RU2461544C1 - 1-(1-adamantyl)ethylamine derivatives and their antiviral activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a 1-(1-adamantyl)ethylamine derivative (remantadin) of general formula:

wherein R is a functional group of the amino acid residue (I-IV) or the lipoic acid residue (V).

(I),

(II),

(III),

(IV),

(V) which possess selective antiviral activity in relation to the influenza A strains, including the viral strains resistant to action of remantadin. The compounds wherein R means the groups (l),(ll),(lll) and (V) are novel.

EFFECT: prepared compounds may be promising as substances as a part of the antiviral drugs and applicable for creating new antiviral drugs.

2 cl, 2 dwg, 3 ex

Description

The invention relates to new biologically active derivative of 1- (1-adamantyl) ethylamine (rimantadine), and may find application in pharmacology and virology.

The influenza virus today remains the most widespread infectious disease, causing enormous economic damage. The most important problem of modern medical virology is overcoming the resistance of viruses to more recently effective chemical drugs.

The antigenic structure of the virus is subject to changes as a result of the selective pressure of the immune system of the host organism and the pressure from the use of chemotherapeutic agents. These two processes lead to the appearance of variants of viruses that can avoid both neutralizing antibodies, and thus overcome the immune system, and overcome the effect of chemotherapeutic drugs, initially aimed at a certain stage of virus reproduction. The complexity of the problem is that each type of virus has its own mechanism for adapting to a chemical drug. It is known that it is possible to inhibit the reproduction of influenza virus at different stages of its life cycle. Currently, drugs are used whose actions are aimed at suppressing the replication of the influenza virus. These are neuraminidase inhibitors - Oseltamivir (Tamiflu) and Zanamivir (Relenza), they act at the stage of release of newly synthesized influenza virus virions from the cell membrane, by blocking the cleavage of viral particles from the cell surface. And selective inhibitors of protein-channel M2 influenza virus functions A - drugs adamantanovonogo series: Rimantadine and amantadine that block the process of acidification in endosomes [1].

Adamantane preparations are much cheaper and easier to manufacture, which makes them more affordable for the treatment and prevention of influenza in the population. However, at present, as a result of the widespread use of adamantane preparations (remantadine and amantadine), their antiviral properties against influenza A viruses have been significantly lost. The loss of activity is mainly associated with a mutation in the transmembrane domain of the influenza virus M2 protein. It has now been established by crystallographic methods that the four subunits of this protein form a channel in the virus membrane through which protons are transported through the membrane of the viral particle. The key element of the proton channel is the histidine amino acid at position 37 (His37). The histidine residues of the four protein chains are combined in the form of a tetramer and form a ring. At high pH (in an alkaline environment) this ring is stationary, and at low pH (in an acidic environment) it changes its configuration due to the fact that histidine residues shift their orientation by 45 degrees. Such a transformation occurs at a speed of 50 thousand times per second. This is the operating state of the “pump” for holding protons inward. The suppliers of protons from the host cell are H ₃ O ⁺ ions. They fit into the M2 channel and are connected by hydrogen bonds with the imidosol ring of histidine, and when the configuration of the amino acid residue is changed, they are disconnected into the inner space of the viral particle [2].

Protons trigger the process of dissociation of viral proteins and the release of the genetic material of the virus into the cytoplasm of the cell. The mechanism of the antiviral effect of remantadine and amantadine is that the adamantane carbocycle is fixed in the phospholipid membrane of the virus, and the only amino group is able to interact with amino acid residues both inside and outside the proton-conducting channel, thereby disrupting the normal flow of protons.

