A deuterated piperazine compound and a preparation method and application thereof

Abstract

The application provides a kind of deuterated piperazine compound and its preparation method and application.The compound has the structure shown in formula I.The application also relates to the preparation method of the compound containing formula I structure, pharmaceutical composition and the application of the above-mentioned compound in the preparation of anti-SARS-CoV-2M pro drug.

Description

Technical Field

[0001] This invention relates to a derivative and its preparation method, specifically to a deuterated piperazine compound, a composition containing the compound, the preparation of the compound, and its application in the field of anti-coronavirus drugs, belonging to the field of organic synthesis and pharmaceutical application technology. Background Technology

[0002] SARS-CoV-2 poses a serious threat to human life and health, impacting socio-economic development, and the COVID-19 pandemic is far from over. SARS-CoV-2 mutates rapidly and possesses significant immune evasion capabilities, enabling it to breach the immune barrier formed by vaccines or infection, leading to repeated infections. It is highly likely that COVID-19 will coexist with humans for a long time, much like the influenza virus. Although several drugs are currently available, COVID-19 is highly prone to mutation and rapidly develops drug resistance. The continuous mutation of SARS-CoV-2 and the interspecies similarity of coronaviruses highlight the urgent need to design and develop broad-spectrum anti-drug drugs against drug-resistant coronaviruses. It is necessary to develop anti-coronavirus drugs targeting broadly conserved targets.

[0003] SARS-CoV-2 main protease (M) pro Cys is a homodimeric cysteine ​​protease composed of two mutually perpendicular monomers, whose catalytic center consists of the Cys145 and His41 groups (attached). Figure 1 The main protease plays a crucial role in the viral replication cycle and is a common protease with high similarity among coronavirus species, including SARS-CoV-2 and SARS-CoV-2 M. pro It exhibits 96% sequence homology. Furthermore, M... pro The high degree of conservation in its sequence and structure makes it an ideal target for the development of anti-coronavirus drugs. To date, several M... pro Inhibitors have been reported successively, mainly including peptide-mimicking compounds, such as the currently marketed peptide-mimicking M. pro Covalent inhibitors include nelmatvir, cenutvir, and lerretvir; non-peptide, non-covalent inhibitors include encetevir and GC-78 (discovered by the applicant). Additionally, FB2001, currently in clinical trials, is also a peptide-like covalent inhibitor, but these inhibitors have a simple structure and are highly homogeneous. Furthermore, due to the high mutagenicity of the novel coronavirus and increased drug use, SARS-CoV-2 has rapidly developed drug resistance, and nelmatvir-resistant strains (E166V / T21I and E166V / L50F double mutant strains) have already appeared clinically. Therefore, there is an urgent need to discover anti-coronavirus drugs with unique backbones and broad-spectrum anti-resistance properties, while also preparing drug reserves and technical support for potential future outbreaks.

[0004]

[0005] Dichlorophenylpiperazine compound GC-78 (IC 50 =0.46μM, EC 50 =0.89μM) is a novel non-peptide main protease inhibitor of SARS-CoV-2 with a novel backbone; however, its activity is still inferior to that of the marketed drug nematvir, and its pharmacokinetic properties are poor (t 1 / 2 =1.64h; F<20%), further optimization and improvement are needed. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a deuterated piperazine compound, a composition comprising the compound, and a method for preparing the same. This invention also provides the above compound as a SARS-CoV-2 malignant material. pro Inhibitor activity screening results and their applications.

[0007] The technical solution of the present invention is as follows:

[0008] 1. Deuterated piperazine compounds

[0009] Deuterated piperazine compounds, or pharmaceutically acceptable salts thereof, have the structure shown in general formula I:

[0010]

[0011] in,

[0012] R1 and R2 are: independently methoxy, deuterated methoxy, or methylthio;

[0013] The compound shown in Formula I has an S configuration.

