Derivative of6-alkylguanine and pharmaceutical composition

 

(57) Abstract:

Describes new derivatives of O6-guanidine of the General formula I, where Y is hydrogen, ribosyl or deoxyribosyl, which may be substituted by hydroxy or C1-C4alkoxygroup; R1represents hydrogen, C1-20alkyl or hydroxy WITH1-20alkyl; R represents (i) a cyclic group having a five-membered heterocyclic ring containing at least one heteroatom selected from O, N or S, optionally condensed with a benzene ring, pyridine or naftalina or six-membered heterocyclic ring containing at least one atom of N, not necessarily with the S atom, optionally condensed with one or two benzene rings, where this cyclic group may be optionally substituted in the heterocyclic () the ring(s) and/or carbocyclic () the ring(Oh) groups, selected from C1-C5of alkyl, halogen, cyano, nitro, azido,1-C5alkoxy, aryl, SOnR"" where R"" is1-C5alkyl and n is 0, 1, 2, COOR5where R5represents N or C1-C5alkyl or N-oxides. These compounds exhibit the ability to reduce active analogues. Also described composition based on compounds of the formula I. 3 C. and 11 C.p. f-crystals, 14 tab., 7 table.

The invention relates to O6substituted derivatives of guanine, method of their production and to their use for the treatment of tumor cells. The invention in particular relates to a derivative of guanine with getaline or naphthylethylene the substituents in the O6-position, these compounds have the ability to reduce the activity of O6-alkylguanine-DNA alkyltransferase (Atasi) in tumor cells.

Background of the invention

It was proposed to use ABOUT6-alkyl derivatives of guanine, which are able to reduce the activity OF6-alkylguanine-DNA alkyltransferase, to enhance the effectiveness of chemotherapeutic alkylating agents, which are used to kill cancer cells. There is increasing evidence that the toxic and mutagenic effects of alkylating agents on mammalian cells is largely the result of alkylation OF6-position of guanine in DNA. Recovery OF6-alkylguanine contributes Ataza - protein-reducing agent, which affects ABOUT6-alkali is in its active site of the protein reductant, in autoactivation process. Important role Atasi in protecting cells from the biological effects of alkylating agents is most clearly been demonstrated by transfer and expression of genes Atasi or complementary DNA in cells with a deficit Atasi: this increases the resistance to the action of various agents, mainly those, which was identified in or chloranilic DNA. Although the mechanism of cell killing under the action of O6-methylguanine in cells with a deficit Atasi still not clarified, the destruction of cells under the action of O6-chloroethylamine occurs due to the formation of miaocheng links to the rest of cytosine opposite chains through cyclic Atamanenko intermediate compounds, and this process is inhibited due to stimulated Atasoy removal chlorethylene group or formation of the complex.

The use OF6-methylguanine and6-batillaria to weakening activity Atasi described (Dolan et al., Cancer Res., 1989, 46, p. 4500; Dolan et al., Cancer Chemother. Pharmacol, 1989, 25, p. 103). It is proposed to use derivatives OF6-benzylguanine to weakening activity Atasi, to make cells expressing Atasu, more susceptible to cytotec patent application WO 91/13898 (Moschem et al.) provides a method of reducing the content OF6-alkylguanine-DNA transferase in the tumor cells of the recipient, which is the appointment to the recipient an effective amount of a composition containing ABOUT6-benzylidene derivatives of guanine following formula:

< / BR>
where Z denotes a hydrogen atom, or

< / BR>
or

< / BR>
Radenotes a benzyl group or substituted benzyl group.

Benzyl group may contain in the ortho-, meta - or para-position such substituents as a halogen atom, nitro, aryl, such as phenyl or substituted phenyl, alkyl containing 1-4 carbon atoms, alkoxy containing 1-4 carbon atoms, alkenyl containing up to 4 carbon atoms, quinil containing up to 4 carbon atoms, amino, monoalkylamines, dialkylamino, trifluoromethyl, hydroxy, hydroxymethyl, and SOnRbwhere n is 0, 1, 2 or 3, and Rbdenotes a hydrogen atom, alkyl containing 1-4 carbon atoms, or aryl. Article Mi-Joung Chae et al., J. Med. Chem., 1994, 37, pp. 342-347 published after the priority date of the present patent application describes tests analogues OF6-benzylguanine containing in position 9 benzyl rings of increasing the size of the substituents. Connection N 6 there adnaco in the section Results and Discussion results on pages 342-343 article Chae et al. it is underlined that the Connection 6 may be of interest, and included in the group "other connections" that have intermediate activity" (page 343, line 12-15 in the text of article). The authors confirmed the previously made observations (J. Med. Chem., 1992, 35, R. 4466) that only allyl or benzyl substituents in the O6-position of guanine effectively inactivate Atasu (page 343, line 21-23 in the text of article).

ABOUT6-Benzylguanine has limited use as desactivate Atasi. It is more stable than is necessary, which leads to the fact that he has long maintained in the animal, which he entered. This compound is potentially toxic alone or in combination with chloroethylamine agents, which also is undesirable and may be associated with survival time.

Compounds of the present invention possess excellent from the6-benzylguanine inactivating properties in relation to Atase, and in some cases their activity up to 8 times higher than the activity of O6-benzylguanine. We have also studied different half existence and toxic properties. Thus, an object of the present invention are novel compounds useful on tiliroside or meteorous antitumor agents.

Another objective of the present invention are pharmaceutical compositions containing compounds that are useful for weakening activity Atasi. Another objective of the present invention is a method of reducing the activity of Atasi in tumor cells. Finally, another aim of the present invention is a method of treating tumor cells of the recipient.

Description of the invention

Thus, the present invention relates to a derivative OF6-alkylguanine formula

< / BR>
where Y is hydrogen, ribosyl or deoxyribosyl, which may be substituted by hydroxy or C1-C4alkoxygroup, R' represents hydrogen, C1-C20alkyl or hydraxis1-C20alkyl;

R stands for

(i) a cyclic group having a five-membered heterocyclic ring containing at least one heteroatom selected from O, N or S, optionally condensed with a benzene ring, pyridine or naphthalene, or six-membered heterocyclic ring containing at least one atom of N, not necessarily with the S atom, optionally condensed with one or two benzene rings, where this cyclic group may be neobyazatel>-C5of alkyl, halogen, cyano, nitro, azido, C1-C5alkoxy, aryl, SOnR"" where R"" represents a C1-C5alkyl and n is 0, 1, 2, COOR5where R5represents H or C1-C5alkyl or N-oxides,

(ii) naphthyl, optionally substituted by halogen or C1-C5alkoxy, or their pharmaceutically acceptable salts.

