TW202139995A - Use of ovatodiolide against sars-cov-2 - Google Patents

Abstract

This subject invention is directed to a compound of formula I (Ovatodiolide) which is safe and effective to use in a pharmaceutical composition for inhibition of SARS-CoV-2. The pharmaceutical composition comprising a safe and effective amount of a compound of formula I (Ovatodiolide) or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier, which has great potential to treat SARS-CoV-2 induced COVID-19 based on a safe and effective amount of a compound of formula I (Ovatodiolide).

Description

Use of Idylactone against the new coronavirus

The focus of the present invention is the use of the compound of formula I-Ovatodiolide (Ovatodiolide; Ova) against new coronaviruses; however, in the research and development process, the preparation, identification and analysis of the natural compound of formula I-Ovatodiolide (Ova) Basic toxicological tests, molecular docking simulation of the mechanism of action of the compound of formula I-dorphyllide against the new coronavirus, and the biochemical test confirmation of the compound of formula I-dorphyllide against the new coronavirus, etc., to discuss and confirm the formula in order The compound I-Codylactone can be produced in appropriate quantities and is safe and sound. It has an anti-new coronavirus mechanism and practical effect; the compound of Formula I-Codylactone is indeed a potential inhibitor of the new coronavirus (SARS-CoV). -2) Infected natural products can be used to develop medicines for the prevention and treatment of severe and special infectious pneumonia (COVID-19).

At present, because the world has not developed an effective preventive vaccine or effective treatment for the new coronavirus; the new coronavirus has spread across all continents, causing severe and special infectious pneumonia to be a global pandemic, and many countries and various multinational pharmaceutical companies are actively doing it. Research and develop vaccines or drugs that can be effectively prevented.

Anisomeles indica O.Kuntze is a commonly used herbal medicine in Taiwan, and it is also known as guest moss (Jiaoling, Meixian, Guangdong), golden sword grass, and ageratum. Taiwan's Ministry of Health and Welfare has included the fish needle grass in the comprehensive list of raw materials available for food, and the whole plant is edible. Fish needle grass is one of the annual or perennial herbaceous plants of the Labiatae family. Fish needle grass is mainly distributed in southwest China, India, the Philippines, Indonesia, Java and Sumatra. It can be found in the plains of Taiwan to low-altitude mountainous areas. Taiwan Hualien Yuli is also sporadically cultivated for medicinal purposes. Generally, folk medicinal use is collected in summer and autumn, uprooting the whole grass or cutting off the ground parts, washing it, and using it fresh or sun-dried. The whole herb has antipyretic, wind-removing, dehumidification, stomach invigorating, detoxification, pain relief, and antibacterial effects. It is commonly used in the folk to treat colds and fever, abdominal pain and vomiting, cholera, stomach pain, gastroenteritis, neurodermatitis, rheumatism, bone pain, eczema, swelling toxins, sores, defecation, poisonous snake bites.

Our research and development team has been breeding needle grass for a long time (GenBank: GU726292), and continues to carry out a series of researches on the whole plant extracts of fish needle grass grown on the farm, especially focusing on the preparation of the pure crystalline substance of the compound of formula I-needle grass lactone ( Figure 2), specifically performed extraction, separation, purification, analysis and identification, as well as anti-inflammatory, anti-virus, anti-Helicobacter pylori, anti-cancer, anti-cancer stem cell and other pharmacological studies. In recent years, our team has completed the test experiment of the compound of formula I-Idylactone in the treatment of type A and type B influenza drugs "Keflu" (Roche Pharmaceuticals: Tamiflu) Idylactone has a good effect on inhibiting influenza virus (a kind of coronavirus). It was recently learned at the end of 2019 that AIDS drugs showed a positive response to the treatment of patients with new coronavirus infections; press, according to the literature report that avidin can inhibit HIV AIDS (Fitoterapia, 2000, 71(5): 574-576) .) . In addition, existing studies have also found that Idrelactone can inhibit the gastritis caused by Helicobacter pylori in the stomach wall, and can also inhibit the inflammatory response mediated by NF-κB and STAT3. Therefore, the compound of formula I-Idrelactone may also alleviate Symptoms of pneumonia caused by the new coronavirus.

The present invention is based on the discovery that the compound of formula I-avidin or its pharmaceutically acceptable salt, can be used to inhibit new coronavirus infection, and even treat or prevent severe and special infectious lungs. inflammation. In particular, the present invention provides a pharmaceutical composition for inhibiting novel coronavirus infection, and even treating or preventing pneumonia, comprising a safe and effective amount of the compound of formula I-avidin or its pharmaceutically acceptable salt, and A pharmaceutically acceptable carrier.

The present invention mainly provides a pharmaceutical composition which takes the compound of formula I-avidylactone as the effective ingredient for inhibiting the infection of the novel coronavirus. The simulation results of the molecular docking of the compound of formula I-Idylactone to the receptor-binding domain (RBD) of the new coronavirus surface spike glycoprotein receptor-binding domain (RBD) showed that the compound of formula I-Idylactone (Ova) binds To RBD several hydrophobic amino acids (L455, F456, Y489, F490) constitute a hydrophobic pocket, and form hydrogen bonds with Y489 and Q493 (Figure 3). The binding site is at the interface where the new coronavirus spike glycoprotein RBD binds to the human cell membrane receptor angiotensin-converting enzyme2 (ACE2), and it is predicted that the compound of formula I-Ova lactone (Ova) It can block or interfere with the direct binding of the viral spike glycoprotein receptor binding domain (RBD) to the receptor (ACE2). The binding of the new coronavirus surface spike glycoprotein to the human cell membrane receptor angiotensin-converting enzyme-2 (ACE2) is a key step in mediating the virus to invade the host. Blocking or interfering with the binding of the virus to the receptor is a potential prevention and treatment Strategy.

