CA2468181A1 - Hydroperylene derivatives - Google Patents
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- C07D303/34—Compounds containing oxirane rings with hydrocarbon radicals, substituted by sulphur, selenium or tellurium atoms
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Abstract
The invention relates to compounds of formula (I) which are suitable for producing medicaments used for the prophylaxis and therapy of illnesses characterised by increased blood platelet aggregation.
Description
Description Hydroperylene derivatives The invention relates to hydroperylene derivatives which are formed by the fungus ST003367, DSM 14452, during fermentation, or are subsequently derivatized, to processes for preparing them and to their use as pharmaceuticals and inhibitors of the blood platelets.
Hydroperylene derivatives, such as hexahydroperylene derivatives or octahydroperylene derivatives, are formed by various microorganisms. Examples of known hydroperylene derivatives are:
~ Altertoxins I, II and III (M. E. Stark et al. J. Nat. Prod. 49, 866-871, 1986), ~ Alteichin (D. Robeson et al. Experientia, 40, 1248-1250, 1984), ~ the Alterlosins (A. Stierle et al. J. Nat. Prod. 52, 42-47, 1989) and ~ Alterperylenol from Alternaria species (T. Okuno et al. Tetrahedron t_ett.
24, 5653-5656, 1983);
~ Stemphyltoxins and Stemphypyrenol, which come from cultures of Stemphylium botryosum (A. Arnone et al. J. Chem. Soc. Perkin Trans.1, 1986, 525-530).
The altertoxins and the stemphyltoxins are phytotoxins which, as a result of their mutagenic effect, can also diminish the value of human foodstuffs. They have therefore been thoroughly described in more than 70 publications (T. J. Schrader et al.
Teratog., Carcinog., Mutagen. 21, 261-274, 2001 ). The dangers which emanate from the altertoxins and stemphyltoxins are naturally dealt with in this connection; a result of the investigations is the wide distribution, and the cancer-eliciting effect, of these compounds, which contain epoxides (oxiranes). In addition to this, alterperylenol has been reported to have a weak telomerase-inhibiting effect (K.-I. Togashi et al. Oncol.
Res. 10, 449-453, 1998); however, at 30 NM, the ICSO value, which is characteristic for inhibition of the enzyme, is low.
Thromboembolic diseases are the most frequent cause of death, particularly in the western industrial nations. An effective prophylaxis and therapy for these diseases is therefore of exceptional importance. A thrombus is understood as being a blood clot which has been formed intravitally and intravascularly. Thrombi are formed following thrombocyte aggregation in arteries, in particular. Damage to the blood vessel wall, retarded blood flow and accelerated clotting all favor thrombus formation.
The thrombocytes (blood platelets) are discus-shaped, anucleate blood cells which ensure hemostasis and blood coagulation when injury occurs. Thrombocytes bring about hemostasis by means of aggregation in a complicated process; thrombocyte aggregation is consequently an essential process for homeotherms.
Nlypofunction r:f the thrombocytes leads to severe hemorrhages, even in the case of relatively small injuries;
on the other hand, an increased tendency towards coagulation increases the danger of thrombosis and embolism. Since platelet hyperfunction, in particular, frequently has fatal consequences, several substances have already been employed as inhibitors of thrombocyte aggregation. Those which are well known are acetylsalicylic acid (Aspirin~), ticlopidine (Tiklyd~) and the related clopidogrel; however, because of side-effects, the use of all these preparations is restricted. For this reason, there is a great need for inhibitors of intracellular platelet activation which can be used for the therapy and long-term prophylaxis of arterial thromboembolic events. Agents of this nature can be employed, for example, in connection with myocardial infarction, in connection with unstable angina or in connection with strokes.
In the endeavor to find effective compounds for preventing or treating blood coagulation diseases it has now been found that thioperylenol, which is formed by the fungal strain ST 003367, DSM 14452, and other hydroperylene derivatives, are able to effectively inhibit blood platelet aggregation.
The invention therefore relates to the compound of the formula I
\ R4 R10 \ ~ J ~a and/or all stereoisomeric forms of the compound of the formula I andlor mixtures of these forms in any ratio, andlor to a physiologically tolerated salt of the compound of the .. formula I, where R1, R2, R3, R4, R5, R6, R8, R10 and R11 are, independently of each other, 1. a hydrogen atom, 2. (C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by 2.1 -OH, 2.2 =O, 2.3 -O-(C,-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by 2.3.1 -CN, 2.3.2 -NH2, 2.3.3 =N-OH or 2.3.4 =N-O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched, 2.4 -O-(C2-Cs)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.5 -O-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.6 -aryl, in which aryl is unsubstituted or substituted once, twice or three times, independently of each other, by 2.6.1 halogen, 2.6.2 -(C~-C4)-alkyl, in which alkyl is straight-chain or branched, 2.6.3 -O-(C~-C6}-alkyl, in which alkyl is straight-chain or branched, 2.6.4 -OH or 2.6.5 -(C,-C4)-alkyl, in which alkyl is straight-chain or branched and is substituted once, twice or three times by halogen, 2.7 -C(O)-OH, 2.8 -C(O)-O-NH2, 2.9 -C(O)-O-(C,-C4)-alkyl, in which alkyl is straight-chain or branched, 2.10 -NH-(C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4.
2.11 -NH-(C2-Cs)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.12 -NH-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.13 -NH2 or 2.14 halogen, 3. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 4. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 5. -O-R9, in which R9 is 1. (C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 2. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, or 3. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above-under 2.1 to 2.14, .
Hydroperylene derivatives, such as hexahydroperylene derivatives or octahydroperylene derivatives, are formed by various microorganisms. Examples of known hydroperylene derivatives are:
~ Altertoxins I, II and III (M. E. Stark et al. J. Nat. Prod. 49, 866-871, 1986), ~ Alteichin (D. Robeson et al. Experientia, 40, 1248-1250, 1984), ~ the Alterlosins (A. Stierle et al. J. Nat. Prod. 52, 42-47, 1989) and ~ Alterperylenol from Alternaria species (T. Okuno et al. Tetrahedron t_ett.
24, 5653-5656, 1983);
~ Stemphyltoxins and Stemphypyrenol, which come from cultures of Stemphylium botryosum (A. Arnone et al. J. Chem. Soc. Perkin Trans.1, 1986, 525-530).
The altertoxins and the stemphyltoxins are phytotoxins which, as a result of their mutagenic effect, can also diminish the value of human foodstuffs. They have therefore been thoroughly described in more than 70 publications (T. J. Schrader et al.
Teratog., Carcinog., Mutagen. 21, 261-274, 2001 ). The dangers which emanate from the altertoxins and stemphyltoxins are naturally dealt with in this connection; a result of the investigations is the wide distribution, and the cancer-eliciting effect, of these compounds, which contain epoxides (oxiranes). In addition to this, alterperylenol has been reported to have a weak telomerase-inhibiting effect (K.-I. Togashi et al. Oncol.
Res. 10, 449-453, 1998); however, at 30 NM, the ICSO value, which is characteristic for inhibition of the enzyme, is low.
Thromboembolic diseases are the most frequent cause of death, particularly in the western industrial nations. An effective prophylaxis and therapy for these diseases is therefore of exceptional importance. A thrombus is understood as being a blood clot which has been formed intravitally and intravascularly. Thrombi are formed following thrombocyte aggregation in arteries, in particular. Damage to the blood vessel wall, retarded blood flow and accelerated clotting all favor thrombus formation.
The thrombocytes (blood platelets) are discus-shaped, anucleate blood cells which ensure hemostasis and blood coagulation when injury occurs. Thrombocytes bring about hemostasis by means of aggregation in a complicated process; thrombocyte aggregation is consequently an essential process for homeotherms.
Nlypofunction r:f the thrombocytes leads to severe hemorrhages, even in the case of relatively small injuries;
on the other hand, an increased tendency towards coagulation increases the danger of thrombosis and embolism. Since platelet hyperfunction, in particular, frequently has fatal consequences, several substances have already been employed as inhibitors of thrombocyte aggregation. Those which are well known are acetylsalicylic acid (Aspirin~), ticlopidine (Tiklyd~) and the related clopidogrel; however, because of side-effects, the use of all these preparations is restricted. For this reason, there is a great need for inhibitors of intracellular platelet activation which can be used for the therapy and long-term prophylaxis of arterial thromboembolic events. Agents of this nature can be employed, for example, in connection with myocardial infarction, in connection with unstable angina or in connection with strokes.
In the endeavor to find effective compounds for preventing or treating blood coagulation diseases it has now been found that thioperylenol, which is formed by the fungal strain ST 003367, DSM 14452, and other hydroperylene derivatives, are able to effectively inhibit blood platelet aggregation.
The invention therefore relates to the compound of the formula I
\ R4 R10 \ ~ J ~a and/or all stereoisomeric forms of the compound of the formula I andlor mixtures of these forms in any ratio, andlor to a physiologically tolerated salt of the compound of the .. formula I, where R1, R2, R3, R4, R5, R6, R8, R10 and R11 are, independently of each other, 1. a hydrogen atom, 2. (C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by 2.1 -OH, 2.2 =O, 2.3 -O-(C,-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by 2.3.1 -CN, 2.3.2 -NH2, 2.3.3 =N-OH or 2.3.4 =N-O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched, 2.4 -O-(C2-Cs)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.5 -O-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.6 -aryl, in which aryl is unsubstituted or substituted once, twice or three times, independently of each other, by 2.6.1 halogen, 2.6.2 -(C~-C4)-alkyl, in which alkyl is straight-chain or branched, 2.6.3 -O-(C~-C6}-alkyl, in which alkyl is straight-chain or branched, 2.6.4 -OH or 2.6.5 -(C,-C4)-alkyl, in which alkyl is straight-chain or branched and is substituted once, twice or three times by halogen, 2.7 -C(O)-OH, 2.8 -C(O)-O-NH2, 2.9 -C(O)-O-(C,-C4)-alkyl, in which alkyl is straight-chain or branched, 2.10 -NH-(C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4.
