Application of BMS-265246 in the preparation of anti-herpes virus drugs and pharmaceutical compositions

Abstract

The present invention belongs to the field of medical technology, and in particular to the application of BMS-265246 in the preparation of anti-herpes virus drugs and pharmaceutical compositions. The present invention provides a novel small molecule compound drug for herpes simplex virus, and the effective drug concentration of BMS-265246 for anti-herpes virus is low, and the safety is good, the selection index at the cell level is high, and the therapeutic index at the animal level is high. It can improve the survival rate of mice infected with a lethal dose of herpes virus, reduce the weight loss of mice, reduce lung tissue pathological damage, and have a good therapeutic effect on diseases caused by herpes virus infection, and has a broad-spectrum and efficient anti-herpes virus effect; in addition, BMS-265246 targets host cell proteins, and basically does not produce virus resistance problems, and provides new ideas and treatment schemes for inhibiting herpes virus replication and clinically preventing and / or treating diseases caused by herpes virus infection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of BMS-265246 in the preparation of anti-herpes virus drugs and a pharmaceutical composition. Background Art

[0002] Herpes virus is a typical representative of the herpesviridae family. Currently, there are eight types of herpes viruses that can infect humans, namely: herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpes virus 6 (HHV-6), human herpes virus 7 (HHV-7) and Kaposi's sarcoma-associated herpesvirus (KSHV). Based on the differences in genome sequence, structure and physical and chemical properties, human herpes viruses can be divided into three subfamilies: alpha herpesvirus subfamily, beta herpesvirus subfamily and gamma herpesvirus subfamily. The representative symptom of herpes virus is the establishment of latency after infection, that is, the virus has the ability to remain dormant in host cells. The cells in which different subfamilies establish latency are different. Most alpha herpes viruses establish latency in neurons, beta herpes viruses establish latency in non-neuronal cells, and gamma herpes viruses establish latency in B lymphocytes and T lymphocytes. Among them, the herpes simplex virus (HSV) of the alpha herpesvirus subfamily is a typical representative of herpes virus, including HSV-1 or HSV-2, which has the characteristics of short replication cycle, wide range of infected hosts, long-term latency, repeated attacks and tropism for neural tissue. Mature HSV consists of the following parts: (1) Viral DNA: 152kb linear double-stranded DNA, encoding at least 74 genes; (2) Capsid: DNA is encapsidated in a capsid composed of 162 capsid particles; (3) Tegument: The capsid is surrounded by 20-23 different viral envelope proteins with structural and regulatory functions; (4) Envelope: The outermost layer is covered by an envelope containing at least 12 different glycoproteins: gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM and gN. A small number of them exist as heterodimers (gH / gL and gE / gI), and the vast majority exist as monomers. Once in contact with a suitable host, the viral glycoproteins will attach to the host cell surface receptors, then induce the fusion of the viral envelope with the host cell membrane, thereby delivering the viral contents into the host cell.

[0003] HSV infection is quite common in humans. Studies have shown that more than 90% of people have been infected with HSV. When the body's resistance is reduced, the latent HSV in the body is activated and the disease occurs, and there are various clinical manifestations. HSV-1 mainly infects mucosal epithelial cells and establishes lifelong infection in sensory neurons. The most common symptom of HSV-1 infection is the formation of painful blister-like lesions in the mouth and face. Symptoms include tingling, itching or burning sensations. These symptoms may recur regularly, and the frequency varies from person to person. In severe cases, they can cause herpes encephalitis and keratitis, and even cause blindness. HSV-2 is often associated with genital infection or neonatal infection. Genital herpes can be asymptomatic in mild cases, or there may be a tingling or burning sensation without skin lesions. In severe cases, there may be recurrent genital ulcers. When symptoms occur, genital herpes is characterized by one or more genital or anal blisters or ulcers. In addition, symptoms of new HSV infection usually include fever, body aches, and swollen lymph nodes. The first attack may be severe and symptoms may recur. Genital herpes caused by herpes simplex virus type 1 tends not to recur often, but genital herpes caused by herpes simplex virus type 2 often has recurring symptoms. HSV-1 and HSV-2 infections have become an important factor affecting people's lives and health.

