阅读说明：本技术 含有环丙基骨架的化合物在制备治疗和/或预防冠状病毒感染药物中的应用 (Application of compound containing cyclopropyl skeleton in preparation of medicine for treating and/or preventing coronavirus infection ) 是由 岑山 吕凯 刘明亮 赵建元 于 2021-03-10 设计创作，主要内容包括：本发明涉及含有环丙基骨架的化合物在制备治疗和/或预防冠状病毒感染药物中的应用,该类含有环丙基骨架的化合物为GSK-LSD1·2HCl,ORY-1001·2HCl,DDP-38003·2HCl,OG-L002,体外抗冠状病毒活性和酶活性水平评价显示该类化合物具有较好的抗冠状病毒活性。(The invention relates to application of a compound containing a cyclopropyl skeleton in preparation of a medicine for treating and/or preventing coronavirus infection, wherein the compound containing the cyclopropyl skeleton is GSK-LSD1 & 2HCl, ORY-1001 & 2HCl, DDP-38003 & 2HCl and OG-L002, and evaluation of in vitro coronavirus resistant activity and enzyme activity level shows that the compound has better coronavirus resistant activity.)

1. The application of the compound containing the cyclopropyl skeleton in preparing the medicine for treating and/or preventing the coronavirus infection is characterized in that the compound containing the cyclopropyl skeleton has any one of the following structures:

。

2. use according to claim 1, wherein the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-NL 63.

3. Use according to claim 1, characterized in that the compound comprising a cyclopropyl skeleton is used as the sole active ingredient in the manufacture of a medicament for the treatment and/or prevention of a coronavirus infection; or

The compound containing cyclopropyl skeleton and other medicine are used together as active component in preparing medicine for preventing and/or treating coronavirus infection.

4. Use according to any one of claims 1 to 3, wherein the compound comprising a cyclopropyl skeleton comprises the same and pharmaceutically acceptable salts and esters, solvates, isomers, polymorphs, isotopically labelled compounds, metabolites or prodrugs thereof.

5. The use of claim 4, wherein the pharmaceutically acceptable salts comprise inorganic and organic acid salts.

6. The use according to claim 5, wherein the pharmaceutically acceptable salt is hydrochloric acid.

7. Use according to claim 5, characterized in that the compound comprising a cyclopropyl skeleton is chosen from the following compounds:

。

8. use according to any one of claims 1 to 3, wherein the anti-coronavirus drug comprises at least one pharmaceutically acceptable carrier or excipient.

9. The use according to claim 1, wherein the anti-coronavirus drug is a tablet, capsule, granule, powder, suspension, emulsion, powder, oral liquid, gel, syrup, pill, tincture, medicated wine, electuary, lozenge, mixture, suppository, injection, inhalant or spray.

10. The use according to any one of claims 1 to 3, wherein the coronavirus infection comprises a respiratory infection, a pulmonary infection or a complication caused by a coronavirus.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a compound containing a cyclopropyl skeleton in preparation of a therapeutic and/or prophylactic medicine.

Background

The current outbreak of the novel Coronavirus pneumonia epidemic (SARS-CoV-2) and the SARS epidemic outbreak in 2002 are caused by previously unknown Coronavirus (CoV). Coronavirus can be transmitted through droplets and respiratory tract secretion due to unpredictability, and has serious consequences, and the coronavirus becomes one of the great threats affecting human health.

The SARS-CoV-2 genome is 29.8kb-29.9kb in length and encodes 16 non-structural proteins (nsp1-nsp 16). Some of these 16 nsps are enzymes essential for SARS-CoV-2 replication. Including papain-like protease (nsp3), chymotrypsin-like protease (3CL protease, nsp5), primer-enzyme complex (nsp7-nsp8), RNA-dependent RNA polymerase RdRp (nsp12), helicase (nsp13), and exonuclease (nsp14), which are potential targets for anti-SARS-CoV-2 drug development.

The research is screened by a compound library of the applicant, and a plurality of compounds containing cyclopropyl skeleton structures are found to have excellent anti-coronavirus activity and SARS-CoV-2RdRp inhibiting activity. These compounds are potential drugs for the treatment of new crown infections.