2.11 -NH-(C2-Cs)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.12 -NH-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.13 -NH2 or 2.14 halogen, 3. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 4. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 5. -O-R9, in which R9 is 1. (C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 2. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, or 3. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above-under 2.1 to 2.14, .
6. -NH-R9, in which R9 is defined as above under 5., 7. -NH-C(O)-H, 8. -NH-C(O)- R9, in which R9 is defined as above under 5., 9. -NH-aryl, in which aryl is unsubstituted or substituted once, twice or three times 5 by R9, 1 ~. =N-OH, 11. =N-O-R9, in which R9 is defined as above under 5., 12. -S-H, 13. -S-R9, in which R9 is defined as above under 5., 14. -S(O)-R9, in which R9 is defined as above under 5., 15. -S(O)2-R9, in which R9 is defined as above under 5., or 16. -S02, or R4 and R5 or R10 and R11, together with the carbon atoms to which they are in each case bonded, form a 3-, 4-, 5- or 6-membered ring system which is aromatic or saturated and contains one or two heteroatoms from the series oxygen, nitrogen and sulfur, R7 has the meaning of R1 as defined above for 2. to 16., and the bond between -Coq-C,5- is a single bond or a double bond.
The invention also relates to a compound of the formula I, where R1, R2, R3 and R6 are in each case -OH, R4 and R5, together with the carbon atom to which they are in each case bonded, form an epoxide, R7 is the radical -S-CH2-CHOH-COOH, R8 is =O, R10 and R11 are in each case a hydrogen atom and the bond between -C,4-C~5- is a single bond, and/or to physiologically tolerated salts of the compound of the formula I.
The invention also relates to optically pure compounds of the formula I and to their stereoisomeric mixtures, such as enantiomeric mixtures and diasteromeric mixtures, in .- any ratio to each other.
The term "halogen" is understood as meaning fluorine, chlorine, bromine or iodine. The term "(C,-Cs)-alkyl" is understood as meaning hydrocarbon radicals whose carbon chain is straight-chain or branched and contains from 1 to 6 carbon atoms, for example methyl, ethyl, propyl, i-propyl, butyl, tertiary butyl, pentyl and hexyl. The term "(C2-C6)-alkenyl" is understood as meaning hydrocarbon radicals whose carbon chain is straight-chain or branched and contains from 2 to 6 carbon atoms, and which exhibits one, two or three double bonds, for example the radicals allyl, crotyl, 1-propenyl, yenta-1,3-dienyl, pentenyl and hexenyl. The term "(C2-C6)-alkynyl" is understood as meaning hydrocarbon radicals whose carbon chain is straight-chain or branched and contains from 2 to 6 carbon atoms and which exhibits one or two triple bonds, for example the radicals propynyl, butynyl and pentynyl.
The term "aryl" is understood as meaning the radicals phenyl, benzyl and 1-naphthyl and 2-naphthyl.
The expression "R4 and R5, together with the carbon atoms to which they are in each case bonded, form a 3-, 4-, 5- or 6-membered ring system which is aromatic or saturated and which contains one or two heteroatoms from the series oxygen, nitrogen or sulfur" is understood as meaning radicals such as epoxide, aziridine, azetidine, azetine, pyrrole, pyrrolidine, pyridine, piperidine, tetrahydropyridine, pyrazole, imidazole, pyrazoline, imidazoline, pyrazolidine, imidazolidine, pyridazine, pyrimidine, pyrazine, piperazine, pyran, oxazole, isoxazole, 2-isoxazoline, isoxazolidine, morpholine, oxothiolane, thiopyran, thiazole, isothiazole, 2-isothiazoline, isothiazolidine or thiomorpholine.
The invention furthermore relates to the compound of the formula II
OH OH
,o ,s \ 20 ..
~~ O
..,, '2 ~~~\ 8 OH
H off s~~
~a 5 '6 ~~~,, ' " COOH
15 2, 23 _ In that which follows, the compound of the formula II is termed thioperylenol (empirical formula: C23H2pNOgS; molecular weight 472.47) and to the physiologically tolerated salts thereof.
The invention furthermore relates to a process for preparing the compound of the formula I, and/or a stereoisomeric form of the compound of the formula !
andlor mixtures of these forms in any ratio, andlor a physiologically tolerated salt of the compound of the formula I, which process comprises a) culturing the microorganism DSM 14452, or its mutants or variants, in an aqueous nutrient medium and isolating and purifying the compound thioperylenol, or b) converting thioperylenol, by means of chemical derivatization, into a compound of the formula I, or c) resolving a compound of the formula I, which has been prepared by methods a) or b) and which, because of its chemical structure, appears in enantiomeric forms, into the pure enantiomers by forming salts with enantiomerically pure acids or bases, by chromatography on chiral stationary phases or by derivatizing with chiral, enantiomerically pure compounds such as amino acids, separating the diastereomers which are thus obtained and eliminating the chiral auxiliary groups, or d) either isolating the compound of the formula I which has been prepared by the methods a), b) or c) in free form or, when acidic or basic groups are present, converting it into physiologically tolerated salts.
The microorganism DSM 14452 (internal designation ST003367) belongs to the fungal group and possesses a white substrate mycelium and very little aerial mycelium and was deposited on August 3, 2001, under the conditions of the Budapest Treaty, in the DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen [German collection of microorganisms and cell cultures] GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, under the number DSM 14452.
Variants of DSM 14452 are understood as being DSM 14452 strains which have been obtained by isolating individual colonies from a culture of DSM 14452 on the proviso that they produce thioperylenol. Mutants of DSM 14452 are understood as being DSM
strains which have been obtained from a culture of DSM 14452 after mutation, with the _ proviso that they produce thioperylenol. Mutants of DSM 14452 can be generated, in a manner known per se, by physical means, for example irradiation, such as ultraviolet or X-ray radiation, or by using chemical mutagens, for example ethyl methanesulfonate (EMS); 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-vitro-N-nitrosoguanidine (MNNG).
The mutants are found, for example, by taking samples from the culture medium and determining the inhibitory effect of thioperylenol.
Thioperylenol is produced by culturing DSM 14452. The nutrient solution contains carbon sources, such as sucrose, corn starch, dextrose, lactose, D-mannitol, molasses or malt extract, and nitrogen sources, such as soybean flour, peanut flour, proteins, peptones, peptides, tryptones, meat extract, yeast extract or ammonium salts or nitrates.
The nutrient solution also contains inorganic salts such as sodium hydrogen phosphate, sodium chloride, calcium chloride, calcium sulfate, calcium carbonate, magnesium sulfate or potassium hydrogen phosphate. In addition, it is also possible to add fat, such as methyl oleate or soybean oil, to the nutrient medium. Besides that, trace elements, such as iron salts, manganese salts, copper salts, zinc salts or cobalt salts, or other metal salts, are also added.
A preferred nutrient solution contains from about 0.05% to 5%, preferably from 1 % to 3%, potato dextrose and from about 0.05% to 3%, preferably from 0.05% to 1 %, yeast extract. The percentages relate to the weight of the total nutrient solution.
DSM 14452 is cultured at temperatures of from 18°C to 35°C, preferably at from 23°C to 28°C, and at pH values of from 3 to 10, preferably from 5 to 9, particularly preferably at values of from 4 to 6. The culture is carried out aerobically, initially in shaking flasks and, after that, in a fermenter while stirring and aerating with air or pure oxygen. The microorganisms are cultured in the fermenters for a period of from 48 to 720 hours, preferably of from 72 to 144 hours.
The formation of thioperylenol reaches its maximum after from about 96 to 144 hours.
The fungal strain DSM 14452 also forms mixtures of several compounds of the formula I
in the nutrient solution. The quantitative proportion of one or more of the compounds of the formula I can vary depending on the composition of the nutrient solution.
In addition, the composition of the medium can be used to direct the synthesis of individual compounds of the formula I such that the microorganism either does not produce individual compounds of the formula I at all or only produces them in a quantity which is below the limit of detection.
Thioperylenol is either isolated directly from the nutrient solution or isolated after the cells have been separated off, for example by means of centrifugation or filtration. The thioperylenol can be isolated by extracting with solvents or by adsorbing on resins such as XAD 16, HP 20, MCI Gel~ CHP20P or ion exchangers.
It is purified, for example, by chromatography on adsorption resins such as on Diaion~
HP-20 (Mitsubishi Casei Corp., Tokyo), on Amberlite~ XAD 7 (Rohm and Haas, USA), or on Amberchrom~ CG (Toso Haas, Philadelphia, USA). The separations can be carried out over a wide pH range. The range of pH 1 to pH 9 is preferred, with the range of pH 2 to pH 8 being particularly preferred. In addition to this, reverse phase supports, which are used within the context of high pressure liquid chromatography (HPLC), are also suitable. Another isolation method is that of using molecular sieves such as Fractogel~
TSK HW-40S or Sephadex~ LH-20.
