JP2005517710A - Phenalenone derivatives, their preparation and use - Google Patents

Abstract

The present invention relates to the production of phenalenone derivatives by fermenting the phenalenone derivative, the microorganism Penicillium herquei Bainer & Sartory, DSM 14142, and derivatizing the phenalenone produced during such fermentation, as well as oncogenic It relates to the use of such phenalenone derivatives as medicaments for the particular treatment of diseases, bacterial infections and / or mycoses and rheumatic diseases.
[Chemical 1]

Description

  The present invention relates to a phenalenone derivative, a process for its production, and its use as a medicament.

  Cancer is a human and animal disease that usually follows a lethal course and results from the uncontrolled growth of endogenous cells. Cancer is a term for malignant growth (malignoma), formation of neoplasms (tumors and carcinomas), or malignant degeneration and leukemia disorders of leukocytes (leukemia, blood cancer). Cancer or tumor cells are formed by the conversion of endogenous cells. The malignancy of cancer cells is an autonomy of growth, that is, an invasion mode, is not classified into organ composition plans, and is expressed by non-suppressive proliferation ability accompanied by tissue destruction. A sure sign of malignancy is the formation (metastasis) of distant tumor deposits after hematogenous or lymphatic spread of tumor cells. Cancer is the most common cause of death in humans, and therefore there is a great need for methods and means for the treatment or treatment of malignant degeneration.

  The potential for treatment of malignant tumors includes radiotherapy with X-rays, α-, β-, and γ-rays, immunotherapy and chemistry, in addition to the radical surgical removal of tumors where possible. There is therapy. Immunotherapy can currently be used only to a limited extent. Tumor chemotherapy is understood to mean the administration of cytotoxins (cytostatic agents) for the treatment of tumors and local surgical treatment or residual tumor cells after irradiation. These substances specifically intervene in specific processes of cell division, so that tissues with a high proportion of dividing cells, such as rapidly growing tumor tissues, react more sensitively. Alkylating compounds such as cyclophosphamide (The Merck Index, 12th edition, page 463), antimetabolites such as methotrexate (The Merck Index, 12th edition, page 1025), alkaloids such as vincristine (The Merck Index, 12th edition, 1704) and antibiotics such as daunomycin (The Merck Index, 12th edition, page 479) and adriamycin (The Merck Index, 12th edition, pages 581-582) are used. However, all these drugs have significant disadvantages due to significant side effects, and as a result only delay rather than avoid the death of affected humans. Furthermore, degenerated (cancer) cells develop resistance to the drugs used, so that current drugs are no longer cytostatic and act toxic because of side effects. In addition, combined or sequential administration of cytostatic agents has been shown to exceed the activity of individual cytostatic agents (monotherapy), and this may not add any side effects that should be considered in combination chemotherapy. It becomes possible. For all these reasons, new chemotherapeutic agents are urgently needed and are being pursued worldwide.

Natural substances having the parent structure of phenalenone have already been described. Phenalene is a condensed, incomplete aromatic ring structure that decomposes in air. Phenalenone is an oxidation product having a carbonyl group at the 1-position.
Patent application WO 99/60992 describes that all positions other than C1 are replaced by hydrogen or C ₁ -C ₄ -alkyl, preferably methyl, or C ₁ -C ₄ -alkoxy, preferably methoxy, for use as hair coloring agents. A comprehensive description of possible phenalenones is given.

Japanese Laid-Open Patent Publication No. 60-199849 is a phenalenone derivative that is effective as a phosphodiesterase (PDE) inhibitor and can be used for the treatment of arteriosclerosis, bronchial asthma, diabetes and cancer, 2,7,8,9-tetrahydroxy-4 -Methoxy-5-methylphenalen-1-one is described.
DA Frost & GA Morrison; J. Chem. Soc., Perkin Trans. 1,20, 2388-2396, 19
73 of norxantherquein (2,3,4,7,8,9-hexahydroxy-5-methyl-phenalen-1-one) from Penicillium herquei Bainer & Sartory Isolation is described.

DHR Barton et al. (Tetrahedron, 6, 48, 1959) describe the isolation of atrobenetin from Penicillium atrovenetum and Penicillium hercule bayner and sultry. Atrobenetin is described as an antioxidant in Y. Ishikawa et al. (J. Am. Oil Chem. Soc. 68, 666-668, 1991) and as a cytostatic agent with antitumor activity in WO00 / 45165. .
N. Narasimhachi et al. (J. Antibiotics, 25, 155, 1972) and J. Simpson (Chem. Soc.
Perkin Trans. 1, 1979, 1233-1238) is a compound of the compound deoxyherquainone (2-O-methylatrobenetin) that can be isolated from Penicillium herquay and exhibits antibacterial activity against gram-positive microorganisms. Mutant forms are described.

