WO1997036852A1 - Novel benzaldehyde derivatives from hericeum erinaceus - Google Patents

Abstract

The present invention relates to a compound of formula (I) R?1 to R10¿ are hydrogen or methyl, where two R?1 to R10¿ on adjacent carbon atoms together with a C-C single bond may stand for a C-C double bond, provided that, in the carbon chain substituted by R?1 to R10¿, only one of the substituents R?1 to R10¿ is methyl and only an adjacent pair of said substituents contribute to a C-C double bond, a process for its production and its use as a medicament, especially for treating diseases produced by an HIV infection.

Description

New benzaldehyde derivatives from Hericeum erinaceus

The invention relates to new benzaldehyde derivatives, a process for their preparation and their use for the manufacture of a medicament, in particular for the treatment of a disease which is caused by an HIV infection.

The diseases of the human body that are caused by the Human Immunodeficiency Virus (HIV) are life-threatening and are associated with a large number of secondary diseases. The severity of the disease is directly linked to the virus titer, i.e. the number of viruses present in the body. Reducing the virus titer is one way to prevent the onset of the disease or to positively influence the course of the disease. The enzyme reverse transcriptase is essential for the multiplication of the virus. Inhibitors of this enzyme have been shown to significantly slow virus replication (e.g. azidothymidine)

It is an object of the present application to provide inhibitors of HIV reverse transcriptase and thus for the treatment of diseases which are caused by HIV infections.

A wide variety of synthetic and natural substances are known to inhibit HIV reverse transcriptase (see, for example, the overview by GTTan et al, Biochem. Biophys. Res. Commun. (1992), 185, 370-378) and thus meet a basic requirement for the treatment of HIV infections. In addition, it is known that Basisiomyceten secondary metabolites with a biological effect such. B. form the strobilurins (see, for example, S. Zapf et al. Angew. Chemie (1995), 107, 255). Especially the fruiting body of the basisiomycete Hericeum erinaceus forms the Hericenone and the as secondary metabolites

CONFIRMATION COPY Hericerin, the effects of which are described as stimulating cytotoxicity or stimulating neurite outgrowth from nerve cells (cf. H. Kawagishi et al, Tetrahedr. Lett. (1990), 31, 373).

Surprisingly, it has now been found that secondary metabolites can also be isolated from the Mγcel of the basisiomycete Hericeum erinaceus, which inhibit HIV reverse transcriptase and are therefore suitable for the treatment of HIV infections. The compounds and their physiologically tolerable salts are hitherto unknown and are characterized by the following structure (formula I)

in which R ¹ to R ^{10 are} hydrogen or methyl, where two R ¹ to R ¹⁰ located on adjacent carbon atoms together with a CC single bond can represent a CC double bond, with the proviso that in the group with R ¹ to R ¹⁰ substituted carbon chain each means only one of the substituents R ¹ to R ^{10 is} methyl and in each case only an adjacent pair of these substituents contributes to a CC double bond.

R ⁵ in formula I is preferably methyl.

R ³ and R ⁶ (for example formula 1) or R ⁶ and R ⁷ (for example formula 2) are particularly preferably together with that between those of them substituted carbon atoms lie CC single bond a CC double bond.

The double bond of these compounds is preferably in the Z configuration.

Formula 1 Hericenal A Formula 2 Hericenal B

The new compound of formula I, in particular compounds 1 and 2, and their physiologically tolerable salts are distinguished by their action against HIV reverse transcriptase, which is clearly superior to that of other metabolites from basisiomycetes.

Investigation of substances for inhibition of HIV "reverse transcriptase"

The activity of the reverse transcriptase (RT) was determined using a "scintillation proximity assay" (SPA). The reagent kit for the RT-SPA was obtained from Amersham / Buchler (Braunschweig). The enzyme RT (cloned from HIV in E. coli) was from HT-Biotechnology LTD, Cambridge, UK. Approach:

The test was carried out according to the method manual of the manufacturer Amersham - with the following modifications:

Bovine serum albumin was added to the "assay" buffer at the final concentration of 0.5 mg / ml.

The test was carried out in Eppendorf reaction vessels with a 100 I batch volume.

The manufacturer's RT concentrate (5000 U / ml) was dissolved in Tris-HCl buffer 20 mM; pH 7.2; Diluted 30% glycerol to an activity of 15 U / ml. The incubation time for the batches was 60 min (37 C). After stopping the reaction and "developing" with the pearl suspension, 130 ml batch in 4.5 ml Tris-HCl buffer, 10 mM; pH 7.4; 0.1 5 M NaCl transferred and the tritium activity measured in a β-counter.

Substance testing:

For a preliminary test of the inhibitor activity, the substances were dissolved in water (stock solution c == 1 mg / ml) and tested in various dilutions (10 ^" , 10 ^{~ 2} , 10 ^" 3 etc.).

To determine IC 50 values, the inhibitor stock solutions were further diluted in water and tested in suitable concentrations. The concentration associated with a 50% enzyme inhibition was determined from the graphical representation of RT activity against the logarithm of the concentration of the respective test substance. Substance IC ₅₀ UvM]

(HIV-RT)

Hyphodontal 77

Mniopetal B 91

Mniopetal D 54

Hericenal A 0.37

The present invention furthermore relates to a process for the preparation of the compounds mentioned. A process for the preparation of the compounds mentioned is characterized in that the microorganism Hericeum erinaceus, preferably Hericeum erinaceus DSM 10600, is cultivated in an aqueous medium and the target compounds are then isolated and purified.

The microorganism Hericeum erinaceus DSM 10600 was deposited on March 20, 1996 with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Marscheroder Weg 1 b, D-38124 Braunschweig, according to the provisions of the Budapest Treaty.

Instead of the strain DSM 10600, its mutants and variants can also be used, provided that they can synthesize compounds of the formula I. Such mutants can be known in a manner known per se by physical means, for example radiation, such as with ultraviolet or X-rays, or chemical mutagens, for example ethyl methanesulfonate (EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methγl- N ^' -nitro-N-nitrosoguanidine (MNNG) are generated. The fermentation conditions described below apply to Hericeum erinaceus and the deposited isolate. The fermentation can take place on a laboratory scale (milliliter and liter scale) or on an industrial scale (cubic meter scale).

Henricium erinaceus, in particular H. erinaceus DSM 10600, produces the compound of the formula I in a nutrient solution which contains a carbon source and a nitrogen source and the customary inorganic salts.

As preferred carbon sources for aerobic fermentation, assimilable carbohydrates and sugar alcohols, such as glucose, lactose or D-mnanite, and carbohydrate-containing natural products, such as, for. B. malt extract. Suitable nitrogenous nutrients are: amino acids, peptides and proteins and their degradation products, such as peptones or tryptones, also meat extracts, ground seeds, for example from corn, wheat, beans, soybeans or the cotton plant, distillation residues from alcohol production, meat flours or yeast extracts, but also Ammonium salts and nitrates. The inorganic solution of inorganic salts can contain, for example, chlorides, carbonates, sulfates or phasphates of the alkali or alkaline earth metals, iron, zinc, cobalt and manganese.

The formation of the compound of formula I with H. erinaceus, preferably DSM 10600, proceeds particularly well in a nutrient solution which contains 0.1 to 2%, preferably 0.5 to 1%, cornstep, 0.5 to 10%, preferably 1 to Contains 5% tomato paste, 0.1 to 5% preferably 0.5 to 3% oatmeal 0.1 to 5% preferably 0.5 to 3% glucose and trace elements (% details based on the weight of the entire nutrient solution). The trace element solution can have the following composition:

FeSO ₄ x 7 H ₂ O 0.01 - 1% preferably 0.05 - 0.2%

MuSO ₄ x 1 H ₂ O 0.01 - 1% preferably 0.05 - 0.2% CuCI ₂ x 2 H ₂ O 0.0005 - 0.01% preferably 0.001 - 0.005%

CaCI ₂ x 2 H ₂ O 0.001 - 0.1% preferably 0.005 - 0.05%

H ₃ BO ₃ 0.005 - 0.01% preferably 0.002 - 0.0075%

(NH ₄ ) 6 Mo ₇ O ₂₄ x 4 H ₂ O 0.0005 - 0.01% preferably 0.001 - 0.005%

ZnSO ₄ x 7 H ₂ 0 0.001 - 0.1% preferably 0.001 - 0.05%

The cultivation is carried out aerobically, for example submerged with shaking or stirring or in fermenters, if appropriate with the introduction of air or hydrogen. It will be carried out in a temperature range from 15 to 30 ° C, preferably from 20 to 30 ° C, in particular from 23 to 28 ° C. The pH is from pH 1 to 7, advantageously from pH 2.5 to 4.5. The fungus is cultivated under these conditions over a period of 100 to 300 hours, preferably 120 to 168 hours.

Cultivation is advantageously carried out in several stages, i. H. one or more precultures are first prepared in a liquid nutrient medium, which are then inoculated into the actual production medium, the main culture, for example in a volume ratio of 1:10. The preculture is obtained e.g. B. by inoculating mycelium in a nutrient solution and growing for about 100 to 200 hours, preferably 120 to 168 hours. The mycelium can be obtained, for example, by allowing the strain to grow on a solid or liquid nutrient medium, for example yeast-malt agar or potato-dextrose agar, for 7 to 40 days, preferably 10 to 20 days.

The course of the fermentation can each be based on the pH of the culture or the mycelium volume as well as by chromatographic methods, such as. B. thin layer chromatography or high pressure liquid chromatography, are monitored. The compound of formula I is predominantly contained in the culture filtrate. The isolation of the formal I compound from the respective culture medium is carried out according to known methods, taking into account the chemical, physical and biological properties of the products. It can e.g. B. the adsorption, ion exchange or gel filtration process can be applied.

Ethyl acetate / methanol / water mixtures are used as mobile solvents in concentrations known to those skilled in the art. Detection in thin-layer chromatographic separation can, for example, also bring UV quenching and coloring reagents such as anisaldehyde, tetraozole blue or orcine into the suitable administration form and / or additives and / or auxiliary substances.

The invention is further explained in the following examples. Percentages relate to the weight, mixing ratios for liquids relate to the volume if no other information has been given.

Hericenals can be found in both the mycelium and the culture filtrate, usually the majority is in the culture filtrate. It is therefore advisable to separate the cell mass from the filtrate by filtration or centrifugation. The filtrate is lyophilized and the lyophilisate extracted with a solvent such as methanol or acetonitrii or 1-butanol. Hericenals can be extracted from the mycelium in the same way. The extractions can be carried out over a wide pH range, a range from pH 3.0 to pH 7.5 is appropriate.

Another method of isolation is the solution distribution in a manner known per se. Another method of purification is chromatography on adsorption resins such as on Diaion * HP-20 (Mitsubishi Casei Corp., Tokyo) on Amberlite ^® XAD 7 (Rohm and Haas, USA) to Amber Chrome ^® CG (Toso Haas, Philadelphia, USA) or on similar supports. Numerous reversed-phase supports, for example RP-18, are also suitable, as are generally used in hop pressure liquid chromatography. Another cleaning option for the Hericenale is the use of so-called straight-phase carriers such. B. silica gel or aluminum oxide, or other in a conventional manner. Many solvents, such as chloroform / methanol or dichloromethane / n-butanol mixtures, are suitable for elution.

An alternative method for isolating the texenomycins is the use of so-called. Molecular sieves such as Fractogel ^® TSK HW-40, Sephadex ^® LH-20 and others in a known manner. It is also possible to obtain the Texenomycins from enriched material by crystallization. Suitable for this purpose are, for example, organic solvents and their mixtures, anhydrous or with added water. Additions of acids such as trifluoroacetic acid, sulfuric acid, hydrochloric acid, formic acid or others can also be advantageous.

The compounds of formula I and their physiologically tolerable salts are also suitable as therapeutic agents against diseases which are caused by an HIV infection. Accordingly, the present invention also relates to the compound of formula I or a physiologically tolerable salt thereof for use as a medicament, preferably for the manufacture of a medicament for the treatment of a disease which is caused by an HIV infection. The medicaments can contain one or more compounds of the formula I or their physiologically tolerable salts and pharmaceutical auxiliaries.

The present invention further relates to Hericeum erinaceus DSM 10600 and its mutants and variants.

Examples

1. a) Production of mycelium from the producer strain H. erinaceus DSM 10600

100 ml nutrient solution (20 g malt extract, 2 g yeast extract, 10 g glucose, 0.5 g (NH ₄ ) ₂ HPO ₄ in 1 l tap water, pH value before sterilization 6.0) in a 500 ml sterile Erlenmeyer flask are mixed with Inoculated the strain DSM 7427 and incubated for 240 hours at 25 ° C and 140 rpm on a rotating shaker. Subsequently, 20 ml culture liquid in a sterile 500 ml Erlenmeyer flask with the nutrient medium [potato infusion 4.0 g / 1 (infusion from 200 g potato in 1000 ml H ₂ O), 20.0 g D-glucose, pH before sterilization 5, 6] to which an additional 15 g agar / l was added for solidification, evenly distributed and decanted. The cultures are incubated at 25 ° C for 10 to 21 days. The mycelium of a flask that was formed after this time is removed with a sterile inoculation needle, used immediately or kept in water at - 4 ° C.

b) Preparation of a preculture of the producer strain

A sterile 500 ml Erlenmeyer flask with 100 ml of the nutrient solution described under a) is inoculated with a culture grown on a slant tube or with a 1 square centimeter piece of agar and incubated on a shaker at 140 rpm and 25 ° C. The maximum production a compound of formula I is reached after about 168 hours. A 1 68 hour old submerged culture (inoculation amount approx. 5%) from the same nutrient solution is sufficient to inoculate 4 (500 ml in 200 ml Erlenmeyer).

2. Preparation of the compounds of the formulas 1 and 2

A 2000 ml Erlenmeyer shake flask is operated under the following conditions:

5 g / l corn steep 10 g / 1 glucose

4 g / l tomato paste 10 ml / 1 trace element solution

10 g / l oatmeal pH 6.8 (before sterilization)

The pH is adjusted with aqueous 2 N NaOH or HCl.

Incubation time: 168 hours

Incubation temperature: 25 ° C Shaking speed: 140 rpm

Trace element solution

Composition: Quantity in percent

FeSo ^ H ₂ O 0.100

MnSO ₄ _1 H ₂ O 0.100

CuCI ₂ _2 H ₂ O 0.0025

CaCl ₂ _2 H ₂ O 0.010

H3BO ₃ 0.0056

(NH ₄ ) 6Mo7024_4 H ₂ O 0.0019

ZnSO ₄ _7 H ₂ O 0.020 3. Isolation of the Hericenale

The culture solution obtained according to Example 2 is centrifuged off and the cell mass is freeze-dried. The clear culture filtrate is adsorbed on Mitsubishi Diaion ^® CHP20 (column dimensions 30 x 6 cm) and eluted with an isopropanol gradient 0 - 70% isopropanol (flow 50 ml / min). The fractions containing the Hericenale are combined, concentrated to the water phase in vacuo and lyophilized.

4. Cleaning the Hericenale

270 mg of the crude mixture obtained in Example 3 are dissolved in 20% aqueous Acetonitrii and initially on a column of Nucleosil 100-12 ^® C ₁₈ with a gradient 25-40% Acetonitrii in water with 0.05% trifluoroacetic acid (column dimensions 250 x 32 mm, Flow 50 ml / min) chromatographed. The fractions containing the Hericenale are pooled, concentrated in vacuo and Nucleosil 100-10 ^® C ₁₈ AB with the same gradient cleaned on (column dimensions 250 x 20 mm, flow 25 ml / min). After concentration of the fractions and lyophilization, 17.6 mg of a mixture of Hercenal A and Hericenal B are obtained. The mixture is obtained by column chromatography on silica gel 60 (Merck, 0.015-0.040 mm, column volume 5 ml) with dichloromethane / n-butanol 20: 1 as Eluens separated into the individual components.

INTERNATIONAL FORM

Hoechst AG

Central pharmaceutical research

Building H 780

65926 Frankfurt / Main RECEIPT CONFIRMATION FOR THE FIRST DEPOSIT, issued in accordance with Rule 7.1 by the INTERNATIONAL DEPOSIT AGENT specified below

I. LABELING OF THE MICROORGANISM

Reference number assigned by the DEPOSIT: INPUT NUMBER assigned by the INTERNATIONAL DEPOSIT:

HAG 000 951

DSM 10600

II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC NAME

With the microorganism designated under I.

() a scientific description

(X) submitted a proposed taxonomic name (check where applicable).

III. ENTRANCE AND ACCEPTANCE

This international depository accepts the microorganism referred to under 1, which it received on 1996-03-20 (date of first deposit) ¹ .

IV. RECEIPT FOR CONVERSION

The microorganism referred to under I was received by this international depository on (date of first deposit) and an application for conversion of this first deposit including a deposit in accordance with the Budapest Treaty was received on (date of receipt of the application for conversion).

V. INTERNATIONAL DEPOSIT

Name: DSMZ-GERMAN COLLECTION OF SIGNATURES) to represent the international depository

MIKROORGANISMEN UND ZELLKULTUREN GmbH authorized person (s) or the employee (s) authorized by them:

Address Mascheroder Weg lb D-38124 Braunschweig L>. UJ ^ ύ *

Date: 1996-03-26

'If Rule 64 (d) applies, this is the time at which the status of an internal depository was acquired Form DSMZ-BP / 4 (single page) 0196 INTERNATIONAL FORM

Hoechst AG

Central pharmaceutical research

Building H 780

65926 Frankfurt / Main LIFE QUALITY CERTIFICATE issued in accordance with Rule 102 of the below

INTERNATIONAL DEPOSIT

I. DEPOSIT II. LABELING OF THE MICROORGANISM

Name: Hoechst AG From the INTERNATIONAL DEPOSIT

Central Pharma Research assigned INPUT NUMBER: Address: Building H 780 DSM 10600

65926 Frankfurt / Main Date of deposit or transfer ': 1996 - 03 - 20

in. VITALITY CERTIFICATE

The viability of the microorganism mentioned under II was checked on 1996 - 03 - 20 ^* . At that time the microorganism was

(X) ¹ viable

() 'no longer viable

(V. CONDITIONS UNDER WHICH VIABILITY EXAMINATION HAS BEEN CARRIED OUT ^*

V. INTERNATIONAL ACCOUNTING OFFICE

Name: DSMZ-GERMAN COLLECTION OF Signature (s) to represent the international depository

MIKROORGANISMEN UND ZELLKULTUREN GmbH authorized person (s) or the authorized person (s):

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 1996 - 03-26

Indication of the date of the first deposit. If a new deposit or a transfer has been made, the date of the last new deposit or transfer is given.

In the cases provided for in Regie 102 (a) (ii) and (iii), the most recent viability test.

Tick the appropriate.

Complete if the information has been requested and if the test results are negative.

Form DSMZ-BP / 9 (single page) 0196

Claims

Claims: 1. Compound of formula I.

or a physiologically acceptable salt thereof, in which R ¹ to R ^{10 are} hydrogen or methyl, where two R ¹ to R ¹⁰ located on adjacent carbon atoms together with a CC single bond can represent a CC double bond, with the proviso that in the ¹ to R ¹⁰ substituted carbon chain in each case only one of the substituents R ¹ to R ¹⁰ is methyl with R, and only one adjacent pair of these substituents contributing to a carbon-carbon double bond. Compound according to claim 1, characterized in that R ^{5 is} methyl. Compound according to Claim 2, characterized in that R ³ and R ⁶ together with the CC single bond located between the carbon atoms they substitute mean a CC double bond. 4. A compound according to claim 3, characterized in that the double bond is in the Z configuration. 5. A compound according to claim 2, characterized in that R ⁶ and R ⁷ together with the CC single bond between the carbon atoms substituted by them mean a CC double bond. 6. A compound according to claim 5, characterized in that the double bond is in the Z configuration. 7. A method for producing a compound according to any one of claims 1 to 6, characterized in that the microorganism Hericeum erinaceus is cultivated in an aqueous medium and the target compound is then isolated and purified. 8. The method according to claim 7, characterized in that Hericeum erinaceus DSM 10600 is used. 9. The method according to claim 8, characterized in that mutants or variants of the microorganism Hericeum erinaceus DSM 10600 are used. 10. A compound or a physiologically acceptable salt thereof according to any one of claims 1 to 6 for use as a medicament. 11. Use of a compound according to any one of claims 1 to 6 or a physiologically acceptable salt thereof for the manufacture of a medicament for the treatment of a disease which is caused by an HIV infection. 12. Medicament containing at least one compound according to one of claims 1 to 6 or a physiologically acceptable salt thereof and pharmaceutical auxiliaries. 13. Hericeum erinaceus DSM 10600 and its mutants and variants.