prism 8 was used to plot and calculate the EC 50 Values. Among them, a: SARS-CoV-2 delta infected Vero E6 cells, added P-gP inhibitor;

[0151] b: SARS-CoV-2 WT, infected Vero E6 cells, without addition of P-gP inhibitor;

[0152] c: SARS-CoV-2 WT, infected Vero E6 cells, with the addition of P-gP inhibitor.

[0153] The experimental results are shown in Tables 11, 12 and Figure 4-5 .

[0154] Compound toxicity test method: Vero E6 cells were plated at 1×10 4 / well of a 96-well plate, after overnight attachment, the supernatant was removed, 100 μl of the compound diluted 3 times with DMEM medium containing 2% fetal bovine serum (FBS) was added to the cell wells, and then an equal volume of DMEM medium containing 2% fetal bovine serum (FBS) was added. After 4 days of continuous culture, the cell CPE was detected with Celltiter-Glo (Promega), the cell survival rate was calculated, and Graphpad Prism 8 was used to plot and calculate CC 50 The experimental results are shown in Table 11.

[0155] BEAS-2B / A549 cells in the logarithmic growth phase were cultured at 2.7×103 / well in a 96-well plate, after overnight attachment, the supernatant was removed, and 200 μl of compound diluted 2-fold in DMEM medium containing 10% fetal bovine serum (FBS) was added to the cell wells. After 48 hours of compound treatment, 20 μL of CCK8 was added to each well. The absorbance value was measured at 450 nm using a thermo scientific MultiskanFC microplate reader. Graphpad prism 8 was used to draw a graph and calculate CC 50 The experimental results are shown in Table 11.

[0156] Table 11 In vitro antiviral activity and cytotoxicity of compounds

[0157]

[0158] Table 12 Antiviral activity of compounds against novel coronavirus mutant strains

[0159]

[0160] The results showed that the representative compounds G1, G2, G4, and G5 of the present invention exhibited good antiviral activity at the cellular level, and the EC 50 The EC values ​​of compounds G1 and G5 against SARS-CoV-2 WT were between 0.325 and 0.779 μM. 50 In the absence of P-gP inhibitors, the EC of G1 50 It remained at 0.678 μM, while the positive control Nirmatrelvir decreased by about 100 times, and the EC 50 The compounds G1 and G5 also have good inhibitory effects on SARS-CoV-2 mutant strains Omicron BA.2 and XBB.1, and their EC 50 Between 0.258-0.389 μM.

[0161] We used Vero E6, BEAS-2B, and A549 cells to test the cytotoxicity of G1, G2, G4, and G5. 50 The values ​​are shown in Table 11, except for CC of G1 and G2 on Vero E6 50 The values ​​were 26.08 and 17.05 μM, and the rest of CC 50 All of them were above 50 μM and had low cytotoxicity.

[0162] In summary, the polysubstituted piperazine compounds containing a chiral center of the present invention are non-peptide-like non-covalent inhibitors targeting SARS-CoV-2Mpro, have significantly improved activity compared to the lead compound GC-78, and have lower cytotoxicity. Compared with the marketed peptidomimetic covalent drugs such as Nirmatrelvir, they have potential advantages in pharmacokinetic properties and safety, and can be further developed as lead compounds against SARS-CoV-2.

[0163] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A compound of general formula (I'), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, polymorph or isotopic variant thereof, and a mixture thereof, characterized in that: (I’) Wherein, R1 is selected from H, C1-C6 alkyl; R2 is selected from C1-C8 alkyl, -R2'OR2'', -R2'OOR2'' and -R2'C(O)NR2''; Said R2' is selected from C1-C6 alkyl; The R2'' is selected from H, C1-C6 alkyl, 3-8 membered heterocyclic group and 3-8 membered halogenated heterocyclic group; R3 is selected from C1-C8 alkyl, phenyl and its derivatives, 3-8 membered heterocyclic group containing N and / or S atoms and its derivatives; the derivatives are those in which one or more hydrogen atoms are replaced by fluorine atoms, chlorine atoms, hydroxyl groups, amino groups, -OR3'; and R3' is selected from C1-C6 alkyl.

2. A piperazine compound, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, polymorph or isotopic variant thereof, and a mixture thereof: any one of the compounds of the following structures; (Ⅱ) (Ⅵ) (Ⅶ-1) (Ⅶ-2) (Ⅷ-1) (Ⅷ-2)； in, In the general formula (II), R1* is selected from C1-C4 chain alkyl, C3-C6 cycloalkyl, -R 1-1 OR 1-2 and -R 1-1 OOR 1-2 ; said R 1-1 is selected from C1-C3 alkyl; said R 1-2 Selected from H, C1-C4 alkyl; In the general formula (VI), R7 is selected from C1-C5 alkyl, phenyl and its derivatives, 3-8 membered heterocyclic groups containing N and / or S atoms and their derivatives, and ; The derivative is one in which one or more hydrogen atoms are replaced by fluorine atoms, chlorine atoms, hydroxyl groups, amino groups, -OR3'; R3' and **R are independently selected from C1-C4 alkyl groups; In the general formula (VII-1) or (VII-2), R8 and R9 are independently selected from H, C1-C5 alkyl and -R8'OR8''; R8' is selected from C1-C3 alkyl; R8'' is selected from H, C1-C4 alkyl; 10 is selected from C1-C5 alkylene, C1-C5 haloalkylene, and a 3-6 membered heterocyclic group containing O, S or N; In the general formula (VIII-1), R 11 Selected from F and Cl, R 12 is selected from H, F and Cl; or R 11 and R 12 connected to form a C3-C6 cycloalkyl group or a C3-C6 halogenated cycloalkyl group; and the C3-C6 cycloalkyl group or the C3-C6 halogenated cycloalkyl group may be selected from a saturated or unsaturated structure; R 13 Selected from H, C1-C4 alkyl, C1-C4 haloalkyl; In the general formula (VIII-2), R 11 is selected from F and Cl; R 11 ' and R 11 '' is independently selected from H, C1-C4 alkyl, C1-C4 haloalkyl.

3. A compound of formula (III), (IV) or (V-2), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, polymorph or isotopic variant thereof, and a mixture thereof: (Ⅲ) (Ⅳ) (Ⅴ-2) ； in, In the general formula (III), R 2’ , R 2’’ R is independently selected from H, C1-C4 alkyl and C3-C6 cycloalkyl; or 2’ and R 2’’ The C3-C6 cycloalkyl group formed by connection; In the general formula (IV), the R3* is selected from C1-C5 alkylene or C1-C5 alkylene in which one or more hydrogen atoms are replaced by hydroxyl groups, chlorine atoms or fluorine atoms; In the general formula (V-2), R6* is selected from C1-C5 alkylene, or C1-C5 alkylene in which one or more hydrogen atoms are replaced by hydroxyl groups, chlorine atoms or fluorine atoms.

4. A compound of general formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, polymorph or isotopic variant thereof, and a mixture thereof, characterized in that: (I) The R is selected from: .

5. A compound, characterized in that Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

6. A method for preparing the compound according to claim 1, characterized in that: The synthetic route includes: 。 7. A method for preparing the compound according to claim 1, characterized in that: The synthetic route includes: 。 8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of an Mpro enzyme inhibitor.

10. Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of an anti-coronavirus drug.

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