RU2791523C1 - 5'-o-(3-phenylpropionyl)-n4-hydroxycytidine and its application - Google Patents

Abstract

FIELD: chemistry; pharmaceuticals; medicine.

SUBSTANCE: group of inventions relates to a new chemical compound, a derivative of N4-hydroxycytidine which can be used to inhibit the replication of RNA and DNA-containing viruses (coronavirus, arbovirus and orthoreovirus). A group of inventions relates to the field of organic chemistry and pharmaceuticals, namely to a new derivative of N4-hydroxycytidine, which can be used to inhibit the replication of RNA and DNA-containing viruses. A compound is disclosed, which is a derivative of N4-hydroxycytidine of the presented formula or a pharmaceutically acceptable salt thereof. Also the use of the above compound or a pharmaceutically acceptable salt thereof for inhibiting the replication of an RNA or DNA-containing virus is disclosed.

EFFECT: effective inhibition of RNA or DNA-containing virus replication.

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Description

Technical field

SUBSTANCE: group of inventions relates to chemistry, pharmaceuticals and medicine, namely to a new chemical compound, a derivative of N4-hydroxycytidine, which can be used to inhibit the replication of RNA and DNA-containing viruses (coronavirus, Arbovirus and Orthopoxvirus).

State of the art

Viruses carried by arthropods (arboviruses) such as mosquitoes, ticks and flies mainly belong to the families Flaviviridae , Togaviridae and Bunyaviridae and are transmitted by them to humans or other vertebrates [10.1007/3-211-29981-5_4]. These viruses are transmitted to vertebrates via saliva when an infected arthropod vector feeds on blood. There are more than 250 types of arboviruses, and at least 80 of them cause human diseases, including hemorrhagic fever, encephalitis, arthritis, and meningitis [10.1099/0022-1317-82-8-1867]. Arbovirus diseases account for the majority of vector-borne diseases and 80% of the world's population lives in areas where one is endemic [https://www.who.int/publications/i/item/9789240013155]. Outbreaks of mosquito-borne viral diseases are of concern to the international community and continue to have a major impact on global health and socio-economic systems.

Currently, there are regular outbreaks of a new coronavirus infection (COVID-19 or SARS-Cov-2) all over the world. Despite intensive countermeasures around the world, morbidity and mortality remain high, and many countries are facing new waves of infection. Vaccines are an important tool in the fight against COVID-19, but the development of antivirals is also a priority, especially with the emergence of options that may partly evade vaccines. Therefore, the clinic urgently needs a safe and effective drug for the prevention and treatment of a new coronavirus infection. Based on the fact that existing antiviral drugs have more or less disadvantages, the creation of antiviral drugs with a better therapeutic effect is a problem that needs to be urgently addressed at the present time.

Poxviruses are widespread in almost all countries of the world. The genus of orthopoxviruses, in addition to the smallpox virus, includes 12 more representatives. Some representatives of this genus are zoonoses, however, normally circulating among animals, they can cause diseases in humans. Of greatest concern in the last decade is the increase in the incidence of monkeypox (4). Widespread vaccination was discontinued after the cessation of circulation of the variola virus. Available data from studies already conducted indicate the existence of cross-immunity in vaccinated people (Gilchuk I. Et al., Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections. Cell. 2016 Oct 20;167(3):684-694). .e9.doi: 10.1016/j.cell.2016.09.049, PMID: 27768891; PMCID: PMC5093772). Studies indicate that cross-immunity among orthopoxviruses (including vaccinia, variola, monkeypox, etc.) is so strong that it allows one to count on successful extrapolation of the obtained data for protection against monkeypox. The cessation of mass vaccination for the prevention of smallpox has increased the number of people living in the natural focus of monkeypox in Africa, who do not have immunity. Of greatest concern is the current outbreak of monkeypox, which began in April 2022. In less than three months, monkeypox has been confirmed in more than 30,000 people (8). One case of monkeypox has also been identified in Russia (9). As a result, the WHO declared the 2022 monkeypox outbreak a public health emergency. Thus, the development of drugs with a specific action is an extremely urgent task.