[0004] There is currently no effective vaccine for HSV. The drugs used to treat herpes virus infections are mainly nucleoside analogs that rely on phosphorylation by viral thymidine kinase, such as acyclovir, valacyclovir, famciclovir and penciclovir. These nucleoside analogs can be phosphorylated by viral thymidine kinase, but cannot be phosphorylated by host cell enzymes. The product nucleotides are incorporated into the replicated DNA, causing the termination of DNA chain synthesis, thereby inhibiting viral DNA replication in virus-infected cells to achieve the effect of treating herpes virus infection. Long-term use of these drugs to treat HSV infection is likely to induce drug-resistant virus strains. Acyclovir-resistant HSV isolates have been identified as the cause of pneumonia, encephalitis, esophagitis and skin and mucosal infections in immunocompromised patients. Due to the limitations that the targets of nucleoside analog drugs are mainly viral proteins and the virus is prone to mutation and escape events, it is very necessary to develop new drugs, especially those with high safety that target non-viral targets that reduce viral escape, for the treatment of HSV. Summary of the invention

[0005] In view of the problems that there is no specific drug for treating HSV in the prior art or that HSV is resistant to nucleoside drugs, the present invention provides the use of BMS-265246 in the preparation of anti-herpes virus drugs, and provides a pharmaceutical composition containing BMS-265246.

[0006] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides the use of BMS-265246 in the preparation of anti-herpes virus drugs.

[0008] Furthermore, the herpes virus is one or more of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7) and Kaposi's sarcoma-associated virus (KSHV).

[0009] Furthermore, the herpes virus is herpes simplex virus type 1 and / or herpes simplex virus type 2.

[0010] Furthermore, the application includes at least one of an application in preparing a drug for inhibiting herpes virus replication and an application in preparing a drug for preventing and / or treating herpes virus infection diseases.

[0011] Furthermore, the herpes virus infection disease includes one or more of herpes labialis, genital herpes, herpetic stromal keratitis, meningitis and herpes simplex encephalitis.

[0012] The second aspect of the present invention provides a pharmaceutical composition for use against herpes virus, the pharmaceutical composition comprising a therapeutically effective amount of BMS-265246 and a pharmaceutically acceptable excipient.

[0013] Furthermore, the pharmaceutically acceptable excipients include one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents and carriers.

[0014] Furthermore, the dosage form of the pharmaceutical composition includes tablets, capsules, granules, pills, liquid preparations, decoctions, suppositories, gels, aerosols or patches.

[0015] The advantages and positive effects of the present invention are:

[0016] The present invention provides a novel small molecule compound drug for herpes simplex virus, namely BMS-265246, which has a half inhibitory concentration (IC50) against herpes simplex virus such as HSV-1 and HSV-2 at the cell level of Vero cells and Hep G2 cells. 50 ) were all below 1.5 μM, and the median lethal concentration (CC 50) is above the 500μM level, the drug has a low effective concentration, good safety, a high selectivity index at the cellular level, and a high therapeutic index at the animal level. It can improve the survival rate of mice infected with a lethal dose of herpes virus, reduce the weight loss of mice, and reduce the pathological damage of their lung tissue. Therefore, BMS-265246 has a good therapeutic effect on diseases caused by herpes virus infection, has a broad-spectrum and efficient anti-herpes virus effect, is conducive to shortening the medication cycle, and provides a new idea for inhibiting the replication and infection of herpes viruses in the lytic period. In addition, BMS-265246 is a CDK1 / 2 inhibitor that targets host cell proteins rather than viral proteins, and basically does not cause viral resistance problems. It has efficient antiviral activity against herpes viruses that are resistant to conventional drugs targeting viral proteins, and provides a new treatment plan for the clinical prevention and / or treatment of diseases caused by drug-resistant herpes virus infection, which has broad application prospects and important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 The cytotoxicity test results of BMS-265246 in the present invention are shown in Figure A and Figure B, respectively, are cell survival rate curves of Vero cells and Hep G2 cells treated with different concentrations of BMS-265246;

[0019] Figure 2 The results of in vitro antiviral activity test of BMS-265246 against herpes simplex virus HSV-1 in the embodiment of the present invention are shown in Figure A and Figure B are respectively the inhibition rate curves of herpes simplex virus HSV-1 in Vero cells and Hep G2 cells after being treated with different concentrations of BMS-265246;

[0020] Figure 3 The results of in vitro antiviral activity test of BMS-265246 against herpes simplex virus HSV-2 in Vero cells in the present invention are as follows;

[0021] Figure 4 This is a curve diagram of the survival rate of BALB / c mice infected with herpes simplex virus HSV-1 after being treated with BMS-265246 in an embodiment of the present invention;

[0022] Figure 5This is a graph showing the weight change of BALB / c mice infected with herpes simplex virus HSV-1 after being treated with BMS-265246 according to an embodiment of the present invention;

[0023] Figure 6 This is a lung tissue pathological section of BALB / c mice infected with herpes simplex virus HSV-1 in an example of the present invention after being treated with BMS-265246 for 5 days. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0026] In order to better understand the present invention rather than limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention indicating dosage, percentage, etc., should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods. In addition, it should be noted that the "and / or" used in the present invention should be regarded as the specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as (i) A, (ii) B and (iii) A and B.

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below.

