Benzotropolone derivatives, pharmaceutical composition; method of inhibition of virus replication

FIELD: chemistry.

SUBSTANCE: present invention refers to the new benzotropolone derivatives of general structural formula (A) as well to their pharmaceutically acceptable salts which are able to inhibit the replication of HIV-virus, to pharmaceutical composition thereof and to the method of inhibition of HIV-virus replication. formula (A) where R1 is hydrogen; R2 is OC1-6alkyl; each of R3, R4, R5 independently is hydrogen; G is selected from the group consisting of the structures I, II; R6 is hydrogen; R7 is (CH2)bCOOR9 where b takes up the integer values from 1 to 5; R8 is selected from C1-6alkyls substituted with one or more halogen atoms; W is O; R9 is selected from hydrogen or C1-6alkyl. The structures I, II are represented in the formula of invention.

EFFECT: claimed compounds inhibit the replication of HIV-virus.

4 cl, 6 dwg, 7 ex

 

The present invention describes new chemical compounds, pharmaceutical compositions and methods for the treatment or prevention of a viral infection. In particular, the invention describes compounds of benzotropine, their pharmaceutically acceptable salts, which are useful for the prevention or treatment of painful conditions mammals, caused by replication of the viruses expressing the metal-containing enzymes.

Viruses are the etiological agents causing a lot of diseases in mammals and other animal groups. Not being alive in the full sense of the word, the virus is capable of extended reproduction (replication) only if it penetrates into sensitive cell. This replication of the virus that is harmful to the cells of a mammal or other organism, where the replication occurs, is the cause of the viral infection that can cause significant harm to the health of a mammal, or cause his premature death. Prevention or treatment of viral infection, therefore, should be aimed at preventing or slowing down the replication of the virus.

Such prevention or deceleration can be achieved by inhibition of the enzymatic systems involved in the rap process the paths of the virus. These enzyme systems can be divided into a virus-specific enzyme system and enzymatic systems of the host cell. Inhibition of the enzymatic systems of the host cell can cause a number of side effects, as these systems may be involved in maintaining vital for living cell functions. At the same time, inhibition of virus-specific enzymes, i.e. those that appear in the cell only after the entry of the virus particle, seems to be the most selective method of inhibiting virus replication.

Regardless of the type of virus replication mechanism will involve one or more virus-specific enzyme that produces synthesis of polynucleotide sequences or manipulation with them. For example, RNA-containing viruses, such as influenza virus (A, B, C), hepatitis C virus, caliciviruses, coronaviruses, and others, use RNA-dependent RNA polymerase to synthesize RNA sequences in the matrix of viral RNA. Retroviruses use RNA-dependent DNA polymerase (reverse transcriptase, revertase) for synthesis of viral DNA in the matrix of viral RNA. In addition, in the replication cycle of retroviruses involved integrase, making the integration of viral DNA into the DNA sequence of the host cell.

Virus-specific enzymes production is handled synthesis of polynucleotide sequences and manipulate them, have active center containing one or more ions of divalent metal, usually magnesium. Such ionic active centers are in the gap or the formation of phosphate-ester bonds in the molecules of nucleic acids. Blocking ion centers leads to inhibition of enzyme activity and, as a consequence, the inhibition of virus replication.

Currently, there are many available antiviral drugs that can counteract the infection. So, to counter retroviral infection caused by HIV are competitive inhibitors espartignac protease (e.g., saquinavir, indinavir, ritonavir, nelfinavir and APV), nucleotide reverse transcriptase inhibitors (zidovudine, didanosine, stavudine, lamivudine and abacavir), dinucleotide reverse transcriptase inhibitors (nevirapine, delavirdine, efavirenz). To combat influenza A viruses are the neuraminidase inhibitors (oseltamivir, zanamivir), inhibitors of ion channels (rimantadine, amantadine). To combat infections caused by herpes viruses and cytomegalovirus, use of acyclovir and ganciclovir. However, the effect of the drugs is temporary, because the virus can easily develop resistance to all known agents. In addition, there are a large number of viral infections, on the I counter which is still not developed specific anti-virus (hepatitis C, hepatitis B, West Nile fever and other).

Many antiviral drugs have toxic side effects. In fact, approximately 50% of patients ultimately do not take antiviral therapy. Treatment failure in most cases caused by the emergence of viral resistance. Viral resistance, which in turn caused a rapid turnover of virus in the manifestation of infection in combination with a high rate of viral mutation. Under these circumstances, incomplete viral suppression, caused by insufficient activity of the medicinal product, - the lack of response to the many complex medicines, as well as internal pharmacological barriers in the interaction form the basis for the emergence of drug resistance drug administration. The emergence of new strains of known viruses that are resistant to standard antiviral therapy, along with having a huge number of viral infections, for which specific therapy is generally not developed, makes the development of new antiviral agents is essential.

The technical task of the present invention is the search for new compounds and drugs with antiviral activity.

To solve this problem by the applicant have created new chemical compounds is based derivatives of benzotropine.

There is currently no known antiviral drugs, based on the derivatives of benzotropine described in the present invention. Some derivatives of benzotropine were described as having other therapeutically useful activity.

Thus, the patent WO 92/20332 describes pharmaceutical compositions containing purpurogallin

and its mono and diglucoside as antioxidants and protectors.

In the patent US 2006/0241154 described purpurogallin acid

and some other natural and synthetic benzotropine as an effective antioxidant and anti-inflammatory drugs.

Patent US 5650439 describes natural benzotropine composition:

as an effective inhibitor of catecholamine-O-methyltransferase.

Patent JP 2004-359575 describes benzotropine:

and similar compounds as substances that induce apoptosis of malignant cells and is suitable for chemotherapy of cancer.

