阅读说明：本技术 含有环丙基骨架的甲基转移酶抑制剂在治疗黄病毒属病毒感染药物中的应用 (Use of methyl transferase inhibitors containing a cyclopropyl backbone in a medicament for the treatment of a flavivirus infection ) 是由 吕凯 刘明亮 汪阿鹏 于 2021-03-31 设计创作，主要内容包括：本发明涉及医药技术领域,具体公开了一类含有环丙基骨架的甲基转移酶抑制剂在制备治疗黄病毒属病毒感染药物方面的应用,该类含有环丙基骨架的化合物包括GSK-LSD1,ORY-1001,DDP-38003,OG-L002中的一种或多种,其体外显示了较好的抗黄病毒活性。(The invention relates to the technical field of medicines, and particularly discloses application of a methyl transferase inhibitor containing a cyclopropyl skeleton in preparation of a medicament for treating flavivirus infection, wherein the compounds containing the cyclopropyl skeleton comprise one or more of GSK-LSD1, ORY-1001, DDP-38003 and OG-L002, and the compounds show better anti-flavivirus activity in vitro.)

1. The application of the methyl transferase inhibitor containing cyclopropyl skeleton in the preparation of the medicine for treating flavivirus infection is characterized in that the methyl transferase inhibitor containing cyclopropyl skeleton has any one of the following structures:

2. the use according to claim 1, wherein the flavivirus virus comprises Zika virus, anti-dengue virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus.

3. Use according to claim 1, wherein the methyl transferase inhibitor having a cyclopropyl backbone is used as the sole active ingredient in the manufacture of a medicament for the treatment of a flavivirus virus; or

The cyclopropyl skeleton methyl transferase inhibitor and other medicine are used together as active component in preparing medicine for treating flavivirus.

4. Use according to any one of claims 1 to 3, wherein the cyclopropyl skeleton-containing methyltransferase inhibitor comprises a cyclopropyl skeleton-containing methyltransferase inhibitor, 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, wherein the cyclopropyl skeleton-containing methyltransferase inhibitor is selected from the following compounds:

8. the use according to any one of claims 1 to 3, wherein the medicament for the treatment of a flavivirus infection comprises at least one pharmaceutically acceptable carrier or excipient.

9. The use according to claim 1, wherein the medicament for treating flavivirus infection is in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, oral liquids, gels, syrups, pills, tinctures, vints, ointments, lozenges, mixtures, suppositories, injections, inhalants or sprays.

Technical Field

The invention relates to the technical field of medicines, in particular to application of a methyl transferase inhibitor containing a cyclopropyl skeleton in a medicine for treating flavivirus virus infection.

Background

Flaviviridae family flaviviridae is a group of insect-borne viruses which are mainly transmitted by vectors such as mosquitoes, ticks and the like, and comprises more than 70 kinds of dengue viruses 1-4(DENV 1-4), Zika virus (ZIKV), Yellow Fever Virus (YFV), West Nile Virus (WNV), Japanese Encephalitis Virus (JEV), tick-borne encephalitis virus (TBEV) and the like. Such viruses can cause a variety of diseases including fever, hepatitis, hemorrhagic fever, and the like, seriously threatening human health, and even leading to death. Worldwide Health Organization (WHO) reports about 5000 to 1 million cases of DENV infection each year worldwide, resulting in about 2.2 million deaths. Currently, there is no clinically available drug for treatment of flavivirus infections.

Methyltransferases are widely present in prokaryotes to eukaryotes, and play important roles in many important physiological processes, such as gene expression and closure, DNA damage repair, synthesis and degradation of intermediates in physiological processes in vivo, and the like, by catalyzing methylation reactions of DNA, RNA, and proteins. In recent years, methyltransferase inhibitors have become a hot spot for research and development in the anti-tumor field, and the related research progress can be found in chem.rev.2018, 118, 989-1068. Among them, EZH2 (histone lysine N-methyltransferase) inhibitor EPZ-6438 (Tazemetostat, SAM competitive inhibitor) was marketed in early 2020 for the treatment of metastatic or advanced epithelioid sarcoma that cannot be surgically excised; EPZ5676(Pinometostat) is in phase II clinical study for the treatment of acute myeloid leukemia; GSK3326595 was in phase I clinical study for myelodysplastic syndrome and acute myeloid leukemia; ORY-1001 is in phase I clinical study for treatment of relapsed or refractory acute leukemia, etc. In addition, more structural types of compounds have been reported to exhibit excellent antitumor activity at a cellular or animal level, and more than 60 MTase inhibitors are commercially available.

