RU2641297C1 - Cyclohexanoxycarbonyl-dipeptide and its antiviral activity against hepatitis c virus - Google Patents

Abstract

FIELD: pharmacology.SUBSTANCE: invention relates to new synthetic compounds, namely to the N-acyl derivative of dipeptide, which is cyclohexyloxycarbonyl-prolyl-tryptophan (Cho-Pro-Trp-OH). Cho-Pro-Trp-OH inhibits reproduction of the hepatitis C virus (HCV), and also has a virucidal effect against HCV. The compound has a low toxic effect on the monolayer of the transplantable kidney cell line in the pig embryo (SPEV)..EFFECT: compound can be recommended for creation of new antiviral drugs, used both as an individual agent and as part of compositions for hepatitis C treatment.3 cl, 6 dwg, 3 tbl, 5 ex

Description

The invention relates to medicine and pharmaceutical production and relates to a new synthetic compound, namely cyclohexyloxycarbonyl-prolyl-tryptophan (Cho-Pro-Trp-OH),

which can be used to create a new antiviral drug against hepatitis C virus to be used both as an individual drug and as part of complex therapy for the treatment of hepatitis C.

The functions of the proteins of the viral ion-selective channels are involved in many stages of the replication cycle of the virus. These channels are localized in the viral membrane or membranes of the host cell and play an important role in the process of penetration of viruses into the cell, as well as in the assembly and release of newly formed virions. Viral ion channels are not always required for the reproduction of the virus, although their assistance usually significantly increases the increase in the number of virions. This family of proteins is called viroporins, it includes proteins that contain at least one hydrophobic transmembrane domain (TM domain) in the form of an α-helix [1]. As a rule, viroporins are represented by small proteins (50-120 a.a.), which tend to form homo-oligomers. Studies of the molecular architecture of viral ion channels have shown that most of them are formed by tetramers, although penta- and hexamer structures for viroporins are also characteristic. A typical representative of tetrameric ion-selective viroporins is the M2 channel of influenza A. This unique viroporin is specific for the transport of hydrogen ions into the virion, while most viroporins exhibit moderate ion selectivity, i.e. they do not differ in preference for any particular ion. Pentameric viroporin, represented by the Vpu protein from HIV-1, shows moderate cationic selectivity in solutions of NaCl and KCl electrolytes [2]. This is also true for the viral hepatitis C hexamer p7 viral hepatitis C (HCV), which carries monovalent cations (Na ⁺ and K ⁺ ) [3].

The high ionic selectivity of viroporin M2 of influenza A virus is due to the special structure of its TM domain, which contains histidine residues at position 37 (His37) in the form of tetrameric conjugation of their imidazole rings. Protons from the environment (intracellular) protonate three or all four imidazole rings of histidines. This leads to a conformational change as a result of electrostatic repulsion, which expands the channel in the His37 region and allows protons to penetrate into the cell [4]. The indole group of tryptophan-41 residue plays an important role in stabilizing the open state of the channel pore. Thus, the molecular model of the M2 ion channel is a “gateway mechanism” [5].

Amantadine (Symmetrel. 1-adamantylamine hydrochloride) was one of the first studied proton channel inhibitors of M2 influenza A. It has been used in clinical practice against influenza A virus since 1966 [6]. Later, in 1985, it was shown that certain mutations in the hydrophobic sequence of the M2 protein can lead to the resistance of the virus to amantadine and its analogue rimantadine (Flumantadine, 1- (1-adamantyl) ethylamine hydrochloride). These data suggested that the M2 protein may be the target for the adamantane series drugs [7], and in 1992 these assumptions were confirmed [8].

Recent studies have shown that the ionic activity of the hexameric channel of the HCV p7 protein can also be inhibited by in vitro adamantane preparations [9, 10]. This gives hope that the p7 channel may be a good target for anti-HCV drugs based on adamantane and other carbocycles. The non-structural protein p7 of HCV consists of 63 a.o. and has two transmembrane domains (TM1 and TM2) [11]. This viroporin, which is localized mainly in intracellular membranes, in vitro demonstrates the function of ion channels necessary for virus assembly and optimal exit from infected cells by changing the acid-base balance of intracellular vesicles. P7 membrane protein is a new target for antiviral therapy for HCV [12].

The essence of the invention is to create a new synthetic compound, which is a derivative of the prolyl-tryptophan dipeptide, protected at the amino group by a cyclohexyloxycarbonyl group, which inhibits the reproduction of HCV and has a virucidal effect against HCV. The compound is low toxic in vitro. The compound Cho-Pro-Trp-OH has the following structural formula:

Technical result: a new low-toxic compound is obtained: (2S) -3- (1H-idol-3-yl) [({1 - [(cyclohexyloxy) carbonyl] pyrrolidin-2-yl} carbonyl) amino] propanoic acid having antiviral, including virucidal activity against HCV to create a new antiviral drug against HCV.

