Azacytidine analogues and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to a novel azacytidine analogue of formula (I): where R is H, R5C(O); R1 is or where the crossing dashed line denotes the formed bond joining R1 to a molecule of formula (I); R2 and R3 are independently OH or H provided that R2 and R3 are not OH at the same time; R4 is H or R5C(O) provided that R and R4 are not H at the same time; and R5 is C3-C26 alkenyl, or a pharmaceutical salt thereof.

EFFECT: compounds have anti-cancer and anti-inflammatory activity.

15 cl, 6 tbl, 7 dwg, 15 ex

 

This application claims priority based on provisional application for U.S. patent No. 60/975437, filed in the U.S. patent office on September 26, 2007, the contents of which are fully incorporated into the present description by reference.

The technical field

The present invention relates to analogs of azacitidine and their application.

The level of technology

Nucleoside analogues, derivatives of natural nucleosides, which are found as building blocks of DNA and RNA, are effective in the clinical treatment of cancer or viral diseases in humans, though these compounds were considered as drugs. Such compounds are registered on the market for over 40 years, and currently about 35 products used daily. Natural nucleosides, are shown in table 1 below, consist of nitrogenous bases of two classes, namely purines (adenine and guanine) and pyrimidines (thymine, uracil and cytosine), and monosaccharide ribose or deoxyribose.

Table 1

All natural nucleosides exist in the so-called β-D configuration, as shown below in formula A. Nitrogenous base and hydroxymethylene side chain in the cycle of sugar are on the same side (CIS from the plane of the sugar ring.

The formula A

With the purpose of receiving nucleoside derivatives with anticancer or antiviral activity were produced by chemical modification in nitrogenous base and/or monosaccharide. For example, the introduction of the nitrogenous base halogen atoms or other functional groups, the introduction of an additional nitrogen atoms, or stereochemical replacement ring monosaccharide with ribose at the arabinose or removing a hydroxyl group from obtaining desoxyribose can lead to the formation of products with therapeutic potential. Many products monosaccharide ring is retained, whereas in the other ring sugar can be transformed into the circuit. Nucleoside analogues are small molecules, which have excellent solubility in water.

Numerous efforts on research and development in the field of nucleoside analogs, caused a worldwide epidemic of AIDS, contributed to the accumulation of fundamental knowledge base and understanding of the mechanism of action, changes in the profile of activity due to chemical modification, etc. apply equally to the field of cancer treatment.

A common shortcoming of many drugs, including nucleoside analogues, is low activity and low specificity in the treatment of concreteobserver disease. Some of these problems can be attributed to the activity intrinsic to drug, some can be attributed to specific resistance mechanisms (both congenital and acquired in the course of treatment, such as multi-drug resistance (MDR) in cancer treatment). Some problems can be attributed to the slow transport or cell capture and activation mechanisms. Some problems can be attributed to the rapid inactivation and/or excretion of the drug.

The efficacy of nucleoside analogues depends largely on their ability to imitate the natural nucleosides and, thus, interact with viral and/or cellular enzymes, and also to prevent leakage of the key processes in the metabolism of nucleic acids (or inhibition). In order to give effect to their antiviral or anticancer activity, nucleoside analogues should be transformed through their mono - and diphosphate in their respective triphosphates under the action of viral and/or cellular kinases. In General, the active agent is Tris, but for some products, such as gemcitabine, even the impact diphosphate may be clinically significant.

In order to reach the affected cancerous or virus-infected cells and the tissues and after enteral or parenteral administration, nucleoside analogues should have appropriate pharmacokinetic characteristics. In addition to the rapid excretion of the drugs introduced, many nucleoside analogues can be deactivated in the bloodstream and in tissues. For example, derivatives of cytosine, even at the level of monophosphate, can be quickly diaminononane under the action of a class of enzymes called deaminase, to inactive analogue of uracil. Cell capture and, thus, the high therapeutic effectiveness of many nucleoside analogues strongly depends on membrane proteins engaged in the transport of nucleosides (called concentrating and balancing nucleoside vector). Therefore, the object of the search are connections that do not require such specific capture mechanism. Another factor limiting activity, especially in the field of cancer treatment, is a mechanism of cell recovery. When monophosphate anticancer nucleoside analogue is incorporated into cellular DNA, it is not removed from the DNA of the cancer cells under the action of the endonuclease associated with the p53 protein. However, the removal of nucleoside analogue of the DNA of healthy cells is desirable in order to limit the side effects of the drug.

For many years, we developed many nucleoside EN the logs which largely overcomes some or many of the factors limiting the activity. As an example, illustrating the connection of high specificity, can lead to acyclovir (ACV). ACV-monophosphate can be formed only under the influence of viral kinases, and this means that ACV may not be activated in uninfected cells. Despite this fact, ACV is not only an active product. In order to circumvent common rate-limiting stage in the activation of nucleoside analogue, i.e. the intracellular formation of monophosphate nucleoside analogue, were developed several phosphonates, such as cidofovir, or even monophosphate products. To enable oral administration or desired disposition of drugs in the body, were obtained specific prodrugs, such as Hepsera.

In addition to structural changes in nucleoside analogues to achieve improved clinical applicability, were made further modifications to improve activity. There are several examples of modified nucleoside analogs obtained by attaching a lipid part (U.S. patent No. 6153594, 6548486, 6316425 and 6384019; application to the European patent number EP-A-56265 and EP-A-393920; and WO 99/26958). The lindens joining the ne part can be achieved by attaching a fatty acid, for example, by ester, amide, carbonate or urethane linkages. You can get more perfect products such as phospholipid derivatives of nucleoside analogues. Cm. Eur. J. Pharm. Sci. 11b Suppl. 2: 15-27 (2000); European patent No. 545966; canadian patent No. 2468099; and U.S. patent No. 6372725 and 6670341. In the above document describes that such analogues have antiviral activity, especially suitable for the treatment and prevention of infections caused by DNA, RNA or retroviruses. They are also suitable for the treatment of malignant tumors. Lipid derivatives of nucleoside analogues can serve several purposes. They can be considered as a prodrug, which is not a substrate for diamines, preventing thus nucleoside analogues from deactivation during transport in the bloodstream. Lipid derivatives can also be more efficiently transported across the cell membrane, leading to increased intracellular concentrations of nucleoside analogue. Lipid preparations may also be more suitable for use in dermal preparations, oral products (see U.S. patent No. 6576636 and WO 01/18013), or specific compositions, such as liposomes (see U.S. patent No. 5223263), designed to impact on the tumor.

It was shown that in the case of nucleoside analogues with preserved β-D Conf is the tenant monosaccharide rings or nucleoside analogues with acyclic side chain antiviral or anticancer activity can be most effectively improved by obtaining the derivative of monounsaturated ω-9 C18 and C20 fatty acids. Cm. Antimicrobial Agents and Chemotherapy, Vol., 53-61 (1999); Cancer Research 59: 2944-2949 (1999); Gene Therapy, 5: 419-426 (1998); Antiviral Research, 45: 157-167 (2000); and Biochemical Pharmacology, 67: 503-511 (2004). It is desirable that monounsaturated derivatives were not only more active than polyunsaturated counterparts, but also that they are more easily subjected to crystallization and were more resistant to oxidation of the lipid chain. Thus, these derivatives are more preferred compounds from the standpoint of chemical and pharmaceutical production. It was also shown that monounsaturated fatty acids ω-9 C18 and C20 are suitable for improving therapeutic activity of a large number of biologically active substances of a non-nature (see European patent No. 0977725).

A relatively new subgroup nucleoside analogues are the so-called Aza-C derivatives. The compounds of this class CH-group in position 5 of pyrimidine base is replaced by a nitrogen atom as shown in formula B.

Formula B

Potential targets for reactivation with new chemotherapeutic agents are genes tumor suppressors that were turned off due to abberant DNA methylation. Derivatives of Aza-cytidine and 5-Aza-2'-deoxycytidine (5-Aza-C and 5-Aza-CdR, Decitabine), which is a strong inhibitor and DNA methylation and protivoanemicakimi means, can reactivate disabled suppressor gene tumor. At high concentrations these compounds are cytotoxic, but at lower concentrations hypomethylation leads to the differentiation of cell lines. These compounds require metabolic activation by deoxycytidine and implement ingibirovanie DNA-methyltransferase. One of the obstacles for their therapeutic action is rapid in vivo inactivation involving tsitidindeaminazy (CD). Instability in aqueous solutions, and the combination of side effects limit the clinical activity of these compounds.