Disclosure of Invention

The invention aims to provide application of a compound containing cyclopropyl skeleton in preparation of anti-coronavirus medicines

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides an application of a compound containing a cyclopropyl skeleton in preparing an anti-coronavirus medicament, wherein the compound containing the cyclopropyl skeleton has any one of the following structures:

as some embodiments of the invention, the coronavirus includes SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL 63.

As some embodiments of the present invention, the use of said compound comprising a cyclopropyl scaffold as the sole active ingredient for the preparation of a medicament for the treatment and/or prevention of a coronavirus infection.

As some embodiments of the present invention, the compound containing a cyclopropyl skeleton is used together with other medicines as an active ingredient for preparing a medicine for treating and/or preventing coronavirus infection.

Based on their effectiveness for coronavirus treatment, they may be used in combination with one or more other active ingredients for the treatment, prevention, inhibition, or amelioration of a disease or condition, wherein the combination of drugs is safer or more effective than either drug alone.

As some embodiments of the invention, the compounds containing a cyclopropyl backbone include the compounds and pharmaceutically acceptable salts and esters, solvates, isomers, polymorphs, isotopically labeled compounds, metabolites or prodrugs thereof.

As some embodiments of the invention, the pharmaceutically acceptable salts include inorganic acid salts and organic acid salts.

The inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid.

The organic acid includes formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, diglucosic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, succinic acid, and the like, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid.

Preferably, the pharmaceutically acceptable salt is hydrochloric acid.

Preferably, the structure is as follows:

as some embodiments of the invention, the anti-coronavirus drug comprises at least one pharmaceutically acceptable carrier or excipient. The preparation can be tablet, capsule, granule, powder, suspension, emulsion, powder, oral liquid, gel, syrup, pill, tincture, medicated wine, soft extract, lozenge, mixture, suppository, injection, inhalant or spray.

As used herein, "pharmaceutically acceptable carrier or excipient" includes: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffers, it will be understood by those skilled in the art that certain pharmaceutically acceptable excipients may be used in more than one function and in alternative functions, depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

For example: when administered orally, it can be formulated into oral preparations such as tablets, capsules, granules, pills, etc., and contains fillers (e.g., saccharide derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose; acacia; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose, polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, calcium sulfate, etc.), disintegrants (e.g., sodium carboxymethyl cellulose, polyvinylpyrrolidone, sodium alginate, sodium, Magnesium stearate, spermaceti, boric acid, sodium benzoate, leucine), stabilizers (methylparaben, propylparaben, etc.), flavoring agents (e.g., conventional sweeteners, acidulants, flavors, etc.). When used parenterally, it may be formulated into injections, including sterile powders for injection and solvents for injection, using carriers or excipients including sterile water, ringer's solution and isotonic sodium chloride solution, and, depending on the nature of the drug, suitable additives such as antioxidants, buffers and bacteriostats. When used for rectal administration, the medicament may be formulated as a suppository or the like. For pulmonary administration, the medicament may be formulated as an inhalant or a spray.

There are many sources available to those skilled in the art which describe pharmaceutically acceptable excipients and which can be used to select suitable pharmaceutically acceptable excipients, for example, books of the pharmaceutical universe of ramiden, the annual book of pharmacy of china, pharmacy, etc.

In the technical scheme of the invention, the coronavirus infection comprises respiratory tract infection, lung infection or complication caused by coronavirus.

The invention has the beneficial effects that:

the verification of the invention proves that the provided compound containing cyclopropyl skeleton has an inhibiting effect on coronavirus, can be used as a medicine for treating and/or preventing diseases caused by coronavirus infection, and has a good development prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of antiviral activity of DDP-38003.2 HCl against HCoV-NL63 in example 2 of the present invention;

FIG. 2 is a diagram showing the construction of a cellular level CoV-RdRp-Gluc reporter system in example 3 of the present invention;

FIG. 3 is a graph showing the expression amounts of nsp12, nsp7 and nsp8 in Western Blot in example 3 of the present invention;

FIG. 4 is a graph showing the results of measurement of Gaussia luciferase activity in example 3 of the present invention;

FIG. 5 is a graph showing the results of the detection of the Z' factor by the oV-RdRp-Gluc reporter system for high throughput screening in example 3 of the present invention;

FIG. 6 is a graph showing the result of the inhibitory activity of DDP-38003 & 2HCl against SARS-CoV-2RdRp in example 3 of the present invention.