At the same time, it is understood that the host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L play a key role in the fusion process of the new coronavirus. The results of simulated molecular docking also showed that the compound of formula I-Ova may also bind to the catalytic pockets of endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L. The compound of formula I-fish needle lactone (Ova) binds to the hydrophobic S2 site of Cathepsin B composed of Y75, P76, A173, A200, and E245 with a hydrophobic alicyclic ring, and is formed by the exocyclic olefin and the catalytic cysteine C29 The covalent complex thus inhibits the activity of Cathepsin B (Figure 4A). On the other hand, the compound of Formula I-Ova lactone (Ova) binds Cathepsin L with a hydrophobic alicyclic ring to the hydrophobic S2 composed of L69, M70, Y72, A135, and M161 Site, and through the exocyclic olefin and the catalytic cysteine C25 to form a covalent complex to inhibit the activity of Cathepsin L (Figure 4B). Since the endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L play a key role in the fusion process of the new coronavirus, the compound of formula I-Ova may potentially block the invasion and fusion process of the new coronavirus .

This study specifically implements the new coronavirus pseudovirus inhibitory activity detection system developed by the laboratory of Professor Zhang Linqi, director of the Comprehensive AIDS Research Center of Tsinghua University, Beijing, specifically to evaluate whether the compound of formula I-avidylactone blocks the process of host cell infection by the new coronavirus . The experimental results show that the compound of formula I-codylactone is specifically different from chloroquine (chloroquine) or remdesivir (Remdesivir) in the molecular mechanism of inhibiting the new coronavirus. The compound of formula I-codylactone is micromolar The grade significantly shows the inhibitory effect on the new coronavirus infection (Figure 5).

The compound of the formula I-midrolactone may possess one or more opposing centers, and therefore have various stereoisomeric forms. The compound of formula I mentioned in the present invention-avidactone includes all these isomers; in addition, it also includes derivative compounds containing the main structure of the compound of formula I-avidactone, and these derivative compounds are effective in inhibiting The molecular mechanism of the novel coronavirus is the same as the molecular docking mechanism disclosed by the present invention that combines the surface spike glycoprotein receptor binding region of the novel coronavirus, or combines the molecular docking mechanism of the host endosome cysteine proteolytic enzymes Cathepsin B and Cathepsin L, Those with similar effects. The compound of formula I-avidylactone has the effect of selectively inhibiting new coronavirus infection; because of its extremely small molecular weight, a lower dose of the compound of formula I-avidrolactone or its pharmaceutically acceptable salt can be used, With a pharmaceutically acceptable carrier, the desired therapeutic effect can be obtained. The present invention is a pharmaceutical composition for inhibiting novel coronavirus infection, and even treating or preventing severe special infectious pneumonia (COVID-19). It combines a safe and effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof with the medicine Acceptable carrier, used to suppress the new coronavirus, or administered to the lung with severe special infectiousness Patients with symptoms of COVID-19, for the purpose of curing, restoring, alleviating, alleviating, changing, treating, improving, improving or affecting the disease, the symptoms of the disease, or the physique prone to the disease. As used herein, "an effective amount" refers to an effective amount of the compound of formula I-Idylactone or a pharmaceutically acceptable salt thereof, which has an inhibitory or therapeutic effect. The effective amount is changed according to the route of administration, excipient usage, and co-usage with other active agents.

"Severe special infectious pneumonia (COVID-19)" here means the fatal pneumonia caused by the new coronavirus (SARS-CoV-2) invading the human body. The new coronavirus uses the spike glycoprotein on the surface of the coronavirus to recognize the angiotensin converting enzyme-2 (ACE2) on the cell surface, and then infect normal cells in the human body. One possible mechanism is that when the virus invades the body, the immune cells in the body act fiercely, triggering an immune storm in the body, releasing a large amount of free radicals (such as free peroxide), which causes protein denaturation, DNA damage, and excessive production of cytokines, resulting in a large number of cells. Necrosis, severely fatal pneumonia forms in the lungs. The compound of formula I-avidylactone can effectively inhibit the new coronavirus infection, thereby preventing or treating severe special infectious pneumonia (COVID-19).

The compound of the formula I-Idylactone is obtained by extracting the whole grass, above-ground branches and leaves, or leaves of Idlings with organic solvent, and then separating and purifying it by a silica gel column; or it is prepared by chemical synthesis method. For example: "Needleweed extract" refers to the extract of needleweed that is more suitable for growth. In order to obtain the Ichthys nobilis extract, extraction techniques well known in the art can be used. For example, the dried and ground ichthyris may be suspended in a solvent or a mixture of two or more solvents for a sufficiently long period of time. Examples of suitable solvents include, but are not limited to: water, methanol, ethanol, acetone, ether (such as diethyl ether) and ethyl acetate and hexane ( hexane). Afterwards, the solid residue is removed (for example, by filtration) to obtain the fish needle grass extract solution, which can be passed through alumina, silica, silica gel The column purification prepares the compound of formula I-ichthyolactone.

