HK1098131A1 - Bengamide derivatives, method for the production thereof and use thereof for the treatment of cancer - Google Patents

Abstract

Preparation of bengamide compounds (3-alkenoylamino-hexahydroazepin-2-one derivatives) (A) involves fermenting Myxococcus virescens ST200611 (DSM 15898) (or its variants and/or mutants). Some of the bengamide derivatives (A) (i.e. 3-(2-methoxy-8-methyl-3,4,5-trihydroxydec-6E-enoylamino-hexahydroazepin-2-ones) are new. Preparation of bengamide compounds of formula (A) or their salts involves fermenting Myxococcus virescens ST200611 (DSM 15898) (or its variants and/or mutants) in a culture medium, isolating the obtained (A) from the culture medium and optionally derivatizing and/or salifying the product. [Image] R 1H or 1-6C alkyl; R 2H or OH; R 3H or 2-7C alkanoyl; R 4Me or Et. Independent claims are included for: (1) bengamide derivatives of formula (B) (this is structure (A) where R 4 = Et) and their salts as new compounds; (2) the new microorganism Myxococcus virescens ST200611 (DSM 15898); and (3) the use of Myxococcus virescens ST200611 (DSM 15898) (or its variants and/or mutants) for the production of (A). ACTIVITY : Cytostatic. A specific diastereomer of 3-(2-methoxy-8-methyl-3,4,5-trihydroxydec-6E-enoylamino)-hexahydroazepin-2-one (A1) had TC 5 0 values of 6 mu M and 10 mu M for inhibition of proliferation of Hep-G2 and Colo 205 cells respectively. MECHANISM OF ACTION : Mitosis inhibitor; Cell cycle regulator.

Description

Pumamide derivative, preparation method thereof and application thereof in treating cancer

Cancer is one of the diseases of humans and animals, which is mostly fatal and is caused by uncontrolled growth of endogenous cells. Cancer is the term used to describe the malignancy (malignomas) and neoplasia (tumor or cancer) or malignant degeneration and disturbed maturation of white blood cells (leukemia, blood cancer). Cancer cells or tumor cells are generated by transformation of endogenous cells. The malignancy of a cancer cell is manifested by its autonomy of growth, i.e., the cell grows in an uninhibited and incompatible manner with the organ structure, and also grows in an invasive manner, thereby causing tissue destruction. After the tumor cells have spread through the blood or lymph, they form scattered areas (metastases) at a certain distance from the tumor, which is a definite signal for the appearance of malignant tumors. Cancer is one of the most common causes of death in humans, and thus, there is a particular need for methods and means to eliminate or treat malignant degeneration in humans.

In addition to the fundamental treatment of resection by surgery, if possible, other means that can be used to treat malignancies include radiation therapy using X-rays, alpha-rays, beta-rays and gamma-rays, as well as immunotherapy and chemotherapy. Currently, immunotherapy is very limited in its application. Tumor chemotherapy refers to the administration of cytotoxic drugs (cytostatic agents) to treat tumors and tumor cells that are still present, usually after local surgical treatment or irradiation. These drugs can specifically interfere with certain processes of cell division, that is to say tissues in which a high proportion of dividing cells are present in the body, for example rapidly growing tumour tissues, which can respond more sensitively. Cytostatic agents used are alkylating compounds (e.g. cyclophosphamide), antimetabolites (e.g. methotrexate), alkaloids (e.g. vincristine), and antibiotics (e.g. daunorubicin and doxorubicin). However, all of these drugs have the serious drawback of having a large number of side effects, so that they can only delay and cannot prevent the death of the patient. In addition, the degenerated (cancerous) cells develop resistance to the drugs used; the drugs used at present no longer have any cytostatic effect, and are toxic to the patient due to their side effects. It has also been found that the combined or sequential use of multiple cytostatics provides a greater effect than that achieved by the use of a single cytostatic agent (monotherapy), and that the serious side effects associated with multiple chemotherapeutic agents may not be additive. For all these reasons, there is an urgent need for new chemotherapeutic agents, and as such, there is a strong search for such chemotherapeutic agents worldwide.