Derivatives of N4-hydroxycytidine are known that are used to treat or prevent viral infections, in particular, Eastern, Western and Venezuelan equine encephalitis (EEE, WEE and VEE, respectively), Chikungunya fever (CHIK), Ebola fever, influenza, seasonal and pandemic coronaviruses (MERS and SARS), respiratory syncytial virus (RSV), and Zika virus [WO2019113462, WO2016106050, US20190022116]. However, for most N4-hydroxycytidine derivatives, activity against new variants of SARS-CoV-2, as well as for other viruses, remains unknown. The activity of some N4-hydroxycytidine prodrugs against recombinant SARS-CoV-2 has been studied [CN111548384A]. Known compound 5'-isopropyl ester of β-D-N4-hydroxycytidine (Molnupiravir), which inhibits the replication of SARS-CoV-2. The drug Lagevrio based on this compound is used for the treatment of COVID-19 [10.1038/s41564-020-00835-2, https://clinicaltrials.gov/ct2/show/NCT04405739, 10.3389/fimmu.2022.855496, 10.1056/NEJMoa2116044]. This solution was chosen as a prototype.

The main disadvantages of Molnupiravir are low antiviral activity against SARS-CoV-2, unknown activity against other representatives of Arbovirus infections, and unstudied activity against Orthopoxviruses.

Thus, there is a need in the technical field to create a connection devoid of these disadvantages.

Disclosure of the essence of the invention

The technical result of the claimed invention is to obtain an ester derivative of N4-hydroxycytidine with a carboxylic acid, which has high activity against SARS-CoV-2 coronavirus, arboviruses and exhibits an antiviral effect against Orthopoxviruses.

The specified technical result is achieved by the fact that a compound has been created, which is a derivative of N4-hydroxycytidine, and corresponding to the formula:

5'-O-(3-phenylpropionyl)-N4-hydroxycytidine, or a pharmaceutically acceptable salt thereof.

Also, the technical result is achieved by the proposed use of the developed compound for inhibiting the replication of RNA and DNA-containing viruses.

In a particular case of using the developed compound, SARS-CoV-2 virus replication is inhibited, Arbovirus replication is inhibited, and Orthopoxvirus replication is inhibited.

A special case of application is also proposed, in which the inhibition of the replication of RNA-containing viruses occurs in the body of mammals.

Brief description of the drawings

In FIG. 1 shows the preparation of N4-trityloxy-2',3'-O-isopropylidenecytidine (3).

In FIG. 2 shows the preparation of 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine(5).

In FIG. 3 shows dose-response curves and IC ₅₀ (μM) values of CPE inhibition of various SARS-CoV-2 variants by test compound.

In FIG. 4 shows dose-response curves and IC ₅₀ (μM) values of CPE inhibition of various Arbovirus variants by test compound.

In FIG. 5 shows dose-response curves and IC ₅₀ (μM) values of CPE inhibition of variola vaccine strain (Lister) by test compound.

In FIG. 6 shows the efficacy of the resulting SN_9 compound against SARS-CoV-2 infection in vivo . The dynamics of changes in the weight of animals before and after infection (A), changes in viral load (B) and virus titer (C) in lung tissues in the control group of animals and animals treated with the studied drugs are shown. Kruskal-Wallis test: * p < 0.05 - statistically significant.

Implementation of the invention

Obtaining 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine consists of several stages.

At the first stage, the vicinal 2' and 3' hydroxyl groups of β-D-N4-hydroxycytidine (NHC) were blocked using acetonide protection. In this case, the optimal reaction conditions were the use of p-toluenesulfonic acid (p-TSA) as a catalyst, carrying out the reaction in acetone at room temperature for 2 h.

At the second stage, the hydroxyl group was blocked at the 4 position of N4-hydroxycytidine. For these purposes, a trityl (Tr) protecting group was chosen, the choice being due to the ease of introduction of the protection, as well as its acid lability, which makes it possible to deblock all the protecting groups under acidic conditions. The reaction was carried out in methylene chloride by the action of trityl chloride (Tr-Cl) in the presence of 4-dimethylaminopyridine (DMAP) as a catalyst, as well as triethylamine.

At the next stage, N4-dimethoxytrityloxy-2',3'-O-isopropylidenecytidine was reacted with phenylpropionic acid in the presence of 1,3-dicyclohexylcarbodiimide in methylene chloride, stirring was carried out at room temperature for 2 h. The intermediate product was purified on silica gel and subjected to acid hydrolysis with 80% aqueous formic acid, stirred at room temperature for 20 hours. The compound was isolated by flash chromatography on silica gel in chloroform:methanol (5% methanol). As a result of the work, a new compound was obtained - 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine.

It was shown that the resulting compound is capable of inhibiting the replication of the SARS-CoV-2 coronavirus, as well as viruses from the Arbovirus group, with an efficiency significantly higher than that of the prototype. In addition, this compound has a fundamentally new property in comparison with the prototype - antiviral activity against viruses of the genus Orthopoxvirus. This property of 5'-O-(3-phenylpropionyl)-N4-hydroxycytidinane is evident from the prior art and is not obvious to the average person skilled in the art.