[0028] BMS-265246 is a potent and selective inhibitor of cyclin-dependent kinases CDK1 and CDK2 with an IC 50) are 6nM and 9nM, respectively. CDK1 and CDK2 are serine / threonine protein kinases that play a key role in cell cycle regulation; CDK1 selective inhibitors can reversibly block the G2 / M phase of the cell cycle; CDK2 mainly targets the G1 / S phase of the cell cycle, during which cells make proteins required for mitosis and replicate their DNA. Existing studies have shown that BMS-265246 can promote iTreg cell differentiation in a dose-dependent manner; as a CDK2 inhibitor, BMS-265246 can inhibit E2-induced expression of EGF3, AREG and CXCL12 in MCF7 cells, and can synergize with tamoxifen to induce apoptosis of MCF7 cells. It has also been reported that BMS-265246, as a CDK1 inhibitor, induces apoptosis of cells in the G2 phase. As a CDK1 and CDK2 inhibitor, BMS-265246 can eliminate the ability of CHI3L1 to stimulate epithelial cell ACE2 and SPP, which plays a key role in the pathogenesis of novel coronavirus infection (COVID-19), making it an attractive therapeutic target for the treatment of COVID-19. However, there have been no reports to date on BMS-265246 inhibiting herpes virus replication.

[0029] In the present study, it was found that BMS-265246 can inhibit the replication of herpes viruses, especially herpes simplex viruses (HSV-1 and HSV-2) in a dose-dependent manner. The half inhibitory concentration (IC50) of BMS-265246 on Vero cells and Hep G2 cells for HSV-1 is 50 ) were only 0.95 μM and 1.027 μM, respectively, and the IC values ​​of HSV-2 on Vero cells were 50 The drug has a low effective concentration of 1.058 μM and has a broad spectrum of anti-herpes virus replication activity. In addition, Vero cells and Hep G2 cells were used to detect cell survival rates. The half lethal concentration (CC 50 ) are all greater than 500μM, and no obvious toxicity is observed at 50μM. The drug safety threshold is high and the drug safety is good; calculated by the above parameters, the selectivity index (SI) of BMS-265246 for HSV-1 on Vero cells and Hep G2 cells is greater than 526.32 and greater than 486.85, respectively, and the SI for HSV-2 on Vero cells is greater than 472.59, indicating that it has a good foundation for drug research and development. Further experiments in mice found that treatment with BMS-265246 can increase the survival rate of mice infected with a lethal dose of HSV-1, reduce the weight loss of mice, and reduce pathological damage to lung tissue, confirming that BMS-265246 also has anti-herpes virus activity in vivo, expanding its indications to the treatment of herpes simplex virus infection, with a shorter medication cycle and better expected safety.

[0030] Based on the above findings, the present invention provides an application of BMS-265246 in the preparation of anti-herpes virus drugs. The application includes at least one of the application in the preparation of drugs for inhibiting herpes virus replication and the application in the preparation of drugs for preventing and / or treating herpes virus infection diseases.

[0031] BMS-265246 has the structure shown in Formula I:

[0032]

[0033] The present invention provides a safer and more effective small molecule compound, BMS-265246, for the prevention and / or treatment of herpes virus. BMS-265246 can effectively inhibit the replication of herpes virus in the lytic phase within a non-toxic range, has a broad-spectrum, efficient antiviral activity, and has a significant therapeutic effect on the infection caused by herpes virus. In addition, the drug BMS-265246 used in the present invention is a CDK1 / 2 inhibitor, which is a drug targeting host cell proteins rather than viral proteins, so herpes virus will not basically develop resistance to BMS-265246, BMS-265246 can inhibit the adsorption and entry of the HSV-1 replication cycle, and has antiviral activity for HSV-1 that is resistant to a variety of conventional antiviral drugs (i.e., drugs targeting viral proteins). Therefore, the BMS-265246 provided by the present invention has the potential for clinical application, and provides a new idea for the clinical prevention and / or treatment of herpes virus infection, including drug-resistant herpes virus infection-induced diseases.

[0034] Optionally, the herpes virus described above is one or more of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpes virus type 6 (HHV-6), human herpes virus type 7 (HHV-7) and Kaposi's sarcoma-associated virus (KSHV). Preferably, the herpes virus is herpes simplex virus type 1 (HSV-1) and / or herpes simplex virus type 2 (HSV-2).

[0035] Optionally, the herpes virus infection diseases illustratively include but are not limited to: cold sores, genital herpes, herpes stromal keratitis, meningitis and herpes simplex encephalitis.

[0036] Another embodiment of the present invention provides a pharmaceutical composition for use against herpes virus, the pharmaceutical composition comprising a therapeutically effective amount of BMS-265246 and a pharmaceutically acceptable excipient.

