Synthesis of polymer conjugates of indolocarbazole compounds

FIELD: chemistry.

SUBSTANCE: invention relates to a method of obtaining a polymer conjugate of an indolocarbazole compound of formula (I), where R1, R2, R3, W1 and W2 represent hydrogen, X represents methoxy-polyethyleneglycol. The method includes the interaction of a polymer compound of formula (II) with an indolocarbazole compound of formula (III), where Y stands for a methoxygroup. The nvention also relates to a polymer conjugate of formula (I), a pharmaceutical composition, containing the conjugate of formula (I) as an active ingredient, and to the application of the polymer conjugate of formula (I).

EFFECT: obtaining the polymer conjugate of the formula with a high output, the polymer conjugate of the formula for treatment of skin pathologies and HMGB1-associated pathologies.

48 cl, 7 dwg, 7 tbl, 15 ex

 

The technical field to which the invention relates.

The present invention relates to a method for producing polymer conjugates indolocarbazole compounds, in particular, the polymer conjugates of K-a and their derivatives, by synthesis, which results in a product of high purity and high yield.

Additional plan present invention relates to new polymer conjugates of K-a and their derivatives, in which a chemical group linking the polymeric link connection To a or derived compounds To a, characterized by a 5-membered oxazolidinediones cyclic structure. These new polymer conjugates obtained by synthesis with high purity and high yield.

The level of technology

The literature describes therapeutic potential of K-a and its derivatives for the prevention, relief and treatment of pathologies associated with a kinase, in particular pathologies associated with HMGB1, such as neurological disorders, diseases of the nervous system and neurodegenerative disorders of the Central and peripheral nervous system (for example, from document PCT/ER/008258, Annual Rev. Pharmacol. Toxicol. 2004;44:451-74; Newochem. Int. 2001 Nov-Dec;39(5-6):459-68; Neuroport. 2000 Nov 9;11(16):3453-6; Neuroscience. 1998 Sep; 86(2):461-72; Brains Res. 1994 Jul 4;650(1): 170-4). In addition, in the prior art disclosed therapeutic efficacy of these indolocarbazole soy is ineni for warnings, the relief and treatment of skin diseases, especially skin diseases associated with excessive proliferation of keratinocytes, such as psoriasis (for example, from the document WO 2005/014003, Raychaudhuri, etc., J. Invest. Dermatol. 122:812-819, 2004). In addition, the prior art it is also known that K-a and its derivatives are used as an active tool against pain associated with NGF (see, for example, Koizumi and others, J. Neurosci. 8:715-721, 1988; Doherty and others, Neurosci. Lett. 96:1-6, 1989; Matsuda and others, Neurosci. Lett. 87:11-17, 1988, Winston J. H. and others J. Pain (2003) 4:329-337). Therefore, the biological significance and therapeutic activity indolocarbazoles connection To a and its derivatives are well covered in the literature (see, for example, Kim and others, Biol. Pharm. Bull. 21:498-505, 1998, Schneider and others, Org. Lett. 7:1695-1698, 2005).

Polymer conjugates of K-a and their derivatives, and their use as active agents in pharmaceutical compositions used for preventing, alleviating and curing the above pathologies are disclosed in the application WO 2007/022999. The description of the specified application included in the present invention as a reference. According to the document WO 2007/022999 the purpose of conjugation to the polymer, and in particular tahilramani (accession Page) active indolocarbazole derivatives connection To a is the development of such forms for such active compounds that provide improved pharmacokinetic and that is elegicheskie characteristics, in order to achieve better bioavailability To-a or its derivatives in various possible ways of application.

Described in document WO 2007/022999 synthetic approach for the production of polymer conjugates of K-a and its derivatives include covalent joining of polymer fragment to indolocarbazoles link structure To a or its derivatives. In particular, in document WO 2007/022999 describes the interaction of activated isocyanate polymer with a hydroxyl group in position tertrahydrofuran ring Sz fragment in a or its derivative in a suitable reaction conditions, the result is a urea bond in the form covalent connection between the polymer fragment and active connection.

Since the polymer conjugates of K-a and their derivatives having a high purity, highly demanded for use in medicine, the aim of the present invention is to develop a method of obtaining a polymer conjugates active indolocarbazole compounds, which leads to the reaction product of high purity obtained with high appropriate output. Moreover, an additional aim of the invention is the elimination of complex stages of purification and ensuring ease of cleaning and extraction of target polymer conjugated compounds in order to bring to Maxi the mind's reaction efficiency of polymer conjugation.

Unexpectedly, the inventors have found that the interaction-a or its derived compounds with ω-1-H-imidazol-carboxamide polymer fragment, was used as the source of polymeric reagent reactions of conjugation, provides a controlled process of conjugation, thus, indolocarbazole-polymer conjugate is obtained with the desired high yield and high purity.

Disclosure of inventions

Therefore, the present invention relates to a method for producing a polymer conjugate indolocarbazoles the compounds of formula (I)

where

R1and R2are the same or different residues, each independently selected from the group consisting of:

(a) hydrogen, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower quinil, hydroxy-, lower alkoxy-, carboxy group, lower alkoxycarbonyl, acyl, nitro, carbamoyl, lower alkylaminocarbonyl, the group-NR5R6in which each R5and R6independently selected from hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower quinil, substituted or unsubstituted aryl, Zam is on or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted lower alkylaminocarbonyl, substituted or unsubstituted lower arylenecarborane, alkoxycarbonyl, carbamoyl, acyl, or R5and R6in combination with the nitrogen atom form a heterocyclic group,

(b) -CO(CH2)jR4where j denotes 1-6 and R4selected from the group consisting of:

(i) hydrogen, halogen, -N3,

(ii) group-NR5R6where R5and R6are the same as above,

(iii) group-SR7where R7selected from the group consisting of hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower quinil, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, the group -(CH2)aCO2R10(in which a represents 1 or 2 and where R10selected from the group consisting of hydrogen and substituted or unsubstituted lower alkyl) and -(CH2)aCO2NR5R6,

(iv) -OR8, -OCOR8where R8selected from hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower quinil, substituted or asamese the aqueous aryl, substituted or unsubstituted heteroaryl

(c) -CH(OH)(CH2)jR4where j and R4are as described above;

(d) -(CH2)dCHR11R12or -(CH2)dCHR11CONR5R6where d is 0-5, R11is hydrogen, a group-CONR5R6or-CO2R13where R13means hydrogen or substituted or unsubstituted lower alkyl, and R12is hydrogen or substituted or unsubstituted lower alkyl;

(e) -(CH2)kR14where k is 2 to 6 and R14represents halogen, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COOR15, -OR15(where R15represents hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower quinil, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or acyl), -SR7(where R7is the same as above), -CONR5R6, -NR5R6(where R5and R6are such as defined above) or a group-N3;

(f) -CH=CH(CH2)mR16where m means 0-4, and R16represents hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted bottom of the s quinil, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COOR15, -OR15(where R15is the same as above) -CONR5R6or-NR5R6(where R5and R6are as specified above);

(g) -CH=C(CO2R12), where R12is the same as described above;

(h) -C=C(CH2)nR16where n means 0-4 and R16is the same as described above;

(i)- (CH2OR22where R22represents three(lower)alkylsilane, in which three groups of lower alkyl are the same or different, or in which R22has the same meaning as R8.

(j) -CH(SR23)2and-CH2-SR7where R23represents lower alkyl, lower alkenyl or lower quinil and where R7is the same as above; and R3means hydrogen, halogen, acyl, carbarnoyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted of alkenyl, substituted or unsubstituted lower quinil or amino group; and

W1and W2independently mean hydrogen, hydroxy group, or W1and W2together represent oxygen;

and where X is a polymer fragment

which includes the interaction of ω-1H-imidazol-carboxamide polymeric compounds of General formula (II)

in which X has the above meaning,

with indolocarbazole compound of General formula (III)

where the substituents R1, R2, R3, W1and W2have the above values and are not protected by a protective group, and where Y means tsepliaeva group and, in addition, necessarily involves the removal of the protective groups of the optionally protected R1, R2, R3, W1and W2in order to obtain the compound of formula (I).

The conjugation reaction in the method according to the invention, in order to synthesize the polymer conjugate compounds of formula (I) is catalyzed by a base in an organic solvent. Preferably the specified base is a strong base. In a preferred embodiment of the present invention, the base is chosen from the group of hydrides of alkali metals, tertiary amines and/or alkoxides. In a very preferred embodiment of the present invention the base catalyzing the process of polymer conjugation according to the invention, is a sodium hydride. However, it can also be used other bases, such as sodium methoxide or triethylamine.

Preferably the molar ratio of the basic catalyst to the compound of formula (III) approximately the away between 1:1 and 4:1, most preferably from about 1:1 to 1.5:1 and most preferably about 1:1.

In addition, the reaction according to the invention is carried out in an organic solvent, preferably anhydrous conditions, i.e. in drained organic solvent. Preferably, the water content in the solvent mixture of the process of conjugation is equal to or less than 200 parts per million (ppm). The organic solvent can be selected from the group of dichloromethane, chloroform, N,N-dimethylformamide. In a very preferred embodiment of the present invention the organic solvent is dichloromethane, even more preferably anhydrous dichloromethane.

In addition, according to the invention it is preferable that the conjugation reaction was carried out in an atmosphere of inert gas such as nitrogen or argon.

Moreover, the reaction according to the invention preferably is carried out at a temperature of approximately -10° to approximately 60°C., more preferably from about 0° to 25°C and most preferably at room temperature after the initial phase at 0°C.

After receiving the target compounds of formula (I) according to the method according to the invention the polymer conjugate of formula (I) can then be isolated from the reaction mixture and purified. According to the preferred is arianto the invention, the compound of formula (I) is obtained by purification of the crude mixture using flash chromatography. Preferably incorporates an automated gradient flash purification, which is supplied to the appropriate column and a solvent. Preferred cleaning method selected from the columns with reversed-phase and linear phase, and the solvent conditioning/elution preferably selected from dichloromethane, water, methanol, acetonitrile, buffer solution of ammonium formate at different proportions in the mixture. In a very preferred embodiment of the invention indolocarbazole-polymeric compound of the formula (I) is purified using flash chromatography with reversed phase using the cartridge 18, and the cleaning is performed under conditions isocratic elution with a mixture of 40:60 acetonitrile/buffer solution of ammonium formate (5 mmol/l, pH 3.5) (as shown in example 3). In addition, the preferred embodiment of the invention indolocarbazole-polymeric compound of the formula (I) is purified using flash chromatography with normal phase (as described in examples 4 and 5.3).

Then the product can be dried, for example over sodium sulfate and filtered, and the solvent is removed by evaporation under reduced pressure, at 25°C. Purification of the target product is carried out using conventional techniques known to the expert in this field of technology.

After the stage of purification of p is obtained polymeric compound of the formula (I) has a purity at least about 95%. More preferably, after the stage of purification of the compound of formula (I) has a purity of at least about 98%. In an even more preferred embodiment, the resulting polymer compound has a purity of 98.5%, 99% or even 99.5%pure.

Moreover, the method of the present invention leads to the exit of the total mass of the compounds of formula (I) from about 40% to 98% by weight, preferably from about 50% to 95% by weight, based on the weight of the reacting compounds of the formula (III).

The rest of Y of formula (III) is tsepliaeva group, i.e. a group which under the reaction conditions and polymer conjugation of the present invention, is removed from the structure of the compounds of formula (III) in order to get oxazolidinediones cycle of compounds of formula (I), which covalently links the polymer fragment with indolocarbazoles connection structure To a or its derivative. According to the invention imidazole ring compounds of General formula (II) is also removed during the reaction of conjugation from a slice of polymeric reagent to obtain the compound of formula (I).

In a preferred embodiment of the present invention tsepliaeva group Y of the formula (III) is selected from the group consisting of triftoratsetata (triplet), tosilata, nelfinavir, sulfate, halogen, hydrox is - or low alkoxygroup. In a particularly preferred embodiment, tsepliaeva group Y of the formula (III) represents a lower alkoxygroup or hydroxy-group. Most preferably, tsepliaeva group Y is lower alkoxygroup, in particular the methoxy group.

Polymer segment covalently attached to indolocarbazoles connection according to the method of the present invention and which, for example, represented in the General formulas (I) and (II) the group X must be biologically compatible, can be of natural or semi-synthetic, or synthetic origin, and may have a linear or branched structure. Preferably, in the present invention, the polymer X is chosen from poly(alkalisation), particularly (polyethylene)oxides. However, additional examples of polymers include, without limitation, polyacrylic acid, polyacrylate, polyacrylamide or its N-alkyl derivatives, polymethacrylic acid, polymethacrylates, polyethylacrilate acid, policyactivity, polyvinylpyrrolidone, poly(vinyl alcohol), polyglycolic acid, poly(lactic) acid, poly(lactic-coglycolide) acid, dextran, chitosan, polyaminoamide, hydroxyethyl-starch.

For the purpose of providing input into the method of the present invention, in particular with the aim of functionalization of the obtained polymer forms the crystals (II) in the method according to the invention the above polymer fragment must contain terminal functional amino group or must be functionalized for to contain terminal functional amino group. Therefore, the polymer segment should be amino-activated polymer of the General formula X-NH2.

In fact, the original polymeric reagent of the formula (II) is obtained by reacting the amino group of the polymer fragment with 1,1-carbonyldiimidazole connection in order to obtain ω-1H-imidazol-carboxamide polymeric compound of General formula (II).

Preferably obtain ω-1H-imidazol-carboxamide polymeric compounds of the formula (II) is carried out in an organic solvent, such as dichloromethane, chloroform, N,N-dimethylformamide. In a highly preferred embodiment, the organic solvent is dichloromethane, even more preferably anhydrous dichloromethane.

In addition, according to the invention it is preferable that the activation of the ω aminopolymers conducted in an atmosphere of inert gas such as nitrogen or argon.

Moreover, it is preferable that the interaction with the formation of ω-1H-imidazol-carboxamide polymer compound of the present invention was carried out at a temperature from about 10° to 60°C, more preferably from about 15° to 25°C and most preferably at room temperature.

