RU2073000C1 - Derivatives of griseolic acid or their pharmaceutically acceptable salts or their esters (variants) - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: product: derivatives of griseolic acid or their pharmaceutically acceptable salts or their esters were used in biochemistry, medicine, veterinary science as inhibitors of phosphodiesterase. EFFECT: enhanced effectiveness. 3 cl, 2 tbl

Description

 The invention relates to a number of new derivatives of griseolic acid to intermediates providing these compounds and methods and compositions for their use.

 Griseolic acid is a nucleoside type compound having an adenine base and two carboxylic acid groups. It was first described inter alia in the specification of European patent application N 293229A, but its structure was not known at this stage. Its structure was first described in US Pat. No. 4,460,765. Some derivatives of griseolic acid were subsequently described in US Patent Application No. 664866 of October 25, 1984, and the structure of griseolic acid is also described there. Other derivatives of griseoleic acid, specifically dihydrodeoxyhydroxy acid and its salts and esters are described in US patent application N 734868 of May 16, 1985.

 In accordance with the recommendations of the International Association of Pure and Applied Chemistry (IURAC), the compounds of the invention are named as derivatives of griseoleic acid (or dihydrodeoxygryzeoleic acid), taking griseolic acid as the starting structure. The numbering system used is presented in US patent application N 664866.

 Griseolic acid and the derivatives of griseoleic acid from US patent applications N 664866 and N 734868, as well as derivatives of this invention, are able to inhibit the activity of phosphodiesterase specific for various cyclic nucleotides, for example, 3 ', 5'-cyclic adenosine monophosphate (cAMP) phosphodiesterase ( PDE) or 3 ', 5'-cyclic guanosine monophosphate (cGMP) PDE and can thus increase the level of the cyclic nucleotide, for example cAMP or GMP, in the cells of the patient to which the compound is applied.

 It is well known that cAMP, which is very widespread in animal tissues, functions as a second carrier and occupies an intermediate position in action for a large number of hormones; as a result, this cAMP has various very important physiological and biochemical values. In addition, it is known that it acts on or takes part in: cell division, proliferation and differentiation; systological system, especially myocardium, hematoposis; various functions of the central nervous system; immune responses; and the release of insulin and histamine. Its concentration in the tissues, and therefore its effect on these various functions, depends on the balance between the enzyme that synthesizes cAMP (i.e. adenylate cyclase) and the enzyme that breaks down cAMP, cGMP PDE. An anti-cAMP inhibitor will increase the level of cAMP in the cells and, thus, can be used for various therapeutic purposes, for example: in the treatment of cardiovascular diseases; as an anti-asthma agent; as a muscle relaxant; as a psychotropic or neutrotropic agent; as an anti-inflammatory agent; in cancer therapy; for the treatment of diabetes.

 The effects of other cyclic nucleotides, such as cGMP, have been less studied to date. However, they are believed to have a region of activity that is the same, although not identical, to cAMP. Therefore, inhibition of PDEs specific for other cyclic nucleotides will lead to a range of therapeutic effects similar to those due to inhibition of cAMP PDE. Since the functions of the effects of such other cyclic nucleotides are under explanation, there is a need to inhibit PDEs associated with such other nucleotides, the inhibitors of which exhibit increased specificity for one or another PDE of other nucleotides, and not for cAMP PDE; indeed, the development of such inhibitors may even help or stimulate the study of such other cyclic nucleotides.

, 129 (1979)]
Был открыт ряд соединений, которые относятся к гризеолевой кислоте и дигидродезоксигризеолевой кислоте и которые обладают активностью гризеолевой кислоты и дигидрорезоксигризеолевой кислоты. Некоторые из этих соединений неожиданно проявляют увеличенную активность против сGMP PDE, чем против сАМР PDE. Соединения изобретения, сохраняя целевую активность, являются также ценными как промежуточные в получении других соответствующих целевых соединений.In addition to griseolic acid and its derivative, other compounds are known to inhibit cAMP and cGMP PDEs, and these compounds include papaverine, dipyridamo, and some compounds that are compound nucleic acid bases, such as terfilin or MSB 22, 948 [Kukovetz et al. Naunyn-Schmiedeberg's etrch. Pharmakol

129 (1979)]
A number of compounds have been discovered that relate to griseoleic acid and dihydrodeoxygryzoleic acid and which have the activity of griseolic acid and dihydroresoxygryzoleic acid. Some of these compounds unexpectedly show increased activity against cGMP PDE than against cAMP PDE. The compounds of the invention, while maintaining the target activity, are also valuable as intermediates in the preparation of other relevant target compounds.

 The aim of the invention is to obtain a substance of a new composition, namely derivatives of griseolic acid and salts and esters of these compounds.

где R⁵ и R⁶ оба представляют собой дополнительную углерод-углеродную связь между углеродными атомами, к которым они присоединены;
R²⁹ представляет группу ОН;
R³⁰ представляет собой C₁-C₄-алкил или арил (С₁-C₄)-алкил;
R³¹ и R³² одинаковые или различные представляют собой атом водорода или карбокси защитную группу;
и его фармацевтически приемлемые соли и его сложные эфиры.In addition, it is an object of the invention to provide intermediates for the preparation of griseolic acid derivatives which exhibit the ability to inhibit PDE activity, which degrade cyclic nucleotides, for example cAMP or cGMP PDE, and have the following formula I:

where R ⁵ and R ⁶ both represent an additional carbon-carbon bond between the carbon atoms to which they are attached;
R ²⁹ represents a OH group;
R ³⁰ represents C ₁ -C ₄ -alkyl or aryl (C ₁ -C ₄ ) -alkyl;
R ³¹ and R ^{32, the} same or different, represent a hydrogen atom or a carboxy protecting group;
and its pharmaceutically acceptable salts and its esters.

 Compounds of formula I may be formulated into pharmaceutical compositions comprising a phosphodiesterase inhibitor in admixture with a pharmaceutically acceptable carrier or diluent, wherein said phosphodiesterase inhibitor is selected from the group consisting of compounds of formula I as defined above and pharmaceutically acceptable salts and esters of these compounds.

 The proposed compounds are used in the treatment of animals, especially a mammal, for example, a person suffering from a disorder caused by an imbalance in phosphodiesterase levels, by applying a phosphodiesterase inhibitor to said animals, wherein said phosphodiesterase inhibitor is selected from the group consisting of compounds of formula I as defined above, and pharmaceutically acceptable salts and esters of these compounds.

 An additional objective of the invention is the provision of a method for producing compounds of formula I.

где R⁵ и R⁶ оба представляют собой атомы водорода или вместе представляют дополнительную углерод-углеродную связь между атомами углерода, к которым они присоединены;
R²² и R²³ одинаковые или различные, и каждый представляет карбоксизащищающую группу;
R²⁹ гидроксильная группа;
R³⁰ представляет собой алкильную или аралкильную группу, необязательно замещенную в арильной части, по крайней мере, одной нитро, метокси или галогено группой.The compounds of the invention used as intermediates are compounds of formula II:

where R ⁵ and R ⁶ both represent hydrogen atoms or together represent an additional carbon-carbon bond between the carbon atoms to which they are attached;
R ²² and R ^{23 are the} same or different, and each represents a carboxy protecting group;
R ^{29 is a} hydroxyl group;
R ³⁰ represents an alkyl or aralkyl group optionally substituted in the aryl part of at least one nitro, methoxy or halogen group.

Where the name “aryl” groups appears in the description, either by themselves or as part of a larger group (eg, “aralkyl group”), this means a carbocyclic aryl group, preferably having 6 14, more preferably 6 10, ring carbon atoms (e.g., a phenyl group, or a 1- or 2-naphthyl group), which may be substituted or unsubstituted. If the group is substituted, then the substituents are usually selected from C _1-4 alkoxy groups, halogen atoms, nitro groups.

 In the compounds of the invention where a halogen atom is mentioned, it means a fluorine, chlorine, bromine or iodine atom.

 The compounds of formula I and formula XL contain two carboxyl groups and can thus form mono- or di-salts and mono- or di-esters. In the case of di-salts or di-esters, the cationic moieties of the salts or alcohol moieties of the esters may be the same or different. However, in practice, it is easiest to prepare di-salts or di-esters in which two cationic moieties or even alcohol moieties are the same.