The present invention is directed to overcoming these and other drawbacks in the art.

Brief description of the invention

One aspect of the present invention is directed to a compound according to formula (I)

where R represents H, R5C(O); R1represents a

where the crossing dashed line depicts the resulting bond attaching R1to the molecule of formula (I), R2and R3independently represent OH or H, provided that R2and R3are not both OH, R4represents H or R5C(O), provided that R and R4are not both H, R represents a C3-C26alkenyl; or a pharmaceutical salt specified connection.

Another aspect of the present invention is directed to a pharmaceutical composition comprising a compound of formula (I) and pharmaceutical excipient, diluent and/or carrier.

Another aspect of the present invention is directed to a method of treating neoplastic syndrome patients. This method involves selecting a patient with a neoplastic syndrome and introducing him to the compounds of formula (I)as described above, or a pharmaceutical salt of this compound, under conditions effective for the treatment of neoplastic syndrome in a patient.

Another aspect of the present invention is directed to a method of treating inflammatory syndrome in patients. This method involves selecting a patient with an inflammatory syndrome and introducing him to the compounds of formula (I)as described above, or a pharmaceutical salt of this compound, under conditions effective for the treatment of inflammatory syndrome in a patient.

The instability of Aza-C in buffer solution and blood plasma are well known (see Israili et al., Cancer Research 36, 1453-1461 (1976); Rudek et al., J. Clin. Oncol., 23:17, 3906-3911 (2005); Rustum et al., J. Chromat., 421:12, 387-91 (1987); Zhao et al., J. Chromat B, 813, 81-88 (2004), fully incorporated into the present application by reference). Reported average end-of-life period for Aza-C, with the bringing of 1.50±2.30 hours in clinical plasma samples (see Rudek et al., J. Clin. Oncol., 23:17, 3906-3911 (2005), this source is fully incorporated into the present application by reference). In vitro studies was marked by a loss of 20% Aza-C even at -60°C after 4.5 days of storage, and the loss of 10% for 0.5 hours when stored at room temperature (see Zhao et al., J. Chromat B, 813, 81-88 (2004), this source is fully incorporated into the present application by reference). Assume that the primary instability of Aza-C caused a rapid (the first stage is reversible) disclosure of cycle 5-Aza-pyrimidine ring with a subsequent decay of formic acid (see Chan et al., J. Pharma Sci., 68;7, 807-12 (1979), this source is fully incorporated into the present application by reference). It is assumed that other ways of collapse due to diaminononane amino group in the 4 position and the hydrolysis of glycosidic bonds with the formation of D-ribose and 5-azacytosine. It was unexpectedly discovered that the preferred lipid derivatives of Aza-C have a much better profile of sustainability in the plasma as compared to Aza-C Connection is stable (fraction remaining from the original ≥94%) in the control matrix human plasma at room temperature for at least 4 hours in experimental conditions, and in the post-extraction the supernatant after precipitation of plasma proteins there were no significant degradation products. Steadily the diamonds in the preferred plasma lipid compounds was studied further in terms of storage at 37°C. It was shown that the possibility of disclosure ring Aza-fragment or other dissolution of the connection is greatly reduced, if Aza-C attached lipid side chain.

The rapid collapse of Aza-C is an obstacle for clinical application Aza-C. High stability in plasma lipid derivatives as compared to Aza-C can provide a high and sustainable level of lipid derived in the plasma of the patient. This may lead to a better distribution in the tissue/organ/tumor and exposure of cells to drug action, as well as the absorption of drugs than for the Aza-C, on the one hand, and, hence, to the best exposure to the DNA of tumor cells to the action of Aza-C after intracellular hydrolysis of the Aza-C-5'-ester bonds.

In embodiments of the present invention by modifying azacitidine and deoxycytidine (for example, 5-Aza-2'-deoxycytidine) proposed new molecules with properties that are unexpectedly different from the properties of azacitidine and deoxycytidine (for example, 5-Aza-2'-deoxycytidine). This provides a series of compounds having activity substantially greater than the anticancer activity of azacitidine and deoxycytidine (for example, 5-Aza-2'-deoxycytidine), which is limited to hematological malignancy. These new connections about adut anticancer action directed against a broad spectrum of solid tumors, including breast cancer and cervical cancer. These compounds also exhibit unexpected activity against those cancers that are resistant to treatment, and thus, they can be useful in the treatment of solid tumors in cases where the current selection of therapeutic agents is limited. The embodiments of the present invention therapeutically applicable in the treatment of cancer in those cases where options and the effectiveness of treatment is limited, and these implementations meet unmet need.

The proposed connection show earlier manifestation of activity after a limited time of exposure, and therefore is only effective short-term treatment with their use in clinical situations. This leads to shorter, less frequent therapeutic effects and reduce the associated with drug toxic effects, compared to the original drug. This provides an increased therapeutic index.

When you change the structure by introducing a lipid (including esters and amides) of the component is saved azole cytidine ring and, therefore, the effect of molecules on epigenetic mechanisms. Epigenetic modulation give the em important mechanism of change in gene expression in cancer and inflammation. These new compounds are active at lower concentrations than azacytidine and, thus, are more active. These compounds with a modified spectrum of activity can modulate epigenetic targets in solid tumors and inflammatory diseases.

Epigenetic mechanisms are important in Pro-inflammatory conditions which include, but are not limited to, inflammatory condition of the lung, connective tissue, gastrointestinal tract and vascular system. Data connection through effects on epigenetic mechanisms can reduce or reverse the development of inflammatory processes responsible for these diseases.

Brief description of drawings

On figa-B depicts a chart of comparative cytotoxic activity of 5-azacytidine 5'-elaidate (CP) and 5-Aza-C in leukemia and solid-tumor cell lines. Cytotoxicity was determined during the analysis of WST-1. Expected IC50in the software environment CalcySyn. Cells were treated for 24 hours (figa) or cells were treated for 24, 48 and 72 hours (pigv).

On figa-B shows a graph of the induction of apoptosis under the action of 5-azacytidine 5'-elaidate in leukemia cells. On figa cells HL60, K562 and U937 not been treated or were treated with 5-azacytidine 5'-elaidate (CP) (0.5 to 4 μm) for 24 hours. P is ocent of apoptotic cells was determined using a fluorescent microscope after staining with acridine orange and ethidiumbromid. On figv as HL60 and K562 not been treated or were treated with 5-azacytidine 5'-elaidate (1, 2, or 4 μm) for 24 hours. The percentage of apoptotic cells were determined using flow cytometry with the use of staining with Annexin-V and PI.

Figure 3 shows graphs of the effect of treatment with 5-azacytidine 5'-elaidate (CP) on the cell cycle. Cells were cultured for the same period of time before you can analyze the DNA content using flow cytometry. The numerical values on the chart represent the regulated population of cells with sub-G1 DNA region.

On figa-C show graphs of differentiation, including the effect of 5-azacytidine 5'-elaidate and 5-Azas. K562 and U937 were subjected to 5-azacytidine 5'-elaidate (CP) or 5-Azas (0,125-1 μm) for 14 days. After the initial treatment day 0 cells were exposed to a new portion of medicines in day 4, day 7 and day 10. Differentiation was assessed by NBT staining (U937) (figa) or benzidine (K562) (pigv).

Figure 5 is a diagram showing stimulated by lipopolysaccharide (LPS) NFκB luciferase activity measured in U937 cells exposed to a complex ester of the acid and Aza-C or 5'-Aza-C-5'-elaidic.

Detailed description of the invention

One aspect of the present invention is directed is a compound according to formula (I)

where R represents H, R5C(O); R1represents a

where the crossing dashed line depicts the resulting bond attaching R1to the molecule of formula (I), R2and R3independently represent OH or H, provided that R2and R3are not both OH, R4represents H or R5C(O), provided that R and R4are not both H, R5represents a C3-C26alkenyl or its pharmaceutical salt.

