Application of alpha, alpha-gem-dihalogenated carbonyl compound in inhibiting activity of SARS-CoV-2 main protease

Abstract

The invention discloses an alpha, alpha-gem-dihalogenated carbonyl compound through analysis of a structure and a Miao compound, and the alpha, alpha-gem-dihalogenated carbonyl compound can be used for inhibiting the activity of SARS-CoV-2 main protease. The invention also provides a pharmaceutical composition and an application of the pharmaceutical composition in inhibiting the activity of the SARS-CoV-2 main protease. The invention makes contributions to promotion of verification of preclinical animal-level curative effect, final clinical drug research and realization of discovery of drugs for inhibiting new coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of α,α-geminal dihalogenated carbonyl compounds in inhibiting the activity of SARS-CoV-2 main protease. Background Art

[0002] The research on the main protease has gone through three stages, namely the structure and lead compound discovery stage, the preclinical animal level efficacy verification stage, and the drug clinical research stage. Structural analysis stage: The team of Academician Rao Zihe of ShanghaiTech University analyzed the crystal structure of compound N3 and the main protease, mainly the crystal structure, N3 is a covalent inhibitor, and warhead is a Michael receptor. The team of Rolf Hilgenfeld of the University of Lübeck analyzed the crystal structure of compound 13b and the main protease. The antiviral activity of the compound is not strong, mainly the crystal complex structure, 13b is a covalent inhibitor, warhead is a ketoamide, and it has been successfully used in HCV protease inhibitors. The team of Liu Hong of the Shanghai Institute of Materia Medica analyzed the crystal structure of compounds 11a and 11b and the main protease. The two compounds have good antiviral activity. In Vero-E6 cells, the EC50 for SARS-CoV-2 is 530nM and 720nM, respectively, and when injected intravenously, mice, rats and beagles show good pharmacokinetic properties. Therefore, it is crucial to find suitable compounds to inhibit the activity of the SARS-CoV-2 main protease. Summary of the invention

[0004] In order to overcome the defects of the prior art, the purpose of the present invention is to provide an α,α-geminal dihalogenated carbonyl compound for use in inhibiting the activity of the main protease of SARS-CoV-2.

[0005] Wherein, the structure of the compound is shown in the following formula (I) and (II):

[0006]

[0007] in,

[0008] Ar is an aromatic ring or a heterocyclic ring (wherein the heterocyclic ring is selected from pyridyl, thienyl, furyl, etc.);

[0009] X is a halogen;

[0010] R 1 is an alkyl group, an aromatic ring, a heterocyclic ring (wherein the heterocyclic ring is selected from pyridyl, thienyl, furyl, etc.), a halogen, a nitro group, a cyano group, a trifluoromethoxy group, an ester group, an amide bond, an alkoxy group, an arylamine, an alkylamine, etc.; and R 1 Link sugars, peptides, amino acids, etc. through ester or amide bonds;

[0011] R 2 is H, an alkyl group, an aromatic ring, or a heterocyclic ring (wherein the heterocyclic ring is selected from pyridyl, thienyl, furyl, etc.).

[0012] Preferably,

[0013] Ar is a benzene ring;

[0014] X is fluorine, chlorine or bromine;

[0015] R 1 is C1-10 alkyl, aromatic ring, heterocyclic ring (wherein the heterocyclic ring is selected from pyridyl, thienyl, furyl, etc.), halogen, nitro, cyano, trifluoromethoxy, ester group, amide bond, C1-10 alkoxy, arylamine, alkylamine, etc.; and R 1 Link sugars, peptides, amino acids, etc. through ester or amide bonds;

[0016] R 2 is H, C1-10 alkyl;

[0017] More preferably,

[0018] Ar is a benzene ring;

[0019] X is fluorine, chlorine or bromine;

[0020] R 1 is methyl, aromatic ring, heterocyclic ring (wherein the heterocyclic ring is selected from pyridyl, thienyl, furyl, etc.), halogen, nitro, cyano, trifluoromethoxy, ester group, amide bond, methoxy, arylamine, alkylamine; and R 1 Link sugars, peptides, amino acids, etc. through ester or amide bonds;

[0021] R 2 It is H, C1-10 alkyl.