[0014] Pharmaceutically acceptable salt types include inorganic acid salts such as commonly used hydrochloride, sulfate, or phosphate, as well as organic acid salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate, or acetate.

[0015] According to the present invention, the deuterated piperazine compound is one of the following compounds:

[0016]

[0017] The term "pharmaceutically acceptable salt" as used in this invention refers to a salt of a compound that, within the scope of reliable pharmaceutical evaluation, is suitable for contact with tissues of humans or lower animals without undue toxicity, irritation, or allergic reactions, possesses a reasonably reasonable benefit-risk ratio, is typically water- or oil-soluble or dispersible, and is effectively used for its intended purpose. This includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are suitable for the intended use and chemically compatible with compounds of formulas I and II. For a list of suitable salts, see SM Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19. Further preferred salt types are inorganic acid salts such as hydrochlorides, sulfates, or phosphates, and organic acid salts such as methanesulfonates, p-toluenesulfonates, fumarates, citrates, or acetates.

[0018] 2. Preparation methods of deuterated piperazine compounds

[0019] The preparation method of deuterated piperazine compounds includes the following steps: using 3,4-dichlorophenylboronic acid 1 and (S)-Boc-piperazine carboxylate 2 as starting materials, and dichloromethane as the reaction solvent, intermediate 3 is obtained through Chan-Lam coupling reaction catalyzed by copper acetate; then, the methyl ester is hydrolyzed by lithium hydroxide to obtain intermediate 4; subsequently, intermediate 4 is dissolved in an appropriate amount of dichloromethane and condensed with various R1s under the action of condensing agent HATU to obtain intermediate 5; intermediate 5 is then deprotected by trifluoroacetic acid to obtain intermediate 6; then intermediate 6 is amide condensed with various substituted nicotinic acids in dichloromethane under the action of HATU to obtain the target products;

[0020] The synthesis route is as follows:

[0021]

[0022] Reagents and conditions: (i) Acetate, oxygen, pyridine, dichloromethane, room temperature; (ii) Lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (iii) Deuterated p-methoxyaniline or p-methoxyaniline, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; (iv) 2M HCl-dioxane, dichloromethane, room temperature; (v) Deuterated methoxynicotinic acid or methylthionicotinic acid, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature.

[0023] Among them, R1 and R2 are: methoxy, deuterated methoxy and methylthio groups, each independently;

[0024] The room temperature described in this invention is 20-30℃.

[0025] According to a preferred method of the present invention for preparing a deuterated piperazine compound, the specific steps are as follows:

[0026] (1) Add (S)-1-tert-butyl-3-methylpiperazine-1,3-dicarboxylic acid (1) and 3,4-dichlorophenylboronic acid (2) to dichloromethane and dissolve until nearly clear during stirring; add anhydrous copper acetate and 2 equivalents of pyridine to the solution at once; react for 24 h under an oxygen atmosphere and detect the reaction by TLC with ethyl acetate / petroleum ether = 1:2, v / v; quench the reaction with water after the reaction is complete; separate the liquid and wash repeatedly with distilled water until the organic phase is free of blue color; wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter it, concentrate it under reduced pressure, and purify the crude product by silica gel column chromatography to obtain intermediate 3, which is a colorless oily liquid;

[0027] (2) Intermediate 3 was dissolved in a mixture of methanol and tetrahydrofuran. An aqueous solution of lithium hydroxide was added dropwise under an ice bath, and the reaction was carried out at room temperature. After stirring for 4 hours, the solution was concentrated under reduced pressure to remove the organic solvent. 1M dilute hydrochloric acid was added dropwise to the remaining aqueous solution under an ice bath to adjust the pH to below 4, and a large amount of milky white solid precipitated out. After filtration, washing, and drying, intermediate 4 was obtained as a white powder.