In a specific embodiment, the implementation of R5may submit C9-C26alkenyl.

In a preferred variant of realization of R is an R5C(O)R1represents, R2represents H, R3represents OH, R4represents H and R5represents CH3-(CH2)7-CH=CH-(CH2)7-.

In a broader aspect of the present invention is directed to a compound according to formula (I),'

where R represents H, R5C(O)R5CH2OC(O) or R5CH2NHC(O)R1represents a

where the crossing dashed line depicts the formed communication, PR is connecting R 1to the molecule of formula (I)', R2and R3independently represent OH or H, provided that R2and R3are not both OH, R4represents H, R5C(O)R5CH2OC(O) or R5CH2NHC(O), provided that R and R4are not both H, and R5represents a C3-C26alkenyl or its pharmaceutical salt.

In a preferred embodiment, the implementation of the compounds according to formula (I),' k equal to 7, and n is 7. In some embodiments of R1represents a

where the crossing dashed line depicts the resulting bond attaching R1to the molecule of formula (I)'. In some embodiments of R is an R5C(O), k is 7, m is 0, n is 7, R2represents H, and R3represents OH. In some embodiments of R5represents a C9-C26alkenyl.

Another aspect of the present invention is directed to a pharmaceutical composition comprising a compound of formula (I), and pharmaceutical excipient, diluent and/or carrier.

Proposed according to the present invention means can be administered orally, parenterally, for example subcutaneously, intravenously, intramuscularly, administered intraperitoneally, by instillation into the nose is whether by application to mucous membranes, for example, to slimy nose, throat and bronchial tubes. They can be administered separately or together with pharmaceutical carriers, and they can be in solid or liquid form, for example in the form of tablets, capsules, powders, solutions, suspensions or emulsions.

Proposed according to the present invention the active tool can be administered orally, for example, with an inert diluent or with assimilable edible carrier, or they may be enclosed in capsules, which are solid or soft shell, or they may be compressed into tablets, or they can be entered directly with food in accordance with diet. In the case of oral therapeutic introduction of these active substances can be entered together with excipients and apply in the form of tablets, capsules, elixirs, suspensions, syrups, etc. Such compositions and preparations should contain at least 0.1% of active funds. The share of funds in these compositions may, of course, be varied and may preferably be in the range from about 2% to about 60% by weight of the dosage form. The amount of active ingredient in such therapeutically applicable compositions is such as to make a suitable dose. Preferred compositions according to the present invention are prepared so that the standard drug Faure is for oral administration contain from about 1 to 250 mg of active substance.

Tablets, capsules and the like may also contain binders, such as tragacanth gum, gum Arabic, corn starch or gelatin; excipients such as dicalcium phosphate; a leavening agent such as corn starch, potato starch, alginic acid; a lubricant substance, for example, magnesium stearate; and a sweetener, such as sucrose, lactose or saccharin. If the standard dosage form is a capsule, in addition to the above types of materials it may contain a liquid carrier such as fatty oil.

Can be many other materials as the shell or to modify the physical form of a standard dosage forms. For example, tablets may be coated with shellac, sugar or both. A syrup may contain, in addition to the active ingredient, sucrose as a sweetener, methyl and propylparaben as preservatives, a dye and flavoring, such as cherry flavor or orange.

These active funds can also enter parenteral. Solutions or suspensions of these active agents can be prepared in water suitably mixed with a surface-active substance, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid propylene glycols and their oil is mixtures. Examples of oils can serve as oil, petroleum, animal, vegetable, synthetic origin, such as peanut oil, soybean oil or mineral oil. In General, the preferred liquid carriers, particularly in the case of injection solutions are water, saline, aqueous dextrose and related sugar, and glycols such as propylene glycol or polyethylene glycol. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for use for injection include sterile aqueous solutions or suspensions, as well as sterile powders for the preparation of sterile solutions or dispersions for injection for immediate use. In all cases the form must be sterile and have fluidity in such a way, that it could easily enter the syringe. It must be stable under conditions of manufacture and storage and must be protected from contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixture of these components or vegetable oil is.

Proposed according to the present invention means can also be entered directly into the respiratory tract in the form of an aerosol. For use in aerosol form proposed according to the present invention means in the form of a solution or suspension may be packaged in a pressurized aerosol container together with suitable gases-plungers, for example, hydrocarbon gases-displacers such as propane, butane or isobutane, together with suitable adjuvants. The substances according to the present invention can also enter in an unpackaged form, for example, using a spray gun or spray.

Another aspect of the present invention is directed to a method of treating neoplastic syndrome in the patient. This method involves selecting a patient with a neoplastic syndrome and the introduction of a given patient, the compounds of formula (I)as described above, or a pharmaceutical salt under conditions effective for the treatment of neoplastic syndrome in a patient.

In some embodiments of neoplastic syndrome is a cancer. Cancer may be a solid tumor or hematological cancer or a malignant tumor. Cancer may pose a leukemia, lymphoma, multiple myeloma or myelodysplastic syndrome.

is some embodiments of the solid tumor may represent cancer tissue, for example, tissues of the breast, ovary, prostate, brain, bladder and lungs.

Another aspect of the present invention is directed to a method of treating inflammatory syndrome in a patient. This method involves selecting a patient with an inflammatory syndrome and the introduction of a given patient, the compounds of formula (I)as described above, or a pharmaceutical salt under conditions effective for the treatment of inflammatory syndrome in a patient.

In some embodiments of the inflammatory syndrome is an inflammatory condition of the lung, connective tissue, gastrointestinal tract, or vascular system.

In materials of this application, if not defined otherwise, technical and scientific terms used in connection with the present application, have their ordinary meaning, well known to the average person skilled in the art. Further, if the context is not specified, the terms in the singular shall include their plural form, and the terms in the plural include the form in the singular.

Examples

Example 1 - Reagents, cell lines and cell culture

5-azacytidine 5'-elaidate (CP or CP-4200) was obtained from the company Clavis Pharma AS a reagent for cell proliferation WST-1 was obtained from Roche Applied Science (Mannheim, Germany), PI and kit for determination of apoptosis Annexin V-FITC apoptosis kit was ordered BD Biosciences, Palo Alto, CA, 5-azacytidine (5-Azas), ethidiumbromid (EB), acridine orange (AO), nicrosini tetrazolium (NBT), phorbol 12-myristate 13-acetate (TPA) were ordered from Sigma Chemical Co (St. Louis, MO).

Line human promyelocytic leukemia cells HL60, human histocytes lymphoma U937, human chronic myelogenous leukemia K562, T-cell line Jurkat human mammary adenocarcinoma (MCF-7, carcinoma of the bladder 5637, prostate carcinoma DU-145 were ordered from American Type Culture Collection. All cell lines, except Jurkat, were kept in RPMI medium 1640 (Gibco, Glasgow, United Kingdom), containing 10% V / V heat inactivated fetal calf serum (FCS), 100 U/ml penicillin 100 mg/ml streptomycin in an atmosphere of 5% CO2at 37°C. Cell line Jurkat were cultured in medium RPMI 1640, containing 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mm sodium pyruvate and 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin.

Example 2 - Analysis of cytotoxicity

Cytotoxicity of lipid 5-azacytidine determined during colorimetric analysis based on the decomposition of salts of tetrazole WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolyl]-1,3-benzodiacepinas) by mitochondrial dehydrogenase in viable cells. Cells were sown when the initial concentration of 1×106/ml (HL60 cells) or 1.25×105/ml (U937, 562 and Jurkat) in the environment, not containing or containing different concentrations of lipid 5-azacitidine in 96-well flat-bottomed microplate, and were cultured for 24 to 72 hours. Cells MCF-7, DU-145 and 5637 (1×104/ml) were sown in a Cup and left for adhesion and growth for 24 hours. Added various concentrations of lipid 5-azacytidine, after which the cultures were maintained for 24 to 72 hours. Cultures were incubated with the reagent WST-1 for 1 hour. Education formazan was measured using microplate reader (Bio-Tek Instruments, Elx 800) at 450 nm with wavelength comparison of 650 nm. The growth inhibition was determined by comparison with untreated cells (%). The values of the IC50was determined using CalcuSyn (Biosoft).

