RU2524216C1 - Peptide derivatives of 1-(1-adamantyl)ethylamine and their antiviral action - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to novel biologically active derivatives of 1-(1-adamantyl)ethylamine (remantadine) representing adamantyl peptides having antiviral action. The invention may find application in medicine, pharmacology and virology as new medicinal products to treat Hepatitis C.

EFFECT: obtained new low-toxic compounds have selective antiviral activity against Hepatitis C virus.

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Description

The invention relates to new biologically active derivatives of 1- (1-adamantyl) ethylamine (remantadine), which are adamantyl peptides with an antiviral effect, primarily against hepatitis C. The invention may find application in medicine, pharmacology and virology as new drugs to treat hepatitis C.

Hepatitis C is known to be a serious public health problem. According to the WHO, 3% of the population (about 170 million people) are infected with hepatitis C virus (HCV), which in most cases leads to chronic slowly progressive lesions of the liver, as well as other organs and tissues of a person. Cirrhosis and hepatocellular carcinoma are the most common complications of HCV infection. HCV is the leading cause of death due to liver damage and one of the most common causes of donor liver transplantation.

HCV belongs to the genus Hepacivirus, the family Flaviviridae [1]. The viral genome consists of single-stranded positive RNA with a length of about 9.6 Kb. It encodes a protein precursor of the order of 3000 amino acid residues, which is cut post-translationally in the endoplasmic reticulum by cellular and viral proteases, which leads to the formation of 10 proteins [2]. They are divided into structural, capsid proteins and two shell glycoproteins E1 and E2, which are responsible for binding to co-receptors and incorporation into hepatocytes. Non-structural p7 short membrane proteins are also formed, which are believed to act as viroporins [3]. At least, it has been proven that in a cell culture, p7 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 [4]. In vivo, p7 is of great importance for the manifestation of the infectious properties of the virus [5].

It is known that, for the treatment of HCV, the most effective medications are interferon (IFN), or IFN in combination with ribavirin. However, in most patients infected with HCV, and in particular genotype 1b, which dominates circulation in the Russian Federation, a stable therapeutic effect cannot be achieved with these drugs. The low availability of these drugs because of their high cost, the severity of side effects with prolonged use continues to stimulate interest in finding new affordable and effective anti-HCV drugs. To solve this problem, researchers are looking for new classes of drugs that act differently from IFN and ribavirin, and are aimed directly at the viral particle.

The main goal is to find "direct-acting" drugs that attack various mechanisms of the vital functions of the virus and minimize the risk of resistance.

The data of crystallographic studies show that the ion-selective channel present in the protein coat of the HCV contains 6 identical p7 subunits and is a hexamer. Each p7 HCV monomer consists of 63 amino acid residues that are mostly hydrophobic and positively charged. The secondary structure of the monomer consists of two transmembrane helices and a short loop. Moreover, the loop faces the cytoplasm, and the N- and C-ends of the polypeptide to the endoplasmic reticulum.

An important role in the reproduction of HCV viral particles is played by the p7 ion channel and represents a new important therapeutic target [6]. It was previously determined that the functions of the p7 proton channel can be blocked by small molecule inhibitors, which will lead to blockade of the reproduction of viral particles [7].

The function of HCV p7 protein as an ion channel in an artificial lipid bilayer was confirmed. In vitro experiments demonstrated the possibility of termination of the channel formed by the p7 protein by amantadine [8]. Amantadine (1-aminoadamantane) and remantadine (1- (1-adamantyl) ethylamine) are known to be used to treat and prevent influenza infection. Their antiviral activity is associated with inhibition of the function of viroporin, formed by the M2 protein in the protein coat of influenza A virus, which regulates proton transport through the membrane of the virus into the virion. The presence of a bulk adamantane carbocycle in the pore of the virus membrane leads to disruption of proton transport due to the formation of a hydrogen bond between the amantadine amino group and serine oxy group at position 31 in the transmembrane domain [9]. Many researchers believe that ion-selective channels can be considered as the "Achilles heel" of viruses. The principle of action of the p7 ion channel in cell membranes and its blocking by amantadine confirm the fact that this viroporin can be considered as a target for future antiviral chemotherapy.