Detailed Description

The present invention is described below in conjunction with specific examples, which are not intended to limit the scope of the present invention, but rather to provide guidance to those skilled in the art in understanding and practicing the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The eukaryotic codon-optimized plasmid pCOVID19-nsp7, eukaryotic codon-optimized plasmid pCOVID19-nsp8, eukaryotic codon-optimized plasmid pCOVID19-nsp10, eukaryotic codon-optimized plasmid pCOVID19-nsp12 and eukaryotic codon-optimized plasmid pCOVID19-nsp14 described in the detailed description of the present invention are plasmids obtained by seamlessly cloning nsp7, nsp8, nsp10, nsp12 and nsp14 genes into pCMV6-entry (available from ORIGEN) vectors by XhoI, respectively.

nsp7, nsp8, nsp10, nsp12 and nsp14 genes are referred to in chip.med.j.2020; 00: 00-00. doi:10.1097/CM 9.0000000000000722.

The cyclopropyl skeleton containing compounds GSK-LSD1 & 2HCl, ORY-1001 & 2HCl, DDP-38003 & 2HCl, OG-L002 used in this example are commercially available and are available from Medchemex press in this study and have the structure shown in Table 1:

TABLE 1 cyclopropyl skeleton-containing Compound Structure

EXAMPLE 1 antiviral Activity of Compounds containing a cyclopropyl backbone on HCoV-OC43

HCT-8 cells at 1.0X 10⁴Inoculating each cell in 96-well plate at a concentration of 100 μ L per well, culturing in DMEM medium containing 10% FBSAnd culturing for 48 hours. The supernatant was discarded and replaced with fresh DMEM medium containing 2% FBS, and HCoV-OC43 (ATCC: VR-1558) virus was infected at MOI of 0.1. Gradient dilutions of cyclopropyl scaffold containing compound (2 fold dilution from 25 μ M, 7 gradient dilutions) were added to each well at 1 μ L, negative control group was added to each well at 1 μ L DMSO (dimethyl sulfoxide), and the broad spectrum antiviral nucleoside inhibitor ridciclovir was used as a positive control (2 fold dilution from 25 μ M, 7 gradient dilutions) at 1 μ L per well, followed by further incubation at 33 ℃ for 60 hours. 20 μ L of Cell Proliferation Assay (MTS) reagent was added and allowed to act for 3 hours at 37 ℃. Finally, the absorption peak generated at 490nm wavelength is detected. Formula for inhibition (relative infectivity): inhibition rate (virome-sample group)/(virome-blank group) × 100%, EC was obtained by statistical analysis using GraphPad Prism 5.0₅₀The results are shown in Table 2.

TABLE 2 EC for HCoV-OC43₅₀

Numbering	EC50(μM)
		GSK-LSD1·2HCl	28.54
ORY-1001·2HCl	24.35
		DDP-38003·2HCl	1.59
OG-L002	10.34
		Ruidexiwei (Ridexil)	1.05

Example 2 antiviral Activity of DDP-38003 & 2HCl on HCoV-NL63

LLC-MK2 cells at 1.0X 10⁴Each cell was inoculated in a 96-well plate at 100. mu.L/well in a DMEM medium containing 10% FBS for 48 hours. The supernatant was discarded and replaced with fresh DMEM medium containing 2% FBS, and HCoV-NL63 virus was infected at MOI of 0.01. DDP-38003 & 2HCl (2-fold dilution from 25. mu.M, 7-gradient dilution) was added to each well in a gradient, 1. mu.L of DMSO (dimethyl sulfoxide) was added to each well in a negative control, and 1. mu.L of Reidsivir, a broad-spectrum antiviral nucleoside inhibitor, was used as a positive control (2-fold dilution from 25. mu.M, 7-gradient dilution) was added to each well, followed by further incubation at 33 ℃ for 60 hours. 20 μ L of Cell Proliferation Assay (MTS) reagent was added and allowed to act for 3 hours at 37 ℃. Finally, the absorption peak generated at 490nm wavelength is detected. Formula for inhibition (relative infectivity): inhibition rate (virome-sample group)/(virome-blank group) × 100%, EC was obtained by statistical analysis using GraphPad Prism 5.0₅₀The results are shown in FIG. 1.