Example 3 - Quantitative assessment of apoptotic cells

Apoptotic cells were identified using morphological criteria and fluorescently-activated cell sorting (FACS) after staining with Annexin V-FITC. For morphological analysis were added 1 μl of the prepared solution containing 100 µg/ml AO and 100 μg/ml EB to 25 μl of cell suspension. Apoptotic cells and apoptotic bodies were analyzed using fluorescence microscopy. The percentage of apoptotic cells was determined after counted the total number of all cells equal to 300. For FACS analysis, 2×105up to 5×106cells were washed with p the power of PBS, and then were labeled with Annexin V-FICS and propitiated (PI) in the binding environment of the reagent according to the instructions supplied by the manufacturer of the kit for determination of apoptosis Annexin V-FITC apoptosis detection kit. Fluorescent signals FITC and PI were detected, respectively, at 518 nm and 620 nm on a FACSCAN (Becton Dickinson, San Jose, California). Record fluorescence of Annexin V-FITC displayed on the X-axis and record the values of PI fluorescence was displayed on the y-axis. Data were processed using the CellQuest program (Becton Dickinson). For each analysis the recording was made, 10000 cellular events.

Example 4 - the Cell cycle

Cells were besieged during centrifugation and washed twice with PBS, fixed with 70% (vol./about.) cold ethanol (-20°C) and kept at 4°C for at least 24 hours. Cells were washed with PBS. Clot cells were stained with staining solution PI/RNase. Cell suspension was incubated in the dark at room temperature for 30 minutes, the DNA Content was determined using flow cytometry FACSCalibur (Becton Dickinson, Mount View, CA). The percentage of cells in stages of Sub-G1, G1, S, and G2/M cell cycle was determined using the program, licenciada charts DNA (Becton Dickinson). Recorded a minimum of 10,000 events per sample.

Example 5 - Synthesis of ester Aza-C-5'-oleic acid

Olein is a new acid (1.77 mmol, 500 mg) was dissolved in toluene (3 ml). Added first DMF (10 μl), and then oxalicacid (3.6 mmol, 457 mg) for 10 min at room temperature. After 3 hours, toluene was removed under vacuum.

Aza-C (1,75 mmol, 427 mg) suspended in DMA (6 ml), was added HCl (1 M in Et2O, 2.0 mmol, 2.0 ml) and after 5 min at room temperature was removed Et2O in vacuum. The resulting cloudy solution was cooled in an ice-water bath was added the acid chloride, dissolved in DMA (2 ml)for 2 h the Reaction mixture was stirred overnight while the temperature did not reach slowly room temperature, and then it was heated at 30°C for 2 hours After cooling to room temperature the reaction mixture was divided between saturated aqueous NaHCO3and EtOAc (25 ml each). The aqueous phase was extracted with additional 3×25 ml EtOAc. The organic phases were combined, washed with brine and dried (MgSO4). After removal of the solvent in vacuum the crude oily product was purified using flash chromatography (SiO2CH2Cl2with 2.5, 5 and 10% MeOH). Yield: 110 mg (13%).

Example 6 - Synthesis of ester 5-azacytidine 5'-elaidic acid

5-azacytidine (4.1 mmol, 1.00 m g) suspended in dry DMA (15 ml) and was slowly added a solution of HCl in diethyl ether (4.9 mmol, 1 M, 4.9 ml) at room temperature with the formation of prozrachnoj the solution. The ether was then removed in vacuum, which led to the formation of a slightly turbid solution. The solution elaidinised (4.8 mmol, 1.44 g) in dry DMA (8 ml) was added for 1 h at room temperature. The reaction mixture was then heated overnight at 30°C, cooled to room temperature and extinguished using methanol (0.05 ml). After at least 1 h the reaction mixture was concentrated in approximately 10-2mbar, and the residue was divided between saturated aqueous NaHCO3and ethyl acetate. The aqueous phase was extracted with ethyl acetate. The organic phases were then combined, washed (brine), dried (Na2SO4) and concentrated in vacuum. After purification using flash chromatography (SiO2CH2Cl2with 0-10% methanol) was obtained 0.9 g (43%) of product.

Example 7 - In vitro effect of 5-azacytidine 5'-elaidate on tumor cells

Compared the cytotoxic activity of 5-azacytidine 5'-elaidate and 5-Azas in relation to the lines of the leukaemic cells and cells of solid tumors. Compared with 5-Azas, the cytotoxic effect of 5-azacytidine 5'-elaidate was higher in all studied cell lines. Compared with solid tumors, leukemia cell lines were more sensitive to 5-azacytidine 5'-elaidate (CP)and 5-Azas, see table 2 (below) and figa-B.

Table 2
24 hours (µm)48 hours (µm)72 hours (µm)
U937CP0,440,600,87
5-Azas1,632,374,77
K562CP1,260,920,42
5-Azas8,2715,51of 5.83
HL60CP3,193,602,68
5-Azas7,284,924,71
JurkatCP1,190,880,84
5-Azas9,55,324,77
MCF-7CP19,1to 9.329,99
5-Azas>16093,753,00
DU145CP6,79of 6.31 5,88
5-Azas20,947,117,20
5637CP9,395,953,52
5-Azas24,566,72to 5.21
Table 2. Comparative cytotoxic activity of 5-azacytidine 5'-elaidate (CP) and 5-Azas in relation to the lines of the leukaemic cells and cells of solid tumors. Values (IC50). Cells were treated for 24, 48 and 72 hours and cytotoxicity was determined during the analysis of WST-1. IC50was calculated using the CalcuSyn program.

A striking result is an early manifestation of the activity in the case of 5-azacytidine 5'-elaidate, which was not observed for azacitidine.

Example 8 Induction of apoptosis in leukemia cells

Cells HL60, K562 and U937 were not treated or were treated with 5-azacytidine 5'-elaidate (0.5 to 4 μm) for 24 h, the Percentage of apoptotic cells was determined using a fluorescent microscope after staining with acridine orange and ethidiumbromid is Yes (see Fig. 2A). In another series of experiments, HL60 and K562 were not treated or were treated with 5-azacytidine 5'-elaidate (1, 2, or 4 μm) for 24 hours. The percentage of apoptotic cells were determined using flow cytometry with the use of staining with Annexin-V and PI. Both methods demonstrated that 5-azacytidine 5'-elaidate induced a concentration-dependent increase in the number of apoptotic cells (see figa and 2B).

Example 9 - Course of cell cycle

Studied the effect of exposure to 5-azacytidine 5'-elaidate on the cell cycle. Cells were cultured for a specified period of time, after which they were analyzed for the composition of DNA using flow cytometry. The numerical values on the chart represent the population of cells, giving the signal above the threshold value with the contents of sub-G1 DNA. The most significant changes were observed after 24 hours of treatment. Compared with the control group (8.3%) of the percentage of sub-G1 phase (apoptotic cells) was increased up to 33,6% in cells treated with 2 μm 5-azacytidine 5'-elaidate, and decreased in G1, S and G2/M phase. The results are summarized in figure 3 and in table 3, below. These results confirmed the induction of apoptosis under the action of 5-azacytidine 5'-elaidate and demonstrated that the effect of 5-azacytidine 5'-elaidate on the cell cycle was concentration-dependent nature of the EP.

Table 3
The percentage of cells
24 hoursDose (μm)Sub G1G1SG2/M
1Control0compared to 8.2638,6133,35a 12.7
2cp0,59,4143,6628,1511,05
3121,0236,1718,988,82
4233,5923,459,142,64
48 hours
1Control04,841,131,14MT 18 : 34
2cp0,515,1434,7942,962,53
3114,3428,0730,0617,91
4224,3422,5115.62 wide7,78
72 hours
1Control09.28 are 47,7227,3914,64
2cp0,2510,8840,1428,1318,25
30,510,5438,7229,1818,82
4110,7540,6728,7718,26
Table 3. Effect of 5-azacytidine 5'-elaidate (CP) on the cell cycle. Cells were cultured for a specified period of time, after which they were analyzed for the composition of DNA using flow cytometry. These numbers represent the percentage of cells in different phases.