Examples of the first class of pumpamides (bengamides) are lauroyl-substituted pumpamides a and B on the caprolactam ring, isolated from the sponge (sea sponge) jasps cf. coiaceae, Choristida B artophorida order (Adamczewski et al, journal of organic chemistry (j. org. chem.)1986, 51, 4497-4498), and reported to be biologically toxic to eukaryotic cells, nematodes and bacteria.

It has been shown that the formula Paamad E

And its N-methylated derivative, Pagaserod F, are examples of Pagaserod derivatives having antitumor activity. Pumad E inhibits cell proliferation by halting cell division at the G1/S restriction site and G2/M phase of the cell cycle. The pumpamide B derivative can inhibit MDA-MB-435 breast cancer cell proliferation (Kinder et al, J.Med.chem. 2001, 44, 3692-.

It is known that a common feature of the derivatives of pumparound is that they are isolated from sponges of the genus Jaspis sp or Pahastissa sp (Thale et al, J. org. chem. 2001, 66, 1733-1741).

It has now been found that the microbial strain Myxococcus virescens ST200611(DSM15898) is capable of producing novel derivatives of pumparound which inhibit cell proliferation at low concentrations and are therefore suitable for the treatment and/or prophylaxis of cancer diseases.

The present invention therefore relates to a compound of the formula (I) or a physiologically tolerable salt of a compound of the formula (I):

in the formula:

R₁is H or (C)₁-C₆) -an alkyl group,

R₂is H or OH, and

R₃is H or-C (═ O) - (C)₁-C₆) -an alkyl group.

Independently of one another, R₁Preferably a hydrogen atom or a methyl group, or a salt thereof,and R is₃Preferably H.

Preferably the present invention relates to compounds of formula (I) as defined below, wherein:

R₁is a group selected from the group consisting of H and methyl,

R₂is H or OH, and

R₃is H.

(C₁-C₆) Alkyl means a straight or branched alkyl group having up to 6 carbon atoms, such as methyl (Me), ethyl, n-propyl, isopropyl, tert-butyl or n-hexyl, preferably methyl.

Furthermore, the present invention relates to a compound of formula (I), which can be characterized by the following formula (II), the following formula (III), the following formula (IV):

the invention also relates to all the obvious chemical equivalents of the compounds of formula (I) according to the invention. These chemical equivalents show only minor chemical differences and they have the same pharmacological effect or they can be converted under mild conditions into the compounds of the invention. Said equivalents also include, for example, salts, reduced products, oxidized products, esters, ethers, acetals or amides of the compounds of formula (I) and equivalents which can be prepared by a person skilled in the art using standard methods and, in addition, include all optical enantiomers and diastereomers and all stereoisomeric forms.

The invention also relates to a method for producing a compound of the formula (V) below or a physiologically tolerated salt of the compound of the formula (V):

in the formula:

R₁is H or (C)₁-C₆) -an alkyl group,

R₂is a compound of formula (I) which is H or OH,

R₃is H or-C (═ O) - (C)₁-C₆) -alkyl, and

R₄is a methyl group or an ethyl group,

the method comprises the following steps:

1. fermenting the strain Myxococcus virens ST200611(DSM15898) or one of its variants and/or mutants in a culture medium under suitable conditions until one or more compounds of formula (V) naturally grow in the culture medium,

2. isolating the compound of formula (V) from the culture medium, and

3. if appropriate, the compounds of the formula (V) are derivatized and/or converted into physiologically tolerable salts.

Preferably the present invention relates to the preparation of compounds wherein R₄A process for preparing a compound of formula (V) which is ethyl. The product of this process corresponds to the compound of formula (I) as described previously.

Particularly preferred the invention relates to a process for the preparation of compounds of the formula (V) in which R are independently of one another₁Is H or methyl, R₃Is H, and R₄Is ethyl.

Furthermore, the present invention relates to processes for the preparation of compounds of formula (II), compounds of formula (III) and compounds of formula (IV) as well as the pumpamide derivatives E and F.

Unless otherwise indicated, the chiral centers of the compounds of formula (I) and (V) may be in the R configuration or in the S configuration. The present invention relates to optically pure compounds and stereoisomeric mixtures, such as enantiomeric mixtures and diastereomeric mixtures.