The microbiologically produced thioperylenol is. used as the starting material for preparing the thioperylenol derivative. The compounds of formula I are prepared in a manner known per se; for example, the epoxide group of the thioperylene can be converted by hydrolysis or solvolysis into alcohols or esters. Epoxides are very reactive compounds which, in addition to water and acids, also add on other nucleophilic reagents, such as alcohols, thiols, amines and Grignard compounds, in the presence of acidic or basic catalysts. The addition of hydrogen cyanide leads further on to f3-hydroxy-propionitrile derivatives. In addition to this, epoxides can be rearranged to give carbonyl-derivatives, with this rearrangement being catalyzed, for example, by Lewis acids. In addition, the reaction products can be subjected to further reactions. These reactions are known per se and are described, for example, by Jerry March, Advanced Organic Chemistry, John Wiley & Sons, 4t" Edition, 1992.
Other derivatives are obtained if the 3-thio-lactic acid radical of the thioperylenol is reacted reductively or oxidatively. It can also be advantageous to use the sulfide as what is termed a leaving group and replace it with other suitable radicals in a manner known from the literature. In order to carry out reactions selectively, it can be advantageous to introduce suitable protecting groups, in a manner known per se, prior to the reaction.
_ The protecting groups ace eliminated after the reaction and the reaction product is then purified.
Pharmacologically tolerated salts of the compounds of the formula I are understood as 5 being both inorganic and organic salts, as are described in Remington's Pharmaceutical Sciences (17th edition, page 1418 (1985)). Physiologically tolerated salts are prepared in a manner known per se from compounds of the formula I, including their stereoisomeric forms, which are capable of forming salts. With basic reagents, such as hydroxides, carbonates, hydrogen carbonates, alcoholates and ammonia, or organic bases, for 10 example trimethylamine, triethylamine, ethanolamine or triethanolamine, or else basic amino acids, for example lysine, ornithine or arginine, the carboxylic acid forms stable alkali metal salts, alkaline earth metal salts or, where appropriate, substituted ammonium salts. If the compound of the formula I possesses basic groups, it is also possible to prepare stable acid addition salts with strong acids. Both inorganic acids and organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, benzenesulfonic acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, 4-bromobenzenesulfonic acid, trifluoromethylsulfonic acid, cyclohexylamidosulfonic acid, acetic acid, oxalic acid, tartaric acid, succinic acid and trifluoroacetic acid, are suitable for this purpose.
The invention also relates to pharmaceuticals which are characterized by an effective content of at least one compound of the formula I and/or a physiologically tolerated salt of the compound of the formula I and/or an optionally stereoisomeric form of the compound of the formula I, together with a pharmaceutically suitable and physiologically tolerated carrier substance, additive and/or other active compounds and auxiliary substances.
On account of their pharmacological properties, the compounds of the formula I
are suitable for the prophylaxis and therapy of all those diseases in which high blood platelet aggregations occur and which are caused by thrombus formation or embolisms.
These diseases include, for example, myocardial infarction, unstable angina pectoris, stroke, transitory ischemic attacks and peripheral arterial occlusion diseases (peripheral vascular disease) such as intermittent claudication.
The invention also relates to the use of at least one compound of the formula I andlor all stereoisomeric forms of the compound of the formula I andlor mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur where R1, R2, R3, R4, R5, R6, R7, R8, R10 and R11 are, independently of each other, 1. a hydrogen atom, 2. (C,-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by 2.1 -OH, 2.2 =O, 2.3 -O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by , 2.3.1 -CN, 2.3.2 -N H2, 2.3.3 =N-OH or 2.3:4 =N-O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched, 2.4 -O-(C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.5 -O-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.6 -aryl, in which aryl is unsubstituted or substituted once, twice or three times, independently of each other, by 2.6.1 halogen, 2.6.2 -(C~-C4)-alkyl, in which alkyl is straight-chain or branched, 2.6.3 -O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched, 2.6.4 -OH or 2.6.5 -(C,-C4)-alkyl, in which alkyl is straight-chain or branched and is substituted once, twice or three times by halogen, 2.7 -C(O)-OH, _, 2.8 -C(O)-O-NH2, 2.9 -C(O)-O-(C,-C4)-alkyl, in which alkyl is straight-chain or branched, 2.10 -NH-(C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.11 -NH-(C2-Cs)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.12 -NH-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.13 -NH2, or 2.14 halogen, 3. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 4. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 5. -O-R9, in which R9 is 1. (C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 2. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, or 3. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 6. -NH-R9, in which R9 is defined as above under 5., 7. -N H-C(O)-H, 8. -NH-C(O)- R9, in which R9 is defined as above under 5., 9. -NH-aryl, in which aryl is unsubstituted or substituted once, twice or three times by R9, 10. =N-OH, 11. =N-O-R9, in which R9 is defined as above under 5., 12. -S-H, 13. -S-R9, in which R9 is defined as above under 5., 14. -S(O)-R9, in which R9 is defined as above under 5., 15. -S(O)2- R9, in which R9 is defined as above under 5., or 16. -S02, or R4 and R5 or R10 and R11, together with the carbon atoms to which they are in each case bonded, form a 3-, 4-, 5- or 6-membered ring system which is aromatic or saturated and which contains one or two heteroatoms from the series oxygen, nitrogen or sulfur, and the bond between -C,4-C~5- is a single bond or a double bond.
The invention furthermore relates to the use of at least one compound of the formula I
for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur, where R1, R3 and R8 are, independently of each other, -OH or =O, R2 is -OH, R4 and R5 are, independently of each other -OH or a hydrogen atom, or R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6, R10 and R11 are, independently of each other, -OH or a hydrogen atom, or R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 is a hydrogen atom or the radical -S-CH2-CHOH-COOH, and the bond between -C~4-C~5- is a single bond or a double bond.
The invention furthermore relates to the use of at least one compound of the formula I
for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur, with thioperylenol, a compound of the formula I, in which R1, R2, R3 and R6 are in each case -OH, R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 is the radical -S-CH2-CHOH-COOH, R8 is =O, R10 and R11 are in each case a hydrogen atom and the bond between -C~4-C~5- is a single bond, alterperylenol, a compound of the formula I, in which R1, R2, R5 and R6 are in each case -OH, R3 and R8 are in each case =O, R4, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C~4-C~5- is a double bond, altertoxin I, a compound of the formula I in which R1, R2, R5 and R6 are in each case -OH, R3 and R8 are in each case =O, R4, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C~4-C~5- is a single bond, altertoxin II, a compound of the formula I, in which R1, R2 and R6 are in each case -OH, R3 and R8 are in each case =O, R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C~4-C~5- is a single bond, and altertoxin III, a compound of the formula I, in which R1 and R3 are in each case =O, R2 and R8 are in each case -OH, R4 and R5 and R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6 and R7 are in each case a hydrogen atom, and the bond between -Coq-C~5- is a single bond, being employed.
The invention also relates to a process for producing a pharmaceutical, which process comprises bringing at least one compound of the formula I, together with a pharmaceutically suitable and physiologically tolerated excipient and, where appropriate, other suitable active compounds, additives or auxiliary substances, into a suitable form for administration.
Examples of suitable solid or galenic preparation forms are granules, powders, sugar-coated tablets, tablets, (micro)capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and preparations having a protracted release of the active compound, in the production of which use is made of customary adjuvants such as carrier substances, disintegrants, binders, coating agents, swelling agents, glidants or lubricants, flavorings, sweeteners and solubilizers. Frequently employed auxiliary substances which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils, such as cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol and solvents, such as sterile water and monohydric 5 or polyhydric alcohols, such as glycerol.
The pharmaceutical preparations are preferably produced and administered in dosage units, with each unit containing, as the active constituent, a particular dose of the 'compound of the formula I according to the invention. In the case of solid dosage units, 10 such as tablets, capsules, sugar-coated tablets or suppositories, this dose can be from 0.1 mg/kg of body weight to 1 000 mg/kg of body weight, preferably from 0.2 mg/kg of body weight to 100 mg/kg of body weight. They are expediently administered in dosage units which contain at least the effective daily quantity of the compound of the formula I, for example up to 1 000 mg, preferably, however, from about 50 to 300 mg and, in the 15 case of injection solutions in ampoule form, up to about 300 mg, preferably, however, from about 10 to 100 mg.
Daily doses of from about 20 mg to 1 000 mg, preferably from about 100 mg to 500 mg, of active compound, depending on the activity of the compound according to formula I, are indicated for treating an adult patient of about 70 kg in weight. However, it may possibly also be appropriate to use higher or lower daily doses. The daily dose can be administered either by means of a once-only administration in the form of a single dosage unit or of several smaller dosage units or by means of a repeated administration of subdivided doses at particular intervals.
The microorganism DSM 14452 is also part of the subject matter of the invention.
The following examples are intended to clarify the invention without there being any wish to restrict the scope of the invention in any way.
Example 1 Preparing a glycerol culture of the fungal strain ST 003367, DSM
30 ml of nutrient solution (malt extract, 2.0%, yeast extract, 0.2%, glucose, 1.0%, (NH4)2HP04, 0.05%, pH 6.0) in a sterile 100 ml Erlenmeyer flask were inoculated with the fungal strain ST 003367, DSM 14452, and incubated at 25°C and 140 revolutions per minute (rpm) on a rotating shaker for 6 days. 1.5 ml of this culture were subsequently diluted with 2.5 ml of 80% glycerol and stored at -135°C.