There are a number of publications describing the derivative of benzotropine as a potential medicinal substance.

In the article "Inhibition of methylation of oestradiol benzotropine: kinetics and modeling in silico" (Bioorganic and Medicinal Chemistry, Vol.13,p.2501-2507, 2005), the authors describe the natural and synthetic derivatives of benzotropine, which are inhibitors of methylation of hydroxyestradiol the catecholamine-O-methyltransferase.

In the article "New derivatives of tropolone, damaging DNA, isolated from Goupia glabra" (Eur. J. Med. Chem., 2003, p.4243-4247) the authors describe the selection of benzotropine:

and its action as genotoxin, as well as its potential application as anticancer drugs.

In the article "Discovery, characterization and study the relationship between structure and activity of polyphenols, causing apoptosis and affect proteins In cell Lyphocyte/Leukemia-2" (J. Med. Chem., Vol.46, p.4259-4264, 2003) have shown that purpurogallin and its derivatives are able to induce apoptosis of malignant cells.

Some derivatives of benzotropine are formed in the oxidation of catechin compounds in fermentation in the process of making black tea. Such compounds have been called theaflavin and their share may take up to 2-6% of the dry weight of quality black tea.

Recently theaflavin attracted considerable attention since it was discovered that they have a rich spectrum of biological activity, including the following effects on the body:

anti-inflammatory and cytotoxic (Bioorganic and Medicinal Chemisty, Vol.12, p.459-467, 2004);

antimutagenic (Mutat. Res. Vol.389, p.167, 1997);

the suppression of activity of isoforms of cytochrome P450 1A1 in cell culture (Agric. Food Chem., Vol.50, p.213, 2002);

anticlastogenic effect on the bone marrow cells of mice (Life Sci., Vol.69, R, 2001);

suppression of cell-cell interactions and development of cells (Carcinogenesis, Vol.20, R, 1999);

anti-inflammatory and cancer preventive action (Biochem. Pharm., Vol.59, R, 2000).

All of these publications do not describe proposed by the applicant derivative of benzotropine, their pharmaceutically acceptable salts, pharmaceutical compositions on their basis intended for inhibiting viral replication, expressing metal-containing enzymes.

Methods of obtaining derivatives of benzotropine (or their isolation from natural sources) are known and have been published in relevant scientific literature (J. Am. Chem. Soc, Vol.52, 1930, R; J. Chem. Soc., 1948, R; J. Chem. Soc., 1951, p.1313; J. Cem. Soc., 1951, p.1318; J. Chem. Soc., 1951, p.1325; J. Chem. Soc., 1952, p.3705; J. Chem. Soc. Japan (Pure Chem. Sect.), Vol.75, 1954, p.620; J. Chem. Soc. Japan (Pure Chem. Sect.), Vol.77, 1956, p.305; Chem., Bd.69, 1957, p.723; Naturforsch., Bd.l4b, 1959, p.742; Chem. Ber., Bd.97, 1964, p.307; Chem. Ber., Bd.97, 1964, p.312; Chem., Bd.98, 1967, p.872; Tetrahedron, Vol.23, 1967, p.2829; Eur. J. Org. Chem., 2003, p.4243).

In accordance with one method, benzotropine the kernel can be obtained by the joint oxidation of a mixture of catecholase and pyrogallol component suitable about what isliteral in water or an organic or aqueous-organic medium:

or

The preferred oxidizing agent is potassium Iodate or sodium, but many other organic and inorganic oxidizing agents are also suitable for this reaction include potassium ferricyanide, hydrogen peroxide, hydrogen peroxide in the presence of the enzyme catalase or peroxidase, nitric acid, visatec sodium, visatec potassium, polyphenoloxidase, potassium bichromate, potassium permanganate, barium ferrate, para-benzoquinone, tetrachloro-ortho-benzoquinone, tetrabrom-ortho-benzoquinone, etc.

Having synthesized benzotropine the core, other derivatives of benzotropine described in this invention can be prepared by standard methods of organic chemistry - electrophilic substitution in benzotropine core, modification of functional groups, oxidation, recovery, removing the protective groups, the formation or hydrolysis of complex or simple esters, amides, oximo, hydroxamic acids, sulfonamides, etc.

In the present invention, unless defined otherwise, the following definitions are used.

"C1-6alkyl" represents an alkyl group containing unbranched or branched hydrocarbon chain containing from 1 to 6 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, Tr is t-butyl and so on.

OC1-6alkyl is a CNS group containing an unbranched or branched hydrocarbon chain containing from 1 to 6 carbon atoms, for example methoxy, ethoxy, n-propoxy, isopropoxy and so on.

"C1-6alkyl, substituted by one or more halogen atoms"represents an alkyl group containing unbranched or branched hydrocarbon chain containing from 1 to 6 carbon atoms, substituted by one or more halogen atoms, such as vermeil, deformity, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl and so on.

OS1-6alkyl, substituted by one or more halogen atoms" is a CNS group containing an unbranched or branched hydrocarbon chain containing from 1 to 6 carbon atoms, substituted by one or more halogen atoms, for example, formatosi, deformedarse, triptoreline, chloromethoxy and so on.

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

Pharmaceutically acceptable may be considered any salt formed by an active compound of the Formula (A), if it is not toxic and does not interfere with the adsorption and pharmacological action of the active compounds. This salt can be obtained by the action on the compound of the Formula (A) organic or inorganic bases such as hydroxide intothree is, the potassium hydroxide, ammonium hydroxide, methylamine, ethylamine and the like.