The above data give the right to assume that the aminoadamantane carbocycle molecule, provided with additional functionally active groups, in the process of interaction with the transmembrane domain of the p7 hexamer protein can disrupt the ion transport through the virus membrane. The source of such functionally active groups can be peptide residues introduced into remantadine by peptide synthesis methods. It is assumed that the nature of the amino acid residue functional group, as well as the distance between it and the adamantane carbocycle, can influence the occurrence of inhibitory properties with respect to the p7 HCV ion channel.

The objective of the present invention is the development and synthesis of new derivatives of adamantane as compounds that selectively inhibit the reproduction of HCV.

The proposed compounds belong to the new adamantane derivatives of 1- (1-adamantyl) ethylamine (remantadine) and have the following formulas:

I - Di (tert-butyloxycarbonyl) lysyl-glycyl-glycyl-1- (1-adamantyl) ethylamine;

II - Di (tert-butyloxycarbonyl) lysyl-glycyl-glycyl-glycyl-1- (1-adamantyl) ethylamine;

III - Di (tert-butyloxycarbonyl) lysyl-prolyl-prolyl-glycyl-1- (1-adamantyl) ethylamine.

EFFECT: new low-toxic compounds with selective antiviral activity against HCV are obtained. Moreover, the virucidal activity of compound II was revealed, which reduces HCV infectivity by five logarithms compared with the viral control (logTsID50 = 5.0).

The essence of the invention lies in the creation of new compounds that are peptide derivatives of 1- (1-adamantyl) ethylamine and have antiviral effects against HCV. Compound II has a virucidal effect.

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.

Figure 1 shows the synthesis scheme of the compound Di (tert-butyloxycarbonyl) -lyl-glycyl-glycyl-1- (1-adamantyl) ethylamine by the mixed anhydride method. Compounds contribute in equimolar amounts. The starting components were obtained by classical peptide synthesis in solution.

Figure 2 presents in the form of Table 1 physico-chemical constants of the proposed compounds.

Figure 3 presents in the form of Table 2 data on the antiviral and cytotoxic activity of compounds with tissue cytopathic dose 10 (TCD10).

Figure 4 presents in the form of Table 3 data on the antiviral and toxic activity of the compounds in TCD100.

Figure 5 presents in the form of Table 4 data on the virucidal activity of compound II at various concentrations and dilutions on the monolayer SPEV.

Figure 6 presents in the form of Table 5 the cytotoxic effect of the proposed adamantyl peptides on the monolayer of cell culture SPEV.

To obtain compounds I-III, the following synthetic approach can be used on the example of compound I (figure 1).

Esters of N-acyldi and tripeptides were obtained by classical peptide synthesis in solution. Esters of N-acyldi and tripeptides were saponified by 0.1 N during the synthesis. NaOH (acetone-water, 1: 1). The formation of a peptide bond between 1- (1-adamantyl) ethylamine containing an amino group and peptides protected at the amino group with tert-butyloxycarbonyl groups (Isch-) was carried out in one step under the reaction conditions of mixed anhydrides in an equimolar ratio. Subsequently, the tert-butyloxycarbonyl group was removed by the action of saturated ethyl acetate 4n. HCl, at room temperature. The resulting adamantyl peptide hydrochloride was condensed with di (tert-butyloxycarbonyl) lysine by reaction of mixed anhydrides.

In the synthesis of peptides, L-amino acids of Nova Biochem (USA) were used. In the synthesis of adamantane derivatives, racemic remantadine from Zhejiang Kangyu Pharmaceutical Co. (China) was used. Iso-butyl chloroformate (IBCF) from Fluka (Switzerland), N-methylmorpholine (NMM) from Koch Light Laboratories) were used; (England). Thin layer chromatography (TLC) was performed 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), iso-propanol - water - 3% ammonia (7: 1: 2) (D), which allow us to state the complete absence of traces of remantadine in the test samples. Physico-chemical constants of the proposed compounds are presented in table 1 (figure 2).

