RU2281297C2 - Polyanionic norbornan derivatives, method for production thereof and reproduction inhibitors of human immunogenicity virus based on the same - Google Patents

Abstract

FIELD: polymers, immunology.

SUBSTANCE: invention relates to polyanionic norbornan derivatives of formula I:

, wherein

or is residue of another oxygen-containing monomer, such as

; n>=15. Preferably n = 35-100 (polymerization ratio); M is H, Na, K, NH₄; X is OH, OM, ONH₃-Y-Nb, NH-Y-Nb, wherein not less 3 % of total X represent sum of ONH₃-Y-Nb and NH-Y-Nb (modification ratio with norbornan), and spacer group -Y- represents - (Y_i)_m [Y_i is CH , CH(CH₃), CH(CH₂CH₃); m>=0, preferably m = 1-3). And norbornan pharmacophor has formula

, wherein

represents bicyclic norbornan carbon skeleton including seven carbon (C) atoms in 1-7 sites and one (any) substituent from R¹, R^2a, R^2b, R^3a, R^3b, R⁴, R^5a, R^5b, R^6a, R^6b, R^7a, R^7b, represents covalent bond between Nb and Y; and the rest eleven substiruents represent H or alkyl, or two by two represent covalent chemical bond in bicyclic hydrocarbon skeleton, i.e. additional double bond between carbon atoms. Method for production of formula I compounds also is disclosed. Said compounds have high selectivity index in relation to HIV-1 strains, having resistance to known azidotimidine pharmaceutical agent.

EFFECT: polyanionic norbornan derivatives useful as active ingredients of antiviral pharmaceutical formulation.

3 cl, 6 ex, 2 tbl, 4 dwg

Description

The present invention relates to the field of organic chemistry and virology and relates to new biologically active derivatives of norbornane (bicyclo [2.2.1] heptane) - its polyanionic modifications, a method for the preparation and antiviral activity of compounds of this series, which can be used in medicine.

In a number of known norbornane compounds, a pronounced antiviral activity is registered in the case of 2- (1-aminoethyl) norbornane, in particular in the form of hydrochloride (deutiforin):

However, deutiforin does not have HIV inhibitory activity, and its antiviral effect (and indications for medical use) are limited, mainly to influenza A virus [1a].

According to the chemical structure, biological activity and clinical indications, deutiforin is a close analogue of remantadine (1- (1-aminoethyl) adamantane hydrochloride)

which is also highly active against a number of strains of the influenza A virus [1b, 2], but, like other low-molecular-weight derivatives of adamantane, does not inhibit the reproduction of HIV in cell cultures. We previously described and patented new adamantane compounds chemically modified with polymer (or oligomeric) polyanions, which, in contrast to the known prototypes of the adamantane series, have pronounced HIV-1 inhibitory activity, inhibiting the penetration of the virus into the cell through the plasma membrane [3, 4]. However, compounds of the adamantane series (frame tricycles) have a more complex chemical structure than norbornane analogues (bicycles), which leads to a significantly more complex synthesis and lower availability of adamantane compounds compared to norbornane compounds. In addition, adamantane abiotics are generally more toxic than similar derivatives of norbornane, which forms the basis of a number of natural bicyclic terpenoids (camphor, camphene, borneol, isobornoleol, fenchen, fenkhon, etc.) [5].

The essence of the invention is to obtain new polyanionic derivatives of the norbornane series, general formula I. The compounds of the invention possess antiviral effects, primarily against human immunodeficiency virus type 1 (HIV-1) and are chemically more accessible than adamantane prototypes. These compounds highly selectively inhibit the reproduction of HIV-1 and are effective against strains that are resistant to the basic drug of anti-HIV / AIDS therapy - azidothymidine (AZT).