[0037] The application advantages of the pharmaceutical composition for use against herpes virus are the same as the advantages of the application of BMS-265246 in the preparation of anti-herpes virus drugs over the prior art as described above, and will not be repeated here.

[0038] The term "pharmaceutical composition" refers to a preparation of the compound represented by Formula I of the present invention (BMS-265246) and a medium generally accepted in the art for delivering biologically active compounds to mammals (e.g., humans), the medium including pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of the organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0039] The term "pharmaceutically acceptable excipient" refers to a component that does not interfere with the efficacy of the biological activity of the compound BMS-265246 and is not significantly toxic to the body at the therapeutically effective concentration of the compound, including any one or a combination of at least two of solvents, dispersants, diluents, fillers, wetting agents, adhesives, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, etc. and carriers. It is well known in the art to use the aforementioned components for pharmaceutically active substances. For example, the solvent includes, but is not limited to, water, dimethyl sulfoxide (DMSO) and sodium carboxymethyl cellulose (CMC-Na), and the carrier includes, but is not limited to, polyethylene glycol, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, gypsum powder, sucrose, cyclodextrin, amylose, magnesium stearate, pectin, gum arabic, stearic acid or lower alkyl ethers of cellulose. Each component can be used alone or in combination with multiple components.

[0040] The terms "effective amount" and "therapeutically effective amount" refer to the amount of a compound, agent, preparation or composition sufficient to relieve one or more symptoms of the disease or condition being treated to some extent after administration, and the intended goal may be the reduction and / or relief of symptoms or causes, or any other desired changes in the body. For example, a therapeutically effective amount is the amount of a pharmaceutical composition comprising a compound disclosed in the present invention required to provide a significant symptom relief effect clinically, and techniques such as a dose escalation test can be used to determine the effective amount suitable for any individual case. The therapeutically effective amount will vary depending on the activity of the compound, the condition caused by the viral infection and its severity, and the size and health of the individual to be treated. Exemplarily, the therapeutically effective amount in a mouse model may be 1 mg / kg-150 mg / kg, preferably 1 mg / kg-100 mg / kg, and more preferably 5 mg / kg-30 mg / kg, such as 10 mg / kg or 20 mg / kg, twice a day.

[0041] The term "treatment" and its analogous words encompass any therapy for animals other than humans or humans, and treatment can be directed to existing illnesses, or can be preventive (preventive treatment), which includes cure, relief or preventive effects. Treatment can also include cure, relief or prevention of symptoms associated with a disease rather than acting on the potential cause of the disease. The term "prevention" and its analogous words include making the patient reduce the possibility of the occurrence or deterioration of a disease or illness, such as for the medication period in which a herpes virus infection has not yet occurred after exposure to a herpes virus environment, and treatment can be for the treatment of an infection after primary infection or latent virus activation.

[0042] Pharmaceutical composition provided by the present invention can be adapted to any form of administration to prevent and / or treat anti-herpes virus, including but not limited to oral, nasal, percutaneous, intravenous and enteral administration, preferably administered by oral route. Those skilled in the art can select suitable formulations according to the administration, for example, when used for oral administration, conventional solid preparations and liquid preparations can be made. In some specific embodiments, dosage form includes but is not limited to: tablet, capsule, granule, drop pill, liquid preparation, decoction, suppository, gel, aerosol or patch etc.

[0043] The pharmaceutical composition provided by the present invention can be in a single dose or multiple dose form. Optionally, the final concentration of BMS-265246 in the pharmaceutical composition is 0.01 μM-500 μM. This concentration refers to the final concentration of BMS-265246 in a pharmaceutical composition such as a liquid preparation, and the amount of drug taken during treatment is adjusted according to the final concentration, dosage form and individual to be treated to ensure that the therapeutically effective amount is reached.

[0044] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are generally carried out according to the conditions recommended by the manufacturer.

[0045] Example

[0046] Current cell-based efficacy evaluation technologies include: (1) phenotypic endpoint detection, including cytopathic effect (CPE) and plaque assay; (2) cell-based single-target detection, which mainly reflects the level of viral replication by detecting viral structural proteins or nucleic acids; (3) reporter gene detection, which uses reporter viruses or reporter cells to detect viral replication levels; (4) viral titer detection, which uses plaque assay or half tissue culture infectious dose (TCID 50 ) detection experiments to determine the production of infectious viruses. These methods can be used to evaluate the antiviral efficacy of drugs. This study used the plaque assay to detect viral titers to reflect the level of viral replication. The plaque assay is the gold standard for detecting titers.