  The present invention is based on the object of providing other phenalenone derivatives that can be used in the treatment of tumors.

もしくは式（Ｉ−Ｂ）

｛式中、Ｘは式（Ｉ−Ｃ）

もしくは式（Ｉ−Ｄ）

のいずれかの基であり、
そして、Ｒ¹および存在するのであればＲ²は同時に、
１.０ Ｈ、もしくは
２.０ Ｃ₁〜Ｃ₆−アルキル、Ｃ₂〜Ｃ₆−アルケニルもしくはＣ₂〜Ｃ₆−アルキニル［
式中、アルキル、アルケニルおよびアルキニルは直鎖もしくは分岐鎖であり、そして、
２.１ −ＯＨ、
２.２＝Ｏ、
２.３ −Ｏ−Ｃ₁〜Ｃ₆−アルキル（式中、アルキルは直鎖もしくは分岐鎖である）
、
２.４ −Ｏ−Ｃ₂〜Ｃ₆−アルケニル（式中、アルケニルは直鎖もしくは分岐鎖であ
る）、
２.５ −アリール、
２.６ −ＮＨ−Ｃ₁〜Ｃ₆−アルキル（式中、アルキルは直鎖もしくは分岐鎖である
）、
２.７ −ＮＨ−Ｃ₂〜Ｃ₆−アルケニル（式中、アルケニルは直鎖もしくは分岐鎖で
ある）、
２.８ −ＮＨ₂、もしくは
２.９ハロゲン
（式中、置換基２.１〜２.９もまた−ＣＮ、−アミドもしくは−オキシム官能基によりさらに置換され得る）により場合により一置換もしくは二置換される］｝
の化合物、ならびに／または式（Ｉ−Ａ）もしくは式（Ｉ−Ｂ）の化合物の立体異性体、ならびに／またはあらゆる割合におけるこの異性体の混合物、ならびに／または式（Ｉ−Ａ）もしくは（Ｉ−Ｂ）の化合物の生理学的に許容し得る塩に関する。
Ｒ¹およびＲ²は、好ましくはＨまたはＣ₁〜Ｃ₆−アルキルである。 The present invention is a compound of the formula (IA)

Or the formula (IB)

{Wherein X is the formula (IC)

Or formula (ID)

Any of the groups
And R ¹ and, if present, R ² are simultaneously,
1.0 H, or 2.0 C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl or C ₂ -C ₆ -alkynyl [
Where alkyl, alkenyl and alkynyl are straight or branched and
2.1-OH,
2.2 = O,
2.3 —O—C ₁ -C ₆ -alkyl (wherein alkyl is linear or branched)
,
2.4 —O—C ₂ -C ₆ -alkenyl (wherein alkenyl is linear or branched),
2.5-aryl,
2.6 —NH—C ₁ -C ₆ -alkyl (wherein alkyl is linear or branched),
2.7 -NH-C ₂ ~C ₆ - alkenyl (wherein alkenyl is linear or branched),
2.8 —NH ₂ , or 2.9 halogen (wherein the substituents 2.1 to 2.9 can also be further substituted by —CN, —amide or —oxime functional groups) optionally mono- or di-substituted. Is replaced]}
And / or stereoisomers of compounds of formula (IA) or (IB) and / or mixtures of these isomers in any proportions and / or formulas (IA) or (I -Physiologically acceptable salts of the compounds of B).
R ¹ and R ² are preferably H or C ₁ -C ₆ -alkyl.

  A chiral center exists in the R or S configuration unless otherwise specified. The present invention relates to both optically pure compounds and mixtures of stereoisomers in all proportions, for example enantiomeric mixtures and diastereomeric mixtures.

  The compounds described in the present invention differ from substances known from the literature in polarity, chemical structure, biological activity or even physical properties.

ならびに、まとめて式（III）の化合物として以下に示される、式（III−Ａ）および（III−Ｂ）のアトロベネチンを生成する。

Penicillium Herkuei Beiner & Saltley strain, DSM 14142 is a nutrient solution containing glucose, starch, oat flakes or glycerin, which is shown below as a compound of formula (II), together with formulas (II-A) and (II-B) ) A compound called penilenone,

In addition, atrobenetins of the formulas (III-A) and (III-B), which are collectively shown below as compounds of the formula (III), are produced.

R ¹ is different from the formula (I-A) and the H (I-B) compounds are isomers, the other can be separated individually, R ¹ of the case further example R ¹ is H The other isomers of the compounds of formula (IA) or (IB) in which R ¹ is different from H, are subsequently derivatized starting with other tautomers in each case Can be converted to each other.
The compounds of formula (IA) and (IB) are collectively shown below as compounds of formula (I).