Example 10 Induction of differentiation

Studied the differentiation on the basis of morphology. As a single exposure (96 hours) had no effect, cells were exposed to 5-azacytidine 5'-elaidate or 5-Azas for 11 (HL60 cells) or 14 (cells U937 and K562) days. After the touch processing at day 0 cells were exposed to new portions of medicines in day 4, 7 and 10. Differentiation was assessed by examining changes narasinga of tetrazole (U937 and HL60) or by staining with benzidine (K562). Despite the fact that 5-azacytidine 5'-elaidate was somewhat more effective, both reagents did not cause significant differentiation. Cm. figa-B.

Example 11 - Metabolic stability of ester 5-Aza-5'-elaidic acid pooled human plasma

Ester 5-Aza-C-5'-elaidic acid was placed in pulirovaniya human plasma at five concentration levels (0,1; 1; 3; 10 and 30 µm, respectively). The mixture was incubated in a water bath shaker at 37°C. were Selected triple (n=3) aliquots (100 µl) encouragea solution through the respective incubation periods (0, 15, 30, 60 and 120 minutes), and immediately laid siege to plasma protein using acetonitrile containing 0.1% formic acid (300 μl). Negative controls were prepared with the test compound and Aza-C in the buffer for analysis (PBS, pH 7,4) at the same concentration incubation (1 μm). After centrifugation the supernatant was directly injected for analysis of LC-MS-MS. Cm. table 4.

Concentration (µm)
Table 4
Metabolic stability of ester 5-Aza-5'-elaidic acid pooled human plasma
% remaining from the original (mean ± STD., n=3)The half-life (min)
0 min15 min30 min60 min120 min
0,1100the 95.8±3,095,0±2,0to 92.1±3,459,0±1,6161
1100to 93.3±1,188,9±1,179,8±2,652,5±3,1130
3100a 99.0±3,596,0±3,085,4±3,456,6±1,6141
1010096,0±2,0br93.1±0,077,1±3,139,1±1,786
3010097,7±35 to 90.4±2,678,5±1,747,4±1,1109

Example 12 - Cytotoxicity of Aza-C and 5-Aza-C-5'-elaidic acid

Cytotoxicity of Aza-C and 5-Aza-C-5'-elaidic acid was determined in line cancerous breast cells MT-3 in adriablastin-resistant cell line MT-3/ADR. For MT-3/ADR characterized by overexpression of MDR-1/p-glycoprotein. Cells were planted in 96-well tablets 5×103cells per well in RPMI medium 1640 with 2 mm glutamine and 10% FBS. Cells were incubated for 24 hours. The compounds were dissolved in DMSO and further diluted medium immediately before use. For analysis of the same concentration used six holes. Cells were incubated with the test compound for 24 hours. Added 20 µl of freshly prepared MTT solution to each well and incubated for 4 hours. The value of the IC50determined from growth curves, plotted on the basis of 8 different concentrations ranging from 0.01 μm to 100 μm. The results are presented in table 5. The same activity were obtained for Aza-C and 5-Aza-C-5'-elaidic acid on line cancerous breast cells MT-3, but in the case of the resistant cell line MT-3/ADR activity of Aza-C was gone. Activity was not observed in the tested interval end is crazy up to 100 μm, while 5-Aza-C-5'-elaidic acid was active with the same value IC50as in the resistant cell line, and not to be resistant line MT-3. This can be very important in the treatment of resistant cancer. Cm. table 5.

Table 5
The cytotoxic effect of Aza-C and 5-Aza-C-5'-elaidic acid on cell line breast carcinoma MT-3, with or without multilocational resistance
Aza-C
IC50(µm)
5-Aza-C-5'-elaidic acid
IC50(µm)
MT-3 carcinoma of breastbr12.62±2,3517,03±of 15.75
MT-3/ADR resistant carcinoma of breast>10014,82±12,76

Example 13 the Effect of inhibiting nucleoside carrier Aza-C and 5-Aza-C-5'-elaidic acid

Assessed the effect of inhibiting nucleoside carrier on the cytotoxic activity in mutant cells carcinoma of the cervix Hela for Aza-C and 5-Aza-C-5'-elaidic acid. Used dipyridamole as an inhibitor of uranov shivaya nucleoside vectors hENT1 and hENT2. Cells were planted in 96-well tablets 5×103cells per well in RPMI medium 1640 with 2 mm glutamine and 10% FBS. The cells were preincubated within 24 hours. Added dipyridamole (10 μm) to the cells for 30 minutes before adding the investigated compounds. The compounds were dissolved in DMSO and further diluted medium immediately before use. For analysis of the same concentration used six holes. Cells were incubated with the test compound for 72 hours. Added 20 µl of freshly prepared MTT solution to each well and incubated for 4 hours. The value of the IC50determined from growth curves, plotted on the basis of 8 different concentrations ranging from 0.01 μm to 100 μm. The results are presented in table 6. The activity of Aza-C decreased in 3 times by adding an inhibitor of nucleoside transport of dipyridamole, indicating that the inflow and outflow of Aza-C partly dependent on nucleoside vectors hENT1 and hENT2. Cytotoxic activity of 5-Aza-C-5'-elaidic acid not only survived, but has grown 50 times when nucleoside vectors hENT1 and hENT2 were blocked with dipyridamole. Increased activity in the cells, where the nucleoside carriers blocked with dipyridamole, is unexpected and may indicate greater activity in patients with resistant the awn to therapy, due to lack of nucleoside vectors. Cm. table 6.

Table 6
Cytotoxic activity of Aza-C and 5-Aza-C-5'-elaidic acid in the mutant cell carcinoma of the cervix Hela add (or without) an inhibitor of nucleoside transport
Azacytidine IC50(µm)5-Aza-C-5'-elaidic acid
IC50(µm)
Hela4,3212,00
Hela dipyridamole12,770,23

Example 14 - Activation of nuclear transcription factor-κB (NFκB)

Determined the activation of nuclear transcription factor-κB (NFκB) to assess the impact on inflammation. NFκB is involved in the regulation of a large number of both immune and inflammatory responses, developmental processes, cellular growth and apoptosis.

Used genes reporters NFκB-luciferase to determine the effect of Aza-C and 5-Aza-C-5'-elaidic acid on NFκB-induced luciferase activity in cell line human monocytes U937, stably transfected with a luciferase reporter containing t and NFκB-binding site. The induction of NFκB was performed using the lipopolysaccharide (LPS) with a concentration of 1 μg/ml for 30 minutes before adding to the environment Aza or 5-Aza-C-5'-elaidic acid at a concentration of 10 nm. Cells were cultured in medium RPMI-1640 containing 10% fetal calf serum. Before stimulation with LPS, the cells are transferred to medium containing only 2% fetal serum cows. Luciferase activity was measured using the display in the display System IVIS 100 (Xenogen Corp., USA) via 1,3 or 6 hours. LPS stimulation increased with time during the 6 hours that observed for control cells treated with DMSO. The effects of 10 nm Aza-C further increased LPS-induced NFκB activation, compared to DMSO treated control. 5-azacytidine-5'-elaidate did not differ from control DMSO in terms of LPS-induced NFκB activity. As a rule, positive stimulation of NFκB observed for azacitidine, is not positive, but it's good that LPS stimulation does not increase further under the influence of 5-Aza-C-5'-elaidic acid. Cm. figure 5.

Example 15 - gene Expression of estrogen receptor β (ERβ) in line cancerous breast cells after treatment with the help of azacytidine or 5-azacytidine 5'-elaidate

Gene expression (determined by the level of RNA) of estrogen receptor beta were determined using quantitative real-time PCR (TaqMan). To EDI breast carcinoma MCF-7 were grown in an environment with a lack of estrogen (not containing phenol red RPMI medium with 2% glutamine and 10% fetal serum, cow, processed loaded dextran coal dust). Cells were sown in the flask 25 cm2and left for adhesion for 24 hours before treatment with 1 μm of azacytidine or 5-azacytidine 5'-elaidate. One untreated sample was included as a control. Cells were collected after 5 days from the beginning of exposure of compounds; they were collected by trypsinization, washed and subjected to shock frozen in liquid nitrogen.