Studies of the genome of influenza viruses resistant to the action of remantadine showed that the mutation affected only one amino acid substitution in the transmembrane region of the M2 protein. This was enough for remantadine and amantadine to lose their antiviral activity. The loss of antiviral activity of adamantane preparations (remantadine, amantadine) is associated with the replacement of the serine residue at position 31 with asparagine, less often with aspartic acid. Such a mutation makes it impossible to fix aminoadamantane in the pore of the M2 protein channel, since no hydrogen bond is formed between the amino group of the adamantane carbotricycle and the hydroxyl group of the serine residue [3]. The strains resistant to the drug rimantadine were initially less than 10% (1995-2002), then the resistance quickly increased to 58% in 2003, in 2004 it was already fixed at 74%, and in 2005-2006 it was 92 % [4, 5].

In some cases, the resulting resistance of influenza A strains can be overcome by introducing some structural changes in the environment of the adamantane carbocycle. At present, at least two directions are being developed to overcome drug resistance to adamantane derivatives. The first approach is the composition of remantadine with other drugs or substances. There are known complexes that promote the prolongation of the properties of remantadine with sodium alginate [6] or with no-shpa [7], as well as compositions that reduce the toxicity of remantadine, for example, a complex with 3,6-dioxocyclohexa-1,4-diene-1,2,4 , Sodium 5-tetrasulfate [8].

The second approach is the synthesis of a new antiviral compound based on the remantadine carbocycle. A patent of the Russian Federation is known [9], in which a complex drug is proposed that inhibits the reproduction of influenza A viruses. This compound of remantadine and polymer matrices called Polymer has a toxicity 5-10 times lower than that of remantadine [10].

One way to restore the antiviral properties of aminoadamantane compounds is to provide them with additional functionally active groups, which, in the process of interaction with the transmembrane domain, would be able to disrupt the process of proton transport through the virus membrane. The source of such functionally active groups may be amino acid residues introduced into peptides by peptide synthesis methods.

The objective of the invention is the creation of new derivatives of remantadine as compounds that selectively inhibit the reproduction of the influenza virus and are effective against strains resistant to the action of remantadine.

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

where R is the functional group of the amino acid residue (I-IV) or the remainder of lipoic acid (V):

I - ditretbutyloxycarbonyl-ornithyl-1- (1 -adamantyl) ethylamine,

II - tert-butyloxycarbonyl-sarcosyl-1- (1-adamantyl) ethylamine,

III - tert-butyloxycarbonyl-histidyl-1- (1-adamantyl) ethylamine

IV - 1- (1-adamantyl) ethylamide-2-aminoethanesulfonic acid

V - 1- (1-adamantyl) ethylamide-α-lipoic acid

To obtain compounds I-V, the following synthetic approach can be used on the example of compound I:

The formation of a peptide bond between the carbocycle containing an amino group and amino acids protected at the amino group by a tert-butyloxycarbonyl group (Boc-) or with lipoic acid (3- (4-carboxybutyl) -1,2-dithiolane) in compound V was carried out in one step under the reaction conditions of mixed anhydrides in an equimolar ratio. If necessary, the tert-butyloxycarbonyl group was removed by the action of ethyl acetate saturated with 4 n HCl at a temperature of 20-24 ° C.

Only L-amino acids produced by Nova Biochem (USA) were used in the work. The solvents used for condensation were prepared according to standard procedures. Analysis of thin-layer chromatography (TLC) was performed on Silufol plates (Czech Republic) in systems: sec-butanol - 3% ammonia (100: 44) (A), methanol-chloroform (13:60) (B), butanol-acetic acid-water -pyridine (30: 3: 12: 10) (C), allowing to state the complete absence in the test samples of traces of remantadine. The present invention is illustrated by the following examples.

Example 1. Synthesis of Boc-Sar-Rem (tert-butyloxycarbonyl-sarcosyl-1-adamantylethylamine) (R = —CO — CH ₂ —NH (Boc) —CH ₃ ) and its isolation.