The implementation of the invention is confirmed by the following examples.

Example 1 Synthesis of 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine.

The compound was obtained in 4 stages. The synthesis scheme is shown in Fig. 1 and FIG. 2. At the first stage, the intermediate product 2',3'-O-isopropylidene-N4-hydroxycytidine (2) was prepared (Fig. 1). To do this, 3.7 g (19.69 mmol) of p-toluenesulfonic acid was added to 1.7 g (6.56 mmol) of N4-hydroxycytidine (1) in dry acetone (240 ml), the resulting mixture was stirred at room temperature for 2 h. The reaction was neutralized by adding triethylamine, solvents distilled off, the residue was purified by flash chromatography on silica gel in the system chloroform:methanol (5% methanol).

Yield of compound (2) 1.6 g (85%), Rf 0.42, T mp. = 195-196°C.

¹ H-NMR spectrum (CDCl ₃ ), δ, ppm: 1.32 (2s, 6H, 2CH ₃ ), 2.00 (s, 1H, NHO H ), 3.65-3.94 (m, 2H, 5'- CH ₂ ), 4.14-4.27 (m, 1H, 4'-CH), 4.87-5.08 (m, 2H, 2' and 3'-CH), 5.38 (d, J=2.6 Hz, 1H, 1'-CH ), 5.62 (d, J=7.8 Hz, 1H, H-5 Cyt), 6.65 (d, J=8.1 Hz, 1H, H-6 Cyt), 8.13 (s, 1H, N H OH).

At the second stage, the intermediate product N4-trityloxy-2',3'-O-isopropylidenecytidine (3) was obtained (Fig. 2). To do this, to a suspension of 1.6 g (5.37 mmol) of 2',3'-О-isopropylidene-N4-hydroxycytidine (2) in dichloromethane (100 ml) with stirring at room temperature was added 1.1 ml (8.06 mmol) of triethylamine and 7 mg (0.054 mmol) of 4-dimethylaminopyridine, the mixture was stirred for 15 min. Then a solution of 1.5 g (5.37 mmol) of trityl chloride in dichloromethane (50 ml) was added dropwise to the reaction flask, stirred at room temperature overnight. The solvent was distilled off on a rotary evaporator, the residue was purified by flash chromatography on silica gel in a gradient of systems - hexane:ethyl acetate:triethylamine (7:3:0.25) followed by elution with a chloroform:triethylamine system (2.5% triethylamine).

Yield of compound (3) 2.1 g (72%), Rf 0.47, T mp. = 132-133°C.

¹ H-NMR spectrum (CDCl ₃ , δ, ppm): 1.29 (s, 3H, CH ₃ ), 1.47 (s, 3H, CH ₃ ), 3.66-3.99 (m, 2H, 5'-CH ₂ ), 4.12-4.25 (m, 1H, 4'-CH), 4.89-5.11 (m, 2H, 2' and 3'-CH), 5.34 (d, J=3.1 Hz, 1H, 1'-CH) , 5.51 (d, J=8.1 Hz, 1H, H-5 Cyt), 6.46 (d, J=8.2 Hz, 1H, H-6 Cyt), 6.79-6.86, 7.16-7.25 , 7.28-7.33 (3m, 15H in Tr), 9.97 (s, 1H, N H OH).

At the next stage, a solution of 145.2 mg (0.268 mmol) of N4-trityloxy-2',3'-O-isopropylidenecytidine (3), 60.3 mg (0.402 mmol) of 3-phenylpropanoic acid and 99.4 mg (0.482 mmol) of 1,3-dicyclohexylcarbodiimide in methylene chloride (15 ml) was stirred at room temperature for 2 h. The reaction mass was prepurified on silica gel in the system chloroform:hexane:triethylamine (7:3:0.25).

In the last step, intermediate (4) was exposed to 80% aqueous formic acid (5 ml), stirred at room temperature for 20 h, evaporated and purified by flash chromatography on silica gel in chloroform:methanol (5% methanol).

Yield of compound (5) 58 mg (52%), R _f 0.33, T pl. = 144-146°C.