[0028] (3) Under ice bath conditions, intermediate 4 and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were added to dichloromethane. After activation for 30 min, N,N'-diisopropylethylamine and deuterated p-methoxyaniline or p-methoxyaniline were added. The mixture was then reacted at room temperature for 20 hours. The reaction was monitored by TLC until it was complete. The reaction was quenched with water. After separation of the dichloromethane phase, the mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate 5.

[0029] (4) Dissolve intermediate 5 in dichloromethane in an ice-water bath, and add a 2M HCl-dioxane and dichloromethane mixture dropwise. After the addition is complete, the reaction is brought to room temperature. After about 6 hours, the reaction solution is concentrated under reduced pressure to obtain a light red oily substance. After adding ethyl acetate, a large amount of white solid precipitates. After filtration, the obtained solid is washed with ethyl acetate / petroleum ether and dried to obtain the hydrochloride salt of intermediate 6.

[0030] (5) Add 4-deuterated methoxynicotinic acid or 4-methylthionicotinic acid and HATU to dichloromethane, activate in an ice-water bath for 20 min, then add N,N,-diisopropylethylamine and the hydrochloride of intermediate 6, react at room temperature for 12 h, and monitor by TLC; after the reaction is completed, add water, and wash the dichloromethane phase obtained by separation with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter and separate by Flash column to obtain the target compound.

[0031] 3. Bioactivity and applications of deuterated piperazine compounds

[0032] This invention discloses the activity screening results of deuterated piperazine compounds and their first application as main protease inhibitors. Experiments demonstrate that the deuterated piperazine compounds of this invention can be used as main protease inhibitors in the preparation of anti-coronavirus drugs. This invention also provides the application of the above compounds in the preparation of anti-coronavirus drugs.

[0033] Experiment on the anti-SARS-CoV-2 main protease activity of the target compound

[0034] The four deuterated piperazine compounds synthesized according to the above method were tested for their SARS-CoV-2 main protease target activity. Their anti-main protease activity data are listed in the appendix. Figure 2 In this study, the lead compound GC-78 and the marketed drug nematvir were used as positive controls.

[0035] The newly synthesized deuterated piperazine compounds of this invention all exhibit excellent main protease inhibitory activity. Among them, GC-78d shows particularly outstanding activity (IC50). 50 =0.21 μM). Screening results for antiviral activity on Vero E6 cells showed that GC-78d exhibited the best activity (EC). 50 =0.25μM), compared to the lead compound GC-78 (EC 50 The concentration (0.89 μM) was increased by 3 times. Therefore, deuterated piperazine compounds can serve as lead compounds for further research and development against SARS-CoV-2.

[0036] One class of deuterated piperazine compounds of the present invention can be used as SARS-CoV-2 main protease inhibitors, specifically, as SARS-CoV-2 inhibitors for the preparation of anti-COVID-19 drugs.

[0037] An anti-COVID-19 pharmaceutical composition comprising the deuterated piperazine compound of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0038] This invention provides novel deuterated piperazine compounds and their preparation methods. It also provides screening results of the compounds' anti-SARS-CoV-2 main protease activity and their first application in the antiviral field. The deuterated piperazine compounds of this invention can be used as SARS-CoV-2 main protease inhibitors and have high application value. Specifically, this invention, through structural optimization, discovered a main protease inhibitor with higher activity and a novel structure, which can be used as a SARS-CoV-2 main protease inhibitor in the preparation of anti-COVID-19 drugs. Attached Figure Description

[0039] Figure 1 It is M pro Three-dimensional structure and its active center diagram;

[0040] Figure 2 It is SARS-CoV-2M pro A schematic diagram illustrating the drug screening principle targeting specific targets. Detailed Implementation

[0041] The following examples help to understand the present invention, but do not limit the scope of the invention. All percentages are mass percentages.

