WO2024123207A1 - 5'-о-(3-phenylpropionyl)-n4-hydroxycytidine and use thereof - Google Patents

Abstract

The group of inventions relates to chemistry, pharmaceutics and medicine, and more particularly to a novel chemical compound which is a derivative of N4-hydroxycytidine and can be used to inhibit the replication of RNA and DNA-containing viruses (coronavirus, arbovirus and orthopoxvirus). The claimed invention makes it possible to produce a novel chemical compound that is highly active against the SARS-CoV-2 coronavirus and arboviruses and has an anti-viral effect against orthopoxviruses.

Description

5'-0-(3-phenylpropionyl)-№4-hydroxycytidine and its use.

Technical field

The group of inventions relates to chemistry, pharmaceuticals and medicine, namely to a new chemical compound derived from M4-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 through saliva when an infected arthropod vector feeds on blood. There are more than 250 species 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]. Diseases caused by arboviruses 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 international concern and continue to have a major impact on global health and socioeconomic systems.

Currently, there are regular outbreaks of new coronavirus infection (COVID-19 or SARS-Cov-2) throughout 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 antiviral drugs is also a priority, especially as variants emerge that may partially evade vaccines. Therefore, the clinic urgently needs a safe and effective drug for the prevention and treatment of new coronavirus infection. Based on the fact that existing antiviral drugs have more or less drawbacks, the creation of antiviral drugs with better therapeutic effect is a problem that requires urgent solutions at present.

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 zoonotic; however, when circulating normally among animals, they can cause diseases in humans. The greatest concern over the past decade has been the increase in the incidence of monkeypox (4). Widespread vaccination was stopped after the variola virus stopped circulating. 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). Research suggests that cross-immunity among orthopoxviruses (including cowpox, smallpox, monkeypox, etc.) is so strong that it can be successfully extrapolated to protect against monkeypox. The cessation of mass vaccination to prevent smallpox has increased the number of people living in the natural outbreak of monkeypox in Africa who do not have immunity. Of greatest concern is the current monkeypox outbreak, 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 specific effects is an extremely urgent task.

M4-hydroxycytidine derivatives are known to be used for the treatment or prevention of 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, W02016106050, 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]. The compound 5'-isopropyl ester of r-E-H-hydroxycytidine (Molnupiravir) is known, which inhibits the replication of SARS-CoV-2. The drug Lagevrio based on this compound is used to treat 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 its low antiviral activity against SARS-CoV-2, unknown activity against other representatives of Arboviral infections and unstudied activity against Orthopoxviruses.

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

Disclosure of the invention

The technical result of the claimed invention is the production of an ester derivative of H4-hydroxycytidine with a carboxylic acid, which has high activity against the SARS-CoV-2 coronavirus, arboviruses and exhibits an antiviral effect against Orthopoxviruses.

This technical result is achieved by creating a compound that is a derivative of N4-hydroxycytidine and corresponds to the formula:

5'-O-(3-phenylpropionyl)-H4-hydroxycytidine, or a pharmaceutically acceptable salt thereof.

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

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

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

Brief description of drawings

In fig. 1 shows the preparation of M4-trityloxy-2',3'-O-isopropylidenecytidine (3).

In fig. 2 shows the preparation of 5'-O-(3-phenylpropionyl)-M4-hydroxycytidine(5). In fig. Figure 3 shows dose-dependent curves and 1C5o(μM) values of CPE inhibition of various variants of SARS-CoV-2 using the test compound.

In fig. Figure 4 shows dose-dependent curves and ICso(pM) values of CPE inhibition of various Arbovirus variants using the test compound.

In fig. Figure 5 shows the dose-dependent curves and 1C5o(μM) values of inhibition of CPE of the smallpox vaccine strain (Lister)BHpyca using the test compound.

In fig. Figure 6 presents data on the effectiveness of the resulting compound SN 9 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 receiving the studied drugs are shown. Kruskal-WallisTecT: *p<0.05 - statistically significant.

Carrying out the invention

The preparation of 5'-O-(3-phenylpropionyl)-4-hydroxycytidine consists of several steps.

At the first stage, the vicinal 2' and 3' hydroxyl groups of P-P-4-hydroxycytidine (NHC)npn were blocked using acetonide protection. In this case, the optimal reaction conditions were the use of para-toluenesulfonic acid (p-TSA) as a catalyst and the reaction in acetone at room temperature for 2 hours.