There is no particular restriction on the nature of the alcohol fragment of the ester, provided that when the compound is intended for therapeutic use, this fragment must be “pharmaceutically acceptable”, which, as is well known to specialists in this field of science, means that it does not reduce or decreases to an acceptable degree, the activity of the compound or its toxicity, and all esters suitably prepared from compounds of this type, can be formed with the compounds of the invention. If esters are not intended for therapeutic use (for example, as intermediates), even this limitation is not required. Examples of such esters include: C _1-6 esters, especially methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl esters; aralkyl esters, especially benzyl, para-nitrobenzyl, ortho-nitro-benzyl, tri-ferinmethyl, bis / ortho-nitrophenyl / methyl, 9-antrylmethyl, 2,4,6-trimethylbenzyl-para-bromobenzyl, para-methoxybenzyl, piperonyl and benzhydryl esters; C _1-6 haloalkyl esters which may have 1 or more halogens (e.g. chlorine, bromine, fluorine or iodine), up to complete perhalogenation, e.g. 2,2,2-trichloroethyl, 2-chloroethyl, 2-bromoethyl, 2-fluoroethyl, 2-iodoethyl and 2,2-dibromethyl esters; alkoxymethyl esters, where the alkoxy portion preferably consists of C _1-4 , for example methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl and butoxymethyl esters; aliphatic acyloxyalkyl esters (especially acyloxymethyl and acyloxyethyl esters) such as acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, 1-acetoxyethyl, 1-propionyloxyethyl, 1-butyryloxyethyl, and 1-butyloxyethyl; (C _1-4 -alkyl) hydroxycarbonyloxyethyl esters such as 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl, 1-propoxycarbonyloxyethyl, 1-isopropoxycarbonyloxyethyl, 1-butoxycarbonyloxyethyl and 1-isobutoxycarbonyloxyethyl esters; heterocyclic esters, such as phthalidyl esters; heterocyclyl methyl esters in which the heterocyclic group is preferably a group, for example (5-methyl-2-oxo-1,3-dioxolen-4-yl) methyl esters; and esters that are readily hydrolyzable in vivo, the class of which includes some esters of the classes indicated above, for example, aliphatic acyloxyalkyl esters, lower alkoxycarbonyloxyethyl esters, (5-methyl-2-oxo-1,3-dioxolen-4-yl / methyl esters of ifthalidyl esters.

 There is no particular restriction on the nature of the cations used to form salts of the compounds of the invention, provided that, if intended for therapeutic use, they are pharmaceutically acceptable. On the other hand, if they are not intended for therapeutic use (for example, as intermediates), even these restrictions are not required. Preferred salts include alkali metal salts (such as sodium or potassium) or with alkaline earth metals (such as calcium).

 The nature of the acid used to form salts is not significant, provided that if they are intended for therapeutic use, they are pharmaceutically acceptable. On the other hand, if they are not intended for therapeutic use (for example, as intermediates), even such a restriction is not required. Examples of such acids include: inorganic acids, such as hydrochloric acid, sulfuric acid and phosphoric acid; organic carboxylic acids such as acetic acid, oxalic acid, maleic acid, succinic acid, citric acid, tartaric acid, fumaric acid, lauric acid, stearic acid and palmitic acid; and organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and para-toluenesulfonic acid.

 The compounds of the invention have a number of asymmetric carbon atoms in their molecules and can therefore exist in the form of various stereoisomers. The invention includes both individual isomers and mixtures of these isomers. Griseolic acid, being a natural product, is the only isomer in which both 2 'and 7' carbon atoms are in the R-configuration; compounds derived from griseoleic acid may retain the same confusion or may have an inverted configuration at one or more asymmetric carbon atoms. For example, when R 'represents a group or atom other than hydrogen, the configuration of the compounds at the 2'-position may be alpha or beta.

When R ²⁹ represents a group or an atom other than hydrogen, the configuration at the 7'-position may be RS, R or S.

 As mentioned above, griseoleic acid is a known compound disclosed, for example, in European Patent Specification No. 29.329 or in US Patent Specification No. 4,460,765. Dihydrodeoxygryzoleic acid is disclosed in published European Patent No. 0162715. Both griseolic acid and dihydrodeoxygryzeolic acid can be obtained by culturing suitable microorganisms belonging to the Streptomyces species, especially Streptomyces griseodyrantidems ANK 63479 (deposited on October 9, 1979 at the Fermentation Research Institute, Aga Institute of Industrial Science and Technology, Japan, from where they can be made available to the consumer under catalog number FE R M-P5223 and October 22, 1980 at the Agricultural Research Service, Peoria, USA, from where they can be delivered under catalog number NRRL 12314). All details regarding the characterization of Streptomyces griseoduranteducs SANK 63479 are given in European Patent Publication No. 29329A and US Pat. No. 4,460,765.

где R⁵, R⁶ имеют указанные выше значения;
R²² и R²³ могут иметь одинаковое или различные значения и каждый из них представляет собой карбоксизащищающую группу;
R³⁰ представляет собой алкильную группу или аралкильную группу;
R³¹ и R³² могут иметь одинаковые или различные значения и каждый из них представляет собой атом водорода или карбокси-защищающую группу.The preparation of a compound of formula I, as well as the use of a compound of formula XL as an intermediate, are shown in the following reaction schemes:

where R ⁵ , R ⁶ have the above meanings;
R ²² and R ²³ may have the same or different meanings and each of them represents a carboxy protecting group;
R ³⁰ represents an alkyl group or an aralkyl group;
R ³¹ and R ³² may have the same or different meanings and each of them represents a hydrogen atom or a carboxy protecting group.

 The compound of formula XL is prepared from N'-oxide (XXXIX) by reaction of the N'-oxide (XXXIX) with lower alkyl halide or alkyl halide or aralkyl halide, as shown in the above scheme (step 30).

The reaction is carried out in the presence of an organic base and, preferably, in the presence of a solvent. The nature of the solvent used is not critical, provided that it does not adversely affect the course of the reaction and can dissolve the reagents, at least to some extent. Suitable solvents include acid amides, such as dimethylformamide or dimethylacetamide. Suitable organic bases include trialkylamines, for example triethylamine. There is no particular limitation on the nature of the halide compound, provided that it is capable of alkylating or aralkylating a hydroxy group. Suitable lower aralkyl halides for this purpose include benzyl bromide and p / nitrobenzyl bromide. The reaction can be carried out in a wide temperature range, although it is usually most convenient to carry out it at a temperature lying in the range of 0-100 ^{° C.} , more preferably at a temperature close to room temperature. The time required to carry out such a reaction can vary over a wide range depending on many factors, mainly on the nature of the reactants and the reaction temperature; however, it is usually sufficient to carry out the reaction for 1 to 20 hours.

 After completion of the reaction, the target product can be isolated from the reaction mixture by conventional means. For example, if necessary, the reaction mixture is washed with water, and then the solvent is distilled off under reduced pressure. The residue can be purified in various ways, such as recrystallization and various chromatographic techniques, for example on a chromatographic column or using preparative thin layer chromatography to obtain the target compound.

 Stage 31 and all the following are given to illustrate the use of the compounds of formula (XL) to obtain the final product of formula I.

 Stage 31.

The compound of formula XL obtained as described above (with respect to step 30) is converted to a compound of formula I (step 31). Such a reaction is preferably carried out as follows. First, a compound of formula XL is treated with alkali in a solvent medium at a pH in the range of 12 13 in order to open the pyrimidine ring (carboxy protecting groups are also sometimes removed). Then the pH of the reaction mixture was re-adjusted to a value of 7.0 and the mixture was heated to recyclize to give a compound of formula I which contains unprotected carboxy groups (i.e., both R ³¹ and R ³² are hydrogen atoms ) Preferred alkalis for the first stage of such a reaction can be aqueous solutions of alkali metal hydroxides, for example sodium and potassium hydroxides. There are no particular restrictions on the nature of the solvent used, provided that it does not impede the course of the reaction and is capable of dissolving the starting compounds, at least to some extent. It is preferable to use water or a mixture of water with a lower alcohol, for example methanol or ethanol. Both reactions can be carried out over a wide temperature range and the choice of a specific temperature is not critical. A temperature close to room temperature is preferred for the ring opening reaction, and at this temperature and pH value of about 12, the reaction usually completes in 30-60 minutes. The cyclization reaction is preferably carried out at a temperature of from 0 ^o to 100 ^o C and at this temperature, the reaction is usually completed in 1 to 5 hours

 If desired, during such reactions, carboxy groups can be protected by an esterification process.