The term "pharmaceutically acceptable carrier" means the carrier must be compatible with other ingredients of the composition and not to cause harm to its recipient, that is to be nontoxic to the cell or mammal in those doses and concentrations in which it is used. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acid salts, antioxidants including ascorbic acid; polypeptides with low molecular weight (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; hepatoblastoma agents such as EDTA; sugar alcohols such as mannitol or sorbitol.

Therapeutically effective amount is the amount needed to achieve the effect of suppressing the replication of HIV in the body of a mammal.

"Mammal", as used here, includes representatives of the Primate order (for example, h is the rights, apes, nonhuman apes, the lower monkeys), the order of predators (e.g. cats, dogs, bears), rodent (e.g. mouse, rat, squirrel), some insectivorous (e.g., shrew, mole) and other

As a result of solving the above technical problem, the applicant proposes to protect new chemical compounds, derivatives of benzotropine the General structural formula (A),

where R1represents hydrogen;

R2represents OC1-6alkyl;

R3, R4, R5each independently represents hydrogen;

G is chosen from the group consisting of structures I-II):

where k=0, 1;

R6represents hydrogen;

R7represents a group (CH2)bCOOR9;

R8choose from C1-6of alkyl, substituted by one or more halogen atoms;

W represents O;

R9selected from hydrogen, C1-6of alkyl;

b accepts integer values in the range from 1 to 5;

or their pharmaceutically acceptable salts.

Most preferred are derivatives of benzotropine or their pharmaceutically acceptable salts, where

R1represents hydrogen;

R2 choose from co3;

R3, R4, R5represent hydrogen;

G represents the structure (I);

R6represents hydrogen;

R7represents CH2Y;

where Y is chosen from the group consisting of COOH, SOOS1-6alkyl.

The present invention also describes pharmaceutical composition intended for the treatment and/or prevention of pathological States caused by replication in the body of a mammal viruses expressing the lifecycle process of metal-containing enzymes containing a therapeutically effective amount of a derivative of benzotropine in claim 1 or its pharmaceutically acceptable salts, and pharmaceutically acceptable carrier.

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

The pharmaceutical compositions can be obtained in accordance with known methods using known and easily obtainable ingredients. Such compositions of the present invention can be designed to provide quick, sustained or delayed introduction of the active ingredient after administration to a patient in accordance with applicable procedures well known to the average expert. Upon receipt of the pharmaceutical component is icii active ingredient is usually mixed with a carrier, or diluted by a carrier, or enclosed in the carrier, which can be in the form of capsules, pills or tablets. When the carrier is a diluent, it may be solid, semi-solid, or liquid material which acts as a binder or excipient for the active ingredient.

Thus, the medicinal product on the basis of the pharmaceutical compositions can be in the form of tablets, pills, powders, balls, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, suppositories, injectable solutions and the like Medicines and pharmaceutical compositions are conventional technological methods. Drugs can be used as internal or external medical devices. Drugs can be used for oral, parenteral or intranasal.

When using compounds described in this invention in the form of pharmaceutical compositions, they may be mixed with suitable additives, inert fillers, diluents, dispersing agents, stabilizers, preservatives, buffer compositions, emulsifiers, fragrances, dyes, sweeteners, and other well-known pharmaceutical additives, such as water, vegetable oils, alcohols (for example, ethanol, benzyl alcohol, etc.), al is lepicol, the glycerol triacetate, gelatin, polyethoxysiloxane vegetable oils, carbohydrates (e.g. lactose, starch, magnesium stearate, talc, lanolin, petrolatum, etc.

The present invention also includes a method of inhibiting viral replication, expressing in the lifecycle process of metal-containing enzymes, including the introduction of a mammal a therapeutically effective amount of a compound according to claim 1 or its pharmaceutically acceptable salt.

Described in the present invention derivatives of benzotropine formula (A) can block ion centers of the enzyme through the formation of chelate complexes with one or more ions, for example, magnesium ions. Thus, inhibiting the activity of metal-containing virus-specific enzymes described in the present invention derivatives of benzotropine formula (A) inhibit the replication of viruses and can be used as antiviral agents.

On the basis of the present invention also describes a method for the treatment and/or prevention of painful conditions mammals caused by replication in the body of viruses expressing the lifecycle process of metal-containing enzymes, including introduction to the mammal a therapeutically effective amount of a compound according to claim 1 or its pharmaceutically acceptable salt.

Use Jaimie doses depend on the age of the mammal, its weight, symptoms, effect of treatment, method of administration and the like Usually the dose is from 0.1 mg to 5 g, preferably from 10 mg to 2 g per individual mammal in one introduction. Such introductions can be from one to 10 during the day, either orally or by intravenous, intramuscular or subcutaneous injection, or intravenous infusion.

Description of the invention accompanied by the drawings.

Figure 1 shows schematically the 3'-processing of U5-duplex carried out by HIV-1 integrase.

Figure 2 shows radioautogram denaturing polyacrylamide gel showing the effect of inhibitors on the 3'-processing activity of integrase in accordance with the method described below in Example 5.

Figure 3 shows the graphical dependence of inhibition of 3'-processing on the concentration of inhibitor obtained in the processing of experimental results, which are determined by the parameters of the exponential curve of inhibition of the first order, best passing through the experimental points. The parameters of the exponential curve is used to determine the value of the IC50inhibition of 3'-processing activity of HIV-1 integrase by this inhibitor.

Figure 4 shows schematically the process of transferring a polynucleotide chain, carried out by HIV-1 integrase.

Figure 5 show radioautogram denaturing polyacrylamide gel, demonstrating the effect of the inhibitor on the activity of integrase by reference to the stage of transfer circuit in accordance with the method described below in Example 5.