Total RNA was extracted from approximately 106the MCF-7 cells exposed shock freezing. Determined the concentration and purity of RNA was transcribable RNA into cDNA using reverse transcription reagents (N808-0234). Conducted quantitative evaluation in real time according to standard protocols using standard pre-mixed reagents for PCR. Mix primer-probe, ER β (ID Hs00230957_m1), hydroxymethylbilane synthase constitutive gene HMBS (ID Hs00609297_m1) was ordered from Applied Biosystems. Gene expression was calculated using the comparative Delta-Delta Ctmethod. Induction of expression of ER-β was 5,26-fold after exposure to 5-azacytidine 5'-elaidate, compared with only of 2.51-fold after exposure of azacitidine. Cm. table 7. This can be very essential for hormone-refractory tumors, in which you can restore sensitivity to the hormone. Cm. table 7.

Table 7
Ct ER βCt HMBSDeltaDelta
Delta
x-fold induction of ERβ gene
Rawup RUB 35.3625,919,45
1 µm 5 Aza-C-5'-elaidic acid32,9725,927,06-2,405,26
1 μm Aza-C34,3126,188,13-1,32of 2.51

Although in the present application is shown and discussed in detail preferred embodiments, those skilled in the art it is obvious that various modifications, additions, substitutions and the like are also within the present invention are described in the paragraphs following claims.

1. The compound of formula (I)

where
R performance, which defaults to a N or R 5C(O);
R1represents a
or
where the crossing dashed line indicates the resulting bond attaching R1to the molecule of formula (I);
R2and R3independently represent IT or N, provided that R2and R3are not simultaneously;
R4represents N or R5C(O) provided that R and R4are not both H; and
R5represents a C3-C26alkenyl,
or a pharmaceutical salt specified connection.

2. The compound according to claim 1, where R5represents a C9-C26alkenyl.

3. The compound according to claim 1, where R is an R5C(O)R1represents a

R2represents H, R3HE is a, R4represents N and R5represents CH3-(CH2)7-CH=CH-(CH2)7-.

4. The pharmaceutical composition intended for the treatment of a condition selected from the neoplastic state and inflammatory status containing the compound according to claim 1 and a pharmaceutical excipient, diluent and/or carrier.

5. A method of treating a neoplastic condition in a patient, comprising selecting a patient with a neoplastic condition, the introduction of the criminal code is related to a patient compounds of the formula

where
R represents H, or R5C(O);
R1represents a
or
where the crossing dashed line indicates the resulting bond attaching R1to the molecule of formula (I);
R2and R3independently represent IT or N, provided that R2and R3are not simultaneously;
R4represents N or R5C(O), provided that R and R4are not both H; and
R5represents a C3-C26alkenyl,
or pharmaceutical salts of the compounds under conditions effective for the treatment of this neoplastic condition in a patient.

6. The method according to claim 5, where R5represents a C9-C26alkenyl.

7. The method according to claim 5, where the specified neoplastic condition is a cancer.

8. The method according to claim 7, where the cancer is a solid tumor or hematological cancer, or malignant.

9. The method according to claim 7, where the specified cancer is a leukemia, lymphoma, multiple myeloma or myelodysplastic syndrome.

10. The method of claim 8, where the solid tumor is a cancer of a tissue selected from the group of tissues consisting of tissue Molo is Noah gland, ovary, prostate, brain, bladder and lungs.

11. The method according to claim 7, where R is an R5C(O)R1represents a

R2represents H, R3HE is a, R4represents N and R5represents CH2-(CH2)7-CH=CH-(CH2)7-.

12. A method of treating an inflammatory condition in a patient, comprising selecting a patient with an inflammatory condition and the introduction of the indicated patient compounds of the formula

where
R represents H, or R5C(O);
R1represents a
or
where the crossing dashed line indicates the resulting bond attaching R1to the molecule of formula (I);
R2and R3independently represent IT or N, provided that R2and R3are not simultaneously;
R4represents N or R5C(O) provided that R and R4are not both H; and
R5represents a C3-C26alkenyl,
or pharmaceutical salts of the compounds under conditions effective for the treatment of this inflammatory condition.

13. The method according to item 12, where R5represents a C9-C26alkenyl.

14. Pic is b-12, where the specified inflammatory condition is an inflammatory condition of the lung, connective tissue, gastrointestinal tract, or vascular system.

15. The method according to item 12, where R is an R5C(O)R1represents a

R2represents H, R3HE is a, R4represents N and R5represents CH3-(CH2)7-CH=CH-(CH2)7-.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new compounds of formula or to pharmaceutically acceptable salts thereof for treating the Flaviviridae infection, as well as to a pharmaceutical composition thereof, and to the use thereof in preparing a drug. In the compound of formula (IVa), the base* represents a purine or pyrimidine base; R1 and R2 together form a cyclic 3',5'-phosphate ester; R7 represents halogen, particularly F or Cl; Y3 independently represents H, F, Cl, Br or I.

EFFECT: preparing the compounds for treating the Flaviviridae infection.

22 cl, 32 ex, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are described new N-oxycarbonyl-substituted 5'-deoxy-5-fluorocytidines of general formula I wherein R1 represents a saturated normal or branched hydrocarbon radical wherein a number of carbon atoms in the longest direct chain makes three to seven, or a redical of formula -(CH2)n-Y, wherein n = 0 - 4; provided Y represents cyclohexyl, or n = 2 - 4, provided Y represents phenyl; R2 represents a hydrogen atom. What is described is an anti-cancer preparation based on the compounds of formula T.

EFFECT: compounds show the excellent high-safety pharmacokinetic properties in treating tumours.

6 cl, 7 tbl

FIELD: biochemistry.

SUBSTANCE: invention refers to derivatives of 3'-ethynilcytidine represented by the formula (1); (where X is hydrogen atom, alkylcarbonyl group where alkyl fragment is non-branched or branched C1-C6alkyl group which can include as substitute(s) mono-or di-substituted with non-branched or branched C1-C6alkyl group aminogroup or alkoxycarbonyl group where alkoxy-fragment is a branched or non-branched C1-C6 alkoxygroup; One of Y and Z is a hydrogen atom or group (R1)(R2)(R3)Si-, and another is a group (R4)(R5)(R6)Si-; and each R1, R2, R3, R4, R5 and R6 that may be similar or different are non-branched or branched C1-C10 alkyl group or C6-C14aryl group) or to its salts. The invention also refers to the derivative of 3'-ethynilcytidine chosen out of compounds (1)-(17), to the pharmaceutical composition, to oncologic drug, to oral oncologic drug, to the use of derivative of 3'-ethynilcytidine, and to the method of tumor treatment.

EFFECT: new biologically active compounds with antitumor activity.

14 cl, 18 ex, 12 tbl

FIELD: chemistry.

SUBSTANCE: nucleic base (e.g. uracil, cytosine, adenine, guanine, hypoxanthine, xanthine or similar) reacts with perfluoroalkyl halide in the presence of sulphoxide, peroxide and an iron compound to obtain a perfluoroalkyl-substituted nucleic base.

EFFECT: high cost effectiveness as an intermediate compound for producing medicinal agents.

15 cl, 6 tbl

FIELD: chemistry.

SUBSTANCE: method enables to obtain 4-amino-1-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one of formula (IV), which is a strong inhibitor of NS5B polymerase of hepatitis C virus (HCV).

EFFECT: high yield.

2 cl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to phosphoramidite derivatives of general formula where Bx denotes adenine, guanine, cytosine, thymine or uracil, where the amine group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl and phenoxyacetyl; R1 is a substitute of general formula in which R11, R12 and R13 are identical or different, and each denotes hydrogen or alkoxy; R2a and R2b are identical or different, and each denotes alkyl; and WG1, WG2 denote a cyano group. The invention also pertains to a multistep method of producing the said compounds. The invention also relates to intermediate compounds of the said method, namely: an intermediate ether compound of general formula where L is a halogen or a C1-C5alkylthio group; WG1 is a cyano group; an intermediate compound of general formula where Bx denotes adenine, guanine, cytosine, thymine or uracil, where the amine group of adesine, guanine and cytosine can be optionally protected by a protective group selected from an acetyl group and a phenoxyacetyl group; and WG1 denotes a cyano group; an intermediate compound of general formula where Bx is as described above; R1 is a substitute of general formula (2); an intermediate compound of general formula where Bx is as described above; A is a silicon-containing substitute of general formula or where R6 denotes alkyl and WG1 denotes a cyano group. The invention also relates to a method of producing an oligonucleotide of general formula where each B independently denotes adenine, guanine, cytosine, uracil or thymine; each R independently denotes H or hydroxyl and at least one of R denotes hydroxyl; Z denotes H or a phosphate group; and n is an integer between 1 and 100, involving steps A-G, characterised by use of said phosphoramidite derivatives as a monomer compound of nucleic acid at step B.