[0047] The in vitro antiviral activity of BMS-265246 was analyzed in depth using African green monkey kidney cell line Vero and human liver cancer cell line Hep G2 as cell models. Herpes viruses can infect a variety of cells, mainly including epithelial cells, neuronal cells, cells and lymphocytes. Due to the obvious susceptibility of HSV-1 to Vero cells, the obvious cytopathic effect (CPE), and the ability to express HSV-1 corresponding receptor proteins on infected Vero cells, the present invention selects Vero cells as the main in vitro experimental research object, and selects human related cells such as Hep G2 cells to detect the antiviral activity of HSV-1, so as to exclude the antiviral effect being limited to specific cell types, and to conduct a more real and systematic evaluation of the in vitro anti-herpes simplex virus effect of BMS-265246.

[0048] BALB / c mice were further used as an animal model to evaluate the antiviral activity of BMS-265246 in vivo. Tail vein injection of BALB / c mice can cause systemic acute infection in mice, supporting viral replication in multiple organs, such as high levels of viral replication in brain tissue and lungs, and a relatively high incidence of fatal encephalitis. In vivo medication is administered by gavage, and the drug has a therapeutic effect on multiple organs in the body. The efficacy is mainly reflected in the recovery of mouse weight, the increase in survival rate, and the reduction of pathological damage.

[0049] The cell line Vero cells required in this example were purchased from the American Type Culture Collection (ATCC); the experimental animals required were 5-week-old BALB / c mice purchased from the Guangdong Medical Experimental Animal Center; the virus strains HSV-1H129 and HSV-2G used were from the Microbial Bacteria (Virus) Collection Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0050] The drug BMS-265246 required in this example was purchased from TargetMol. When conducting cell experiments, the drug was dissolved in DMSO, and when conducting animal experiments, the drug was diluted with 2% CMC-Na. The required DMEM culture medium was purchased from Corning; fetal bovine serum (FBS) was purchased from Meiluncell; and the Cell Titer-Glo cell proliferation detection kit was purchased from Promega.

[0051] The instruments required in this example, the Varioskan LUX multifunctional microplate reader, were purchased from Thermo Company; the CO2 constant temperature incubator was purchased from Thermo Company; and the biosafety cabinet was purchased from ESCO Company.

[0052] 1. Cytotoxicity testing of BMS-265246

[0053] 1. Cell culture

[0054] After three passages, the cells were cultured in DMEM medium containing 10% fetal bovine serum and double antibodies (penicillin 100 U / mL and streptomycin 100 μg / mL) with a seeding density of no less than 1.5 × 10 4 cell / mL, the subculture density should not be higher than 5×10 4 cell / mL.

[0055] 2. Drug treatment of cells

[0056] Take 100 μL of Vero cells or Hep G2 cells and culture them at a volume of 2×10 4 Cells / well were inoculated in a 96-well cell culture plate and cultured for 18 hours until the confluence of the cell wells reached about 85%; BMS-265246 was prepared with 100 μL of culture medium (DMEM medium + 2% serum) per well and added to the corresponding cell wells for mixing. The drug was set to 8 concentration gradients, with 3 replicates for each gradient concentration, and the final concentrations were 0.23 μM, 0.69 μM, 2.06 μM, 6.17 μM, 18.52 μM, 55.56 μM, 166.67 μM and 500 μM, and cultured for 72 hours in an incubator at 37°C with 5% CO2.

[0057] 3. Calculate the toxicity of drugs to cells in each test well

[0058] Remove the supernatant and add 100 μL of a mixture of Cell Titer-Glo and PBS in a ratio of 1:2 to each well, and react at room temperature in the dark for 10 minutes. Take 80 μL of the above reaction solution from each well and place it in a new black-walled, transparent-bottom 96-well plate, place it on an ELISA reader to read its chemiluminescence value, and calculate the cell survival rate to detect the cytotoxicity of BMS-265246.

[0059] Cell survival rate (%) = drug-treated group / untreated control group*100%.

[0060] Graphpad Prism 8 software was used to plot the relative cell viability (cell viability) against the logarithm of the drug concentration and calculate the half cytotoxic concentration (CC) of the drug. 50 ), the result is as follows Figure 1 As shown, Figure A and Figure B are cell survival rate curves of Vero cells and Hep G2 cells treated with different concentrations of BMS-265246, respectively.

[0061] As can be seen from the figure, after BMS-265246 was treated with the highest concentration of 500 μM for 72 hours, the cell viability was significantly reduced compared with the control group, but it did not decrease to 50%, indicating that BMS-265246 had a certain weak toxicity to Vero cells and Hep G2 cells at this concentration, and its half toxic concentration CC 50 Greater than 500μM. 2. In vitro antiviral activity of BMS-265246 against herpes simplex virus HSV-1

[0062] 1. Cell culture

[0063] After three passages, the cells were cultured in DMEM medium containing 10% fetal bovine serum and double antibodies (penicillin 100 U / mL and streptomycin 100 μg / mL) with a seeding density of no less than 1.5 × 10 4 cell / mL, the subculture density should not be higher than 5×10 4 cell / mL.