  Compounds of formula (II-A) and (II-B) are tautomers and cannot be separated from each other under normal conditions (eg room temperature). The compounds of formula (II-A) and (II-B) are collectively shown below as compounds of formula (II).

  The compounds of formula (III-A) and (III-B) are likewise tautomeric and cannot be separated separately from each other under normal conditions (eg room temperature). The compounds of formula (III-A) and (III-B) are collectively shown below as compounds of formula (III).

Compounds of the formulas (IA) and (IB) in which R ¹ is H are known in a manner known per se, eg the book Advanced Organic Chemistry by John Kerry March, John Wiley & Sons, 4th edition,
As described in 1992, it can be selectively derivatized according to the present invention by reacting the hydroxyl group with an alkylating agent. The alkylating agent is, for example, a diazomethane derivative, preferably trimethylsilyldiazomethane. In order to carry out the reaction selectively, it may be advantageous to introduce suitable protecting groups in a manner known per se before the reaction. The protecting group is removed after the reaction and the reaction product is then purified.

  To date, the selective alkylation of phenalenone to obtain the compounds described in the present invention has not been described. For example, when dezoxyherquineinone is reacted with diazomethane according to Suga et al. (Bull. Chem Soc. Jpn., 56, 3661-3666, 1983), dezoxyherquainone dimethyl ether or isomeric endo-atrobenetine trimethyl ether Is obtained.

Compounds of the formula R ¹ is H (I-A) and (I-B), for example, be prepared by cleavage of the ether compound of R ¹ is different from H Formula (I-A) and (I-B) . The cleavage of ethers can be carried out in a manner known per se, as described, for example, in the book Advanced Organic Chemistry, John Wiley & Sons, 4th edition, 1992 by Jerry March.

  The invention therefore further relates to a compound of formula (I) having the formula (II) or a pharmaceutically acceptable salt of a compound of formula (II).

Therefore, the present invention adds to that,
1. Culturing the microorganism Penicillium herculae Beiner & Saltry, DSM 14142, or one of its variants or mutants in an aqueous nutrient medium,
2. Separating and purifying the compound of formula (II), and
3. It relates to a process for the preparation of a compound of formula (II), which optionally comprises converting a compound of formula (II) into a pharmacologically acceptable salt.

The present invention further includes
1. Culturing the microorganism Penicillium herculae Beiner & Saltry, DSM 14142, or one of its variants or mutants in an aqueous nutrient medium,
2. a) separating and purifying the compound of formula (II), or
b) separating and purifying the compound of formula (III),
3. a) derivatizing a compound of formula (II) to give a compound of formula (I), or
b) derivatizing the compound of formula (III) to give a compound of formula (I), and
4). It relates to a process for the preparation of a compound of formula (I) comprising, in some cases, converting the compound of formula (I) into a pharmacologically acceptable salt.

  Penicillium Herkuei Beiner & Saltley strain, DSM 14142, produces penilenone and secondary products in nutrient solutions containing glucose, starch, oat flakes or glycerin.

  The Penicillium Herkuei Beiner & Saltry isolates are in accordance with the rules of the Budapest Treaty and are Number: DSM 14142, deposited on March 6, 2001.

  Penicillium Herkuei Beiner & Sartorium, DSM 14142, has a gray to luminescent green basal mycelium and very little aerial mycelium. The leachate does not form on the malt medium and no pigment is excreted into the medium. In culture, the strains form a dense spore that is rough, 200-400 × 3.5-4.0 μm characteristic of the genus Penicillium. The “metulae” is relatively short, usually 4-6, 10-12 × 3.0-5.0 μm, and in the shape of a club. The phialida is arranged in an ampoule-shaped 7-10 × 3.0 μm 6-10 “varticilli”. The conidia are 3.5-5.0 × 3.0-3.5 μm “apiculate” ellipses with smooth cell walls. The conidia form parallel chains up to 100 μm in length.

Such a process comprises a medium containing Penicillium Hervai Beiner & Saltry, DSM 14142, variants and / or mutants thereof under aerobic conditions, at least in each case one carbon and nitrogen source, inorganic salts and optionally trace elements. Culturing in a.
Cultivation is preferably carried out at a temperature of 20 to 35 ° C. and at a pH of 2 to 9.

  Instead of the DSM 14142 strain, its variants and mutants can also be used as long as they produce the compounds described in the present invention. Such mutants may be irradiated by physical means such as irradiation using ultraviolet light or X-rays, or chemical mutagens such as ethyl methanesulfonate (EMS), 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl. It can be made in a manner known per se with -N'-nitro-N-nitrosoguanidine (MNNG).