In particular, the invention relates to a compound of formula I, where R contains a heterocyclic ring condensed with the benzene ring, and where O6-alkylguanine group attached to R or heterocyclic, or a benzene ring.

Preferred is a compound of formula I, where R is a five-membered heterocyclic ring containing one sulfur atom.

For example, R can be selected from a thiophene ring, furan ring or their substituted derivatives.

Preferred is also a compound of formula I, where R is a cyclic group substituted with halogen, cyano, SOnR"" where R"" represents a C1-C5alkyl and n = 0, 1, or 2, or-COOR5where R5represents C1-C5alkyl. For example, R can be selected from a thiophene ring, furan ring and the Association of the formula I, where-CHRR' is ABOUT6-tanila or bromine-substituted derivatives.

Preferred is also a compound of formula I, selected from

ABOUT6-analgoidea;

ABOUT6-(3-trimethyl)guanine;

ABOUT6-piperidylamine;

ABOUT6-furfurylamine;

ABOUT6-(3-furylmethyl)guanine;

ABOUT6-(2-benzo[b]thienylmethyl)guanine;

O6-(2-benzofuranyl)guanine;

ABOUT6-(5-triazolylmethyl)guanine;

ABOUT6-(5-ethoxycarbonylphenyl)guanine;

ABOUT6-(5-bromanil)guanine;

ABOUT6-(5-cyanophenyl)guanine;

ABOUT6-(2-benzo[b]thienylmethyl)guanosine;

ABOUT6-(4-picolyl)guanine;

ABOUT6-(2-naphthylmethyl)guanine.

The proposed compounds of formula I can be used to obtain a pharmaceutical composition having the ability to weaken the activity OF6-alkylguanine-DNA alkyltransferase in tumor cells. Preferred is ABOUT6-tanilgan or its substituted derivative.

Also proposed pharmaceutical composition having the ability to weaken the activity OF6-alkylguanine-DNA alkyltransferase the owner, containing a derivative of guanine and fctive amount of the compounds of formula I or its pharmaceutically acceptable salt according to any one of the preceding paragraphs.

The pharmaceutical composition may further comprise an alkylating agent, which may be, for example, selected from 1,3-bis(2-chloroethyl)-1-nitrosoanatabine (BCNU) and temosolomida.

In addition, the method for obtaining derivatives OF6-alkylguanine formula I, described above, consists in the fact that carry out the following stages: interaction of sodium hydrate solution RR'OH, where R and R' above, in an organic solvent, adding a chloride of 2-amino-N,N,N-trimethyl-1H-purine-6-amine or 2-amino-6-glucuronate, the processing of a weak acid and ether and extraction of the target product.

Examples of compounds of the present invention, together with the compounds Century 4214 and C. 4218, which are not included in the scope of the invention) shown in table 1.

Among the compounds listed in table 1, the compounds B. 4214 and C. 4218 are not compounds of the present invention. Connection Century. 4210 (i.e., compound of formula I in which R represents 2-pyridyl, R' denotes H, and Y denotes H) is not the preferred compound of the present invention.

The most preferred compounds of the present invention include:

Century 4205 ABOUT6tanilgan

Century 4206 ABOUT6-(3-tie ABOUT6-(2-benzofuranyl)guanine

Century 4275 ABOUT6-(5-triazolylmethyl)guanine

and compounds substituted in the heterocycle group R by halogen atoms, cyano or ester group, including

Century 4229 ABOUT6-(5-ethoxycarbonylphenyl)guanine

Century 4269 ABOUT6-(5-bromanil)guanine

Century 4273 ABOUT6-(5-cyanophenyl)guanine.

Other preferred compounds include

Century 4209 ABOUT6-(3-furylmethyl)guanine

B. 4276 ABOUT6-(2-benzo[b]thienylmethyl)guanosin

B. 4277 ABOUT6-(4-picolyl)guanine.

The most preferred compounds according to the invention are those connections that inactivate Atasu in vitro and/or in mammalian cells and/or tumor xenografts is more efficient than6-benzylguanine (BeG), and which stimulate mammalian cells and/or tumor xenografts at the destruction of cells or stimulate the inhibitory effect of nitrosamine and/or meteorous agents more effectively than BeG. The preferred connection must have compared to BeG less toxic to normal tissues and/or body as a whole when combined with these agents. Predpochtitelnye in doses required for inactivation of Atasi; moreover, should not be any toxic products of the hydrolysis is the preferred connection, which is not chemically resistant. Although the invention is not limited to any theoretical conclusions, it may be that the preferred connection must be less persistent than BeG, so that they could undergo spontaneous destruction soon after achieving the maximum degree of inactivation of Atasu: in this case, any impact of the exchange processes on the agent, leading to the formation of toxic derivatives, could be minimized. Preferred compounds have less to increase the sensitivity of human bone marrow or other types of normal cells to the toxic effects of alkylating agents, so that they do not reinforce known to be toxic or not would lead to the emergence of the toxicity of these agents in normal human tissues.

Preferred compounds of the present invention include compounds having relatively low values of I50below in table 4 (in particular, below 1.0 μm, more preferably less than 0.04 mcm), and/or with the conditions in vitro) and/or phosphate buffered physiological saline solution (an example of a physiological environment), listed below in table 4 (in particular, less than 20 h in Buffer I or less than 16 hours in phosphate buffered saline solution).

A relatively short period of existence may be an indication that the connection according to the invention is less stable than the O6-benzylguanine due to the reactivity of group RR'CH - and has a tendency to collapse during the hydrolysis in physiological environment.

Group influence RR'CH - in the compounds of formula I, allowing them to act as inhibitors of Atasi, defined by the electronic, steric and physicochemical factors. Steric factors may be associated with properties of the nearest environment of the site cysteine receptor Atasi. Mainly, R' denotes a hydrogen atom. It was shown that the second carbon atom attached to the O6(as in DL derivatives B. 4214 or B. 4217), significantly weakens inactivating ability, likely due to the large size of the Deputy.

A cyclic group R does not contain a methyl group in vicinal position (as in Century 4222), although the influence of vicinal Deputy, obviously, much less in the case when R is a heterocycle, than in the case of naphthionate for distribution in the water - lipid important when selecting compounds for use in vivo and affect the composition, absorption and transfer connections. The choice of compounds can influence their different ability distribution in various tissues.