The present invention provides polyanionic derivatives of norbornane (bicyclo [2.2.1] heptane) of general formula I

Where

or the remainder of another oxygen-containing monomer, for example:

n≥15, preferably n = 35-100 (degree of polymerization);

M = H, Na, K, NH ₄ ;

-X = -OH, -OM, -ONH ₃ -Y-Nb, -NH-Y-Nb,

moreover, in the series of the indicated modifications of the chemical moiety -X, at least 3% of the total number -X is the combined share of the variants -NH ₃ -Y-Nb and -NH-Y-Nb (degree of modification with norbornane) in which the spacer group is -Y - = - (Y _i ) _m - where Y _i = CH ₂ , CH (CH ₃ ), CH (CH ₂ CH ₃ ), m≥0, (preferably m = 1-3), and "norbornane pharmacophore" is described by the formula :

Where

frame bicyclic carbon skeleton of norbornane, including seven carbon atoms (C) in positions 1-7,

where in the series of the above twelve substituents R ¹ , R ^2a , R ^2b , R ^3a , R ^3b , R ⁴ , R ^5a , R ^5b , R ^6a , R ^6b , R ^7a , R ^7b one (any) of them is - (covalent bond between Nb and Y), while the remaining eleven = H, or alkyl, or in pairs form a covalent chemical bond within the bicyclic hydrocarbon core, that is, an additional (double) bond between carbon atoms.

The compounds of formula I are new derivatives of norbornane, modified oligo - or polymer derivatives of succinic acid. Moreover, in the aqueous phase, carboxyl groups (or their salt modifications): - COOM and -COX (where X = OH, OM) are capable of dissociation with the formation of carboxy anions bearing a negative electric charge

At sufficiently high degrees of polymerization (n≥15), a polyanionic modification of norbornane derivatives is achieved, which provides excellent solubility of the compounds of formula I in aqueous (physiological) media (unlike unmodified norbornane compounds) and leads to a significant modulation of the biological activity of norbornane pharmacophores. In particular, the compounds of formula I exhibit specific activity against HIV-1, including virus resistant strains of AZT.

The proposed mechanism of the HIV-inhibitory effect of the compounds of formula I is a synergistic combination of two main functions: 1) the electrostatic effect of polyanions on positively charged sites of virus-specific proteins on amino acid residues of lysine, arginine or histidine (preferably at the stage of HIV cell adsorption, in the region of V3 loop of glycoprotein of the viral envelope gp120) and 2) protection of cell membranes due to membranotropic norbornane (Nb) pharmacophores (preferably at the stage of membrane fusion and cell membrane virus).

The synthesis of compounds of formula I can be carried out:

Method A. Attachment of the amino derivatives of norbornane of the formula NH ₂ -Y-Nb to the corresponding oligo- or polymeric (with a polymerization degree n of at least 15) succinic anhydride analogues (PA), followed by hydrolysis of unreacted anhydride units and salt modification of the carboxy-acid groups formed as a result of hydrolysis:

Or a similar result is achieved

Method B. Attachment of the same amine reagents to the carboxyl groups not of the anhydride but of the acid form of the corresponding oligo- or polymer analogues of succinic acid (PC), but in the presence of condensing agents (CA):

The present invention is illustrated by the following examples.

Example 1. Synthesis, isolation and purification of compound Ia, formula I,

Where

mass average value n = 70 ± 20

M = Na

X = OH (≤86%), ONH ₃ -Y-Nb (≤7%, ammonium form), NH-Y-Nb (≥7%, amide form)

2-substituted norbornane without separation of stereo- (exo-, endo- and optical isomers), that is: one of the group R ^2a , R ^2b , R ^3a , R ^3b forms a covalent bond between Nb and Y, and all other substituents, including also R ¹ , R ⁴ , R ^5a , R ^5b , R ^6a , R ^6b , R ^7a , R ^7b are hydrogen atoms (H).

Obtaining compound Ia according to method A, option A-1.