Example 2 Preparing a preliminary culture of the fungus ST 003367, DSM 14452, in an Erlenmeyer flask 100 ml of nutrient solution (malt extract, 2.0%, yeast extract, 0.2%, glucose, 1.0%, -(NH4)2HP04, 0.05%, pH 6) in a sterile 300 ml Erlenmeyer flask were inoculated with the fungal strain ST 003367, DSM 14452, and incubated at 25°C and 140 rpm on a rotating shaker for 4 days. 2 ml of this preliminary culture were subsequently required for preparing the main cultures.
Example 3 Preparing thioperylenol by culturing the fungal strain ST 3367, DSM 14452.
In the flask:
A sterile 300 ml Erlenmeyer flask containing 100 ml of the following nutrient solution:
potato dextrose, 2.4%, yeast extract, 0.2 %, pH 5.1, was inoculated with a culture which had been grown on a sloping tube (same nutrient solution but containing 2%
agar), or with 2 ml of a preliminary culture, as described in example 2, and incubated at 25°C and 140 rpm on a shaker. The maximum production of one or more compounds of the formula I was achieved after about 96 hours.
In the fermenter:
The preliminary culture of the strain ST 003367, DSM 14452, was grown at 25°C and 140 rpm in a 2 L Erlenmeyer flask (volume in the flask, 500 mL). The fermenter was inoculated after 72 h. The strain was fermented in 8 L fermenters. The conditions for the fermentation were set as follows and led to the production of the compound according to the invention thioperylenol:
Temperature = 25°C; gassing = 0.5 vvm; rotational speed = 200-220 revolutions per minute (rpm); inoculum = 6%; culturing time = 96 hours (h).
The invention also relates to a compound of the formula I, where R1, R2, R3 and R6 are in each case -OH, R4 and R5, together with the carbon atom to which they are in each case bonded, form an epoxide, R7 is the radical -S-CH2-CHOH-COOH, R8 is =O, R10 and R11 are in each case a hydrogen atom and the bond between -C,4-C~5- is a single bond, and/or to physiologically tolerated salts of the compound of the formula I.
The invention also relates to optically pure compounds of the formula I and to their stereoisomeric mixtures, such as enantiomeric mixtures and diasteromeric mixtures, in .- any ratio to each other.
The term "halogen" is understood as meaning fluorine, chlorine, bromine or iodine. The term "(C,-Cs)-alkyl" is understood as meaning hydrocarbon radicals whose carbon chain is straight-chain or branched and contains from 1 to 6 carbon atoms, for example methyl, ethyl, propyl, i-propyl, butyl, tertiary butyl, pentyl and hexyl. The term "(C2-C6)-alkenyl" is understood as meaning hydrocarbon radicals whose carbon chain is straight-chain or branched and contains from 2 to 6 carbon atoms, and which exhibits one, two or three double bonds, for example the radicals allyl, crotyl, 1-propenyl, yenta-1,3-dienyl, pentenyl and hexenyl. The term "(C2-C6)-alkynyl" is understood as meaning hydrocarbon radicals whose carbon chain is straight-chain or branched and contains from 2 to 6 carbon atoms and which exhibits one or two triple bonds, for example the radicals propynyl, butynyl and pentynyl.
The term "aryl" is understood as meaning the radicals phenyl, benzyl and 1-naphthyl and 2-naphthyl.
The expression "R4 and R5, together with the carbon atoms to which they are in each case bonded, form a 3-, 4-, 5- or 6-membered ring system which is aromatic or saturated and which contains one or two heteroatoms from the series oxygen, nitrogen or sulfur" is understood as meaning radicals such as epoxide, aziridine, azetidine, azetine, pyrrole, pyrrolidine, pyridine, piperidine, tetrahydropyridine, pyrazole, imidazole, pyrazoline, imidazoline, pyrazolidine, imidazolidine, pyridazine, pyrimidine, pyrazine, piperazine, pyran, oxazole, isoxazole, 2-isoxazoline, isoxazolidine, morpholine, oxothiolane, thiopyran, thiazole, isothiazole, 2-isothiazoline, isothiazolidine or thiomorpholine.
The invention furthermore relates to the compound of the formula II
OH OH
,o ,s \ 20 ..
~~ O
..,, '2 ~~~\ 8 OH
H off s~~
~a 5 '6 ~~~,, ' " COOH
15 2, 23 _ In that which follows, the compound of the formula II is termed thioperylenol (empirical formula: C23H2pNOgS; molecular weight 472.47) and to the physiologically tolerated salts thereof.
The invention furthermore relates to a process for preparing the compound of the formula I, and/or a stereoisomeric form of the compound of the formula !
andlor mixtures of these forms in any ratio, andlor a physiologically tolerated salt of the compound of the formula I, which process comprises a) culturing the microorganism DSM 14452, or its mutants or variants, in an aqueous nutrient medium and isolating and purifying the compound thioperylenol, or b) converting thioperylenol, by means of chemical derivatization, into a compound of the formula I, or c) resolving a compound of the formula I, which has been prepared by methods a) or b) and which, because of its chemical structure, appears in enantiomeric forms, into the pure enantiomers by forming salts with enantiomerically pure acids or bases, by chromatography on chiral stationary phases or by derivatizing with chiral, enantiomerically pure compounds such as amino acids, separating the diastereomers which are thus obtained and eliminating the chiral auxiliary groups, or d) either isolating the compound of the formula I which has been prepared by the methods a), b) or c) in free form or, when acidic or basic groups are present, converting it into physiologically tolerated salts.
The microorganism DSM 14452 (internal designation ST003367) belongs to the fungal group and possesses a white substrate mycelium and very little aerial mycelium and was deposited on August 3, 2001, under the conditions of the Budapest Treaty, in the DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen [German collection of microorganisms and cell cultures] GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, under the number DSM 14452.
Variants of DSM 14452 are understood as being DSM 14452 strains which have been obtained by isolating individual colonies from a culture of DSM 14452 on the proviso that they produce thioperylenol. Mutants of DSM 14452 are understood as being DSM
strains which have been obtained from a culture of DSM 14452 after mutation, with the _ proviso that they produce thioperylenol. Mutants of DSM 14452 can be generated, in a manner known per se, by physical means, for example irradiation, such as ultraviolet or X-ray radiation, or by using chemical mutagens, for example ethyl methanesulfonate (EMS); 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-vitro-N-nitrosoguanidine (MNNG).
The mutants are found, for example, by taking samples from the culture medium and determining the inhibitory effect of thioperylenol.
Thioperylenol is produced by culturing DSM 14452. The nutrient solution contains carbon sources, such as sucrose, corn starch, dextrose, lactose, D-mannitol, molasses or malt extract, and nitrogen sources, such as soybean flour, peanut flour, proteins, peptones, peptides, tryptones, meat extract, yeast extract or ammonium salts or nitrates.
The nutrient solution also contains inorganic salts such as sodium hydrogen phosphate, sodium chloride, calcium chloride, calcium sulfate, calcium carbonate, magnesium sulfate or potassium hydrogen phosphate. In addition, it is also possible to add fat, such as methyl oleate or soybean oil, to the nutrient medium. Besides that, trace elements, such as iron salts, manganese salts, copper salts, zinc salts or cobalt salts, or other metal salts, are also added.
A preferred nutrient solution contains from about 0.05% to 5%, preferably from 1 % to 3%, potato dextrose and from about 0.05% to 3%, preferably from 0.05% to 1 %, yeast extract. The percentages relate to the weight of the total nutrient solution.
DSM 14452 is cultured at temperatures of from 18°C to 35°C, preferably at from 23°C to 28°C, and at pH values of from 3 to 10, preferably from 5 to 9, particularly preferably at values of from 4 to 6. The culture is carried out aerobically, initially in shaking flasks and, after that, in a fermenter while stirring and aerating with air or pure oxygen. The microorganisms are cultured in the fermenters for a period of from 48 to 720 hours, preferably of from 72 to 144 hours.
The formation of thioperylenol reaches its maximum after from about 96 to 144 hours.
The fungal strain DSM 14452 also forms mixtures of several compounds of the formula I
in the nutrient solution. The quantitative proportion of one or more of the compounds of the formula I can vary depending on the composition of the nutrient solution.
In addition, the composition of the medium can be used to direct the synthesis of individual compounds of the formula I such that the microorganism either does not produce individual compounds of the formula I at all or only produces them in a quantity which is below the limit of detection.
Thioperylenol is either isolated directly from the nutrient solution or isolated after the cells have been separated off, for example by means of centrifugation or filtration. The thioperylenol can be isolated by extracting with solvents or by adsorbing on resins such as XAD 16, HP 20, MCI Gel~ CHP20P or ion exchangers.
It is purified, for example, by chromatography on adsorption resins such as on Diaion~
HP-20 (Mitsubishi Casei Corp., Tokyo), on Amberlite~ XAD 7 (Rohm and Haas, USA), or on Amberchrom~ CG (Toso Haas, Philadelphia, USA). The separations can be carried out over a wide pH range. The range of pH 1 to pH 9 is preferred, with the range of pH 2 to pH 8 being particularly preferred. In addition to this, reverse phase supports, which are used within the context of high pressure liquid chromatography (HPLC), are also suitable. Another isolation method is that of using molecular sieves such as Fractogel~
TSK HW-40S or Sephadex~ LH-20.