In order to protect the carboxy groups, it is preferable to react griseolic acid with a diazo compound, for example with a diazomethane or diphenyldiazomethane, or with a triazine compound, especially with a p-tolyltriazine derivative, for example with N-methyl-p-tolyltriazine. Such a reaction is preferably carried out in the presence of a solvent, the nature of which is not critical provided that it does not adversely affect the reaction, and also provided that the starting materials can be dissolved in such a solvent, at least to some extent. Suitable solvents for this purpose include: ketones, for example acetone; esters, for example tetrahydrofuran; amides, for example dimethylformamide; as well as mixtures of water with one or more of the above organic solvents. The reaction can take place over a wide temperature range and the choice of a specific reaction temperature is not critical, although it is usually preferred to carry out the reaction in the temperature range from -20 ^° C to +50 ^° C. The time required for the reaction will vary depending on many factors , especially from the nature of the starting materials and the reaction temperature; however, for example, at room temperature, it usually takes 1 to 24 hours to carry out such a reaction.

 Stage 32.

At this stage, the R ³⁰ alkyl or aralkyl group is removed and replaced with a hydrogen atom by catalytic reduction. The reaction is preferably carried out in the presence of a solvent, the nature of which is not critical, provided that it does not have a harmful side effect on the course of the reaction and is capable of dissolving the starting materials, at least to some extent. Organic carboxylic acids, especially fatty acids such as acetic acid, may be preferred solvents. There is no particular restriction on the nature of the catalyst, and at this stage any catalyst commonly used for catalytic reduction processes can be used, it is preferable to use platinum oxide, platinum on carbon, or palladium on carbon, even more preferred is palladium chloride adsorbed on active charcoal. Such a reaction is preferably carried out by mixing the catalyst mixture with the compound of formula I in the medium of the selected solvent in a hydrogen atmosphere. The reaction can be carried out over a wide temperature range and the choice of a specific temperature is not critical. As a rule, it is most convenient to carry out such a reaction at a temperature of 5-60 ^° C. The time required to carry out such a reaction can vary over a wide range depending on many factors, including the nature of compound I and the reaction conditions, especially the reaction temperature; however, under the above conditions, it is usually sufficient to carry out the reaction for 1 to 10 hours. If desired, the resulting compound may be subjected to an optional further conversion, further described for step 34.

 Stage 33.

At this stage, ring opening and recyclization reactions are carried out similar to those described for stage 3 without affecting the carboxy protecting groups. Such a reaction is preferably carried out in the presence of a solvent, the nature of which is not critical, provided that it does not impede the course of the reaction and is capable of dissolving, at least to some extent, the starting compound. Preferably, the solvent is capable of maintaining the pH of the reaction solution in the range of 6-8 and, accordingly, it is preferable to use a mixture of a buffer solution with a pH of 6-8 and a lower alcohol, for example methanol or ethanol, preferably a mixture of a buffer with a pH of 7.0 and methanol. The reaction can be carried out in a wide temperature range, for example in the range of 30 150 ^o C. The time required for the reaction may vary depending on many factors, mainly on the reaction temperature, pH and the nature of the starting materials and solvents; however, under the conditions proposed above, such a reaction is sufficient to carry out for 3 to 10 hours. If desired, the resulting compound may be subjected to the optional operation described below in step 34.

 Stage 34.

At this stage, the alkoxy or aralkyloxy group —OR ^{30 is} replaced with a hydrogen atom by treating the compound with an active metal in the presence of a solvent. The nature of the solvent is not critical, provided that it does not adversely affect the course of the reaction and is capable of dissolving the starting materials, at least to some extent. A suitable solvent for this purpose is a mixture of a dilute aqueous solution of an acid, for example a dilute aqueous solution of hydrochloric acid, and a water-miscible organic solvent, for example acetone. A preferred solvent is a mixture of equal volumes of 1 N. an aqueous solution of hydrochloric acid and acetone. There is no particular limitation on the nature of the active metal, provided that it can be used in the reduction reaction, where the acid serves as a proton donor. Active metals such as Raney nickel are preferred. The reaction is carried out over a wide temperature range, although it is usually most convenient to carry out such a reaction at temperatures of 0 ^{° to} 50 ^° C. The time required for the reaction to take place can vary over a wide range depending on many factors, mainly on the nature of the reactants and the reaction temperature; however, under the above conditions, it is usually sufficient to carry out the reaction for 1 to 10 hours

 If desired, the protecting groups can be removed, and as is well known, the nature of the deprotection reaction depends on the nature of the protecting group.

 In the case where a lower alkyl group is used as a carboxy protecting group, it can be removed by treating the compound with a base, especially an alkali metal hydroxide, for example sodium hydroxide. It is preferable to use an aqueous solution of alkali metal hydroxide, for example 1 N. an aqueous solution of sodium hydroxide. The reaction is preferably carried out in the presence of a solvent, the nature of which is not critical to the invention, provided that it does not adversely affect the course of the reaction. As a rule, an aqueous solution is effective for this purpose. The reaction can be carried out over a wide temperature range, although it is usually more convenient to carry out it at a temperature close to room temperature. The time required for the reaction can vary widely, however, usually a period of time from 30 minutes to 10 hours is sufficient.

When the carboxy protecting group is an aralkyl group or a lower haloalkyl group, they are preferably removed by reduction. Preferred reducing agents include: in the case of lower haloalkyl groups, a zinc-aqueous solution of acetic acid; and in the case of aralkyl groups, hydrogen and a catalyst (e.g. palladium on charcoal or a plate) or an alkali metal sulfide (e.g. potassium sulfide or sodium sulfide). The reaction is preferably carried out in the presence of a solvent, the nature of which is not critical, provided that it does not adversely affect the course of the reaction. Examples of suitable solvents include alcohols such as methanol or ethanol; esters such as tetrahydrofuran or diaxane; fatty acids, for example acetic acid; or mixtures of one or more organic solvents with water. The time required for the reaction is not critical, although it is usually preferable to carry out such a reaction at a temperature lying in the range from 0 ^{° C.} to room temperature. At this reaction temperature, it is usually sufficient to carry out for a period of time from 5 minutes to 12 hours.

In the case where the carboxy protecting group is an alkoxymethyl group, such a group can be removed by treating the compounds with an acid in a solvent. Preferred acids include: hydrochloric acid; a mixture of acetic acid with sulfuric acid; or a mixture of p-toluenesulfonic acid with acetic acid. There are no particular restrictions on the nature of the solvent used in this reaction, provided that it does not adversely affect the course of the reaction. Suitable solvents for this purpose include: alcohols such as methanol or ethanol; esters such as tetrahydrofuran or dioxane; and mixtures of one or more of these solvents with water. There is no particular restriction on the reaction temperature, although it is usually most convenient to carry out such a reaction at a temperature lying in the range of 0-50 ^{° C.} , and the reaction is usually sufficient to carry out for a period of time from 10 minutes to 18 hours.

 If the carboxy protecting group is removed by treatment with aqueous ammonia, this operation usually results in the conversion of the carboxy groups at positions 8 ′ and 7 ′ into carbamoyl groups.

If desired, the free carboxylic acid can be converted into a salt, for example an alkali metal salt, by conventional methods. For example, a suitable reaction for this purpose is to dissolve the acid in a mixture of water and a water-immiscible organic solvent, such as ethyl acetate, add an aqueous solution of the corresponding alkali metal carbonate or bicarbonate (such as potassium carbonate or sodium bicarbonate) at an appropriate temperature, for example at temperatures ranging from 0 ^o C to room temperature, and subsequently adjusting the pH of the system, for example at a value equal to 7, so that the resulting salt can be isolated on azhdeniem.