To a solution of a polyanhydride copolymer of the formula PA-1

0.80 g (0.0060 g-equiv. Of anhydride reaction centers) in 9.0 ml of dry dimethyl sulfoxide (DMSO) at a temperature of 25 ± 3 ° C with vigorous stirring add 0.12 g (0.00085 mol) of 2- (1-aminoethyl) norbornane in 0.9 ml of dry toluene . The reaction solution is stirred for 1 hour at 40 ° C and 24 hours at 25 ± 3 ° C. The polymer product is planted in excess hexane (50 ml), filtered and residual organic solvents are removed in vacuo at 40 ° C for 10 hours. To the resulting powdery product was added 0.52 g (0.006 moles) of sodium bicarbonate in 25 ml of double-distilled water and stirred at room temperature until a homogeneous solution was formed. The temperature of the solution was raised to 40-45 ° C and stirred for 2 hours, the solution was cooled to room temperature and incubated for 48 hours in a nitrogen atmosphere. Low molecular weight impurities are removed by ultrafiltration on membranes with a permeability of 1 KD. Removal of high molecular fractions with simultaneous sterilization of the product is carried out by ultrafiltration on membranes with a permeability of 100 KD. The final purified product is isolated from the aqueous phase by freeze drying in the form of a highly porous white powder. Yield 0.95 g (83%).

¹ H NMR (D ₂ O, δ, ppm): 4.49 (H,> CH-O-CH <at positions 2.5 of the furan ring of fragment -Z-), 3.43 (2H, CH ₂ at position 7 of Nb-2- exo), 3.04 (1H, CH at position 1 of Nb-2-exo), 2.90 (2H, CH ₂ at position 7 of Nb-2-endo), 2.9-2.7 (1H,> CH-CO- in the composition of succinic acid residues ), 2.40-2.35 (CH ₂ at the 5-endo and 6-exo positions of Nb-2-exo, as well as within the fragment -Z-), 2.07 (1Н, СН at position 1 Nb-2-endo), 2.01 ( 1H, CH at position 4 of Nb-2-endo), 1.94 (1H, CH ₂ at position of 2-exo Nb-2-endo), 1.83 (1H, CH ₂ at position of 3-exo Nb-2-endo), 1.74 (1H, CH ₂ at the 6-exo position Nb-2-exo), 1.62 (1H, CH ₂ at the 2-end position Nb-2-exo), 1.60 (1H, CH ₂ at the 5-exo position Nb-2- endo), 1.57 (1H, CH ₂ at the 6-exo position of Nb-2-endo), 1.40 (1H, CH ₂ at the 5-endo position of Nb-2-endo), 1.23 and 1.15 (2H, CH ₂ at positions 7a and 7b of Nb-2-endo) 1.04 (1H, CH ₂ at the 6-endo position of Nb-2-exo), 0.97 (1H, CH ₂ at the 6-endo position of Nb-2-endo), 0.78 (1H, CH ₂ at the 3-endo position of Nb- 2-exo), 0.53 (1H, CH ₂ at position 3-endo Nb-endo)

Obtaining compound 1A according to method A, option A-2.

To a solution of a polyanhydride copolymer of the formula PA-1, 1.17 g (0.0088 g-equiv. Of anhydride reaction centers) in 20.0 ml of dry N-methylpyrrolidone (N-MP) at a temperature of 20 ° C was added 0.23 g (0.0013 mol) of 2- (1-aminoethyl ) norbornane hydrochloride in 2.0 ml of dry N-MP, mix for 10 minutes and add 0.13 g (0.0013 mol) of triethylamine. The reaction solution is stirred for 3 hours at 40 ° C and left for 24 hours at 25 ± 3 ° C. The polymer product is precipitated in excess precipitating solution, for example: pentane (50 ml) + diethyl ether (30 ml) + acetic acid (1 ml), filtered, washed with pentane and residual organic solvents are removed in vacuo at 40 ° C for 10 hours. To the resulting powdery product was added 0.71 g (0.0089 mol) of sodium bicarbonate in 45 ml of double-distilled water and stirred at room temperature until a homogeneous solution was formed. The temperature of the solution was raised to 40-45 ° C and stirred for 2 hours, the solution was cooled to room temperature and incubated for 48 hours in a nitrogen atmosphere. Low molecular weight impurities are removed by dialysis on membranes with a permeability of 1-3 KD. Removal of high molecular fractions with simultaneous sterilization of the product is carried out by ultrafiltration on membranes with a permeability of 100 KD. The final purified product is isolated from the aqueous phase by freeze drying in the form of a highly porous white powder. Yield 1.28 g (77%). The spectral and physico-chemical characteristics of the product are identical to those for the product obtained by method A, option A-1.