To 1.5 g (0.0079 mol) of tert-butyloxycarbonyl-sarcosine (Boc-Sar-OH) in 15 ml of CHCl _{3 are} added 0.87 ml (0.0079 mol) of N-methylmorpholine (MMM). It is cooled to -20-25 ° C and 1.08 ml (0.0079 mol) of isobutyl chloroformate (IBCF) is added to the reaction mass with stirring. Mix for 10 to 15 minutes. Then, the second component, 1- (1-adamantayl) ethylamine hydrochloride 1.71 g (0.0079 mol) in 15 ml CHCl ₃ with 0.87 ml (0.0079 mol) is added, previously prepared and cooled to -20 ° C. ) NMM. Stirred for 30 minutes, then another 1 hour at 0 ° C and 2 hours at 24 ° C.

The reaction mass is evaporated from CHCl ₃ in vacuo. The residue was dissolved in 35 ml of ethyl acetate. Sequentially extracted with H ₂ O (5 ml × 1), 0.5 N NaHCO ₃ (10 ml × 2), 10% citric acid (4 ml × 1) and H ₂ O (5 ml × 1). The organic layer was dried over anhydrous Na ₂ SO ₄ . Ethyl acetate was removed in vacuo to give white crystals. Yield: 2.55 g (83%), R _f ^A = 0.86; B _f ^B = 0.68; R _f ^C = 0.91, [α] ^D ₂₀ = -2.5 °, mp. 116 ° C.

In a similar manner, compounds I, III, IV and V were obtained, the physicochemical constants of which are presented in Table 1 (Figure 1).

Example 2. The study of the antiviral activity of the compounds of formula I-V against influenza A.

Remantadine-resistant influenza viruses were used in the work: a pandemic strain of the influenza virus A / IIV-Moscow / 01/2009 (H1N1) v [11], and an epidemic strain of the influenza virus A / Moscow / 26/2009 (H3N2).

The antiviral activity of remantadine and new compounds (derivatives of remantadine) was studied on 96-well panels with a formed monolayer of MDCK tissue culture cells. At the same time as infection, remantadine derivatives were introduced into the cell monolayer: compound I or II, or III, or IV, or V at a concentration of 5 μg / ml, and remantadine at the same concentration was introduced on each panel as a control. The panels were incubated for 24 hours at 37 ° C, the reaction was stopped by fixing the cells with 80% acetone in phosphate buffer. The statement of the method of enzyme-linked immunosorbent assay (ELISA) was carried out according to the method described previously [12]. The percentage inhibition of viral activity by compounds was determined as the ratio:

where OP is the optical density at 492 nm, OD _{cell k} - OD ₄₉₂ cell control, OD _vir. - OD ₄₉₂ viral control.

It was found that the synthesized compounds (I-V) showed a high percentage of inhibition of the reproduction of influenza A virus strains resistant to the action of remantadine and amantadine. The data are presented in table 2 (Figure 2).

Example 3. Determination of the cytotoxic effect of the compounds of formula I-V.

The toxicity of the compounds was studied by adding their concentrations (5, 20, 40 and 80 μg / ml) to the monolayer of MDCK tissue culture cells in 96-well panels and incubation at 37 ° C. The state of the cell monolayer was checked under a microscope. The concentration of the substance that causes the degeneration of 50% of the cells compared to the control is taken as the average toxic concentration (CT ₅₀ ). The smallest concentration of a substance that causes cell degeneration is considered the minimum toxic concentration (MTK). The maximum tolerated concentration (MTK) is considered to be half the concentration of the compound, which does not have a toxic effect on the cells. Assessment of cytotoxicity was carried out by the colorimetric method. After incubation for 72 hours at 37 ° C, the monolayer was washed with a solution of PBS (phosphate buffer). The number of viable cells was determined by comparing the intensity of staining the solution in the control and experimental wells with the addition of neutral red on an automatic spectrophotometer at a wavelength of 450 nm. The concentration of the drug, inhibiting the value of OD ₄₅₀ by 50% compared with the cell control, is taken as a 50% cytotoxic dose (CD ₅₀ ). The data are presented in table 2 (Figure 2). It was noted that the toxic effect of the action on the cells of compounds (IV) did not exceed the effect of remantadine. Moreover, the cytotoxic effect of compounds IV and V is significantly less than that of remantadine.