Physiologically tolerated salts of the compounds of the formulae (I) and (V) are to be understood as meaning their organic and inorganic salts, as described in Remington's Pharmaceutical Sciences (17 th edition, p. 1418 (1985)). For reasons of better physical and chemical stability and higher solubility, sodium, potassium, calcium and ammonium salts are preferred for compounds containing acid groups; for compounds containing a basic group, salts of hydrochloric acid, sulfuric acid or phosphoric acid, or salts of carboxylic acids or sulfonic acids, such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid, are preferred.

The culture medium is a nutrient solution or a solid medium which contains at least one customary carbon and nitrogen source and customary inorganic salts. If hydroxylysine is added to the culture medium, the strain Myxococcus virens ST200611(DSM15898) is able to produce a strain in which R is present due to the digestion of hydroxylysine₂Compound (V) which is OH.

Thus, part of the subject matter of the present invention relates to the preparation of compounds as described above in which R is₂A process for the preparation of a compound of formula (V) which is OH and wherein the culture medium of step 1 contains hydroxylysine.

The process of the invention can be used for both laboratory scale (milliliter to liter scale) fermentations as well as industrial scale (cubic meter scale) fermentations.

Suitable carbon sources for the fermentation are assimilable carbohydrates and sugar alcohols, such as glucose, lactose, sucrose or D-mannitol, and also carbohydrate-containing natural products, such as wort or yeast extract. Examples of nitrogen-containing nutrients are amino acids, peptides and proteins, but also degradation products thereof, such as casein, peptone or tryptone; meat extract, yeast extract, gluten, ground seeds, such as seeds of corn, wheat, beans, soybeans, or cotton plants; distillation residue from the production of alcohol; meat meal; yeast extract, ammonium salt, nitrate. Preferred nitrogen sources are one or more peptides, which may be obtained synthetically or biosynthetically. Examples of inorganic salts are chlorides, carbonates, sulfates or phosphates of alkali metals, alkaline earth metals, iron, zinc, cobalt and manganese. Examples of trace elements are cobalt and manganese.

Suitable conditions for preparing the inventive pumpamides are as follows: the pumamiad of the invention is preferably produced in a culture medium containing 0.05-5%, preferably 0.1-2.5% yeast extract; 0.2-5.0%, preferably 0.1-2% tyrose peptone; 0.02-1.0%, preferably 0.05-0.5% CaCl₂·2H₂O; 0.02-1.5%, preferably 0.05-0.7% MgSO₄·7H₂O and 0.00001-0.001% cyanovitamin B₁₂. The percentage values given in each case are based on the total weight of the total nutrient solution.

The microorganisms are cultured aerobically, i.e.for example by submerged cultivation with shaking or stirring in shaker flasks or fermenters, or aerobically in a solid medium, if appropriate with introduction of air or oxygen. The microorganisms can be cultured at temperatures of about 18 to 35 ℃, preferably 20 to 32 ℃ and particularly preferably 27 to 30 ℃. The pH can be in the range from 4 to 10, preferably from 6.5 to 7.5. In general, the time for culturing the microorganism under these conditions is 2 to 10 days, preferably 72 to 168 hours. It is advantageous to culture the microorganisms in several steps, i.e.initially to prepare one or more preliminary cultures from a liquid nutrient medium and then to inoculate these preliminary cultures into the actual production medium, i.e.the main culture, for example in a ratio of 1: 10 to 1: 100 by volume. For example, a strain in the form of vegetative cells or fruit bodies is inoculated into a nutrient solution and allowed to grow for about 20 to 120 hours, preferably 48 to 96 hours, thereby obtaining a preliminary culture. For example, vegetative cells and/or fruit bodies can be obtained by growing the strain on a solid or liquid nutrient substrate, such as yeast agar, for about 1 to 15 days, preferably 4 to 10 days.

From the culture medium, a pumpamide derivative of formula (V) can be isolated or purified using known methods and taking into account the chemical, physical and biological properties of the natural substance. The concentration of the pumpamide derivative in the culture medium or in the individual separation steps is determined by HPLC, while the amount of the substance formed is expediently compared with a calibration solution.

Upon isolation, the culture broth or culture is freeze-dried together with a solid medium, after which the pumparound derivative is extracted from the lyophilizate using an organic solvent, such as methanol or 2-propanol. The organic solvent phase contains the natural substance of the present invention; where appropriate, it is concentrated in vacuo and then subjected to further purification.