The microbiologically produced thioperylenol is. used as the starting material for preparing the thioperylenol derivative. The compounds of formula I are prepared in a manner known per se; for example, the epoxide group of the thioperylene can be converted by hydrolysis or solvolysis into alcohols or esters. Epoxides are very reactive compounds which, in addition to water and acids, also add on other nucleophilic reagents, such as alcohols, thiols, amines and Grignard compounds, in the presence of acidic or basic catalysts. The addition of hydrogen cyanide leads further on to f3-hydroxy-propionitrile derivatives. In addition to this, epoxides can be rearranged to give carbonyl-derivatives, with this rearrangement being catalyzed, for example, by Lewis acids. In addition, the reaction products can be subjected to further reactions. These reactions are known per se and are described, for example, by Jerry March, Advanced Organic Chemistry, John Wiley & Sons, 4t" Edition, 1992.
Other derivatives are obtained if the 3-thio-lactic acid radical of the thioperylenol is reacted reductively or oxidatively. It can also be advantageous to use the sulfide as what is termed a leaving group and replace it with other suitable radicals in a manner known from the literature. In order to carry out reactions selectively, it can be advantageous to introduce suitable protecting groups, in a manner known per se, prior to the reaction.
_ The protecting groups ace eliminated after the reaction and the reaction product is then purified.
Pharmacologically tolerated salts of the compounds of the formula I are understood as 5 being both inorganic and organic salts, as are described in Remington's Pharmaceutical Sciences (17th edition, page 1418 (1985)). Physiologically tolerated salts are prepared in a manner known per se from compounds of the formula I, including their stereoisomeric forms, which are capable of forming salts. With basic reagents, such as hydroxides, carbonates, hydrogen carbonates, alcoholates and ammonia, or organic bases, for 10 example trimethylamine, triethylamine, ethanolamine or triethanolamine, or else basic amino acids, for example lysine, ornithine or arginine, the carboxylic acid forms stable alkali metal salts, alkaline earth metal salts or, where appropriate, substituted ammonium salts. If the compound of the formula I possesses basic groups, it is also possible to prepare stable acid addition salts with strong acids. Both inorganic acids and organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, benzenesulfonic acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, 4-bromobenzenesulfonic acid, trifluoromethylsulfonic acid, cyclohexylamidosulfonic acid, acetic acid, oxalic acid, tartaric acid, succinic acid and trifluoroacetic acid, are suitable for this purpose.
The invention also relates to pharmaceuticals which are characterized by an effective content of at least one compound of the formula I and/or a physiologically tolerated salt of the compound of the formula I and/or an optionally stereoisomeric form of the compound of the formula I, together with a pharmaceutically suitable and physiologically tolerated carrier substance, additive and/or other active compounds and auxiliary substances.
On account of their pharmacological properties, the compounds of the formula I
are suitable for the prophylaxis and therapy of all those diseases in which high blood platelet aggregations occur and which are caused by thrombus formation or embolisms.
These diseases include, for example, myocardial infarction, unstable angina pectoris, stroke, transitory ischemic attacks and peripheral arterial occlusion diseases (peripheral vascular disease) such as intermittent claudication.
The invention also relates to the use of at least one compound of the formula I andlor all stereoisomeric forms of the compound of the formula I andlor mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur where R1, R2, R3, R4, R5, R6, R7, R8, R10 and R11 are, independently of each other, 1. a hydrogen atom, 2. (C,-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by 2.1 -OH, 2.2 =O, 2.3 -O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by , 2.3.1 -CN, 2.3.2 -N H2, 2.3.3 =N-OH or 2.3:4 =N-O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched, 2.4 -O-(C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.5 -O-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.6 -aryl, in which aryl is unsubstituted or substituted once, twice or three times, independently of each other, by 2.6.1 halogen, 2.6.2 -(C~-C4)-alkyl, in which alkyl is straight-chain or branched, 2.6.3 -O-(C~-C6)-alkyl, in which alkyl is straight-chain or branched, 2.6.4 -OH or 2.6.5 -(C,-C4)-alkyl, in which alkyl is straight-chain or branched and is substituted once, twice or three times by halogen, 2.7 -C(O)-OH, _, 2.8 -C(O)-O-NH2, 2.9 -C(O)-O-(C,-C4)-alkyl, in which alkyl is straight-chain or branched, 2.10 -NH-(C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.11 -NH-(C2-Cs)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.12 -NH-(C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.3.1 to 2.3.4, 2.13 -NH2, or 2.14 halogen, 3. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 4. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 5. -O-R9, in which R9 is 1. (C~-C6)-alkyl, in which alkyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 2. (C2-C6)-alkenyl, in which alkenyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, or 3. (C2-C6)-alkynyl, in which alkynyl is straight-chain or branched and is unsubstituted or substituted once, twice or three times, independently of each other, by as described above under 2.1 to 2.14, 6. -NH-R9, in which R9 is defined as above under 5., 7. -N H-C(O)-H, 8. -NH-C(O)- R9, in which R9 is defined as above under 5., 9. -NH-aryl, in which aryl is unsubstituted or substituted once, twice or three times by R9, 10. =N-OH, 11. =N-O-R9, in which R9 is defined as above under 5., 12. -S-H, 13. -S-R9, in which R9 is defined as above under 5., 14. -S(O)-R9, in which R9 is defined as above under 5., 15. -S(O)2- R9, in which R9 is defined as above under 5., or 16. -S02, or R4 and R5 or R10 and R11, together with the carbon atoms to which they are in each case bonded, form a 3-, 4-, 5- or 6-membered ring system which is aromatic or saturated and which contains one or two heteroatoms from the series oxygen, nitrogen or sulfur, and the bond between -C,4-C~5- is a single bond or a double bond.
The invention furthermore relates to the use of at least one compound of the formula I
for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur, where R1, R3 and R8 are, independently of each other, -OH or =O, R2 is -OH, R4 and R5 are, independently of each other -OH or a hydrogen atom, or R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6, R10 and R11 are, independently of each other, -OH or a hydrogen atom, or R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 is a hydrogen atom or the radical -S-CH2-CHOH-COOH, and the bond between -C~4-C~5- is a single bond or a double bond.
The invention furthermore relates to the use of at least one compound of the formula I
for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur, with thioperylenol, a compound of the formula I, in which R1, R2, R3 and R6 are in each case -OH, R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 is the radical -S-CH2-CHOH-COOH, R8 is =O, R10 and R11 are in each case a hydrogen atom and the bond between -C~4-C~5- is a single bond, alterperylenol, a compound of the formula I, in which R1, R2, R5 and R6 are in each case -OH, R3 and R8 are in each case =O, R4, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C~4-C~5- is a double bond, altertoxin I, a compound of the formula I in which R1, R2, R5 and R6 are in each case -OH, R3 and R8 are in each case =O, R4, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C~4-C~5- is a single bond, altertoxin II, a compound of the formula I, in which R1, R2 and R6 are in each case -OH, R3 and R8 are in each case =O, R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C~4-C~5- is a single bond, and altertoxin III, a compound of the formula I, in which R1 and R3 are in each case =O, R2 and R8 are in each case -OH, R4 and R5 and R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6 and R7 are in each case a hydrogen atom, and the bond between -Coq-C~5- is a single bond, being employed.
The invention also relates to a process for producing a pharmaceutical, which process comprises bringing at least one compound of the formula I, together with a pharmaceutically suitable and physiologically tolerated excipient and, where appropriate, other suitable active compounds, additives or auxiliary substances, into a suitable form for administration.
Examples of suitable solid or galenic preparation forms are granules, powders, sugar-coated tablets, tablets, (micro)capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and preparations having a protracted release of the active compound, in the production of which use is made of customary adjuvants such as carrier substances, disintegrants, binders, coating agents, swelling agents, glidants or lubricants, flavorings, sweeteners and solubilizers. Frequently employed auxiliary substances which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils, such as cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol and solvents, such as sterile water and monohydric 5 or polyhydric alcohols, such as glycerol.
The pharmaceutical preparations are preferably produced and administered in dosage units, with each unit containing, as the active constituent, a particular dose of the 'compound of the formula I according to the invention. In the case of solid dosage units, 10 such as tablets, capsules, sugar-coated tablets or suppositories, this dose can be from 0.1 mg/kg of body weight to 1 000 mg/kg of body weight, preferably from 0.2 mg/kg of body weight to 100 mg/kg of body weight. They are expediently administered in dosage units which contain at least the effective daily quantity of the compound of the formula I, for example up to 1 000 mg, preferably, however, from about 50 to 300 mg and, in the 15 case of injection solutions in ampoule form, up to about 300 mg, preferably, however, from about 10 to 100 mg.
Daily doses of from about 20 mg to 1 000 mg, preferably from about 100 mg to 500 mg, of active compound, depending on the activity of the compound according to formula I, are indicated for treating an adult patient of about 70 kg in weight. However, it may possibly also be appropriate to use higher or lower daily doses. The daily dose can be administered either by means of a once-only administration in the form of a single dosage unit or of several smaller dosage units or by means of a repeated administration of subdivided doses at particular intervals.
The microorganism DSM 14452 is also part of the subject matter of the invention.
The following examples are intended to clarify the invention without there being any wish to restrict the scope of the invention in any way.
Example 1 Preparing a glycerol culture of the fungal strain ST 003367, DSM
30 ml of nutrient solution (malt extract, 2.0%, yeast extract, 0.2%, glucose, 1.0%, (NH4)2HP04, 0.05%, pH 6.0) in a sterile 100 ml Erlenmeyer flask were inoculated with the fungal strain ST 003367, DSM 14452, and incubated at 25°C and 140 revolutions per minute (rpm) on a rotating shaker for 6 days. 1.5 ml of this culture were subsequently diluted with 2.5 ml of 80% glycerol and stored at -135°C.