Virological studies were performed with a cytopathogenic variant of HCV genotype 1b isolated from the blood serum of patients with chronic hepatitis C [10]. 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 compounds I, II, III was studied on a pig embryo kidney cell culture (SPEV) sensitive to HCV production. The cultures were grown as a two-day monolayer in 48-well plastic culture panels in medium 199 (production of the MP Chumakov Research Institute of Poliomyelitis and Viral Encephalitis RAMS) with the addition of 100 PIECES / ml of penicillin and streptomycin and 10% cattle serum . The control of culture cells was carried out using a light-optical microscope.

For a more specific understanding 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 a direct inactivating effect 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 1.0 or more logarithms (1g) 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.

To more fully illustrate the present invention, examples are provided below. However, these examples should not be construed as a limitation on the scope of the present invention in all respects.

Example 1. Synthesis of Boc-Lys (Boc) -Gly-Gly-Gly-Rem (II) (di-tert-butyloxycarbonyl) lysyl-glycyl-glycyl-glycyl-1- (1-adamantyl) ethylamine) and its isolation.

0.24 ml (0.0022 mol) of NMM are added to 0.76 g (0.0022 mol) of di-tert-butyloxycarbonyl-lysine in 10.0 ml of CHCl ₃ . It is cooled to -25 ° C and 0.30 ml (0.0024 mol) of IBCF isobutyl chloroformate are added to the reaction mass with stirring. Stirred for 15 minutes. Then, a solution of 0.85 g (0.0022 mol) of glycyl-glycyl-glycyl-1- (1-adamantyl) ethylamine hydrochloride in 10.0 ml CHCl ₃ with 0.24 ml (0.24 ml) was prepared and cooled to -20 ° C. 0.0022 mol) NMM. Stirred for 30 minutes, then another 1 hour at 0 ° C and 2 hours at room temperature.

The reaction mass is freed from CHCl ₃ in vacuo. The residue was dissolved in 35.0 ml of ethyl acetate. Sequentially extracted with H ₂ O (5.0 ml × 1), 0.5 N. NaHCO ₃ (10.0 ml × 2), 0.5N H ₂ SO ₄ (4.0 ml × 1) and H ₂ O (5.0 ml × 1). The organic layer was dried with anhydrous Na ₂ SO ₄ .

Ethyl acetate was removed in vacuo to give a white foam, which crystallized in hexane-ether (1: 1).

.Yield: 1.22 g (82%), mp. 107-108 °, R _f = 0.89 (A); R _f = 0.68 (B); R _f = 0.95 (C), R _f = 0.96 (D), $${[\alpha ]}_{\mathrm{twenty}}^D=-{\mathrm{one}}^{\circ }$$

.

Compounds I and III were similarly prepared.

Example 2. The study of the antiviral activity of compounds I-III. The antiviral activity of the compounds of the invention was determined in HCV-infected SPEV cell cultures. Compounds were introduced at the time of infection of the cells (experiment 1) and 20 hours after infection (experiment 2) at concentrations of 5.0; 2.5; 1.25; 0.62; 0.15; 0.07 and 0.035 mg / ml. The panels were incubated at 37 ° C for 24 hours. 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 the reference drug (remantadine) at the same concentrations and control infected cultures (without compound). The study was carried out with tissue cytopathic dose 10 (TCD10) (figure 3) and with TCD100 (figure 4).

The synthesized compounds (I-III) were found to show a high degree of inhibition of HCV reproduction. It should be noted the pronounced antiviral effect of the compounds and when they are introduced into the cell culture 20 hours after infection with HCV. The most active antiviral effect is possessed by compound II, which effectively suppressed HCV replication already at a concentration of 0.15 mg / ml both in experiment 1 and in experiment 2. (Figs. 3 and 4).

Example 3. The study of the virucidal activity of compound II against HCV.

Virucidal activity is illustrated by the example of compound II. In the experiment we used the compound at concentrations of 5.0, 2.5 and 1.25 mg / ml, which were mixed with the virus as follows - 200.0 μl of a solution of the compound with the addition of 100.0 μl of HCV-containing material in the initial concentration. Exposure to virus-containing material was carried out at room temperature for 30 minutes. Then, the infectious activity of HCV was titrated in each experiment in cultures of SPEV cells at various dilutions of a mixture of compound II with HCV. A compound was characterized by virucidal activity only if the decrease in the HCV infectious titer for SPEV cells after exposure was 100 or more times.

As a result, a high virucidal activity of compound II 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 4 (figure 5).

