RU2515413C2 - 1,2,5-oxadiazole derivatives, having anti-hiv activity, pharmaceutical composition, method of inhibiting hiv-1 integrase - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to use of nucleoside derivatives - 1,2,5-oxadiazoles of general structural formula I

where R¹ and R² are selected from phenylsulphonyl, substituted with one or more halogen atoms, nitro groups, carboxy groups, alkyl halides, CH₃, OCH₃, OCF₃; X is selected from N or N→O; or R¹ and R² form a

group, where R', R", R'" and R'''' are independently selected from hydrogen; halogens; nitro groups, hydroxy group, carboxy group, CH₃; CH₂Br; OCH₃; phenylsulphonyl; phenylthio group; or the following groups:

R' and R" can also be merged into one of the following common rings

for inhibiting human immunodeficiency virus (HIV) replication. The invention also relates to a pharmaceutical composition based on compounds of formula I and a method of inhibiting HIV-1 subtypes A and B integrase, including forms which are resistant to raltegravir.

EFFECT: detecting novel activity in compounds of formula I, which can be used in medicine as HIV replication inhibitors.

3 cl, 5 tbl, 4 ex

Description

The present invention relates to molecular biology, biochemistry, medicine, in particular to antiviral therapy, and describes the use of compounds of a non-nucleoside nature: derivatives of 1,2,5-oxadiazoles (furazans), N-oxides of 1,2,5-oxadiazoles (furoxanes), 1,2,5-benzoxadiazoles (benzofurazans), N-oxides of 1,2,5-benzoxadiazoles (benzofuroxans) of the following general structural formula

and pharmaceutical compositions based thereon for inhibiting the replication of the human immunodeficiency virus (HIV), or preventing or treating an infection caused by HIV, or for preventing, treating or delaying the development of Acquired Immunodeficiency Syndrome (AIDS).

The HIV-1 virus is one of the retroviruses and can infect cells containing the CD-4 surface receptor. It is an etiological agent causing Acquired Moon Deficiency Syndrome and degenerative changes in the nervous system. Therefore, compounds that are capable of eliminating the virus from the patient’s body or slowing down its reproduction in the patient’s body can be effective in treating or preventing HIV-1 infection and AIDS.

Like other retroviruses, after attachment and penetration into the host cell, HIV-1 virus releases viral RNA and three enzymes necessary for virus replication into the cell cytoplasm: reverse transcriptase, integrase and viral protease. Reverse transcriptase synthesizes viral DNA on a viral RNA template. This viral DNA then penetrates the nucleus and integrates into the genome of the host cell using HIV-1 integrase. Viral DNA, integrated into the genome of the cell, is then involved in the processes of transcription and translation, carried out using the usual enzymatic reactions of the host cell. The viral proteins synthesized in this way are finally processed with HIV-1 protease, after which the viral proteins and viral RNAs form new units of the virus that leave the infected cell.

Thus, HIV-1 specific enzymes — reverse transcriptase, protease, and integrase — are necessary for virus replication and are natural targets for possible antiretroviral therapy.

Currently, there are many available antiviral drugs that can counteract infection. These drugs can be divided into three classes, based on the viral protein that is their target, as well as on their mode of action. In particular, saquinavir, indinavir, ritonavir, nelfinavir and amprenavir are competitive inhibitors of the aspartyl protease expressed by HIV. Zidovudine, didanosine, stavudine, lamivudine and abacavir are nucleotide reverse transcriptase inhibitors that behave like substrate mimetics and that stop the synthesis of viral DNA. Non-nucleotide reverse transcriptase inhibitors, such as nevirapine, delavirdine and efavirenz, inhibit viral DNA synthesis through a non-competitive mechanism. The use of these drugs is effective only to reduce the replication of the virus. The effect is only temporary, since the virus easily develops resistance to all known agents. However, combination therapy has proven to be highly effective in both attenuating viral replication and suppressing resistance in many patients. In the USA, where combination therapy is widely used, the number of deaths associated with HIV has decreased (Palella, FJ; Delany, K.M .; Morman, A.C .; Loveless, MO; Futher, J .; Satten, GA; Aschman, DJ; Holmberg, SDN Engl. J. Med. 1998, 338; 853).