[0064] 2. Drug treatment of cells

[0065] Take 100 μL of Vero cells or Hep G2 cells and culture them at a volume of 2×10 4 Cells / well were inoculated in a 96-well cell culture plate and cultured for 18 hours until the cell well confluence reached about 85%; 0.01 MOI (multiplicity of infection) of HSV-1 virus was added to the infection group to infect Vero cells or Hep G2 cells, and at the same time, drugs of various gradient concentrations (final concentrations were 0.02 μM, 0.07 μM, 0.21 μM, 0.62 μM, 1.85 μM, 5.56 μM, 16.67 μM and 50 μM, 3 replicates per gradient) were added to a total volume of 100 μL of culture medium (DMEM medium + 2% serum), and cultured in a cell culture incubator at 37°C for 48 hours.

[0066] 3. Plaque method to detect infectious virus particles in cell supernatant

[0067] The number of dilutions required varies according to the expected titer of the virus in question, using culture medium as the virus diluent. The diluted sample supernatant was used to infect a 12-well plate filled with a monolayer of Vero cells at 200 μL / well at 4°C for 2 hours. A negative control of cells was used with culture medium without virus supernatant. The plate was gently shaken every 15 minutes to ensure uniform coverage and prevent the cell monolayer from drying. After infection, the supernatant was discarded and the cells were washed twice with PBS to remove free virus that was not attached to the cells. A 1:1 mixture of culture medium (containing a final concentration of 2% FBS) and microcrystalline cellulose (final concentration of 1.5%) was mixed by pipetting and 1.5 mL was added to a 12-well plate. Incubate in a 37°C, 5% CO2 incubator for 48 hours. After discarding the microcrystalline cellulose overlay, the cells were fixed with 4% paraformaldehyde (0.5 mL / well) for 30 minutes. The cells were covered with 0.5% (w / v) crystal violet solution for about 30 minutes, after which the crystal violet was gently washed off with running water. The plaque method was used to detect the infectious virus particle titer levels in the cell supernatant of the drug-treated group, the non-drug-treated group, and the cell negative control group to evaluate the replication level of herpes simplex virus HSV-1 after treatment with different concentrations of BMS-265246.

[0068] 4. Calculate the inhibition rate of the drug on the virus in each test well

[0069] Count the number of plaques in each well, the negative control should have a uniform monolayer. Determine the viral titer of the stock sample by taking the number of plaques after the dilution and the inverse of the total dilution factor.

[0070] The virus titer PFU calculation formula is: PFU / mL = [dilution factor × n] / V; where n is the plaque of a certain dilution, and V is the volume of the virus added to the well plate after dilution.

[0071] The inhibition rate was calculated by the following formula: inhibition rate (%) = (1-PFU of drug-treated group / PFU of non-drug-treated group)*100%.

[0072] Graphpad Prism 8 was used to plot the inhibition rate against the logarithm of the drug concentration and calculate the half-maximal inhibitory concentration (IC) of the drug against herpes simplex virus HSV-1. 50 ), the results are shown in Figure 2 , where Figure A and Figure B are respectively graphs of the HSV-1 inhibition rate in Vero cells and Hep G2 cells after treatment with different concentrations of BMS-265246.

[0073] As can be seen from the figure, BMS-265246 significantly inhibited the formation of herpes simplex virus HSV-1 virions in Vero cells and Hep G2 cells in a dose-dependent manner. Its half effective concentration IC 50 0.95 μM and 1.027 μM respectively.

[0074] 5. Calculation of drug selection index

[0075] The drug selection index (SI) is used to determine the safe range of drug effects. The selection index is greater than 3 for effective drug effects. The larger the index, the greater the safe range. The calculation formula is: SI = CC 50 / IC 50 .

[0076] Combined with the above data, the selection index of BMS-265246 for herpes simplex virus HSV-1 on Vero and Hep G2 cells is greater than 526.32 and greater than 486.85, respectively, and it has effective anti-HSV-1 activity.

[0077] III. In vitro antiviral activity of BMS-265246 against herpes simplex virus HSV-2

[0078] 1. Cell culture

[0079] After three passages, the cells were cultured in DMEM medium containing 10% fetal bovine serum and double antibodies (penicillin 100 U / mL and streptomycin 100 μg / mL) with a seeding density of no less than 1.5 × 10 4 cell / mL, the subculture density should not be higher than 5×10 4 cell / mL.