One or more compounds of the invention are further purified by chromatography on suitable materials, preferably, for example, molecular sieves, silica gel, alumina, ion exchangers or adsorbent resins or reverse phase chromatography (RPs). The pump-amomidide derivative is isolated by this chromatography. These pumpamide derivatives are chromatographically separated using an aqueous buffer solution or a mixture of aqueous and organic solutions.

The mixture of aqueous solution and organic solution is understood to be any water-miscible organic solvent, preferably methanol, 2-propanol or acetonitrile, the concentration of which is 5 to 95%, preferably 5 to 40%, or any aqueous buffer solution which is miscible with organic solvents. The buffer used was the same as indicated previously.

Based on their different polarity, reverse-phase chromatography may be used, for example using(adsorption resin of Mitsubishi, Japan) or Amberlite(TOSOHMS), or other hydrophobic materials, such as RP-8 or RP-18 phases. In addition, such separation can be carried out by normal phase chromatography, for example, using silica gel, alumina, or the like.

These pumpamide derivatives are chromatographically separated using buffered alkaline or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other water-miscible organic solvents. It is preferred to use acetonitrile and methanol as organic solvents.

Buffered alkaline or acidified aqueous solutions are understood to be, for example, water, phosphate buffer, ammonium acetate and citrate buffer, in concentrations of up to 0.5M, and formic acid, acetic acid, trifluoroacetic acid, ammonia and triethylamine, or commercially available acids and bases known to the person skilled in the art, preferably in concentrations of up to 1%. In the case of an aqueous buffer solution, 0.1% ammonium acetate is particularly preferred.

The chromatographic separation is carried out using a gradient starting with 100% water and ending with 100% solvent, preferably a linear gradient from 5% to 95% acetonitrile.

Alternatively, gel chromatography or chromatography of the hydrophobic phase may be performed. Gel chromatography is carried out using polyacrylamide or copolymer gels, e.g. Biogel-P(Biorad) or FractogelTSK(Merck, Germany). The order of the chromatographic separation steps described above may be reversed.

If the pumpamides are present as diastereomers, they can be separated by known methods, for example, by chiral column separation.

By methods known per se (J.March, Advanced organic chemistry, John Wiley&Sons, 4 th edition, 1992), the compounds of the formulae (I) and/or (V) (R in each case)₃All H) side chain OH groups to give an acyl radical (in each case R)₄is-C (═ O) - (C)₁-C₆) -an alkyl group). For example Adamczeski et al, J.Am.chem.Soc. 1989, 111, 647-654 describe reaction with acetic anhydride to give a compound of formula (I) and/or (V), wherein R is₃is-C (═ O) -CH₃。

Likewise, methods known per se (J.March, advanced organic chemistry, John Wiley)&Sons, 4 th edition, 1992), for example by reaction with Me₂CO₃Or Me₂SO₄By reaction with (C) in the presence of a base to prepare the corresponding N-methylated derivatives₁-C₆) Reaction of alkyl bromides with compounds of the formulae (I) and/or (V) (in each case R)₁Is H) is alkylated with the NH groups in the caprolactam.

The isolate of the microorganism strain Myxococcus virens ST200611 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Germany under the provisions of the Budapest treaty at 11/9, and was deposited under the number DSM15898, Mascheroder Weg 1B, 38124 Braunschweig.

The vegetative cells of strain DSM15898 are rod-shaped, a characteristic of Myxococcus virescens. On a solid nutrient substrate, myxococcus virescens ST200611(DSM15898) forms orange-yellow fruiting bodies containing round myxospores.

In addition to the strain Myxococcus virens ST200611(DSM15898), it is also possible to use mutants and/or variants of this strain which are capable of synthesizing one or more compounds according to the invention.

A mutant is a microorganism in which one or more genes in the genome have been modified by a gene or genes that determine the production of the compounds of the invention by the organism, while still maintaining functionality and heritability.

Such mutants can be produced in a known manner by physical methods, for example irradiation, such as with ultraviolet radiation or X-rays, or by chemical mutagens, for example Ethyl Methanesulfonate (EMS), 2-hydroxy-4-Methoxybenzophenone (MOB) or N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), or as described by Brock et al in microbiology (Biology of Microorganissm), Prentice Hall, pp.238-247 (1984).