The stage of integration of viral DNA into the cellular DNA is one of the key in the replicative cycle of HIV; therefore, the viral enzyme integrase (IN) catalyzing it is one of the most attractive targets for the development of HIV-1 inhibitors. It was shown that a virus containing a defective IN that is unable to integrate viral DNA does not propagate in cell culture. In addition, IN does not have a cellular equivalent, and therefore, inhibitors that specifically inhibit its catalytic activity should not affect cellular processes and, as a result, should be less toxic to the cell and the entire organism as a whole than inhibitors of other stages of the replicative cycle HIV Integration proceeds in two stages: first, integrase catalyzes the 3'-terminal processing reaction, which is the cleavage of the GT dinucleotide from the 3'-end of each viral cDNA strand; then IN embeds the ends of the viral DNA into the cellular DNA. According to the mechanism of action, integration inhibitors can be divided into two groups: inhibitors of the 3'-terminal processing and inhibitors of the incorporation of viral DNA into the cell genome [Prikazchikova TA, Sycheva AM, Agapkina Yu.Yu., Aleksandrov DA, Gottikh M. B. Advances in Chemistry, 2008, 77, 445-459]. The drug Isentress or raltegravir, approved for use as a component of anti-retroviral therapy, is an inhibitor of embedding. This drug showed less toxicity than HIV-1 reverse transcriptase and protease inhibitors, it is better tolerated by patients.

Thus, a large number of HIV-1 reverse transcriptase and protease inhibitors and only one HIV-1 IN inhibitor have been introduced into medical practice, although IN is undoubtedly an extremely attractive target for the treatment of HIV-1 infection. All of these drugs are used in the form of multicomponent therapy. Such multicomponent therapy is today the main way to combat Acquired Immunodeficiency Syndrome.

Unfortunately, not all patients are susceptible to multicomponent therapy, and its use can often be unsuccessful, in addition, many anti-HIV drugs have toxic side effects. It is known that approximately 30-50% of patients ultimately do not receive combination therapy, and the main problems are usually associated with the appearance of viral resistance. Obviously, the emergence of new multi-resistant strains of HIV-1 that are resistant to standard multicomponent therapy makes the development of new anti-HIV antiviral agents extremely necessary. It should also be noted their high cost and the absence (with rare exceptions) of domestic drugs. Thus, the search for new compounds with anti-HIV activity both against the wild strain of HIV-1 and against resistant isolates of the virus, with the aim of creating medicinal anti-HIV drugs, is an extremely important and promising problem of modern virology and medical chemistry.

The scope of the development is the treatment of HIV infection and AIDS. HIV-1 ID inhibitors can be effective drugs for the prevention and treatment of HIV-1 infection, as HIV-1 ID is a key enzyme necessary for HIV replication.

Currently, there are a large number of patent publications relating to various aspects of the synthesis of new substances that can be used against the human immunodeficiency virus. However, the use of all these substances has limitations, the main of which is the formation of stable forms of the virus during therapy, which makes it necessary to constantly change drugs. The drug substance must be effective against a huge number of different types of HIV-1, while most of the known compounds are not sufficiently effective against mutant forms of HIV. Thus, there is a great need to develop new structures that could be used in resistant mutations of the virus.

The development of HIV-1 integration inhibitors has been ongoing for over 10 years, but so far only one integration inhibitor, raltegravir, is approved for use as a medicine. This inhibitor chelates metal ions located in the active center of the enzyme and blocks one of the reactions catalyzed by integrase, chain transfer. Raltegravir successfully passed all stages of clinical trials and in October 2007 was approved by the FDA as a drug for patients who became resistant to HAART components [FDA approves raltegravir tablets. AIDS Patient Care STDS 2007, 21, 889], and in July 2009, the FDA authorized the use of raltegravir in the initial treatment of HIV-infected patients [FDA notifications. Raltegravir indication extended for treatment-naive patients. AIDS Alert 2009.24, 93; WO 2003/035077; WO 2006/060730]. One of the disadvantages of therapy using this drug is that in some patients the virus quickly becomes resistant to raltegravir. Several other HIV-1 integration inhibitors are in the clinical trials phase. However, all of them are analogues of raltegravir both in structure and in mechanism of action. The emergence of the resistance of the virus to some of them is caused by mutations in integrase, similar to mutations that cause resistance to raltegravir.