In a very preferred embodiment of the present invention is alimamy fragment X is polietilenglikolya (PEG) block, in which the terminal Oh-group may not necessarily be modified, for example, C1-C6-alkyl or C1-C5-acyl groups, preferably C1-With2- or3-alkyl groups or With1-With2- or3groups. Preferably, the modified polyethylene glycol is a terminal alkoxy-substituted polyethylene glycol, more preferably a methoxy-polyethylene glycol (MPEG).

According to the present invention is used, the polymer has a molecular weight in the range from approximately 100 to 100,000 units Dalton (Da), preferably from about 200 to 50,000 and more preferably from about 500 to 10,000 Da. In accordance with one preferred design of the invention the polymer is a poly(ethylene glycol) with a short chain, preferably a terminal alkoxy-substituted PEG, such as methoxy-substituted poly(ethylene glycol) with molecular weight in the range from approximately 200 to 1500 Da, preferably from about 400 to 1200 and even more preferably from about 550 to 1100. In the most preferred embodiment, the PEG with a short chain or MPEG has an average molecular weight of approximately 550 Yes or approximately 1100 Yes. In accordance with the second preferred semiclassically polymer is a long chain poly(ethylene glycol), preferably the terminal alkoxy-substituted PEG, such as methoxy-substituted poly(ethylene glycol)with molecular weight in the range from approximately 4,000 to 6,000 and preferably from about 4500 to 5500 Yes. In the most preferred embodiment of this idea of the invention, the PEG with a long chain or MPEG has an average molecular weight of about 2000 Da, or about 5000 Da.

Used above, the term "approximately" when referring to values and ranges of molecular weight portion of the polymer of the invention means that the above values and/or ranges can vary in the range ±20%, preferably in the range of ±10%.

Except other briefly mentioned cases, each of the following terms used in the present invention, will be described below. The term "lower alkyl" for individual use or in combination with other groups, means a lower alkyl group with straight chain or branched, containing from 1 to 6 carbon atoms, preferably from 1 to 5, more preferably from 1 to 4 and particularly preferably 1-3 or 1-2 carbon atoms. These groups include, in particular, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, amyl, isoamyl, neopentyl, 1-ethylpropyl, hexyl, and things under the Noah. A fragment of the lower alkyl in "lower alkoxy", "lower alkoxycarbonyl", "lower alkylaminocarbonyl", "lower hydroxyalkyl" and "the three(lower)alkylsilanes" groups has the same meaning indicated above for "lower alkyl".

Group "inferior alkenyl" is defined as C2-C6-alkeline group which may have a straight or branched chain and may be in Z - or E-form. Such groups include vinyl, propenyl, 1-butenyl, Isobutanol, 2-butenyl, 1-pentenyl, (2)-2-pentenal, (E)-2-pentenyl, (7)-4-methyl-2-pentenal, (E)-4-methyl-2-pentenyl, pentadienyl, such as 1,3 - or 2,4-pentadienyl, and the like. More preferred With1-C6-alkenylamine groups are2-C5-With2-C4-alkeline group, and even more preferred With2-C3-alkeline group.

The term "lower Alchemilla" group refers to C2-C6-alkynylaryl groups, which may be straight or branched chain and include ethinyl, PROPYNYL, 1-butynyl, 2-butynyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl and the like. More preferred With3-C6-alkenylamine groups are2-C5-With2-C4-alkyline group, and more preferred From2-C3-alkyline group.

T is pmin "aryl" group is a C 6-C14-aryl groups containing from 6 up to 14 ring carbon atoms. These groups can be mono-, bi - or tricyclic and represent a condensed ring. Preferred aryl groups include phenyl, biphenyl, naphthyl, anthracene, phenanthrene and the like. Aryl fragment "arylcarboxylic" and "allamericanheroes" groups have the same meanings as above.

The term "heteroaryl" refers to a group that may contain 1 to 3 heteroatoms independently selected from nitrogen, sulfur or oxygen, and is a C3-C13-heteroaryl groups. These groups can be mono-, bi - or tricyclic. Heteroaryl group3-C13the present invention includes heteroaromatic and saturated and partially saturated heterocyclic group. These heterocyclic groups may be monocyclic, bicyclic, tricyclic. Preferred 5-or 6-membered heterocyclic groups are thienyl, furyl, pyrrolyl, pyridyl, pyranyl, morpholinyl, pyrazinyl, methylpyrrole and pyridazinyl. Group C3-C13-heteroaryl may be a bicyclic heterocyclic group. Preferred bicyclic heterocyclic groups are benzofuran, benzothiazyl, indolyl, imide is alil and pyrimidinyl. The most preferred3-C13-heteroaryl groups are furyl and pyridyl.

The term "lower alkoxy" includes alkoxygroup containing from 1 to 6 carbon atoms, preferably from 1 to 5, more preferably from 1 to 4 and particularly preferably from 1 to 3, or 1-2 carbon atoms, and groups can be straight or branched chain. These groups include methoxy, ethoxy-, propoxy-, butoxy, isopropoxy-, tert-butoxy, pentox, hexose and the like.

The term "acyl" includes lower alkanoyl, which contains from 1 to 6 carbon atoms, preferably from 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms and may be straight or branched chain. Preferably these groups include formyl, acetyl, propionyl, butyryl, isobutyryl, tertiary, butyryl, pentanoyl and hexanoyl. Acyl fragment "acyloxy-"group has the same meanings as above.

The term "halogen" includes fluorine, chlorine, bromine, iodine and the like.

The term "kalkilya" group is a C7-C15-aralkyl, in which an alkyl group substituted by aryl. The above alkyl group and aryl may be selected from C1-C6alkyl groups and C6-C14-aryl groups defined above, in which the number of carbon atoms is between 7 and 15. Preferred C7-C15-Uralkalij groups are benzyl, phenylethyl, phenylpropyl, phenylisopropyl, phenylbutyl, diphenylmethyl, 1,1-diphenylether, 1,2-diphenylether. Uralkaliy fragment "Arakelov-" group has the same meanings as above.

Substituted lower alkyl, alkeline and alkyline groups have from 1 to 3 substituents independently selected from such as lower alkyl, hydroxy-, lower alkoxy-, carboxy, lower alkoxycarbonyl, nitro, halogen, amino, mono - or di-(lower)alkylamino, dioxolane, dioxane, ditiolan, dition. Substituting the fragment of the lower alkyl substituted in the lower alkyl, alkenylphenol and alkenylphenol groups and a fragment of a lower alkyl lower alkoxy group, lower alkoxycarbonyl and Vice mono - or di-lower alkylamino group in the substituted lower alkyl, alkenylphenol and alkenylphenol groups have the same meanings as above for "lower alkyl".

Each substituted aryl, substituted heteroaryl and substituted kalkilya group has from 1 to 3 independently selected substituents, such as lower alkyl, hydroxy-, lower alkoxy-, carboxy-, low alkoxycarbonyl, nitro-, amino-, mono - or di-(lower)alkylamino-, and halogen. Among the substituents of the lower alkyl fragment of lower alkyl, lower alkoxy, lower alkoxycarbonyl the La, and mono - or di-(lower) alkylamino-group have the same meanings as lower alkyl as indicated above.

Heterocyclic group which is formed by the radicals R5and R6in combination with the nitrogen atom, comprises pyrrolidinyl, piperidinyl, piperidine, morpholine, morpholine, thiomorpholine, N-methylpiperazine, indolyl and isoindolyl.

Preferably, R1and R2independently selected from the group consisting of hydrogen, halogen, nitro, -CH2HE, -(CH2)kR14, -CH=CH(CH2)n,R16, -C=C(CH2)jR15, -CO(CH2)jR4where R4represents-SR7CH2O-(substituted or unsubstituted) lower alkyl (where the substituted lower alkyl preferably denotes methoxymethyl, methoxyethyl or ethoxymethyl), -NR5R6.

In the above preferred values of R1and R2the remainder R14preferably selected from the group consisting of phenyl, pyridyl, imidazolyl, thiazolyl, tetrazolyl, -COOR15, -OR15(where R15preferably selected from hydrogen, methyl, ethyl, phenyl or acyl), -SR7(where R7preferably selected from substituted or unsubstituted lower alkyl, 2-thiazoline and pyridil) and-NR5R6(where R5and R6preferably selected from hydrogen, methyl, ethyl, Hairdryer is La, carbamoyl and lower alkylaminocarbonyl). Moreover, the remainder R16preferably selected from hydrogen, methyl, ethyl, phenyl, imidazole, thiazole, tetrazole, -COOR15, -OR15and-NR5R6(where the remains of R15, R5and R6have the preferred meanings indicated above). In the above preferred values of R1and R2the remainder R7preferably selected from the group consisting of substituted or unsubstituted lower alkyl, substituted or unsubstituted phenyl, pyridyl, pyrimidinyl, thiazole and tetrazole. In addition, k preferably represents 2, 3 or 4, j is preferably 1 or 2 and t, and preferably n is independently 0 or 1.

Preferably R represents hydrogen or acetyl, most preferably hydrogen. In addition, each W1and W2preferably is hydrogen.

The preferred implementation of the present invention relates to connection-a, anywherefrom polymer fragment. Even more preferred variant implementation relates to polymeric conjugates of K-a and their derivatives, where the chemical group linking the polymeric link connection To a or derived compounds To a, characterized by a 5-membered oxazolidinediones cyclic structure. Therefore, in in the CIMA preferred embodiment of the present invention the polymer conjugate of formula (I) is represented by the connection, in which R1, R2, R3, W1and W2represent hydrogen and X is a polymer fragment. According to this very preferred embodiment of the invention the polymer fragment is a fragment of polyethylene glycol (PEG) or a methoxy-polyethylene glycol (MPEG). Even more preferably, the polyethylene glycol or methoxy-polyethylene glycol is the preferred option of the invention is a polymer PEG or MPEG long chain with an average molecular weight of about 2000 Da, or about 5000 Da. Similarly preferred is polyethylene glycol or methoxy-polyethylene glycol with a short chain with an average molecular weight of approximately 550 Yes or approximately 1100 Yes.

The method of the present invention includes an optional stage protective group of one, several or all of the substituents R1, R2, R3, W1and W2indolocarbazoles connection To a or derived compounds. In this context, the term "protective group" refers to any derivative of the substituents R1, R2, R3, W1and W2known from the prior art, which can be used if necessary to mask R1, R2, R3, W1The W 2during the synthesis process, and which can then be removed under conditions that lead to reverse the receipt of the substituents R1, R2, R3, W1and W2without unwanted transformations in the rest of the molecule. In particular, if necessary, the protective group is introduced in one, several or all of the substituents R1, R2, R3, W1and W2during the process of conjugation of the present invention to implement a chemically selective conjugation of the polymer in position C3 indolocarbazoles patterns To a or derived compounds. Upon completion of the conjugation reaction, one or more protective groups can be reversibly removed in order to regain the original functional groups of substituents R1, R2, R3, W1and W2obtaining indolocarbazoles conjugated compounds of the formula (I).

According to the present invention, this purpose may be used any suitable protective group known from the prior art. The selection of a suitable protective group, and any suitable means and conditions for introduction and removal of protecting the substituents R1, R2, R3, W1and W2can be carried out by a specialist in this field of technology on the basis of General knowledge in the field of organic synthesis is. The used means and conditions for the introduction and removal of the protection depends on the nature of the available functional groups of R1, R2, R3, W1and W2. Protective groups for the hydroxy-, amino-and/or carboxy residues preferably selected from the acetonide, ethylidene-methoxymethyl, 2-methoxyethoxymethyl, benzoyloxymethyl, tetrahydropyranyl, methyl, ethyl, isopropyl, tert-butyl, benzyl, triphenylmethyl, tert-butyldimethylsilyl, triphenylsilane, methoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, acetyl, benzoyl, toluensulfonyl, dimethoxybenzyl, nitrophenylacetylene, nitrobenzisoxazole, allyl, fluorenylmethyl, tetrahydrofuranyl, Fenella, acetol, phenyl, trimethylsilyl, pyrrolidyl, indolyl, hydrazine and other protective groups which are known in the prior art, for example, those that can be found in the book by Greene, T. W. and other "Protective groups in organic synthesis", 4th ed., John Wiley and Son, New York, NY (2007). In particular, the reagents and conditions for reaction of the introduction and removal of protection chosen for their suitability selective when attaching and removing the protective group without undesirable effects on the rest of the connection part. Usually suitable conditions and reagents are known from practice specialist in this field of technology.

Under this and the finding of the compounds of formula (I) can also be obtained in the form of pharmaceutically acceptable salts, including salts of inorganic acids such as hydrochloric, uudistoodetena, Hydrobromic, phosphoric, metaphosphoric, nitric acid and sulfuric acid, and salts of organic acids such as tartaric, acetic, citric, lactic, benzoic, glycolic, gluconic, succinic, arylsulfonyl (for example, p-toluensulfonate, benzolsulfonat acid), phosphoric acid, malonic acid and the like. Suitable acids for the formation of pharmaceutically acceptable salts are well-known specialists in this field of technology. In addition, pharmaceutically acceptable salts of compounds of formula (I) may be formed with pharmaceutically acceptable cation. Pharmaceutically acceptable cations are known to specialists in this field of technology and include alkali cations (Li+, Na+, K+), alkaline earth cations (Mg2+CA2+BA2+), ammonium and organic cations such as the cations of Quaternary ammonium.

An additional aspect of the present invention are new polymer conjugates of K-a and their derivatives, in which a chemical group linking the polymeric link connection To a or derived compounds To a, characterized by a 5-membered oxazolidinediones cyclic structure. These polymer conjugates obtained using the new synthetic is on the way disclosed in the invention.

Therefore, in particular, an additional aspect of the present invention is a polymer conjugate indolocarbazoles the compounds of formula (I)

in which R1, R2, R3, W1and W2and polymer fragment X are as described above, or their pharmaceutically acceptable salts.