[0022] Specifically, the compound structure is as follows:

[0023]

[0024] The present invention also provides a pharmaceutical composition, which comprises an α,α-geminal dihalogenated carbonyl compound as shown in formula (I) or (II) and a pharmaceutically acceptable carrier.

[0025] The present invention also provides the use of the pharmaceutical composition in inhibiting the activity of the main protease of SARS-CoV-2.

[0026] The beneficial effect of the present invention is that the main protease, also called 3CL protease (abbreviated as Mpro or 3CLpro), has received great attention for the new coronavirus. The present invention provides an α,α-geminal dihalogenated carbonyl compound through the analysis of the structure and lead compound, which can be used to inhibit the activity of the main protease of SARS-CoV-2. The present invention contributes to promoting the verification of the efficacy at the preclinical animal level, and ultimately conducting clinical drug research to achieve drug discovery for inhibiting the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Compounds containing halogen warheads, thiophene rings or benzene rings were designed to inhibit the activity of the main protease of SARS-CoV-2. (AE) The half maximum inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 2. (B) Compound 3. (C) Compound 5. (D) Compound 6. (E) Compound 9.

[0028] Figure 2 .Compounds designed with monohalogen and polyhalogen substitutions on the benzene ring of the dibromoacetophenone aromatic ring skeleton for inhibiting the activity of the SARS-CoV-2 main protease. (AR) Maximum half inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 10. (B) Compound 11. (C) Compound 12. (D) Compound 13. (E) Compound 14. (F) Compound 15. (G) Compound 16. (H) Compound 17. (I) Compound 18. (J) Compound 19. (K) Compound 20. (L) Compound 21. (M) Compound 22. (N) Compound 23. (O) Compound 24. (P) Compound 25. (Q) Compound 26. (R) Compound 27.

[0029] Figure 3 .Compounds designed by simple substitution on the benzene ring of the dibromoacetophenone aromatic ring skeleton for inhibiting the activity of the SARS-CoV-2 main protease. (AG) Maximum half inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 28. (B) Compound 30. (C) Compound 31. (D) Compound 33. (E) Compound 34. (F) Compound 35. (G) Compound 36.

[0030] Figure 4 .Compounds with ortho- and meta-substitutions on the benzene ring of the dibromoacetophenone aromatic ring skeleton were designed to inhibit the activity of the SARS-CoV-2 main protease. (AF) Maximum half inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 32. (B) Compound 37. (C) Compound 38. (D) Compound 39. (E) Compound 40. (F) Compound 41.

[0031] Figure 5 .Compounds with amide substitution, aminoacyl substitution or para-amide substitution on the benzene ring of the dibromoacetophenone aromatic ring skeleton were designed to inhibit the activity of the SARS-CoV-2 main protease. (AL) Maximum half inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 42. (B) Compound 43. (C) Compound 45. (D) Compound 46. (E) Compound 47. (F) Compound 48. (G) Compound 49. (H) Compound 50. (I) Compound 51. (J) Compound 52. (K) Compound 53. (L) Compound 54.

[0032] Figure 6 .Compounds with aminoacyl substitution skeletons, sulfonamide skeletons and phosphoramide skeletons were designed on the benzene ring of the dibromoacetophenone aromatic ring skeleton to inhibit the activity of the main protease of SARS-CoV-2. (AF) Maximum half inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 55. (B) Compound 56. (C) Compound 57. (D) Compound 58. (E) Compound 59. (F) Compound 60.

[0033] Figure 7 .Compounds with different substituents designed on the aromatic ring warhead of dibromoacetophenone to inhibit the activity of SARS-CoV-2 main protease. (AF) Maximum half inhibitory concentration (IC50) values ​​were determined by FRET. (A) Compound 61. (B) Compound 62. (C) Compound 63. (D) Compound 64. (E) Compound 65. (F) Compound 66.