In one of the embodiments of the present invention is claimed pharmaceutical composition comprising a compound of formula I, where the values of Y, R and R' are indicated previously, or its pharmaceutically acceptable salt and pharmaceutically acceptable filler. The composition optionally may also contain an alkylating agent, such as chloranilide or metymirumi agent.

In another embodiment, the present invention claims a method of reducing the activity of Atasi the recipient, which is to assign to the recipient an effective amount of a composition containing a compound of formula I, where the values of Y, R and R' are indicated previously, or its pharmaceutically acceptable salt, in particular, the above composition. This method otherwise you can define as a way of weakening stimulated Atasoy activity recovery of DNA from the recipient.

In the invention hereinafter claimed a method of treating tumor cells in the body, consisting of R and R' are indicated previously, or its pharmaceutically acceptable salt, in particular, the above composition, and appointment to the recipient an effective amount of a composition containing an alkylating agent. The method can be used to treat tumors that are sensitive to the action of alkylating agents, in particular melanoblastoma and gliomas and other in which resistance to the action alone alkylating agents can be overcome by the use of the activator of the present invention.

In the claimed invention is also a method of obtaining compounds of formula I, comprising the following stages: interaction of sodium hydride with a solution RR'CHOH (where R and R' are listed above) in an organic solvent, mainly at room temperature or a temperature lower than room temperature, the addition of chloride of 2-amino-N,N,N-trimethyl-1H-purine-6-amine or 2-amino-6-globulinemia; treating the reaction mixture with a weak acid in ether; and extraction of the target product.

Brief description of drawings

The invention is explained in more detail with reference to the accompanying drawings, of which:

In Fig. 1 graphically presents the residual activity of purified recombinant Atasi man followed in the t average of three values. Line corresponding to the 50% activity, is used to calculate the value of I50, i.e. the concentration of deactivator, and needed to reduce by 50% the activity of Atasi.

In Fig. 2 shows two graphs of the dependence of cell growth on the concentration of alkylating agent (μg/ml), which show the sensitizing effects OF6-benzylguanine (BeG) and6-teilhardina (Century 4205) at two different concentrations (0.1 and 1.0 μm) on BCNU in sensitization of Raji cells. Line corresponding to the 90% rate is used to calculate the values of D90i.e. dose BCNU, at which there is 90% growth compared with non-exposed control samples, i.e., corresponds to growth inhibition by 10%.

In Fig. 3 shows four graphs of the dependence of cell growth, expressed in percent, from the concentration of alkylating agent (μm), which show the effect of four different concentrations of 0.1, 0.5, 1.0 and 5.0 μm) and BeG Century 4205 temozolomide when sensitization of Raji cells.

Fig. 4 is a graph obtained by extrapolation of the values shown in Fig. 3, for values of D50(μm) (i.e., dose temosolomida at which there is 50% increase, compared to not paderna Fig. 5 shows two graphs of the dependence of cell growth, expressed in percent, of the concentration of inactivator, which show an increase inhibitory effects OF6-furfurylamine (Century 4203) and Century 4205 cells V79 Chinese hamster (RKJO) and compared to (+120).

In Fig. 6 shows two graphs of the dependence of cell growth, expressed in percent, of the concentration of inactivator, which show an increase inhibitory effects Century 4203 Century and 4205 of the subclones Xeroderma pigmentosum.

In Fig. 7 shows four graphs of the dependence of cell growth, expressed in percent, of the concentration of inactivator (μm), which show the influence of the decay products of inactivation Century 4203, Century 4205 Century and 4212 (O6-piperidylamine) and Century 4226 (O6-[2-benzo(b)thienylmethyl]guanine) on the growth of Raji cells.

Fig. 8 is a diagram activity Atasi (FM/mg) time (in hours), which shows the attenuation of the activity of Atasi in xenografts A naked mice for non-exposed control samples treated with corn oil, and extracts, processed BeG (30 mg/kg) and Century 4205 (30 mg/kg).

In Fig. 9-13 shows the graphs of the results of the study xenografts. the days xenografts A naked mice exposed to one BCNU, BeG in combination with BCNU and Century 4205 in combination with BCBU. Bottom graph in each figure shows the number of surviving mice in each exposed group depending on time (in days) after treatment, are shown in the top graph.

The drawings have the following differences (table. 2)

In Fig. 14 shows three graphs of the dependence of the survival rate on the concentration temosolomida (μm), which indicate the survival of bone marrow cells with subsequent processing inactivation (10 μm) or DMSO (control samples) in combination with increasing doses temosolomida.

Ways of carrying out the invention

Derivative OF6-getalllanguages with inactivating abilities in relation to Atase can be obtained by adapting standard techniques, which are presented below.

Chloride 3-amino-N,N,N-trimethyl-1H-purine-6-amine receive in accordance with the methodology described in article Kiburis et al., J. Chem. Soc. (C), 1971, 3942. Detailed conditions for the reaction of this Quaternary salt benzisoxazol sodium (with the formation OF6-benzylguanine), not mentioned in the article Mc Coss, Chen and Folman, Fetrahedron Zett., 1985, 26, 1815, in the European so applicants was developed following the standard technique.

The standard technique to obtain ABOUT6-getallalbumsindb (Formula I, Y = H)

Sodium hydride (60% in oil; 0.8 g, 20 mmol) are added to a solution RR'CHOH (56 mmol, approx. 5 ml) in DMSO (5 ml) and the mixture is stirred at room temperature for 1 hour. For solid alcohols or alcohols with high molecular weight instead of 5 ml can be used up to 10 ml DMSO. Add the chloride of 2-amino-N, N, N-trimethyl-1H-purine-6-ammonium (to 2.29 g, 10 mmol) and stirring is continued for 1 hour. The change in UV absorption ends (max312 ---> 284 nm) and almost clear solution is treated with acetic acid (1.7 ml). After cooling and dilution with ether (300 ml), the mixture is left to stand (2 hours) and solid (A) is filtered off. Triturated with water to give the target compound. The second fraction can be obtained after evaporation of the filtrate ether-DMSO and trituration of the residue successively with ether and water. Otherwise, the product may be extracted from the solids (A) warm acetonitrile. Recrystallized compounds give a single spot according to thin-layer chromatography (TLC) (benzene:methanol, 4:1) and confirmed by the elemental analysis data and NMR spectroscopy. They often contain crystallization spectroscopy are shown in table 3. Spectra 1H NMR obtained on a spectrometer Bruker WP80 and MSL 300.