Figure 6 shows the graphical dependence of the inhibition reactions of the transfer circuit from the concentration of inhibitor obtained in the processing of experimental results, which are determined by the parameters of the exponential curve of inhibition of the first order, best passing through the experimental points. The parameters of the exponential curve is used to determine the value of the IC50characterizing the inhibitory activity of the substance in relation to the migration process chain, catalyzed by HIV-1 integrase.

To the subject of the present invention more comprehensible, the following are some examples of obtaining derivatives of benzotropine (Examples 1-4) and their use as integrase inhibitors and antiviral drug compounds. The examples are illustrative, and the content of the present invention is in no way limited to the examples presented.

Example 1

Synthesis of methyl ester of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid:

(i) methyl ester 3,4,5-trihydroxybenzoate acid

Boil 1.130 g 5 mmol) 3,4,5-trimethoxyphenylacetic acid in 25 ml conc. HBr in an argon atmosphere for 12.5 hours. The solvent is then distilled off in vacuum (10 Torr, 80-90°C). The residue is dried under vacuum (2 Torr, 20°C) over NaOH for 3 days. Technical product containing 3,4,5-trihydroxybenzene acid, dissolved in methanol (10 ml) and cooled to 0÷+5°C. To the cooled methanol solution was added 0.51 ml (0.833 g, 7 mmol) of thionyl chloride. Boil the reaction mixture under reflux for 2.5 hours, then the solvent is distilled off, the residue is dried under vacuum (2 Torr, room temperature) over NaOH for 2 hours. The obtained methyl ester 3,4,5-trihydroxybenzoate acid can be used in the subsequent synthesis without additional purification.

(ii) methyl ether 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid

Dissolve methyl ester 3,4,5-trihydroxybenzoate acid obtained in the previous phase and 0.686 g (4.900 mmol) 3-methoxypyridine in a mixture of water (25 ml) and acetone (5 ml). To the resulting solution at room temperature and vigorous stirring, a solution of 1.049 g (4.900 mmol) of potassium Iodate in 35 ml of water. The reaction mixture becomes brown and soon begins the evolution of carbon dioxide and precipitation of the reaction product. The stirring is continued for 1.5 hours and then leave the reaction mixture to stand for another 30 minutes. The precipitate is filtered off, washed with whom it three times a small amount of water (3×5 ml) and air-dried.

Technical benzotropine dissolved in acetone and evaporated to 2 ml perlite, the dried mixture in a vacuum of 2 mm RT. Art. the mixture was charged in an apparatus for continuous extractive chromatography (neutral silica gel 100/160 multiples, 3 ml) and extracted with hexane for 2 days. Get 0.300 g (20%) of pure methyl ester of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid. Melting point 172-173°C.1H-NMR (500 MHz; DMSO-D6): δ 3.65 (s, 3H, 4-SOON3), 3.60 (s, 2H, 4-CH2), 3.97 (s, 3H, 7-och3), 7.09 (s, 1H, 3-H), 7.10 (s, 1H, 5-H), 7.50 (s, 1H, 6-H), 9.38 (s, 1H, 8(9)-HE), 9.53 (s, 1H, 9(8)-HE), 15.00 (s, 1H, 2-OH).

Example 2

Synthesis of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid:

Boil 0.240 g (0.667 mmol) of the methyl ester of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid in a mixture of 3 ml of acetic acid and 0.1 ml conc. hydrochloric acid for 4 hours, then evaporated in vacuum (10 Torr) at 90-95°C and air-dried. Get 0.129 g (66%) of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid. Melting point 201-203°C.

Example 3

Obtaining the sodium salt of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid

Dissolve 0.278 g (1 mmol) 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid in a solution of sodium methylate, prepara the hinnon in advance of 0.023 g of sodium and 30 ml of methanol. The methanol is distilled off on a rotary evaporator, the residue is dried in vacuum (10 Torr). Obtain 0.3 g of sodium salt of 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid.

Example 4

Synthesis 7,8,10-trihydroxy-6-methoxy-1-trichloromethyl-2-oksobenzo[f]azulene-3,9-dione

To a solution of 0.7 g (5.000 mmol) of o-methoxypyridine and 1.498 g (5.000 mmol) 4,5,6-trihydroxy-3-trichloromethyl-3H-isobenzofuran-1-it (synthesized according to J. Chem. Soc. 119, 208 (1921)) in 6 ml of acetone under stirring rapidly added a solution of 1.070 g (5 mmol) of potassium Iodate in 60 ml of water; the mixture becomes a dark purple color, and then begins to fall out the product and produce carbon dioxide. Stir the mixture for 1.5 h, allowed to stand without stirring 30 min, the precipitate is filtered off, washed three times in 5 ml of water and dried in air. Technical benzotropine dissolved in acetone and evaporated to 2 ml perlite, the dried mixture under vacuum (2 Torr) for 1 hour at 90°C. the mixture was charged in an apparatus for continuous extractive chromatography (neutral silica gel 100/160 multiples, 5 ml) and extracted with MTBE 7 h, get 1.427 g 7,8,10-trihydroxy-6-methoxy-1-trichloromethyl-2-oksobenzo[f]azulene-3,9-dione red. It is washed on the filter twice in 0.2 ml acetone, then three times in 0.2 ml of hexane and dried in air. Get 0.588 g (57%) of pure material. Decomposition temperature >253°.sup> 1H-NMR (500 MHz; DMSO-D6): δ 4.00 (s, 3H, 6-och3), 6.57 (s, 1H, 1-H), 7.57 (s, 1H, 5-H), 8.19 (s, 1H, 4-H), 10.15 (USS, 1H, 7(8)-HE), 11.03 (s, 1H, 8(7)-HE), 14.50 (s, 1H, 10-HE).