In the most preferred embodiment, the invention relates to a new polymer conjugate compounds of formula (I)in which R1, R2, R3, W1and W2represent hydrogen and the polymer fragment X is a polyethylene glycol (PEG) or a terminal alkoxy-substituted PEG, for example, preferably methoxy-polyethylene glycol (m-PEG). The specified connection corresponds polymer anywherefrom connection To a according to the present invention. Preferably the polymer fragment is a polyethylene glycol with a long chain, more preferably the terminal alkoxy-substituted PEG, such as methoxy-polyethylene glycol (m-PEG)with average molecular weight of about 2000 Da, or about 5000 Da. Similarly, preferably, when the polymer fragment is a polyethylene glycol with a short chain, even more preferably t is Renaldo alkoxy-substituted PEG, such as methoxy-polyethylene glycol (m-PEG)with average molecular weight of approximately 550 Yes or approximately 1100 Yes.

Unexpectedly, the authors of the present invention found that, compared with representatives indolocarbazole compounds and in particular as compared to K-a or its derivatives that do not contain a polymer corresponding to the polymer conjugated to the compounds of formula (I) have improved pharmacokinetic and Toxicological characteristics, due to their high solubility, which leads to improved biological availability of these therapeutically and biologically active compounds. In another aspect of the present invention it has been unexpectedly discovered that conjugated polymer indolocarbazole the compounds of formula (I) have limited systemic absorption of the local application due to their larger molecules and amphipathicity, and thus, increasing their local therapeutic and biological efficacy and reduced systemic toxicity and/or side effects due to the local application.

In addition, unexpectedly, the authors of the present invention found that indolocarbazole-polymer conjugates of the formula (I) demonstrate a significant increase in selectivity for inhibiting activity of the faithful is in the tyrosine kinase TrkA compared to non-selective inhibitory kinase activity for themselves indolocarbazole compounds and in particular for K-a and its derivatives, containing no polymer. Thus, the conjugation indolocarbazole compounds, and in particular To-a, to the polymer molecule according to the invention leads to the production of active agents that are selective against therapeutic targets consistent with the reduction of unwanted side effects.

Therefore, an additional aspect of the present invention is the use of compounds of formula (I) as active agents in medicinal products. In a preferred design of the invention the compounds of formula (I) are used as active agents in medicinal remedy for systemic use and treatment. In such preferred the idea of the present invention relates to the use of compounds of formula (I) as active agents in local medicines.

In particular, the conjugated polymer compound of the present invention are used as active agents in medicinal product, suitable for the prevention, relief and treatment of pathologies associated with HMGB1. Pathology associated with HMGB1 represents the condition of the patient, which in biological fluids and tissues is increased, the concentration of the nuclear protein HMGB1 and/or HMGB1-homologous proteins in acetylated or deacetylating form than the s concentration in normal objects, where these nuclear protein HMGB1 is almost impossible to detect. Extracellular HMGB1 acts as a strong chemotactic Pro-inflammatory chemokines. Therefore, the pathology associated with HMGB1, represent a pathology with a strong inflammatory basis, i.e. pathologies that arise from the stimulation of cytokine, such as TNF-alpha, IL-1, IL-6 and others, or pathologies that arise from toxic events, such as intoxication, infection, burn, etc. In particular, high concentrations of HMGB1 proteins and homologous proteins were found in plasma of patients with common blood, plasma and synovial fluid of patients with rheumatoid arthritis, the brain of patients with Alzheimer's disease, plasma and tissues of patients with melanoma, in the plasma of patients with systemic lupus erythematosus, atherosclerosis platelets of patients with atherosclerosis and other identification and proof of HMGB1 protein and/or homologous proteins in biological fluids and tissues can be obtained by conventional means of diagnosis, which are known to experts in this field of technology, for example, including enzyme-linked immunosorbent assay in solid - phase ELISA and other

Therefore, many diseases are characterized by a relevant presence of extracellular HMGB1, which, in particular, include (but not limiting who are) inflammatory diseases, stenosis, restenosis, atherosclerosis, rheumatoid arthritis, autoimmune diseases, tumors, infectious diseases, blood poisoning, acute inflammatory lung injury, systemic lupus erythematosus, neurodegenerative diseases, diseases of the Central and peripheral nervous system and multiple sclerosis. In a particularly preferred embodiment, conjugated polymer compound of formula (I) are used for prevention, relief and treatment of cardiovascular diseases, particularly atherosclerosis and/or restenosis that occurs during or after reconstruction of vessels. More preferably, the drug is used for blocking, inhibiting, and/or weakening of the regeneration of connective tissue in the restenosis during or after reconstruction of vessels.

In a particularly preferred aspect of the invention conjugated polymer compound of formula (I) are effective when used as an active agent in medicine for the prevention, relief and treatment of neurological disorders, diseases of the nervous system and neurodegenerative disorders of the Central and peripheral nervous system.

In addition, the inventors have demonstrated that the new polymer conjugate compounds are able to reduce and/is to inhibit the secretion of plasma cytokine under the action of systemic therapy. Therefore, the polymer conjugate compounds are used as active agents in medicinal remedy for systemic injections, which are suitable for preventing, alleviating and/or treating pathologies involving increased secretion of plasma cytokine. These pathology preferably represent a pathology, which mainly involved the secretion of TNF-α, IFN-γ, MCP-1, MIP-1 and/or RANTES.

In particular, in the context of the present invention pathologies that are associated with increased secretion of plasma cytokines include, but are not limited to inflammatory diseases, autoimmune diseases, systemic syndrome inflammatory response, reperfusion injury after organ transplantation, cardiovascular lesions, obstetric and gynecological complications, infectious diseases, allergic and atopic diseases, pathology of solid and liquid tumors, diseases, transplant rejection, congenital diseases, dermatological diseases, neurological diseases, General atrophy, renal disease, condition iatrogenic intoxication, metabolic and idiopathic diseases and ophthalmological diseases.

In the most preferred embodiment, compounds of the present invention is applied is I as active agents in medicinal remedy for systemic therapy, suitable for preventing, alleviating and/or treating disease behceta, Sjogren syndrome, vasculitis, uveitis, retinopathy.

In other particular ideas of the invention is preferred in order conjugated polymer compound of the present invention was used as active agents in local medicines are suitable for the prevention, relief and/or treatment of skin diseases. The authors of the present invention demonstrated that described in the invention conjugated polymer compounds are advantageously used as local medicines, since the introduction through the skin for them are not harmful or toxic effects (e.g. irritation) or any phototoxic effects (for example, photomutagenicity, phototoxicity or photosensitization) (as shown by studies described in the subsequent examples).

Skin pathology, preferred in the context of the present invention, represent a pathology characterized by hyperproliferative keratinocytes, such as psoriasis, atopic dermatitis, chronic eczema, acne, red hairy pytilias, keloids, hypertrophic scars and skin tumors, such as keratoakantoma, squamous cell carcinoma, basal cell cancer. In a more preferred VA is iante implementation of the compounds of the present invention are used as active agents in local medicines suitable for preventing, alleviating and curing psoriasis.

Thanks to the high selectivity of the compounds of the invention in the inhibition of TrkA additional purpose of the invention is the use of these conjugated compounds for the prevention, relief and treatment of pathologies in which TrkA plays a crucial role in the pathophysiological mechanism that causes the development of these pathologies. In this regard, in a highly preferred variant of the invention, the conjugated polymer compound K-a formula (I), (II) and/or (III) used as the active agent in medicine for the prevention, relief and treatment of pain and increased pain sensitivity associated with NGF.

Therefore, an additional aspect of the present invention is the use of compounds of formula (I)defined above, optionally together with pharmaceutically acceptable carriers, adjuvants, diluents and/or additives for the manufacture of a medicinal product for preventing, alleviating and/or treating the pathologies indicated above.

The compounds of formula (I) or their pharmaceutically acceptable salts can be administered as such or in the form of various pharmaceutical compositions according to the pharmacological activity and destination. Another aspect of us is Vashego of the invention is a pharmaceutical composition, which contains an effective amount of at least one of the compounds of formula (I), optionally together with pharmaceutically acceptable carriers, adjuvants, diluents and/or additives. Pharmaceutical carriers, adjuvants, diluents and/or additives known to specialists in this field of technology and therefore can be used in the formulation of pharmaceutical compositions containing the compound of the present invention.

Compounds of the present invention can be used as the sole active agent in the pharmaceutical composition. Alternatively, the compounds of formula (I) can be used in combination with one or more active agents, such as other active pharmaceutical agents in the treatment of the above disorders.

In particular, the polymer conjugate compounds of the invention can be used in combination with at least one steroidal anti-inflammatory drug and/or one additional agent which is able to inhibit early mediator of the inflammatory cytokine cascade, such as an antagonist or inhibitor of a cytokine, which is selected from the group consisting of TNF, IL-1α, IL-1β, IL-Rα, IL-8, MIP-1α, MIF-1β, MIP-2, MIF and IL-6. Particularly suitable anti-inflammatory drugs are selected from the deep is opinaca of clobetasone, amcinonide, beclomethasone dipropionate, betamethasone benzoate betamethasone dipropionate betamethasone betamethasone sodium phosphate, betamethasone phosphate and sodium acetate, betamethasone valerate, clobetasol butyrate, clobetasol propionate, pialat of clocortolone, cortisol (hydrocortisone), acetate cortisol (hydrocortisone), butyrate cortisol (hydrocortisone), cortisol (hydrocortisone) cypionate, cortisol (hydrocortisone) phosphate sodium, cortisol (hydrocortisone) succinate sodium valerate cortisol (hydrocortisone, cortisone acetate, desonide, desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diacetate diflorasone, valerate of diflucortolone, acetate fludrocortisone, fludroxycortide, pialat flumetazon, flunisolide, acetonide fluoqinolona, fluocinonide, fluocortolone, formation, flurandrenolide, fluticasone propionate, halcinonide, propionate halobetasol, Madison, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, mometazon furoate, acetate of paramethasone, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetate,triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide. Suitable antagonists or inhibitors of cytokine selected from infliximab, etanercept or adalimumab.

Additional agents that may be used in combination with polymeric compounds of the invention are, for example, antagonists and/or inhibitors of RAGE, antagonists and/or inhibitors of HMGB1 antagonists and/or inhibitors of the interaction of the receptor, such Toll (TCR), HMGB1, a functional N-terminal domain similar to lectins (D1) of thrombomodulin and/or synthetic Dunaeva nucleic acid or molecule is similar nucleic acid with curved structure, as described in the application for international patent WO 2006/002971, which is included in the present invention as a reference.

The pharmaceutical composition of the present invention can be introduced conveniently, as is known to experts in this field of technology, such as therapists. In particular, the pharmaceutical composition of the invention may be introduced by injection or infusion, in particular by intravenous, intramuscular, subcutaneous or intraperitoneal injection or infusion, or through the mucous membrane, and/or by inhalation, oral, local, cutaneous, nasal, aerosol, and/or rectal, and other Composition can be injected locally or systemically. Preferably, the introduction of compounds and pharmaceutical compositions of the invention can be carried out by parenteral destination, especially in the form of the LM is of such solutions or suspensions; or by oral destination, especially in the form of tablets or capsules, or through the nose, especially in the form of powders, nasal drops, or aerosols; or through the skin, for example, using ointments, creams, oils, liposomes or dermal patches.

According to one aspect of the invention the pharmaceutical composition is assigned systematically. In particular, the polymer conjugate compounds may be introduced by injection or infusion, in particular intravenously, intramuscularly, or through the mucous membrane, by subcutaneous or intraperitoneal injection or infusion, and/or by oral administration.

In an even more preferred embodiment, the pharmaceutical composition of the present invention is administered by local purposes, in particular by applying to the skin. In the case of applying to the skin the introduction of the compounds of the present invention can be implemented in the form of liposomes.

In addition, in the preferred embodiment of the invention the pharmaceutical composition is introduced in a reversible fixed condition on the surface of the medical device, in particular by bonding, coating and/or embedding compound and the composition of the invention on the medical device such as (but not limited to) artificial vessel, catheter, surgical instruments, cannulas, Klah is Ana heart or vascular prostheses. After contacting the medical device with body fluids or body tissues results in the release reversible fixed connection. Consequently, the coated medical device functions as a feeder of the drug, and the drug eluted, and thus, can be controlled kinetics receipt of a medicinal product, providing, for example, immediate release or adjustable, delayed or long-term supply of a drug. Coating technology medical devices are well known to specialists in this field of technology.

The pharmaceutical composition of the present invention can be used for diagnostic or therapeutic applications. For diagnostic applications, the compound of formula (I) may be present as a labeled compound, for example, in the form containing the isotope, such as a radioactive isotope or an isotope, which can be detected by the method of nuclear magnetic resonance. In the case of local application the preferred therapeutic use is the prevention, relief and treatment of psoriasis and dermatitis, while in the case of a system application is the prevention, relief and treatment of connective tissue regenerated when restenosis.

Conc is tion of the compounds of the present invention in the pharmaceutical composition may vary. The concentration will depend on such factors as the total dosage of the medicinal product to be injected, the chemical characteristics (e.g., hydrophobicity) of the compounds used, the method of taking the drug, age, body weight and symptoms of the patient. Typically, compounds of the present invention are provided in an aqueous physiological buffer solution containing about 0.1 to 10% (weight/volume) of the compound for parenteral administration. A typical dose range is from about 1 μg to 1 g/kg body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day and preferably from about 0.1 to 20 mg/kg, one to four times per day. Preferably, the dosage of the medications of the drug is likely to depend on such variables as the type and degree of development of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the selected compound and formulation of compounds with filler, and method of taking the drug.

An improved method of synthesis according to the present invention must also be explained using the following figures and examples, which however shall not limit the subject matter of the present invention.

A brief description of what their drawings

Figure 1 shows a preferred variant of the method according to the present invention. Indolocarbazole connection To a interacts with α-methoxy-ω-1H-imidazol-carboxamid-polyethylene glycol (MPEG-NH-CO-Im) to obtain methoxypolyethyleneglycol conjugate connection To a according to the invention. In this preferred compound of the invention methoxy-polyethylene glycol covalently attached to the active connection To a using a 5-membered oxazolidinediones a cyclic structure.