Example 3 resistance test of DDP-38003 & 2HCl to the New coronavirus exonuclease nsp12

Step 1: establishment of cell level CoV-RdRp-Gluc report system

The luciferase reporter system specifically initiated by the novel coronavirus SARS-CoV-2RdRp is called CoV-RdRp-Gluc for short. The pCoV-Gluc plasmid was constructed by inserting Gaussia luciferase (Gluc) coding sequence (shown in SEQ ID NO. 1) between the 5 'UTR and 3' UTR of the novel coronavirus, then inserting it between pRetrox-light-Pur (Clontech) vectors BamHI-HF (# R3136L, NEB) and Notl (NotI-HF, NEB) sites, and primers in forward (5'-GGC GGA TCC ATT AAA GGT TTA TAC-3', shown in SEQ ID NO. 2) and reverse (5'-TTA GCG GCC GCG TCA TTC TCC TAA GAA-3', shown in SEQ ID NO. 3). Under the action of the CMV promoter, Gluc (mRNA) of the positive strand is transcribed and translated to produce Gluc protein. When a novel coronavirus RNA-dependent RNA polymerase (nsp12) is simultaneously expressed in the system, RdRp first synthesizes negative-strand vRNA using positive-strand Gluc as a template, and then the vRNA is transcribed into positive-strand Gluc (mrna) and finally translated into Gluc protein. Thus, the increased Gluc after addition of RdRp reflects the activity of SARS-CoV-2 RdRp. The schematic construction is shown in FIG. 2.

In view of the involvement of nsp7 and nsp8 in the functioning of nsp12, three sets of tests were set up for comparison in this example: CoV-Gluc (pCoV-Gluc plasmid constructed above) (10ng) was expressed alone, CoV-Gluc (10ng) and eukaryotic codon optimized plasmid pCOVID19-nsp12(nsp12) (200ng) were co-expressed, and CoV-Gluc (10ng), nsp12(200ng), eukaryotic codon optimized plasmid pCOVID19-nsp7(nsp7) (600ng) and eukaryotic codon optimized plasmid pCOVID19-nsp8(nsp8) (600ng) were co-expressed, and in order to ensure the same total transfection amount of the plasmids, empty vector plasmid pCMV6-entry was added to each group for complementation in the experiment. The expression result detection method comprises the following steps:

(1) western Blot experiment

Mixing 2.5X 10⁵HEK 293T cell suspension per mL was seeded at 2mL per well in 6-well plates. The culture media used by the HEK 293T cells are DMEM culture media containing 10% Fetal Bovine Serum (FBS); the cells were cultured in a constant temperature incubator containing 5% carbon dioxide at 37 ℃. When the cells grew to 80%, the HEK 293T cell group was transfected with plasmid per well as designed in the experimental group above. The medium was changed to DMEM medium containing 10% Fetal Bovine Serum (FBS) 4 hours after transfection; the culture was continued for 24 hours. Discard the medium, add 80. mu.L of RIPA lysate per well, transfer the lysate to 1.5ml EP tubes for 20 min on ice, add 20. mu.L of 5 Xprotein loading buffer per tube, and cook in a metal bath at 100 ℃ for 30 min. SDS-PAGE gel electrophoresis separation, and Western Blot to detect the expression levels of nsp12, nsp7 and nsp 8. The results are shown in FIG. 3.

(2) Gaussia luciferase Activity assay

Dissolving 250 μ g of substrate Coelenterazine-h lyophilized powder in 600 μ L of anhydrous ethanol to obtain substrate mother liquor with concentration of 1.022mM, and storing at-20 deg.C; before measurement, the stock solution was diluted in PBS at a ratio of 1:60 to prepare a substrate working solution. Standing at room temperature for 30min to stabilize the working solution, since the substrate is shown inLight is unstable, and the whole process needs light-proof treatment; mu.L of the cell culture supernatant (cell supernatant after 24 hours of culture after transfection in the Western Blot experiment described above) was transferred to a white opaque 96-well plate, and the substrate working solution incubated in the dark was added to each well in an amount of 60. mu.L per well by using a microplate reader Centro XS3 LB 960 as an autosampler, and the signal was collected for 0.5 seconds, and the measurement results were expressed in Relative Light Units (RLU). Three sets of replicates were set up and statistical analysis was performed, where<0.01，***P<0.001 is referred to the group expressing CoV-Gluc alone. Experimental data onExpressed and plotted using GraphPad Prism 5.0 and statistically analyzed. The results are shown in FIG. 4.