The variant is a phenotype of said microorganism. Microorganisms have the ability to adapt to the environment and therefore have a high physiological plasticity for development. All cells of a microorganism are involved in this phenotypical adaptation, whose altered properties are not genetically regulated under altered conditions, but are reversible (H.Stolp, Microbial ecology: organisms, growth environment, vitality (Microbial biology: organismus, halotats, activites), Cambridge university Press, Cambridge, GB, page 180, 1988).

Mutants and/or variants which synthesize one or more compounds of the invention are selected according to the following scheme:

-freeze-drying the fermentation medium;

-extracting the seed lyophilizate with an organic solvent;

-extracting the compound from the culture filtrate using a solid phase;

analysis by HPLC, TLC or by assay for biological activity.

The described fermentation conditions can be applied to myxococcus virens ST200611(DSM15898) and to mutants and/or variants thereof.

The invention also relates to the use of the microorganism myxococcus virens ST200611(DSM15898) or a mutant and/or variant thereof for the preparation of a compound of formula (V), in particular a compound of formula (IV) or a physiologically acceptable salt thereof as described before.

Inhibition of cell proliferation was tested using assays based on measuring intracellular ATP concentrations. Known tumor cell lines, such as Hep-G2 and Colo205, can be used. In this assay, the ATP content of metabolically active cells was used as a measure of the number of viable cells in the luciferase reaction.

The single doses of the compounds of formulae (II) - (VI) used in this assay were 0.3-40. mu.M, as TC₅₀The values give a dose dependence, in each case (IIA) and (IIB) representing a diastereomer of the compound of formula (II):

the invention therefore also relates to the use of a compound of the formula (I) or a physiologically acceptable salt thereof as a human or animal medicament, in particular in the treatment and/or prophylaxis of cancerous diseases. Preferably the present invention relates to the use of a compound of formula (I) or a physiologically acceptable salt thereof for the treatment of breast, bowel, stomach, liver, brain, ovarian, esophageal, kidney and muscle cell tumors, in particular head and neck muscle cancer.

Furthermore, the invention relates to a medicament containing at least one compound of the formula (I) or a physiologically tolerable salt thereof, it being possible for one or more compounds of the formula (I) to be administered per se, but preferably in the form of a mixture with one or more generally pharmacologically suitable carrier substances or auxiliary substances.

The compounds of the invention are stable in the solid state and also in solutions at a pH of 2 to 9, in particular 5 to 7, and can therefore be formulated as conventional galenic preparations.

Although the medicaments according to the invention can be administered orally or parenterally, rectal administration is also possible in principle. Examples of suitable solid or liquid galenical forms are granules, powders, tablets, sugar-coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, aerosols or ampoules, and also preparations of the active compounds with a slow release, in the preparation of which pharmacologically suitable carrier substances or auxiliary substances, such as disintegrants, binders, coatings, bulking agents, glidants, lubricants, flavorings, sweeteners or stabilizers, such as magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starches, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycols and polyols, can be used.

If desired, the oral dosage units may be microencapsulated to provide delayed release or extended reach of a relatively long duration, e.g., by coating or embedding the active compound in a suitable polymer, wax, or the like.

Pharmaceutical preparations of dosage units, each unit containing a defined dose of one or more of the compounds of formula (I) as active ingredient, are preferably manufactured and administered. In the case of solid dosage units, such as tablets, capsules and suppositories, the dosage may be up to about 500mg, preferably 0.1 to 200mg per day, and in the case of ampoule injections, the dosage may be up to about 200mg, but preferably about 0.5 to 100mg per day.

The daily dose administered depends on the body weight, age, sex and condition of the mammalian subject. However, it will be appreciated that higher or lower daily doses may also be suitable. Such daily doses may be administered as a single dosage unit or divided into several smaller dosage units and the sub-doses divided into multiple administrations at defined intervals.

The medicaments according to the invention can be produced by adding one or more compounds of the formula (1) according to the invention to a suitable administration form, optionally with the addition of one or more customary carrier substances or auxiliary substances.

The following examples serve to explain the invention in more detail, but without restricting the scope of protection of the invention in any way.