The following examples 5-7 illustrate the measurement of inhibitory capacity described in the present invention derivatives of benzotropine in relation to biochemical reactions catalyzed by metalloproteinase expressed by viruses, in particular HIV-1 integrase, and measuring the inhibitory ability of these compounds against HIV replication in cultured human lymphoid cells. Also provides an example of the tests described in the present invention compounds on acute lethality in mice with intraperitoneal injection.

Example 5

The ability of compounds to inhibit HIV-1 integrase was determined by the following method.

The impact on the 3'-processing activity of integrase.

Free from detergent recombinant HIV-1 integrase were established in the culture of Escherichia coli, subsequent isolation and purification were performed in accordance with the methodology: Leh, N., Brodin, P., Bischerour, J., Deprez, E., Tauc, P., Brochon, J.C., LeCam, E., Coulaud, D., Auclair, C. & Mouscadet, J.F., 2000,

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase. Biochemistry 39, 9 285-9294. In oligonucleotide U5B (10 pmol) was introduced radioactive label using T4 polynucleotide kinase (Fermentas) and 50 µCi [γ-32P]ATP (300 Ci/mmol). After incubation at 37°C for 1 hour T4 polynucleotide inaktivirovanie the addition of EDTA and heated to 65°C for 5 minutes. Then he made the annealing of the oligonucleotide U5B with equimolar amounts of complementary oligonucleotides bound U5A. The formed duplex U5B/U5A finally washed from unreacted [γ-32P]ATP in column (MicroSpin G-25 (Amersham Biosciences).

The impact on the 3'-processing activity of integrase in the conditions of competitive inhibition.

In 10 μl of buffer (20 mm HEPES pH 7.5, 1 mm DTT, 7.5 mm magnesium chloride) was prepared 3 nm solution32P-labeled substrate U5B/U5A containing radioactively labeled processed by the circuit U5B (mixture I) and the test inhibitor in the concentration range 0.1-100 μm. 10 μl of 200 nm solution of integrase in the same buffer was added to the mixture and I was kept in the incubator for 2 hours at 37°C. On completion of the reaction, to the mixture was added 80 μl of stop mix (9 mm Tris-HCl, 6 mm EDTA, 0.4 M CH3COONa, 0.125 mg/ml glycogen), the enzyme was extracted with 100 μl of a mixture of phenol/chloroform/isoamyl alcohol (25:24:1). Nucleotide material was besieged by a 5-fold excess of ethyl alcohol at a temperature of 0°C and analyzed by electrophoresis in 20%denaturing SDS page followed by visualization of the gel on the instrument STORM TM Phosphorlmager (Molecular Dynamics, USA) and counting by means of the program ImageQuant 4.1. The degree of proceeding of the reaction is AI 3'-end processing to be judged by the appearance of radioautography strip, the corresponding shortened to two nucleotide chains U5B (19-tier product). The ratio of the radiation intensities of the bands corresponding to the 21 - and 19-tier oligonucleotides, determined the efficiency of processing. On the obtained values of the efficiency was calculated concentration of the reaction product. Data were averaged for three independent experiments. The values of the IC50inhibitors were calculated from the experimental dependences of inhibiting the catalytic conversion of U5 substrate integrase concentration taken inhibitor. When assessing the degree of conversion of the substrate was taken into account, the total concentration of DNA exposed 3'end processing, and followed by phase transfer chain [R.]Σ. Adding to the reaction mixture of the inhibitor resulted in reduced total product concentration as a result of suppression of the catalytic conversion of the substrate. The residual concentration of products with the addition of an inhibitor in taken the greatest concentration denoted as [P]fin. The experimental dependence of the formation of products of catalytic conversion U5 substrate concentration of inhibitor was approximatively exponential function of the form:

[P]Σ=[P]fin+A×exp(-[I]/),

where [I] and [R] is the total concentration of the inhibitor and the amount of the reaction products, respectively, and a, b - you are Islamia parameters.

The empirical parameters a and b were used for finding the values of the IC50were calculated IC50inhibitors, as

IC50=A×ln(2A/(A[P]fin)).

The process of determining the IC50for inhibiting the activity of 3'-processing of HIV-1 integrase illustrated in figures 1, 2 and 3, which show the obtained gel and graphic dependence of inhibition of 3'-processing one of the compounds. Figure 2 shows radioautogram gel, and track And corresponds to the original32P-labeled oligonucleotide chain, the track corresponds to the reaction products in the presence of integrase without added inhibitor and DMSO, the track With the reaction products in the presence of integrase without added inhibitor, but in the presence of 10% DMSO. The rest of the tracks correspond to reaction products in the presence of integrase and inhibitors in a sequentially increasing concentrations (shown by a triangle). As measurements involving inhibitors are carried out in the presence of 10% (volume) DMSO, the experiment represented by the path necessary to control the activity of integrase in the presence of 10% DMSO.

The impact on the 3'-processing activity of integrase in the conditions of the pre-formed complex of the DNA integrase.

For the formation of integrase complex with substrate DNA 21-tier labeled substrate32P-U5B/U5A (3 nm) were incubated with 100 nm integrase for 30 minutes at room temperature in 18 μl of buffer (20 mm HEPES pH 7.5, 1 mm DTT, 7.5 mm magnesium chloride). Then to the pre-formed so complex integrase with the substrate was added 2 μl of inhibitor solution in increasing concentrations so that after addition of a solution concentration of integrase was 100 nm, the substrate concentration is 3 nm, and the concentration of inhibitor 0.1-100 μm. After addition of inhibitor, the reaction mixture was incubated at 37°C for 2 hours. Subsequent operations are similar to those described above in the section "Impact on the 3'-processing activity of integrase in a competitive inhibition".