[0034] Figure 8 .The interaction relationship between the structure of Mpro-58 complex and other inhibitors, and the geminal dihalogenation reaction mechanism. (A) Comparison of the binding mode of compound 58 to the main protease with the binding mode of other reported inhibitors; the inhibitors are shown as stick graphs. The four secondary sites S1', S1, S2 and S4 are marked. (B) Proposed geminal dihalogenation reaction mechanism. DETAILED DESCRIPTION

[0035] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0036] Example 1 Preparation of the α,α-geminal dihalogenated carbonyl compound represented by the active compound formula (II)

[0037]

[0038] Condition A: NaBr (0.5 mmol, 2.5 equiv.), NaHSO 4. H2O (0.7mmol, 3.5equiv.) and H2O (108.0mg, 30.0equiv.), the reaction solution was stirred for 8 hours under blue LED (6W, 460nm) and air atmosphere, and the reaction was complete when detected by thin layer chromatography. After filtration, the organic phase was concentrated by vacuum distillation. The crude product was purified by silica gel chromatography and eluted with PE:EtOAc to obtain the desired target product.

[0039] Condition B: LiCl (0.6 mmol, 3.0 equiv.), NaHSO 4. H2O (0.8mmol, 4.0equiv.) and H2O (108.0mg, 30.0equiv.), the reaction solution was stirred for 8 hours under blue LED (6W, 460nm) and air atmosphere. The reaction was completely detected by thin layer chromatography. After filtration, the organic phase was concentrated by vacuum distillation. The crude product was purified by silica gel chromatography and eluted with PE:EtOAc to obtain the desired target product.

[0040] The compounds numbered 1-3, 7 in the following table are α-monohalogenated carbonyl compounds represented by formula (I). Some of these compounds are commercially available. For specific synthesis methods, please refer to the Chinese invention patent document with application number: 202011380064.0 and invention name: A class of α, α-geminal dibromocarbonyl compounds and their synthesis methods.

[0041] The compound No. 4 is an α,α-geminal difluorocarbonyl compound, and the compound No. 8 is an acetone compound, which are commercially available. The compounds No. 5-6, 9-66 are the target products prepared in Example 1 of the present invention;

[0042]

[0043]

[0044] Example 2 Cloning, protein expression and purification of SARS-CoV-2 Mpro

[0045] The full-length gene encoding the SARS-CoV-2 main protease (NC_045512) (Note: It is well known and can be queried on the uniprot database) was optimized and synthesized by GeneWeiZ to make it suitable for expression in the E. coli expression system. The optimized gene was cloned into a modified pET-32a vector with an N-terminal thioredoxin tag and a SARS-CoV main protease cleavage site.

[0046] The protein was expressed by competent E. coli BL21 (DE3) cells. The cells were grown at 37°C in LB medium supplemented with 50 μg / mL kanamycin. When OD600 reached 0.6-0.8, the temperature was lowered to 16°C and IPTG was added at a final concentration of 0.2 mg / mL to induce protein expression. After 16 hours of induction, the cells were collected by centrifugation and then frozen at -80°C until needed.

[0047] The cells were resuspended in lysis buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 5% glycerol) and lysed with a high-pressure homogenizer, followed by centrifugation at 25000g for 30 minutes. The supernatant was loaded onto a Ni-NTA affinity column (Qiagen, Germany), then washed with a wash buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 5% glycerol, 20mMimidazole) and eluted with an elution buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 5% glycerol, 300mMimidazole). Concentrate and remove imidazole through a desalting column. Next, human rhinovirus 3C protease was added overnight at 19°C to cleave the His tag at the C-terminus. The next day, another Ni-NTA affinity purification step was performed to remove the remaining His-tagged M pro The flow-through was then loaded onto a Hitrap Q HP (5 mL) ion exchange column pre-equilibrated with 10 mM Tris-HCl pH 8.0, 20 mM NaCl buffer, and the purified protein was then transferred to 10 mM Tris-HCl pH 8.0 buffer and stored at -80°C until needed.