A brief description of the drawings.

For a clearer understanding of the essence of the claimed invention, which is reflected in the claims, as well as to demonstrate its features and advantages, the following is a detailed description with reference to the drawings.

In FIG. 1 shows a synthesis scheme for the compound Cho-Pro-Trp-OH by the mixed anhydride method. Synthesis scheme for cyclohexyloxycarbonyl-prolyl-tryptophan compound, where NMM is N-methylmorpholine, IBCF is isobutylchloroformate.

In FIG. 2 shows mass spectrometry of the compound Cho-Pro-Trp-OMe (cyclohexyloxycarbonyl-prolyl-tryptophan methyl ether), the compound Cho-Pro-Trp-OMe. [M + H] = 442.

In FIG. 3 shows mass spectrometry of the compound Cho-Pro-Trp-OH. [M + H] = 428; [M + Na] = 450; [M + K] = 466.

In FIG. 4 in table 1 presents data on the effect of various concentrations of the Cho-Pro-Trp-OH compound on HCV reproduction in a culture of transplantable pig embryonic kidney cells (SPEV) when it is added before infection, simultaneously with the virus and after infection. ID ₅₀ - the concentration at which 50% of the virus is suppressed. 0 - all cells survived, (+) - 15% of the cells died; (++) - 50% died, (++++) - all cells died, K.v. - virus control (without substance).

In FIG. 5 in the form of table 2 presents data on the virucidal activity of the compound Cho-Pro-Trp-OH on the SPEV monolayer at various concentrations of the substance and dilutions of the virus. TTsID ₅₀ - tissue infectious dose of the virus that infects 50% of the exposed cell cultures. 0 - all cells survived; (++) - 50% died; (++++) - all cells died. K.V. - virus control (no connection)

In FIG. 6, Table 3 shows the biological properties of the compound Cho-Pro-Trp-OH. ID ₅₀ - inhibitory dose 50: the minimum dilution of the drug, causing protection of 50% of the cells of the monolayer. IPC - maximum tolerated concentration - half the concentration of the compound, which does not have toxic effects on cells. ID ₉₅ - the minimum concentration of compounds that causes protection of 95% of the cells. Chemotherapy Index (CTI) is the ratio of IPC to ID ₅₀ .

Various approaches can be used to prepare the Cho-Pro-Trp-OH compound. One way to obtain the compound Cho-Pro-Trp-OH is the mixed anhydride method (Fig. 1). However, the proposed synthesis method should not be construed as a limitation of the scope of the present invention in all respects.

The formation of a peptide bond between cyclohexanoxycarbonylproline and tryptophan methyl ester was carried out in a single step in an equimolar ratio.

In the synthesis of compounds used cyclohexanol, triphosgene (bis (trichloromethyl) carbonate) and L-amino acids of the company Sigma-Aldrich (USA). Iso-butyl chloroformate (IBCF) from Fluka (Switzerland), N-methylmorpholine (NMM) from Sigma-Aldrich (USA) were used. All solvents used for condensation and deprotection were pre-absolutized and distilled according to standard procedures. Identification of the obtained compounds was carried out using thin layer chromatography (TLC) on Silufol plates (Czech Republic) in the systems: methanol-chloroform. 13:60 (A), sec-butanol - 3% ammonia, 100: 44 (B), n-butanol - acetic acid - water - pyridine, 30: 3: 12: 10 (C). MALDI mass spectra were obtained on a Bruker autoflex speed mass spectrometer (Bruker Daltonics Inc., Germany) equipped with a solid-state UV laser with λ = 355 nm and a reflectron in the mode of recording positively charged ions. To record the mass spectra of MALDI, the MTP 384 ground steel target (Baiker Daltonics Inc., Germany) was used.

To record the MALDI mass spectra, the sample was mixed with a solution of the CIN matrix (synapinic acid, Sigma-Aldrich (USA)) in a 1: 1 ratio, applied to a steel target, and dried.

Virological studies were performed with a cytopathogenic variant of HCV genotype 1b isolated from the blood serum of patients with chronic hepatitis C [13]. Virus-containing material was used in the form of a sample of culture fluid collected 5 days after infection of cell cultures. The antiviral activity of the compound was studied in a pig embryonic kidney cell culture (SPEV) sensitive to HCV production. The cultures were grown as a one-day monolayer in 48-well plastic culture panels in medium 199 (manufactured by PanEco LLC) with the addition of 100 IU / ml penicillin and streptomycin and 10% cattle serum. The control of culture cells was carried out using a light-optical microscope.