The figure 2 shows the1H-NMR spectrum of the activated polymer of MPEG-NH-CO-Im in the solvent DMSO-d6 at a magnetic field of 400 MHz.

The figure 3 shows the ESI-MS spectrum of MPEG-NH-CO-Im in the mass range 500-1400 m/z, using ionization elektrorazpredelenie with direct infusion ion trap.

The figure 4 shows1H-NMR spectrum of the polymer conjugate It-a figure 1 in the solvent DMSO-d6 at 400 MHz.

The figure 5 shows13C-NMR spectrum of the polymer conjugate It-a figure 1 in the solvent DMSO-d16 at 400 MHz.

The figure 6 shows the ESI-MS spectrum of the polymer conjugate It-a figure 1 in the mass range 500-1400 m/z using ionization elektrorazpredelenie with direct infusion ion trap.

The figure 7 shows the curve of inhibition of TrkA polymer conjugate It-a figure 1.

Done by the pressure inventions

Examples

Example 1. Synthesis of α-methoxy-ω-1H-imidazol-carboxamid-polyethylene glycol (MPEG-NH-CO-Im) (Im=imidazole)

In a round-bottom flask 500 ml dissolve 35,0 g MPEG-NH2(molecular weight 1892) (purity analysis 96%, 17,76 mmol) in 85 ml of dichloromethane in a nitrogen atmosphere. The solvent is removed under reduced pressure and the compound was dried using a mechanical pump for two hours. Then, the substrate is dissolved in 150 ml of dichloromethane in a nitrogen atmosphere and the solution transferred into a round-bottom three-neck flask with a volume of 2 liters

To the solution add 4,80 g of 1,1-carbonyldiimidazole (purity analysis 90%, 26,64 mmol) at room temperature. The mixture is stirred at room temperature under nitrogen atmosphere and analyzed by thin-layer chromatography (TLC, eluent CH2CL2/methanol (Meon) 90:10). The TLC plate is treated with a solution of ninhydrin to detect the presence of primary amino groups (purple staining).

This reaction is finished within two hours. The mixture is cooled at 0°C, and the solid product is deposited on a slow addition of diethyl ether (700 ml for 60 min) with vigorous stirring. The mixture is stirred for 30 minutes at 0°C., and optionally, add 300 ml of diethyl ether. The product was filtered through filter woro the ku with a disk of sintered glass, washed with diethyl ether (100 ml) and dried in vacuum. Get 34,0 g dry solids white (yield 94%).

This product is characterized by methods1H-NMR and ESI-MS.

1H-NMR (DMSO-d6) δ (M. D.): 8,24 (m, 1H, CH), 7,69 (m, 1H, CH), 7,27 (s, 1H, NH), 7,02 (s, 1H, CH), 3,55 (m, CH2PEG), 3,40 (m, 2H, CH2NH), up 3.22 (s, 3H, och3).

ESI-MS (cluster+2)...944,4, 966,4, 988,5, 1010,5, 1032,5... (the increase in the mass +47 relative to the cluster+2 MPEG-ÌÍ2...897,4, 919,4, 941,5, 963,5, 985,5).

Example 2. The conjugation reaction of the polymer with a K-a to get oxazolidinediones conjugate

Diagram of the method shown in figure 1.

In a round-bottom flask 500 ml dissolve 33,0 g MPEG-NH-CO-Im (16,00 mmol) in 85 ml of dichloromethane in a nitrogen atmosphere. The solvent is removed under reduced pressure, and the connection is dried using a mechanical pump for two hours.

In the reactor of 2 l, equipped with block thermocryostatic, mechanical stirrer and thermometer, dissolved 6,21 g K-a (purity analysis 98%, to 13.29 mmol) 1.85 l of dichloromethane in a nitrogen atmosphere and the solution is cooled at 0°C. under nitrogen atmosphere add to 0.53 g of sodium hydride (purity analysis of 60%to 13.29 mmol)and the mixture stirred for 10 minutes. Dried, MPEG-NH-CO-Im is dissolved in 90 ml of dichloromethane, and the solution is added to the mixture To a and NaH in dichloromethane at 0°C under nitrogen atmosphere. The mixture is stirred for 30 mi the ut at 0°C, then heated to 25°C and at this temperature is maintained with stirring for 10 minutes.

The reaction mixture was analyzed by liquid chromatography high resolution (HPLC) to determine the degree of transformation To a and the ratio of compounds in the mixture. After 10 minutes at 25°C. to the reaction mixture of 3.60 g of 1,1-carbonyldiimidazole (purity analysis 90%, 19.93 per mmol) and the solution stirred at 25°C for 30 minutes. The reaction mixture was analyzed by HPLC in order to determine the degree of transformation of amide side product (a conjugate of MPEG through the carboxyl fragment in position 9 connection To a) in desirable oxazolidinediones conjugate. The reaction mixture was neutralized with formic acid (purity analysis 98%) to a final pH 6 (approximately 2 ml, 53 mmol).

The solvent is removed under reduced pressure at 25°C and get to 44.0 g of light yellow crude product.

According to HPLC the purity of the conjugate in the raw product exceeds 90%. The content of the desired product in the crude mixture is approximately 65-70 wt.%.

Example 3. Cleaning oxazolidinediones conjugate To-a method flash chromatography on reversed phase

The crude mixture obtained in stage conjugation of example 2, and purified using flash chromatography on reversed phase. And the use of the system Biotage Horizon, equipped with a flash cartridge 65iM KP-C18. The batch is divided into 15 aliquot of 3.0 g each. These aliquots are treated separately.

Column 18 condition first 200 ml of solvent using the following gradient: from 100% acetonitrile to the mixture 40:60 acetonitrile/water and then 200 ml of a mixture of 40:60 acetonitrile/ammonium formate (5 millimoles/liter (mm) pH 3.5, in isocratic conditions.

The crude product (3.0 g) is dissolved in 3.0 ml of N,N-dimethylformamide (DMF) and the solution is loaded into a column. Cleaning is performed by isocratic elution with a mixture of 40:60 acetonitrile/ammonium formate, 5 mm, pH 3.5. Collected individual fractions analyzed using HPLC, and combine pure fractions. The solvent is removed under reduced pressure at 25°C and receives approximately 2 g of pure moist product.

Each aliquot of purified, following the first method, and finally the fraction of each clean wet product was dissolved in 10 ml of dichloromethane and then combine. The solution is dried over sodium sulfate. The solid is filtered off and the solvent is removed under reduced pressure at 25°C.

The obtained solid product analyzed by NMR spectroscopy and detect approximately 1 mol-equivalent of ammonium formate. To remove this salt, the product is dissolved in 50 ml of dichloromethane and elute through the layer of silicone is El, soaked in dichloromethane. The product is extracted by elution 700 ml of a mixed solvent of dichloromethane/methanol 9:1. The eluate is collected and the solvent is removed under reduced pressure at 25°C. the Product is again dissolved in 80 ml dichloromethane and precipitated at 0°C and vigorous stirring by adding 500 ml of diethyl ether to obtain a solid product. This product is filtered through a filter funnel with a disk of sintered glass, washed with diethyl ether (100 ml) and dried in vacuum for 16 hours.

Obtain 16.0 g of light-yellow powder with a total yield (algae + cleaning) 51%.

The product is characterized by NMR, ESI-MS and HPLC. The degree of purity? determined by NMR using an internal standard, corresponds to 101%of mass.

1H-NMR (DMSO-d6) δ (M. D.): a 9.25 (d, 1H, CH), to 8.70 (s, 1H, NH), 8,11 (d, 1H, CH), 7,95 (d, 1H, CH), of 7.70 (d, 1H, CH), 7,51 (m, 2H, CH), 7,42 (m, 2H, CH), 7,31 (m, 1H, CH), of 5.05 (s, 1H, NHCH2), 3,90 is 3.40 (m, CH2PEG)of 3.25 (s, 3H, och3), to 2.35 (m, 4H, CH3+1H CH2).

13C-NMR (DMSO-d6) δ (M. D.): 172,3, 172,0, 153,7, 139,4, 137,5, 133,5, 128,1, 126,2, 124,9, 124,0, 123,2, 122,3, 121,6, 120,8, 120,4, 117,0, 115,4, 113,5, 109,9, 98,4, 90,6, 85,3, 71,8, 70,0, 68,9, 66,3, 58,5, 45,9, 40,0, 23,16.

ESI-MS (cluster+2)...1128,2, 1150,3, 1172,3, 1194,3, 1216,3... (the increase in the mass +to 230.8 on cluster+2 MPEG-NH2, 897,4, 919,4, 941,5, 963,5, 985,5).

The exact value of the molecular mass (MM): the discrepancy between the values MM between the recorded spectrum and theoretical spectrum corresponds to 2 M. D. Therefore, the obtained polymeric compound has a purity of at least about 98%.

Example 4. Cleaning oxazolidinediones conjugate To-a method flash chromatography on normal phase

The method of synthesis were carried out as described above in examples 1 and 2, and in this mode of synthesis gain of 21.7 g of the crude product.

The crude mixture obtained at the specified phase conjugation, purified using flash chromatography on normal phase using the Biotage Horizon, fitted with SNAP cartridge (size 71×168 mm)filled with silica gel KP-SIL (340 g). The crude product is separated into two aliquots, each of which was purified separately once (in each aliquot contains 10,86 g and 10.8 g of crude material).

The cartridge SNAP is brought to equilibrium in a mixture of 940 ml of dichloromethane/methanol 96:4 by volume. The flow rate of 65 ml/min/

Loading of the sample is performed using a pre-filled sample SNAP cartridge (34 g) by dissolving the crude material in 10 ml of dichloromethane, using the solution in the sample cartridge and inserting the sample in SNAP cartridge.

SNAP the cartridge elute at a flow rate of 65 ml/min with the following formula:

- 705 ml of dichloromethane/methanol 96:4 by volume;

- 1881 ml of dichloromethane/methanol, 93:7 by volume;

- 942 ml of dichloromethane/methanol, 85:15 by volume.

The first 999 ml erwerbende what about the solvent throw, then suirvey solvent is collected in the form of fractions of the volume 111 ml.

Collected individual fractions analyzed by HPLC and combine the fractions containing the compound conjugated product with a purity >98% (HPLC, pure fractions).

The remaining aliquot (10.8 g) of the mixture of the crude material with phase conjugation cleanse likewise.

Selected fractions from the two stages of purification aliquot of the crude mixture unite, the solvent is removed to dryness under reduced pressure and 25°C receives 8,11 g conjugate product, which is again dissolved in 29 ml of dichloromethane, cooled to 2°C, and precipitated under vigorous stirring by adding 150 ml of diethyl ether for 15 minutes. The mixture was stirred at 2°C for 15 minutes, then add 225 ml of diethyl ether. The precipitated solid substance produce by filtering through a filter of sintered glass (G4) and dried in vacuum at 25°C. for 16 hours, receiving of 6.95 g of the test sample in the form of a solid whitish-yellowish color. Purity according to HPLC analysis is 99%.

Example 5. The method of synthesis oxazolidinediones conjugate To-a

1) Synthesis of MeO-PEG-NH-CO-Im (Me is methyl)

Dissolve MeO-PEG-Mog (M, 8.06 g) in dichloromethane (25 ml) under nitrogen atmosphere and the solvent is removed by distillation under reduced pressure at 40°C. Then the OS is atok (MeO-PEG-CRUSHING dried in vacuo (< 40 mbar=4 kPa) at 40°C for more than 2 hours.

Dried MeO-PEG-ÌÍ2 (as described above) dissolved in dichloromethane (35 ml) at 25°C. in a nitrogen atmosphere, add to this a solution of 1,1'-carbonyldiimidazole (1,02 g) and the mixture is stirred at room temperature for more than 2 hours (according to the ion-steam liquid chromatography (IP): the degree of conversion of >95%).

The reaction mixture was cooled to 0°C, then add 230 ml of diethyl ether for 1 hour under vigorous stirring. The mixture is stirred 30 minutes at 0°C and add an additional 69 ml diethyl ether for 25 minutes. The residue on the filter is washed twice with diethyl ether (23 ml) and dried in vacuum at a temperature not exceeding 40°C until constant weight to obtain of 8.25 g of MeO-PEG-NH-CO-Im in the form of a white solid.

2) the Reaction of conjugation with the polymer

Dissolve MeO-PEG-NH-CO-Im (72,0 g) in dichloromethane (185 ml) under nitrogen atmosphere and the solvent is removed by distillation under reduced pressure at 40°C. Then the residue is dried in vacuo (<40 mbar=4 kPa) at 40°C for more than 2 hours.

Dissolve CA (13,11 g) in dichloromethane (3920 ml) and then the solution is cooled to 0°C. sodium Hydride (1,17 g, 60%) are added in parts.

Dried MeO-PEG-NH-CO-Im dissolved in dichloromethane (140 ml)and the solution is added at a temperature below 5°C in the reaction mixture Ka, and the mixture is stirred 30 minutes at 0°C. Then rest the R reaction mixture is heated to 25°C, keeping stirring at this temperature for 10 minutes (Eph 1: the degree of transformation Ka >96%).

Added to the reaction mixture of 1,1'-carbonyldiimidazole (7,11 g) and the solution stirred at 25°C. over 30 minutes (Eph 2: the ratio of the crude product: amide >80/20).

Add formic acid (5 ml)to bring the reaction mixture to pH 6. The solvent is removed by distillation at 25°C under reduced pressure and the residue is dried in vacuum at 25°C until constant weight.

3) isolation and Purification of the polymer conjugate

The crude mixture (81 g) was dissolved in 325 ml of dichloromethane (DHM) at temperatures below 35°C for 15 minutes and filtered through a layer of Celite (3 cm). Specified Celite was washed with dichloromethane (81 ml). The solvent is removed by distillation under reduced pressure and a temperature below 35°C and dried to constant weight at 35°C, receiving of 77.0 g of solid material. This solid material is dissolved in 770 ml of dichloromethane.