The experimental results showed that the groups co-expressing CoV-Gluc and nsp12 and the groups CoV-Gluc, nsp12, nsp7 and nsp8 were 2-fold and 38-fold, respectively, higher than the group expressing CoV-Gluc alone. The above results show that the expression of Gluc protein in the CoV-RdRp-Gluc system constructed by the present invention is specifically dependent on the novel coronavirus RdRp.

The Z' factor detection is carried out on the CoV-RdRp-Gluc report system, and the detection method comprises the following steps:

mixing 2.5X 10⁵A suspension of HEK 293T cells (2 mL/mL) was plated in 6-well plates and cultured for 24h before transfection. The method is carried out in two groups: cells expressing plasmid CoV-Gluc (10ng) were used as a negative control (supplemented with 1.4. mu.g of plasmid pCMV6-entry, purchased from ORIGEN), and cells co-expressing plasmids CoV-Gluc (10ng), nsp12(200ng), nsp7(600ng) and nsp8(600ng) were used as a positive control. After 12h of transfection, both groups of cells were trypsinized, added to DMEM medium to prepare cell suspensions, and seeded in 96-well plates (1X 105 cells/mL) in a volume of 100. mu.l/well. Luciferase activity was measured after 24h incubation. The calculation formula is as follows: the result of the Z 'factor being 1- (3 × positive control relative fluorescence value SD +3 × negative control relative fluorescence value SD)/(positive control relative fluorescence value average-negative control relative fluorescence value average) is shown in fig. 5, from which it is known that the Z' factor is 0.73, which meets the requirement of high throughput screening.

Step 2: test experiments are carried out on DDP-38003 & 2HCl by using a CoV-RdRp-Gluc report system

Mixing 2.5X 10⁵HEK 293T cell suspension per mL was seeded at 2mL per well in 6-well plates. When the cells were 80% long, the HEK 293T cell group was co-transfected with 10ng of pCoV-Gluc constructed in example 1, 200ng of eukaryotic codon-optimized plasmid pCOVID19-nsp12, 600ng of eukaryotic codon-optimized plasmid pCOVID19-nsp7 and 600ng of eukaryotic codon-optimized plasmid pCOVID19-nsp8 per well. The medium was changed to DMEM medium containing 10% Fetal Bovine Serum (FBS) 4 hours after transfection, and the culture was continued for 12 hours. Digesting the cells in a six-well plate to prepare a cell suspension, and preparing the cell suspension according to the proportion of 1.0 multiplied by 10⁴HEK 293T cells were seeded at 100. mu.L/well in 96-well plates per ml. Each well was treated by adding 1. mu.L of the compounds DDP-38003 & 2HCl and Reidesciclovir at a concentration of 10. mu.M, followed by incubation for 24 hours. Wherein the negative control group was added with 1. mu.L DMSO (dimethyl sulfoxide) per well. And finally, detecting the fluorescence value according to the method in the step 1.

The final concentrations of the compounds DDP-38003 & 2HCl and Reidesciclovir were 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40. mu.M, respectively. Experimental groups were run in triplicate, each with reference to the DMSO group of each group. Experimental data onExpressed and plotted using GraphPad Prism 5.0 and statistically analyzed. The results are shown in FIG. 6B. The experimental result shows that the compound DDP-38003 & 2HCl inhibits the EC of SARS-CoV-2RdRp₅₀1.43. mu.M, and the positive compound, Reidesvir₅₀The value was 1.08. mu.M, both EC₅₀(DDP-38003 & 2 HCl/Reidcisvir) equivalent. The experimental result shows that the small molecular DDP-38003 & 2HCl can play a potential role in resisting the new coronavirus by inhibiting SARS-CoV-2 RdRp.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.