For a more specific description of the biological properties of the obtained compounds in the present invention, antiviral and virucidal activity should be understood:

The virucidal activity of the compound is associated with the direct inactivating effect of the compound on the virions in the viral population, as a result of which the infectious activity of the virus is partially or completely lost. To check the virucidal properties of a substance (compound), it is enough to incubate the mixture of virus and substance for a certain time, and then check the infectious properties of the virus without the test substance and the virus mixed with the substance by titration in cell cultures or in the body of laboratory animals. A significant decrease in the infectious activity of the virus by more than 1.0 logarithm (lg) or its complete loss compared with the virus without a substance indicates the manifestation of the virucidal activity of the test compound.

The antiviral activity of a compound, as a rule, includes both the virucidal activity of the substance and its antiviral properties, i.e. the ability to inhibit one or another stage of virus replication: from the adsorption of the virus to cells, its penetration, to the effect on the assembly and exit of the virus from the infected cell. Therefore, the antiviral activity of the test substance is checked by treating the cell monolayer or by administering the substance to laboratory animals before infection with the virus (prophylactic effect of the substance), at the time of infection (therapeutic effect) and after a certain period of time after infection (therapeutic effect). The antiviral effect of the substance is evaluated both by the percentage of viable infected cells or healthy laboratory animals preserved under the conditions of use of the substance, compared with the control group, and by its property to reduce the ability of infected cells or infected animals to produce an infectious virus.

The present invention is illustrated by the following examples. However, these examples should not be construed as a limitation on the scope of the present invention in all respects.

Cho-Pro-Trp-OH was synthesized by the mixed anhydride method, starting from Cho-Pro-OH obtained by the method of [14] and HCl * H-Trp-OMe, followed by saponification. The synthesis scheme for the compound Cho-Pro-Trp-OH is shown in FIG. one.

Example 1. Obtaining Cho-Pro-Trp-OMe (cyclohexyloxycarbonyl-prolyl-tryptophan methyl ester).

Cho-Pro-OH (cyclohexyloxycarbonyl-proline) was obtained according to the procedure described by us earlier [14]. The resulting Cho-Pro-OH was used in the synthesis of the final compound.

To 1.0 g (4.15 mmol) of Cho-Pro-OH in 25.0 ml of CHCl _3, 0.42 ml (4.15 mmol) of NMM was added and with stirring at minus 25 ° C, 0.56 ml (4 15 mmol) IBCF. It was stirred for 10 minutes and a solution of 1.05 g (4.15 mmol) of rimantadine hydrochloride in 10.0 ml of CHCl ₃ with 0.42 ml (4.15 mmol) of NMM was added cooled to minus 20 ° C. Stirred for 30 minutes at minus 20 ° C, then 1 hour at 0 ° C and 10 hours at 20 ° C. The solvent was removed in vacuo, the residue was dissolved in 35.0 ml of ethyl acetate and 10.0 ml of H ₂ O and washed successively with 0.25 M H ₂ SO ₄ (4.0 ml × 1), 0.25 M KHCO ₃ (10, 0 ml × 2) and H ₂ O (5.0 ml × 1). The organic layer was separated and dried with anhydrous Na ₂ SO ₄ . Ethyl acetate was removed in vacuo to give an oily product.

Yield 1.31 g (93%); R _ƒ .98 (A), 0.88 (B), 0.90 (C); [α] _D ²⁰ -30 °; m / z found [M + H] ⁺ : 442.63; calculated M (C ₂₄ H ₃₁ N ₃ O ₅ ) 441.22 atomic unit (au) (Fig. 2)

The N-acyldipeptide ester (Cho-Pro-Trp-OMe) thus obtained was subsequently saponified in a mixture of 0.2 n NaOH - Acetone (1: 1)

Example 2. Obtaining Cho-Pro-Trp-OH (cyclohexyloxycarbonyl-prolyl-tryptophan). To a solution of 1.1 g (2.49 mmol) of Cho-Pro-Trp-OMe in 12.0 ml of acetone was added 16.0 ml (4.0 mmol) of 0.25 n NaOH. Maintain the reaction mixture at 20 ° C for 45 minutes. Acetone is removed in vacuo, the aqueous fraction is acidified with 10%) citric acid to pH 4, an oily precipitate forms. Transferred to a separatory funnel and add 20.0 ml of ethyl acetate, mix until two transparent layers are formed, the aqueous layer is separated and washed with water (2 × 4 ml). The aqueous layer was again separated, the organic layer was dried with anhydrous Na ₂ SO ₄ . Ethyl acetate is removed in vacuum, a brown oil is formed, weighing 1.66 g. After grinding the resulting oil with ether, an amorphous substance is formed. The ether is decanted. The substance is dried in vacuum until a dense foam forms. Foam is frozen at minus 40 ° C, then collapses and again dried in vacuum.