Example 2 Preparing a preliminary culture of the fungus ST 003367, DSM 14452, in an Erlenmeyer flask 100 ml of nutrient solution (malt extract, 2.0%, yeast extract, 0.2%, glucose, 1.0%, -(NH4)2HP04, 0.05%, pH 6) in a sterile 300 ml Erlenmeyer flask were inoculated with the fungal strain ST 003367, DSM 14452, and incubated at 25°C and 140 rpm on a rotating shaker for 4 days. 2 ml of this preliminary culture were subsequently required for preparing the main cultures.
Example 3 Preparing thioperylenol by culturing the fungal strain ST 3367, DSM 14452.
In the flask:
A sterile 300 ml Erlenmeyer flask containing 100 ml of the following nutrient solution:
potato dextrose, 2.4%, yeast extract, 0.2 %, pH 5.1, was inoculated with a culture which had been grown on a sloping tube (same nutrient solution but containing 2%
agar), or with 2 ml of a preliminary culture, as described in example 2, and incubated at 25°C and 140 rpm on a shaker. The maximum production of one or more compounds of the formula I was achieved after about 96 hours.
In the fermenter:
The preliminary culture of the strain ST 003367, DSM 14452, was grown at 25°C and 140 rpm in a 2 L Erlenmeyer flask (volume in the flask, 500 mL). The fermenter was inoculated after 72 h. The strain was fermented in 8 L fermenters. The conditions for the fermentation were set as follows and led to the production of the compound according to the invention thioperylenol:
Temperature = 25°C; gassing = 0.5 vvm; rotational speed = 200-220 revolutions per minute (rpm); inoculum = 6%; culturing time = 96 hours (h).
Nutrient media:
r Preliminary culture Malt extract 20 g/L
Yeast extract 2 g/L
Glucose 10 g/L
(N H4)2PO4 0.5 g/I
vMain culture Potato dextrose broth 24 g/L
Yeast extract 2 g/L
Desmophene 1.25 mUL
It was possible to suppress foam formation by repeatedly adding ethanolic polyol solution. The maximum production was reached after from about 96 to 144 hours.
Example 4: Isolating the natural product thioperylenol.
15 liters of the culture solution obtained in accordance with example 3 were freed from the cell mass by centrifugation. The mycelium (1.5 liters) is extracted with 5 liters of methanol. The clear alcoholic phase was concentrated down to about 2 liters under reduced pressure and combined with the culture filtrate. The turbid aqueous solution was then loaded onto a 1 L-capacity column which was filled with the absorption resin MCI Gel~ CHP20P. Column dimensions: width x height: 6 cm x 35 cm. The column was eluted with a solvent gradient of 0.1 % ammonium formate buffer, pH 4.5, in water after 2-propanol. The column effluent (60 mUminute) was collected in fractions of in each case 220 mL. The thioperylenol-containing fractions 21 and 22, which were tested by HPLC analyses, were collected and concentrated under reduced pressure. The thioperylenol was obtained in pure form by repeated preparative HPLC on SP
NUCLEOSIL 100-7 C18 HD~ columns (Macherey-Nagel, Duren) using the eluent 0.1 ammonium formate, pH 4.8, in water/95% acetonitrile in a gradient method. The flow rate through the column was 25 mUminute and the thioperylenol was eluted from the separating column with an acetonitrile content of about 25%. 10 mg of crystalline thioperylenol were obtained by slowly concentrating under reduced pressure.
a Example 5: High pressure liquid chromatography (HPLC) of the thioperylenol.
Column: YMC-Pack Pro C18~, AS-303, 250 x 4.6 mm, S-5 Nm;
Mobile Phase: 0 to 2 minutes: 0.02% trifluoroacetic acid (TFA), 2 to 20 minutes: 0% to 100% acetonitrile in 0.1 % TFA, 20 to 25 minutes: 100% acetonitrile.
Flow rate: 1 mL per minute, Detection by UV absorption at 210 nm.
The retention time for thioperylenol was found to be 12.1 minutes.
Example 6: Properties of thioperylenol.
The physicochemical and spectroscopic properties of thioperylenol can be summarized as follows:
Appearance:
Brownish crystals which are soluble in medium-polar and polar organic solvents and in aqueous neutral buffers. Stable in neutral and mildly acidic, non-oxidizing medium but unstable in strongly acidic and strongly alkaline solution.
Empirical formula: C23H2oO9S, molecular weight: 472.47 'H and'3C NMR: see table 1;
UV maxima in water/acetonitrile (1 to 1 ), pH 3.0: 216 nm, 259 nm, 292 nm and 383 nm.
r Preliminary culture Malt extract 20 g/L
Yeast extract 2 g/L
Glucose 10 g/L
(N H4)2PO4 0.5 g/I
vMain culture Potato dextrose broth 24 g/L
Yeast extract 2 g/L
Desmophene 1.25 mUL
It was possible to suppress foam formation by repeatedly adding ethanolic polyol solution. The maximum production was reached after from about 96 to 144 hours.
Example 4: Isolating the natural product thioperylenol.
15 liters of the culture solution obtained in accordance with example 3 were freed from the cell mass by centrifugation. The mycelium (1.5 liters) is extracted with 5 liters of methanol. The clear alcoholic phase was concentrated down to about 2 liters under reduced pressure and combined with the culture filtrate. The turbid aqueous solution was then loaded onto a 1 L-capacity column which was filled with the absorption resin MCI Gel~ CHP20P. Column dimensions: width x height: 6 cm x 35 cm. The column was eluted with a solvent gradient of 0.1 % ammonium formate buffer, pH 4.5, in water after 2-propanol. The column effluent (60 mUminute) was collected in fractions of in each case 220 mL. The thioperylenol-containing fractions 21 and 22, which were tested by HPLC analyses, were collected and concentrated under reduced pressure. The thioperylenol was obtained in pure form by repeated preparative HPLC on SP
NUCLEOSIL 100-7 C18 HD~ columns (Macherey-Nagel, Duren) using the eluent 0.1 ammonium formate, pH 4.8, in water/95% acetonitrile in a gradient method. The flow rate through the column was 25 mUminute and the thioperylenol was eluted from the separating column with an acetonitrile content of about 25%. 10 mg of crystalline thioperylenol were obtained by slowly concentrating under reduced pressure.
a Example 5: High pressure liquid chromatography (HPLC) of the thioperylenol.
Column: YMC-Pack Pro C18~, AS-303, 250 x 4.6 mm, S-5 Nm;
Mobile Phase: 0 to 2 minutes: 0.02% trifluoroacetic acid (TFA), 2 to 20 minutes: 0% to 100% acetonitrile in 0.1 % TFA, 20 to 25 minutes: 100% acetonitrile.
Flow rate: 1 mL per minute, Detection by UV absorption at 210 nm.
The retention time for thioperylenol was found to be 12.1 minutes.
Example 6: Properties of thioperylenol.
The physicochemical and spectroscopic properties of thioperylenol can be summarized as follows:
Appearance:
Brownish crystals which are soluble in medium-polar and polar organic solvents and in aqueous neutral buffers. Stable in neutral and mildly acidic, non-oxidizing medium but unstable in strongly acidic and strongly alkaline solution.
Empirical formula: C23H2oO9S, molecular weight: 472.47 'H and'3C NMR: see table 1;
UV maxima in water/acetonitrile (1 to 1 ), pH 3.0: 216 nm, 259 nm, 292 nm and 383 nm.
Table 1: Chemical shifts of V-2474 in DMSO at 300 K.
Position ' H '3C
1 - 160.14 2 7.01 118.22 3 8.05 132.57 4 - 125.12 - 137.09 6 - 113.86 7 - 122.82 8 7.53 123.75 9 6.79 113.50 - 156.37 11 - 122.66 12 - 128.08 13 - 204.10 14 3.62/3.06 - 39.1 4.29 46.56 16 - 70.80 17 3.64 41.35 18 3.93 49.15 19 ~ 3.44 55.30 5.11 59.67 21 2.92/2.76 36.23 22 3.72 72.96 23 - 174.38 5 Example 7: Mass spectrometric characterization of thioperylenol.
Thioperylenol is assigned the mass of 472 on the basis of the following findings: the ESI+
spectrum gave weak peaks at 490 amu (M+NH4)'' and 962 amu (2M+NH4)+. The ESI' spectrum gave a peak, inter alia, at 471 amu (M-H)~.
Using an FTICR mass spectrometer, a peak was observed, inter alia, at 569.0525 amu in the ESI' mode and in the added presence of phosphoric acid. The measured value agrees well with that calculated for (M+H2P04)' = C23H220~3PS = 569.0524 amu (0.2 ppm difference).
5 MS/MS experiments using an FTICR mass spectrometer led to the following fragmentations in the ESI' mode:
471 amu to 349 amu (-C3H603S), 331 amu (-C3H804S), 313 amu (-C3H~pO5S), 303 amu (-C4H805S), 285 amu (-C4H,oO6S), 261 amu (-C6H,oO6S) and smaller fragments.
Pharmacological examples The inhibition of platelet aggregation can be measured, inter alia, by determining platelet aggregation turbimetrically as described by Born (Born, GV, Nature, 1962, 194:
929).