Obtaining compound Ia according to method B.

To a solution of a polyacid copolymer of the formula PK-1

0.91 g (0.012 g-equiv. Of carboxylic acid reaction centers) in 9.0 ml of dry dimethyl sulfoxide (DMSO) was added as a condensing agent N, N'-dicyclohexylcarbodiimide 1.24 g (0.006 mol) and stirred at 45 ° C for 2 hours. To the resulting reaction mixture, 2- (1-aminoethyl) norbornane 0.12 g (0.00085 mol) in 0.9 ml of dry DMSO was added dropwise with vigorous stirring, stirred for 1 hour at 40 ° C. The polymer product is precipitated in excess precipitant, for example, pentane + ether + acetic acid (respectively: 30 ml + 15 ml + 5 ml), filtered, the filtrate is washed with pentane and the remaining organic solvents are removed in vacuo at 40 ° C for 10 hours. 0.32 g (0.003 mol) of sodium carbonate in 25 ml of double-distilled water is added to the obtained powdery product and stirred at 40-45 ° C for 5 hours. Subsequent operations are performed similarly to those described above by method A. Yield 0.74 g (65%). The spectral and physico-chemical characteristics of the product are identical to those for the product obtained by method A.

Compound Ia is perfectly soluble in aqueous and aqueous-alcoholic media, as well as in DMSO.

Example 2. Synthesis, isolation and purification of compound Ib of formula I,

where the parameters -Z-, n, M and -X are the same as in the previous example (compound Ia), but

5-substituted norbornan-2,3-ene in the exo-isomeric stereo configuration (without separation of the optical isomers), i.e.

-R ^2b -R ^3b - = - (double bond at positions 2, 3), and one of the exo-isomeric R ^5a or R ^6a constitutes a covalent bond between Nb and Y, while all other substituents, including also R ¹ , R ^2a , R ^3a , R ⁴ , R ^5b , R ^6b , R ^7a , R ^7b are hydrogen atoms (H).

The synthesis, isolation and purification of compound Ic is carried out similarly to the procedures described in example 1 for compound Ia, but 5-exo-aminomethylene-2,3-norbornene or its hydrochloride, for example, according to method A, option A-2, is used as an amino reagent .

To a solution of a polyanhydride copolymer of the formula PA-1 (see Example 1) 1.17 g (0.0088 g-equiv. Anhydride reaction centers) in 20.0 ml of dry N-methylpyrrolidone (N-MP) at a temperature of 20 ° C. add 0.21 g (0.0013 mol) 5-exo-aminomethylene-norbornane-2,3-ene hydrochloride in 2.0 ml of dry N-MP, is stirred for 10 minutes and 0.13 g (0.0013 mol) of triethylamine are added. The reaction solution is stirred for 3 hours at 40 ° C and left for 24 hours at 25 ± 3 ° C. The polymer product is precipitated in excess precipitating solution: pentane (50 ml) + diethyl ether (30 ml) + acetic acid (1 ml), filtered, washed with pentane and the remaining organic solvents are removed in vacuo at 40 ° C for 10 hours. To the resulting powdery product was added 0.71 g (0.0089 mol) of sodium bicarbonate in 45 ml of double-distilled water and stirred at room temperature until a homogeneous solution was formed. The temperature of the solution was raised to 40-45 ° C and stirred for 2 hours, the solution was cooled to room temperature and incubated for 48 hours in a nitrogen atmosphere. Low molecular weight impurities are removed by ultrafiltration on membranes with a permeability of 1 KD. Removal of high molecular fractions with simultaneous sterilization of the product is carried out by ultrafiltration on membranes with a permeability of 100 KD. The final purified product is isolated from the aqueous phase by freeze drying in the form of a highly porous white powder. Yield 1.36 g (82%).