In 2005, Japan Tobasso and Gilead Sciences) began clinical trials of an integrase inhibitor called elvitegravir, [Al-Mawsawi, L.Q .; Al-Safi, R.I .; Neamati, N. Expert Opin. Emerg. Drugs 2008, 13, 213; WO2007 / 089030; US 2010/0285122 A1], which is the most active representative of IN inhibitors of the structural class 4-oxoquinoline. According to the results of studies, elvitegravir showed high efficiency comparable to the effectiveness of the widely used antiviral efavirenz.

All known analogues of raltegravir act on HIV-1 integrase by the same mechanism, contain a similar structural motif, and exhibit comparable activity in vitro and in cell trials. Identity of this kind casts doubt on the success of the future use of these inhibitors as medicines. These concerns are caused by the cross-resistance of the virus to these inhibitors. The occurrence of cross-resistance can be explained, first of all, by a similar mechanism of binding of chain transfer inhibitors to a complex of IN and viral DNA [Mouscadet, J.F .; Delelis, O .; Marcelin, A.G .; Tchertanoy, L. Drug Resist Updat. 2010, 13, 139]. These compounds bind in such a way that they “push out” the 3'-terminal hydroxyl of the processed DNA chain from the active center of the enzyme and thereby block integration.

Another problem that can complicate the successful use of integration inhibitors is the lack of our knowledge about the IN polymorphism in HIV-1 of various subtypes. In vitro experiments have shown that the subtype C virus ID containing the E92Q / N155H mutation is 10 times more sensitive to raltegravir and elvitegravir than the HIV-1 subtype B enzyme [Bar-Magen, T .; Donahue, D.A .; McDonough, E.I. AIDS 2010, 24, 2171]. It was also shown that in the virus of the subtype CR F02 AG, mutations of the G140 residue are less likely to occur than in the subtype B [Maiga, A.I .; Malet, I .; Soulie, C; Derache, A .; Koita, V .; Amellal, B .; Tchertanov, L .; Delelis, O .; Morand-Joubert, L .; Mouscadet, J.-F., et al. Antivir. Ther. 2009, 14. 123]. There is evidence that raltegravir is more often ineffective in individuals infected with the non-B-subtype virus [Sichtig, N .; Sierra, S .; Kaiser, R .; Daumer, M .; Reuter, S .; Schulter, E .; Altmarm, A .; Fatkenheuer, G .; Dittmer, U.; Pfister, H., et al. J. Antimicrob. Chemother. 2009, 64, 25]. This result is especially important, given that in Russia the subtype A virus is the most common.

Thus, the object of the present invention is the use of chemical compounds of a non-nucleoside nature of general structural formula I, including derivatives of 1,2,5-oxadiazoles (furazans), N-oxides of 1,2,5-oxadiazoles (furoxans), 1,2,5- benzoxadiazoles (benzofurazans), JV-oxides of 1,2,5-benzoxadiazoles (benzofuroxans), as well as pharmaceutical compositions based on them, to inhibit the replication of human immunodeficiency virus (HIV), to prevent or treat infections caused by human immunodeficiency virus (HIV) as well as for preventing, treating, or delaying the development of Acquired Immunodeficiency Syndrome (AIDS).

where R ¹ and R ² are selected from phenylsulfonyl substituted with one or more halogen atoms, nitro groups, carboxy groups, alkyl halides, CH ₃ , OCH ₃ , OCF ₃ ;

X is selected from N or N → O;

or R ¹ and R ² form a group

where R ′, R ″, R ″ ″ and R ″ ″ ″ are independently selected from hydrogen; halogens; nitro groups, hydroxy groups, carboxy groups, CH ₃ ; CH ₂ Br; OCH ₃ ; phenylsulfonyl; phenylthio groups; or the following groups:

R 'and R' 'can also be combined in one of the following general cycles:

In the present invention, unless otherwise specified, the following definitions are used:

"Halogen" means chlorine, bromine, iodine or fluorine.

The term "prodrug" means a derivative of a compound described in the present invention, which has groups that are easily cleavable as a result of chemical or metabolic processes, and which, after application by a patient in his body, cleaves these groups to give the parent compound of the present invention exhibiting its inherent anti HIV activity in vivo. A “prodrug” of compounds of structural formula I can be obtained in the usual way by modifying the functional groups of the compounds, such as a hydroxy or carboxy group.