In the treatment method of the present invention, the compound of formula I-dorphyllide or its pharmaceutically acceptable salt can be administered simultaneously or separately, orally, parenterally, via inhalation spray or by Implanted reservoir (implanted reservoir) method. "Non-oral" as used here refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, and intrasynovial , Intrasternal (intrasternal) subarachnoid space (intrathecal), disease site (intraleaional) and intracranial (intracranial) injection and perfusion technology. The compound of formula I used in the present invention-avidactone and/or its pharmaceutically acceptable salt can be combined with at least one solid, liquid or semi-liquid excipient or adjuvant to form a suitable pharmaceutical form. The forms include, but are not limited to, tablets, capsules, emulsions, aqueous suspensions, dispersions and solutions. Carriers generally used for lozenges include lactose and corn starch. A lubricating agent, such as magnesium stearate, is also generally added to the lozenge. Diluents used in the capsule form include lactose and dried corn starch. When oral administration is an aqueous suspension or emulsion, the active ingredient can be suspended or dissolved in an oily phase combined with an emulsifying or suspending agent. If necessary, specific sweetening, flavoring and coloring agents can be added. The compound of formula I used in the present invention-midrolactone or its pharmaceutically acceptable salt can also be formulated as a sterile injection component (for example, a water or oil suspension), for example, using techniques known in the art Use suitable dispersing or wetting agents (such as Tween 80) and suspending agents. Sterile injection preparations can also include sterile injection solutions or suspensions in non-toxic non-oral diluents or solvents, such as 1,3-Butanediol (1,3-Butanediol). Available vehicles (Vehicles) and solvents include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are often used as solvents or suspension vehicles (for example, synthetic mono- or di-glycerides). Fatty acids, such as oleic acid and its glyceride derivatives, can also be used in the preparation of injections. They are natural pharmaceutically acceptable oils, such as olive oil and castor oil, especially for their polyoxyethylene. Polyoxyethylated (polyoxyethylated) variants. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersants. The compound of formula I used in the present invention-midrolactone or its pharmaceutically acceptable salts can also be formulated into an inhalation component according to techniques well known in this technical field. For example, it can be made into a salt solution, using benzyl alcohol or other suitable preservatives, bioavailability-enhancing adsorption promoters, fluorocarbons or other additives well known in the art. Soluble or dispersant to formulate. The carrier used in the pharmaceutical composition must be "acceptable", compatible with the active ingredients of the formulation (and preferably have the ability to stabilize the active ingredients) and not harmful to the patient. For example, cosolvents (such as cyclodextrins) (which form specific and more soluble complexes with one or more active compounds of the extract) are used as pharmacological adjuvants for the delivery of effective ingredients. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

In addition, antiviral agents are prone to side effects when administered to patients in high doses. Based on the results of a series of toxicological experiments on the compound of formula I-civic lactone disclosed in the present invention, including a single-dose oral acute toxicity test for rats, a 28-day feeding toxicity test for rats, and an Ames test for Salmonella reverse mutation , Chromosome abnormality analysis test on mammalian cell lines in vitro, on peripheral blood of mice Micronucleus tests, etc., all show that the compound of formula I-avidin is not genotoxic, and provides a safe oral dose range. The pharmaceutical composition of the present invention contains a safe and effective amount of the compound of formula I-ichola lactone, which is used to inhibit new coronavirus infection, wherein the safe and effective amount is for a general adult (60 kg body weight): within 480 mg orally per day, And to continue taking within 28 days as a limit. It is preferably a general adult (60 kg body weight): 20 mg to 40 mg orally orally every day, and it is appropriate to continue taking it for 7 to 14 days. The specific dose administered to an individual patient depends on all possible factors, such as: the activity of the specific compound used, age, weight, general health, gender, eating status, administration time and route, excretion rate, and drug substance The combination, and the severity of the disease to be treated, etc.

The present invention also provides the use of a composition for preparing a drug for inhibiting coronavirus, wherein the composition includes a compound of formula I-Ovatodiolide, or a compound of formula I-the structural isomer of Ovatodiolide Things.

Figure 1. The formula I compound-Ovatodiolide (Ovatodiolide) structural formula.

Figure 2. X-ray ORTEP diagram of the compound of formula I-the crystals of fish needle grass lactone.

Figure 3. The structure of the complex of the new coronavirus spike glycoprotein receptor binding region RBD and the human cell membrane receptor ACE2, and the docking structure of the compound of formula I-Ova and RBD, the compound of formula I-Idylla Lactone (Ova) binds to RBD and interferes with its binding to ACE2. Labels in the figure: ACE2: Angiotensin-converting enzyme 2; nCov S-RBD: Novel coronavirus spike glycoprotein receptor binding domain; Ovatodiolide (Ova): Formula I compound-Idylactone.

Figure 4. The docking structure of the compound of formula I-Ova lactone (Ova) and the cysteine proteolytic enzymes Cathepsin B (A) and Cathepsin L (B), wherein the compound of formula I-Ova lactone (Ova) ) The catalytic cysteine C29 of Cathepsin B with the exocyclic olefin, and Cathepsin L catalyzes the formation of covalent bonds with cysteine C25. Labels in the figure: Ovatodiolide (Ova): the compound of formula I-zinnia lactone; Cathepsin B: cysteine proteolytic enzyme B; Cathepsin L: cysteine proteolytic enzyme L.

Figure 5. The activity curve of the compound of formula I- _{midrolactone in inhibiting the novel coronavirus infection, IC 50} =3.73μM.