EFFECT: high yield.

7 cl, 1 dwg, 21 ex

FIELD: chemistry.

SUBSTANCE: disclosed are α- and β-crystalline forms of 5'-desoxy-N4-carbopentyloxy-5-fluorocytidine of formula (III) , their preparation method through crystallisation of the raw product from a suitable solvent and pharmaceutical compositions based on the said compounds, having anti-cancer activity. The solvent used when preparing the α-modification is an ester or a mixture of ester-containing solvents. The solvent used when preparing the β-modification is a mixture of water and alkanol or a mixture of tetrahydrofuran and diethyl ether or carbon tetrachloride.

EFFECT: obtaining compounds and pharmaceutical compositions based on the said compounds, having anticancer activity.

10 cl, 2 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to new compounds of formula (IX) or to their pharmaceutically acceptable salts having inhibitory activity to hepatitis C, to the related pharmaceutical composition and to their application for making a medical product. In compound of formula (IX) , R1 and R2 independently represent H, phosphate or acyl; X represents O; base* represents pyrimidine base; R12 represents C(Y3)3; Y3 represents H and R13 represents fluorine.

EFFECT: higher efficiency of the composition and treatment method.

31 cl, 14 dwg, 26 ex

FIELD: medicine.

SUBSTANCE: invention relates to method of obtaining gemcitabine hydrochloride, characterised by the following: 2,2-dimethyl-[1,3]-dioxolane-4-carbaldehyde is subjected to interaction with ethyl bromodifluoracetate in presence of zinc in organic solvent medium processing reaction mixture with ultrasound for 5-60 minutes, obtained ethyl 3-hydroxy-2,2-difluoro-3-[2,2-dimethyl-[1,3]dioxolane-4-yl]propionate is subjected to hydrolysis and cyclisation by means of ion-exchange resin in water-alcohol medium obtaining (4R,5R)-4-hydroxy-5-hydroxymethyl-3,3-difluorodihydrofuran-2(3H)-on, which is processed with solution of trimethylchlorosilane in dichloromethane obtaining (4R,5R)-4-trimethylsilyloxy-5-((trimethylsilyloxy)methyl)-3,3-difluorodihydrofuran-2(3H)-on, which is subjected to reduction by means of lithium diisopropylalumohydride in organic solvent medium at cooling to -70°C obtaining (4R,5R)-2-hydroxy-4-(trimethylsilyloxy)-5-((thrimethylsilyloxy)methyl)-3,3-difluorotetrahydrofurane, which is converted into (4R,5R)-2-methylsulphonyloxy-4-(trimethylsilyloxy)-5-((trimethylsilyloxy)methyl)-3,3-difluorotetrahydrofurane by processing with methane sulphonylchloride in solvent medium at cold, obtained (4R,5R)-2-methylsulphonyloxy-4-(trimethylsilyloxy)-5-((trimethylsilyloxy)methyl)-3,3- difluorotetrahydrofurane after optic isomer separation is processed with bis-trimethylsilylacetylcytozine in water-free dichlorethane and boil with trifluoromethane sulphonyloxymethylsilane with further cooling and separation of obtained gemcitabine in form of base or hydrochloride, as well as method of gemcitabine hydrochloride purification by its re-crystallisation from water solution with processing with ultrasound.

EFFECT: invention results in increase of ratio 3-(R)-hydroxy-isomer to 3(S)-hydroxy-isomer.

6 cl, 2 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention refers to anhydrous polymorphic salt representing hemisulphate 1-[4(S)-azido-2(S),3(R)-dihydroxy-4-(hydroxymethyl)-1-(R)-cycloamyl]- cytosine of formula (Ia) possessing extra stability and improved physical properties that facilitates production, transportation of compound and preparation of compositions on the basis of compounds of formula I . Invention also refers to various crystal forms of compounds of formula (Ia), characterised by certain values of lattice constant D (interplanar spacing), to methods of their production from compounds of formula (I), to pharmaceutical compositions on the basis of compounds of formula (Ia) and to method of disease treatment caused by C hepatitis virus, including injection if such treatment is required, of therapeutically effective amount of compound of formula (Ia).

EFFECT: production of compound possessing extra stability and improved physical properties that facilitates production, transportation of compound and preparation of compositions on its basis.

14 cl, 5 dwg, 1 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula (I) or (I'): Z-X1-(-CH2-CH2-O-)n-Yp-D (I), D-Yp-(-CH2-CH2-O-)n-X1-Z (l'), where: Z is a reactive carboxylic ether selected from a group consisting of N-succinimidyl, N-sulphosuccinimidyl, N-phthalimidyl, N-sulphophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulpho-4-nitrophenyl, 3-carboxy-4-nitrophenyl and a trifluorophenyl ester, or haloacetamide; D is maytansinoid; X is an aliphatic structural unit; Y is an aliphatic structural unit linked to the maytansinoid through a thioether bond; where said aliphatic structural unit, represented by X or Y, is a simple or branched alkyl group with 1-20 carbon atoms in the chain, a cyclic alkyl group having 3-10 carbon atoms, a simple or branched alkenyl group, having 2-15 carbon atoms in the chain or a simple or branched alkynyl group, having 2-15 carbon atoms in the chain; 1 equals 0 or 1; p equals 0 or 1; and n is an integer from 1 to 2000. The invention also relates to a conjugate of a cell-binding agent, and cytotoxic maytansinoid, where the cell-binding agent is an antibody.

EFFECT: obtaining compounds and conjugates, as well as pharmaceutical compositions based thereon, which can be used in medicine to treat tumours, autoimmune diseases, graft rejection, graft-versus-host disease, viral infections and parasitic infections.

20 cl, 38 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to imidazo[1,2-b]pyridazine and pyrazolo[1,5-a]pyrimidine derivatives of the following structures (I) and (II) wherein X represents NH or O; R represents H or C1-6alkyl; R1 represents a structure specified in wherein R1' means a substitution in n-, o- or m-positions by one or more substitutes from halogen, -OCF3, -CF3 or -OCH3; R2 represents -(CH2)1;2-piperid-4-yl or substituted -(CH2)1,2-piperid-4-yl, substituted by C1-6alkyl or C(O)cyclopropyl; or R2 represents -(CH2)n-cyclohexyl wherein n means 0 or 1, wherein -(CH2)n-cyclohexyl is optionally substituted by one or more substitutes specified in halogen, oxo, cyano, amino, alkylamino, alkyl, alkoxy, hydroxyalkyl, morpholinyl, -NReC(=O)Rf, -S(=O)2NH2, -NReS(=O)2Rf, wherein Rf and Re are identical or different and independently represent hydrogen, alkyl or morpholinylalkyl. The invention also refers to a composition possessing activity inhibiting Pim-1 kinase activity.

EFFECT: there are produced new compounds and composition on their basis which can find application in medicine for treating cancer expressing Pim-1 kinase.

13 cl, 5 dwg, 9 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of formula (I) or pharmaceutically acceptable salts thereof wherein A, R1, R2, R3 and m are specified in the patent claim. The present invention also refers to the number of specific compounds, and to a pharmaceutical composition containing the above compounds effective for inhibition of kinases, such as glycogen synthase kinase 3 (GSK-3), Rho kinase (ROCK), Janus kinase (JAK), AKT, PAK4, PLK, CK2, KDR, MK2, JNK1, aurora, pim 1 and nek 2.

EFFECT: preparing the specific compounds and pharmaceutical composition containing the above compounds effective for kinase inhibition.

18 cl, 393 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to 4-[3-(4-cyclopropanecarbonyl-piperazin-1-carbonyl)-4-fluor-benzyl]-2H-phthalazin-1-one in the form of a crystalline form L having characteristic peaks on an X-ray powder diffraction pattern presented in the patent claim, to methods for preparing the form L, pharmaceutical formulation containing the form L, and versions of using the form L and formulations containing the form L.