However, there is no report on the use of methyltransferases for treating flavivirus infections.

Disclosure of Invention

The invention aims to provide application of a methyl transferase inhibitor containing cyclopropyl skeleton in preparing a medicament for treating flavivirus infection.

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

the invention provides an application of a methyl transferase inhibitor containing a cyclopropyl skeleton in a medicament for treating flavivirus infection, wherein the compound containing the cyclopropyl skeleton has any one of the following structures:

as some embodiments of the invention, the flavivirus virus includes Zika virus, anti-dengue virus, West Nile virus, Japanese Encephalitis Virus (JEV) and tick-borne encephalitis virus (TBEV).

As some embodiments of the present invention, the use of a compound comprising a cyclopropyl scaffold as the sole active ingredient in the manufacture of a medicament for the treatment of a flavivirus infection.

As some embodiments of the invention, the compound containing cyclopropyl skeleton is used together with other medicines as an active ingredient in the preparation of medicines for treating flavivirus infection.

Based on their effectiveness for treatment with flavivirus viruses, they can be used in combination with one or more other active ingredients to treat, prevent, inhibit or ameliorate 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 medicament for treating flavivirus infection contains 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.

The invention has the beneficial effects that:

experiments prove that the methyl transferase inhibitor containing cyclopropyl skeleton has obvious in-vitro flavivirus virus (Zika virus and dengue virus) activity, can be used for preparing a flavivirus medicament for treatment, and provides a new method for developing a medicament for resisting flavivirus infection.

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 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 Structure of methyltransferase inhibitors containing a cyclopropyl backbone

Example 1 in vitro anti-Zika Virus Activity of a methyl transferase inhibitor containing a cyclopropyl backbone (VERO cells)

Vero cells from non-human primates are taken as sensitive cells, and ZIKV SZ-WIV01(Genbank accession number: KU963796) which is separated from the port entry venereal disease cases in China in 2016 is taken as an infection strain. Firstly, inoculating the cells into a 96-well plate, and paving 70-80% of the cells after 24 hours; further diluting the tested compound by using a DMEM culture solution containing 2% FBS in a multiple ratio, inoculating the diluted tested compound to cells in a 96-well plate, and adding an equal volume of DMEM culture solution containing 2% FBS into a negative control well; culturing 96-well plate in a carbon dioxide incubator at 37 deg.C for 3-7 days (the specific time depends on cell line), and culturingThe Cell activity was measured by luminecent Cell Viability Assay (Promega), and the TC0, TC50 and TC90 of the drug were calculated from the measurement results. On the basis of the compound, the antiviral activity evaluation is carried out: firstly, inoculating cells into a 96-well plate, and paving 70-80% of the 96-well plate after 24 hours; the test compound was diluted in duplicate using DMEM medium containing 2% FBS, starting from the maximum nontoxic concentration of the compound (TC 0); further using 100 times of tissue infectious dose of virus (100TCID50) to infect the compound group and negative control group cells, and adding compounds with different concentrations into the test hole at the same time, wherein the cell control hole is not added with virus and compounds; culturing 96-well plate in carbon dioxide incubator at 37 deg.C for 3-7 daysThe Cell activity was measured by Luminescent Cell Viability Assay, and the dose-effect relationship was fitted using Origin software according to the measurement results to calculate IC50 for each compound, the results are shown in table 1.