Thus, new compounds of formula I-V based on remantadine are obtained that have selective anti-influenza activity with an efficiency exceeding that of remantadine hydrochloride. The presented compounds are able to inhibit the replication of influenza A viruses, including influenza strains resistant to the action of rimantadine preparations. The compounds of formula IV and V in vitro have significantly lower toxicity compared to remantadine. These amino acid, aminosulfonic and sulfhydride derivatives of remantadine described by Formula I-V are capable of efficiently penetrating 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 that of remantadine, whose adamantane carbocycle penetrates the membrane of a viral particle and will play the role of a carrier to which a functionally active group of the corresponding amino acid is “attached”. The resulting compounds can be used to create new antiviral drugs.

Literature

1. Balannik V., Wang J., Ohigashi Y., "Design and pharmacological characterization of inhibitors of amantadine-resistant mutants of the M2 ion channel of influenza A virus." Biochemistry v. 48, p.11872-11882 (2009).

2. Hu F., Luo W., Hong M. "Mechanisms of Proton Conduction and Gating in Influenza M2 Proton Channels from Solid-State NMR." Science, v. 330 no. 6003. p.505-508 (2010).

3. Balannik V., Camevale V., Fiorin G., “Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel”. Biochemistry v. 49, p. 696-708 (2010).

4. Laohpongspaisan C., Rungrotmongkol E., Intharathep P., “Why Amantadine Loses Its Function in Influenza M2 Mutants: MD Simulations” J. Chem. Inf. Model, v. 49, p. 847-852 (2009).

5. Burtseva E.I., Shevchenko E.S., Leneva I.A. et al. “Sensitivity to rimantadine and arbidol of influenza viruses that caused epidemic rises in the incidence rate in Russia in the 2004-2005 season”, Issue. virusol. No. 2., p.24-29 (2007).

6. Kiselev V.I., Kiselev O.I., Yuryev M.Z. "The drug for the treatment and prevention of viral diseases." RF patent No. 2185822 (RU).

7. Ashmarin I.L., Zhilinskaya I.N., Kiselev O.I. Osidak L.V. "A treatment for the flu." RF patent No. 2229877 (RU).

8. Eropkina E.M., Kiselev O.I., Eropkin M.Yu., Tveryanovich I.A. "An agent with antihypoxic and cytoprotective activities used to reduce the toxicity of remantadine in the treatment of influenza infection." RF patent No. 2302236 (RU).

9. Kiselev O.I., Chizhov N.P., Ilyenko V.I. and others. "Antiviral drug POLIREM." RF patent No. 2071323 (RU).

10. Kiselev O.I., Sominina A.A., Kozeletskaya K.N. “Structure and antiviral activity of adamantane-containing polymer drugs”, Vop. virusol., No. 5, pp. 19-26 (2003).

11. Lvov D.K. Burtseva E.I., Prilipov A.G. and others. "The strain of influenza virus A / IIV-Moscow / 01/2009 (H1N1) sw1 for the development of biological protection means and methods." RF patent No. 2412244 (RU).

12. Leneva I.A., Fedyakina I.T., Sokolova M.V., Guskova TA. “The use of enzyme immunoassay to study the effect of an antiviral drug on the reproduction of the respiratory syncytial virus, Q. virusol. No. 2., p. 42-45 (2002).

Claims (2)

1. Derivatives of 1- (1-adamantyl) ethylamine (remantadine) of the general formula:

where R is the functional group of the amino acid residue (I-III)
or the remainder of lipoic acid (V):

2. The compounds according to claim 1 and the compound of the formula according to claim 1, where R - means a group of amino acid residue

possessing selective antiviral activity against influenza A strains, including strains of the virus resistant to the action of remantadine.

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