[0042] Example 1: Preparation of representative compounds

[0043]

[0044] (1) (S)-1-tert-butyl-3-methylpiperazine-1,3-dicarboxylic acid (1, 5.0 g, 20.5 mmol, 1.0 eq.) and 3,4-dichlorophenylboronic acid (2, 7.8 g, 41 mmol, 2.0 eq.) were added to 100 mL of dichloromethane and dissolved nearly clearly with stirring. Anhydrous copper acetate (3.71 g, 20.5 mmol, 1.0 eq.) and pyridine (3.20 g, 41 mmol, 2.0 eq.) were added to the solution in one batch. After uniform resuscitation, the mixture was reacted under an oxygen atmosphere for 24 h. The reaction was detected by TLC with an EA / PE ratio of 1:2, v / v. After the reaction was complete for 16 h, water was added to quench the reaction in the system. The mixture was separated and washed repeatedly with distilled water until the aqueous phase was free of blue color. The organic phase was washed with saturated sodium chloride solution, and the separated organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by Flash column chromatography (EA / PE gradient elution) to obtain intermediate 3, which was 4.5 g of a colorless oily liquid, with a yield of 59%. 17 H 22 Cl2N2O4(388.1). 1 HNMR (400MHz, DMSO-d6) δ7.41(d,J=9.0Hz,1H),7.13(s,1H),6.83(dd,J=47.5,8.8Hz,1H),4.91–4.70(m,1H),4.38(d,J=13. 5Hz,1H),3.96(t,J=16.6Hz,1H),3.60(s,3H),3.50(d,J=11.5Hz,1H),3.24–2.89(m,2H),1.40(d,J=1.6Hz,9H).ESI-MS:m / z 389.2[M+H] + .C 17 H 22 Cl2N2O4(388.1).

[0045] (2) Intermediate 3 (4.0 g, 7.5 mmol, 1.0 eq.) was dissolved in a mixture of 50 mL methanol and 50 mL tetrahydrofuran. An aqueous solution of lithium hydroxide (1.57 g, 37.5 mmol, 5.0 eq.) was added dropwise under ice bath conditions. The reaction was carried out at room temperature, and after stirring for 12 h, a portion of the organic solvent was concentrated under reduced pressure. A 1 M dilute hydrochloric acid solution was added dropwise to the remaining aqueous solution under ice bath conditions to adjust the pH to 4, resulting in the precipitation of a large amount of milky white solid. The solid was filtered, washed several times with 100 mL of water, and dried under vacuum to obtain approximately 2.5 g of intermediate 4, with a yield of 89.3%, as a pale yellow powder. ESI-MS: m / z 373.1 [MH] - .C 16 H 20 Cl2N2O4 (374.0).

[0046] (3) Under ice-water bath conditions, intermediate 4 (2.4 g, 6.4 mmol, 1.0 eq.) and HATU (3.65 g, 9.6 mmol, 1.5 eq.) were added to 100 mL of dichloromethane. After activation for 30 min, diisopropylethylamine (DIPEA, 2.5 g, 19.2 mmol, 3.0 eq.) and deuterated methoxyaniline (1.2 g, 9.7 mmol, 1.0 eq.) were added, and the mixture was reacted at room temperature for 20 h. The reaction was monitored by TLC until completion. The reaction was quenched with water, extracted with dichloromethane, washed successively with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by silica gel column chromatography to obtain 2.4 g of the target product, with a yield of 79.1%. ESI-MS: m / z 483.2 [M+H] + .C 21 H 25 Cl2N3O3S(482.1).

[0047] (4) Intermediate 5 (2.45 g, 5.1 mmol, 1.0 eq.) was dissolved in 100 mL of dichloromethane under ice-water bath conditions. A mixture of 6 mL of 2 M HCl-dioxane and 30 mL of dichloromethane was added dropwise. After the addition was complete, the mixture was allowed to react at room temperature. After approximately 5 hours, TLC was used to detect the complete reaction. The reaction mixture was then concentrated under reduced pressure to obtain a pale red oil. Upon addition of 50 mL of ethyl acetate, a large amount of white solid precipitated. The solid was filtered, washed twice with 30 mL of ethyl acetate, and dried under vacuum. 1.7 g of the hydrochloride salt of the target product intermediate 6 was obtained, with a yield of 73.5%. ESI-MS: m / z 383.1 [M+H] + .C 16 H 17 Cl2N3OS(382.1).