TABLE 1 in vitro Activity of methyltransferase inhibitors containing a cyclopropyl backbone on Zika

Example 2 in vitro anti-Zika Virus Activity of methyl transferase inhibitors containing a cyclopropyl backbone (BHK cells)

BHK cells were used as sensitive cells, and ZIKV SZ-WIV01(Genbank accession number: KU963796) isolated from the case of entry-induced diseases at the port of China in 2016 was used as an infectious strain. Firstly, inoculating the cells into a 96-well plate, and paving 70-80% of the cells after 24 hours; further diluting the tested compound by using a DMEM culture solution containing 2% FBS in a multiple ratio, inoculating the diluted tested compound to cells in a 96-well plate, and adding an equal volume of DMEM culture solution containing 2% FBS into a negative control well; culturing 96-well plate in a carbon dioxide incubator at 37 deg.C for 3-7 days (the specific time depends on cell line), and culturingThe Cell activity was measured by luminecent Cell Viability Assay (Promega), and the TC0, TC50 and TC90 of the drug were calculated from the measurement results. On the basis of the compound, the antiviral activity evaluation is carried out: firstly, inoculating cells into a 96-well plate, and paving 70-80% of the 96-well plate after 24 hours; the test compound was diluted in duplicate using DMEM medium containing 2% FBS, starting from the maximum nontoxic concentration of the compound (TC 0); further using 100 times of tissue infectious dose of virus (100TCID50) to infect the compound group and negative control group cells, and adding compounds with different concentrations into the test hole at the same time, wherein the cell control hole is not added with virus and compounds; culturing 96-well plate in carbon dioxide incubator at 37 deg.C for 3-7 daysThe Cell activity was measured by Luminescent Cell Viability Assay, and the dose-effect relationship was fitted using Origin software according to the measurement results to calculate the IC50 for each compound, as shown in table 2.

TABLE 2 in vitro Activity of methyltransferase inhibitors containing a cyclopropyl backbone on Zika

Example 3 in vitro anti-dengue Virus Activity of DDP-38003 & 2HCl

BHK cells were used as sensitive cells, and dengue II virus international reference strain (DENV-NGC; Genbank accession number: AF038403.1) was used as an infectious strain. Firstly, inoculating the cells into a 96-well plate, and paving 70-80% of the cells after 24 hours; further diluting the tested compound by using a DMEM culture solution containing 2% FBS in a multiple ratio, inoculating the diluted tested compound to cells in a 96-well plate, and adding an equal volume of DMEM culture solution containing 2% FBS into a negative control well; the 96-well plate was placed in a carbon dioxide incubator at 37 ℃ for 3 to 7 days (the specific time varies depending on the Cell line), the Cell activity was measured using CellTiter-glominnescent Cell Viability Assay (Promega), and TC0, TC50 and TC90 of the drug were calculated from the measurement results. On the basis of the above, the antiviral activity evaluation of the compound DDP-38003 & 2HCl was carried out: firstly, inoculating cells into a 96-well plate, and paving 70-80% of the 96-well plate after 24 hours; the test compound was diluted in duplicate using DMEM medium containing 2% FBS, starting from the maximum nontoxic concentration of the compound (TC 0); further using 100 times of tissue infectious dose of virus (100TCID50) to infect the compound group and negative control group cells, and adding compounds with different concentrations into the test hole at the same time, wherein the cell control hole is not added with virus and compounds; the 96-well plate is placed in a carbon dioxide incubator at 37 ℃ for 3-7 days, Cell activity is measured by using CellTiter-Glo Luminescent Cell vitality Assay, and according to the measurement result, dose-response relationship is fitted by using Origin software, and IC50 of each compound is calculated.

The activity of DDP-38003 · 2HCl on dengue virus (IC50 ═ 5.23 ± 1.21 μ M) was found to be slightly weaker than the activity of the control drug NITD008 (IC50 ═ 0.65 ± 0.12 μ M).

EXAMPLE 4 injection

Dissolving DDP-38003 & 2HCl compound 150g in water, dissolving sodium chloride and ethyl p-hydroxybenzoate in hot water, mixing, and adjusting pH to 5-7. Diluting the injection water to 1000ml, filtering with hollow fiber membrane, bottling, and sterilizing.

The invention develops new medical application of a methyl transferase inhibitor containing a cyclopropyl skeleton, develops new application field of the compound in preparing medicaments for treating flavivirus infection, has stronger inhibition effect on Zika, dengue fever and other viruses, and provides an alternative for clinically treating diseases caused by flavivirus infection. The compound can be prepared into a preparation form suitable for clinical use by adopting a modern pharmaceutical process, and the treatment accuracy and the patient compliance are improved.

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.