  Suitable preferred carbon sources for fermentation are assimilable carbohydrates and sugar alcohols such as glucose, lactose, sucrose or D-mannitol, and natural products containing carbohydrates such as malt extract or yeast extract. Suitable nitrogen-containing nutrient sources include ground amino acids, peptides and proteins, and their degradation products such as casein, peptone or tryptone, and meat extracts, yeast extracts such as corn, wheat, kidney beans, soybeans, rice or cotton Distillation residues of seeds, alcohol products, meat flour or yeast extract, as well as ammonium salts and nitrates, in particular peptides obtained synthetically or biosynthetically. Inorganic salts that the nutrient solution may contain are, for example, alkali metal or alkaline earth metal, iron, zinc, cobalt and manganese chlorides, carbonates, sulfates or phosphates.

The production of the compounds according to the invention is, for example, about 0.05-5%, preferably 1-2% malt extract; and 0.05-3%, preferably 0.05-1% yeast extract; 0.2 It progresses particularly well in nutrient solutions containing -5%, preferably 0.5-2% glucose; and 0.5-3%, preferably 1.5-3% autoflakes. The percentage details are based on the total nutrient weight in each case.

  In this nutrient solution, Penicillium Herkuei Beiner & Saltley, DSM 14142 produces a mixture of compounds described in the present invention. Depending on the composition of the nutrient solution, the measurable content of one or more compounds according to the invention can vary. Furthermore, the synthesis of individual compounds can be controlled by the composition of the culture so that no compound is produced at all or in an amount below the detection limit of the microorganism.

  The cultivation of the microorganisms is carried out aerobically, i.e. with shaking or stirring in a shaker flask or fermenter, for example, in liquid or on solid medium, optionally with air or oxygen. The culture can be carried out at a temperature in the range of about 15-30 ° C, preferably about 20-30 ° C, in particular 25-30 ° C. The pH range should be 4-10, preferably 6.5-7.5. The microorganism is generally cultured under these conditions for 48 to 720 hours, preferably 72 to 350 hours. Cultivation involves several steps: first preparing one or more pre-cultures in a liquid nutrient medium, then into the main culture medium which is the actual production medium, eg in an amount of 1:10 to 1: 100 It is advantageously carried out by inoculating with. The preculture is obtained, for example, by inoculating mycelium in a nutrient solution and allowing it to grow for about 20 to 120 hours, preferably 48 to 72 hours. The mycelium is obtained, for example, by culturing the strain on a solid or liquid nutrient medium such as yeast malt agar, autoflakes agar or potato dextrose agar for about 1 to 42 days, preferably 21 to 35 days.

  The fermentation process and screening for variants and mutants producing the compounds according to the invention can be carried out according to methods known to the person skilled in the art, for example by assaying for biological activity in bioassays or by thin layer chromatography (TLC). Alternatively, it can be monitored by a chromatography method such as high performance liquid chromatography (HPLC).

  Penicillium Herkuei Beiner & Saltry, DSM 14142, can produce the compounds described in the present invention by surface or static culture in solid nutrient media. The solid nutrient medium is prepared, for example, by adding agar or gelatin to an aqueous nutrient medium. Furthermore, it is possible to obtain the compounds according to the invention by a submerse process, ie by fermentation of Penicillium Herkuei Bayner & Saltley, DSM 14142 in an aqueous suspension. The compounds described in the present invention can occur in both mycelium and culture filtrate, but usually the majority is found in the cell body. Therefore, it is appropriate to separate the fermentation broth by filtration or centrifugation. The filtrate is extracted using the adsorbent resin as the solid phase. The mycelium is the same in surface culture, but it is appropriate to extract it using an organic solvent such as methanol or propan-2-ol.

  The extraction can be carried out over a wide pH range, but it is appropriate to work in an environment of neutral or weak acidity, preferably pH 3 to pH 7. The extract can be concentrated and dried, for example in vacuum.

The compounds of formula (II) and formula (III) are anxious substances if no specific treatment is performed during the separation and purification process. 1) Work during reducing and purifying conditions under reducing conditions, for example in the constant presence of ascorbic acid, 2) Separation in an acidic environment with a pH of less than 7, preferably a pH range of 2-5 3) Penilenone from the culture of DSM 14142 strain if only mild agents are used during the purification process, eg adsorbent resin is used as chromatographic support, and 4) the presence of amine is excluded during the whole process. Has been found to be obtained in very good yields.