¹ H NMR (D ₂ O, δ, ppm): 6.06 (H, CH at positions 2 and 3 Nb, double bond), 4.49 (H,> CH-O-CH <at positions 2.5 of the fragment furan cycle - Z-), 3.31, 3.18 (1Н, СН ₂ fragments -Y-), 2.82 (1Н, СН at position 1 Nb), 2.9-2.7 (1Н,> СН-CO- in the composition of succinic acid residues), 2.63 (1Н CH at the 5-endo position of Nb), 2.35 (CH ₂ as part of the -Z- fragment), 1.56 (1H, CH ₂ at the 6-exo Nb position), 1.25, 1.34 (2H, CH ₂ at the 7 Nb position), 1.14 (1H, CH; at the 6-endo position of Nb).

Compound Ib is perfectly soluble in aqueous and aqueous-alcoholic media, as well as in DMSO.

Example 3. Synthesis, isolation and purification of compound Ic of formula I,

where the parameters -Z-, n, M, -X and -Y- are the same as in the previous example 2 (compound Ib), but

5-substituted norbornane-2,3-ene in the endo-isomeric stereo configuration * (without separation of the optical isomers,), i.e.: -R ^2b -R ^3b - = - (double bond at positions 2, 3), and one of the endo the isomeric R ^5b or R ^6b constitutes a covalent bond between Nb and Y, while all other substituents, including also R ¹ , R ^2a , R ^3a , R ⁴ , R ^5a , R ^6b , R ^7a , R ^7b , are hydrogen atoms (H) * Note: for the Nb fragment, compound 1c is an endo-stereoisomeric analogue of compound Ib having an exo-isomeric configuration.

The synthesis, isolation and purification of compound 1c is carried out similarly to the procedures described in examples 1-2 for compounds Ia and Ib, but 5-endo-aminomethylene-2,3-norbornene or its hydrochloride is used as an amino reagent. Yield 69-88%.

¹ H NMR (D ₂ O, δ, ppm): 6.17, 5.95 (H, CH at positions 2 and 3 Nb, double bond), 4.49 (H,> CH-O-CH <at positions 2.5 of the furan ring -Z- moiety), 3.55, 3.46 (1Н, СН ₂ -Y- moieties), 2.82 (1Н, СН at 4 Nb position), 2.9-2.7 (1Н,> СН-CO- in the composition of succinic acid residues), 2.51 (1H, CH at position 1 Nb), 2.35 (CH ₂ in the fragment -Z-), 2.05 (1H, CH at position 5-exo Nb), 1.69 (1H, CH ₂ at position 6-exo Nb), 1.54 1.34 (2H, CH ₂ at position 7 of Nb), 1.24 (1H, CH ₂ at position of 6-endo Nb).

Compound Ic is readily soluble in aqueous and alcoholic liquids, as well as in DMSO.

Example 4. Synthesis, isolation and purification of compound Id, formula I,

Where

mass average value n = 80 ± 20

M = K

X = OH (≤90%), ONH ₃ -Y-Nb (≤5%, ammonium form), NH-Y-Nb (≥5%, amide form)

2-substituted 7,7-dimethylnorbornane without separation of stereo- (exo-, endo- and optical isomers), that is: one of the group R ^2a , R ^2b , R ^3a , R ^3b forms a covalent bond between Nb and Y, R ^7a = R ^7b = CH ₃ , and all other substituents, including R ¹ , R ⁴ , R ^5a , R ^5b , R ^6a , R ^6b are hydrogen atoms (H).