[0048] (5) 4-Deuterated methoxynicotinic acid (0.037 g, 0.24 mmol, 1.2 eq.) and HATU (0.114 g, 0.3 mmol, 1.5 eq.) were added to 10 mL of dichloromethane. After activation for 30 min, diisopropylethylamine (DIPEA, 0.103 g, 0.8 mmol, 4.0 eq.) and the hydrochloride salt of key intermediate 6 (0.091 g, 0.2 mmol, 1.0 eq.) were added. The reaction was carried out at room temperature for 12 h, and monitored by TLC. After the reaction was completed, water was added to quench the reaction, and the dichloromethane phase was separated and washed successively with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the product was filtered and separated by silica gel column chromatography to obtain the target compound GC-78d. The product was a white powder with a yield of 58% and a melting point of 138-139 °C.

[0049] 1 HNMR (600MHz, DMSO-d6) δ10.09(s,1H),9.73(s,1H),8.58–8.18(m,2H),7.42(d,J=10.3Hz,2H),7.31(s,1H),7.16(s,1H),7.05(s,1H),6.92–6. 77(m,3H),4.33(s,1H),3.81–3.72(m,1H),3.67(dd,J=8.3,4.1Hz,1H), 3.63–3.57(m,1H),3.54(dd,J=13.6,4.7Hz,1H),3.45(d,J=11.8Hz,1H). 13 C NMR (150MHz, DMSO) δ168.72,165.11,161.40,156.00,152.66,152.46,150.14,132.02,131.04,121.99,121.88,115 .77,115.59,114.65,114.50,114.30,114.18(C×2),107.78,58.73,54.04,45.58,43.29,41.26,40.52.ESI-MS:m / z 521.54[M+H] + .C 25 H 18 D6Cl2N4O4(520.1).HPLC purity:99.43%.

[0050]

[0051] The procedure is the same as above, except that p-methoxyaniline is used in step 3.

[0052] The product is a pale yellow foamy solid with a yield of 45% and a melting point of 115-116℃.

[0053] 1 HNMR(600MHz,DMSO-d6)δ10.09(s,1H),9.73(s,1H),8.60–7.90(m,2H),7.42 (d,J=10.3Hz,2H),7.31(s,1H),7.16(s,1H),7.05(s,1H),6.87(dd,J=19.3,9 .1Hz,3H),4.33(s,1H),3.75(d,J=4.7Hz,1H),3.72(s,3H),3.68(q,J=4.4Hz, 1H), 3.63–3.56 (m, 1H), 3.54 (dd, J=13.6, 4.7Hz, 1H), 3.45 (d, J=12.2Hz, 1H). 13 C NMR (150MHz, DMSO) δ165.23,165.11,161.42,156.00,152.66,152.47,149.95,132.01,131.04,121.99,121.88,115 .79,115.59,114.66,114.50,114.31,114.19(C×2),107.96,55.66,54.03,45.58,43.29,41.26,40.51.ESI-MS:m / z 518.18[M+H] + .C 25 H 21 D3Cl2N4O4(517.1).HPLC purity:98.82%.

[0054]

[0055] The procedure is the same as above, except that 4-methoxynicotinic acid is used in step 5.

[0056] The product is a white, foamy solid with a yield of 67% at 118-119℃.