[0048] Example 3 Enzyme activity and inhibition experiments

[0049] The enzyme activity and inhibition assay of the SARS-CoV-2 main protease was performed by injecting recombinant SARS-CoV-2 M pro(final concentration of 40nM) was mixed with the compound prepared in Example 1 of the present invention in 50μL of experimental buffer (20mM Tris, pH 7.3, 150mMNaCl, 1mM EDTA, 1% glycerol, 0.01% twee-20) and incubated for 10 minutes. The fluorescent substrate MCA-AVLQSGFRK(DNP)K (GL Biochem, Shanghai) was added to a final concentration of 20μM. Subsequently, the fluorescence signal was measured at 320nm (excitation wavelength) / 405nm (emission wavelength) using an EnVision multi-mode plate reader (Perkin Elmer, USA) for 8 consecutive time points for 8 minutes. When the protease reaction is carried out in a linear manner, the initial velocity is measured.

[0050] Crystallization, data collection and structure determination

[0051] When the SARS-CoV-2 main protease was concentrated to 6 mg / mL, it was incubated with 0.5 mM inhibitor (compound prepared in the embodiment of the present invention) at 25 ° C for 1 hour. Then, the main protease and the compound prepared in the embodiment of the present invention were mixed with a pool solution containing 0.1 M MES (pH 5.4-5.8), 7% polyethylene glycol (PEG) 6000 and 6% DMSO at a ratio of 1: 1 (v / v) at 20 ° C. Crystals were obtained two days later and stored in liquid nitrogen. The cryoprotectant solution was a pool solution plus 20% glycerol. Diffraction data were then collected at 100K using an Eiger X 16M image flat panel detector or a Pilatus 36M image flat panel detector on the BL17U1 and BL19U1 beamlines of the Shanghai Synchrotron Radiation Facility (SSRF). Data integration and scaling were performed using the XDS program. Molecular replacement (MR) was performed using the PHASER program in the Phenix 1.19.2 software package. Using SARS-CoV-2M pro (PDB: 6LU7) was used as a template. The model obtained from MR was then iterated through manual model adjustment in Coot0.8 and refined using Phenix REFINE. The inhibitor was built based on the crystal diffraction electron density map.

[0052] Table 1

[0053]

[0054]

[0055] Table 2

[0056]

[0057]

[0058] Note: The ID codes in PDB are 8JCJ: main protease-compound 18, 8JCK: main protease-compound 32, 8JCL: main protease-compound 52, 8JCM: main protease-compound 55, 8JCN: main protease-compound 58, 8JCO: main protease-compound 65.

[0059] From the above table data, it can be concluded that the structural model of the inhibitor (the compound prepared in the embodiment of the present invention) and the protein crystal complex is accurately constructed, and the error is within a reasonable range.

Claims

1. Application of α,α-dihalogenated carbonyl compounds in inhibiting the activity of SARS-CoV-2 main protease, It is characterized in that The structures of the α,α-geminal dihalogenated carbonyl compounds are shown in the following formulas (I) and (II): in, Ar is an aromatic ring or a heterocyclic ring; X is a halogen; R 1 is an alkyl group, an aromatic ring, a heterocyclic ring, a halogen, a nitro group, a cyano group, a trifluoromethoxy group, an ester group, an amide bond, an alkoxy group, an arylamine, or an alkylamine; and R 1 Link sugars, peptides, and amino acids through ester or amide bonds; R 2 is H, alkyl, aromatic ring, heterocyclic ring; Wherein, the heterocyclic ring is selected from pyridyl, thienyl and furyl.

2. The use according to claim 1, It is characterized in that Ar is an aromatic ring or a heterocyclic ring; X is fluorine, chlorine or bromine; R 1 is C1-10 alkyl, aromatic ring, heterocyclic ring, halogen, nitro, cyano, trifluoromethoxy, ester group, amide bond, C1-10 alkoxy, arylamine, alkylamine, and R 1 Link sugars, peptides, and amino acids through ester or amide bonds; R 2 is H, C1-10 alkyl; Wherein, the heterocyclic ring is selected from pyridyl, thienyl and furyl.

3. The use according to claim 1, It is characterized in that The structure of the α,α-geminal dihalogenated carbonyl compound is as follows:

4. A pharmaceutical composition, It is characterized in that The invention comprises the α,α-geminal dihalogenated carbonyl compound as claimed in claim 1 and a pharmaceutically acceptable carrier.

5. Use of the pharmaceutical composition as claimed in claim 4 in inhibiting the activity of the main protease of SARS-CoV-2.

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