Yield: beige foam, 1.02 g (96%), [α] ^D ₂₀ -20 °, R _f 0.75 (A); R _f 0.72 (B); R _f 0.66 (C), m / z found [M + H] ⁺ : 428.22; calculated M (C ₂₃ H ₂₉ N ₃ O ₅ ) 427.61 au (Fig. 3).

Example 3. The study of the antiviral activity of Cho-Pro-Trp-OH.

The antiviral activity of the proposed compound was determined in HCV-infected cultures of SPEV cells grown as a full one-day monolayer in 96-well plastic culture panels. The compound in concentrations of 10.0; 5.0; 2.5; 1.25; 0.62; 0.15; μg / 200.0 μl was added 6 hours before infection of the cell monolayer with the virus at a dose of 0.1 TCID ₅₀ , immediately after adsorption of HCV on the cells and 6 hours after infection of the cells. The panels were incubated at 37 ° C for 5-6 days, when in the control wells not treated with the compound, the death of 100%) monolayer cells was not observed. The antiviral activity of the compounds was determined by their ability to protect SPEV cells from the cytopathogenic effect of the virus in comparison with control infected cultures (without compound). ID _{50 was} determined.

The synthetic compound Cho-Pro-Trp-OH was found to show a high degree of inhibition of HCV reproduction. It should be noted the pronounced antiviral effect of the compound and when it is introduced into the cell culture 6 hours before infection and simultaneously with HCV infection (Fig. 4).

Example 4. The study of the virucidal activity of the compound against HCV.

In the experiment, the compound at a concentration of 5.0; 2.5; 1.25 μg / ml was mixed with the virus as follows: 900.0 μl of a solution of the compound with the addition of 100.0 μl of HCV-containing material in the initial concentration. As a control, 900.0 μl of saline was added to 100.0 μl of viral material. Exposure to virus-containing material was carried out at room temperature for 30 minutes. After that, HCV infection activity was titrated in each experiment in SPEV cell cultures, infecting cells with 10-fold dilutions of a mixture of virus and a mixture of Cho-Pro-Trp-OH with HCV. A compound was characterized by virucidal activity only if the infectious titer of HCV for SPEV cells decreased by 100 or more times after exposure compared to the control titration.

As a result, a high virucidal activity of the compound Cho-Pro-Trp-OH was detected. The decrease in the infectious titer occurred by more than five logarithms (10,000 times) in relation to the viral control. The data are presented in table 2 (Fig. 5).

Example 5. Determination of cytotoxic effects.

The toxicity of the compound was studied when applied at concentrations of 10.0; 5.0; 2.5 and 1.25 mg / 200.0 μl per cell monolayer of SPEV tissue culture cells in 48-well panels and incubated at 37 ° C for 72 hours. The state of the cell monolayer was checked using a light optical microscope. The maximum tolerated concentration (MIC) is the concentration of the compound that causes toxic death of 50% of the monolayer cells. The BMD for the Cho-Pro-Trp-OH compound was more than 10 μg / 200 μl (Fig. 5.).

Thus, based on the data in Table 3 (Fig. 6), the chemotherapeutic index (CTI) for the compound Cho-Pro-Trp-OH was> 32 - with the prophylactic regimen; > 16 - with the treatment-and-prophylactic regimen of the compound with the virus; and> 8 - with the treatment-introduction regimen.

Proposed connection

может быть использовано для создания новых противовирусных препаратов, ингибиторов функции ионоселективного канала р7 ВГС с использованием как в виде индивидуального лекарства, так и в составе комплексной терапии гепатита C, применяемой в виде лечебной, профилактической и лечебно-профилактической схемах.

can be used to create new antiviral drugs, inhibitors of the function of the ion-selective channel p7 of HCV using both as an individual medicine and as part of hepatitis C complex therapy, used in the form of therapeutic, prophylactic and therapeutic prophylactic regimens.

BIBLIOGRAPHY

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3. Premkumar. A .; Wilson, L .; Ewart, G. D .; Gage, P.W. Cationselective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. // FEBS Lett. 2004. V. 557, P. 99-103.

4. Gazzarrini S., Kang M., Abenavoli A., Romani G., Olivari C, Gaslini D., Ferrara G., Etten JL, Kreim M., Kast SM, Thiel G., Moroni A. Chlorella virus ATCV- 1 encodes a functional potassium channel of 82 amino acids // Biochem. J. 2009. V. 420 P. 295-303.

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Claims (4)

1. The compound Cho-Pro-Trp-OH has the following structural formula:

2. The compound according to claim 1, having antiviral activity against hepatitis C. 3. The compound according to claim 1, having virucidal activity against hepatitis C.

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