[0057] 1HNMR(600MHz,DMSO-d6)δ10.09(s,1H),9.73(s,1H),8.55–8.40(m,1H),8.26(d,J=37.9Hz, 1H),7.42(dd,J=9.1,1.5Hz,2H),7.31(s,1H),7.16(s,1H),7.05(s,1H),6.93–6.78(m,3H) ,4.62–4.55(m,1H),4.33(s,1H),3.87(d,J=12.1Hz,1H),3.74(dd,J=13.8,4.4Hz,1H),3.6 7(dd,J=8.5,4.2Hz,1H),3.61(s,3H),3.54(dd,J=13.7,4.7Hz,1H),3.45(d,J=11.8Hz,1H). 13 C NMR (150MHz, DMSO) δ165.23,165.11,161.42,156.00,152.67,152.48,149.96,132.02,131.04,121.99,121.89,115 .77,115.59,114.66,114.50,114.30,114.18(C×2),107.79,54.00,45.58,43.28,42.26,41.26,40.50.ESI-MS:m / z 518.26[M+H] + .C 25 H 21 D3Cl2N4O4(517.1).HPLC purity:99.52%.

[0058]

[0059] The procedure is the same as above, except that 4-methylthionicotinic acid is used in step 5.

[0060] The product is a pale yellow solid, with a yield of 51% at 124-126℃.

[0061] 1HNMR(600MHz,DMSO-d6)δ9.74(s,1H),8.44(dd,J=48.6,5.0Hz,1H),8.31–8.02(m,1H),7.42(d,J=9.1Hz,2H),7.34(d,J=9.2Hz,2H),7.06(d,J=3.0Hz ,1H),6.92–6.78(m,3H),4.87–4.56(m,1H),4.35(d,J=15.9Hz,1H),3.80(s ,1H),3.73–3.54(m,2H),3.48(t,J=19.1Hz,1H),3.30(s,1H),2.47(s,3H). 13 C NMR (150MHz, DMSO) δ195.95,168.37,165.85,156.00,152.73,152.50,150.04,146.48,132.03,131.05(C×2),121.93,121.66 ,119.41,116.44,115.81,114.56,114.27,114.18(C×2),58.55,55.38,45.60,43.32,40.52,13.63.ESI-MS:m / z534.29[M+H] + .C 25 H 21 D3Cl2N4O3S(533.1).HPLC purity:94.9%.

[0062] Example 3: The target compound's reaction with the main protease (M) pro Inhibition experiment

[0063] Experimental principle:

[0064] The fluorescence resonance energy transfer (FRET) method used had a substrate structure of MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2, where MCA is the fluorescent donor and Dnp is the fluorescent acceptor or fluorescence quencher. The complete sequence contains both a fluorescent group and a fluorescence quencher. Due to the close spatial distance between the two groups, the inhibitory effect of the quencher prevents the fluorescent group from fluorescing. When the SARS-CoV-2 main protease M was added... pro Subsequently, because the main protease can cleave between amino acid residues Q and S, the fluorescent group is moved away from the quenching group, emitting fluorescence at a wavelength of 405 nm under excitation light of 320 nm. M is detected by measuring the fluorescence. pro The activity of the compound, and thus indirectly reflects its inhibitory activity (see Appendix). Figure 2 (Dai Wenhao, et al., Science. 368(6497): 1331-1335, 2020. Qiao Jingxin, et al., Science. 371(6536): 1374-1378, 2021.)

[0065] Experimental methods:

[0066] The inhibitory activity of the target compound against the main protease was tested using fluorescence resonance energy transfer (FRET). MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 was used as the reaction substrate. Under light-protected conditions, 1.5 μM of SARS-CoV-2 M... pro 500 μM of substrate and 10 μM of compound were added to 96-well plates for initial screening. The plates were incubated at 37°C for 10 minutes. Fluorescence intensity of each group was detected using a multi-mode microplate reader with excitation wavelength of 320 nm and emission wavelength of 405 nm. Measurements were taken every 10 seconds for 10 minutes to obtain fluorescence intensity. First, the fluorescence intensity values ​​were converted into the increase in fluorescence intensity per unit time based on the standard curve. The data from the first minute was used to obtain the rate. The change in the initial reaction rate represents the degree of inhibition of enzyme activity by the inhibitor. This is used to study enzyme inhibition. The initial reaction rate of the blank control was V0, and the rate after adding the inhibitor was V... i The degree of inhibition of enzyme activity can be expressed by the following equation: i% = (1 - V) i The experiment was divided into a blank control group, a positive control group, and an experimental group. Compounds GC-78 and nematriberi were used as positive control groups. Compounds with inhibition rates exceeding those of the positive control at a concentration of 10 μM were screened again.