Effects on the activity of integrase in the migration process chain (strand transfer).

Investigation of the effect of inhibitors on the process of transfer of the polynucleotide chain, catalyzed by HIV-1 integrase were carried out in conditions similar to the inhibition of 3'-processing in a competitive environment. The substrate for studying the migration process chain represented processionary similar U5 duplex, labeled32R. the substrate Concentration was 10 nm. All other components were present in the reaction mixture in the above concentrations (see "Impact on the 3'-processing activity of integrase in a competitive inhibition"). The degree of conversion of the substrate was evaluated by the appearance of radioactive bands corresponding to polynucleotides, the possession is under the process of electrophoresis less mobility, the original 19-dimensional polynucleotide chain. The ratio between the radioactivity associated with these slow lanes, and the radioactivity contained in the band, due to the presence of the original 19th-dimensional polynucleotide chain, was used to assess the effectiveness of the response of the transfer chain. The value of the IC50determined in accordance with the above procedure (see "Impact on the 3'-processing activity of integrase in a competitive inhibition"). For the reaction of the transfer circuit used the equation of similar form, except that as a variable [P] considered the concentration of the only products of this reaction. The process of determining the value of the IC50for inhibition stage transfer circuit illustrated in Figure 4, 5 and 6. Figure 5 presents radioautogram gel, and 6 is a graphical dependence of the inhibition stage of the transfer circuit from the concentration of one of the inhibitors. Figure 5 path And corresponds to the original32P-labeled oligonucleotide chain, the track corresponds to the reaction products in the presence of integrase without added inhibitor and DMSO, the track With the reaction products in the presence of integrase without added inhibitor, but in the presence of 10% DMSO.

Compounds described in this invention show inhibitory activity against whom Oseni HIV-1 integrase in all three tests. Some of the compounds described in examples 1-4, characterized by the values of the IC50less than 5 microns in all three tests. So, for 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid (receipt see Example 2) and its sodium salt (receipt see Example 3) obtained IC50=4.8 mm.

Example 6

Inhibiting activity of the compounds described in the present invention, with respect to the reproduction of the virus HIV-1 was determined in accordance with the following procedure. Human lymphoblastoid cells MT-4 (3.0-5.0·105cells / ml) were infected with HIV-1 (BRU strain) at a dose of 100 TCID50and then incubated for 5 days in culture medium RPMI 1640 containing 10% (volume) of serum of newborn calves and 100 μg/ml gentamicin at 37°C in atmosphere containing 5% carbon dioxide at 98% humidity. Simultaneously with the introduction of HIV-1 infection in culture medium was added to the test compound in the form of solutions obtained by serial dilution. After 5 days the number of living cells was determined by the method of staining Trifanova blue. For the tested compounds value EC50was determined as the concentration of this compound in the culture medium, which was suppressed by 50% cytopathic effect caused by the multiplication of the virus in cell cultures MT-4.

Soy is inane, described in this invention show inhibitory activity against HIV-1 replication in the culture of lymphoblastoid cells MT-4.

Some of the compounds described in examples 1-4, characterized by the values of the EU50less than 10 μm in the above-described test for inhibition of reproduction of HIV-1. So, for 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid (receipt see Example 2) and its sodium salt (receipt see Example 3) obtained EC50=7 ám.

Example 7

Compounds described in this invention was tested on acute lethality in mice (females line SV), that is, mortality during the first two hours after intraperitoneal administration of the drug. Compounds were converted into the sodium salt by reacting with an equimolar amount of sodium methylate in methyl alcohol followed by distillation of the solvent, as described in Example 3.

Then sodium salt was dissolved in isotonic sodium chloride to obtain the desired concentration. Each mouse received an injection of 0.5 cm3the resulting solution. Values of the tested doses were chosen in accordance with the Tabular Express method" Webpronews.com "a Practical guide for rapid determination of the mean effective doses and concentrations of biologically active substances", Baikalsk, Spbg is, 1994, 46 C. was Used on two mice per dose.

The calculation of LD50characterizing the degree of toxicity was also conducted in accordance with the Tabular Express method" Webpronews.com.

In the result, it was shown that the compounds described in this invention are of low toxicity. So, for 7-methoxy-8,9-dihydroxybenzophenone-4-methylanthranilic acid (receipt see Example 2) obtained LD50>0.75 g/kg

1. Derivatives of benzotropine, General structural formula (A)

where R1represents hydrogen;
R2is a OS1-6alkyl;
R3, R4, R5each independently represents hydrogen;
G is chosen from the group consisting of structures I-II):



where k=0,1;
R6represents hydrogen;
R7represents a group (CH2)bCOOR9;
R8choose From1-6of alkyl, substituted by one or more halogen atoms;
W is chosen from O;
R9selected from hydrogen, C1-6of alkyl;
b accepts integer values in the range from 1 to 5;
or their pharmaceutically acceptable salts.

2. Derivatives of benzotropine according to claim 1 or their pharmaceutically acceptable salts,
where R1not only is em a hydrogen;
R2choose from OCH3;
R3, R4, R5represent hydrogen;
G represents the structure (I);
R6represents hydrogen;
R7represents CH2Y;
where Y is chosen from the group consisting of COOH, COOC1-6alkyl.

3. Pharmaceutical composition having inhibitory activity against the replication of HIV-1 virus containing a therapeutically effective amount of a derivative of benzotropine according to claim 1 or its pharmaceutically acceptable salts, and pharmaceutically acceptable carrier.