At the second stage, the hydroxyl group in the 4th position of H4-hydroxycytidine was blocked. For these purposes, a trityl (Tg) protecting group was chosen, the choice being due to the ease of introducing the protection, as well as its acid lability, which makes it possible to deblock all protective 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, H4-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 hours. The intermediate product was purified on silica gel and subjected to acid hydrolysis by the action of an 80% aqueous solution of formic acid, stirred at room temperature for 20 hours. The compound was isolated by flash chromatography on silica gel in the system chloroform:methanol (5% methanol). As a result of the work, a new compound was obtained - 5'-O-(3-phenylpropionyl)-H4-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 follows clearly 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 presented in Fig. 1 and Fig. 2. At the first stage, the intermediate product 2',3'-O-isopropylidene-K4-hydroxycytidine (2) was prepared (Fig. 1). To do this, 3.7 g (19.69 mmol) of para-toluenesulfonic acid was added to 1.7 g (6.56 mmol) of N-hydroxycytidine (1) in dry acetone (240 ml), the resulting mixture was stirred at room temperature for 2 hours. 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, mp. = 195-196 °C.

*H-NMR spectrum (CDC1 ₃ ), 5, ppm: 1.32 (2s, 6H, 2CH ₃ ), 2.00 (s, 1H, NHOH), 3.65-3.94 (m, 2H, 5'-CH ₂ ), 4.14-4.27 (m, W, 4'-CH), 4.87-5.08 (m, 2H, 2' and Z'-CH), 5.38 (d, J=2.6 Hz, 1H, G-CH), 5.62 (d, J=7.8 Hz, W, H-5 Cyt), 6.65 (d, J=8.1 Hz, 1H, H-6 Cyt), 8.13 (s, 1H, NHOH)

At the second stage, the intermediate product M4-trityloxy-2',3'-O-isopropylidenecytidine (3) was obtained (Fig. 2). For this purpose, 1.1 ml (8.06 mmol) of triethylamine and 7 mg (0.054 mmol) 4-dimethylaminopyridine, the mixture was stirred for 15 minutes. Then a solution of 1.5 g (5.37 mmol) of trityl chloride in dichloromethane (50 ml) was added dropwise to the reaction flask and 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 the system chloroform: triethylamine (2.5% triethylamine).

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

'H-NMR spectrum (CDCh, 5, ppm): 1.29 (s, 3H, CH3), 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, G- 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 (Zm , 15H in Tr), 9.97 (s, 1H, NHOH).

At the next stage, a solution of 145.2 mg (0.268 mmol) of N-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-dicycle ohexylcarbodiimide in methylene chloride (15 ml) was stirred at room temperature for 2 hours. The reaction mass was prepurified on silica gel in the system chloroform: hexane: triethylamine (7: 3: 0.25).

At the last stage, intermediate product (4) was exposed to an 80% aqueous solution of formic acid (5 ml), stirred at room temperature for 20 hours, evaporated and purified by flash chromatography on silica gel in a solvent mixture of chloroform:methanol (5% methanol).

Yield of compound (5) 58 mg (52%), Rf 0.33, mp. = 144-146 °C.

'H-NMR spectrum (DMSO-d _6.5 , ppm): 2.67 (t, J=7.4 Hz, 2H, CHg-CO), 2.85 (t, J=7.5 Hz, 2H, CHg-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, G-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, 5H, Ph), 9.56 (s, 1H, NHOH), 10.01 (s, 1H, NHOH).

Thus, as a result of the work carried out, 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 resulting 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 Vego E6 cell line (ATCC CRL-1586). Cells were cultured in DMEM growth medium (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, USA), lx antibiotic-antimycotic (CapricomScientificGmbH) and 1x GlutaMAX (Gibco, USA). To study the antiviral effect, Vero E6 cells (2x10 ⁴ cells/well) were seeded in 96-well plates in complete growth medium DMEM the day before the experiment. Then, various dilutions of the test compound were added to the cell monolayer and incubated for 1 hour at 37°C and 5% CO ₂ . After this, infection with the 8AB8-CoY-2 virus was carried out at lOOTCIDso (TCID50 is a tissue cytopathogenic dose that causes the death of 50% of the cells of the monolayer). In this experiment, the following variants of the SARS-CoV-2 virus were used: “Wuhan” V.1.1 (PMVL-4), “Omicron” VA.4.6 (PMVL-55), “Omicron” VA.5 (PMVL-52) and “ Omicron" VA.5.2 (PMVL-54). Inhibition of virus-induced cytological effect (CPE) under the influence of the compound was determined using the MTT test [10.1007/s00018-021-03985-6]. 72 hours after the addition of the 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 media 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 plate spectrophotometer. The results of the experiment are presented in Fig.Z.