This standard methodology (variations are shown as symbols in table 2) is used to obtain the compounds listed in tables 2A and 3A. In compounds Century 4217 and Century 4219 R' denotes methyl; in other compounds R' denotes h O6-Benzylguanine and Compounds Century 4214, C. 4218 Century and 4231 below in table 4, obtained according to this standard methodology for conducting comparative tests.

Variations indicated by the symbols in table 2 indicate the following:

a) For this connection using 5 mmol of sodium hydride in 1 mmol Quaternary salt. If you use the standard number (2 mmol), in the development of the reaction mixture may be recovered 40% of the Quaternary salt.

b) This compound is obtained by hydrolysis of the methyl ester Century 4229 (145 mg, 0.5 mmol) in 2-methoxyethanol (2.5 ml) and water (2.5 ml) by treatment with 2 m sodium hydroxide solution (2.5 ml) for 4 h at room temperature. Neutralize with acetic acid (0,32 ml, 5.5 mmol), carefully evaporated, triturated with water (3 ml) and filtered, obtaining a solid substance, which after extraction with methanol receive acid Century. 4234.

C) Deobhog,3%. Found: Na Of 4.44%.

d) Instead of the standard number of 5.6 mmol use 3 mmol of alcohol RCH2OH on 1 mol of the Quaternary salt.

(e) For alcohols, which are too sensitive to the action of sodium hydride in DMSO at room temperature, DCH2ONa get in DMF (2.5 ml at minus 10oC; 3 mmol RCH2OH; 2 mmol of sodium hydride). After 15-20 minutes, add 1 mmol Quaternary salt and stirring is continued for 2-3 h at room temperature.

f) the Products are extracted with acetonitrile.

Getting riboside (Formula I, Y = ribosyl)

The alkoxide solution obtained by the above standard method of sodium hydride (60% in oil, 120 mg, 3 mmol) and RR'CHOH (4.6 mmol) in dry DMSO (2 ml) for 1 hour, treated with 2-amino-6-globulinemia (302 mg, 1 mmol) and stirred for 5 minutes at room temperature, and then 15 minutes at a temperature of 60 - 65oC. On completion of the reaction by UV-spectrum (max311 ---> 284 nm). Cooling and thorough rubbing with ether (100 + 15 ml) followed by filtration allows to obtain a solid substance, which is treated with water (10 ml). Bring the pH from 11 to 8, long flowing carbon dioxide. Neorganics(four 10 ml, containing a drop of pyridine). After evaporation of the total number of methanol and addition of a small amount of ether to obtain the product, pure according to TLC (benzene:methanol, 4:1). It is recrystallized from methanol containing a trace amount of pyridine, dissolving and pariva at a temperature below 40oC, sometimes at the end add a little ether.

This method is used to obtain the compounds shown in tables 2b and 3b. Trace amounts of impurities difficult to remove, however, the data of NMR spectroscopy show that the purity of the nucleosides is approximately 90%. The outputs are of the order of 30-40%.

The original alcohols RR'CHOH usually get recovery of the corresponding aldehydes, most of which is produced by using sodium borohydride. Another approach used to produce precursors of ester Century 4229 and nitrile Century 4273. Sucrose turn [W. N. Haworth and W. G. M. Jones, J. Chem. Soc., 1944, 667] formed through the intermediate 5-chloromethylphosphonate 5-gidroksimetilfurfural. Oxidation [T. Reichstein, Helv. Chim.Acta, 9, 1926, 1066] this aldehyde get carboxylic acid, which etherification method Bocchi et al. [V. Bocchi, G. Casnati, A. Dossena and R. Marchelli, Synthesis. 1979, 961ym. Chem. Ed., 22, 1984, 863], required for the synthesis Century 4229. The oxime [J. Kiermayer, Chemiker-Zeitunq, 19, 1895, 1004] aldehyde dehydration method Carotti et al. [A. Carotti, F. Campagna and R. Ballini, Synthesis. 1979, 56]; the crude product before extraction with dichloromethane pre-treated with a methanol solution of concentrated aqueous ammonia. After distillation 5-cyanophenyl alcohol [A. J. Floyd, R. G. Kinsman, Y. Roshan-Ali and D. W. Brown, Tetrahedron. 39, 1983, 3881], required for the synthesis Century 4273.

In the synthesis Century 4266 by the reaction of Vilsmeier [V. T. Suu, N. P. Buu-Hoi and N. D. Xuong, Bull. Soc. Ghim. France, 1962, 1875] from benzofuran get 2-aldehyde, which restores to the desired alcohol [T. Reichstein and L. Reichstein Helv. Chim. Acta. 13, 1930, 1275], and the synthesis Century 4226 beginning a lithium derivative, which is treated with dimethylformamide and get 2-aldehyde and alcohol [N. Takeshita, H. Mametsuka and H. Motomura. J. Heterocycl.Chim., 23, 1986, 1211].

In the synthesis Century 4274 dimethyl ester of tartaric acid oxidizes [F. J. Wolf and J. Weijlard, Orq. Synth. Coll. Vol. 4, 1963, 124] methyl ester of Glyoxylic acid, which interacts [A. M. van Leusen, B. E. and H. Hoogenboom Siderius, Tetrahedron Lett., 1972, 2369] toiletrieschoice with methyl ether oxazol-5-carboxylic acid [A. Maguestiau, R. Flammang and F. B. Abdelouahab, Heterocycles, 29, 1989, 103]. Restore it.. 112, 1990, 4070].

In the synthesis Century 4275 from bromoanisole aldehyde [S. Trofimenko, J. Orq. Chem. , 28, 1963, 3243] and thiourea get 2-aminothiazol-5-carboxaldehyde [I. Sawhney and J. R. H. Wilson (Shell Internationale), EP. 395, 174, 1989 (Chem. Abs.. 114, 143410s)]. It will dataminimum the amilnitrita [I. Sawhney and J. R. H. Wilson (Shell Internationale), EP. 395, 174, 1989 (Chem.Abs., 114, 143410s)], followed by reduction with sodium borohydride and get a 5-hydroxymethylimidazole [S. Fallab, Helv. Chim. Acta. 35, 1952, 215].

In the synthesis Century 4271 5-aspiringly alcohol (so pl. 82-84oC) found: C 54,60; H 4,58; N is 9.09; calculated for C7H7NO3: C 54,90; H br4.61; N 9,15%) are obtained from the corresponding aldehyde [F. Dallacker, P. Fechter and V. Mues, Z. Naturforsch., 34b, 1979, 1729].