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): 2.67 (t, J=7.4 Hz, 2H, C H ₂ -CO), 2.85 (t, J=7.5 Hz, 2H, C H ₂ -Ph), 3.92-4.03 (m, 2H, 5'-CH ₂ ), 4.08-4.22 (m, 1H, 4'-CH), 5.21-5.35 (m, 2H, 2' and 3'-CH ), 5.56 (d, J=8.2 Hz, 1H, 1'-CH), 5.70 (d, J=5.5 Hz, 1H, H-5 Cyt), 6.82 (d, J=8.2 Hz, 1H, H-6 Cyt), 7.16-7.28 (m, 5Н, Ph), 9.56 (s, 1H, N H OH), 10.01 (s, 1H, NHO H ).

Thus, as a result of the work performed, a new compound 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine was obtained.

Example 2 Study of antiviral effect against SARS-CoV-2 variants.

The purpose of this experiment is to evaluate the ability of the obtained compound (5'-O-(3-phenylpropionyl)-N4-hydroxycytidine) to inhibit the replication of various variants of the SARS-CoV-2 virus.

The experiment was carried out on the cell line Vero E6 (ATCC CRL-1586). Cells were cultured in DMEM growth medium (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, USA), 1x antimycotic antibiotic (CapricornScientificGmbH), and 1x GlutaMAX (Gibco, USA). To study the antiviral effect, Vero E6 cells (2×10 ⁴ cells/well) were seeded in 96-well plates in complete DMEM growth medium the day before the experiment. Then, various dilutions of the test compound were added to the cell monolayer and incubated for 1 h at 37°C and 5% CO ₂ . After that, infection with the SARS-CoV-2 virus was performed at 100TCID ₅₀ (TCID ₅₀ is a tissue cytopathogenic dose that causes the death of 50% of the monolayer cells). In this experiment, the following variants of the SARS-CoV-2 virus were used: "Wuhan" B.1.1 (PMVL-4), "Omicron" BA.4.6 (PMVL-55), "Omicron" BA.5 (PMVL-52) and " Omicron BA.5.2 (PMVL-54). Inhibition of the virus-induced cytopathic effect (CPE) under the action of the compound was determined using the MTT test [10.1007/s00018-021-03985-6]. 72 hours after the addition of coronavirus, a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) in phosphate-buffered saline was added to each well to a final concentration of 0.5 mg/ml . The method is based on the ability of MTT to be reduced to colored formazan in the presence of mitochondrial enzymes of living cells. After a 2-hour incubation, the medium was removed from the wells and 150 μl of dimethyl sulfoxide was added. Next, the optical density of the formazan solution was measured at a wavelength of 590 nm using a tablet spectrophotometer. The results of the experiment are presented in Fig. 3.

Analysis of the antiviral activity of 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine against four variants of the SARS-CoV-2 virus showed that this compound has ~2 times more activity (Fig. 3) than the prototype, which is approved for treatment for COVID-19.

Example 3 Study of the antiviral effect against Arboviruses.

The purpose of this experiment is to evaluate the ability of the resulting compound (5'-O-(3-phenylpropionyl)-N4-hydroxycytidine) to inhibit the replication of various viruses from the Arboviruses group.

The experiment was carried out on the cell line Vero E6 (ATCC CRL-1586). Cells were cultured in DMEM growth medium (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, USA), 1x antimycotic antibiotic (CapricornScientificGmbH), and 1x GlutaMAX (Gibco, USA). To study the antiviral effect, Vero E6 cells (2×10 ⁴ cells/well) were seeded in 96-well plates in complete DMEM growth medium the day before the experiment. Various concentrations of test compound were added to VeroE6 cells, followed by infection with arbovirus at a dose of 100 pfu (plaque forming units) per well. In this study, the following Arboviruses were used: Sindbis (strain Az16), Mosquito fever of Sicily (SFS 1943), Batken (strain Ast. 247), Inko (strain KN3641), Batai (strain VLG 42) and Tyaginya (strain 92). Inhibition of the virus-induced cytopathic effect (CPE) by the compound was determined using the MTT test 72 hours after infection. The results of the experiment are presented in Fig. 4.

Thus, the antiviral effect of 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine was studied using six members of the Arboviruses group. It was determined that the resulting compound inhibited the replication of all viruses from this group with lower values of IC ₅₀ (half-maximal inhibition concentration), compared with the prototype (Fig. 4).

Example 4 Investigation of the antiviral effect against the pox virus.

The purpose of this experiment is to evaluate the ability of the resulting compound (5'-O-(3-phenylpropionyl)-N4-hydroxycytidine) to inhibit the replication of the pox virus.