The crude material from example 5.2 purified in preparative system Knauer HPLC, using a flash cartridge KP-SIL 75L (75×300 mm, 800 g of silica gel) from the company Biotage. Prior to the filing of the initial solution, the cartridge was washed with n-heptane (1.5 l) and brought to equilibrium in 3 liters of a mixture DHM:Meon=96:4 (by volume). Each time introducing 200 ml of the above initial solution DHM:Meon=96:4 (by volume) (download 20 g) and start the indicate the elution at a flow of 185 ml/min and the following gradient.

Minutes% Meon
0,004
8,304
80,1015
80,1050
108,0050

Each cartridge is used only for one time. The product eluted between 25 and 90 minutes. Faction collect, analyze and combine according to their purity (IP: >98% a/a).

Then the solvent is removed under reduced pressure at 35°C. the Solids are dried to constant weight in vacuo at 35°C to obtain the crude material (37,92 g). This material is dissolved in 140 ml of dichloromethane and cooled to 2°C. Add 700 ml of diethyl ether at 2°C and stirred for 15 minutes. Add 1050 ml of diethyl ether at 2°C.

The suspension was filtered using a vacuum filter. The residue on the filter is washed with mother liquor and dried to constant weight, getting a 32.6 g of substance - cleared drug. According to HPLC analysis, the purity of the obtained product is 98,99%.

In additional examples describe some experiments the tion, made with polymer conjugate indolocarbazole compounds according to the invention, in particular with the polymer conjugate, obtained using the synthesis method of the present invention, for example as described in examples 1-5. The test conjugate connection (also referred to as "test sample") is oxazolidinediones conjugate Ka with polyethylene glycol (1892 MM).

Example 6. Evaluation of in vitro inhibitory 50% concentration (IC50) oxazolidinediones conjugate To-a against TrkA

The aim of this study is to determine the value of the IC50for the conjugate of example 3 against kinase TrkA. The test compound dissolved in dimethyl sulfoxide (DMSO), and then the solution is further diluted with 25 times the analytical buffer solution to obtain a final solution of the test compound. The conjugate was tested at the following concentrations: 30000 nanomoles/l (nm)10000 nm, 3000 nm, 1000 nm, 300 nm, 100 nm, 30 nm, 10 nm, 3 nm and 1 nm.

Reference compound (Staurosporin) for analytical control prepared in the same manner as in the method used to prepare the test connection.

The method of analysis presented by the external device Analysis panning spots (MSA) and is described below:

1) Solution of compound 5 μl×4,5 μl×4 substrate (CSKtide 1000 nm)/adenosine-triphosphate (ATP 75 μm)/the solution of the metal (Mg, 5 mm) and a solution of 10 μl kinase×2 prepared in analytical buffer solution [containing 20 mm HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0,01% Triton X-100, 2 mm DTT (1,2-dithiotreitol), pH 7.5], mixed and cultured in the wells of a polypropylene microtiter plate with 384 wells within 1 or 5 hours* (*depending on kinase) at room temperature.

2) In the hole add 60 ál of the final buffer solution (sorting solution QuickScout contributing to the analysis of the MSA; firm Cama Biosciences).

3) the Reaction mixture is fed into the system analysis LabChip3000 (firm Caliper Life Science), and peptide peaks of the product and substrate are separated and quantitatively analyzed.

4) Interaction of the kinase appreciate the value of the product calculated by the height of the peptide peaks of the product (P) and substrate (S) (P/(P+S)).

The value read control interaction (final reaction mixture) was taken as 0% inhibition, and read the value of the background (the Enzyme(-)) was taken as 100% inhibition, then calculate the percentage of inhibition for each of the test solution. The value of the IC50calculated curves according to percent inhibition concentration, the method of selection of the logistic curve with four parameters. The interaction of the kinase appreciate the value of the product calculated by the height of the peptide peaks of the product (P) and substrate (S) (P/(P+S)).

The value of the IC50for conjugate against TrkA is 202 nm, corresponding to the value of the IC50for reference soybean is inane (Staurosporin) against TrkA is 0,372 nm. These results are summarized in figure 7.

Example 7. Study of acute dermal toxicity in rats

Acute toxicity of the conjugate from example 3 are examined after the introduction of a single dermal dose rats.

A single dose of 2000 mg/kg administered group of 5 animals male 5 female animals during the period of 24 hours. The day before the planned dosing removes hair from the dorsal surface of the trunk over the estimated area of 10% of the entire surface of the body. Necessary precautions to avoid any damage or abrasion of the skin. The test sample is injected locally with the dose volume of 4 ml/kg of body weight immediately after preparation of the sample. The required aliquot of the formulation of the test sample is evenly distributed over the gauze size 2.5×2.5 to see Then on the skin of the animal is placed a gauze pad, and the test substance is in direct contact with the skin. On top of the treated area is placed tape synthetic film, and the whole set is held in place, covering the torso of the animal long elastic adhesive bandage.

After a period of 24 hours band ligation may be removed. Then the treated skin area carefully washed, removing the remains of the test sample using lukewarm water. During this study all animal which x is checked twice a day. Animals are examined for signs of reaction to the batched processing approximately 30 minutes, 2 and 4 hours after entering the doses in 1 day, then every day, just for 14 days. Each animal is weighed in the designated study day-to-day input dose (1st day) and on the 8th and 15th day. After a period of 14 days, all animals killed and subjected to autoprocessor research.

During this study, the deaths did not occur and was not observed clinical signs in animals male or female animals. Changes in body weight observed in animals at the end of the study, were in the expected range for the specified type and age of animals. At necropsy, the animals at the end of the study was not observed internal abnormalities. Also, there were no abnormalities in the treated area.

These results demonstrate that the tested compound has no toxic effect on rats after exposure of the skin over a 24 hour period at the level of 2000 mg/kg No mortality shows that the lethal dose for 50% mortality (LD50) exceeds 2000 mg/kg

Example 8. Study 13-week dermal toxicity in rats after 4-week recovery period

The objective of this research is to evaluate the toxicity of the test sample on rats the donkey daily skin introduction (extract 6 hours) during the period of 13 weeks and the study of possible recovery from any possible effects, related to processing, during the period of 4 consecutive weeks. In addition, it was estimated toxicokinetics profile.

Three groups each of 10 rats male and 10 female rats Sprague Dawley (see table 1: animals male numbered even numbers, females numbered odd numbers)were tested sample by applying to the skin at doses of 0.5, 2.5 and 5 mg/kg/day for 13 consecutive weeks (table 1: number of groups 2-4). The fourth group of similar composition received only the vehicle (purified water) and played the role of the control (table 1: group number 1). Five additional animals of each sex were included in the group of high doses and the control group (table 1: number of groups 4 and 1, respectively) to assess recovery. In addition, three support groups for toxicokinetics studies, including 9 males and 9 females (table 2: numbers of groups 5-8, respectively), and 1 control group, including 3 males and 3 females (table 2: group number 5), animals were treated as in the main groups for toxicokinetics estimates.

Designation of groups and number of animals to be processed, are shown below in tables 1 and 2:

Table 1
(Major groups)
Group numberTreatment(mg/kg/day)LevelThe number of rats
The main phaseThe recovery phase
Males (even)Females (odd)Males (even)Females (odd)
10Control2-201-1922-3021-29
20,5Low32-5031-49
32,5Average52-7051-69
45High72-90 71-8992-10091-99

Table 2
(reference group)
Group numberDosage (mg/kg/day)Processing levelThe number of rats
Males (even)Females (odd)
102 to 106101-105
50Control108-124107-123
60,5Low
72,5Average126-142125-141
85High144-160143-159

The treated area examined every day, approximately 3 hours after the start dose the Finance. Irritation in these areas, compared with neighbouring areas of raw hides, ascribed numerical values in accordance with the table below:

Erythema and scab formationValue
Erythema no0
Very faint erythema (barely perceptible)1
Well-defined erythema2
Erythema moderate to severe3
Severe erythema (beet redness) scabbing,4
preventing classification of erythema
The formation of edemaValue
Edema is absent0
Very weak edema (barely perceptible)1
Weak edema (edges of area well-shaped due
pronounced swelling 2
Moderate edema (swelling approximately 1 mm)3
Severe edema (swelling more than 1 mm, which is4
extends outside the area of impact)

Results

During this study there was no deaths related to treatment (one female of the high dose died on the 40th day of the study), and there was no clinical signs related to treatment. Significant differences in body weight are not registered, and food consumption in the treated animals remained comparable to the control in all the time of research.

It was not observed any signs of irritation in the treated areas (coefficient irritation equal to 0).

In the ophthalmic research, conducted at the end of the treatment period, not registered associated with the processing of defeat.

With hematological point of view, leukopenia observed in animals in the high-dose group and in females in the group average doses were partially reversible at the end of the recovery period. Any other changes of Toxicological significance were observed.

Any changes to Toxicological values were observed according to the clinical chemistry and urine studies.

Final body weight was comparable between control and treated groups.

It was not observed any changes in absolute and relative weight of organs that have Toxicological significance.

Was not observed changes associated with treatment, according to macroscopic and microscopic examination.

As for toxicokinetics data, the first day of the plasma levels of the test sample were usually lower than NPCO (lower limit of quantitation =to 49.9 ng/ml) in males and females received doses of 0.5, 2.5 and 5 mg/kg/day of the test sample, and only one animal from time to time registered values slightly above NPCO, between 2 and 8 hours after the dose. The recorded values were not proportional to dose.

Similar results were obtained at 4 and 13 weeks, when the detected low degree of absorption. This becomes particularly evident at 4 weeks males who have from time to time there are slightly more NPCO between 6 and 8 hours after the dose only in animals that received 5 mg/kg/day of the test sample and at 13 weeks females (figures a little more NPCO between 4 and 8 hours only after receiving a dose of 5 mg/kg/day).

For animals in the group treated only carrier not detected distinct levels. On the basis pointed to by the x above results, after daily oral dose for 13-week period does not accumulate.

Conclusions

If any of the investigated level doses of the test sample (0.5, 2.5 and 5 mg/kg/day) are not observed harmful effects. It is believed that weak leukopenia observed in treated animals when compared with control animals no Toxicological significance, since it has a low value, usually not dose dependent and is not accompanied by any microscopic changes. It is therefore considered that the high dose of 5 mg/kg/day represents the level at which no observed adverse effects (NOAEL) for the test sample after daily skin of rats during the period of 13 weeks.

The results of the analysis of plasma samples showed that the tested sample is only minimally absorbed by the percutaneous route.

Example 9. Study 13-week dermal toxicity in rabbits after 4-week recovery period

The toxicity of the test sample was investigated in rabbits after daily skin injection level doses of 0.5, 2.5 and 5 mg/individual/day for 13-week period and recovery from any potential effects associated with the treatment, during the period of 4 consecutive weeks.

Three groups, each with 6 male and 6 female new Zealand white rabbit is in, does not contain specific pathogens (SPF) (compare table 3: animals male numbered even numbers, females numbered odd numbers)were tested sample by applying to the skin at doses of 0.5, 2.5 and 5 mg/individual/day for 13 consecutive weeks (table 3: numbers of groups 2-4, respectively). The fourth group of similar composition received only the vehicle (purified water) and played the role of the control (table 3: group number 1). Three additional animals of each sex were included in the group of high doses and the control group (table 3: groups 4 and 1, respectively) to assess recovery.

Designation of groups and processing are shown below in table 3:

Table 3
Group numberTreatment (mg/kg/day)LevelThe number of rabbits
The main phaseThe recovery phase
Males (even)Females (odd)Males (even)Females (odd)
10Control2-121-1114-1813-17
20,5Low20-3019-29
32,5Average32-4231-41
45High44-5443-5356-6055-59

Following studies were conducted: daily clinical signs, body weight, food consumption, macroscopic examination of the areas of processing, the study of clinical pathology, final body weight, body weight, post-mortem macroscopic examination and histopathological study.

Blood samples were taken from each animal on the first day of the 1st and 13th week and for toxicokinetics estimates.

Processing abled who are every day, approximately 3 hours after the start of the input dose. Irritation in the processing, when compared to adjacent untreated skin was assessed on a numerical scale as shown in the table below:

Weak edema (edges of area well-shaped due to severe swelling)
Erythema and scab formationValue
Erythema no0
Very faint erythema (barely perceptible)1
Well-defined erythema2
Erythema moderate to severe3
Severe erythema (beet redness) scabbing, preventing classification of erythema4
The formation of edemaValue
Edema is absent0
Very weak edema (barely perceptible)1
2
Moderate edema (swelling approximately 1 mm)3
Severe edema (swelling more than 1 mm, which extends outside the area of impact)4

Results

During this study there was no deaths related to treatment (one male from the control group restore was humanely killed on day 29 of the study), and were observed for clinical signs associated with treatment.

At macroscopic examination of the treated areas was not observed signs of irritation.

During this study no significant differences in body weight and food consumption in the treated animals remained comparable to the control.

In the ophthalmic research, conducted at the end of the treatment period, not registered lesions associated with processing.

According to the Hematology and clinical chemistry is not registered with the changes that have Toxicological importance,

As for toxicokinetics data, in the first week of the plasma levels ispy is been created sample were slightly higher than NPCO (lower limit of quantitation =51,30 ng/ml) in the majority of males that received the dose of the test sample 2.5 mg/kg/day and in females that received a dose of 5 mg/kg/day. Only one male, at doses of 0.5 and 5 mg/kg/day from time to time were registered values slightly above NPCO. The amount of absorption was not proportional to the dose level.

Very low degree of absorption is observed on the 13th week. Usually the indicators more NPCO observed between 2 and 24 hours after the dose. Absorption in females was slightly larger than the males.

For animals in the group treated only carrier not detected distinct levels. Based on the above results, it follows that after daily oral dose for 13-week period does not accumulate.

Final body weight was comparable between control and treated groups, and were not observed any changes in absolute and relative weight of organs that have Toxicological significance.

Was not observed changes associated with treatment, according to macroscopic and microscopic examination.

Conclusions

If any of the investigated level doses of the test sample (0.5, 2.5 and 5 mg/individual/day) no observed adverse effects. It is therefore considered that the high dose of 5 mg/kg/day represents a level at which no observed adverse effects (NOAEL) for the test sample after daily skin of rabbits during the period of 13 weeks.

The results of the analysis of plasma samples showed that the tested sample is only minimally absorbed by the percutaneous route.

Example 10. The study of the acute intravenous toxicity in rats

Acute toxicity of the conjugate of example 3 was investigated after intravenous injection (10 ml/kg in saline) single dose to rats Sprague Dawley subsequent 14-day period of the survey.

A separate group of 5 males and 5 females were injected dose of 2000 mg/kg Animals were treated with dose formulations of the test sample at the desired level immediately after preparation of the sample injected into the tail vein using hypodermal needle attached to a syringe of appropriate volume dose was 10 ml/kg body weight. Throughout the study, animals were checked twice a day.

Animals are examined for signs of reaction to treatment dose approximately 30 minutes, 2 and 4 hours after entering the doses in 1 day, then daily for 14 days. Each animal was weighed on the day appointed research, on the day of input dose (1st day) and on the 2nd, 8th and 15th day. At the end of survey period all animals were killed and subjected to autoprocessor research.

The deaths occurred among male animals and female animals. In all animals at day dose observed CL the clinical signs were reduced activity and piloerection. In one female noted hair loss on his back in the second week of the study.

Changes in body weight observed in animals at the end of the study, were in the expected range for the specified type and age of animals. When autopsies the study all animals were observed internal abnormalities. Also, there were no abnormalities at the site of injection.

These results demonstrate that the conjugate does not have toxic effects on rats after a single intravenous administration at the dose level of 2000 mg/kg body weight. Animals have been observed only minor clinical signs. The test sample is locally tolerant when injected into the tail vein at the tested dose level.

Example 11. Mutation in cells mouse lymphoma L5178Y TK+/-(fluctuation method)

The mutagenic activity of the test sample is investigated by analyzing the induction of resistant 5-cryptorchidism mutants in cells mouse lymphoma L5178Y after treatment in vitro in the absence and in the presence of S-9 metabolic activation, using fluctuation method. The specified method can be used to detect gene mutation, clastogenic and energenie effects.

Used in this study method of analysis of mutations based on the identification of colonies L5178Y that have acquired resistance to oxicash analogue of thymidine - cryptorchidism (TFT). The specified analog may be subject to metabolism by the enzyme timedancing (TC) in nucleosides, which are used in the synthesis of nucleic acids, which leads to the death of the LC-competent cells.

Cells with a deficit of TK, which presumably result from mutations in the gene TC, can't be subjected to metabolism triptorelin and, thus, survive and grow in his presence.

In cells mouse lymphoma L5178Y gene, which encodes TK enzyme is located on chromosome 11. Cells that are heterozygous at the location of TK (TK+/-)may be subject to a separate stage direct mutations in the TK-/- genotype, which has a small TC activity or none at all.

Used cells, L5178Y TK+/-, made from one of the two clones resulting from tumor thymus induced in mice DBA/2 under the action of methylcholanthrene. The system mutations TC cells mouse lymphoma L5178Y well characterized and justified (Clive D., Johnson K. A., Spector J. F., Batson A. G., Brown M. M. Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutat. Res. 1979 Jan.; 59(1):61-108) and accepted by most regulatory authorities.

In the analysis of mouse lymphoma often bimodal size distribution-resistant TFT colonies, designated as small or large. It was found that the accuracy of the data mutations and deletions within the active allele (nutrigenie event) produce large colonies. Small colonies partly arise from lesions that affect not only the active TC alleles, but also on the flanking gene, expression of which modulates the growth rate of cells.

Found that the tested sample is dissolved in complete medium RPMI 1640 at a concentration of 50.0 mg/ml

Conducted a preliminary analysis of cytotoxicity. Based on the results of the solubility of the tested sample was analyzed at the maximum dose of 5000 μg/ml both in the absence and in the presence of S9 metabolism. In a series of processing was included a wide range of doses low: 2500, 1250, 625, 313, 156, 78,1, 39,1 and 19.5 ág/ml

In the absence of S9 metabolic activation using short-term treatment has been a slight decrease in the relative survival at several concentrations, regardless of dose. When using long-term treatment toxicity observed at the two higher concentrations, and the relative survival rate is reduced by approximately 60% of the concurrent negative control.

In the presence of S9 metabolic activation, at any concentration of the test sample is not observed significant toxicity.

Based on the results of toxicity obtained in the previous test, were carried out two independent analyses of the Mut is tion, resistant to 5-cryptorchidism, using the dosage levels specified in the following table 4:

Table 4
Room analysisS9Processing time (hours)Dose level (µg/ml)
1-/+35000, 2500, 1250, 625, 313
2-245000, 2500, 1250, 625, 313
2+35000, 3571, 2551, 1822 and 1302

Not observed a significant increase in the frequency of mutations after treatment of the test sample in the absence or in the presence of S9 metabolism.

In each experiment mutations were included processing solvent and positive controls in the absence and presence of S9 metabolism. The frequency of mutations in the control cultures with the solvent is in the normal range. A noticeable increase in mutations observed in the positive control treatment, indicating correct operation of the analysis system.

The conclusion, is that the test sample does not cause mutations in cells mouse lymphoma L5178Y after treatment in vitro, in the absence or in the presence of S9 metabolic activation, in these experimental conditions.

Example 12. Analysis of photomutagenicity in bacteria (S. typhimurium and E. coli)

For the test sample was investigated photomutagenic activity analysis of reverse mutations in prototrophic in prokaryotic organisms : Salmonella typhimurium and Escherichia coli after exposure to light.

Were used in three of the tested strains of S. typhimurium - TA, ÒÀ98 and TA100 and the test strain E. coli WP2. Bacteria in the tablet along with the test sample in soft agar were irradiated with different doses of UV radiation.

The adopted methodology was proposed Ames and others, 1975, and revised Maron and Ames, 1983. The test sample is applied in the form of a solution in sterile distilled water.

The test sample analyzed in the test of toxicity at the maximum doses of 5000 mg/tablet and four low concentrations, selected approximately semi-logarithmic range: 1580, 500, 158 and 50.0 mg/tablet. They chose two widely separated from each other UV dose for each of the tested strain of bacteria on the basis of the maximum allowable primary dose. Significant toxicity was not observed at any concentration of the test sample, or any dose of UV radiation.

We carried out two independent experiment using the tablet method Inko is of the Corporation.

The test sample is assayed at the maximum dose level of 5000 mg/tablet and four low dose levels selected twofold dilutions: 2500, 1250, 625, 313 µg/tablet. The prepared tablets irradiate the following doses of UVA and UVB (table 5):

Table 5
The test strainUVA (j/cm2)UVB (j/cm2)
0,4-
TA0,2-
0,1-
0,2-
ÒÀ980,1-
0,05-
0,04-
TA1000,02-
0,01 -
0,0040,004
WP20,0020,002
0,0010,001

Results

The test sample does not cause a twofold increase in the number revertant colonies compared to the background UV effect at any dose level of the test sample in any test strain at any dose of UV radiation. Conclusions

It is concluded that the test sample does not cause reverse mutation in Salmonella typhimurium or Escherichia coli, when processing is carried out in the presence of UV radiation.

Example 13. Chromosomal aberrations in Chinese hamster oocytes in vitro (analysis of photomutagenicity)

Analsouth the ability of the test sample to cause chromosomal damage in Chinese hamster oocytes after treatment in vitro in the absence and in the presence of UVA/UVB radiation.

One analysis of chromosomal damage is carried out at the dose levels 5000, 2500, 1250, 625, 313, 156, 78,1 and 39,1 μg/ml, and the analysis carried out both in the absence and in the presence of ultraviolet radiation.

The preparations of the test sample were prepared in a balanced saline solution, Hank (HBSS). Both in the absence and in the presence of the tvii UV radiation cells treated for 3 hours and use the time of collection culture 20 hours, that is approximately half of the cell cycle.

The experiment includes the appropriate negative and positive control. For each test point should prepare two cell culture.

Dose levels for the quantitative evaluation of chromosomal aberrations were selected on the basis of cytotoxicity processing the test sample, which is determined by the decrease in the number of cells at the time of collection culture.

Since appreciable toxicity was not observed in the whole range of dosages for quantitative evaluation were selected following dose levels: 5000, 2500 and 1250 µg/ml in the absence and in the presence of UV radiation.

For chromosomal aberrations from each culture quantify hundred growths metaphases.

Results

After processing the test sample is not observed a statistically significant increase in the percentage of cells with aberrations, including or excluding gaps, compared with the corresponding control values in the absence or in the presence of ultraviolet radiation.

A statistically significant increase in the number of cells with aberrations (including or excluding gaps)was observed after treatment positive control agents Mitomycin-C and 8-Methoxypsoralen that indicates correct operation of the test system.

Conclusions

On on the basis of the above results it is concluded, in these experimental conditions tested sample does not cause chromosomal aberrations in Chinese hamster oocytes after treatment in vitro, in the absence or in the presence of UV radiation.

Example 14. Analysis of phototoxicity cells Balb/C 3t3 (neutral red uptake)

Potential toxicity in vitro test sample is assessed through measurement of the absorption of neutral red (dye) for cellular toxicity in cell cultures of Balb/c 3T3 treated with various doses of the test sample and subjected to UVA irradiation. The preparations of the test sample prepared using a balanced salt solution, Earle (EBSS).

Was performed a preliminary search experiment in the range of doses, in the presence (+UVA) and in the absence (-UVA) irradiation in order to choose the appropriate dose levels for the main analysis. The test sample was analyzed at the maximum dose of 1000 µg/ml (the upper limit specified in the study Protocol) and in a wide range from low doses: 500, 250, 125, 62,5, 31,3, 15,6 and 7,81 µg/ml Because the value of the IC50cannot be calculated in the presence and in the absence of UVA irradiation, also cannot calculate the coefficient of futurestate (PIF). In this case, suppose that the sample is not phototoxic. The same range of d is C used for the primary analysis.

The basic experiment is performed with the use of the next level doses: 1000, 500, 250, 125, 62,5, 31,3, 15,6 and 7,81 µg/ml On the survival curves in the presence and in the absence of UV radiation, there is a similar profile, which confirms the results obtained in the previous search experiment in the range of doses. Factor value futurestate (PIF) cannot be calculated because there are no values IC50for both curves. The average value of the photoelectric effect (MPE) is 0,089 that corresponds to the range of a lack of phototoxicity.

Since in this experiment received clearly negative results, additional experiments were not conducted.

Positive control agent Chlorpromazine caused acceptable positive response to the magnitude PIF 21,9 that indicates correct operation of the analysis system.

Due to the inability of the calculation of the PIF, or the value of MPE<0,1 allows us to predict the lack of phototoxicity on the basis of the obtained results, it is concluded that the tested sample should be classified as "phototoxicity" in the specified conditions of the experiment.

Example 15. The study futurestate/photosensitivity for the test sample, 0.1%cream, Guinea pigs

The potential ability of the test sample (0.1%cream) call photoallergic and/and and Tatarskaya reactions after topical application on the skin, in connection with exposure to ultraviolet radiation, estimated using the model of the Guinea pig.

The study is divided into two phases.

In the first phase assesses Tatarskaya characteristics of the test sample in 6 groups of animals. These characteristics are used as justification for appropriate concentrations of the test sample for use in sensibilisation analysis and more information on photo-induced irritation. The animals are treated in the following way (table 6):

Table 6
Group numberprocessingUV-irradiationThe number of animals
1Media + Control sample5
2Media + Control sampleno5
3Media + test sample5
4Media + Ispy is been created sample no5
5Media + 8-methoxypsoralenno5
6Media + 8-methoxypsoralen5

In the second phase of the study assessment of sensitization, where all 5 groups treated as follows (table 7):

Table 7
Group numberProcessing the inductionTreatment with stimulatingThe number of animals
7F. C. A.1+ media10
Media + test sample5 out of 10
Media + control sample5 out of 10
8F. C. A. + control the way the Media control sample10
9F. C. A. + a tested sampleThe media tested sample10
10F. C. A. + mediaMedia Musk Ambrette3
11F. C. A. + Musk AmbretteMedia Musk Ambrette5
1Complete Freund adjuvant (Freund's Comlete Adjuvant)

Photoestrogens

Test futurestate carried out using two (irradiated) groups, each with 5 animals treated with control cream and placebo cream containing 0.1% of the test sample (see table 6, group 1 and 3) and two groups of similar composition (see table 6, group 2 and 4), in which animals are treated in the same way, but not irradiated. The undiluted aliquots of the test or control sample (100%) in two concentrations (20% and 50% purified water) of the test and control samples and one medium (purified water) evenly distribute on certain parts of the skin, prepared on the back of animals. In the irradiated groups (groups 1 and 3) animals subjected to irradiation with UVA (10 j/cm 2) and UVB (0.1 j/cm2after a dose. The reference substance positive control, 8-methoxypsoralen, research using these techniques in concentrations of 0.001%, 0.01% to 0.1%in five subjects (irradiated) and five control (non-irradiated) animals (see table 6, group 6 and 5 respectively). Approximately 1, 4, 24, 48 and 72 hours after exposure to the control, the test or reference samples, treated areas are checked for the presence of a reaction of irritation from handling.

Results

Slight irritation was observed in one out of five animals on the plots treated with cream 0.1% of the test sample and irradiated with UV. Four of the 5 animals on the plots treated with the test sample, but not UV irradiated irritation was observed, ranging from minor to quite specific.

A minor response was also observed in one of the 5 animals treated with the control sample (cream placebo) not irradiated with UV.

On plots treated with one carrier, the reaction was not observed/

The irritation observed in animals treated with the test sample, is not induced, as it is seen in animals irradiated with UV light, and also in non-irradiated animals (mostly).

Minor periodic reaction was also observed in non-irradiated alive is the shaft, treated control sample.

In animals treated with the positive control substance, 8-methoxypsoralen and then exposed to ultraviolet radiation, was observed erythema, from well-defined to moderate, and weak swelling on the plots treated with two higher investigated concentrations of 0.01% and 0.1%. In animals treated with 8-methoxypsoralen without subsequent exposure to ultraviolet radiation, the reaction was not observed, thus, demonstrated that the observed response was photoinduction.