In the synthesis Century 4278 1-methylpyrrole-2-carboxaldehyde nitrous [R. Fournari, Bull. Soc. Chim. France, 1963, 488] and reduced to alcohol [L. Grehn, Chem.Scr., 16, 1980, 72] using sodium borohydride.

As a concrete example, the methodology for obtaining OF6-teilhardina (Century 4205):

Getting ABOUT6-analgoidea

The solution tinylogo alcohol (3,18 ml, 33.6 mmol) in DMSO (3 ml) is treated with sodium hydride (60% in oil; of 0.48 g, 12 mmol), spending the first careful mixing. After 1 hour, add the chloride of 2-amino-N,N,N-trimethyl-1H-purine-6-amine (1,37 g, 6 mmol) and stirring prodl). 1-2 hour emit particulate matter (2,09 g). After evaporation of the ether and distillation of DMSO and excess tinylogo alcohol (so pl. 48-57oC/0.2 mm RT. Art.) get the balance from which the rubbing with ether to get the second portion of the solid (0.36 g). The received portion of the solid product combined triturated with water (6 ml) and receive the product (1,335 g, 90%), which shows one big spot according to TLC (benzene:methanol, 4: 1) and only trace amounts of impurities. After dissolving in hot ethanol (30 ml), clarification by filtration through celite and evaporation (up to 10 ml using a rotary evaporator receive B. 4205 (1,125 g, 71%, contains 1/3 EtOH at mol in the form of MES).

The compounds of formula I in which Y denotes R"XCHR'" (SECO-nucleosides), can be obtained analogously to the reaction of O6-benzylguanine with esters of alpha-chlorocarbonic acids (MacCoss et al., Fetrahedron Lett.; European patent application 184473) or were synthesized (in particular, Kjellberg, Liljenberg and Johansson, Fetrahedron Lett., 1986, 27, 877; Moschel, McDougall, Dolan, Stine and Pegg, J. Med. Chem., 1992, 35, 4486).

Typical components "sugar", the corresponding R"XCHR'", which lead to the SECO-nucleosides, get the techniques described, in particular, article McCormick and Mcblhinney, J. Chem Soc., Perkin Frans, 1, 1983, 93; Zuce (nucleosides), can be obtained by methods analogous to the synthesis of riboside and 2-desoxyribose O6-benzylguanine (Moschel et al., 1992; Gao, Fathi, Gaffney et al., J. Org. Chem., 1992, 57, 6954; Moschel, Hudgins and Dipple, J. Am. Chem. Soc., 1981, 103, 5489 (see above for obtaining riboside).

Industrial use

The amount used of the compound of the present invention vary depending on the actual amount required for the treatment of tumor cells. A suitable dose is the dose, leading to the creation in cancer cells, the treatment of which lead, such concentration of the compound of the present invention, which will lead prior therapy with an alkylating agent to a weakening of activity Atasi, in particular about 1 to 2000 mg/kg, preferably 1 to 800 mg/kg, most preferably 1 to 120 mg/kg

The pharmaceutical composition of the present invention can be prepared in conventional forms, together with the usual excipients, as described in International application WO 91/13898, the description of which is given here for reference. The composition may contain inactivator of the present invention together with an alkylating agent; or the composition may consist of two parts, one of which is the content of the invention can also be normal, as, for example, as described in the application WO 91/13898. When assigning the patient inactivator of the present invention the pharmaceutical composition may contain the appropriate inactivator in a suitable medium such as a 40% polyethylene glycol 400 in physiological saline solution, or physiological saline or 3% ethanol (physiological solution) for intravenous injection or in the form of a powder in the form of the respective capsules for oral purposes.

Alkylating agents can be assigned in accordance with known techniques in the form of conventional dosage forms, as described, for example, in the application WO 91/13898, or preferably as a single dose immediately or within 24 hours, however, mostly, for about 2 hours after the appointment of inactivating Atasu agent in doses lower than the doses that are used in conventional modes of treatment. Dose reduction may be necessary because, as expected, inactivator usually increase the toxicity of alkylating agents. Examples glorafilia agents are 1,3-bis(2-chloroethyl) - 1-nitrosoanatabine (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1 - nitrosoanatabine (CCNU), fotemustine, mitozolomide and Claeson and agents, Cancer Dnig Des., 1993, 8, 115. Examples meteorous agents include temozolomide (Patent UK 2104522) and dacarbazine, procarbazine and streptozocin.

Methods

Cloning of complementary DNA and overexpression of Atasi person already described previously [Fan, C.-Y., Potter, P. M. Rafferty, J. A. Watson, A. J., Cawkwell, L., Searle, P. F., O'connor, P. J. and Margison, G. P. (1991) Nucleic Acids Res. 18, 5723-5727] . Purification of recombinant proteins is carried out either by affinity chromatography on a column of DNA-cellulose as described by Wilkinson et al. [Wilkinson, M. C. , Potter P. M., Cawkwell, L., Georgiadis, P., Patel, D., Swann, P. F. and Margison, G. P. (1989) Nucleic Acids Res. 17, 8475-8484.] [Wilkinson, M. C., Cooper, D. P., Southan, C., Potter, P. M. & Margison, G. P. (1990) Nucleic Acids Res., 18, 13-16], or ion-exchange chromatography on DEAE-cellulose. In the latter case, the protein Atasu before to be placed in the column of DEAE-cellulose partially purified by precipitation with ammonium sulfate (30-60%) and dialysis against a solution containing 10 mm Fris - HCl with pH 7.5, 1 mm dithiothreitol, 2 mm add, 10% glycerol. Then Atasu elute in a gradient of 0-0,1 M NaCl. Purified Atasa person retains its activity when stored in a concentrated form at a temperature of minus 20oC in buffer 1 [50 mm Fris - HCl (pH 8,3)/3 mm dithiothreitol/1 mm etc] and can be thawed and re-frozen several times without significant loss of its activity.

6-benzylguanine or the test compound with a total volume of 200 μl IBSA containing 10 μg DNA of the thymus gland of a calf, for 1 hour at a temperature of 37oC. Add [3H] -methylated DNA as a substrate (100 ál contains 6.7 ág DNA and 100 fmol O6-methylguanine) kept in the incubator for 1 hour at a temperature of 37oC until the reaction is complete. After acid hydrolysis of the DNA, as described previously [Morten, J. E. N. & Margison, G. P. (1988) Carcinogenesis 9, 45-49], [3H]-methylated protein isolate and determine the amount by means of scintillation in the liquid. Usually carried out on two samples, and the experiments repeated several times. I50is the concentration of inactivator required to reduce the activity of Atasi 50%.

Cell culture and production of extracts

Mammalian cells are grown in standard conditions. For example, Raji cells (line limp the Leno 10% horse serum. Cells AS are human melanoma cells from which they receive the above xenografts and then injected them subcutaneously naked mice; WiDr cells are a cell line of cancer cells human colon; Hamster cells +120 are selected using mitozolomide subline cell line V 79 fibroblast Chinese hamster lung, which denote RJ KO; cells Yoshita represent cell line Karpinsky rats, a Yrbus represent her subline, resistant to the action of busulfan; XP cells are transformed under the action of SV 40 cell line fibroblast derived from the skin of the patient, the patient Xeroderma pigmentosum; cells pHMGhAT2b are a clone of these cells that infect expression vector mammalian cell containing complementary DNA Atasi person, and cells pHMG1a are a clone, which infect only expression vector (i.e., does not contain DNA Atasi person). Tablets cells again suspended in cold (4oC) Buffer 1 containing 2 μg/ml leupeptin, exposed to ultrasound for 10 seconds at intervals of 12 microseconds between pulses. After cooling with ice, the cells exposed to ult volumes of a solution of 6.7 mg/ml phenylmethanesulfonyl in ethanol and treated with ultrasound mixture is centrifuged with acceleration 15000g for 10 minutes at a temperature of 4oC to separate cellular debris in the form of tablets. The liquid above the sediment retain for determining the activity of Atasi (see below).

The stability of inactivation at 37oC

Inactivator (10 mm in DMSO) were diluted to a concentration of 0.1 mm in a preheated divisioona Buffer 1 (1 mm etc, 50 mm Fris, pH 8,3) or phosphate buffered saline (pH 7-7,2). Buffered phosphate saline saline solution is a solution containing 0.8% of sodium chloride, 0.02% potassium chloride, and 0.15% sodium dihydrophosphate, 0.02% potassium dihydrophosphate with a pH of 7.2. Samples are immediately transferred to the spectrophotometer CARY 13 (block ditch is at a temperature of 37oC) and scanned on a suitable wavelength (in accordance with the spectral properties of the connection) for up to 80 hours with intervals of 3-10 minutes. The results are expressed as the percentage change in the absorption depending on the time and the values of T1/2 (half life) is obtained from them by extrapolation.

The inhibition activity Atasi in mammals

Cells were diluted to a concentration of 106/ml in the culture medium culture containing either the appropriate concentration inactivator, or the equivalent amount is stripperwear, twice washed with phosphate buffered saline and received the tablet from the cells (1-2 x 107on the tablet) stored at a temperature of minus 20oC.

Activity Atasi determine, as indicated previously, in two series of cell extracts subjected to ultrasound, and expressed as percent residual activity relative to the activity present in untreated control samples (e.g., 350-450 FM/mg in Raji cells). The values of I50(i.e., the concentration of inactivator required to reduce the activity of Atasi 50%) is obtained from them by extrapolation.

Sensitization of cells Raji and A375M to action BCNU and temosolomida

The sensitivity of Raji cells to the cytotoxic effects of BCNU and temosolomida after pre-treatment for 2 h with inactivation studied using analysis of HTT [Scudiero, D. A., Shoemaker, R. J., Paul, R. D., Monks , A., Tierney , S., Nofziger I.e., Currens, M. J., Seniff D & Boyd, M. R. (1988), Cancer Research 48, 4827-4833]. In brief, cells are placed in quantities of 1000 on the cell plates with 96 cells before addition of medium containing either inactivator in the appropriate concentration, or the equivalent volume of medium, incubated in the incubator for 30 minutes at a temperature of 37oC. Avivamento number of media, and allow the cells to grow for 6 days. At the end of the specified term, add a solution of XTT and incubated the cells in the incubator for 4 hours at a temperature of 37oC. Obtained as the reaction product formazan red-orange color is quantitatively determined by measuring the absorption at a wavelength of 450 nm using an apparatus for reading microtitration tablets.

The sensitivity of A375M cells to the cytotoxic effects of BCNU studied using the MTT assay [Carmichael, J., DeGraff, W. C., Gazdar A. F., Minna, J. D. , Mitchell J. B. (1987). Evaluation of a tetrazolium-based semiautomated colorimetric assay : assessment of chemosensitivity testing. Cancer Res. 47: 936-942] , which has the following differences from the above analysis of HTT. A375M cells (1000 per cell) give to grow for 24 h, then treated with inactivation, and after 2 hours with BCNU. After 6 days add the MTT solution (4 mg/ml) and incubated the cells in the incubator for 3 hours at a temperature of 37oC. the Medium is removed and the resulting pink crystals formazan dissolved in DMSO (120 ml) and viable cells determined by modifying the absorption at the wavelength of 450 nm using an apparatus for reading microtiter plates.

From the obtained data, calculate the percentage of cell growth in relation to contreviennent of Raji cells to BCNU (D90. C/D90.I) determine the quotient of the values of D90(i.e., the dose at which there is 90% growth compared to non-exposed control samples, or inhibition of growth by 10%), calculated from results by using one BCNU (D90. C), and a value of D90for BCNU plus inactivator (D90. I ). The value of one (I) indicates, therefore, in the absence of sensitization under the influence of inactivator. The sensitivity of Raji cells to temozolomide and sensitivity of A375M cells to BSNU determined from the corresponding values of D50(i.e., the dose at which there is 50% inhibition of growth).

The sensitivity of mammalian cells to the action of inactivation or products of their hydrolysis,

To study the effect of inactivation cells Xeroderma pigmentosum cells and V79 Chinese hamster on them for 2 hours at a temperature of 37oC, effect of increasing concentrations (up to 600 μm) selected inactivator. In some cases, inactivator previously subjected to hydrolysis for 20 hours at a temperature of 37oC, and then added to Raji cells to study the possible degree of inhibition of cell growth products Rastogi of the brain to temosolomida (analysis of GM-CFC)

For the analysis of granulocyte/macrophage colony-forming cells (analysis of GM-CFC) get the initial samples of human bone marrow from patients undergoing cardiothoracic surgery. After removal of the samples of erythrocytes, the cells are placed in a quantity of 1-2 x 105/ml on Wednesday Iscove 300mOsM containing 10 μm of inactivator or equivalent volume of DMSO, 20% serum fetal cows, 10% conditioned medium 5637 as a source of growth factors and 0.3% purified agar in Petri dishes containing the appropriate dose temosolomida, and incubated in an incubator at a temperature of 37oC in an atmosphere containing 5% carbon dioxide and 95% air. After 9 days identify colonies containing more than 50 cells. Colonies are cells-the precursors of granulocyte/makropoulou line differentiation (huGM-CFC). Survival determined as the percentage of colonies at zero concentration temosolomida.

The study xenografts

Animals

Male rats BALB-C with congenital absence of the thymus gland (nu/nu) weighing 25-35 g is obtained from cultivated under the conditions of the premises of the colony of Petersenovskaya Institute for cancer research, Christie Hospital NHS Frust, Wilmslow Road, Manchester M20 9BX, England) (mouse ASU). Harlan-Olac, Harlan UK Ltd, Shawis Farm, Blackthorn, Bicester, Oxon, OX6 OPT. England (mouse OLAC). The weight of these mice is 17-23 g; it later turned out that these mice are more sensitive to the toxic effects when combined (inactivator and BCNU) treatment. For this reason, use a smaller dose.

Cells

A375M cells (human melanoma) grown in modified Eagle medium of Dulbecco containing 10% serum fetal cows. Cells receives for in vivo inoculation, soaking in the incubator with 0.01% trypsin.

Tumor

Cells (106) in 100 μl of phosphate buffered physiological saline injected subcutaneously in the right flank of nu/nu mice aged 8-10 weeks. Give these cells for 3-4 weeks to grow into the tumor for transplantation experimental animals. Blocks of tumors with a size of 2 mm x 2 mm x 2 mm are implanted subcutaneously into the right side. These animals 7-10 days ready for experiments with inactivation.

Treatment of tumors

Mice nu/nu intraperitoneally administered either 30 or 60 mg/kg of O6--benzylguanine or B. 4205 and the corresponding number of media control for 60 to 90 minutes before the introduction of 10-25 mg/kg BCNU (intraperitoneally). O6-Benzylguanine and B. 4205 prepared in the form of a solution with the concentration of the intent of tumors

Measurement xenoplanets spend every 1-2 days employees with a digital compass. Tumor volume is calculated by the formula (height x width x length) /6. Experimental animals also weighs every 1-2 days. Measurements continued until until tumors in control animals reach the maximum allowable volume (i.e. 1 cm x 1 cm x 1 cm).

Results

In tables 4, 5 and 6 presents the physical, biochemical properties and obtained in vivo data for each inactivator. The above tests are explained in the Methods section.

In Fig. 1 shows the results of analysis on the inactivation of Atasi in vitro using the four compounds. Connection B. 4206 few more efficiently than BeG, however, B. 4212 and B. 4205 in terms of the analysis show significantly better results. B. 4203 not as affective as BeG.

In Fig. 2 shows that the concentration of 0.1 μm inactivator B. 4205 more effective than BeG in sensitization of Raji cells to the growth inhibitory effect of BCNU; at a concentration of 1.0 μm B. 4205 and BeG equally effective.

Fig. 3 shows that when the concentration of inactivator 0.1 μm, B. 4205 more effectively, che the dose of inactivator sensitization using BeG becomes more effective and sensitization with B. 4205 remains at the same level. The absence of such a response under the action of B. 4205 and a clear dose-dependent BeG the response can be clearly seen from Fig. 4.

Fig. 5 shows that despite some growth inhibition of V79 cells under the action of B. 4203 at doses of more than 100 microns (i.e., at least 100 times larger than the dose of I50for this connection, such effects for B. 4205 not observed even at the highest investigated concentration.

Fig. 6 shows that the XP cells are sensitive to the growth inhibitory effect of B. 4203, however, these cells are more sensitive than cells V79, to step B. 4205. However, the dose required to increase the inhibition of at least 100 times higher than the dose needed to inactivate Atasi. We can conclude that the potential inhibitory effect of these inactivation will not make a significant contribution to sensitization of cells to increasing the inhibitory effects of alkylating agents.

In Fig. 7 shows that a significant growth inhibitory effect was not observed in Raji cells, when these compounds lead to hydrolysis (see Methods), before they are added to the cells without further dobavljeni will not contribute to the increase of inhibition, which is observed when using these tools in combination with alkylating agents.

Fig. 8-13 show more detail the results of the study xenografts. The weakening and recovery activity Atasi after action O6-benzylguanine or B. 4205 measured in extracts of tumor xenografts A375 (Fig. 8), obtained from animal tissues, dead after 2 or 24 hours after the appointment of inactivator. Fig. 9-13 indicate sensitization A375 xenografts in naked mice as O6-benzylguanine and B. 4205 in combination with BCNU, as described in the Methods section. In each figure, the top graph shows the increase in tumor volume, expressed as a percentage, in the course of the experiment. The bottom graph shows the number of animals in each test group, which survive after exposure. The graphs show that B. 4205 comparable to the O6-benzylguanine (BeG) in the reduction of tumor volume to is significantly less toxic in combination with BCNU than BeG in combination with BCNU. Patients-people cutaneous malignant melanoma treated with BCNU, in particular in the USA (C. M. Balch, A. Houghto & L. Peters (1989) "Cutancous Melanoma", in: Cancer: Principles and Practise of Oncology, V. T. De Vita, S. Helman & S. A. Rosenberg (eds.). pp. 1499-1542, the school system, which is clinically highly reliable.

Fig. 14 shows the results of tests for sensitization of cells of human bone marrow to action temosolomida, as described above in the Methods section. Bone marrow cells receive from three patients, designated as C, D and E, respectively. Survival curves show the relation to the cytotoxic effects of the combined impact of inactivator/temozolomide. It is desirable that this effect was decreased. The results for patients C and E show that B. 4205 has less sensitizing effects than the O6-benzylguanine (O6BeG); for patients D differences not observed, however, this result can be considered as a permissible deviation when conducting scientific experiments with human material.

Table 7 shows the toxic effect of inactivator on bone marrow cells obtained from five patients A-E, including the same patients C-E, the data for which are shown in Fig. 14. The results for B. 4205 comparable with the results for O6-benzylguanine. Note that the B cells of the patient are almost twice as sensitive to the action as BeG, and B. 4205, compared with other samples.

Brief imaginig conditions when conducting in vitro assays. In these conditions, two of the compounds (B. 4214 and B. 4217) does not inactivate Atasu up to the highest concentrations used, however, are compounds in which R' denotes methyl. Other compounds inactivate Atasu, with I50range from 0,0045 to 95 µmol. Eight compounds (B. 4205, B. 4206, B. 4212, B. 4226, B. 4266, B. 4269, B. 4273, B. 4275) have values of I50smaller than BeG (table 4).