[0067] Secondary screening: 1.5 μM SARS-CoV-2M was selected. pro The IC50 of the compound was measured using a substrate of 500 μM and four concentration gradients (0.1 μM, 0.5 μM, 1 μM, 5 μM). 50 Each group was configured with 3 replicates and incubated at 37°C for 10 minutes. The fluorescence intensity of each group was detected using a multi-mode microplate reader with an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Measurements were taken every 10 seconds for 10 minutes. Finally, IC50 analysis was performed using a GraphPad Prism 8 microplate reader based on the inhibition rate at different concentrations. 50 The calculations were performed. The experimental results are shown in Table 1.

[0068] Table 1. Results of secondary screening for inhibition of SARS-CoV-2 main protease by deuterated piperazine compounds

[0069]

[0070]

[0071] a IC 50 (μM): The concentration of the compound required to achieve 50% inhibition of the enzyme, i.e., the half-maximal inhibitory concentration.

[0072] Analysis of experimental conclusions:

[0073] The newly synthesized deuterated piperazine compounds of this invention all exhibit excellent main protease inhibitory activity. The activities of compounds GC-78d, GC-78d-S1, GC-78d-S4, and GC-87 are comparable to those of the lead compound GC-78, with GC-78d showing particularly outstanding activity (IC50). 50 =0.33±0.007μM); therefore, deuterated piperazine compounds have value for further research and development.

[0074] Example 4 Evaluation of cellular-level anti-SARS-CoV-2 activity - Immunofluorescence assay

[0075] Experimental materials:

[0076] SARS-CoV-2 virus strain; Calu-3 cells; test compound; PBS buffer; EMEM 10% fetal bovine serum (FBS); anti-spike protein (S) antibody; goat anti-mouse IgG; positive controls: nematvir, GC-78;

[0077] Experimental methods:

[0078] Calu-3 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells approximately 24 hours before infection. 5 Cell wells. After treatment with the test compound (50 μL / well) in increments, cells were infected with an equal volume of SARS-CoV-2 (final multiplicity of infection MOI 0.01) and cultured at 37°C for 2 days in a biosafety level 3 laboratory. Cells were fixed and infiltrated with acetone / methanol (1:3) at room temperature for 10 min. Viral S protein was detected using an anti-spike protein S antibody (GTX632604; Genetex, USA) and Alexa Fluor 488 conjugated with goat anti-mouse IgG antibody (Invitrogen, USA), while cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI, Invitrogen). Fluorescence images were quantitatively analyzed using the Operetta high-content screening system (PerkinElmer, USA) and the built-in Harmony software. The 50% effective concentration (EC50) was calculated by determining the chemical concentration required to reduce the intensity of the viral S protein-derived green fluorescence by 50%. 50 EC50 The values ​​were generated using curve fitting with Graphpad Prism 8.0. Each sample was measured at least three times in parallel, and the average and standard deviation were taken. The experimental results are shown in Table 2.

[0079] Activity Results and Discussion

[0080] Table 2. Results of intracellular anti-SARS-CoV-2 activity of deuterated piperazine compounds.

[0081]

[0082] a EC 50 (μM): Half-maximal effective concentration.

[0083] Analysis of experimental conclusions:

[0084] Representative compounds with good enzyme inhibitory activity from this invention were selected for anti-SARS-CoV-2 activity testing in Calu-3 cells. GC-78d showed particularly outstanding activity, exhibiting a 3-fold increase in anti-SARS-CoV-2 activity compared to the lead compound GC-78, and superior to the marketed drug nematvir; therefore, deuterated piperazine compounds have further research and development value.