Photosensitivity

Test photosensitivity was performed using two groups of 10 animals (see table 7, group 8 and 9), induced by the control sample and the test sample, and one control group of 10 animals (see table 7, group 7), induced by the selected carrier (purified water). When you try to induce sensitization of animals was injected intradermally emulsion of complete Freund adjuvant. The test and control samples at a concentration of 100% locally applied to the surface around the injection site of the stimulator, a total of 6 times over a two week period. After applying a dose animals were irradiated as UVA (10 j/cm2)and UVB (0.1 j/cm2) radiation. Control group animals were treated in the same way, but have you used the early media (purified water) instead of the test or control sample. Approximately 2 weeks after the final inducing impacts animals were stimulated by applying media, as well as the test or control sample in concentrations of 20% and 50%, respectively. These concentrations were chosen because it is believed that they do not irritate the skin in combination with ultraviolet irradiation, on the basis of the results obtained from testing futurestate. Animals from the two test groups and the control group is exposed to UVA (10 j/cm2) and UVB (0.1 j/cm2radiation after any dose. Each animal was locally processed extra skin by the media, as well as the test or control sample, however, after treatment the animals were not subjected to UV irradiation. Approximately 24, 48 and 72 hours after stimulation treated areas are checked for the presence of a reaction of irritation from handling.

The positive control reference sample, Musk Ambrette, was investigated using the same method to prove the applicability of the test system. One group of 5 animals (see table 7, group 11) induced by the specified substance in a concentration of 15% in acetone. A control group of 3 animals (see table 7, group 10) was treated in the same way, one carrier (AC is tone). For stimulation was selected concentration of 10% of the reference sample (Musk Ambrette) in acetone.

Results

Stimulation control sample, cream placebo, at a concentration of 50%, followed by ultraviolet irradiation results in response to the control sample in all groups of 10 animals (group 8). The response to the control sample was observed in six of the 10 animals in group 8 on the treated but not irradiated areas. In addition, it was observed the reaction of 5 out of 5 animals in the control group (group 7) on plots treated with stimulation of a control sample, with subsequent UV irradiation and in 4 of 5 animals in the control group on the plots treated with the control sample, but not irradiated. Reaction to one medium was not observed.

Stimulation test sample (0.1%cream at a concentration of 20%), followed by ultraviolet irradiation resulted in response to the test sample in 8 of 10 animals of this group (group 9). The reaction on the test sample was not observed in animals of group 9 in the previously treated areas, but not irradiated. In addition, it was observed the reaction of 5 out of 5 animals in the control group (group 7) on plots treated with stimulation test sample, followed by ultraviolet irradiation and 1 out of 5 animals in the control group in areas obrabotannaya sample but not irradiated. Reaction to one medium was not observed.

Based on the above results it is clear that the response observed in animals treated or the test or control sample. The reaction, which is observed in animals from the control group (not induced by the test sample), most likely caused by irritating substances than sensitization. Furthermore, the reaction can also occur in areas not exposed to UV.

In the conduct of the second stimulation (re-promote) the test and control sample at low concentration of 5%.

The response is not observed in any animals from groups 8 and 9 with repeated stimulation test and control sample at a concentration of 5%, followed by ultraviolet irradiation. The response to the test or control sample is not observed in animals from groups 8 or 9 on the treated but not irradiated areas. The reaction does not occur on any parts of animals from the control group (group 7), processed by the stimulation of the test and control sample. Reaction to one medium was not observed.

Stimulating animals of the positive control reference sample (Musk Ambrette) at a concentration of 10%, followed by ultraviolet irradiation gives a response (erythema from very weak to negligible) in 4 out of 5 alive who were in this group (group 11). The response of the reference sample is not observed in animals of group 11 on the treated but not irradiated areas. The response of the reference sample is not observed in animals of group 10, induced by the media. This shows that the test system is able to detect photoallergic characteristics of the substances.

Conclusions

The results imply that the tested sample is 0.1%cream may not cause photoestrogens or photoallergic reaction after exposure to the skin in combination with ultraviolet radiation.

1. A method of obtaining a polymer conjugate indolocarbazoles the compounds of formula (I)

where
R1and R2are the same and represent hydrogen atoms, and
R3means hydrogen, and
W1and W2mean hydrogen, and
where X represents a methoxy-polyethylene glycol (MPEG),
which includes the interaction of ω-1H-imidazol-carboxamide polymeric compounds of General formula (II)

in which X has the above meaning,
with indolocarbazole compound of General formula (III)

where R1, R2, R3, W1and W2have the above values and where Y means tsepliaeva group selected from metoxygroup.

2. The method according to the .1, which is carried out in the presence of a base in an organic solvent.

3. The method according to p. 2, in which the molar ratio of the base to the compound of formula (III) is between about 1:1 and 4:1.

4. The method according to p. 3, in which the molar ratio of the base to the compound of formula (III) is from about 1:1 to 1.5:1.

5. The method according to p. 4, in which the molar ratio of the base to the compound of formula (III) is about 1:1.

6. The method according to p. 2, wherein the base is selected from the group hydrides of alkali metals.

7. The method according to p. 6, in which the base is selected from sodium hydride.

8. The method according to p. 1, which is carried out in an organic solvent selected from the group of dichloromethane, chloroform and N,N-dimethylformamide.

9. The method according to p. 8, which is carried out in an anhydrous organic solvent.

10. The method according to p. 1, which is carried out in an atmosphere of inert gas.

11. The method according to p. 10, which is carried out in an atmosphere of nitrogen or argon.

12. The method according to p. 1, which is carried out at a temperature of from about 10 to 60°C after the initial phase at 0°C.

13. The method according to p. 12, which is carried out at a temperature from about 15° to 25°C after the initial phase at 0°C.

14. The method according to p. 13, which is carried out at room temperature after the initial phase at 0°C.

15. The method according to p. 1, which additionally includes a step of chromatographic the Russian scavenging polymer conjugate compounds of formula (I).

16. The method according to p. 15, in which the cleaning polymer conjugate compounds of formula (I) is conducted in a solvent selected from dichloromethane, water, methanol, acetonitrile, buffer solution of ammonium formate at different proportions in the mixture.

17. The method according to p. 1, in which the polymer X has a molecular weight of from about 100 to 100,000 Da.

18. The method according to p. 1, in which the polymer X has a molecular weight of from about 200 to 50,000 Da.

19. The method according to p. 1, in which the polymer X is MPEG with an average molecular weight from about 500 to 10,000 Da.

20. The method according to p. 1, in which the polymer X is MPEG with an average molecular weight of approximately 550 Yes.

21. The method according to p. 1, in which the polymer X is MPEG with an average molecular weight of approximately 1100 Yes.

22. The method according to p. 1, in which the polymer X is MPEG with an average molecular weight of about 2000 Da.

23. The method according to p. 1, in which the polymer X is MPEG with an average molecular weight of about 5000 Da.

24. Polymer conjugate indolocarbazoles the compounds of formula (I)

where R1, R2, R3, W1, W2and X are as defined in paragraph 1 or PP.17-23, or its pharmaceutically acceptable salt.

25. Polymer conjugate according to p. 24, where the polymerase is X is MPEG with an average molecular weight from about 500 to 10,000 Da.

26. Polymer conjugate according to p. 24, where the polymer X is MPEG with an average molecular weight of approximately 550 Yes.

27. Polymer conjugate according to p. 24, where the polymer X is MPEG with an average molecular weight of approximately 1100 Yes.

28. Polymer conjugate according to p. 24, where the polymer X is MPEG with an average molecular weight of about 2000 Da.

29. Polymer conjugate according to p. 24, where the polymer X is MPEG with an average molecular weight of about 5000 Da.

30. Polymer conjugate according to any one of paragraphs.24-29 for use in a medicinal product for preventing, alleviating and/or treating skin pathologies selected from psoriasis, atopic dermatitis, chronic eczema, acne, red nails of pityriasis, keloids, hypertrophic scars and skin tumors.

31. Polymer conjugate under item 30 for use in a medicinal product for local use.

32. Polymer conjugate under item 30 for use in medicine for system use, for example, by injection, infusion or inhalation.

33. Pharmaceutical composition for preventing, alleviating and/or treating skin pathologies selected from psoriasis, atopic dermatitis, chronic eczema, acne, red nails of pityriasis, keloids, hypertrophies is their scars and skin tumors containing at least one polymer conjugate according to any one of paragraphs.24-29 as the active ingredient, optionally together with pharmaceutically acceptable carriers, adjuvants, diluents and/or other active in the treatment of pathologies of pharmaceutical agents.

34. Pharmaceutical composition for p. 33 for preventing, alleviating and/or treating skin pathologies selected from psoriasis, atopic dermatitis, chronic eczema, acne, red nails of pityriasis, keloids, hypertrophic scars and skin tumors.

35. The application of a polymeric conjugate according to any one of paragraphs.24-30 for the manufacture of a medicinal product for preventing, alleviating and/or treating pathologies associated with HMGB1.

36. The application of p. 35, where the pathology associated with HMGB1, choose from stenosis, restenosis, atherosclerosis, rheumatoid arthritis, autoimmune diseases, tumors, infectious diseases, General blood poisoning, acute inflammatory lung injury, systemic lupus erythematosus, neurodegenerative diseases, diseases of the Central and peripheral nervous system and multiple sclerosis.

37. The application of p. 35, where the polymer conjugate reversibly attached to the surface of the medical device.

38. The application of a polymeric conjugate according to any one of paragraphs.24-30 for production of the medicinal product for the prevention, facilitate and/or treatment of neurological disorders, diseases of the nervous system and neurodegenerative disorders of the Central and peripheral nervous system.

39. The application of a polymeric conjugate according to any one of paragraphs.24-30 for the manufacture of a medicinal product for preventing, alleviating and/or treating skin disorders.

40. Application on p. 39, where skin diseases are characterized by hyperproliferative keratinocytes.

41. Application on p. 39, where skin diseases are psoriasis, atopic dermatitis, chronic eczema, acne, red hairy pytilias, keloids, hypertrophic scars and skin tumors.

42. The application of p. 41, where cutaneous pathology represents psoriasis.

43. Application on p. 39, where the drug is intended for local administration.

44. The application of p. 43, where the introduction is carried out in the form of liposomes.

45. The application of a polymeric conjugate according to any one of paragraphs.24-30 for the manufacture of a medicinal product for preventing, alleviating and/or treating pain and increased pain sensitivity associated with NGF.

46. The application of a polymeric conjugate according to any one of paragraphs.24-30 for the manufacture of a medicinal product for preventing, alleviating and/or treating inflammatory diseases, autoimmune diseases, systemic syndrome vospitatel the CSO response reperfusion injury after organ transplantation, cardiovascular injuries, obstetric and gynecological complications, infectious diseases, allergic and atopic diseases, pathology of solid and liquid tumors, diseases, transplant rejection, congenital diseases, dermatological diseases, neurological diseases, General atrophy, renal diseases, conditions iatrogenic intoxication, metabolic and idiopathic diseases and ophthalmological diseases.

47. The application of a polymeric conjugate according to any one of paragraphs.24-30 for the manufacture of a medicinal product for preventing, alleviating and/or treating disease behceta, Sjogren syndrome, vasculitis, uveitis, retinopathy.

48. The application of p. 45, where the medicinal product is intended for system administration.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to method of obtaining polymer compounds, containing imidazolium ionic groups, which can be used as dispersers. Method consists in performing interaction of α-dicarbonyl compound, aldehyde, at least, one amino compound, and hydrogen acid, α-dicarbonyl compound representing compound of formula (I) R1-CO-CO-R2, in which R1 and R2 independently on each other represent hydrogen atom or organic residue with 1-20 carbon atoms. Aldehyde represents aldehyde of formula (II): R3-CHO, in which R3 stands for hydrogen atom or organic residue with 1-20 carbon atoms. Amino compound has, at least, two primary amino groups, and represents alkylene diamine with 2-20 carbon atoms, or aromatic diamine, or triamine. Hydrogen acid represents carboxylic acid, sulfonic acid, phosphoric acid or phosphonic acid, and acid groups of sulfonic acid, phosphoric acid or phosphonic acid can be partially etherified. Carboxylic groups of α-dicarbonyl compound and aldehyde, in case of necessity, can be present in form of hemiacetal, acetal, respectively, hemiketal or ketal groups.

EFFECT: invention makes it possible to simplify technological process and increase output of polymer compounds.

9 cl, 1 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to production of binder for polymer composite materials, used to make structures based on carbon fibre filler with operating temperature of 200-400°C, and can be used in aircraft, aerospace, motor car and ship-building and other industries. The polymer binder for composite materials consists of, pts.wt: tetranitrile of aromatic tetracarboxylic acid 100, polyester imide thermoplastic 2-10, amine curing agent 2-6. Also disclosed is a prepreg containing the disclosed polymer binder and fibre filler, with the following ratio of components, wt %: polymer binder 30.0-40.0, fibre filler 60.0-70.0. The fibre filler used is glass fibre or carbon fibre filler.

EFFECT: making high-strength articles at high temperature.

3 cl, 3 tbl

FIELD: chemistry.

SUBSTANCE: method consists of two steps, where at the first step, starting bisphenol A reacts with a cyanuric halide (cyanuric chloride or cyanuric bromide) to form a bisphenol A monocyanate prepolymer, and at the second step, the bisphenol A monocyanate prepolymer reacts with a cyan halide (cyan chloride or cyan bromide) to obtain the desired bisphenol A cicyanate prepolymer.

EFFECT: disclosed method enables partial replacement of the hazardous and poisonous cyan halide with much less hazardous cyanuric halide and also avoid the use at the second step of high temperatures and metal-containing polymerisation catalysts which cut the storage life of the bisphenol A cicyanate prepolymer.

7 ex

FIELD: construction.