In order to make the above-mentioned and other objects, features, and advantages of the present invention more obvious and understandable, the following specifically refers to preferred embodiments, which are described in detail as follows:

Example 1. The preparation and analysis of the compound of formula I-Idrelactone: Take the roughly dried leaves (800g) of Idrel (annual, harvested in autumn in Yuli, Hualien, Taiwan), and put it in an oven (40°C) for drying ( 24 hours), can get dried fish needle grass leaves (500g). Put the dried leaves (500g) of acupuncture needles in a 20-liter bucket (PE material), and add 10 liters of 95% alcohol to ensure that all leaves are soaked in the solvent, and then seal the bucket in a cool place for 7 days. After 7 days, the 95% alcohol layer and leaves were separated by filtration and concentrated by a rotary evaporator to obtain a yellow-green extract (10 g). Purification was performed by alumina column chromatography (neutral alumina: 300 g). The yellow-green extract (10 g) was dissolved with 10 mL of acetone and added to the packed alumina column. Solvent extraction ratio Hexane: Ethyl acetate/100%: 0%, gradually increase the polarity to Hexane: Ethyl acetate/70%: 30%. Through the screening and comparison of thin-layer chromatography test paper (TLC), the compound of formula I-dorphyllactone can flow out of the column in the third section. After the volatile matter is drained by a rotary evaporation concentrator, 15 mL of acetone is added to re-dissolve it, and the solvent diffusion method is used to grow crystals. After seven days, 1.56 g of the crystals of the transparent compound of formula I-Idylactone can be obtained, and the yield is about 0.3 %. It is identified by X-ray, NMR, IR, Mass, HPLC and other instruments. The purified crystals were determined to be the compound of formula I-avidin. Through drying, extraction, purification and identification, we succeeded in isolating the compound of formula I-ovatodiolide (ovatodiolide) from the needle grass, and its yield to the leaves was about 3000 ppm, but the leaves only accounted for the needle grass. Less than 7% of the whole plant, and it is not easy to collect. The compound of formula I-ovatodiolide analysis and identification: X-ray Crystals of ovatodiolide are Orthorhombic, space group P2 ₁ 2 ₁ 2 ₁ , with a=10.7714(3), b=12.8674(3), c=13.0829(3 ),V=1813.29(8)Å ³ ,D(calculated)=1.203Mg/m ³ ,Z=4,Formula weight=328.39,Goodness-of-fit on F ² =1.056,R indices(all data): R1 =0.0347,wR2=0.0942. ORTEP diagram is depicted as Figure 2 (Figure 2). ¹ HNMR(400MHz,CDCl ₃ ): 1.59(s,3H),1.62(m,1H),1.64(m,1H),1.72(s,3H),2.04 (m, 1H), 2.12 (m, 1H), 2.19 (m, 1H), 2.26 (dd, 1H), 2.39 (m, 1H), 2.45 (m, 1H), 2.52 (m, 1H), 2.80 ( m, 1H), 2.86 (dd, 1H), 4.81 (m, 1H), 4.85 (bd, 1H), 5.08 (m, 1H), 5.12 (bd, 1H), 5.57 (bs, 1H), 6.12 (bs , 1H), 6.98 (bs, 1H). ¹³ CNMR (125MHz, CDCl ₃ ): 15.1, 19.3, 23.7, 24.9, 33.3, 36.3, 40.3, 42.7, 77.9, 78.8, 122.9, 125.0, 129.1, 131.2, 134.3, 134.5, 139.6, 147.4, 170.4, 173.0. FTIR (KBr pellet): 3100,2900,1740,1650,1430,1395,1320,1200,1110,1045,1080,980,960,930,910,880,860,820,750,625cm ¹ and HRMS(ESI) m/z calcd.for C ₂₀ H ₂₄ O ₄ (M+ ) 328.1675, found 328.1672.

Example 2. Single-dose oral acute toxicity test of the compound of formula I-dorphyllactone in rats: This example is a safety test of the single-dose oral acute toxicity test of the compound of formula I-dorphyllide in rats , Provide reference for food safety assessment. The test is based on the Health Food Safety Assessment of Taiwan's Ministry of Health and Welfare-Single-dose oral acute toxicity test, US Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, OPPTS 870.1100, Acute oral toxicity, US EPA 712-C-98-190.In: OPPTS) Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 420: Acute Oral Toxicity-Fixed Dose Procedure, No. 423: Acute Oral Toxicity-Acute Toxic Class Method, No. 425: Acute Oral Toxicity-Up and Down Method) and other test specifications for single-dose oral acute toxicity test. In this experiment, a single-dose oral acute toxicity test of the compound of formula I-dawthorn lactone in rats (Sprague-Dawley, SD strain) was carried out. The compound of formula I-dorphyllactone is yellowish crystalline, and the test purity is 99.95%. The concentration of the solution is 0.1g/mL with 10% DMSO during the test, and the feeding volume of each rat is 10mL/kg body weight , Rats were orally fed according to their body weight on the same day, and the final dose was 1g/kg-body weight in total, and they were observed for 14 consecutive days after administration. The results showed that after oral administration of the compound of formula I-avidin, all the rats had no symptoms of poisoning or died. In terms of weekly weight change (g), there was no significant difference in the weekly weight and weight gain of male and female rats in the treatment group compared with the control group. After the test, the blood value changes of male and female rats in the treatment group included: total white blood cell (WBC count), total red blood cell (RBC count), hematocrit ratio (Hct), average red blood cell volume (MCV), average hemoglobin ( MCH), mean hemoglobin concentration (MCHC), the total number of platelets and the classification of white blood cells have no obvious abnormalities. Serum liver and kidney enzyme values of male and female rats in the treatment group, including: aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), and creatinine (creatinine) No effect. In terms of the absolute weight of internal organs (g) and the percentage of organ weight, there was no significant difference between the treatment group and the control group in the adrenal gland, brain, heart, kidney, liver, spleen, thymus, testicle, or ovary. After examination of internal organs, the adrenal gland, brain, heart, kidney, liver, spleen, thymus, testicles or ovaries in the treatment group had no macroscopic lesions. The results of histopathological examination showed that none of the important organs in the compound of formula I-dorphyllide treatment group had any histopathological changes related to the test substance. Comprehensive test results show that the single-dose oral acute toxicity test of 1g/kg-body weight (converted to a human applicable dose of about 50mg/kg-body weight) of the compound of formula I-dorphyllide did not cause acute poisoning or Death, and no pathological changes related to toxic reactions in tissues and organs were caused to important organs in the body.