[0085] Example 5: Evaluation of pharmacokinetic activity in rats

[0086] Objective: This study aimed to observe the plasma concentration of the test drug GC-78d in rats after intravenous injection and gavage administration, and to estimate the corresponding pharmacokinetic parameters and absolute bioavailability.

[0087] Methods: Six rats were randomly divided into two groups, receiving GC-78d via intravenous injection and oral gavage, respectively. In the intravenous injection group, blood samples (approximately 0.25 mL) were collected from the jugular vein at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration. In the oral gavage group, blood samples (approximately 0.25 mL) were collected at the same times. The concentration of GC-78d-HCl in rat plasma samples was determined using LC-MS / MS, and pharmacokinetic parameters were calculated using WinNolin software.

[0088] Results: The main pharmacokinetic parameters of rats after a single intravenous injection of 2 mg / kg GC-78d and gavage administration of 10 mg / kg GC-78d are shown in Table 3.

[0089] Table 3. Pharmacokinetic parameters of GC-78d in rats a

[0090]

[0091] a PK parameters (mean±SD, n=3); b Administered by injection at 2 mg / kg; c Administer 10 mg / kg by gavage.

[0092] Analysis of experimental conclusions:

[0093] GC-78d, a representative compound with good antiviral activity in this invention, was selected for pharmacokinetic evaluation in rats. The results showed that compared with the lead compound GC-78, GC-78d exhibited significantly improved pharmacokinetic properties in rats, with the oral half-life extended from 1.7 h to 4.83 h; oral bioavailability increased to 40.7%, fully meeting the requirements for oral formulations. Therefore, the deuteration strategy not only improved antiviral activity but also significantly enhanced pharmacokinetic properties. Deuterated piperazine compounds have further research value.

Claims

1. A deuterated piperazine compound, or a pharmaceutically acceptable salt thereof, characterized in that, is one of the following compounds:

2. The deuterated piperazine compound according to claim 1, wherein The pharmaceutically acceptable salt type is the commonly used hydrochloride, sulfate or phosphate salt, and the methanesulfonate, p-toluenesulfonate, fumarate, citrate or acetate salt.

3. The method for preparing the deuterated piperazine compound according to claim 1 or 2, comprising the following steps: using 3,4-dichlorobenzenboronic acid 1 and (S)-Boc-piperazine formate 2 as starting materials, dichloromethane as a reaction solvent, and copper acetate as a catalyst, an intermediate 3 is obtained by Chan-Lam coupling reaction; then the methyl ester is hydrolyzed by lithium hydroxide to obtain an intermediate 4; then the intermediate 4 is dissolved in an appropriate amount of dichloromethane, and deuterated p-methoxyaniline or p-methoxyaniline is condensed under the action of condensing agent HATU to obtain an intermediate 5; the intermediate 5 is deprotected by trifluoroacetic acid to obtain an intermediate 6; then the intermediate 6 is subjected to amide condensation with deuterated methoxy nicotinic acid 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) lithium hydroxide, methanol, tetrahydrofuran, water, room temperature; (iii) deuterated p-methoxyaniline or p-methoxyaniline, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; (iv) 2M HCl-dioxane, dichloromethane, room temperature; (v) deuterated methoxy nicotinic acid, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; wherein R1 is methoxy and deuterated methoxy.

4. The deuterated piperazine compound of any one of claims 1-2 for use in the preparation of a medicament for the treatment of SARS-CoV-2 Mpro.

5. An anti-coronavirus pharmaceutical composition comprising the deuterated piperazine compound of any one of claims 1-2 and one or more pharmaceutically acceptable carriers.

6. The anti-coronavirus pharmaceutical composition of claim 5, wherein the coronavirus is specifically the novel coronavirus.

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