[0080] 2. Drug treatment of cells

[0081] Take 100 μL of Vero cells and plate 2×10 4 Cells / well were inoculated in a 96-well cell culture plate and cultured for 18 hours until the cell well confluence reached about 85%; the infection group was added with 0.01 MOI (multiplicity of infection) of HSV-2 virus to infect Vero cells, and at the same time, drugs of various gradient concentrations (final concentrations were 0.02 μM, 0.07 μM, 0.21 μM, 0.62 μM, 1.85 μM, 5.56 μM, 16.67 μM and 50 μM, 3 replicates per gradient) were added to a total volume of 100 μL of culture medium (DMEM medium + 2% serum), and cultured in a cell culture incubator at 37°C for 48 hours.

[0082] 3. Plaque method to detect infectious virus particles in cell supernatant

[0083] The number of dilutions required varies according to the expected titer of the virus in question, using culture medium as the virus diluent. The diluted sample supernatant was used to infect a 12-well plate filled with a monolayer of Vero cells at 200 μL / well at 4°C for 2 hours. A negative control of cells was used with culture medium without virus supernatant. The plate was gently shaken every 15 minutes to ensure uniform coverage and prevent the cell monolayer from drying. After infection, the supernatant was discarded and the cells were washed twice with PBS to remove free virus that was not attached to the cells. A 1:1 mixture of culture medium (containing a final concentration of 2% FBS) and microcrystalline cellulose (final concentration of 1.5%) was mixed by pipetting and 1.5 mL was added to a 12-well plate. Incubate in a 37°C, 5% CO2 incubator for 48 hours. After discarding the microcrystalline cellulose overlay, the cells were fixed with 4% paraformaldehyde (0.5 mL / well) for 30 minutes. The cells were covered with 0.5% (w / v) crystal violet solution for about 30 minutes, after which the crystal violet was gently washed off with running water. The plaque method was used to detect the titer of infectious viral particles in the cell supernatant of the drug-treated group (infected group), the non-drug-treated group, and the cell negative control group, in order to evaluate the replication level of herpes simplex virus HSV-2 after treatment with different concentrations of BMS-265246.

[0084] 4. Calculate the inhibition rate of the drug on the virus in each test well

[0085] Count the number of plaques in each well, the negative control should have a uniform monolayer. Determine the virus titer of the stock sample by taking the number of plaques after the dilution and the inverse of the total dilution factor, and calculate the inhibition rate.

[0086] Graphpad Prism 8 was used to plot the inhibition rate against the logarithm of the drug concentration and calculate the half-maximal inhibitory concentration (IC) of the drug against herpes simplex virus HSV-2. 50 ), the results are shown in Figure 3 .

[0087] As can be seen from the figure, BMS-265246 significantly inhibited the formation of herpes simplex virus HSV-2 virions in Vero cells in a dose-dependent manner. Its half effective concentration IC 50 It is 1.058μM.

[0088] 5. Calculation of drug selection index

[0089] Combined with the above data, the selection index of BMS-265246 for herpes simplex virus HSV-2 on Vero cells is greater than 472.59, and it has effective anti-HSV-2 activity.

[0090] 4. Evaluation of the antiviral activity of BMS-265246 against herpes simplex virus HSV-1 in animals

[0091] 1. Mouse labeling and grouping

[0092] All BALB / c mice were separated into cages before the formal experiment, with 5 mice in each cage. The mice were kept for about 7 days after separation to allow them to better adapt to the living environment. The average weight of the mice before the experiment was about 20g. The right ears of the mice were marked by ear tags. After marking, the mice were weighed and the weight data were recorded.

[0093] 2. Virus infection in mice

[0094] Mice were infected by tail vein injection. The total amount of virus used was calculated according to the experimental needs. Each mouse was intravenously injected with a lethal dose of 625 PFU of virus, and the volume of the injected virus solution was 125 μL.

[0095] 3. Drug administration in mice

[0096] The drug BMS-265246 was diluted with 2% CMC-Na to 10 mg / kg and 20 mg / kg, respectively. A control group was also set up, and the dosing solution was 2% CMC-Na. The dosing was performed by gavage, and the gavage dose was 0.2 mL / mouse, twice / day, for 4 consecutive days.

[0097] 4. Data Recording

[0098] Observe the mice every day and record their weight. When a mouse dies, remove the body from the cage in time and handle it properly. Record the number of mice that survive. Use Graphpad Prism 8 to plot the survival rate and weight change rate (%) against the number of days infected with the virus. Draw the survival curve and weight change curve of the drug against HSV-1 infection. The results are shown in the figure. Figure 4-5 shown.

[0099] The treatment groups with 10mg / kg and 20mg / kg of BMS-265246 increased the survival rate of mice infected with a lethal dose of herpes simplex virus HSV-1 by 10% and 50% respectively compared with the non-drug treated group (control group), in a dose-dependent manner. In addition, the treatment groups reduced the weight loss of mice.