The resulting carboxylic acid salt, if desired, can be converted to ether, the carboxyl groups of which are protected by easily hydrolyzable in vivo protecting groups. First, the acid salt is dissolved in an appropriate solvent, for example in an ether such as tetrahydrofuran; or in a polar solvent such as dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide or triethyl phosphate. Then, at least two equivalent bases, for example, an organic base, such as triethylamine or dicyclohexylamine, an alkali metal hydride, for example sodium hydride, or an alkali metal carbonate or bicarbonate, such as sodium carbonate, sodium carbonate or sodium bicarbonate, are reacted with salt formation, and a reaction is performed between the resulting salt and a lower aliphatic acyloxymethyl halide, for example, acetoxymethyl chloride or propionyloxymethyl bromide, with lower alkoxycarbo iloasietil halide, such as 1-metoksikarboniloksietilhloridom or 1-ethoxycarbonyloxyethyl iodide, a phthalidyl halide or a (2-oxo-5-methyl-1,3-dioxolen-4-yl) methyl halide. The reaction is preferably carried out in the presence of a solvent, the nature of which is not critical, provided that it does not adversely affect the course of the reaction. Suitable solvents may be the polar solvents referred to above. The reaction can be carried out over a wide temperature range, although it is usually most convenient to carry out such a reaction at a temperature of 0-100 ^° C. The time required for the reaction can vary widely; however, under the conditions proposed above, it is usually sufficient to carry out the reaction for a period of from 30 minutes to 10 hours. The above step can be used, if desired, in each of the steps of steps 32, 33, 34, 36, 37, 38, 50 and 54 .

 After completion of any of the reactions previously described, the target product from each of these steps can be isolated from the reaction mixture by conventional methods. For example, if necessary, the reaction mixture is washed with water, and then the solvent is distilled off under reduced pressure. The residue can be purified by various methods, such as recrystallization and various chromatographic techniques, for example, on a chromatographic column or using preparative thin layer chromatography to obtain the target compound.

 Inhibitory activity of phosphodiesterase (PDE).

 Some compounds of the invention were tested (hereinafter indicated by the numbers of examples) along with theophylline and compounds of the Kaneko patent (US patent N 463706, Kenako corresponds to USSR patent application N 38085/04) as a comparative compound.

The test was carried out practically in accordance with a method similar to the method of AL Pichard and WI Cheung (Journal of biological chemistry), 251, 5726 5737, 1976). The crude enzyme solution obtained from rat brain was used as a source of cAMP PDE. As a substrate, ¹⁴ C-labeled cAMP was used. It was used in a buffer 0.1 M Tris-hydrochloric acid solution (pH 8.0) in an amount sufficient to obtain a final concentration of 0.14 μM. “Tris” is a tris (aximethyl) aminomethane. The substrate solution is mixed with the appropriate amount of the compound used, dissolved in a volume of 2.0 5.0 μl, dimethyl sulfoxide and 20 μl of snake venom solution and 40 μl of the crude enzyme solution. A sufficient amount of Tris-hydrochloric acid buffer is added until a total volume of 100 μl is obtained. The resulting mixture was allowed to react at 30 ^{° C.} for 20 minutes. At the end of this period of time, the reaction mixture is treated with Amberlit (brand) IRP-58 resin and the level of remaining adenosine radioactivity in the product is determined. The experiment is carried out for a number of different concentrations for each active compound, and from here the values of 50 inhibitions are calculated (I ₅₀ ).

The experiment was repeated, except that cyclic guanosine monophosphate (cGMP) was used instead of cAMP. Again I ₅₀ values are calculated depending on the cGMP PDE.

The results are presented in table. 1 and 2, where the values of I _{50 are} presented in micromoles.

Theophylline given in table. 1 is a known compound used for comparison as well as the compound of the Kaneko patent (see table 2). Theophylline is known to inhibit both cAMP PDE and GMP PDE, and is used therapeutically for this purpose. The least effective of the tested compounds of the invention has I ₅₀ values that are approximately an order of magnitude smaller than the corresponding values for theophylline, although the most effective of the tested compounds of the invention have I ₅₀ values of 3 to 4 orders of magnitude lower, which indicates that the activity of the compounds of the invention in as PDE inhibitors are exceptionally large. The differences in activities against cAMP PDE and cMP PDE are also clearly visible.

 Thus, various therapeutic uses of the compounds are possible, for example, in the treatment of cardiovascular diseases; as anti-asthma agents; as relaxants of smooth muscles; as psychotropic or neurotropic agents; as anti-inflammatory agents in the treatment of cancer and in the treatment of diabetes.

 The compounds of formula I can also be used as therapeutic agents for various cerebral circulatory disorders, such as cerebral consequences of a stroke, and as activators of metabolism in the brain, for example, in the treatment of senile paralysis or traumatic brain injuries. The compounds of formula I can be administered orally or non-orally, for example subcutaneously or intramuscularly.

 The compounds of formula I can be administered orally in the form of solid preparations, which may optionally contain various kinds of conventional additives. These additives include: diluents such as sugars and cellulose preparations; binders such as starch, resins, and methylene cellulose; and distribution agents. Doses may vary depending on the symptoms of the disease, the age and weight of the patient. For example, in the case of adult patients, a suitable daily dose may be a dose of 0.1 to 100 mg / kg of active compound, which can be taken either immediately or in divided doses.

 The preparation of various compounds of the invention is illustrated further in the examples. The preparation of some starting materials is illustrated in the following examples.

 Example 1

1 / a / Dimethyl-1'-deadenino-1 ⁶ β-acetoxy-4'5'-dihydro-0 ² , O ^{7 ′} -diacetylgryseolate.

2 ml of concentrated sulfuric acid is added to a solution of 500 mg of dimethyl-6-desamino-6-hydroxy-4′β, 5′-dihydro-O ^{2 ′} , O ^{7 ′} diacetyl griseolate (obtained by the method of Production Example 6 as described in Preparation 2 ) in 100 ml of a mixture of 4 1 by volume of acetic acid and acetic anhydride, and the resulting mixture was allowed to stand at room temperature for 14 hours under nitrogen. 15 g of sodium acetate were added to the reaction mixture, and the resulting solvent was evaporated under reduced pressure. The residue was dissolved in saturated aqueous sodium bicarbonate and the resulting solution was extracted three times with methylene chloride. The methylene chloride extracts are combined and dried over anhydrous magnesium sulfate. Then, the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a mixture of cyclohexane ethyl acetate 2 1. Evaporation of the solvent from the second fraction after elution gave 292 mg of the title compound.

NMR spectrum (CDCl ₃ ), δ, ppm 2.44 (1H, doublet of doublets, J = 6.4 & 14.7 Hz); 2.65 (1H, doublet of doublets, J = 14.7 & 3.0 Hz); 4; 98 (1H, doublet of doublets, J 2.5 & 6.4 Hz); 5.02 (1H, doublet of doublets, J = 2.9 & 4.0 Hz); 5.15 (1H, doublet of doublets, J = 4.0 & 2.5 Hz); 5.72 (1H, singlet); 6.29 (1H, doublet, J = 2.9 Hz).

 Mass spectrum (m / e): 474 (M + МЗ).

 Mass spectrum (hereinafter abbreviated as Mass spectrum with fast electron bombardment: FAB Mass spectrum) with fast electron bombardment (m / e): 431 (M +).

(b) Dimethyl-1'-deadenino-1'-a-acetoxy-4 ', 5'-dihydro-O ^{2 ′} , O ^{7 ′} -diacetylgryseolate
The first fraction isolated by column chromatography as described in Example 1 / a / was concentrated by evaporation under reduced pressure to obtain 33 mg of the title compound.

NMR spectrum (CDCl ₃ ) δ, ppm 2.67 (1H, doublet of doublets, J = 7.4 & 15.1 Hz); 2.98 (1H, doublet of doublets, J = 15.1 & 2.9 Hz); 4.94 4.99 (2H, multiplet); 5.05 5.07 (1H, multiplet); 5.61 (1H, singlet); 6.38 (1H, doublet, J = 4.9 Hz).

 Mass spectrum (m / e): 474 (M + M3).

 FAB mass spectrum (m / e): 431 (M +).

1 / s /. Dimethyl-1'-diadenino-1 'b-acetoxy-4', 5'-dihydro-Ο ^{2 ′} , Ο ^{7 ′} -dibenzoylgryseolate.