Example 3. A 28-day feeding toxicity test of compound of formula I-dorphyllide to rats: This example is to test the repeated dose oral toxicity safety test of compound of formula I-dorphyllide to rats to establish The material safety data sheet provides a reference for the clinical safety assessment of repeated use of the human body. The test is based on the 28-day feeding toxicity test (1999) and drug non-clinical trial safety regulations (2014) of the health food safety assessment of the Taiwan Ministry of Health and Welfare, and complies with the U.S. Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, OPPTS 870.1100, Repeated Dose 28) -Day Oral Toxicity Study in Rodents.In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No.407 : Repeated Dose 28-day Oral Toxicity Study in Rodents) and other test specifications. This experiment is to investigate whether the compound of formula I-aviola lactone may cause potential side effects to humans, for clinical safety assessment, and to observe the clinical side effects of the compound of formula I-aviola lactone on the 28-day repeated dose oral toxicity in rats test. The compound of formula I-dorphyllactone is yellowish crystals, and the test purity is regarded as 99.95%. The sample is prepared with 5% DMSO during the test. Rats (Sprague-Dawley, SD strain) are divided into control group (5% DMSO), low-dose group (10mg/kg-body weight), medium-dose group (25mg/kg body weight) and high-dose group (50mg/kg) -body weight) and other 4 groups, each with 20 rats, half male and half, each rat was fed with a volume of 10 mL/kg-body weight, and the rats were orally fed according to their body weight on the same day for 28 consecutive days. The test results showed that after 28 days of continuous oral administration of the compound of formula I-avidin, all the rats had no symptoms of poisoning or death due to the test substance. After the test, the weight change, feed consumption, urine, blood value, serum enzyme value and organ weight of the male and female rats in each treatment group of the compound of formula I-dorphyllide were compared with those of the control group. There is a slight increase or decrease due to individual differences, but it is still within the normal range, or there is no correlation between dose and response between groups, and has no clinical pathological significance, and has nothing to do with the test substance. There were no obvious gross pathological changes in the whole body organs of the rats in each group. The results of histopathological examination showed that the high-dose group did not cause organ toxicity-related pathological changes to the various organs in the body. Based on the above inspection results, it is shown that the compound of formula I-dorphyllide was administered in the low-dose group (10mg/kg-body weight), the medium-dose group (25mg/kg-body weight) and the high-dose group (50mg/kg-body weight). weight) After 28 days of continuous oral feeding to rats, it did not cause toxic reactions in the various organs of male and female rats. The "No Observed Adverse Effect Level" (NOAEL) of the 28-day feeding toxicity test for rats was 50mg/kg-body weight, converted to a human applicable dose of about 8mg/kg-body weight.

Example 4. Ames test of the compound of formula I-the reversal mutation of Salmonella typhimurium against Salmonella typhimurium: This example is to test the reversion of Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 The Ames test is used to establish the Material Safety Data Sheet, which provides a reference for the use of safety assessment. The test is based on the genotoxicity study (1999) of the "Health Food Safety Assessment Method" of the Ministry of Health and Welfare of Taiwan (1999), the U.S. Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, Bacterial Reverse Mutation Test, US EPA 712-C-98- 247.In: OPPTS Harmonized Test Guidelines, Series 870.5100, 1998) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 471: Bacterial Reverse Mutation Test, 2002) and other test specifications Variability test. In this experiment, the Ames test of the compound of formula I-sauroplaston against Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 strains back mutation. In the test, the concentration of 1.25, 2.5, and 5 mg/plate of the compound of formula I-squirrel lactone, etc., was used together with the strain for 18-20 hours to conduct a bacterial toxicity test. The results showed that the compound of formula I-codylactone was not significantly toxic to the TA102 strain below 5mg/plate, but it was toxic to the TA98, TA100, TA1535 and TA1537 strains; therefore, the compound of the formula I-codylactone was used for Concentrations such as 0.63, 1.25 and 2.5mg/plate were combined with TA98, TA100, TA1535 and TA1537 strains for 18 hours to conduct bacterial toxicity test. The results showed that the compound of formula I-codola lactone below 2.5 mg/plate had no significant toxicity to the TA98, TA100, TA1535 and TA1537 strains. The highest concentration of the compound of formula I-zodiac lactone that has no significant toxicity to the TA102 strain was continuously diluted by 2 times, and five concentrations of 0.31, 0.63, 1.25, 2.5 and 5mg/plate were selected as the formal Ames test; and TA98, The highest concentration of TA100, TA1535 and TA1537 strains without significant toxicity was continuously diluted down by 2 times, and 5 concentrations of 0.16, 0.31, 0.63, 1.25 and 2.5 mg/plate were selected as the formal Ames test, and the compound of formula I-Yu needle grass The lactone is directly mixed with the rat liver activating enzyme extract (S9) and then acts on the Salmonella mutant strain, thereby mimicking the metabolites of the compound of formula I-dorphyllide after being metabolized by the liver enzyme (S9) in the animal’s body. For the genetic variability of the strains, count the amount of bacteria after 48 hours of co-cultivation. The results showed that, no matter whether the compound of formula I-midrolactone was directly or after S9 action, the number of reverse mutant bacteria on the bacteria was not more than 2 times the number of reverse mutant bacteria in the negative control group. Based on the above results, the compound of the formula I-avidin is not mutagenic to the Ames test of Salmonella reverse mutation, and the result of the bacterial gene mutation test is negative (non genetic mutation in Ames test).

Example 5. Analysis of chromosome abnormality of compound of formula I-dorphyllide on mammalian cell lines in vitro: This example is to test the analysis of compound of formula I-dorphyllide on chromosome abnormality of mammalian cell line in vitro, so as to establish product safety information (Material Safety Data Sheet), provides a reference for the use of safety assessment. The test is based on the in vitro mammalian cell line chromosome aberration analysis test of the Ministry of Health and Welfare of Taiwan, and complies with the U.S. Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, OPPTS 870.1100, In vitro mammalian chromosome aberration test, US EPA 712-C-98-190) .In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366, 1998) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No.473: In vitro mammalian chromosome Aberration test 1997) and other test specifications for genotoxicity tests. In this experiment, the chromosomal aberration test with mammalian cell in culture, the compound of formula I, mirin. The compound of the formula I-dactylactone is yellowish crystals, and was prepared with Dimethyl sulfoxide (DMSO) in the test. The cytotoxicity test is divided into two parts. One part uses CHO-K1 cells to directly interact with the compound of formula I-halide, and the other part uses rat liver activating enzyme extract (S9) to simulate human metabolism. CHO-K1 cells and large The rat liver activated enzyme extract (S9) and the compound of formula I-dorphyllactone are mixed for action. In the test without rat liver activating enzyme extract (-S9), a 24-hour cytotoxicity test was carried out with 5 test doses of 12.5, 15, 17.5, 20 and 25μM. The results showed that the 17.5μM compound of formula I-Yu needle grass The survival rate of lactone to CHO-K1 cells is about 65.6%. In addition, in the test containing rat liver activating enzyme extract (+S9), a 24-hour cytotoxicity test was carried out at five test doses of 60, 70, 75, 80 and 90 μM. The results showed that 75 μM of the compound of formula I-Yu needle grass The survival rate of the ester on CHO-K1 cells is about 60.3%, which shows that the compound of formula I-dorphyllide is cytotoxic to CHO-K1 cells. This concentration is selected as the highest dose in the formal test. Cell chromosome variation test Take samples of the compound of formula I-halide at 12.5, 15 and 17.5μM (-S9), prepare the sample solution in each cell culture dish, and incubate for 24 hours; the other at 55, 65 and 75μM After co-cultivating with S9 for 3 hours, observe whether the number and structure of cell chromosomes are normal for 24 hours. The results showed that the compound of formula I-dorphyllide was tested at 12.5, 15 and 17.5μM (-S9) and 55, 65 and 75μM (+S9), etc. under or without S9 mixture metabolic activation system test conditions The frequency of chromosomal abnormalities in CHO-K1 cells caused by the three dose groups did not increase significantly compared with the negative control group, and there was no significant change in the location of cell chromosomal mutations. Based on the above results, it is shown that no matter whether it contains the S9 mixture or not, the compound of formula I-halide has no mutagenic effect on the chromosomes of the mammalian cell line CHO-K1 in vitro.