EFFECT: preparing the new crystalline form of the above compound which possesses poly-(ADP)polymerase (PARP) inhibitory activity The form L contains no solvent impurities that enables more accurate dosage of the active compound when treating the patient.

17 cl, 4 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology and hematology, and may be used in treating the patients with refractory and recurrent clinical course of Hodgkin's lymphoma. For this purpose, 24 hours before the leucopheresis procedure, a granulocyte colony-stimulating factor is administered to the patient. That is followed by the leucopheresis procedure with using MCS "Haemonetics" blood separator, and the prepared leukapack is divided on 4 portions. The leukapack portions are collected in sterile containers and incubated for 180 minutes at temperature 37°C: portions 1 and 3 with recombinant interleukin-2 5 thousand IU/ml, portions 2 and 4 with interferon-α-2b 10 thousand IU/ml. That is followed by the intravenous, drop-by-drop reinfusions of the leukapack before a course of the polychemotherapy: portions 1 and 2 on the day of the leucopheresis procedure, portions 3 and 4 of the laukapack are kept in a fridge at temperature 0 to 8°C for 24 hours and then incubated with immune preparations in the specified mode and administered to the patient. After the course of the polychemotherapy has been completed, the leucopheresis procedure is conducted again. The above therapy is prescribed for cycles 4 and 6 of the polychemotherapy used for refractory Hodgkin lymphoma and for cycles 2 and 4 cycles of the polychemotherapy used for recurrent Hodgkin's lymphoma, regardless of a response to therapy.

EFFECT: method enables the effective therapy of the given pathology ensured by activation of the proper anti-tumour response.

2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmacy and medicine, and concerns using the pyridopyrazine derivatives for preparing a drug for treating or preventing the physiological and/or pathophysiological conditions associated withPI3K kinase inhibition in mammals.

EFFECT: invention provides high clinical effectiveness.

7 cl, 4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to oncology and can be used in treatment of gall bladder cancer with metastases into liver. Essence of method lies in the following: 1-st course of regional chemotherapy is carried out on the 14-th day after operative intervention. Under conditions of procedure room 20 ml of autoblood were sampled from patient and incubated with gemzar in amount 1000 mg/m2 for 30 minutes at temperature 37.5°C. Chamber of infusion system was punctured with Gruber's needle and preparation is introduced. On the second day introduction of oxalyplatin on 5% glucose solution was introduced in amount 100 mg/m2. Courses of regional autohemochemotherapy were repeated every 14 days.

EFFECT: method provides possibility of carrying out chemotherapeutic treatment in earlier terms of postoperative period, as well as realisation of direct impact on remaining tumour cells and focuses of metastatic affection, in addition method application makes it possible to reduce amount of applied anti-tumour medications and minimise their side toxic manifestations, achieve maximal concentration of chemical preparations in regional blood flow.

1 ex

FIELD: medicine.

SUBSTANCE: what is presented is a nucleic acid construct for treating tumours comprising at least two open reading frames that include sequences encoding a cytotoxic or cytostatic gene product, in particular diphtheria toxin functionally related to various tumour-specific promoters: H19-specific promoter, IGF-II P3 or P4 promoter. What is described is a eukaryotic expression vector comprising the above nucleic acid construct, and methods of treating and inhibiting the tumour development in a human subject by administering the nucleic acid construct under the invention.

EFFECT: invention can find further application in therapy of cancer.

33 cl, 12 ex, 40 dwg

FIELD: chemistry.

SUBSTANCE: described are novel chiral cis-imidazolines selected from a group which includes 2-{4-[(4S,5R)-2-(2-tert-butyl-4-ethoxypyrimidin-5-yl)-4,5-bis-(4-chlorophenyl)-4,5-dimethyl-4,5-dihydroimidazole-1-carbonyl]-piperazin-1-yl}-acetamide, [(4S,5R)-2-(2-tert-butyl-4-ethoxypyrimidin-5-yl)-4,5-bis-(4-chlorophenyl)-4,5-dimethyl-4,5-dihydroimidazol-1-yl]-[4-(1,1-dioxohexahydrothiopyran-4-yl)-piperazin-1-yl]-methanone, [(4S,5R)-2-(2-tert-butyl-4-ethoxypyrimidin-5-yl)-4,5-bis-(4-chlorophenyl)-4,5-dimethyl-4,5-dihydroimidazol-1-yl]-[4-(3-methanesulphonylpropyl)-piperazin-1-yl]-methanone, 2-{4-[(4S,5R)-2-(6-tert-butyl-4-ethoxypyridin-3-yl)-4,5-bis-(4-chlorophenyl)-4,5-dimethyl-4,5-dihydroimidazole-1-carbonyl]-piperazin-1-yl}-N,N-bis-(2-methoxyethyl)-acetamide. 2-{1-[(48;5K)-2-(6-tert-butyl-4-ethoxypyridin-3-yl)-4,5-bis-(4-chlorophenyl)-4,5-dimethyl-4,5-dihydroimidazole-1-carbonyl]-piperidin-4-yl}-acetamide and others described by the general structural formula (I), and pharmaceutical composition containing said compounds.

EFFECT: compounds can be used as anti-cancer agents, particularly as agents for treating solid tumours.

8 cl, 217 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a compound having chemical formula or a salt thereof, where: Ar is an optionally substituted heteroaryl; R1 in each case is independently selected from a group which includes halogen, lower alkyl, optionally substituted with one or more substitutes selected from fluorine, lower alkoxy, fluorine-substituted lower alkoxy, monoalkylamino, dialkylamino, -O-R5, -N(R5)-R6 and -N(R5)-C(X)-R7; m equals 0 or 1; n equals 0, 1 or 2; R2 is hydrogen or a halogen; L2 is -S(O)2-; R3 is a lower alkyl, optionally substituted with fluorine, C3-6 cycloalkyl, optionally substituted with a lower alkyl, a 5- or 6-member nitrogen-containing heterocycloalkyl, optionally substituted with one or more substitutes selected from fluorine, lower alkyl, fluorine-substituted lower alkyl, lower alkoxy, fluorine-substituted lower alkoxy, lower alkylthio or fluorine-substituted lower alkylthio, aryl, optionally substituted with a halogen, lower alkyl, optionally substituted with a halogen or lower alkoxy, optionally substituted with a halogen, or a heteroaryl, optionally substituted with a halogen or a lower alkyl; L1 is selected from a group which includes -O-, -C(R12R13)-X-, -X-C(R12R13)-, -C(R12R13)-N(R11)-, -(R11)-C(R12R13)-, -C(X)-N(R11)-, -N(R11)-C(X)-; X is O; R11 is hydrogen; R4 is hydrogen or a lower alkyl; R5 and R6 in each case are independently selected from a group which includes hydrogen, lower alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, where each is optionally substituted with one or more substitutes selected from fluorine, lower alkoxy, fluorine-substituted lower alkoxy, lower alkylthio, fluorine-substituted lower alkylthio, monoalkylamino, dialkylamino; R7 in each case is independently selected from a group which includes lower alkyl; where the terms "lower alkyl", "lower alkoxy", "lower alkylthio", "monoalkylamino", "dialkylamino", "cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", are as described in the claim. The invention also discloses a pharmaceutical composition for treating Raf kinase mediated diseases which is based on a compound of formula I; use of the compound of formula I to produce a medicinal agent is also disclosed.

EFFECT: novel compound which can be useful in treating diseases and conditions associated with aberrant activity of protein kinases is obtained and described.

9 cl, 13 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: what is presented is the use of paracetamol and ibuprofen in preparing a drug for treating moderate to severe rheumatoid arthritis or moderate or severe osteoarthritis, wherein the drug contains a combined composition containing 125 mg to 150 mg of ibuprofen and 475 mg to 500 mg of paracetamol with the above drug applicable for administering 250 mg to 300 mg of ibuprofen and 950 mg to 1000 mg of paracetamol (versions) and a related method of treating.

EFFECT: what is shown is a synergetic effect of paracetamol and ibuprofen in the declared combinations: analgesic (anti-inflammatory) action of the drug corresponds to a double dose of ibuprofen.

24 cl, 2 dwg

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