Principle of the test: the method is based on the property that the optical density of a particle suspension depends on the number of particles and not on their size.
After a platelet suspension has been stimulated, the platelets begin to aggregate.
This leads to the appearance of relatively large platelet aggregates and to an increase in light transmission. This increase is registered photometrically, and recorded in curve form, continuously. The degrees to which the thrombocytes can be aggregated, or to which this aggregation can be inhibited, can be deduced from the changes in light transmission. The method of staining cells with calcein was used for determining cytotoxicity (Hollo Z et al., Biochim Biophys Acta 11;1191 (2):384-8, 1994).
Living cells incorporate the substrate calcein acetoxymethyl ester (C-AM), which is hydrolyzed in the cells by nonspecific esterases. C-AM is colorless and does not fluoresce. In the living cell, it is converted into the fluorescent hydrolysis product and the vitality of the cells can be measured on the basis of the fluorescence (~eX:485nm ~em:''~38nm).
By way of example, table 2 summarizes the inhibitory effects of some hydroperylene derivatives as ICSO values. The IC5o value indicates the concentration which inhibits thrombocyte aggregation by 50% under defined conditions.
Table 2. The concentrations of some hydroperylene derivatives which inhibit thrombocyte aggregation by 50% (ICso).
Compound IC5o (platelet aggregation) ICSO (toxicity) Thioperylenol 7.2 NM 61 ~rM
Alterperylenol 1.6 NM > 100,uM
Altertoxin I 29 ~M > 100 NM
Altertoxin II 2.6 NM 10 NM
Altertoxin III 6.1 NM 20,uM
As can be seen from the table, in addition to inhibiting thrombocyte aggregation, some of the hydroperylene derivatives also exhibit a substantial degree of cell toxicity. The cell toxicity is particularly marked in the case of compounds which carry epoxide groups. For this reason, those compounds of the formula I which, while possessing an inhibitory effect on platelet aggregation, are relatively nontoxic are particularly valuable.
Preference is consequently given to hydroperylenol derivatives which are epoxide-free.
Example 8: Test for inhibition of thrombocyte aggregation.
The following reagents are required for implementing the thrombocyte aggregation test:
Materials Supplier Catalogue No.
Water tissue culture Sigma W-3500 grade NaCI Merck 1.06404 KCI Merck 1.04933 CaCl2 Merck 1.02083 Albumin, bovine (BSA)Sigma A-6003 Calcein AM Molecular Probes C-1430 Collagen reagent Nycomed 5368 Human alpha Thrombin Haemochrom DiagnosticaHT1002A
The assay was carried out as follows:
Tyrode buffer:
NaCI 120 mM
KCI 2.6 mM
NaHC03 12 mM
NaH2P04 x H20 0.39 mM
Hepes 10 mM
Glucose 5.5 mM
BSA 0.35%
Add fresh glucose and BSA daily Adjust pH to 7.4 Calcein AM: 1 mg of Calcein AM in 1 ml of DMSO (1 mM) Human a-thrombin (stock solution): 1 000 units in 1 ml of 0.9% NaCI
Final concentration of activator: 0.05 U of thrombin/ml or 1 Ng of collagen/ml Wortmannin, stock solution: 1 mg in 233.43 girl of DMSO (10 mM).
Aggregation in aggregometer (PAP 4) - Cell number, 3 x 105 thrombocytes/,ul - Mixture in siliconized glass microtubes - Total volume, 400,u1/tube Per microtube:
- 320 NI of thrombocytes - 20 NI of 10 mM CaCl2 (f.c. 0.5 mM) - 20 girl of test substance - 40 NI of agonist Controls:
- Blank: 400 NI of buffer (Tyrode, 0.35% BSA) - Negative control: 320 NI of thrombocytes NI of CaCl2 5 mM (f.c. 0.5 mM) 40 ,ul of buffer - Positive control: 320 ,ul of buffer 40 NI of 5 mM CaCl2 (f.c. 0.5 mM) 40 ,ul of activator The blank value for the channels of the aggregometer is first of all adjusted using buffer.
The thrombocytes are then pipetted into microtubes and, after the thrombocytes have been added, heated at 37°C for a few minutes in the aggregometer. After a magnetic stirrer has been added to each reaction tube, the substances, and CaCl2, are added and a start is made in recording the course of the curve in the aggregometer.
After 2 minutes of incubation, the activator, or buffer in the case of the control, is added.
The course of the curve is then recorded for a further 6 min at 37°C and at a stirrer speed of 1 050 revolutions per minute.
Position ' H '3C
1 - 160.14 2 7.01 118.22 3 8.05 132.57 4 - 125.12 - 137.09 6 - 113.86 7 - 122.82 8 7.53 123.75 9 6.79 113.50 - 156.37 11 - 122.66 12 - 128.08 13 - 204.10 14 3.62/3.06 - 39.1 4.29 46.56 16 - 70.80 17 3.64 41.35 18 3.93 49.15 19 ~ 3.44 55.30 5.11 59.67 21 2.92/2.76 36.23 22 3.72 72.96 23 - 174.38 5 Example 7: Mass spectrometric characterization of thioperylenol.
Thioperylenol is assigned the mass of 472 on the basis of the following findings: the ESI+
spectrum gave weak peaks at 490 amu (M+NH4)'' and 962 amu (2M+NH4)+. The ESI' spectrum gave a peak, inter alia, at 471 amu (M-H)~.
Using an FTICR mass spectrometer, a peak was observed, inter alia, at 569.0525 amu in the ESI' mode and in the added presence of phosphoric acid. The measured value agrees well with that calculated for (M+H2P04)' = C23H220~3PS = 569.0524 amu (0.2 ppm difference).
5 MS/MS experiments using an FTICR mass spectrometer led to the following fragmentations in the ESI' mode:
471 amu to 349 amu (-C3H603S), 331 amu (-C3H804S), 313 amu (-C3H~pO5S), 303 amu (-C4H805S), 285 amu (-C4H,oO6S), 261 amu (-C6H,oO6S) and smaller fragments.
Pharmacological examples The inhibition of platelet aggregation can be measured, inter alia, by determining platelet aggregation turbimetrically as described by Born (Born, GV, Nature, 1962, 194:
929).
Principle of the test: the method is based on the property that the optical density of a particle suspension depends on the number of particles and not on their size.
After a platelet suspension has been stimulated, the platelets begin to aggregate.
This leads to the appearance of relatively large platelet aggregates and to an increase in light transmission. This increase is registered photometrically, and recorded in curve form, continuously. The degrees to which the thrombocytes can be aggregated, or to which this aggregation can be inhibited, can be deduced from the changes in light transmission. The method of staining cells with calcein was used for determining cytotoxicity (Hollo Z et al., Biochim Biophys Acta 11;1191 (2):384-8, 1994).
Living cells incorporate the substrate calcein acetoxymethyl ester (C-AM), which is hydrolyzed in the cells by nonspecific esterases. C-AM is colorless and does not fluoresce. In the living cell, it is converted into the fluorescent hydrolysis product and the vitality of the cells can be measured on the basis of the fluorescence (~eX:485nm ~em:''~38nm).
By way of example, table 2 summarizes the inhibitory effects of some hydroperylene derivatives as ICSO values. The IC5o value indicates the concentration which inhibits thrombocyte aggregation by 50% under defined conditions.
Table 2. The concentrations of some hydroperylene derivatives which inhibit thrombocyte aggregation by 50% (ICso).
Compound IC5o (platelet aggregation) ICSO (toxicity) Thioperylenol 7.2 NM 61 ~rM
Alterperylenol 1.6 NM > 100,uM
Altertoxin I 29 ~M > 100 NM
Altertoxin II 2.6 NM 10 NM
Altertoxin III 6.1 NM 20,uM
As can be seen from the table, in addition to inhibiting thrombocyte aggregation, some of the hydroperylene derivatives also exhibit a substantial degree of cell toxicity. The cell toxicity is particularly marked in the case of compounds which carry epoxide groups. For this reason, those compounds of the formula I which, while possessing an inhibitory effect on platelet aggregation, are relatively nontoxic are particularly valuable.
Preference is consequently given to hydroperylenol derivatives which are epoxide-free.
Example 8: Test for inhibition of thrombocyte aggregation.
The following reagents are required for implementing the thrombocyte aggregation test:
Materials Supplier Catalogue No.
Water tissue culture Sigma W-3500 grade NaCI Merck 1.06404 KCI Merck 1.04933 CaCl2 Merck 1.02083 Albumin, bovine (BSA)Sigma A-6003 Calcein AM Molecular Probes C-1430 Collagen reagent Nycomed 5368 Human alpha Thrombin Haemochrom DiagnosticaHT1002A
The assay was carried out as follows:
Tyrode buffer:
NaCI 120 mM
KCI 2.6 mM
NaHC03 12 mM
NaH2P04 x H20 0.39 mM
Hepes 10 mM
Glucose 5.5 mM
BSA 0.35%
Add fresh glucose and BSA daily Adjust pH to 7.4 Calcein AM: 1 mg of Calcein AM in 1 ml of DMSO (1 mM) Human a-thrombin (stock solution): 1 000 units in 1 ml of 0.9% NaCI
Final concentration of activator: 0.05 U of thrombin/ml or 1 Ng of collagen/ml Wortmannin, stock solution: 1 mg in 233.43 girl of DMSO (10 mM).