2) Inactivator undergo hydrolysis in aqueous solutions at different speeds (half existence from 0.17 to > 80 hours), but they are not related to their effectiveness as inactivation Atasi (table 4).

3) Compounds that are effective in the inactivation of Atasi in vitro (I50< 1,0 µm), inactivate Atasu in human cells, with values of I50in the General case is only slightly higher (1.5 times) than the values found when using recombinant protein in in vitro assay (table 4).

4) B. 4205 with equal efficiency inactivates Atasu in human cells, rat and Chinese hamster (the values of I50make 0,02-0,03). For B. 4203 specified interval is 0.04 and 0.12. In cell lines using B. 4205 and B. 4203 almost seven times more effective than BeG cells V79, however, at concentrations that are at least 100 times greater than the concentrations at which there is a sensibility to the action of BCNU (Fig. 5 and 6).

6) Compounds effective in the inactivation of Atasi in vitro (I50< 1,0 µm) and Raji cells (I50< 1,0 µm), sensibiliser Raji cells and A375M to increasing the inhibitory effects of BCNU in the process of conducting the relevant tests (table 4).

7) At concentrations of inactivator 0.1 μm, B. 4205 more effective than BeG , sensitization of Raji cells with respect to increasing the inhibitory effect temosolomida (Fig. 13).

8) Analysis of in vitro can be used to predict which compounds are most likely to be effective for sensitization of mammalian cells to increasing the inhibitory effects of BCNU and similar cytotoxic agents.

9) B. 4205 similar BeG or slightly more effective than BeG, when sensitization xenografts of human melanoma grown in naked mice, increasing inhibitory effects of BCNU (Fig. 9-13).

10) B. 4205 as effective as and BeG, with inactivation of Atasi in the xenografts Melli Atasi in xenografts can be used for predictions what compounds most likely to be efficient sensitizers of melanoma xenografts to the growing influence of BCNU and similar cytotoxic agents.

12) In contrast to BCNU, which causes mortality to 70% of affected animals, B. 4205 has very little effect on the sensitivity of Nude mice with xenografts of human melanoma, acute toxic effects of BCNU in the conditions of testing (Fig. 9-13).

13) BeG sencibilisiruet GM-CFC in three samples of human bone marrow, tested for toxic effects temosolomida, however, in two of these samples sensitization under the action of B. 4205 is weak or not observed. The specified analysis can therefore be used to predict possible mielosupressivne effects inactivation Atasi when used in a clinical setting in combination with BCNU and related compounds (Fig. 14).

1. Derivative OF6-alkylguanine formula

< / BR>
where Y is hydrogen, ribosyl or deoxyribosyl, which may be substituted by hydroxy or C1-C4alkoxygroup;

R' represents hydrogen, C1-20alkyl illico, containing at least one heteroatom selected from O, N or S, optionally condensed with a benzene ring, pyridine or naphthalene, or six-membered heterocyclic ring containing at least one atom of N, not necessarily with the S atom, optionally condensed with one or two benzene rings, where this cyclic group may be optionally substituted in the heterocyclic () the ring(s) and/or carbocyclic () the ring(Oh) groups selected from C1-C5of alkyl, halogen, cyano, nitro, azido,1-C5alkoxy, aryl, SOnR"" where R"" is1-C5alkyl and n is 0, 1, 2, R5where R5represents N or C1-C5alkyl or N-oxides, (ii) naphthyl, optionally substituted with halogen or1-C5alkoxy,

or their pharmaceutically acceptable salts.

2. Connection on p. 1, where R contains a heterocyclic ring condensed with the benzene ring and where O6-alkylguanine group attached to R or heterocyclic, or a benzene ring.

3. Connection under item 1 or 2, where R is a five-membered heterocyclic ring containing one sulfur atom.

4. ameenah derivatives.

5. The compound according to any one of the preceding paragraphs, where R is a cyclic group substituted with halogen, cyano, SOnR"" where R"" is1-C5alkyl and n = 0, 1, or 2, or-R5where R5is1-C5alkyl.

6. The compound according to any one of the preceding paragraphs, where R is selected from thiophene ring, furan ring, and their substituted derivatives, which are selected from their bromine and cyanoderivatives.

7. Connection on p. 6, RR' is ABOUT6-tanila or poslednym derived.

8. Connection on p. 1, selected from O6-analgoidea;6-(3-trimethyl)guanine;6-piperidylamine;6-furfurylamine;6-(3-furylmethyl)guanine;6-(2-benzo[b] thienylmethyl)guanine;6-(2-benzofuranyl)guanine;6-(5-triazolylmethyl)guanine;6-(5-ethoxycarbonylphenyl)guanine;6-(5-bromophenyl)guanine;6-(5-cyanophenyl)guanine;6-(2-benzo[b] thienylmethyl)guanosine;6-(4-picolyl)guanine;6-(2-naphthylmethyl)guanine.

9. The compound according to any one of paragraphs.1 - 8 be used to obtain the pharmaceutical composition having the ability to weaken the act the excitation OF6-tanilgan or its substituted derivative under item 1.

11. Pharmaceutical composition having the ability to weaken the activity OF6-alkylguanine-DNA alkyltransferase the owner, containing guanine derivative and a pharmaceutically acceptable carrier, characterized in that as a derivative of guanine it contains an effective amount of the compounds of formula I or its pharmaceutically acceptable salt according to any one of the preceding paragraphs.

12. The pharmaceutical composition according to p. 11, characterized in that it further comprises an alkylating agent.

13. The pharmaceutical composition according to p. 12, characterized in that the alkylating agent is selected from 1,3-bis(2-chloroethyl)-1-nitrosoanatabine (BCNU) and temosolomida.

14. The method of obtaining derivatives OF6-alkylguanine formula I under item 1, characterized in that carry out the following stages: interaction of sodium hydrate solution RR'OH, where R and R' are specified in paragraph 1, in an organic solvent, adding a chloride of 2-amino-N,N,N-trimethyl-1H-purine-6-amine or 2-amino-6-glucuronate, the processing of a weak acid and ether and extraction of the target product.

Priority points:

08.06.1993 - PP. 1 - 4, 12, 14, and 8, 9, 11, 10 with Asim from PP. 5 to 7, and p. 13 for temosolomida.

 

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