[0100] 5. Study on the degree of pathological damage of lung tissue in mice infected with herpes simplex virus HSV-1 treated with BMS-265246

[0101] 1. Mouse labeling and grouping

[0102] All BALB / c mice were separated into cages before the formal experiment, with 5 mice in each cage. The mice were kept for about 7 days after separation to allow them to better adapt to the living environment. The average weight of the mice before the experiment was about 20g. The right ears of the mice were marked by ear tags. After marking, the mice were weighed and the weight data were recorded.

[0103] 2. Virus infection in mice

[0104] Mice were infected by tail vein injection. The total amount of virus used was calculated according to the experimental needs. Each mouse was intravenously injected with a lethal dose of 625 PFU of virus, and the volume of the injected virus solution was 125 μL.

[0105] 3. Drug administration in mice

[0106] The drug BMS-265246 was diluted with 2% CMC-Na to 20 mg / kg. A control group was also set up, and the drug solution was 2% CMC-Na. The drug was administered by gavage, and the gavage dose was 0.2 mL / mouse, twice / day, for 4 consecutive days.

[0107] 4. Data Recording

[0108] The mice were observed every day. Five days after infection, the lung tissues of the mice were sliced ​​and stained with HE to detect the degree of pathological damage of the lung tissues of the mice infected with HSV-1 by BMS-265246. The results were as follows: Figure 6 shown.

[0109] Pathological examination of lung tissue sections showed that the lung sections of mice in the virus infection group had strong cell damage, destroyed alveolar structure, obvious infiltration of inflammatory cells, tissue fibrosis, mild degeneration and shedding of bronchial epithelial cells, and other inflammatory pathological manifestations. In contrast, the alveolar structure of the lung tissue of mice in the group treated with 20 mg / kg of BMS-265246 was relatively intact, and inflammatory pathological damage was significantly reduced.

[0110] In summary, the present invention detected the cytotoxicity of BMS-265246 on the African green monkey kidney cell line Vero and the human liver cancer cell line Hep G2, and determined the inhibitory effect of BMS-265246 on herpes simplex virus type 1 (HSV-1) in the Vero cell line and the Hep G2 cell line and on herpes simplex virus type 2 (HSV-2) in the Vero cell line. The results showed that the small molecule compound BMS-265246 exhibited significant dose-dependent anti-herpes simplex virus activity against both HSV-1 and HSV-2. Therefore, the anti-HSV-1 virus activity of BMS-265246 in vivo was further detected at the animal level. The results showed that BMS-265246 had a dose-dependent anti-HSV-1 activity in mice, improved the survival rate of mice, reduced weight loss, and improved the degree of pathological damage to lung tissue, and had high biosafety.

[0111] The in vivo and in vitro antiviral activity of BMS-265246 against herpes simplex virus HSV-2 is basically the same as that of HSV-1. This is because HSV-2 and HSV-1 have similarities in genome and glycoprotein structure, and the antiviral effects of HSV-2 and HSV-1 at the cellular level are almost the same. The in vivo antiviral activity against herpes simplex virus HSV-2 is also basically the same as that of HSV-1. This part of the data will not be repeated here.

[0112] In summary, BMS-265246 provided by the present invention is a new type of antiviral drug for herpes simplex virus, which has the advantages of good safety, high selectivity index, broad-spectrum anti-herpes virus, etc. It can be used to develop drugs for preventing and / or treating herpes virus infections, especially HSV-1 and HSV-2 infections, and expand the treatment options for herpes virus-infected diseases. It has broad application prospects and important clinical significance.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 present invention.

Claims

1. Use of a compound in the preparation of an anti-herpes virus drug, characterized in that: The herpes virus is herpes simplex virus type 1 and / or herpes simplex virus type 2; The structural formula of the compound is shown in Formula I: Formula I.

2. Use of the compound according to claim 1 in the preparation of anti-herpes virus drugs, characterized in that: The application is selected from at least one of an application in preparing a drug for inhibiting herpes virus replication and an application in preparing a drug for preventing and / or treating herpes virus infection diseases.

3. Use of the compound according to claim 2 in the preparation of anti-herpes virus drugs, characterized in that: The herpes virus infection disease is selected from one or more of herpes labialis, genital herpes and herpes simplex encephalitis.

4. Use of the compound according to claim 2 in the preparation of anti-herpes virus drugs, characterized in that: The medicine is prepared from a therapeutically effective amount of the compound and pharmaceutically acceptable auxiliary materials.

5. Use of the compound according to claim 4 in the preparation of anti-herpes virus drugs, characterized in that: The pharmaceutically acceptable excipient is selected from one or more of a dispersant, a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifier and a flavoring agent.

6. Use of the compound according to claim 4 in the preparation of anti-herpes virus drugs, characterized in that: The dosage form of the drug is selected from tablets, capsules, granules, pills or liquid preparations.

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