A suitable method for the isolation of the compounds according to the invention is solvent partitioning by methods known per se. Another method of purification is an adsorbent resin such as Diaion ^(R) HP-20 (Mitsubishi Chemical Corporation),
Tokyo), Amberlite ^(R) XAD7 (Rohm and Haas, USA), Amberchrom ^(R) CG (Toso Haas, a chromatography using Philadelphia, USA) or similar. Further suitable under the specified conditions are a number of reversed phase carriers such as RP ₈ and RP _18, which are generally known in connection with high performance liquid chromatography (HPLC). Other purification possibilities under the specified conditions include the use of “normal phase” chromatographic supports such as silica gel or Al ₂ O ₃ or others known per se. Another separation process, molecular sieves, for example, Fractogel ^(R) TSKHW-4
0, is the use of Sephadex ^(R) G-25 and per se known methods other things.

  It is further possible to obtain the compounds of formula (I) according to the invention after concentration by crystallization, which can use for example organic solvents and mixtures thereof, anhydrides or water.

  Other steps for the separation and purification of the compounds according to the invention include the use of anion exchangers, preferably in the pH range 4-7, and the use of cation exchangers, preferably in the pH range 2-5. It is. Particularly suitable for this is the use of a buffer to which an organic solvent part has been added.

On the other hand, it is also possible to separate and / or purify the compounds according to the invention by sublimation.
A particularly advantageous purification method for the separation of the compounds according to the invention is crystallization carried out in a manner known per se.

The compounds of formula (I) according to the invention can be converted into the corresponding pharmacologically acceptable salts according to techniques known to those skilled in the art. Pharmacologically acceptable salts of the compounds described in the present invention are understood to mean both inorganic and organic salts as described in Remingtons Pharmaceutical Sciences (17th edition, page 1418 [1985]). . Possible salts include in particular alkali metal salts, ammonium salts, alkaline earth metal salts, salts with physiologically acceptable amines and inorganic or organic acids such as HCl, HBr, H ₂ SO ₄ , maleic acid, fumaric acid There is salt with. On the other hand, possible salts include complexes with metal ions such as calcium, magnesium, zinc, iron or others. The compounds of formula (I) have a pronounced tendency to bind in the form of complexes with ions, preferably cations.

  Surprisingly, the compound of the formula (I) according to the present invention has a strong cytostatic activity, and therefore is suitable for the treatment and / or prevention of diseases caused by disordered tissue or cell proliferation or oncology. Has been found. Of particular note is that the compounds described in the present invention have no cross-resistance to conventional cytostatics.

  Compounds of formula (I) have been found to inhibit protein kinases. The kinase belongs to a transferase that transfers a phosphate group from adenosine triphosphate to another substrate. Proteins and enzymes are phosphorylated by protein kinases, usually on serine, threonine or tyrosine side chains, and their activity is modified, which is recognized as a principle of extensive regulation in metabolism and signaling. In the case of cancer, the affected tissue grows in an unregulated manner and therefore intervention in the regulation of growth controlled by the kinase is desirable. Multiple kinases are involved in the cell proliferation cascade. Some of these kinases are inhibited by the compounds described in the present invention.

Of further importance is the antibacterial activity of the compounds of formula (I) according to the invention against bacteria such as Staphylococcus aureus, Streptomyces murinus, and fungi such as Aspergillus niger which can cause refractory and deadly infections. Sex inhibitory action. Its antibacterial activity can be demonstrated, for example, by the “agar diffusion method”. For example, penilenone forms an 11 mm inhibition circle with a 1 mg / ml solution and an 8 mm inhibition circle with a 0.1 mg / ml solution on an agar plate containing a culture of Streptomyces murinus. Accordingly, the compounds of formula (I) according to the invention are likewise suitable for the treatment and / or prevention of bacterial infections and / or fungal diseases.

The compounds of formula (I) can also be used as antioxidants. Antioxidants (antioxidants) are organic compounds that inhibit or prevent undesirable changes caused by the action of oxygen in a substance to be protected. Antioxidants are needed, for example, in plastics for protection against aging, in fats for protection against rancidity, in oils for resinification, in aromatics for worsening odors, in food and in pharmaceuticals It is. The action of the antioxidant is usually as a radical scavenger for free radicals generated by oxidation. The antioxidant action of atrobenetin (compound of formula (III)) has already been described in Y. Ishikawa et al. J. Am. Oil Chem. Soc. 68, 666-668, 1991. However, microbial antioxidants are often too weak or not necessarily acceptable in their action. Accordingly, there is a considerable need for new, effective and acceptable antioxidants. The compounds of formula (I) are highly effective antioxidants that are significantly superior in their action to atrobenetin. For example, atropenine reacts only slowly with air oxygen in solution and in solid form (eg in hours), whereas for example penilenone of formula (II) binds to oxygen in seconds or in minutes. In fact, this increased affinity of penilenone for oxygen is clearly advantageous for materials that are very susceptible to oxidation.