Unless otherwise indicated, the percentage values refer to weight percentages, while the mixing ratio in the case of liquids refers to volume mixing ratios.

Example 1: storage of Myxococcus viridis ST200611(DSM15898) at-135 ℃

On agar plates (1% of N.baker, 1% CaCl)₂·2H₂O, 20mM HEPES, 0.00005% cyanovitamin B₁₂1.5% agar, pH7.2) to the strain Myxococcus virens ST200611(DSM15898),and cultured at 30 ℃ for about 7 days. The cells in the surface culture were scraped from the agar surface using a sterile spatula and suspended in 0.5ml of tyrose peptone medium (1% tyrose peptone (Difco), 0.15% MgSO 2)₄·7H₂O, pH7.0) and stored at-135 ℃.

Example 2: preparation of a preliminary culture of Myxococcus viridis ST200611(DSM15898) in Erlenmeyer flasks

100ml of nutrient solution (1% of neobaker's yeast, 1% CaCl) in a 300ml sterile conical flask₂·2H₂O, 20mM HEPES, 0.00005% cyanovitamin B₁₂pH7.2) was inoculated with the strain Myxococcus virens ST200611(DSM15898), and the culture was cultured at 30 ℃ for 4 days on a rotary shaker at 180 rpm. A main culture was then prepared using 5ml of this preliminary culture.

Example 3: preparation of a liquid Main culture of Myxococcus viridis ST200611(DSM15898)

In a container containing 100ml of a nutrient solution (0.5% yeast extract, 0.5% casein peptone, 0.1% CaCl)₂·2H₂O、0.2％MgSO₄·7H₂O, 0.00005% cyanovitamin B₁₂pH 7.4) was inoculated with 5ml of the preliminary culture (see example 2) or on fresh agar plates (1% of Saccharomyces neobaker, 1% of CaCl)₂·2H₂O, 20mM HEPES, 0.00005% cyanovitamin B₁₂pH7.2, 1.5% agar), at 30 ℃ on a 180rpm shaker. The maximum amount of pumamed of the present invention is reached after 72-96 hours. A72-96 h old submerged culture (inoculum size about 5-10%) obtained from the same nutrient solution as described in example 2 was sufficient to inoculate a 10-200L fermenter.

Example 4: preparation of liquid Main culture of Myxococcus viridis ST200611(DSM15898) with simultaneous addition of hydroxylysine for production of Pumamid derivative (IV)

In a container containing 100ml of a nutrient solution (0.5% yeast extract, 0.5% casein peptone, 0.1% CaCl)₂·2H₂O、0.2％MgSO₄·7H₂O, 0.00005% cyanovitamin B₁₂1mM hydroxylysine, pH 7.4) into a 300ml sterile Erlenmeyer flask, 5ml of the preliminary culture of example 2 or on fresh agar plates (1% of neobaker's yeast, 1% of CaCl)₂·2H₂O, 20mM HEPES, 0.00005% cyanovitamin B₁₂pH7.2, 1.5% agar) were cultured at 30 ℃ on a shaker at 180 rpm. The maximum amount of the production of the pumpamide derivative (IV) is reached after 72-96 hours. Analysis was performed by HPLC-MS. 72-96 h old submerged cultures (inoculum size about 5-10%) obtained from the same nutrient solution as described in example 2 were sufficient to inoculate a 10-200L fermentor.

Example 5: preparation of a pumpamide derivative in a fermenter

The 10L and 30L fermentors were operated as follows:

inoculum about 5% to about 9%

Fermentation tank 30L 10L

Nutrient media see example 2

The culture temperature is 30 ℃ and 30 DEG C

Stirring speed 112rpm 150rpm

Aeration is carried out at 8L/min and 4L/min

pH adjustment pH 7.8 to pH 7.5 pH 8.1 to pH 7.5

pO₂Adjust nothing

Always using 10% KOH or 10% H respectively₂SO₄The pH is adjusted. Repeated addition of Clerol FBA265(Cognis Deutschland GmbH) can suppress foam formation. Reaches a maximum after about 72 to 96 hoursThe amount produced.

Example 6: isolation of the Pumamiad derivatives (II) and (III) and Pumamiad E and F from shaken culture of Myxococcus viridis ST200611(DSM15898)

After completion of fermentation with Myxococcus virescens ST200611(DSM15898), the culture of example 3(30L culture) was freeze-dried together with the cells, and the freeze-dried product was extracted with methanol (2X 5L). The methanol extract was concentrated to 1.2L under vacuum and then applied to a preparative column packed with about 1.5L of CHP-20P material (aGel, 75-150 μ, Mitsubishi Chemical Corporation). The column was eluted with 95% methanol. The column effluent (120ml/min) was collected and concentrated to 1.5L under vacuum.

Example 7: RP-18 chromatography is used for preparing the pump amide derivatives (II) and (III) and the pump amide E and F

1.5L of the solution obtained as described in example 6 was loaded onto Phenomenex10 μ C18(2) column (size: 50 mm. times.250 mm) with one column10 μ C18(2) front-end column (size: 21.2 mm. times.60 mm) and gradient elution was carried out for 60min using a solution of from 5% to 95% acetonitrile in water (0.1% ammonium acetate, pH4.6, pH adjusted with acetic acid). The flow rate was 150ml/min and the amount of fractions was 200 ml. There are pumparounds in fractions 5-9, 10-11 and 12-14.

Example 8: purification of the Pumilide derivatives (II) and (III) and Pumilide E and F the individual fractions from example 7 were freeze-dried and used with PhenomenexHPLC purification of a 10. mu.C 18(2) column (size: 21 mm. times.250 mm), which had one column, was repeatedPrep MS C1810 μm (Waters, size 19X 10mm) front column. The column was eluted over 40min with a gradient of 5% to 40% acetonitrile in water (0.1% ammonium acetate added, pH 8.8, pH adjusted with triethylamine). The column effluent (50ml/min) was collected in fractions of 7.5ml per fraction. Fractions 5-9 of example 7, containing the compound of formula (III), gave 86mg of pumpamide E (> 95% purity) after chromatographic separation and freeze-drying. After chromatographic separation, fractions 10-11 from example 7 gave 145mg of pumparound (II) (purity > 95%, 70/30 diastereomer mixture) and 5mg of pumparound F (purity > 95%). 35mg of pumparound (III) (purity > 95%) were obtained from fractions 12-14 of example 7 as a diastereoisomeric mixture in a ratio of 75: 25. In each case, the diastereomer is the corresponding C-16 epimer.

Example 9: isolation of Hydroxypump Amides (IV) from shaken culture of Myxococcus viridis ST200611(DSM15898)

After completion of fermentation with Myxococcus virescens ST200611(DSM15898), the culture of example 5(10L culture) was freeze-dried together with the cells, and the freeze-dried product was extracted with methanol (2X 3L). The methanol extract was concentrated to 400ml under vacuum and then applied to a preparative column packed with about 0.6 liter of CHP-20P (Gel, 75-150 μ, Mitsubishi Chemical Corporation). The column was eluted with 5% to 95% aqueous methanol. The column effluent was collected in fractions (100ml/min) over 60 min. Fractions containing the desired derivative (fractions 45-109) were pooled and concentrated to 700ml under vacuum.

Example 10: prepurification of hydroxy pump Amides (IV) by RP-18 chromatography

The solution of example 9 was loaded onto a Phenomenex10 μ C18(2) column (size: 50 mm. times.250 mm) with a Phenomenex10 μ C18(2) front-end column (size: 21.2 mm. times.60 mm), eluted with a gradient of from 5% to 40% acetonitrile in water (0.1% ammonium acetate, pH 8.8, pH adjusted with triethylamine) over 60 min. The flow rate was 150ml/min and the amount of distillate was 225 ml. Fraction 22 contains the desired pumpamide derivative.

Example 11: purification of hydroxyl Pump Amides (IV)

Fraction 22 from example 10 was freeze dried and further processed with PhenomenexHPLC of a 10. mu.C 18(2) column (size: 21 mm. times.250 mm) with a Waters column was purified once morePrep MS C1810 μm pre-column (size 19X 10 mm). The column was eluted with a gradient from 5% to 95% acetonitrile in water for 60 minutes (0.1% ammonium acetate, pH4.6, pH adjusted with acetic acid). The column effluent (50ml/min) was collected in fractions (7.5 ml of each fraction). The fractions containing pumamide (fractions 26-28) were combined, desalted and lyophilized. Thus, 7mg of pumamide (IV) were obtained as a mixture of diastereomers in a ratio of 75: 25.

Example 12: characterization of the compound of formula (II):

empirical formula C₁₈H₃₂N₂O₆

Molecular weight: 372.47

Mixture of diastereomers: 75: 25

Example 13: characterization of the compound of formula (III):

empirical formula C₁₉H₃₄N₂O₆

Molecular weight: 386.49

Mixture of diastereomers: 75: 25

Example 14: characterization of the compound of formula (IV):

empirical formula C₁₈H₃₂N₂O₇

Molecular weight: 388.46

Mixture of diastereomers: 75: 25

Example 15:

characterization of Pumamid E

Empirical formula C₁₇H₃₀N₂O₆

Molecular weight: 358.44

Example 16: characterization of Pumamid F

Empirical formula C₁₈H₃₂N₂O₆

Molecular weight: 372.47

Example 17: separation of diastereoisomers of Compound (II)

A chiral column (AD/H, Daicel, 20X 200mm, 0.5ml flow rate, mobile phase acetonitrile: methanol 4: 1+ 0.1% NH) was used₄Ac) the diastereoisomeric mixture of the compound of formula (II) of example 8 is isolated. An analytical AD/H column (Daicel) (4.6X 250mm, 30 ℃ C., mobile phase acetonitrile: methanol 4: 1+ 0.1% NH) was used₄Ac. Flow rate of 0.75ml, Rt peak 1: 9.9min, Rt Peak 2: 10.9min) to correct the optical purity.

Example 18:

cell proliferation assays for various tumor cell lines

Tumor cell lines Hep-G2(ATCC No. HB-8065) and COLO205 (ATCC No. CCL-222) were used to determine cell proliferation. The cell lines were each present at a rate of 1000 cells/well [ Hep-G2]And 3500 cells/well [ Colo205 ]]Inoculating into cell culture medium, and culturing at 37 deg.C with 5% CO₂The cells were incubated for 4 hours.

Medium of Hep-G2: dulbecco's modified Eagle's Medium/Ham's F12-mix (Gibco); NEAA (10%; non-essential amino acids, Gibco), sodium pyruvate (1%, Gibco), L-glutamine (1%, Gibco), fetal bovine serum (5%; PAA) ].

Media of COLO 205: RPMI 1640(Gibco), L-glutamine (1%, Gibco), HEPES (1%, Gibco), fetal bovine serum (10%; PAA).

After 4 hours, compounds (II), (III) and (IV) and pumpamides E and F were dissolved in DMSO/cell culture medium and added at various concentrations, and the mixtures were incubated at 37 ℃ with 5% CO₂And culturing for 72 hours. The intracellular ATP content was determined using the detection reagent CellTiterGlo (Promega).

The results of the cell proliferation assay are shown in Table 1.

Claims (10)

1. A compound of the following formula (I) or a physiologically tolerable salt thereof:

in the formula:

R₁is H or (C)₁-C₆) -an alkyl group,

R₂is H or OH, and

R₃is H or-C (═ O) - (C)₁-C₆) -an alkyl group,

2. the compound of claim 1, wherein R₁Is H or methyl.

3. The compound of claim 1, wherein R₃Is H.

4. A compound according to any one of claims 1-3, wherein R is₁Is H or methyl, R₂Is H or OH, and R₃Is H.

5. A compound according to any one of claims 1 to 3, characterised in that it is a compound of formula (II):

6. a compound according to any one of claims 1 to 3, characterised in that it is a compound of formula (III):

7. a compound according to any one of claims 1 to 3, characterised in that it is a compound of formula (IV):

8. use of a compound of formula (I) according to any one of claims 1 to 7 or a physiologically tolerable salt thereof in the manufacture of a medicament.

9. Use of a compound of formula (I) according to any one of claims 1 to 7 or a physiologically tolerable salt thereof for the production of a medicament for the treatment and/or prophylaxis of cancer diseases.

10. A medicament containing a compound of formula (I) according to any one of claims 1 to 7 or a physiologically tolerable salt thereof.