¹ H NMR (D ₂ O, δ, ppm): 6.11 (H, -CH = CH- at positions 3.4 of the furan ring of the -Z- fragment), 5.32 (H,> CH-0-CH <at positions 2, 5 furan cycle of fragment -Z-), 3.04 (Н, СН at position 1 of Nb-2-exo), 3.3-2.9 (Н,> СН-CO- in the composition of succinic acid residues), 2.40-2.35 (Н, СН ₂ at the 5-endo and 6-exo positions of Nb-2-exo and as part of the -Z- fragment), 2.07 (Н, СН at position 1 of the Nb-2-endo), 2.01 (Н, СН at position 4 of Nb-2- endo), 1.94 (H, CH ₂ at the 2-exo position Nb-2-endo), 1.83 (H, CH ₂ at the 3-exo position Nb-2-endo), 1.74 (H, CH ₂ at the 6-exo position Nb-2-exo), 1.62 (H, CH ₂ at the 2-endo position of Nb-2-exo), 1.60 (H, CH ₂ at the 5-exo position of Nb-2-endo), 1.57 (H, CH ₂ at position 6 -exo Nb-2-endo), 1.40 (H, CH ₂ at the 5-endo position of Nb-2-endo), 1.04 (H, CH ₂ at the 6-endo position of Nb-2-exo), 0.97 (H, CH ₂ at position 6-endo Nb-2-endo), 0.93 (Н, СН ₂ at position 7 Nb), 0.78 (Н, СН ₂ at position 3-endo Nb-2-exo), 0.53 (Н, СН ₂ at position 3-endo Nb-endo).

The compound Id is perfectly soluble in aqueous and aqueous-alcoholic media, as well as in DMSO.

Example 5. The toxic properties of the compounds of formula I.

5-1. Experimental Cytotoxicity Testing Procedures

Determination of the number of living cells using trypan blue.

To determine the doses of drugs that cause destruction of uninfected cells by 50% (CTD ₅₀ ) and 90% (CTD ₉₀ ), the studied drugs were dissolved in RPMI-1640 medium and introduced into the cell suspension. After 48 hours, the percentage of living and dead cells in the control (cells untreated with drugs) and experimental (cells treated with drugs) samples was determined. The calculation was carried out in a Goryaev chamber after staining with a 0.4% trypan blue solution. The data obtained were statistically processed by the method of Ridd and Mench [6].

Determination of the number of living cells using the MTT test

MTT test is based on the ability of cells to translate soluble yellow 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium (MTT) bromide into insoluble intracellular purplish-blue crystals of MTT-derived formazan. This characterizes the overall dehydrogenase activity, which is directly proportional to the number of viable cells. A concentrated MTT solution (RT MTT; 11 mg / ml) is prepared on the basis of a 10-fold Hanks saline solution [7].

5-2. The results of an experimental assessment of the cytotoxicity of compounds of formula I

Figure 1, as an example, shows the dependence of the cytotoxic effect of the norbornane derivative (compound 1A) on its concentration in the MT-4 cell culture.

Table 1 shows the effect of norbornane derivatives of formula I in comparison with the known reference drug (remantadine) on the viability of MT-4 cells

Table 1.
Cytotoxicity of compounds of formula I against MT-4 cells Preparations CTD ₅₀ (μg / ml) CTD ₉₀ (μg / ml) Compound Ia 1260 2750 Compound Ib 1100 1150 Compound Ic > 1000 - Remantadine 160 340

Table 2 shows the effect of norbornene derivatives on the viability of MAGI cells (HeLa-CD4 + -LTR / β-gal). MAGI cells are modified monolayer HeLa cells containing an integrated CD4 receptor and an integrated copy of the β-galactosidase gene under the control of HIV-1 LTR [8].

Table 2.
Cytotoxicity of compounds of formula I against "MAGI" cells Preparations CTD ₅₀ (μg / ml) CTD ₉₀ (μg / ml) Compound Ia 1260 3750 Compound Ib 1325 2225

The data presented show that the compounds of formula I are non-toxic even in high concentrations and their toxicity is several times less than the toxicity of remantadine, which is used in clinical practice as an anti-influenza drug.

Example 6. Antiviral activity of compounds of formula I as inhibitors of HIV-1 reproduction

HIV-1 inhibitory efficacy of the compounds was evaluated in an experimental virus-cell model (in vitro) in the presence of compounds introduced into the cell culture at various concentrations and at different times relative to the time the virus was introduced.

Infection of lymphoblastoid cells MT-4 HIV-1 was carried out by adding clarified virus-containing liquid to the cell suspension at a concentration of 2 × 10 ⁶ cells / ml and a multiplicity of infection of 0.1-0.2 TCID ₅₀ / CL. The effect of drugs on HIV-1 reproduction was quantified by the level of HIV-1 proteins, determined by electrophoresis in SDS page and immunoblotting. Figure 2 shows an example of the inhibitory effect of compound Ia on HIV-1 replication (strain 899).

The effect of the compounds of formula I on early events during HIV infection, including the stages from the recognition of specific receptors by the virus to the integration of the viral genome into the genome of the host cell, was investigated in a one-cycle infectivity analysis by infecting the monolayer of MAGI indicator cells. To infect cells, a series of twofold dilutions of the virus was prepared from 1: 2 to 1:64. The number of infected cells was evaluated by the level of activation of integrated β-galactosidase in the X-gal hydrolysis reaction. Figure 3 presents an example of an experimental evaluation of the antiviral efficacy of compound Ib.

The results of experimental tests show that the most complete suppression of HIV replication occurs when compounds of formula I are added simultaneously with the virus, while in the pretreatment of cells with these drugs, the inhibition of HIV replication is less significant. When drugs are added 1 hour after the virus is introduced, the level of replication suppression is reduced, and when drugs are added after 4 hours, the replication suppression was not observed. These data indicate that the main inhibitory effect of the compounds of formula I appears during the first hour of infection. Thus, drugs inhibit HIV replication in the early stages of infection, i.e. apparently affect the adsorption of the virus on the target cell. Nevertheless, when drugs were introduced after the adsorption of the virus, suppression of HIV replication was observed, on the basis of which it can be assumed that the drugs affect not only viral adsorption, but also the subsequent early stages of HIV replication. This may be a fusion of viral and cell membranes, stripping of the virus or transport of viral RNA and proteins to the nucleus. Given the lipophilic membranotropic nature of norbornane pharmacophores [2], the most likely therapeutic target is fusion of the virus membrane and cell membrane.

The effect of norbornane derivatives on infection caused by an AZT-resistant strain of HIV-1.

Azidothymidine (AZT) is the most widely used drug for the treatment of HIV infection. Unfortunately, its use is limited to the rapid onset of viral resistance. Therefore, the development and use of drugs with other mechanisms of action that could be applied after prolonged use of AZT, and would effectively suppress viral strains resistant to AZT and other nucleoside reverse transcriptase inhibitors, are of particular importance. In order to show the effectiveness of the compounds in the suppression of infection caused by the AZT-resistant variant, a virus with a high degree of AZT resistance was used. The ability of the compounds to inhibit the replication of the AZT-resistant strain of HIV-1 was tested in a single cycle infectivity analysis, for example, FIG. 4. As can be seen from the drawing, compound Ia at a concentration of 10 μg / ml completely inhibits the replication of the AZT-resistant strain of HIV-1. Compounds Ib and 1c also effectively inhibit the replication of the AZT-resistant strain of HIV-1, but at concentrations of 25-50 μg / ml.

Chemotherapeutic evaluation of compounds of formula I

In order to evaluate the prospects of the compounds described by the present invention as possible chemotherapeutic agents, a selectivity index (IP) was calculated, which most statistically reliably characterizes the specific antiviral activity of the drugs [9]

where CTD ₅₀ is the concentration of the compound, causing a decrease in cell viability by 50% (see example 4),

ID ₅₀ - the concentration of the compound that inhibits HIV replication by 50%.

At the optimal time for making preparations (simultaneously with the virus), the selectivity index of compounds of formula I exceeds 100.

Thus, it was found that the compounds of formula I exhibit pronounced HIV-inhibiting activity in cell cultures, and their antiviral effect is manifested at the earliest stage of the HIV-1 infection cycle - at the initial stages of the virus penetration into the cell. The described compounds have a high selectivity index and are effective against HIV-1 strains that are resistant to the known clinical drug azidothymidine. The data obtained confirm the promise of the polyanionic derivatives of norbornane - compounds of formula I as possible chemotherapeutic agents or biologically active substances in the composition of antiviral dosage forms.

Information sources

1a. Mashkovsky M.D. Medicines, Novaya Volna Publishing House, 2002, Volume 2, p. 326.

1b. Mashkovsky M.D. Medicines, Novaya Volna Publishing House, 2002, Volume 2, pp. 325-326.

2. Kiselev O.I., Blinov V.M., Ilyenko Yu.I., Platonov V.G., Chupakhin O.N., Stukova M.A., Karginov V.A. The molecular mechanism of action of antiviral drugs of the adamantane series. \\ Vestn. Grew up. Acad. Honey. Science 1993 (3): 10-15.

3. Boukrinskaia A.G., Serbin A.V., Bogdan O.P., Stotskaya L.L., Alymova I.V., Klimochkin Yu.N. Polymeric Adamantane Analogues. United States Patent US 005880154 A, 1999, Mar. 9.

4. Burstein M.E., Serbin A.V., Khakhulina T.V., Alymova I.V., Stotskaya L.L., Bogdan O.P., Manukchina E.E., Jdanov V.V., Sharova N.K., Bukrinskaya A.G. Inhibition of HIV-1 replication by newly developed adamantane-containmg polyanionic agents. Antiviral Research. 1999 Apr; 41 (3): 135-44.

5. Muravyova D.A., Samylina I.A., Yakovlev G.P. Pharmacognosy. M .: "Medicine", 2002, 654 p.

6. Reed L.T., Muench H.A. A simple method for estimating fifty percent endpoints.//Amer. J. Hyg. 1938 (27): 493.

7. Vistica D.T., Scehan P., Scudiero D. et al. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. // Cancer Research 1991.51: 2515-2520.

8. Kimpton J., Emerman M. Detection of replication component and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene.//J. Virol. 1992 (66): 2232-39.

9. Novokhatsky A.S., Ershov F.I. Determination of the biosynthetic chemotherapeutic index of antiviral drugs in cell culture. \\ Questions of Virology, 1976 (4): 468-473.

Claims (3)

1. Polyanionic derivatives of norbornane ((bicyclo [2.2.1] heptane) of the general formula:

Where

or the remainder of another oxygen-containing monomer, for example

n≥15, preferably n = 35-100 (degree of polymerization); M = H, Na, K, NH ₄ ; X = OH, OM, ONH ₃ -Y-Nb, -NH-Y-Nb, moreover, in the series of the indicated modifications of the chemical fragment X, not less than 3% of the total number X comprises the total share of the ONH ₃ -Y-Nb and NH-Y-Nb variants (degree of norbornane modification), in which the “spacer” group -Y - = - ( Y ₁ ) _m -, where Y _i = CH ₂ , CH (CH ₂ ), CH (CH ₂ CH ₃ ), m≥0 (preferably m = 1-3), and "norbornane pharmacophore" is described by the formula Where

frame bicyclic carbon skeleton of norbornane, including seven carbon atoms (C) in positions 1-7, where in the row above the twelve substituents R ¹ , R ^2a , R ^2b , R ^3a , R ^3b , R ⁴ , R ^5a , R ^5b , R ^6a , R ^6b , R ^7a , R ^7b one (any) of them is - (covalent bond between Nb and Y), while the remaining eleven = H, or alkyl, or in pairs form a covalent chemical bond within the bicyclic hydrocarbon backbone , that is, an additional (double) bond between carbon atoms. 2. The method of producing compounds according to claim 1, which consists in the chemical modification of the corresponding amino derivatives of norbornane with polyanionic oligomers or polymers comprising succinic anhydride or succinic acid units, followed by the conversion of the resulting compound into a carboxylic acid or carboxylic acid-salt form, isolation and purification using methods separation of substances of various molecular weights. 3. Polyanionic derivatives according to claim 1, having selective antiviral activity against human immunodeficiency virus (at the earliest stages of viral infection of cells), including HIV strains that are resistant to traditional clinical drugs based on azidothymidine.

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