Concentrated sulfuric acid (2 ml) was added while cooling on ice to a solution of 400 mg of dimethyl- (6-desamino-6-hydroxy-4 ', 5'-dihydro-O ^{2 ′} , Ο ^{7 ′} -dibenzoylgryzeolate) obtained by the method similar to those described in production examples 1 3, 5 and 7, however, using 2-benzoyl chloride in the production example instead of acetic anhydride dissolved in 80 ml of a mixture of 4 1 by volume of acetic acid and acetic anhydride, and the resulting mixture was allowed to stand at room temperature for 14 hours. Then the reaction mixture is mixed with 15 mg of sodium acetate and concentrated, evaporating under reduced pressure. The residue was dissolved in a mixture of methylene chloride and saturated aqueous sodium bicarbonate and extracted three times with methylene chloride. The methylene chloride extracts were combined and dried over anhydrous magnesium sulfate, and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with a mixture of 2 1 cyclohexane and ethyl acetate, and the fractions containing the title compound were concentrated by evaporation under reduced pressure to obtain 243 g of the title compound.

Nuclear Magnetic Resonance Spectrum (CDCl ₃ ), d, ppm 2.50 2.60 (1H, multiplet); 2.96 3.04 (1H, multiplet); 5.06-5.16 (2H, multiplet); 5.48 5.52 (1H, multiplet); 5.88 (1 H, singlet), 6.50 6.58 (1 H, multiplet).

Elemental analysis for C ₂₇ H ₂₆ O ₁₂ :
Calculated, C 59.78; H 4.83; N 0.

 Found C, 59.59; H 4.80; N, 0.01.

 Mass spectrum (m / e): 585 / M + 43 /.

 Example 2. Dimethylgryseolate N'-oxide.

 8.14 g of dimethyl griseolate, prepared as described in Preparation 1, are suspended in methanol. 6.90 g of meta-chloroperbenzoic acid was added, and the resulting mixture was stirred at room temperature for 24 hours. Then, the solvent was distilled off under reduced pressure. When the solvent is almost distilled off, 300 ml of diethyl ether are added and the lumps in the solution are ground with a spatula until they completely disappear. The resulting mixture was filtered, then the resulting residue was washed with 100 ml of diethyl ether and dried to obtain 7.81 g of a white powder. This powder is dissolved by heating in 200 ml of methanol and 300 ml of methylene chloride as carefully as possible, and then the solvent is distilled off under reduced pressure with an aspirator to reduce the volume of the remaining solution to approximately 50 ml. The resulting crystals were filtered to obtain 6.31 g of the title compound as white powder crystals.

Nuclear Magnetic Resonance Spectrum (CD ₃ ) ₂ SO), d, ppm 3.63 (3H, singlet), 3.78 (3H, singlet), 4.60 (1H, doublet, J = 5.0 Hz), 4.67 (1H, singlet), 4.15 (1H, doublet, J 2.2 Hz), 5.90 (1H, doublet of doublets, J 2.2 and 5.0 Hz), 6.53 (1H, singlet), 8.53 (1H, singlet), 8.72 ( 1H, singlet).

 Example 3

 Dimethyl-1'-deadenino-1 'b-2-amino (1,2,4) -oxadiazolo- [3,2-i] -purin-7-yl-griseolate hydrobromide.

 8.46 g of dimethylgryseolate-N-oxide obtained by the method of example 2, suspended in 400 ml of methanol. 2.52 g of cyanogen bromide are added, and the resulting mixture is stirred at room temperature for 1.5 hours, and then stirred an additional hour after the addition of an additional 630 mg of cyanogen bromide. The solvent is distilled off under reduced pressure to obtain a volume of about 100 ml of the remaining solution. 100 ml of ethyl acetate was added and distillation under reduced pressure was continued until the liquid volume reached 100 ml. To the remaining solution, 1000 ml of ethyl acetate was gradually added with stirring. The suspension containing crystals is placed in the refrigerator overnight. The crystals are filtered off to obtain 9.66 g of the title compound.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 3.67 (3H, singlet), 3.73 (3H, singlet), 4.67 (1H, doublet, J 5.0 Hz), 4.66 (1H, singlet), 5.26 (1H, doublet, J 2.2 Hz), 5.84 (1H, doublet of doublets, J 2.2 & 5.0 Hz), 6.76 (1H, singlet), 9.07 (1H, singlet), 10.23 (1H singlet).

 Example 4

Dimethyl-N ⁶ -cyanogreezeate-N'-oxide.

 9.77 g of dimethyl-1'-deadenino-1'b- (1,2,4) -oxydiazolo- [3,2-i] -purin-7-yl-griseolate hydrobromide obtained according to the method of Example 3 are dissolved in 50 ml of methanol. To the resulting mixture was added 50 ml of 20 (v / v) ammonia in methanol and the mixture was allowed to stand at room temperature for 60 minutes, then 200 ml of ethyl acetate was added to the reaction mixture and the solvent was distilled off under reduced pressure with an aspirator. When the liquid volume reaches 200 ml, ethyl acetate is added and then it is distilled off under reduced pressure. When the liquid volume reaches 200 ml, the resulting mixture was placed in the refrigerator overnight. The crystals thus obtained were filtered off to obtain 8.53 g of the title compound as yellowish-white fine crystals.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 3.67 (3H, singlet), 3.74 (3H, singlet), 4.63 (1H, doublet, J 5.0 Hz), 6.66 (1H, singlet), 5.16 (1H, doublet, J 2 Hz), 5.93 (1 H, doublet of doublets, J 2.2 & 5.0 Hz), 6.50 (1 H, singlet), 8.33 (1 H, singlet), 8.49 (1 H, singlet )

 Example 5

Dimethyl-N'-benzyloxy-N ⁶ -cyanogreezeolate.

8.53 g of dimethyl-N ⁶ -cyanogrizeolate-N'-oxide obtained by the method of example 4, was dissolved in 100 ml of dimethylformamide. 10 ml of benzyl bromide and 10 ml of triethylamine are added and the resulting mixture is stirred at room temperature for 4 hours. The solvent is distilled off under reduced pressure. 30 ml of ethanol and 30 ml of toluene are added to the residue, and then they are distilled off. This procedure is repeated 4 times. The oily substance thus obtained is mixed with 500 ml of diethyl ether and sonicated with a spatula with stirring until a pale yellow powder is obtained. This substance is filtered off and dissolved in 300 ml of water and 500 ml of ethyl acetate. The organic layer that is released is washed in portions (100 ml each) of a saturated aqueous solution of sodium chloride, 0.2 N aqueous hydrochloric acid and 10 (w / v) aqueous sodium bicarbonate and saturated aqueous sodium chloride in this order, and then dried over anhydrous magnesium sulfate. After treating the solution with activated carbon, the solvent was distilled off under reduced pressure to obtain 8.8 g of the title compound as a yellow caramel-like substance.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 3.68 (3H, singlet), 3.76 (3H, singlet), 4.61 (1H, doublet J, 5.0 Hz), 4.67 (1H, singlet), 5.26 (1H, doublet, J) 2.2 Hz), 5.37 (2H, singlet), 5.83 (1H, doublet of doublets, J 2.2 & 5.0 Hz), 6.57 (1H, singlet) 7.2 7.8 ( 5H, multiplet), 8.56 (1H, singlet), 8.81 (1H, singlet).

 Example 6

Dimethyl-N ⁶ -cyano-N'-methoxygryseolate.

8.3 g of triethylamine and 3.9 g of methyl iodide are added to 40 ml of dimethylformamide containing 4.5 g of dimethyl-N ⁶ -cyanogreezeolate-N'-oxide (obtained by the method of example 4), while cooling on ice. The resulting mixture was stirred at room temperature for 4.5 hours. Then, the solvent was distilled off and 100 ml of diethyl ether was added to the residue to obtain a powder by grinding the mixture with a spatula. The insoluble product is filtered off, washed with 30 ml of diethyl ether and dissolved in a mixture of 80 ml of ethyl acetate and 20 ml of water, and then the resulting solution is extracted again with ethyl acetate. The ethyl acetate layer was dried over anhydrous magnesium sulfate. After the drying agent is filtered off, the solvent is distilled off to obtain 3 g of the title compound.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 3.60, 3.73 (instead of 3H, each singlet), 4.14 (3H, singlet), 4.58 (1H, doublet, J 5.0 Hz), 4.64 (1H, singlet), 5, 21 (1H, doublet, J 2.2 Hz), 5.83 (1H, doublet of doublets, J 2.2 and 5.0 Hz), 6.55 (1H, singlet), 8.53 and 8.93 ( instead of 1H, each singlet).

 Cooking 1.

 Griseolic acid dimethyl ether.

 700 mg of griseolic acid is dissolved in 100 ml of dimethylformamide and the solution is cooled with ice. A solution of 1.0 1.2 mmol of diazomethane in 1 ml of diethyl ether is added with stirring to the first solution until a yellow color appears, indicating the formation of diazomethane. The mixture was then incubated for 10 minutes. Acetic acid was added to decompose the excess diazomethane, then the solvents were removed by evaporation under reduced pressure to give a residue. This residue was dissolved in methanol and the insoluble matter was filtered off, the filtrate was evaporated under reduced pressure to give a residue which was recrystallized from water to give 540 mg of the title compound.

UV absorption spectrum (in methanol), l _max. nm: 258 (3 15600).

 NMR spectrum (in deuterodimethyl sulfoxide), ppm 4.60 (1H, d, J 6.0 Hz) 4.66 (1H, s), 5.12 (1H, d, J 3.0 Hz), 6.06 (1H, dd, J 3.0 and 6.0 Hz) 6.53 (1H, s), 8.33 (1H, s), 8.37 (1H, s).

 Cooking 2.

Dimethyl 6-desamino-6-hydroxy-0 ^{2 ',} 0 ^7' -diatsetil-4'β-bromo-5'-gidrogrizeolovoy acid.

A mixture of 500 mg of 0 ^{2 ′} , 0 ^{7 ′} -diacetyl-6-desamino-6-hydroxygryzeolic acid dimethyl ether (prepared as described in Preparation 1) and 10 ml of acetic acid containing 1 g of hydrobromic acid is placed in a sealed vessel and dissolved, using ultrasound waves for 30 min. Then, the solution was kept at room temperature for 64 hours. The solvent was distilled off under reduced pressure to obtain a residue, to which acetone and toluene were added. Then the solvents are distilled off. These operations are repeated 3 times. A mixture of the resulting residue and 30 ml of ethyl acetate is treated with ultrasound waves and filtered to obtain an insoluble material. This material is dissolved in a mixture of 30 ml of ethyl acetate and 30 ml of an aqueous solution of sodium bicarbonate containing 1.5 g of salt, and the layers are separated. The organic phase layer was washed with saturated aqueous sodium chloride (20 ml) and dried over anhydrous magnesium sulfate. The solution was purified by silica gel column chromatography using methylene chloride containing 3 vol.% As eluent. methanol. The solvent was evaporated from the main fraction under reduced pressure, and the residue was dissolved in benzene. Leopard drying of a benzene solution gives 60 mg of the title compound as a white powder.

 NMR spectrum (in deuterodimethyl sulfoxide), ppm 2.98 (1H, d, J 15.6 Hz), 3.47 (1H, d, J 15.6 Hz), 5.35 (1H, d, J 4.2 Hz), 5.57 (1H singlet), 6.32 (1H, d, J 6.6 and 4.2 Hz), 6.53 (1H, d, J 6.6 Hz), 8.17 (1H, s), 8.47 (1H, s).

 Example 7

 2-amino-6-benzyloxygrycolic acid.

100 ml 0.2 n an aqueous solution of sodium hydroxide is added to 400 ml of methanol containing 8.8 g of dimethyl-N ^1- benzyloxy-N ⁶ -cyanogreezeolate (obtained by the method of example 5) with stirring. The reaction solution was alkalinized to pH 12 - 12.5 by adding 1 N. an aqueous solution of sodium hydroxide, and then left to stand at room temperature for 60 minutes. The solvent is distilled off until the remaining liquid volume reaches about 100 ml. The resulting solution was mixed with 50 ml of water and 150 ml of ethanol and heated at the boil under reflux for 2 hours. The solvent was distilled off again under reduced pressure to a liquid volume of about 100 ml. Add 100 ml of 2 N. an aqueous solution of sodium hydroxide and the resulting solution is left to stand at room temperature for 60 minutes 200 ml of ethyl acetate are added to the solution and acidified to pH 0.5 with concentrated hydrochloric acid with stirring.

 The aqueous and organic layers are separated, the organic layer is washed with 50 ml of 0.1 N. an aqueous solution of hydrochloric acid, and then combined with the aqueous layer and treated with activated carbon. The pH of the solution was adjusted to 2.3 by adding solid sodium bicarbonate with vigorous stirring. The resulting insoluble precipitate is placed overnight in the refrigerator. The resulting powdery substance was filtered off and dried to give 5.29 g of the title compound as a yellowish-white powder.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 4.54 (1H, singlet), 4.55 (1H, doublet, J 5.0 Hz), 5.06 (2H, singlet), 5.08 (1H, doublet, J 2.2 Hz), 5, 83, (1H, doublet of doublets, J = 2.2 & 5.0 Hz), 6.27 (1H, singlet), 7.2 7.6 (5H, multiplet), 7.79 (1H, singlet).

 Example 8

2-amino-N ⁶ -oxygryzeolic acid.

200 mg of 2-amino-N ^6- benzyloxygryzeolic acid obtained by the method of example 6 is heated with 20 ml of methanol and 20 ml of water. The resulting solution was allowed to stand at room temperature and cooled with ice. 50 mg of 10 (w / w) palladium on charcoal was added and the resulting mixture was stirred in a stream of hydrogen. Since the reaction mixture becomes cloudy due to the formation of a white substance in 30 minutes, it is clarified with 5 ml of 1 N. an aqueous solution of hydrochloric acid and the resulting solution was stirred for 1.5 hours. The catalyst was removed by filtration, methanol was distilled off under reduced pressure, and the pH of the solution was adjusted to 2.2 by adding a saturated aqueous solution of sodium bicarbonate with stirring. The reaction mixture was allowed to stand in an ice bath for 30 minutes, and then the resulting yellowish product was filtered off to obtain 100 mg of the title compound.

NMR spectrum ((CD ₃ ) ₂ SO)? d, ppm 4.54 (1H, singlet), 4.75 (1H, doublet, J 5.0 Hz), 5.11 (1H, doublet, J 2.2 Hz), 5.95 (1H, doublet of doublets, J 2 , 2 and 5.0 Hz), 6.31 (1H, singlet), 7.83 (1H, singlet).

 Example 9

 2-aminogrizeolic acid.

100 mg of 2-amino-N ^6- benzyloxygryzeolic acid obtained by the method of example 7 is dissolved in a mixture of 20 ml of 1 N. hydrochloric acid solution and 20 ml of acetone. 1 ml of Raney nickel (W-2) was added and the resulting mixture was stirred at room temperature for 2.5 hours. Raney nickel was filtered off and the pH of the reaction mixture was adjusted to 2.3. Then it is purified on a chromatographic column using RP-8 pre-packed column (Mersk), eluting with 3 (volume / volume) aqueous acetonitrile. The remaining fraction was lyophilized to give 12 mg of the title compound as a white powder.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 4.46 (1H, singlet), 4.55 (1H, doublet, J 5.0 Hz), 5.02 (1H, doublet, J 2.2 Hz), 5.87 (1H, doublet of doublets, J 2 , 2 and 5.0 Hz), 6.28 (1H, singlet), 7.90 (1H, singlet).

Thin layer chromatography (the ratio of R _f to the corresponding value for griseolic acid is taken as 1.0).

 Silica gel plate (Mersk): 0.8 (developing solvent: water methanol acetonitrile 70 15 15 by volume).

Reverse Phase Plate RP-8:
0.79 (developing solvent: water containing 2 (v / v) acetonitrile and 0.02 (v / v) acetic acid).

 Example 10

Dibenzihydride-2-amino-N ^6- benzyloxyhydrizeolate
1.0 g of 2-amino-N ^6- benzyloxygryzeolic acid obtained by the method of example 7, suspended in 100 ml of acetonitrile and 100 ml of water. Diphenyldiazomethane is added until its red color disappears. Then the reaction mixture is stirred while adding 4 ml of 1 N. an aqueous solution of hydrochloric acid and diphenyldiazomethane is added until the disappearance of its red color is no longer observed.