4. Method of inhibiting replication of HIV-1 virus comprising the administration to a mammal a therapeutically effective amount of a compound according to claim 1 or its pharmaceutically acceptable salt.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: present invention refers to the new benzotropolone derivatives of general structural formula (A) as well to their pharmaceutically acceptable salts possessing anti hiv-activity, to the pharmaceutical composition thereof and to the method of HIV-integrase inhibition. formula (A) where R1 is selected from the group consisting of the hydrogen and halogen; R2 is selected from the group consisting of the hydrogen and OC1-6alkyl; each of R3, R4, R5 independently is hydrogen; G is selected from the group consisting of the structures I, II; R6 is hydrogen; R7 is COOR9; R8 is selected from C1-6alkyls substituted with one or more halogen atoms; W is O; R9 is selected from hydrogen or C1-6alkyl. The structures I, II are represented in the formula of invention.

EFFECT: claimed compounds possess anti HIV-activity.

5 cl, 6 dwg, 13 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to a method for preparing beraprost of the formula (I): or its salts, in particular, its sodium salt, and to intermediate compounds of the formulae (IV): and (V): wherein R1 and R2 have values given in the description. Invention provides a new method for preparing beraprost allowing enhancing the yield of the end product and to simplify the process.

EFFECT: improved preparing method.

14 cl, 9 dwg, 7 ex

FIELD: organic chemistry, pharmacy.

SUBSTANCE: invention relates to a novel intermediate compound of the formula (VI)

and its salts wherein R3 means hydrogen atom (-H); R4 means -H, and means a simple or double bond, and its pharmaceutically acceptable salts.

EFFECT: improved preparing method.

4 cl, 12 ex

The invention relates to a previously undescribed 2,8 - dimethyl-3,7-dialkoxy-5-R-furo[c; b] tropylium salts of General formula (1), where R = a hydrogen atom; still;

aryl unsubstituted or substituted mono - or dihydroxy mono - or di(C1- C10) alkoxy, methylenedioxy, di(C1- C6)alkylamino, halogen, nitro group; R1= C1-C6alkyl, which are potentially biologically active compounds, as the most famous tropon('olonne)for UNIX systems and urotropin('olonne)for UNIX systems exhibit various types of physiological activity [1], anti-inflammatory (Muth C

FIELD: chemistry.

SUBSTANCE: present invention refers to the new benzotropolone derivatives of general structural formula (A) as well to their pharmaceutically acceptable salts possessing anti hiv-activity, to the pharmaceutical composition thereof and to the method of HIV-integrase inhibition. formula (A) where R1 is selected from the group consisting of the hydrogen and halogen; R2 is selected from the group consisting of the hydrogen and OC1-6alkyl; each of R3, R4, R5 independently is hydrogen; G is selected from the group consisting of the structures I, II; R6 is hydrogen; R7 is COOR9; R8 is selected from C1-6alkyls substituted with one or more halogen atoms; W is O; R9 is selected from hydrogen or C1-6alkyl. The structures I, II are represented in the formula of invention.

EFFECT: claimed compounds possess anti HIV-activity.

5 cl, 6 dwg, 13 ex

FIELD: chemistry.

SUBSTANCE: novel compounds of formulas , , , , , , (designation of all groups are given in invention formula) are used for treatment of different metabolic diseases, such as insulin resistance syndrome, diabetes, hyperlipidemia, fatty liver, cachexia, obesity, atherosclerosis and arteriosclerosis.

EFFECT: using compounds as biologically active agent and creating pharmaceutical compositions based on said compounds.

124 cl, 52 ex, 17 tbl, 2 dwg

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to photoinitiating agents of phenylglyoxylic acid order used in polymerizing compositions to be subjected for hardening. Invention describes a photoinitiating agent of the formula (I): wherein Y means (C3-C12)-alkylene, butenylene, butinylene or (C4-C12)-alkylene that are broken by groups -O- or -NR2- and not following in sequence; R1 means a reactive group of the following order: -OH, -SH, -HR3R4, -(CO)-OH, -(CO)-NH2, -SO3H, -C(R5)=CR6R7, oxiranyl, -O-(CO)-NH-R8-NCO and -O-(CO)-R-(CO)-X; R2 means hydrogen atom, (C1-C4)-alkyl, (C2-C4)-hydroxyalkyl; R3 and R4 mean hydrogen atom, (C1-C4)-alkyl, (C2-C4)-hydroxyalkyl; R, R and R mean hydrogen atom or methyl; R8 means linear or branched (C4-C12)-alkylene or phenylene; R9 means linear or branched (C1-C16)-alkylene, -CH=CH-, -CH=CH-CH2-, C6-cycloalkylene, phenylene or naphthylene; X, X1 and X2 mean -OH, Cl, -OCH3 or -OC2H5. Also, invention describes a method for synthesis of a photoinitiating agent, polymerizing composition and substrate covered by its. Proposed photoinitiating agent possesses the effective introducing capacity and absence of migration in thermal treatments.

EFFECT: improved and valuable properties of agent.

13 cl, 1 tbl, 16 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to novel intermediate compounds and inmproved method for synthesis of compound of the formula (C): . Proposed method is based on using inexpensive parent substances and provides synthesis of intermediate compounds with the high yield and high purity degree being without carrying out procedures for chromatographic purification and can be realized in large-scale industry manufacture. Invention relates to improved methods for synthesis of compound of the formula (I): , compound of the formula (II): , compound of the formula (III): , compound of the formula (VIII): , compound of the formula (IX): , and to a reagent consisting of boron tribromide and 2,6-dimethylpyridine. Method is used for a sparing and selective splitting a methyl group in aromatic methyl ethers.

EFFECT: improved method of synthesis.

12 cl, 8 ex

FIELD: organic chemistry, perfumery.

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EFFECT: valuable cosmetic properties of compounds.

13 cl, 14 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to synthesis of 1,3-dicarbonyl compounds and to a new method for preparing 4-substituted alkyl-3-oxobutanoates of the formula: wherein: R is C6H5CH2, 2-F-6-ClC6H3CH2, 2,6-Cl2C6H3CH2, 1-C10H7CH2, Ph2CH; Alk is Me; R is 1-AdCH2; Alk is i-Pr that are used precursors of antiviral agents of pyrimidine order. Method involves acylation of 2,2-dimethyl-1,3-dioxane-4,6-dione with acyl chlorides in dichloromethane in the presence of triethylamine followed by alcoholysis of 5-(1-hydroxyethylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione wherein acylation is carried out with acyl chlorides in the presence of trimethylsilyl chloride in the mole ratio 2,2-dimethyl-1,3-dioxane-4,6-dione : acyl chloride : trimethylsilyl chloride : triethylamine = (1-2):1:1.1:3.5, respectively, with formation of an intermediate product 5-[1-(trimethylsilyloxy)ethylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione that is subjected for hydrolysis with formation of 5-(1-hydroxyethylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione and its following alcoholysis and formation of the end product. Method provides enhancing yield and purity of claimed compounds.

EFFECT: improved method for preparing.

2 cl, 8 ex

FIELD: organic chemistry.

SUBSTANCE: invention relates to new method for production of m- or p-substituted α-arylalkanecarboxylic acids of general formula I

from respective α-hydroxylated derivatives using inexpensive reagents and without converting of any reducible groups such as ester or ketone ones in side chains. In formula R is hydrogen, C1-C6-alkyl; R1 is hydrogen, linear or branched C1-C6-alkyl, phenyl, p-nitrophenyl, alkali or earth-alkali cation or cation of pharmaceutically acceptable ammonia salt: A is C1-C4-alkyl, aryl, optionally substituted with one or more alkyl, hydroxy, etc., aryloxy, arylcarbonyl; A is in m- or p-sites; P - linear or branched C1-C6-flkyl, phenyl, nitrophenyl. Claimed method includes the next steps: a) converting of compounds of formula II to compound of formula III either by reaction of II with compound of formula in presence of organic or inorganic base or by reaction of II with thiophene of formula and followed by reaction of obtained product with HNRaRb, wherein Ra andRb are as defined above; b) thermal rearrangement of III to form IIIb ; c) catalytic dehydration of IIIb to form IIIc ; and d) optional hydrolysis of IIIc to obtain target compound of formula I. Also are disclosed new compounds of formulae III and IIIb.

EFFECT: new α-arylalkanecarboxylic acids and intermediates thereof.

6 cl, 5 ex

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The invention relates to an improved method of transesterification of esters-ketocarboxylic acid with the structural formula (I), the alcohol of formula (II) R3HE, and R1, R2, R3means a branched, unbranched or cyclic, saturated or unsaturated C1-C6is an alkyl group or benzyl group, and R1and R2are not the same, which are used as, for example, intermediates for biologically active substances for agro - and pharmaceutical industry, as solvents, and so on

The invention relates to an improved process for the preparation of ester 2-alkylidene-4-bromocatechol acid of the formula (3), where R1and R2each independently from each other represent a lower alkyl group with 1-5 carbon atoms, which is used as an intermediate connection upon receipt of the substances for pharmaceutical purposes, such as antibiotics

FIELD: chemistry.

SUBSTANCE: present invention refers to the new benzotropolone derivatives of general structural formula (A) as well to their pharmaceutically acceptable salts possessing anti hiv-activity, to the pharmaceutical composition thereof and to the method of HIV-integrase inhibition. formula (A) where R1 is selected from the group consisting of the hydrogen and halogen; R2 is selected from the group consisting of the hydrogen and OC1-6alkyl; each of R3, R4, R5 independently is hydrogen; G is selected from the group consisting of the structures I, II; R6 is hydrogen; R7 is COOR9; R8 is selected from C1-6alkyls substituted with one or more halogen atoms; W is O; R9 is selected from hydrogen or C1-6alkyl. The structures I, II are represented in the formula of invention.

EFFECT: claimed compounds possess anti HIV-activity.

5 cl, 6 dwg, 13 ex

FIELD: chemistry.

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EFFECT: using compounds as biologically active agent and creating pharmaceutical compositions based on said compounds.

124 cl, 52 ex, 17 tbl, 2 dwg

FIELD: organic chemistry, pharmacy, pharmacy.

SUBSTANCE: invention relates to novel compounds designated for delivery of active substances to tissues of the following formula: wherein values of radicals R1-R7 are determined in claim 1 of the invention claim, and to their pharmaceutically acceptable salts. Also, invention relates to compositions designated for delivery of active substances to tissues and containing: (A) active substance and (B) at least one compound designated for delivery of active substance to animal tissues of the formula: wherein values of radicals R1-R7 are determined in claims 3-5 of the invention claim. Also, proposed invention relates to a standard medicinal formulation designated for delivery of active substances to body tissues and to a method for preparing indicated compositions and administration of substances for their delivery to body tissues.

EFFECT: valuable properties of compounds.

23 cl, 11 tbl, 11 ex

The invention relates to new derivatives of 4-oxobutanoic acid of formula 1, where groups a and b independently from each other selected from mono - or bicyclic aryl group selected from phenyl and naphthyl, cycloalkyl group having 5 to 8 carbon atoms, a saturated heterocyclic group selected from tetrahydrofuryl groups; group a and b may have 1 to 3 substituents selected from C1-C6alkyl group, a C1-C6alkoxygroup, halogen; or two Deputy together represent methylenedioxy
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