SUBSTANCE: invention relates to the field of production of a liquid thermosetting oligomer on the basis of 2,2-bis(4-cyanatiphenyl)propane modified with a polysulfide rubber used as a filling or impregnation compound, and also a polymer base of a binder for structural glass-fibre plastics of electrotechnical purpose. A liquid oligomer is proposed based on dicyanogen ether of diphenyl propane produced by thermal oligomerisation of 2,2-bic(4-cyanatophenyl)propane (70-90 wt parts) in the presence of a modifying additive - a polysulfide rubber of thiocol II-NT (10-30 wt parts).

EFFECT: higher pot life and phase resistance of oligomer down to negative temperatures for increased reliability of polymer electric insulation.

1 cl, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: prepreg contains the following (wt %): adhesive epoxy composition (30-60) and carbonaceous filler (40-70) treated with epoxy finishing agent based on bundles having not more than 2 twists per metre, linear density of 100-72000 tex and consisting of elementary fibres with strength of 3000-4500 MPa.

EFFECT: prepreg has high tensile, compression and shearing strength and high impact resistance of the article made from said prepreg.

3 cl, 2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of producing tetrazole-containing copolymers, having physiological and catalytic activity, as well as high power capacity. Said copolymers can be used to prepare prodrugs, chemical current sources, catalyst systems, as well as energy-intensive binding components of gas-generating systems and special-purpose blasting powder. Method of producing tetrazole-containing copolymers involves mixing hydroxyl-containing high-molecular compounds in dimethyl formamide with acrylonitrile and a catalytic amount of sodium hydroxide, heating the reaction mixture to temperature 50-60°C, adding a suspension of sodium azide and ammonium chloride in dimethyl formamide and then carrying out the reaction at temperature 100-110°C.

EFFECT: obtaining novel copolymers based on hydroxyl-containing high-molecular compounds, having valuable physical and chemical properties and high energy intensity with low sensitivity to mechanical and thermal loads.

2 cl, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method is described for producing electrocatalytic composition based on polypyrrole, involving polymerisation of pyrrole in the presence of platinised soot and surface-active additive, wherein the process is carried out under the effect of a radical initiator in an organic solvent at temperature of approximately 0°C, where the radical initiator is dicyclohexylperoxy dicarbonate, the surface-active additive is a product from reacting tertiary amine (CH3)2NR (R - aliphatic residue C12-14) and propylene oxide in ethyl cellosolve, containing an ionic component - quaternary ammonium base (CH3)2RN+R1(OH)-, where R1- propylene oxide oligomers and a nonionic component - propylene oligomers, and the organic solvent is ethyl cellosolve. After mixing, the components the mixture undergo vacuum treatment at 10-2 mm Hg, and during the initiation process, the system is exposed to an acoustic field with frequency 20 to 22 kHz. Polymerisation process of pyrrole is carried out until obtaining an electrocatalytic composition system which is soluble in organic solvents. In this process electrocatalytic composition is obtained, with the following ratio of said components, wt %: pyrrole 15 to 17; platinised soot 6 to 8; surface-active additive 8-10; dicyclohexylperoxy dicarbonate 5-7; ethyl cellosolve - the rest.

EFFECT: design of an efficient method of producing electrocatalytic composition based on polypyrrole.

2 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention refers to method of producing heat-resistant heterocyclic polymers to be used as binding agents for heat-resistant black-reinforced, glass-reinforced and organoplastics, as a base for glues, sealants, caulking compounds, paint films. Aromatic bis-o-cyanamine reacts with 4,4'-(m-phenylendioxy)diphthalonitrile at temperature 160±2°C. 4,4'-(m-phenylendioxy)diphthalonitrile is introduced by cycles. Bis-o-cyanamine is 1,3-bis(3-cyano-4-aminophenoxy)benzene, or 3,3'-dicyano-4,4'-diaminodiphenylmethane, or 3,3'-dicyano-4,4'-diaminodiphenyloxide.

EFFECT: invention allows developing economical method of producing heat-resistant heterocyclic polymers, reducing possibility of collateral reactions, improving physical-mechanical properties of polymers.

1 tbl, 4 ex

FIELD: polymer materials.

SUBSTANCE: invention relates to a method of producing polymer material from polymers or composition containing polymers being mixtures of fusible ethers of oligotriazine with 4-18 rings having linear and branched structures. Invention further relates to polymer material and application thereof, to products in the form of finished articles, and to a method of fabrication thereof. Method of preparing polymer material comprises condensation of aminotriazine ethers having following structure: , where R1 represents R4-O-CHR3-NH- or [R4-O-CHR3]2N-; R2 represents -NH2, -NH-CHR3-OR4, -N[CHR3-O-R4]2, -CH3, -C3H7, -C6H5, -OH, phthalimido, or succinimido; and R3 is H or C1-C-7-alkyl, to form polymer in the form of fusible oligotriazine ethers having following structure: . Polymer material may contain up to 75 wt % of fillers and/or reinforcing fibers, ethylene copolymers, maleic anhydride copolymers, modified maleic anhydride copolymers, polymethacrylates, polyamides, polyesters and/or polyurethanes, and up to 2 wt % stabilizers, UV absorbers and/or supplementary agents. Method of producing products in the form of profiled pieces, sheets, swelled material sheets, tubes, laminates, parts, strands, or fibers consists in melting polymer material and further processing thereof by conventional manners suitable for thermoplastic polymers: calendaring, jet molding, extrusion, and the like.

EFFECT: enabled production of polymer material soluble in polar solvents such as C1-C10-alcohols, dimethylformamide, or dimethylsulfoxide and manufacturing of products with good mechanical characteristics.

26 cl, 11 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to a method for synthesis of copolymers of pyridine or methyl-substituted pyridine with vinyl epoxy-compounds. Method involves heating pyridine or methyl-substituted pyridine at 50-145°C for 2-7 h with vinyl epoxy-compound in the mole ratio of reagents from 1:1 to 10:1. Also, invention relates to coupled copolymers of pyridines synthesized by above indicated method of the general formula: wherein R1 means hydrogen atom (H), 2-, 3-, 4-CH3; R2 means -CH2O(CH2)2OCH=CH2, or R2 means -CH2OCH2CH=CH2. Synthesized polymers possess electric conducting and paramagnetic properties. The parent compounds are easily available.

EFFECT: improved method of synthesis.

4 cl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to conjugates of Disorazol with cell adhesion molecules, such as peptides, proteins, hormones, blood proteins, and methods for preparing conjugates.

EFFECT: conjugates of Disorazol may be used as drug preparations for treating various tumours.

19 cl, 17 dwg, 2 tbl, 17 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel crystalline polymorphous forms of the rifaximin antibiotic (INN) known as rifaximin δ and rifaximin ε. The invention also relates to a method for synthesis of such polymorphs, which is realised through hot dissolution of crude rifaximin in ethanol and crystallisation of the product, which is induced by adding water at temperature of approximately 50°C and is carried out for approximately 4-36 hours, with subsequent drying at controlled conditions until attaining given content of water in the end product. Novel polymorphs have properties which enable to control rifaximin absorption when used as an antibiotic. The invention also relates to pharmaceutical compositions containing the novel polymorphous forms of rifaximin.

EFFECT: obtaining novel polymorphous forms of rifaximin.

20 cl, 2 tbl, 3 ex, 2 dwg

Organic compounds // 2394038

FIELD: medicine.

SUBSTANCE: new crystal form II N-benzoilstaurosporin is described, as well as pharmaceutical composition containing it, inhibiting protein kinase C; ways of obtaining crystalline form of compounds, and the use of crystal form of II N-benzoilstaurosporin for treatment neoplastic diseases. Crystalline form of II N-benzoilstaurosporin is a stable form of N-benzoilstaurosporin with low hygroscopicity.

EFFECT: suitability in pharmaceutical compositions.

21 cl, 2 tbl, 4 dwg, 16 ex

FIELD: medicine.

SUBSTANCE: invention refers to a compound of the formula , where R1 and R2 are different independent groups and are selected from the group consisting of OR3 and N (R3') (R3"); or R1 and R2 are different groups connected through a single bond and selected from the group consisting of O and NR3; R3, R3', and R3" are independently selected from the group consisting of H, phenyl, substituted phenyl, where substituents are independently selected from the group consisting of C1-C6 alkyl, halogen; R4 and R4': (a) independently selected from the group consisting of H, OH, a group of the formula ; R5, R6, and R7 are independently selected from the group consisting of OCH3; R8 and R9 are joined by (i) a single bond and represent CH2 or (ii) double bond and are CH; R15 are selected from the group consisting of C=O; n is equal to 2. The invention also refers to method for obtaining these compounds.

EFFECT: obtaining new compounds which can be used in medicine as neurodefensive and neurogenerative, antiproliferative and anti-inflammatory drugs.

43 cl, 7 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: regioselective synthesis of complex rapamycin 42-ether (CCI-779) involves: (a) acylation of 31-silyl rapamycin ether by compound of formula HOOC.CR7R8R9 or its combined anhydride, where: R7 is hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, alkinyl with 2-7 carbon atoms, -(CR12R13)fOR10, -CF3, -F or -CO2R10; R10 is hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, alkinyl with 2-7 carbon atoms, triphenylmethyl, benzyl, alcoxymethyl with 2-7 carbon atoms, chloroethyl or tetrahydropyranyl; R8 and R9 together form X; X is 2-phenyl-1,3,2-dioxaborinane-5-yl or 2-phenyl-1,3,2-dioxaborinane-4-yl, where phenyl can be optionally substituted; R12 and R13 each is independently hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, alkinyl with 2-7 carbon atoms, trifluormethyl or -F; and f=0-6; to obtain 42-ether boronate of 31-silyl rapamycin ether; (b) selective hydrolysis of 42-ether boronate of 31-silyl rapamycin ether in moderately acid environment to obtain rapamycin 42-ether boronate; and (c) diol treatment of rapamycin 42-etherboronate to obtain complex rapamycine 42-ether. Invention also claims new intermediate products applicable in this method.

EFFECT: application as antitumour medication.

48 cl, 3 ex

FIELD: organic chemistry, biochemistry.

SUBSTANCE: invention relates to some new macroheterocyclic compounds that can act as selective inhibitors of kinase or double kinase. Invention describes compounds of the following formulae: formula (Ia1), formula (Ib1), formula (If1), formula (Ii1), and formula (Ij1) wherein values R2, R4 and R5 are chosen by the dependent manner as given in the invention claim. Invention provides preparing new compounds possessing valuable biological properties.

EFFECT: valuable biological properties of compounds.

5 cl, 3 tbl, 22 ex

Derivative to-a // 2205184
The invention relates to new derivatives of K-a (a derivative of indolocarbazole), which are represented by the General formula 1, as well as to a method for improving the functioning and/or increase the survival of cholinergic neurons and the way to improve cell survival at risk of death because of the compounds of formula 1 inhibit production of interleukin-2 and have immunosuppressive activity

The invention relates to novel analogues of camptothecin, in particular to the compounds corresponding to the following formulas (I) and (II), as well as their racemic or enantiomeric forms or combinations of these forms, where the substituents have the values

The invention relates to a new class of chemical compounds, namely to derive a new heterocyclic system - tetrabenazine[3,4-b:3',4'-f: 3", 4"-j: 3"', 4"'-n-[1,4,5,8,9,12,13,16]-actuatable[14.2.2]eicosa-4,8,12-triens General formula (I), where R1- R4= H, lower alkyl or lower alkyl containing functional groups, such as or SIG5, SR5, NR5R6, СОNR5R6or СООR7where R5, R6, R7= H, lower alkyl, or R2+ R3and/or R1+ R4together with the neighboring carbon atoms pieperazinove cycle form an alicyclic, benzene or heterocyclic annelirovannymi cycle

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to benzoimidazole derivatives of formula (I), as well as to their enantiomers, diastereoisomers, racemates and pharmaceutically acceptable salts, where n equals from 2 to 4, each of R1 substituents is independently selected from H, halogen, -C1-4alkyl, -C1-4pergaloalkyl, trifluoro-C1-4alkoxy, -NO2, -CN, CO2H, -OC1-4alkyl, -SC1-4alkyl, -S(C1-4alkyl)-Rc, -S(O)2(C1-4alkyl)-Rc, -S(O)-C1-4alkyl, -SO2-C1-4alkyl, -S-Rc, -S(O)-Rc, -SO2-Rc, -SO2-NH-Rc, -O-Rc, -CH2-O-Rc, -C(O)NH-Rc, -NRaRb, benzyloxy, phenyl, optionally substituted with one-two Rd, cyanobiphenyl-4-ylmethylsulpfanyl, cyanobiphenyl-4-ylmethanesulphonyl, or -S-(CH2)2-morpholine and two adjacent groups R1 can bind with formation of an aromatic 5-6-membered ring, optionally substituted with one methyl group or two atoms of halogen, optionally containing one or two S or N; Ra and Rb each independently represents C1-4alkyl, -C(O)C1-4alkyl, -C(O)-Rc, -C(O)CH2-Re, C1-4alkyl-Re, -SO2-Rc, -SO2-C1-4alkyl, phenyl, benzyl; or Ra and Rb together with a nitrogen atom, which they are bound with, form a monocyclic 5-6- membered heterocycloalkyl ring, optionally containing one heteroatom, selected from O; Rc represents -C3-8cycloalkyl, phenyl, optionally substituted with one-two Rd, benzyl, optionally substituted with one-three Rd; morpholine; Rd independently represents halogen, -OH, -C1-4alkyl or -C1-4perhalogenalkyl, trifluorine C1-4alcoxy, -OC1-4alkyl, or -O-benzyl optionally substituted with halogen, Re represents -C6heterocycloalkyl, optionally containing one or two of O or N atoms, optionally substituted with a methyl group; R2 and R3 both represent H, -CF3 or C1-3alkyl; each of Z represents a C or N atom, on condition that simultaneously not more than two Z represent N. The invention also relates to particular compounds, a pharmaceutical composition, based on formula (I) compound or a particular said compound, a method of treating diseases, mediated by propyl hydroxylase activity.

EFFECT: novel derivatives of benzimidazole, possessing an inhibiting activity with respect to PHD are obtained.

11 cl, 1 tbl, 186 ex

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