The experiment was carried out on the cell line Vero E6 (ATCC CRL-1586). Cells were cultured in DMEM growth medium (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, USA), 1x antimycotic antibiotic (CapricornScientificGmbH), and 1x GlutaMAX (Gibco, USA). To study the antiviral effect, Vero E6 cells (2×104 cells/well) were seeded in 96-well plates in complete DMEM growth medium the day before the experiment. VeroE6. A vaccine strain of variola virus (Lister; Microgen, Russia) and VeroE6 cells were used for experiments. Cells were infected with variola virus at 100TCID50. Inhibition of the virus-induced cytopathic effect by the compound was determined using the MTT test 72 hours after infection. The results of the experiment are presented in Fig. 5.

In the study of antiviral activity against the pox virus, it was found that the developed N4-hydroxycytidine derivative SN_9 inhibits the virus-induced cytopathic effect (CPE) with a value of IC5049.3μM (Fig. 5), while the prototype did not show a pronounced inhibitory effect.

Example 5 Animal study of the effectiveness of the obtained compound against the SARS-CoV-2 virus.

The resulting compound can be administered to mammals by any route. Preliminary studies have shown that the oral route is the optimal route of administration. The maximum dose of the drug was determined as 350 mg/kg.

The experiment used female Syrian hamsters. Animals were divided into two groups of 5 heads, which were injected with:

1) 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine, 200 mg/kg;

2) Phosphate buffered saline (negative control).

Then, animals were intranasally challenged at 10 ⁵ TCID ₅₀ SARS-CoV-2PMVL-4. For 4 days, the animals were injected with the studied preparations twice a day. After the animals were euthanized on the fifth day of the experiment, they were opened and the lungs were taken. Hamster lungs were subjected to homogenization followed by separation of the supernatant by low speed centrifugation. The virus titer was determined in a monolayer of Vero E6 cells grown in 48-well plates. The virus titer for each lung homogenate sample was determined 72 hours later and expressed as PFU/mg lung. Total RNA was isolated from lung homogenates using the ExtractRNA reagent (Evrogen, Russia) according to the manufacturer's instructions. The reverse transcription reaction was performed using a SARS-CoV-2 FRT kit for quantitative determination of SARS-CoV-2 coronavirus RNA using a panel of SARS-CoV-2 amplified fragment characterized by the number of copies of the SARS-CoV-2 amplifiable fragment (N.F. Gamaleya National Research Center for Epidemiology and Microbiology) . The results were expressed as numbers converted to log10 SARS-CoV-2 viral load per mg of lung tissue.

In the course of the study of the effectiveness of the drugs, it was found that the animals from the negative control group lost a significant amount of weight. Animals treated with the resulting compound gained weight during infection (FIG. 6A), indicating efficacy of the study drug and no animal toxicity.

It was also determined that in animals that were administered the resulting compound, there was a decrease in the amount of SARS-CoV-2 RNA in lung tissues (Fig. 6B).

When determining the titer of infectious virus in the lungs of animals, it was found that treatment of animals with the resulting compound reduces the titer of viable virus in the lungs by ~1.5 Lg (Fig. 6B).

Thus, the technical result of the invention is confirmed by the fact that the claimed compound - 5'-O-(3-phenylpropionyl)-N4-hydroxycytidine- differs from the known prototypes of N4-hydroxycytidine derivatives and exhibits a higher antiviral effect against the pox virus, various variants of SARS- CoV-2 and Arboviruses. In addition, this compound exhibits therapeutic activity against SARS-CoV-2 infection in vivo , and can be used for the treatment/prevention of COVID-19.

Industrial Applicability

The resulting compound (5'-O-(3-phenylpropionyl)-N4-hydroxycytidine) can be used in healthcare as a new drug for the treatment of various viral infections. The developed and studied compound has a specific antiviral and polytarget activity. Polytargeting lies in the antiviral effect against various viral infections represented by SARS-CoV-2 coronaviruses, Arboviruses and Orthopoxviruses.

Claims (8)

1. A compound that is a derivative of N4-hydroxycytidine and corresponds to the formula:

or a pharmaceutically acceptable salt thereof. 2. The use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for inhibiting the replication of an RNA or DNA containing virus. 3. Use according to claim 2, wherein the RNA virus is SARS-CoV-2 virus. 4. Use according to claim 2, wherein the RNA-containing virus belongs to the group Arbovirus. 5. Use according to claim 2, wherein the DNA-containing virus belongs to the genus Orthopoxvirus. 6. The use according to claim 2, wherein the inhibition of the replication of RNA-containing viruses occurs in the body of mammals.

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