Example 6. The micronucleus test of compound of formula I-dorphyllactone on peripheral blood of mice: This example is to test the genotoxicity test of compound of formula I-dorphyllide, so as to establish the product safety data (Material Safety Data). Sheet), provides a reference for the use of safety assessment. The test is based on the mouse peripheral blood micronucleus test of the Ministry of Health and Welfare of Taiwan-Genotoxicity Test, and complies with the U.S. Environmental Protection Agency (USEPA) (Mammalian Erythrocyte Micronucleus Test, In: OPPTS Harmonized Test Guidelines, Series 870.5395, EPA 712- C-98-226) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 474: Mammalian Erythrocyte Micronucleus Test, 1997) and other test specifications for genotoxicity testing. In this experiment, the micronucleus test of the compound of formula I-midrolactone in the peripheral blood of mice (ICR strain) was carried out. This test mainly tests the ratio of the compound of formula I-halide to the occurrence of micronuclei in the peripheral blood of rodents, in order to assess the degree of damage caused by gene mutations that directly or indirectly trigger red blood cell chromosomes or mitosis. The test takes ICR mice as the test object, and the test is divided into a negative control group, a positive control group (Cyclophosphamide, 60mg/kg bw ip), a low-dose compound of formula I-dorphyllide (0.25g/kg bw), and a medium-dose 5 groups (0.5g/kg bw) and high-dose (1g/kg bw), 5 mice (male) in each group, and a single feeding of idenoidin via a gastric tube, the test substance was administered for 48 hours And 72 hours later, evaluate the incidence of micronuclei in reticulocytes and reticulocytes in the peripheral blood of the mice (‰). The results showed that 48 hours and 72 hours after administration of the compound of formula I-avidin, there were no toxic symptoms and weight differences in the treatment groups. The reticulated erythrocytes in the peripheral blood of the mouse were stained with 0.1% Acridine orange stain under a fluorescent microscope to be orange-red. In the reticulated erythrocytes, yellow-green fluorescent micronuclei with the size of about 1/20-1/5 of the erythrocytes can be seen. There was no significant difference between the 48-hour and 72-hour reticulated erythrocytes and the number of micronuclei in the reticulated erythrocytes in the treatment groups of the compound of formula I-dorphyllactone and the negative control group. Compared with the negative control group, the number of reticulated red blood cells of mice in the positive control group decreased significantly (p<0.05), and the number of micronuclei in the reticulated red blood cells also increased significantly (p<0.05). Based on the above results, there is no significant difference in the number of reticulated erythrocytes and micronuclei in the reticulated erythrocytes in the peripheral blood of the mice in each dose group of the compound of formula I-dorphyllide and the negative control group, and the test result is negative. Therefore, the compound of the formula I-dorphyllide does not have the toxic effect of chromosomal gene mutation on peripheral red blood cells of mice.

Example 7. The molecular docking simulation study of the compound of formula I-dorphyllide binding to the surface spike glycoprotein receptor binding region of the novel coronavirus: the surface spike glycoprotein of the novel coronavirus and the human cell membrane receptor angiotensin converting enzyme 2 (angiotensin-converting enzyme 2, ACE2) binding is a key step in mediating the virus to invade the host, and blocking or interfering with the binding of the virus to the receptor is a potential prevention and treatment strategy. This example is based on molecular docking to evaluate whether the compound of Formula I-Ova) binds to the surface spike glycoprotein of the new coronavirus and blocks or interferes with the receptor molecule Angiotensin Converting Enzyme 2 (ACE2) Combine to clarify the antiviral mechanism of the compound of formula I-avidin. The specific implementation method is as follows: Take the crystal structure (PDB code: 6M0J) of the receptor-binding domain (RBD) of the new coronavirus surface spike glycoprotein receptor-binding domain (PDB code: 6M0J) as the molecular pair acceptor, and use the MOE software to add hydrogen atoms to the RBD structure. Perform energy optimization. The structure of the compound of the ligand formula I-Ova is also constructed by MOE software, using the standard MMFF94 molecular force field and an energy gradient of 0.0001 kcal/mol as the convergence criterion for energy optimization. The MOE-based molecular docking module performs molecular docking, and the docking structure with the best energy is further optimized for energy and docking mode analysis. The results of molecular docking showed that the compound of formula I-Ova) binds to a hydrophobic pocket composed of several hydrophobic amino acids (L455, F456, Y489, F490) of RBD, and forms hydrogen bonds with Y489 and Q493 (Figure 3) . The binding site is at the interface where the new coronavirus spike glycoprotein RBD binds to the receptor ACE2, and it is predicted that the compound of formula I-Ova (Ova) can block or interfere with the interaction between the viral spike glycoprotein RBD and the receptor ACE2. Directly combine.

Example 8. The compound of formula I-zinnia lactone binds to host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L molecular docking simulation study: host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L pair The fusion process of the coronavirus plays a key role. This example is based on molecular docking to evaluate whether the compound of formula I-avidylactone binds to and inhibits Cathepsin B and Cathepsin L, so as to clarify the antiviral mechanism of the compound of formula I-avidactone. The specific implementation method is as follows: take the crystal structures of human endosomal cysteine proteolytic enzymes Cathepsin B and cathepsin L (PDB code: 3AI8 & 2XU1) as molecular pair acceptors, and use MOE software to add hydrogen atoms to the RBD structure and proceed. Energy optimization. The structure of the compound of the ligand formula I-Ova is also constructed by MOE software, using the standard MMFF94 molecular force field and an energy gradient of 0.0001 kcal/mol as the convergence criterion for energy optimization. The MOE-based molecular docking module performs molecular docking, and the docking structure with the best energy is further optimized for energy and docking mode analysis. The results of molecular docking also showed that the compound of formula I-Ova may also bind to the catalytic pockets of the endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L. The compound of formula I-fish needle lactone (Ova) binds to the hydrophobic S2 site of Cathepsin B composed of Y75, P76, A173, A200, and E245 with a hydrophobic alicyclic ring, and is formed by the exocyclic olefin and the catalytic cysteine C29 The covalent complex thus inhibits the activity of Cathepsin B (Figure 4A). On the other hand, the compound of formula I-Ova is a hydrophobic alicyclic binding to the hydrophobic S2 site of Cathepsin L composed of L69, M70, Y72, A135, and M161, and through the exocyclic olefin and catalytic cysteine The amino acid C25 forms a covalent complex to inhibit the activity of Cathepsin L (Figure 4B). Since the endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L play a key role in the fusion process of the coronavirus, the compound of formula I-Ova) potentially blocks the invasion and fusion process of the new coronavirus.

Example 9. Study on the activity of the compound of formula I-dorphyllide in inhibiting new coronavirus infection: The compound of formula I-dorphyllide described in Example 7 and the binding region of the surface spike glycoprotein receptor of the new coronavirus ( Receptor-binding domain (RBD) molecular docking simulation research results, and the molecular docking simulation research of the compound of formula I described in Example 8. As a result, it is predicted that the compound of formula I-avidylactone can inhibit the infection process of the new coronavirus. This example is based on the new coronavirus pseudovirus inhibitory activity detection system developed in the laboratory of Professor Zhang Linqi, director of the Comprehensive AIDS Research Center of Tsinghua University, Beijing, to evaluate whether the compound of formula I-avidylactone blocks the process of host cell infection by the new coronavirus. The specific implementation method is carried out according to the two steps of new coronavirus pseudovirus construction and new coronavirus infection inhibition detection: Step 1. Use of membrane glycoprotein deletion (Env-defective) and the HIV-1 viral genome plasmid pNL4-3R expressing luciferin protein -E-luciferase, and express new coronavirus full-length surface spike glycoprotein pcDNA3.1/SARS-CoV-2 co-transfected 293T cells, cultured in DMEM medium containing 10% fetal bovine serum for 60 hours. Take the culture supernatant to obtain the virus solution of the novel coronavirus pseudovirus (referred to as SARS-CoV-2 virus solution). Step 2. Take a 96-well cell culture plate, add 100 microliters of the compound of formula I-dalactone diluent and 50 microliters of SARS-CoV-2 virus liquid (in 50 microliters of SARS-CoV-2 virus liquid) to each well The concentration of the virus is 1×10 ⁴ TCID50/mL), so that the concentration of the compound of formula I-dorphyllactone solution in the mixed system is the corresponding diluted concentration, and the mixture is allowed to stand and incubate for 1 hour at 37°C. The DMEM medium with an equal volume of 10% fetal bovine serum was used instead of the compound of formula I-dilphalactone solution dilution as a virus control. An equal volume of DMEM medium containing 10% fetal bovine serum was used instead of the SARS-CoV-2 virus solution as a cell control. Take the cell culture plate and inoculate 100 microliters of Huh7 cell suspension per well (the solvent used to prepare the cell suspension is DMEM medium containing 10% fetal bovine serum, and the concentration of Huh7 cells in the cell suspension is 2×10 ⁵ Cells/mL), and incubate at 37°C for 64 hours. Aspirate and discard the supernatant, add 150 microliters of lysis buffer (Micro Glass Biotechnology, Catalog No. T003, operate according to the instructions) to each well, and incubate at 37°C for 5 minutes. Take the cell culture plate and detect the luciferase activity. Set up multiple multiple holes for each treatment. Inhibitory activity (%)=[1-(fluorescence intensity of test group-fluorescence intensity of cell control)/(fluorescence intensity of virus control-fluorescence intensity of cell control)]×100%. Calculated using Prism 5 software inhibitory activity of compounds of formula I 50% - fish pilosa lactone concentration, i.e. the compound of formula I - IC ₅₀ value fish pilosa lactone (FIG. 5). The results of the study show that the compound of formula I-codylactone is specifically different from chloroquine or Remdesivir in its molecular mechanism of inhibiting the new coronavirus, and it has a significant effect on the new coronavirus infection at the micromolar level. Inhibitory effect.

Claims (10)

The use of a composition for the preparation of a drug for inhibiting the novel coronavirus (SARS-CoV-2), wherein the composition includes a compound of formula I-Ovatodiolide or a compound of formula I-Ovatodiolide The structural isomers.

The use according to claim 1, wherein the composition further comprises a derivative compound comprising the main structure of the compound of the formula I-dorphyllide, and the derivative compound system comprising the main structure of the compound of the formula I-dorphyllide For inhibiting the mechanism of action of the novel coronavirus (SARS-CoV-2) and the molecular docking mechanism disclosed by the compound of formula I-zona lactone, a novel coronavirus surface spike glycoprotein receptor binding region or binding in a host The molecular docking mechanism of body cysteine proteolytic enzymes Cathepsin B and Cathepsin L has similar effects. The use according to claim 1, wherein the composition comprises a safe and effective amount of the compound of the formula I-Idylactone or a safe and effective amount of the compound of the formula I-Idylactone and its pharmaceutically acceptable The combination of the salt or carrier (carrier). The use according to claim 3, wherein the composition can be used to prevent or treat diseases caused by a novel coronavirus (SARS-CoV-2). The use according to claim 3, wherein the composition can be used to prevent or treat severe Special infectious pneumonia (COVID-19). The use according to claim 1, wherein the compound of the formula I-ichonoid lactone is a natural compound obtained by extracting ichonoid from an organic solvent and separating and purifying by a chromatographic column as a component, or The chemical synthesis method prepares the synthetic compound with the natural structure of the compound of the formula I-midrolactone as the component. The use according to claim 2, wherein the derivative compound containing the main structure of the compound of the formula I-zinnia lactone is a compound prepared by chemical synthesis as a component. The use according to claim 3, wherein the effective amount is a general adult (60 kg body weight): within 480 mg orally per day, and the limit is limited to 28 days of continuous administration. The use according to claim 3, wherein the effective amount is a general adult (60 kg body weight): 20 mg to 40 mg orally per day, and it is appropriate to continue taking it for 7 to 14 days. The use of a composition for preparing a drug for inhibiting coronavirus, wherein the composition includes a compound of formula I-Ovatodiolide, or a compound of formula I-structural isomer of Ovatodiolide.

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