Another chemical property of the compounds according to the invention is the ability to form complexes with multivalent, preferably 2- and 3-valent cations, such as Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Fe ^3+. It is. Its ability to form a complex can be advantageous for the production of pharmaceuticals, and thus inhibitors of the enzyme that can bind to zinc of matrix metalloproteinase (MMP) are known, for example. On the other hand, other possibilities of use for diseases whose onset manifests as abnormal metal ion concentrations in the body are also conceivable. It is also possible to make the complexing ability of the compounds described in the present invention available to non-pharmaceuticals, eg for water technology, body care ingredients, and polymerization technology (Ullmans Enzyklopadie der Technischen Chemie [Ullman's encyclopedia of industrial chemistry], 5th edition, A 10, 95-100, 1985-1995).

  The compounds of formula (I) according to the invention can likewise act in the treatment of rheumatic diseases such as rheumatoid arthritis. Active ingredients in reducing oxidative stress by free radical scavengers or antioxidants for rheumatoid arthritis are described, for example, in Ostrakhovitch and Afanas (Biochemical Pharmacology, 2001, 743-746).

  The invention can therefore also be treated by the use of the compounds of formula (I) according to the invention as pharmaceuticals, in particular by oncosis, bacterial infections, mycoses, rheumatic diseases and inhibition of matrix metalloproteinases It also relates to the treatment and / or prevention of disease.

In addition, the invention relates to a medicament containing at least one compound according to the invention.
Such medicaments are prepared by mixing at least one compound of formula (I) with one or more physiologically acceptable additives and making it into a suitable dosage form.

The pharmaceutical agent described in the present invention can be administered enterally (orally), parenterally (intramuscularly or intravenously), rectally or locally (externally). They can be administered in the form of solutions, powders (tablets, capsules including microcapsules), ointments (creams or gels), or suppositories. Possible physiologically acceptable additives for this type of dosage form include liquid or solid excipients and extenders common in formulations, solvents, emulsifiers, lubricants, flavoring agents, colorants and / Or a buffer substance. Suitable dosages include 0.1 to 1000 per kg body weight,
Preferably 0.2-100 mg is administered. They are suitably administered in dosage units containing at least an effective daily dose of a compound according to the invention, for example 30-3000, preferably 50-1000 mg.

  The following examples are intended to provide a more detailed explanation of the invention and do not wish to limit the scope of the invention in any way.

Example 1
Preparation of glycerin culture solution of Penicillium hervai Bayner & Saltley, DSM 14142 Nutrient solution in a sterile 100 ml Erlenmeyer flask (malt extract 2.0%, yeast extract 0.2%, glucose 1.0%, (NH ₄ ) ₂ 30 ml of HPO _{4 (} 0.05%, pH 6.0) is inoculated with Penicillium genus Helena Beiner & Sartley, DSM 14142, and then cultured for 6 days at 25 ° C. and 140 rpm in a rotary shaker. Then 1.5 ml of this culture is diluted with 2.5 ml of 80% strength glycerin and stored at -135 ° C.

Example 2
Preparation of pre-culture solution in Erlenmeyer flask of Penicillium Herkuei Beiner & Saltry, DSM 14142 Nutrient solution in a sterile 300 ml Erlenmeyer flask (malt extract 2.0%, yeast extract 0.2%
, Glucose 1.0%, (NH ₄ ) ₂ HPO ₄ 0.05%, pH 6.0) was inoculated with Penicillium genus Herkuei Beiner & Sartley, DSM 14142, and then in a rotary shaker at 25 ° C. and 140 rpm Incubate for 4 days. Then 2 ml of this preculture is inoculated for the preparation of the main culture.

Example 3
Preparation of the main culture solution of Penicillium genus Hervai Bayner & Saltley, DSM 14142 The following nutrient solution (malt extract 2.0%, yeast extract 0.2%, glucose 1.0%, (NH ₄ ) ₂ HPO ₄ 0.05% , PH 6.0) Sterilized 300 ml Erlenmeyer flask containing 100 ml Inoculated with 2 ml of culture or preculture (see Example 2) cultured in a tilted tube (same nutrient solution except adding 2% agar) After that, the cells are cultured in a shaking incubator at 140 rpm and 25 ° C. The maximum production of one or more compounds of penilenone according to the present invention is achieved after about 144 hours. For inoculation of a 10-200 L fermentor, submerged culture (inoculation amount of about 10%) of 96-144 hours of the same nutrient solution is sufficient. The conditions of this fermenter are as follows.
Temperature 25 ° C
Stirrer speed: 200rpm
Ventilation 15Lmin ^-1
The formation of bubbles can be suppressed by repeatedly adding an ethanolic polyhydric alcohol solution. Maximum production is achieved after about 96-144 hours.

Example 4
Separation of compounds (II) and (III) After 5 liters of the culture broth obtained according to Example 3 was centrifuged, the cell mass (0.5 liters) was diluted with 2 liters of methanol supplemented with 0.1% ascorbic acid. Extracted. Methanolic phase after clear filtration was concentrated 1L in vacuo, and subjected to a column packed with adsorption resin MCl gel ^(R) CHP20P volume of 1L. Column dimensions: width x height: 7 cm x 27 cm.
10% propan-2-ol to 90% propan-2-yl in 0.1% ascorbic acid aqueous solution
Elution was performed using an all solvent gradient. Column eluate (140 ml / min) was collected in 250 ml fractions. Fraction 23-26 (a mixture of compounds of formulas (II-A) and (II-B), collectively referred to as compounds of formula (II)) containing penilenone as confirmed by HPLC analysis and fractions containing atrobenetin Fractions 43-51 (mixtures of compounds of formula (III-A) and (III-B), collectively referred to as compounds of formula (III)) were collected and concentrated in vacuo. The fractions combined in each case were concentrated in vacuo and then stored cold. Penilenone (260 mg of the compound of formula (II)) was crystallized from fractions 23-26, and 1.2 g of atrobenetin (compound of formula (III)) was obtained from fractions 43-51. The crystals were filtered under an argon protective gas atmosphere, and after removing oxygen, the crystals were stored cold.

Example 5
Separation and purification by HPLC Column: Superspher 100RP-18e ^(R) , 250-4, with pre-column Mobile phase:
2 min: 5% acetonitrile in 0.1% phosphoric acid 18 min: gradient from 5% to 100% acetonitrile in 0.1% phosphoric acid, then constant with 100% acetonitrile Flow rate: 1 ml / min Detection by UV absorption at 210 nm A retention time of 13.5 minutes was found for the compound of (II) and 20.5 minutes for the compound of formula (III).

Example 6
Characterization of the compound of formula (II) The physicochemical and spectroscopic properties of penilenone can be summarized as follows.
Appearance: Soluble in yellow crystalline material, polar culture medium and polar organic solvent, hardly soluble in water. The melting point cannot be measured due to decomposition. Stable in slightly acidic solvent under reducing conditions. Under the influence of oxygen, penilenone turns green in neutral drugs or in the presence of amines.
Experimental formula: C ₁₄ H ₁₀ O ₆
Molecular weight: 274.23
Molecular ion 275.2 [M + H] ⁺ was found by ESI + (electrospray ionization) mass spectrometry and 273 [M−H] ⁻ or 271 [M−3H] ⁻ was measured under ESI (negative) conditions. It was.
UV maximum value: 215 nm, 248 (sh) nm, 275 (sh) nm, 389 nm

Example 7
Obtaining penilenone dimethyl ether derivatives of formula (IV-A) and (IV-B) 40 mg of a compound of formula (II) (penilenone, separated according to Example 4) are dissolved in 30 ml of tetrahydrofuran and then 2.0 M (trimethylsilyl). It was treated with 0.5 ml of diazomethane and dissolved in hexane [Aldrich, cat. No. 36,283-2]. After 1 hour, water was added to terminate the reaction, and the solvent was distilled off in vacuo. Then separating the reaction product on Nucleosil ^(Nucleosil) HD ^(R) column (21mm × 250mm). The eluent used was a gradient of 10% to 99% acetonitrile in 0.1% acetic acid. The 20 ml / min column effluent was collected in fractions. The fractions containing the methylated product were combined in each case, concentrated in vacuo and crystallized.
6 mg of penilenone dimethyl ether of formula (IV-A) (empirical formula: C ₁₆ H ₁₄ O ₆ , molecular weight: 302.29) and penilenone dimethyl ether of formula (IV-B) (empirical formula: C ₁₆ H ₁₄ O ₆ , Molecular weight: 302.29) 1 mg was obtained.

Penilenone dimethyl ether of formula (IV-A):
UV maximum: 216 nm, 242 nm, 280 nm (sh), 387 nm

Penilenone dimethyl ether of formula (IV-B):
UV maxima: 213 nm, 241 nm and 390 nm

Example 8
Obtaining Atrobenetine Derivatives (VA) and (VB) 100 mg of the compound of formula (III) (atrobenetin, prepared according to Example 4) is dissolved in 5 ml of tetrahydrofuran and then 1 ml of 2.0 M (trimethylsilyl) diazomethane is used. And dissolved in hexane [Aldrich, cat. No. 36,283-2]. After 15 minutes, water was added to terminate the reaction and the solvent was distilled off in vacuo. The reaction product was then separated on a Nucleosil AB ^(R) column (21 mm x 250 mm). The eluent used was a gradient of 10% to 99% acetonitrile in 0.02% trifluoroacetic acid adjusted to pH 4.5 with ammonium hydroxide. The 15 ml column effluent was collected in fractions. The fractions containing the methylated product were combined in each case, concentrated in vacuo and crystallized.
Atrobenetine monomethyl ether (VA) (empirical formula: C ₂₀ H ₂₀ O ₆ , molecular weight: 35
6.38) 24 mg and atrobenetine monomethyl ether of formula (VB) (VB) (
Empirical formula: C ₂₀ H ₂₀ O _6, molecular weight: 356.38) was obtained 10 mg.

Atrobenetine monomethyl ether (VA):
UV maximum: 218 nm, 260 nm (sh), 394 nm

Atrobenetine monomethyl ether (V-B):
UV maximum value: 222 nm, 282 nm, 385 nm

Claims (15)

Formula (IA)

Or the formula (IB)

{Where
X is the formula (I-C)

Or formula (ID)

Any of the groups
And R ¹ and, if present, R ² are simultaneously,
1.0 H, or 2.0 C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl or C ₂ -C ₆ -alkynyl [
Where alkyl, alkenyl and alkynyl are straight or branched and 2.1-OH,
2.2 = O,
2.3 —O—C ₁ -C ₆ -alkyl (wherein alkyl is linear or branched)
,
2.4 —O—C ₂ -C ₆ -alkenyl (wherein alkenyl is linear or branched),
2.5-aryl,
2.6 —NH—C ₁ -C ₆ -alkyl (wherein alkyl is linear or branched),
2.7 -NH-C ₂ ~C ₆ - alkenyl (wherein alkenyl is linear or branched),
2.8 —NH ₂ , or 2.9 halogen (wherein the substituents 2.1 to 2.9 can also be further substituted by —CN, —amide or —oxime functional groups) optionally mono- or di-substituted. Is replaced]}
And / or stereoisomers of compounds of formula (IA) or formula (IB) and / or mixtures of these isomers in any proportions and / or formula (IA) or formula (I -A physiologically acceptable salt of the compound of B). R ¹ and R ² are H or C ₁ -C ₆ - alkyl, wherein according to claim 1, wherein (I-A) or a compound of formula (I-B). Formula (II-A)

Or formula (II-B)

Or a compound of formula (IA) or (IB) according to claim 1 or 2, characterized in that it has a physiologically acceptable salt thereof. 1. Microbial Penicillium herquei Bayner & Sain (Bainer & Sa
rtory), DSM 14142, or one of its mutants or mutants is cultured in an aqueous nutrient medium,
2. a) Formula (II-A)

Or formula (II-B)

Separating and purifying the compound of
b) Formula (III-A)

Or formula (III-B)

The compound is isolated and purified,
3. a) derivatizing a compound of formula (II-A) or formula (II-B) to give a compound of formula (IA) or formula (IB), or
b) derivatizing a compound of formula (III-A) or formula (III-B) to give a compound of formula (IA) or formula (IB), and
4). Converting a compound of formula (IA) or formula (IB), optionally into a pharmaceutically acceptable salt,
A process for the preparation of a compound of formula (IA) or (IB) according to claim 1 or 2, characterized in that 1. Culturing the microorganism Penicillium herculae Beiner & Saltry, DSM 14142, or one of its variants or mutants in an aqueous nutrient medium,
2. Separating and purifying the compound of formula (II-A) or formula (II-B); and
3. Converting the compound of formula (II-A) or formula (II-B), optionally into a pharmaceutically acceptable salt,
A process for producing a compound of formula (II-A) or (II-B) according to claim 3, characterized in that   The production method according to claim 4, wherein the derivatization is performed using an alkylating agent.   The production method according to claim 6, wherein the alkylating agent is a diazomethane derivative.   Use of a compound according to any of claims 1 to 3 for the manufacture of a medicament.   Use of a compound according to claim 8 for the manufacture of a medicament for the treatment and prevention of oncology.   Use of a compound according to claim 8 for the manufacture of a medicament for the treatment and prevention of bacterial infections and / or fungal diseases.   Use of a compound according to claim 8 for the manufacture of a medicament for the treatment and prevention of rheumatic diseases.   Use of a compound according to claim 8 for the manufacture of a medicament for diseases that can be treated by inhibition of matrix metalloproteinases.   Use of the compound according to any one of claims 1 to 3 as an antioxidant.   A medicament comprising at least one compound according to any of claims 1 to 3 and one or more physiologically acceptable additives.   Penicillium genus Hervai Beiner & Saltley strain, DSM 14142.