“Prodrugs” are used to improve the characteristics of drugs, such as solubility, tissue compatibility, controlled release in the body of a mammal, absorption from the gastrointestinal tract, and transportation of the drug in the body to its site of action.

Prodrugs include acid derivatives well known to those of ordinary skill in the art, such as, for example, esters obtained by reacting the starting acid compound with the corresponding alcohol or amides obtained by reacting the starting acid compound with the corresponding amine. Aliphatic or aromatic esters derived from the acidic side groups of the compounds of the present invention are preferred prodrugs. In some cases, it is desirable to obtain other ether derivatives, such as alkyl substituted esters or ((alkoxycarbonyl) oxy) alkyl substituted esters.

The term "pharmaceutically acceptable" carrier means that the carrier must be compatible with other ingredients of the composition and not harm its recipient, that is, be non-toxic to cells or mammals at the doses and concentrations in which it is used. Often, the pharmaceutically acceptable carrier is an aqueous buffer solution. Examples of physiologically acceptable carriers include buffers, for example solutions of phosphates, citrates and other salts of organic and inorganic acids, antioxidants including ascorbic acid; low molecular weight polypeptides (less than 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol.

A therapeutically effective amount is the amount necessary to achieve the effect of suppressing HIV replication in a mammal.

“Mammal” includes representatives of the order of primates (for example, humans, anthropoids, nonhuman monkeys, lower monkeys), order of predators (eg, cats, dogs, bears), order of rodents (eg, mouse, rat, squirrel), order insectivores (e.g. shrew, mole), etc.

The term “AIDS prevention” refers to the administration of anti-HIV drugs by HIV-positive subjects who, however, have not yet developed Acquired Immunodeficiency Syndrome, as well as the use of anti-HIV drugs by people with AIDS symptoms undergoing antiretroviral therapy whose condition has improved , in order to avoid repeated deterioration, as well as the use of anti-HIV drugs in case of the threat of possible HIV infection.

From the analysis of literature data, a method for producing 4-nitrobenzofuroxanes (4-NBPS) is known [Ghosh R.V., Whitehouse M.W. // J. Med. Chem. 1968, 11 (2), 305-311]. For the synthesis, the oxidation of aromatic ortho-nitroamines with hypochlorites was chosen as the method giving the highest yields, followed by the oxidation of the resulting benzofuroxanes with nitric acid. Thus, the synthesis of 4-NBFS was carried out according to the following scheme:

The preparation of 6-nitrobenzofuroxanes (6-NBFS) by nitration of the corresponding benzofuroxanes, as well as the oxidation of 2,4-dinitroaniline derivatives with hypochlorites, results in a large number of by-products [Terrier F., Lakhdar S., Boubaker T., Goumont R. // J. Org. Chem., 2005, 70, 6242-6253]. Therefore, for the synthesis of IN inhibitors belonging to the 6-NBFS class, it was decided to use the pyrolysis of ortho-nitrophenyl azides obtained by the nucleophilic substitution of the halogen atom in the corresponding dinitrophenyl halagenides with sodium azide in dimethyl sulfoxide [Ghosh RV, Whitehouse M.W. // J. Med. Chem. 1968, 11 (2), 305-311; Mallory F.B., Varinibi S.P. // J. Org. Chem. 1963, 28 (6), 1656-1659]. The general scheme for the preparation of 6-NBFS used for their synthesis is presented below:

To obtain 4,6-dinitrobenzofuroxanes (4, 6-NBFS), as well as 6-nitrobenzofuroxanes, it is advisable to use the pyrolysis of ortho-nitrophenyl azides and then carry out the oxidation with nitric acid [Ghosh R.V., Whitehouse M.W. // J. Med. Chem. 1968, 11 (2), 305-311; Mallory F.B., Varinibi S.P. // J. Org. Chem. 1963, 28 (6), 1656-1659]. Unsubstituted 4,6-dinitrobenzofuroxan and its derivatives were synthesized according to the following scheme:

From the analysis of the literature [Ghosh R.V., Whitehouse M.W. II J. Med. Chem. 1968, 11 (2), 305-311] it is known that 4-nitrobenzofurazans (4-NBFZ) are most conveniently obtained from the corresponding benzofuroxans with their subsequent reduction by the action of trimethylphosphite and nitration with concentrated nitric acid. For the synthesis of integrase blockers, the oxidation of aromatic ortho-nitroamines with hypochlorites was used. The selected scheme for obtaining substances is presented below:

To obtain unsubstituted 5-nitrobenzofurazan (5-NBFZ) we also decided to use the reduction of the corresponding 6-nitrobenzofuroxan according to the procedure [Ghosh R.V., Whitehouse M.W. // J. Med. Chem. 1968, 11 (2), 305-311]. The scheme for obtaining the potential blocker IN 5-nitrobenzofurazan is presented below:

To confirm the declared structures of chemical compounds, ¹ H NMR spectra were studied, and the degree of purity of new compounds was analyzed by UPLC chromatography. The structures of all synthesized compounds were successfully confirmed. Most of the selected compounds according to the results of the analysis showed 80-98% purity.

The present invention also includes pharmaceutical compositions for treating HIV infections, comprising a therapeutically effective amount of the non-nucleoside derivatives of claim 1 and a pharmaceutically acceptable carrier.

The active ingredient in such formulations comprises from 0.1 to 99.9% by weight of the composition, preferably from 5 to 90%.

Pharmaceutical compositions may be prepared in accordance with known techniques using known and readily prepared ingredients. Such compositions of the present invention provide rapid, sustained or delayed administration of the active ingredient after administration to the patient in accordance with applicable procedures known to the average expert. In preparing the pharmaceutical composition, the active ingredient is usually mixed with a carrier or diluted with a carrier or enclosed in a carrier, which may be in the form of a capsule, dragee, or tablet form. When the carrier is a diluent, it can be a solid, semi-solid or liquid material, which acts as a binder or inert filler for the active ingredient. Thus, medicines based on pharmaceutical compositions may be in the form of tablets, pills, powders, beads, suspensions, emulsions, solutions, syrups, aerosols of soft and hard gelatin capsules, suppositories, injectable solutions, and the like. Medicines and pharmaceutical compositions are made by conventional technological methods. Medicines can be used as internal or external medical devices. Medicines can be administered orally, parenterally or intranasally.

When using the compounds described in the present invention in pharmaceutical compositions, they can be mixed with suitable additives, inert excipients, diluents, dispersants, stabilizers, preservatives, buffers, emulsifiers, perfumes, colorants, sweeteners and other known pharmaceutical additives, such as water, vegetable oils, alcohols (e.g. ethanol, benzyl alcohol, etc.), polyethylene glycol, glycerol triacetate, gelatin, polyethoxylated to grow Yelnia oils, carbohydrates (e.g., lactose, starch), magnesium stearate, talc, lanolin, petrolatum and the like.

The present invention also includes a method of inhibiting HIV-1 integrase, comprising administering to the mammal a therapeutically effective amount of a compound of formula I.

A method for preventing or treating HIV infection in a mammal involves administering to the patient a therapeutically effective amount of non-nucleoside derivative formulas I. The doses used depend on the age of the mammal, its weight, symptoms, treatment effect, method of administration, etc. Typically, the doses are from 0.1 mg up to 5 g, preferably from 10 mg to 2 g, per one mammal per administration. Such injections can be from one to 10 during the day, either orally, or by intravenous, intramuscular or subcutaneous injection, or intravenous infusion.

Additionally, other anti-HIV agents can be administered together with the active compound, in particular competitive HIV protease inhibitors (e.g. saquinavir, indinavir, ritonavir, nelfinavir and amprenavir), nucleotide reverse transcriptase inhibitors (e.g. zidovudine, didanosine, stavudine, lamivirudine, ), non-nucleotide reverse transcriptase inhibitors (e.g. nevirapine, delavirdine and efavirenz).

Proven Biological Effect (HIV-1 Integrase Inhibition)

In order to make the subject of the present invention more clear, the following are some examples of the use of non-nucleoside derivatives as integrase inhibitors and anti-HIV drug compounds. The examples are illustrative, and the content of the present invention is in no way limited to the presented examples.

The following examples illustrate measurements of the inhibitory ability of non-nucleoside derivatives described herein with respect to biochemical reactions catalyzed by HIV-1 integrase.

Examples of biological effect studies

Example 1

Effect on the activity of integrase from subtype B virus in 3'-processing B reaction 10 μl of buffer (20 mM HEPES 7.5 pH, 1 mM DTT, 7.5 mM magnesium chloride) prepared a 6 nM solution of ³² P-labeled U5B / U5A substrate containing radioactively labeled a processable U5B chain (mixture I) and a test inhibitor in a concentration range of 0.02-200 μM. 10 μl of a 250 nM integrase solution was added to the resulting mixture in the same buffer, stirred and incubated for 2 hours at 37 ° C. Upon completion of the reaction, 80 μl of the stop mixture (9 mM Tris-HCl, 6 mM EDTA, 0.4 M CH ₃ COONa, 0.125 mg / ml glycogen) was added to the mixture, the enzyme was extracted with 100 μl phenol / chloroform / isoamyl alcohol mixture (25:24 :one). The nucleotide material was precipitated with a 5-fold excess of ethyl alcohol at 0 ° C and analyzed by electrophoresis in 20% denaturing PAAG followed by visualization of the gel on a STORM 840TM Phosphorlmager (Molecular Dynamics. USA) and counting using ImageQuant 4.1 software. The extent of the reaction of the 3'-terminal processing was judged by the appearance on the radio-autograph of a band corresponding to a U5B chain shortened by two nucleotides (a 19-unit product). The ratio of the radiation intensities of the bands corresponding to the 21- and 19-link oligonucleotides determined the processing efficiency. Based on the obtained efficiency values, the concentration of the reaction product was calculated. Data were averaged over three independent experiments. The IC ₅₀ values of the inhibitors were calculated from the experimental dependences of the inhibition of the catalytic conversion of the U5 integrase substrate on the concentration of the taken inhibitor. When assessing the degree of substrate conversion, the total concentration of DNA subjected to the 3'-terminal processing, as well as the chain transfer stage following it, [P] _Σ , was taken into account. The addition of an inhibitor to the reaction mixture led to a decrease in the total concentration of products due to suppression of the catalytic conversion of the substrate. The residual concentration of the products upon addition of the inhibitor at the highest concentration taken is indicated as [P] _fin . The experimental dependences of the formation of the products of the catalytic conversion of the U5 substrate on the inhibitor concentration were approximated by an exponential function of the form

[P] _Σ = [P] _fin + A × exp (- [I] / B),

where [I] and [P] are the total concentrations of the inhibitor and the sum of the reaction products, respectively, and A, B are the calculated parameters.

The empirical parameters A and B were used to find the IC ₅₀ value. IC ₅₀ inhibitors were calculated as

IC ₅₀ = B × ln (2A / (A- [P] _fin )).

The results of the effectiveness of the inhibitors are presented in tables 1-4.

Example 2

Effect on the activity of integrase from subtype A virus in a 3'-processing reaction B 10 μl of a buffer (20 mM HEPES 7.5 pH, 1 mM DTT, 7.5 mM magnesium chloride) prepared a 6 nM solution of ³² P-labeled U5B / U5A substrate containing radioactively labeled a processable U5B chain and a test inhibitor in a concentration range of 0.02-200 μM. 10 μl of a 200 nM integrase solution was added to the resulting mixture in the same buffer, stirred and kept in a thermostat for 2 hours at a temperature of 37 ° C. Upon completion of the reaction, 80 μl of the stop mixture (9 mM Tris-HCl, 6 mM EDTA, 0.4 M CH ₃ COONA, 0.125 mg / ml glycogen) was added to the mixture, the enzyme was extracted with 100 μl phenol / chloroform / isoamyl alcohol mixture (25:24 :one). The nucleotide material was precipitated with a 5-fold excess of ethyl alcohol at 0 ° C and analyzed by electrophoresis in 20% denaturing PAAG followed by visualization of the gel on a STORM 840TM Phosphorlmager (Molecular Dynamics. USA) and counting using ImageQuant 4.1 software. The extent of the reaction of the 3'-terminal processing was judged as described in example 1.

The results of the effectiveness of the inhibitors are presented in tables 1-4.

Example 3

Influence on the activity of integrase from subtype B virus during chain transfer

In a 10 μl buffer (20 mM HEPES 7.5 pH, 1 mM DTT, 7.5 mM magnesium chloride), a 20 μM solution of ³² P-labeled U5B-2 / U5A containing a radioactively labeled U5B-2 processed chain shortened by two nucleotides was prepared and test inhibitor in the concentration range 0.02-200 μM. 10 μl of a 250 nM integrase solution was added to the resulting mixture in the same buffer, stirred and incubated for 2 hours at 37 ° C. Upon completion of the reaction, 80 μl of the stop mixture (9 mM Tris-HCl, 6 mM EDTA, 0.4 M CH ₃ COONa, 0.125 mg / ml glycogen) was added to the mixture, the enzyme was extracted with 100 μl phenol / chloroform / isoamyl alcohol mixture (25:24 :one). The nucleotide material was precipitated with a 5-fold excess of ethyl alcohol at a temperature of 0 ° C and analyzed by electrophoresis in 20% denaturing PAAG followed by visualization of the gel on a STORM 840TM Phosphorlmager instrument (Molecular Dynamics. USA) and counting using imageQuant 4.1 software. The degree of progression of the chain transfer reaction was judged by the appearance of radioactive bands corresponding to oligonucleotides that have less mobility in the process of electrophoresis than the original 19-link oligonucleotide chain. The efficiency of the chain transfer reaction was determined by the ratio of the emission intensities of the bands corresponding to the reaction products with lower electrophoretic mobility and the 19-link starting oligonucleotide. The value of IC _{50 was} determined in accordance with the procedure described in example 1. For the chain transfer reaction, an equation of a similar type was used, except that the concentration of only the products of this reaction was considered as the variable [P]. The results of the effectiveness of the inhibitors are presented in tables 1-4.

Example 4

Effect on the activity of integrase containing amino acid substitutions that confer HIV-1 resistance to raltegravir during chain transfer

A technique similar to that described in Example 3 was used. Integrase preparations from subtype B virus containing amino acid substitutions N155H or G140S / Q148K were used. The activity of the compounds that showed the highest inhibitory activity against integrase without amino acid substitutions was determined (Example 3).

The results of the effectiveness of the inhibitors are presented in table 5.

The compounds described in the present invention demonstrate inhibitory activity against HIV-1 integrase preparations of subtypes A and B in both reactions: 3'-processing and chain transfer. Some of the compounds described below are characterized by IC ₅₀ values of less than 5 μM. The most active integrase inhibitors retain their activity with respect to integrase preparations from subtype B virus containing amino acid substitutions N155H or G140S / Q148K, which confer resistance to raltegravir on the virus.

Table 1

4-Nitro-substituted derivatives of benzofurazans and their N-oxides

table 2

5-Nitro, 6-nitro-substituted derivatives of benzofurazans and their N-oxides

Table 3

Dinitrosubstituted derivatives of benzofurazans and their N-oxides

Table 4

Furoxan derivatives

Table 5

Inhibition of integrase preparations containing amino acid substitutions that confer HIV-1 resistance to raltegravir

Claims (3)

1. The use of compounds of non-nucleotide nature - derivatives of 1,2,5-oxadiazoles, the structural formula of which is presented below:

where R ¹ and R ² are selected from phenylsulfonyl substituted with one or more halogen atoms, nitro groups, carboxy groups, alkyl halides, CH ₃ , OCH ₃ , OCF ₃ ;
X is selected from N or N → O;
or R ¹ and R ² form a group

where R ′, R ″, R ″ ″ and R ″ ″ ″ are independently selected from hydrogen; halogens; nitro groups, hydroxy groups, carboxy groups, CH ₃ ; CH ₂ Br; OCH ₃ ; phenylsulfonyl; phenylthio groups; or the following groups:

R 'and R "can also be combined in one of the following general cycles:

to inhibit the replication of the human immunodeficiency virus (HIV). 2. A pharmaceutical composition having an inhibitory activity against HIV-1 virus replication, comprising a therapeutically effective amount of a non-nucleotide derivative of the nature of Claim 1 and a pharmaceutically acceptable carrier. 3. A method of inhibiting HIV-1 integrase, comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition according to paragraph 2.