Example 4. Determination of the cytotoxic effect of compounds I-III.

The toxicity of the compounds was studied by adding them at concentrations of 5.0, 2.5, and 1.25 mg / ml to the monolayer of SPEV tissue culture cells in 48-well panels and incubation at 37 ° C. The state of the cell monolayer was checked under a microscope. The maximum tolerated concentration (MPC) is considered to be half the concentration of the compound, which does not have a toxic effect on the cells.

It was noted that the cytotoxic effect of compounds I and III is significantly less than that of remantadine; their BMD is about 0.625 mg / ml, and for compound II, the BMD is about 1.25 mg / ml (Fig.6).

T.O. New compounds of the formula I-III based on adamantyl peptides with selective antiviral activity were obtained. The proposed compounds are able to inhibit HCV replication. Compounds of formula I-III in vitro are significantly less toxic than remantadine. Moreover, compound II has the highest BMD, and also exhibits virucidal activity against HCV. These amino acid derivatives of adamantane are able to efficiently penetrate the membrane phospholipid bilayer of the virus. The mechanism of the antiviral action of the obtained compounds is not fully understood. The main proposed mechanism of antiviral action is probably similar to the effect of amantadine on the HCV p7 ion channel, which was discovered by C. Griffin and co-workers [8]. The adamantane carbocycle penetrates the membrane of the viral particle, where the ion-selective channel p7 of the HCV is located, which represents the most likely target for the proposed compounds. Adamantane in this case, obviously, plays the role of a carrier to which the functionally active group of the corresponding peptide is “attached”. The proposed compounds can be used to create new antiviral drugs, inhibitors of the function of the ion-selective channel of HCV using both as an individual drug and as part of complex therapy, similar to the highly active antiretroviral therapy (HAART), which is now used to treat HIV infection.

Literature

1. Jones C.T., Murray C.L., Eastman D.K. et. al. "Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus." J. Virol. 2007, v. 81 (16), p. 8374-8383.

2. Cook G.A., Opella S.J. "NMR studies of p7 protein from hepatitis C virus." Eur. Biophys J. 2010, V.39, p.l097-1104.

3. Tedbury P., Welbourn S., Pause A. et al. "The subcellular localization of the hepatitis C virus non-structural protein NS2 regulated by an ion channel-independent function of the p7 protein." J. Gen. Virol. 2011, v. 92 (4), p. 819-30.

4. Murphy S., Martin S., Parton R.G. "Lipid droplet-organelle interactions; sharing the fats. " Biochim Biophys Acta. 2009.v.1791 (6), p.441-447.

5. Martin S, Parton RG. “Lipid droplets: a unified view of a dynamic organelle)). Nat. Rev. Mol. Cell. Biol. 2006. v. 7 (5), p. 373-378.

6. Griffin S. D. C., Harvey R., Clarke D. S., et al. “A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria)). J. Gen. Virol. 2004. v.85, p.451-461.

7. T.L. Foster, M. Verow, A. L. Wozniak, et al. “Resistance Mutations Define Specific Antiviral Effects for Inhibitors of the Hepatitis C Virus p7 Ion Channel)). Hepatolodgy. 2011. v. 54 (l), p. 79-90.

8. Griffin, S.D., Beales, L.P., Clarke, D.S., et. al. “The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, amantadine)). FEBS Lett. 2003. v.30, p. 34-38.

9. Cady S. D., Mishanina T. V., Hong M. et. al. “Structure of amantadine-bound M2 transmembrane peptide of influenza A in lipid bilayers from magic-angle-spinning solid-state NMR: The role of Ser31 in amantadine binding)) J. Mol. Biol. 2009. v.385, p.1127-1141.

10.P.G.Deryabin, D.K. Lvov. "Highly productive variant of the hepatitis C virus. Isolation, characterization, identification." Reports of the Academy of Sciences of the Russian Federation, 1998, v. 358, N.5, p. 688-691.

Claims (3)

1. Peptide derivatives of 1- (1-adamantyl) ethylamine and having the following formulas:

2. Compounds I-III according to claim 1, having selective antiviral activity against hepatitis C. 3. Compounds II according to claim 1, having virucidal activity against hepatitis C.

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