 The resulting mixture was stirred for 60 minutes. Acetone is removed by distillation under reduced pressure, and water is removed by decantation. The residue was dissolved in a mixture of 50 ml of ethyl acetate and 50 ml of water, the organic layer was washed with 30 ml of a 5 weight / volume aqueous solution of sodium bicarbonate and 30 ml of a saturated aqueous solution of sodium chloride and then dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was dissolved in 30 ml of ethyl acetate, and the resulting solution was poured into 500 ml of hexane with stirring. The resulting insoluble matter was collected by filtration and purified on a pre-packed chromatography column with silica gel (Megg), eluting with methylene chloride containing 2.5 vol / vol of methanol. A fraction is collected from the two main fractions, which comes later, evaporated to dryness and lyophilized from benzene to obtain 430 mg of the title compound as a white powder.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 4.63 (1H, doublet, J = 5.0 Hz) 4.99 (1H, singlet), 5.07 (2H, singlet) 5.31 (1H, doublet, doublet, J = 2.2 Hz) 5.97 ( 1H, doublet of doublets, J = 2.2 & 5.0 Hz), 6.33 (1H, singlet), 6.75 (1H, singlet), 6.81 (1H, singlet) 7.74 (1H, singlet) .

 Example 11

 Dibenzhydryl-2-hydroxygryseolate.

 0.6 g of dibenzhydryl-2-aminogrizeolate is dissolved in 50 ml of 80 (v / v) water with acetic acid. After replacing the air with nitrogen in the container, 1N was added to the resulting mixture. a solution of sodium nitrite under ice cooling and the resulting mixture was allowed to react at room temperature for 1.5 hours. The solvent was distilled off and the residue was mixed with water, the water was distilled off again. The substance remaining in the precipitate is suspended in water and collected by filtration. The residue was dissolved in 15 ml of acetone and the pH of the solution was adjusted to 9-10 by adding concentrated aqueous ammonia. The resulting solution was allowed to stand at room temperature for 20 minutes, and then the acetone was distilled off. The residue is mixed with 50 ml of ethyl acetate and 50 ml of water and mixed thoroughly. The resulting precipitate was filtered off and purified on a silica gel column chromatography (eluting with 10 (v / v) ethanol and methylene chloride) to obtain 0.35 g of the title compound. The separated ethyl acetate layer was washed with 10 (w / v) aqueous sodium bicarbonate and saturated aqueous sodium chloride and dried over anhydrous magnesium sulfate. The drying agent was removed by filtration and the solvent was distilled off. The resulting residue was purified by column chromatography using methylene chloride containing 10 (v / v) methanol as eluent to obtain 0.04 g of the title compound.

Nuclear Magnetic Resonance Spectrum ((CD ₃ ) ₂ SO), d, ppm 4.59 (1H, doublet, J = 5.0 Hz), 4.90 (1H, singlet), 5.25 (1H, doublet, J = 2.2 Hz) 6.06 (1H, doublet of doublets, J 2, 2 and 5.0 Hz), 6.30 (1H, singlet), 6.67 and 6.73 (together 1H, each singlet), 7.0 7.5 (2H, multiplet), 7.93 (1H, singlet).

 Example 12

 2-hydroxygryzeolic acid.

 0.37 g of dibenzhydryl ester of 2-hydroxygryzeolic acid (dissolved as described in Example 11) is dissolved in 3 ml of anilose. Under ice-cooling, 3 ml of trifluoroacetic acid was added and the mixture was allowed to stand for 10 minutes at room temperature.

 Then, toluene is added and after that the solvent is distilled off. Then add a mixture of acetone and toluene, which is subsequently distilled off. This technique is repeated twice, and the resulting mixture is suspended in 2 ml of acetone, which is poured into 100 ml of hexane with stirring. The precipitated substance is collected by filtration. The residue was dissolved in sodium bicarbonate solution (10 g in 100 ml) and this solution was acidified to pH 1.2 by the addition of concentrated hydrochloric acid. The resulting mixture was purified by chromatography using a pre-filled RP-8 column (Merck). Elution with water gives 0.17 g of the title compound.

NMR spectrum (in deuterodimethyl sulfoxide), ppm 4.46 (1H, singlet), 4.47 (1H, doublet, J 5.0 Hz), 5.05 (1H, d, J 2.2 Hz), 5.89 (1H, doublet pair, J 2 , 2 and 5.0 Hz), 6.24 1 H), s), 7.95 (1 H, s). Thin layer chromatography analysis (relative Rf) compared to griseolic acid taken 10): on an RP-8 plate for the reverse phase (Merck) R _f 1.32 (elution solvent: water containing 2 vol. Acetonitrile and 0 , 02 vol. Of acetic acid).

 Example 13

 6-Desamino-2-6-dihydroxygryzeoleic acid.

 Dissolve in 1 ml of anisole 40 mg of dibenzhydryl ester of 6-desamino-2,6-dihydroxygryzoleic acid. 1 ml of trifluoroacetic acid was added under ice-cooling, and the mixture was allowed to stand for 10-15 minutes at room temperature. These operations of adding and distilling off toluene, then adding and distilling off a mixture of acetone and toluene are repeated twice. The residue is suspended in 0.5 ml of acetone. After adding 20 ml of hexane, the precipitated substance was collected by filtration. The residue was dissolved in an aqueous solution of sodium bicarbonate (10 g / 100 ml). The solution was acidified to pH 0.6 by the addition of concentrated hydrochloric acid. The resulting solution was purified by chromatography using a pre-filled RP-3 column (reverse phase, Merck type). Eluting with water, obtaining 25 mg of the substance indicated in the workpiece.

 NMR spectrum (in deuterosulfoxide), ppm 4.49 (1H, singlet), 4.58 (1H, doublet, J 5.0 Hz), 5.12 (1H, d, J 2.2 Hz), 5.53 (1H, dd, J 2, 2 and 5.0 Hz), 6.41 (1H, s), 7.91 (1H, s).

Thin layer chromatography (values of R _f relative to griseolic acid, taken as 1.0): on platinum with silica gel (Merck company) R _f 0.57 (eluting solvent water methanol acetonitrile: 70 15 15 by volume), on a plate RP-8 for reverse phase (Merck) R _f 1.65 (water eluting solvent containing 2 vol. acetonitrile and 0.02 vol. acetic acid).

 Example 14

N ₁ -benzyloxy-2- (N ⁶ , N ² -dimethylaminomethylene) -aminogryzeolic acid dimethyl ester.

528 mg of 2-amino-N ^1- benzyloxy-gryzeolic acid dimethyl ester is dissolved in 10 ml of dimethylformamide. 0.24 ml of dimethylformamide dimethyl acetate was added and the mixture was allowed to stand for 2 hours at room temperature. After the disappearance of the starting material, as confirmed by thin layer chromatography, the solvent was distilled off under reduced pressure. The residue was dissolved in 40 ml of methylene chloride and 40 ml of water, and the organic layer was washed with 20 ml of a saturated aqueous solution of sodium chloride. All aqueous layers were combined and then mixed with 2 ml of an aqueous sodium bicarbonate solution (5 g in 100 ml) and extracted twice with 20 ml of methylene chloride. The organic layers are combined, dried with anhydrous magnesium sulfate and the solvent is distilled off under reduced pressure. The residue is subjected to chromatographic purification on a column of silica gel (Merck), eluted with methylene chloride containing 3 vol. methanol. After purification, the obtained substance is subjected to freeze drying from benzene. 322 mg of the title compound is obtained in the form of a white powder.

 NMR spectrum (in deuterodimethyl sulfoxide), ppm 3.04 (3H, singlet) 3.19 (3H, s), 3.67 (3H, s), 3.72 (3H, C) 4.47 (1H, doublet, J = 5.0 Hz) 4, 63 (1H, d) 5.01 (2H, s) 5.12 (1H, d, J 2.2 Hz) 6.00 (1H, dd, J 2.2 and 5.0 Hz) 6.38 ( 1H, s) 7.2 7.6 (5H, multiplet) 7.83 (1H, s) 8.50 (1H s).

 Example 15

 2-N ', N'-dimethylaminomethylene) aminogrizeolic acid dimethyl ester.

583 mg of N ⁶ -benzyloxy-2-N ', N'-dimethylaminomethylene) aminogrizeolic acid dimethyl ester (prepared as described in Example 14) was dissolved in 100 ml of acetone and 75 ml of hydrochloric acid (1 N). Add 10 ml of Raney Nickel (brand W-2) suspended in water, and the resulting mixture was stirred at room temperature for 30 minutes, maintaining the pH not lower than 1.0, analyzing the acidity with a pH meter. Raney Nickel (catalyst) is removed by evaporation under reduced pressure. When acetone is almost completely removed, the resulting mixture is stirred with 200 ml of methylene chloride and neutralized with an aqueous solution of sodium bicarbonate. The remaining insoluble precipitate is removed by filtration. After separation of the organic layer, the aqueous layer was extracted 3 times, each time with 50 ml of methylene chloride. These organic layers are combined and dried over anhydrous magnesium sulfate. The solvent is distilled off under reduced pressure, and the residue is subjected to purification on a chromatographic column (company Merck), eluted with methylene chloride containing 5 vol. methanol. The resulting substance is purified and subjected to freeze drying from benzene. 180 mg of the title compound are obtained in the form of a white powder.

 NMR spectrum (deuterodimethyl sulfoxide), ppm 3.04 (2H, s) 3.15 (3H, s) 3.67 (3H, s) 3.73 (3H, s) 4053 (1H, d J 5.0 Hz) 4.64 (1H, s ) 5.13 (1H, d J 2.2 Hz) 6.06 (1H, dd, J 2.2 and 5.0 Hz) 6.47 (1H, s) 7.33 (1H, s).

 Example 16

 6-Desamino-2- (N ', N'-dimethylaminomethylene-amino-6-hydroxygryzeolic acid dimethyl ester.

 477 mg of 2- (N ', N'-dimethylaminomethylene) -aminogrycelic acid dimethyl ester (prepared as described in Example 15) was dissolved in 50 ml of an aqueous solution of acetic acid (80 vol.).

 While cooling with ice, 1.34 g of sodium nitrite was added and the mixture was allowed to stand for 17 hours at room temperature. After the disappearance of the starting material, which is controlled by thin layer chromatography, the solvent is distilled off under reduced pressure.

 Ethanol is added and then it is distilled off, and these addition and distillation operations are repeated until the smell of acetic acid disappears. The residue is dissolved in a mixture of 50 ml of methylene chloride, 20 ml of water and 5 ml of an aqueous solution of sodium bicarbonate (5 g in 100 ml). The organic layer was separated and extracted 3 times, 30 ml of methylene chloride each time and the extracts combined.

 The solvent was distilled off under reduced pressure. The residue is purified using a chromatographic column (Merck company), pre-filled with silica gel and eluted with methylene chloride containing 10 vol. methanol. The main fractions were collected and freeze-dried from benzene to give 310 mg of the title compound as a white powder.

UV absorption spectrum (in methanol), l _max nm: acid 292, neutral
300, core 279.

 Example 17

 2-amino-6-desamino-6-hydroxygryzeolic acid.

 130 mg of 6-desamino-2- (N ', N'-dimethylamino-methylene) -amino-6-hydroxy-gryzeolic acid dimethyl ester (prepared as described in Example 16) was dissolved in 20 ml of concentrated aqueous ammonia and the mixture was allowed to stand at room temperature for 3 hours. The solution was evaporated dry under reduced pressure and the residue was dissolved in 10 ml of water. The resulting solution was acidified to pH 2.3, and then subjected to chromatographic purification on a pre-filled column RP-8 (Merck), which was washed with water and then eluted with water containing 5 vol. acetonitrile. The main fractions were collected and lyophilized to give 67 mg of the title compound as a white powder.

Spectrum-UV absorption (in water), λ _max , nm: acid 255, 273 (shoulder) neutral 253, 278 (shoulder); core 264.

 NMR spectrum (in deuterodimethyl sulfoxide), ppm 4.48 (1H, singlet) 4.53 (1H, doublet, J 4.9 Hz); 5.07 (1H, d. J 2.4 Hz); 5.79 (1H, doublet of doublets, J = 2.4 & 4.9 Hz); 6.25 (1H, C); 7.87 (1H, s).

Thin layer chromatography (relative value of R _f , for griseolic acid, R _{f is} taken as 1.0):
for silica gel plate (Merck company) 0.80 (eluting solvent water intanol acetonitrile 70 15 15 by volume) for RP-8 plate with reverse phase (Merck) 1.44 (eluting solvent water containing 2 vol. of acetonitrile and 0, 02 vol. Acetic acid).

 Example 18

By the procedure of Example 9, but using 2-amino-N ^6- methoxy-gryzeolic acid instead of 2-amino-N ^6- benzyloxy-gryzeolic acid, the same final product, 2-amino-gryzeolic acid, was obtained, yield 30 as a white powder.

 NMR data and biological activity data are similar to those for the compound of Example 9.

 Biological activity data are similar to those of Example 9.

 Example 19

 The procedure of Example 5 was repeated, but changing the reaction time for 1 h and then separately for 5 h, the finished product was obtained with practically the same yield as in Example 5.

 Preparative example.

 Dihydrodeoxygryzeolic acid.

Prepare 30 l of medium having a pH of about 7.0 before sterilization, the following composition, mass-percent by weight:
Glucose 5
Soya flour 1
Yeast extract 0.1
Polypepton 0.4
Meat extract 0.4
Sodium Chloride 0.25
Calcium Carbonate 0.25
Water Up to 100
15 liters of this medium are loaded into each of two 30-liter vibratory fermenters. The medium is then sterilized under pressure at 120 ^° C. for 30 minutes. The culture medium is cooled and then 150 ml (1 by volume) of Streptomyces geiseoausantiacns APK 63479 culture broth are inoculated into each fermenter, which is previously incubated in the medium described above in a rotary shaker cultivator at 28 ^° C. for 72 hours. Then, cultivation is carried out at 28 ^o C for 96 hours with aeration at a speed of 15 l of air per minute and stirring at a speed of 200 rpm

 Then, two culture broths are filtered to remove the mycelial cake and the combined filtrates (pH 7.0) with a total volume of 28 L are passed through a Diaion HP 20 ion exchange resin column (a trademark of the resin manufactured by Mitsubishi Chemical Industries Ltd.) and then adsorbed onto activated carbon column. This column is washed with water and then the adsorbed material is eluted with a mixture of 40-60 (vol.) Acetone and water. Acetone was evaporated from the resulting solution under reduced pressure, and the remaining aqueous solution was concentrated by evaporation under reduced pressure, and then freeze-dried to obtain 150 mg of a crude powder.

This crude powder is dissolved in a small amount of distilled water and then adsorbed on a 1 x 4 Dowex ion exchange resin (in the form of CI ^- , trademark resin produced by Dow Chemical Co.). At this stage, the product is a mixture of griseolic acid and dihydrodeoxygryzeolic acid. This mixture was subjected to gradient elution with a gradient of sodium chloride in order to separate the two components. Then, the eluate is fed to a chromatographic column filled with Sephadex IH-20 resin (a trademark of a product of Pharmacia Co.), whereby dihydrodeoxyhydrize acid is eluted with water. The fractions containing this substance are combined and acidified to pH 2.5 by adding 1 N. aqueous hydrochloric acid. Then the product is adsorbed on a column with Diaion RH 20 resin washed with water, and then eluted with a mixture of 60 to 40 vol. acetone and water. The eluate was allowed to stand overnight at 4 ^° C., after which the dihydrodeoxygryzeolic acid was isolated in the form of plates. These plates are separated from the solution, and a total of 1.87 mg of dihydrodeoxygryzeolic acid is obtained in the form of white plates melting at 160 ^° C. (with decomposition accompanied by a color change to brown). This compound gives one spot when chromatographed by thin layer chromatography on silica gel (silica gel article 5715, a product from Merck & Co., Inc.).

Claims (2)

1. Derivatives of griseolic acid of General formula I

where R ⁵ and R ⁶ both represent an additional carbon-carbon bond between the carbon atoms to which they are attached; R ^{2 9} OH group;
R ^{3 0} (C ₁ -C ₄ ) alkyl or aryl (C ₁ -C ₄ ) alkyl,
R ^{3 1} and R ^{3 2, the} same or different, represent a hydrogen atom or a carboxy protecting group, or pharmaceutically acceptable salts thereof, or esters thereof. 2. Derivatives of griseolic acid of General formula II

where R ⁵ and R ⁶ both represent a hydrogen atom or together represent an additional carbon-carbon bond between the carbon atoms to which they are attached;
R ^{2 2} and R ^{2 3 are the} same or different and each represents a carboxy-protecting group;
R ^{2 9 is a} hydroxyl group;
R ^{3 0} alkyl or aralkyl optionally substituted in the aryl moiety with at least one nitro, methoxy or halo group;
or their pharmaceutically acceptable salts and their esters.

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