Analysis of the antiviral effect of 5'-O-(3-phenylpropionyl)-H4-hydroxycytidine against four variants of the SARS-CoV-2 virus showed that this compound has ~ 2 times greater activity (Fig. 3) than the prototype, which is approved for treatment of 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 Arbovirus group.

The experiment was carried out on the Vero E6 cell line (ATCC CRL-1586). Cells were cultured in DMEM growth medium (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, USA), 1x antibiotic-antimycotic (CapricomScientificGmbH) and 1x GlutaMAX (Gibco, USA). To study the antiviral effect, Vero E6 cells (2x10 ⁴ cells/well) were seeded in 96-well plates in complete growth medium DMEM the day before the experiment. Various concentrations of the test compound were added to VegoEb cells, after which they were infected with arbovirus at a dose of 100 PFU (plaque-forming units) per well. The following Arboviruses were used in this study: Sindbis (strain Az 16), Sandfly fever 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 representatives of the Arbovirus group. It was determined that the resulting compound inhibited replication of all viruses from this group with lower IC50 values (half-maximal inhibition concentration) compared to the prototype (Fig. 4).

Example 4: Study of the antiviral effect against the smallpox 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 smallpox virus.

The experiment was carried out on the Vego E6 cell line (ATCC CRL-1586). Cells were cultured in DMEM growth medium (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, USA), lx antibiotic-antimycotic (CapricomScientificGmbH) and 1x GlutaMAX (Gibco, USA). To study the antiviral effect, Veto E6 cells (2x104 cells/well) were seeded in 96-well plates in complete growth medium DMEM the day before the experiment. Two-fold dilutions of the test compounds were prepared in 96-well plates, after which they were transferred to a monolayer of VegoEb cells. For the experiments, a vaccine strain of the smallpox virus (Lister; Microgen, Russia) and VegoEb cells were used. The cells were infected with the smallpox virus at 100 TCID50. 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.

When studying the antiviral activity against the smallpox virus, it was found that the developed 4-hydroxycytidine derivative SN 9 inhibits the virus-induced cytopathic effect (CPE) with a value of 1C5049.3cm (Fig. 5), while the prototype did not show a pronounced inhibitory effect.

Example 5. Study of the effectiveness of the resulting compound against the SARS-CoV-2 virus in animals.

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

Female Syrian hamsters were used in the experiment. The animals were divided into two groups of 5 animals, which were administered:

1) 5'-O-(3-phenylpropionyl)-4-hydroxycytidine, 200 mg/kg;

2) Phosphate-buffered saline (negative control).

Then, intranasal infection of animals was carried out at 10 ⁵ TCID5oSARS-CoV-2PMVL-4. Over the course of 4 days, the animals were administered the study drugs twice a day. day. After euthanasia of the animals on the fifth day of the experiment, they are autopsied and the lungs are collected. Hamster lungs were homogenized, 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 after 72 hours 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 set of reagents for the quantitative determination of RNA of the coronavirus SARS-CoV-2 “SARS-CoV-2 FRT” using a panel characterized by the number of copies of the amplified fragment of SARS-CoV-2 (N.F. Gamaleya Research Center for Epidemiology and Microbiology). . The results were expressed as numbers converted to log 10 SARS-CoV-2 viral load per mg of lung tissue.

During the study of the effectiveness of the drugs, it was found that animals from the negative control group lost a significant amount of weight. Animals that were treated with the resulting compound gained weight during the course of infection (Figure 6A), indicating that the study drug was effective and not toxic to the animals.

It was also determined that animals treated with the resulting compound experienced a reduction in the amount of SARS-CoV-2 RNA in their lung tissue (Figure 6B).

When determining the titer of infectious virus in the lungs of animals, it was found that treatment of animals with the resulting compound reduced 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 - phenyl propionyl)-N4-hydroxycytidine - differs from the known prototypes of H4-hydroxycytidine derivatives and exhibits a higher antiviral effect against the smallpox 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)-H4-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 multitarget effect. Multi-targeting consists of an antiviral effect against various viral infections represented by coronaviruses SARS-CoV-2, Arboviruses and Orthopoxviruses.

Claims

Claim 1. A compound that is a derivative of 4-hydroxycytidine and corresponds to the formula:

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