Aggregation in aggregometer (PAP 4) - Cell number, 3 x 105 thrombocytes/,ul - Mixture in siliconized glass microtubes - Total volume, 400,u1/tube Per microtube:
- 320 NI of thrombocytes - 20 NI of 10 mM CaCl2 (f.c. 0.5 mM) - 20 girl of test substance - 40 NI of agonist Controls:
- Blank: 400 NI of buffer (Tyrode, 0.35% BSA) - Negative control: 320 NI of thrombocytes NI of CaCl2 5 mM (f.c. 0.5 mM) 40 ,ul of buffer - Positive control: 320 ,ul of buffer 40 NI of 5 mM CaCl2 (f.c. 0.5 mM) 40 ,ul of activator The blank value for the channels of the aggregometer is first of all adjusted using buffer.
The thrombocytes are then pipetted into microtubes and, after the thrombocytes have been added, heated at 37°C for a few minutes in the aggregometer. After a magnetic stirrer has been added to each reaction tube, the substances, and CaCl2, are added and a start is made in recording the course of the curve in the aggregometer.
After 2 minutes of incubation, the activator, or buffer in the case of the control, is added.
The course of the curve is then recorded for a further 6 min at 37°C and at a stirrer speed of 1 050 revolutions per minute.
Claims (9)
1. A compound of the formula I
and/or all stereoisomeric forms of the compound of the formula I and/or mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I, where R1, R3 and R8 are, independently of each other, -OH or =O, R2 is -OH, R4 and R5 are, independently of each other -OH or a hydrogen atom, or R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6, R10 and R11 are, independently of each other, -OH or a hydrogen atom, or R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 IS the radical -S-CH2-CHOH-COOH, and the bond between -C14-C15- is a single bond or a double bond.
and/or all stereoisomeric forms of the compound of the formula I and/or mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I, where R1, R3 and R8 are, independently of each other, -OH or =O, R2 is -OH, R4 and R5 are, independently of each other -OH or a hydrogen atom, or R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6, R10 and R11 are, independently of each other, -OH or a hydrogen atom, or R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 IS the radical -S-CH2-CHOH-COOH, and the bond between -C14-C15- is a single bond or a double bond.
2. A compound of the formula I as claimed in claim 1, where R1, R2, R3 and R6 are in each case -OH, R4 and R5, together with the carbon atoms to which they are in each case bonded, farm an epoxide, R7 is the radical -S-CH2-CHOH-COOH, R8 is =O, R10 and R11 are in each case a hydrogen atom, and the bond between -C14-C15- is a single bond, and/or physiologically tolerated salts of the compound of the formula I.
3. A compound of the formula I as claimed in claim 1 or 2, wherein the compound of the formula I is the compound of the formula II
4. A process for preparing the compound of the formula I as claimed in one or more of claims 1 to 3, and/or a stereoisomeric form of the compound of the formula I
and/or mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I, which comprises a) culturing the microorganism DSM 14452, or its mutants or variants, in an aqueous nutrient medium and isolating and purifying the compound thioperylenol, or b) converting thioperylenol, by means of chemical derivatization, into a compound of the formula I, or c) resolving a compound of the formula I, which has been prepared by methods a) or b) and which, because of its chemical structure, appears in enantiomeric forms, into the pure enantiomers by forming salts with enantiomerically pure acids or bases, by chromatography on chiral stationary phases or by derivatizing with chiral, enantiomerically pure compounds such as amino acids, separating the diastereomers which are thus obtained and eliminating the chiral auxiliary groups, or d) either isolating the compound of the formula I which has been prepared by the methods a), b) or c) in free form or, when acidic or basic groups are present, converting it into physiologically tolerated salts.
and/or mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I, which comprises a) culturing the microorganism DSM 14452, or its mutants or variants, in an aqueous nutrient medium and isolating and purifying the compound thioperylenol, or b) converting thioperylenol, by means of chemical derivatization, into a compound of the formula I, or c) resolving a compound of the formula I, which has been prepared by methods a) or b) and which, because of its chemical structure, appears in enantiomeric forms, into the pure enantiomers by forming salts with enantiomerically pure acids or bases, by chromatography on chiral stationary phases or by derivatizing with chiral, enantiomerically pure compounds such as amino acids, separating the diastereomers which are thus obtained and eliminating the chiral auxiliary groups, or d) either isolating the compound of the formula I which has been prepared by the methods a), b) or c) in free form or, when acidic or basic groups are present, converting it into physiologically tolerated salts.
5. A microorganism DSM 14452.
6. A pharmaceutical which has an effective content of at least one compound of the formula I as claimed in one or more of claims 1 to 3, and/or of a physiologically tolerated salt of the compound of the formula I and/or of an optionally stereoisomeric form of the compound of the formula I, together with a pharmaceutically suitable and physiologically tolerated carrier substance or additive and/or other active compounds and auxiliary substances.
7. The use of at least one compound of the formula I as claimed in claim 1 and/or all stereoisomeric forms of the compound of the formula I and/or mixtures of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula I, for producing pharmaceuticals for the prophylaxis and therapy of diseases in which high blood platelet aggregations occur, where R1, R3 and R8 are, independently of each other, -OH or =O, R2 is -OH, R4 and R5 are, independently of each other -OH or a hydrogen atom, or R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6, R10 and R11 are, independently of each other, -OH or a hydrogen atom, or R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 is a hydrogen atom or the radical -S-CH2-CHOH-COOH, and the bond between -C14-C15- is a single bond or a double bond.
8. The use as claimed in claim 7, where thioperylenol, a compound of the formula I, in which R1, R2, R3 and R6 are in each case -OH, R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7 is the radical -S-CH2-CHOH-COOH, R8 is =O, R10 and R11 are in each case a hydrogen atom and the bond between -C14-C15- is a single bond, alterperylenol, a compound of the formula I, in which R1, R2, R5 and R6 are in each case -OH, R3 and R8 are in each case =O, R4, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C14-C15- is a double bond, altertoxin I, a compound of the formula I in which R1, R2, R5 and R6 are in each case -OH, R3 and R8 are in each case =O, R4, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C14-C15- is a single bond, altertoxin II, a compound of the formula I, in which R1, R2 and R6 are in each case -OH, R3 and R8 are in each case =O, R4 and R5, together with the carbon atoms to which they are in each case bonded, form an epoxide, R7, R10 and R11 are in each case a hydrogen atom, and the bond between -C14-C15- is a single bond, altertoxin III, a compound of the formula I, in which R1 and R3 are in each case =O, R2 and R8 are in each case -OH, R4 and R5 and R10 and R11, together with the carbon atoms to which they are in each case bonded, form an epoxide, R6 and R7 are in each case a hydrogen atom, and the bond between -C14-C15- is a single bond, is used.
9. The use as claimed in one or more of claims 7 or 8, wherein the diseases in which high blood platelet aggregations occur are myocardial infarction, unstable angina pectoris, stroke, transitory ischemic attacks and peripheral arterial occlusion diseases such as intermittent claudication.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10158402.4 | 2001-11-28 | ||
| DE10158402A DE10158402A1 (en) | 2001-11-28 | 2001-11-28 | Hydroperylene derivatives |
| PCT/EP2002/012802 WO2003045905A1 (en) | 2001-11-28 | 2002-11-15 | Hydroperylene derivatives |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2468181A1 true CA2468181A1 (en) | 2003-06-05 |
Family
ID=7707291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002468181A Abandoned CA2468181A1 (en) | 2001-11-28 | 2002-11-15 | Hydroperylene derivatives |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP1451151B1 (en) |
| JP (1) | JP4452078B2 (en) |
| AT (1) | ATE420854T1 (en) |
| AU (1) | AU2002358015B2 (en) |
| CA (1) | CA2468181A1 (en) |
| DE (2) | DE10158402A1 (en) |
| IL (1) | IL162108A0 (en) |
| MX (1) | MXPA04004661A (en) |
| WO (1) | WO2003045905A1 (en) |
-
2001
- 2001-11-28 DE DE10158402A patent/DE10158402A1/en not_active Withdrawn
-
2002
- 2002-11-15 EP EP02791683A patent/EP1451151B1/en not_active Expired - Lifetime
- 2002-11-15 IL IL16210802A patent/IL162108A0/en unknown
- 2002-11-15 MX MXPA04004661A patent/MXPA04004661A/en active IP Right Grant
- 2002-11-15 CA CA002468181A patent/CA2468181A1/en not_active Abandoned
- 2002-11-15 DE DE50213225T patent/DE50213225D1/en not_active Expired - Lifetime
- 2002-11-15 AT AT02791683T patent/ATE420854T1/en not_active IP Right Cessation
- 2002-11-15 WO PCT/EP2002/012802 patent/WO2003045905A1/en active Application Filing
- 2002-11-15 JP JP2003547357A patent/JP4452078B2/en not_active Expired - Fee Related
- 2002-11-15 AU AU2002358015A patent/AU2002358015B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP1451151B1 (en) | 2009-01-14 |
| ATE420854T1 (en) | 2009-01-15 |
| EP1451151A1 (en) | 2004-09-01 |
| DE10158402A1 (en) | 2003-06-12 |
| AU2002358015B2 (en) | 2007-09-20 |
| JP4452078B2 (en) | 2010-04-21 |
| WO2003045905A1 (en) | 2003-06-05 |
| DE50213225D1 (en) | 2009-03-05 |
| JP2005510560A (en) | 2005-04-21 |
| AU2002358015A1 (en) | 2003-06-10 |
| IL162108A0 (en) | 2005-11-20 |
| MXPA04004661A (en) | 2004-08-13 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |