Bengamide derivatives, making process and application in cancer therapy

FIELD: chemistry.

SUBSTANCE: invention refers to bengamide derivatives produced by fermented microorganism Myxococcus virescens ST200611 (DSM 15898), to application in cancer therapy and/or prevention, to medical products containing bengamide derivatives, making process of bengamide of formula . In addition, the invention refers to compound of formula .

EFFECT: new bengamide derivatives are characterised with useful biological properties.

15 cl, 7 tbl, 18 ex

 

Cancer is a disease of man and animal, occurring most often fatal and is caused by the uncontrolled growth of homologous cells. Cancer called malignant tumors (malignoma), neoplasms (tumors, respectively carcinoma or malignant degeneration (malignancy), and impaired maturation of white blood cells (leukemia, blood cancer). Cancer or tumor cells arise from the conversion of homologous cells. Malignancy of a cancer cell is expressed in the autonomy of growth, i.e. its ability ingeltrude grow freely and randomly in terms of the structure of organs and the tissue destruction. Reliable sign of malignancy is the formation of further tumor metastezirovanii (metastases) due to hematogenous or lymphogenous spread of tumor cells. Cancer is one of the most frequent causes of mortality of man, and therefore there is a great need in the ways and means to cure or therapy of malignant tumors.

The opportunity the treatment of cancer includes, along with surgical removal of the tumor, if possible radical removal, radiation therapy using x-rays, α-, β-, γ-rays, immunotherapy and chemotherapy. Immunotherapy currently use the ima only to a limited extent. Under the chemotherapy of tumors see the introduction of cytotoxins (drugs) for the treatment of tumors and remained tumor cells often after local surgical therapy or radiation. These substances are specifically mediate certain processes of cytokinesis, so react more sensitive tissues with a high proportion of dividing cells, as, for example, rapidly growing tumor tissue. Use of alkylating compounds, such as cyclophosphamide, antimetabolites, as methotrexate, alkaloids like vincristine, and antibiotics, as daunomycin or adriamycin. All of these agents, however, because of significant side effects have big disadvantages, so that only the moves, but do not prevent death. Moreover, in the case of degenerated (cancerous) cells having resistance against related tools, then modern medicines do not apply more than cytotoxic, and are probably due to toxic side effects.

Also found that the combined or sequential use of drugs is superior to the performance of specific cytostatic agent (monotherapy), and therefore it is possible that significant side effects in chemotherapy are not cumulative. For all these reasons, it is highly necessary and why in the world from sbivautsa new chemotherapeutic agents.

As the first examples of benjaminov from the marine sponge Jaspis cf. Coriacea (family Coppatiidae, detachment Choristidia B Astrophorida) allocated dodekanision in caprolactame cycle benhamadi a and b (Adamczewski and others, J. Org. Chem.,51, 4497-4498 (1986)) and is described as biotoxin with respect to eukaryotic cells, nematodes and bacteria.

Examples of derivatives benhamed, in which case the detected antitumor activity are benhamed E

and its N-methylated derivative - benhamed F. Benhamed E inhibits cell proliferation due to the fact that stops cytokines in the restriction site G1/S and G2/M-phase of the cell cycle. Derivatives benhamed To inhibit cell proliferation in breast cancer MDA-MB-435 (Kinder and others, J. Med. Chem.,44, 3692-3699 (2001)).

Common to the known derivatives benhamed is that they are isolated from marine sponges of the genus Jaspis sp. or Pachastrissa sp (Thale, etc., J. Org. Chem.,66, 1733-1741 (2001)).

Currently, we discovered that the microorganism strain Myxococcus virescens ST200611 (German collection of microorganisms (DSM 15898) can produce a new kind of derivative benhamed, which in low concentrations inhibit cell proliferation and is therefore suitable for the treatment and/or prevention of cancer.

The present invention relates to joint the formula (I):

and

R1means H or (C1-C6)-alkyl;

R2means H or HE

R3means H or-C(=O)-(C1-C6)-alkyl;

or physiologically acceptable salts of the compounds of formula (I).

Independently from each other, R1means preferably H or methyl, and R3means preferably N.

The invention preferably relates to a compound of formula (I), where

R1means H or methyl;

R2means H or HE

R3means N;

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

Further, the invention relates to the compound of formula (I), characterized in that it is a compound of formula (II):

the compound of formula (III):

and the compound of formula (IV):

The present invention relates further to all the obvious chemical equivalents proposed according to the invention compounds of formula (I). Such equivalents are compounds that have a slight chemical difference and have the same pharmacological action or agcih conditions become proposed according to the invention compounds. To these equivalents include, for example, also salts, reduction products, oxidation products, esters, ethers, acetals or amides of compounds of formula (I), and equivalents, which can be obtained by standard methods, moreover, all the optical antipodes, diastereoisomers and all stereoisomeric forms.

The invention relates further to a method for obtaining compounds of formula (V):

and

R1means H or (C1-C6)-alkyl;

R2means H or HE;

R3means H or-C(=O)-(C1-C6)-alkyl; and

R4means methyl or ethyl;

or physiologically acceptable salts of the compounds of formula (V),

characterized in that

1) strain Myxococcus virescens ST200611 (DCM 15898) or one of its variants and/or mutants in suitable conditions are fermented in a culture medium until the accumulation of one or more of the compounds of the formula (V) in culture medium;

2) the compound of formula (V) was isolated from the culture medium;

3) the compound of formula (V), if necessary, derivatizing and/or, if necessary, converted into a physiologically acceptable salt.

Preferably the invention relates to a method for obtaining compounds of formula (V), and R4means ethyl. The product of such method corresponding to the compound of formula (I), as explained above.

Particularly preferably, the invention relates to compounds of formula (V), and, independently of one another, R1means H or methyl, R3mean N and R4means ethyl.

Further, the invention relates to a method for obtaining compounds of formula (II), the compounds of formula (III), compounds of formula (IV), as well as derivatives benhamed E and F.

Centers of chirality in the compounds of formulas (I) and (V), unless nothing else can be in the R - or S-configuration. The invention relates as to optically pure compounds, and also mixtures of stereoisomers, as a mixture of enantiomers and mixtures of diastereomers.

Under physiologically acceptable salts of the compounds of formulas (I) and (V) understand how their organic and inorganic salts, which are described in Remington''s Pharmaceutical Sciences (17th edition, S. 1418 (1985)). On the basis of physical and chemical stability and solubility for acid groups, among others, preferred salts are sodium, potassium, calcium and ammonium; for the main group, among others, preferred salts of hydrochloric acid, sulfuric acid, phosphoric acid or of carboxylic acids or sulfonic acids, such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluensulfonate.

Cultural cf the DOI is a nutrient solution or solid medium with at least one common source of carbon and nitrogen, as well as the usual inorganic salts. If culture medium add hydroxylysine, the strain of Myxococcus virescens ST200611 (DCM 15898) due to the restriction by hydroxylysine produces the compound of formula (V), where R2means IT.

The object of the present invention is also a method of obtaining the compounds of formula (V), as described above, and R2means HE, and cultural environment in stage 1 contains hydroxylysine.

Proposed according to the invention method can be used for fermentation at laboratory scale (scale from milliliters to liters) and in industrial scale (scale cubic meters).

As carbon sources for the fermentation of suitable assimilated carbohydrates and reduced sugar, such as glucose, lactose, sucrose or D-mannitol, as well as containing carbohydrates, natural products, such as malt extract or yeast extract.

Nitrogen-containing nutrients are, for example, amino acids; peptides and proteins, as well as the products of their degradation, such as casein, peptone or tripton; meat extracts; yeast extracts; gluten; milled seeds, such as corn, wheat, beans, soybean or cotton; residue after distillation in obtaining alcohol; meat fodder flour; ammonium salt; nitrates.

Preferably the source of AZ is that is one or more synthetic or biosynthetic derived peptides. Inorganic salts include, for example, chlorides, carbonates, sulfates or phosphates of alkali or alkaline earth metals, iron, zinc, cobalt and manganese. Trace elements are, for example, cobalt and manganese.

Suitable conditions for the formation of a proposed according to the invention benjaminov are the following: education proposed according to the invention benjaminov preferably occurs in the culture medium, which contains 0.05-5%, preferably 0.1 to 2.5% yeast extract; 0.2 to 5.0 percent, preferably 0.1 to 2% casitone; 0,02-1,0%, preferably of 0.05-0.5% CaCl2x 2 H2O; of 0.02 to 1.5%, preferably 0.05 to 0.7% of MgSO4x 7H2O, and also from 0.00001% to 0.001% of cyanocobalamin. Data in percent refer to the weight of the entire nutrient solution.

Cultivation of the microorganism aerobic exercise, such as submerged cultures with shaking or stirring in shake flasks or fermenters or on solid medium, if necessary, with the introduction of air or oxygen. The cultivation can be carried out in the temperature range from about 18°C to 35°C, preferably at from about 20°C to 32°C, in particular, at 27-30°C. the range of values of pH should be between 4 to 10, preferably from 6.5 to 7.5. The microorganism under these conditions, cultivate, in General, within lane is an ode to time from 2 days to 10 days, preferably from 72 hours to 168 hours. Preferably cultivated in several stages, i.e. first aqueous nutrient medium receive one or more preculture, which are then subcultured directly producing environment, the main culture, for example, in a volume ratio from 1:10 to 1:100. Preculture receive, for example, the fact that the strain in the form of vegetative cells or fruit bodies subcultured on nutrient solution and leave to rise for about 20-120 hours, preferably lasts for 48-96 hours. Vegetative cells and/or fruiting bodies can be obtained, for example, the fact that the strain within approximately 1-15 days, preferably 4-10 days, leaves grow on a solid or liquid nutrient medium, such as yeast agar.

Selection, respectively cleanup derived benhamed formula (V) of the culture medium carried out by known methods with respect to chemical, physical and biological properties of natural substances. To determine the concentration of the corresponding derivatives benhamed in culture medium or in the individual stages of the selection using high-performance liquid chromatography (HPLC), and the number of formed substances, it is advisable compared to the reference solution.

To highlight the culture broth or culture together with the solid medium leofiles the shape, then the derived benhamed extracted from freeze-dried using an organic solvent, such as methanol or 2-propanol. The organic phase solvent contains a proposed according to the invention a natural substance, and, if necessary, concentrated in vacuo and purified further.

Further purification of one or more compounds according to the invention is carried out by chromatography on suitable materials, preferably, for example, using molecular sieves, silica gel, alumina, ion exchangers or adsorbent resins, respectively reversed phase (RP). With this chromatography share derivatives benhamed. Chromatography derivatives benhamed carried out using a buffered aqueous solutions or mixtures of aqueous and organic solutions.

Under mixtures of aqueous or organic solutions understand all miscible with water and organic solvents, preferably methanol, 2-propanol and acetonitrile, at a concentration of from 5% to 95% of solvent, preferably from 5% to 40% solvent, or all buffered aqueous solutions, which are mixed with organic solvents. Used buffers are as specified above.

The separation of the derivatives benhamed on the basis of their different polarity is carried out by chromatogr is the philosophy with reversed phase (RP), for example, MCI®(adsorbing resin, Mitsubishi, Japan) or Amberlite XAD®(TOSOHAAS), or other hydrophobic materials, such as, for example, RP-8 or RP-18-phases. In addition, the separation can be performed using chromatography on normal phase, for example, on silica gel, aluminum oxide and the like.

Chromatography derivatives benhamed carried out using a buffered, alkaline or acidic aqueous solutions or mixtures of aqueous solutions with alcohols or other, miscible with water and organic solvents. As the organic solvent preferably used acetonitrile and methanol.

Under buffered, alkaline or acidic aqueous solutions, see, for example, water, phosphate buffer, ammonium acetate, citrate buffer, at a concentration of up to 0.5 M, as well as formic acid, acetic acid, triperoxonane acid, ammonia, triethylamine or all of the usual, well-known specialist acids and bases, preferably at a concentration of up to 1%. In the case of buffered aqueous solutions are especially preferred is 0.1%solution of ammonium acetate.

Chromatographic using a gradient starting with 100% water and ends with 100% of solvent, preferably using a linear gradient of 5-95% acetonitrile.

Alternatively, the implementation is by gel chromatography or chromatography on hydrophobic phases. Gel chromatography is carried out on polyacrylamide or copolymer gels, such as Biogel-P 2®(Biorad) or Fractogel TSK HW 40®(Merck, Germany). The sequence of the above chromatography is reversible.

If benhamadi are in the form of diastereomers, they can be divided by the known methods, for example by separation on a column with a chiral phase.

The derivatization Oh-groups in the side chain of the compounds of formula (I) or (V) (R3accordingly, means N) to the acyl group (R4accordingly, means-C(=O)-(C1-C6)-alkyl) carry out itself by known methods (J. March, Advanced Organic Chemistry, John Wiley and Sons, 4th edition, 1992), for example, by entering into interaction with the acid anhydride. Interaction with acetanhydride with obtaining the compounds of formula (I) or (V), where R3means-C(=O)=CH3described, for example, Adamczeski etc., J. Am. Chem. Soc.,111, 647-654 (1989).

Alkylation of the NH-group in caprolactame cycle of compounds of formula (I) or (V) (R1accordingly, means N) are also in itself known methods (J. March, Advanced Organic Chemistry, John Wiley and Sons, 4th edition, 1992), for example, by reaction with IU2CO3or IU2SO4to obtain the corresponding N-methylated derivatives, or by reaction with (C1-C6-alkylbromides in prisutstvie the base.

The isolate of the microorganism strain Myxococcus virescens ST200611 deposited in the German collection of microorganisms and cell cultures GmbH (DSMZ), Mascheroder Weg 1B, 38124 Braunschweig, Germany, under the provisions of the Budapest agreement dated 11.09.2003 under the following number: DSM 15898.

Vegetative cells of strain DSM 15898 are characteristic of Myxococcus virescens rod-like shape. On solid nutrient media Myxococcus virescens ST200611 (DSM 15898) forms an orange-yellow fruit bodies, which contain all mixo disputes.

Instead of strain Myxococcus virescens ST200611 (DSM 15898) you can also use its mutants and/or variants that synthesize one or more of the offer according to the invention compounds.

A mutant is an organism in which the modified one or more genes of the genome, and functionally saved or inherited a gene or genes that are responsible for the body's ability to produce proposed according to the invention the connection.

Such mutants can be obtained in itself known through physical means, for example irradiation, as irradiation with ultraviolet or x-rays, or chemical mutagens, such as ethylmethanesulfonate (EMS); 2-hydroxy-4-methoxybenzophenone (MOU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or as described by Brock and others in the book “Biology of Microorganism”, Prentice Hall c. 238-247 (1984).

Option is the phenotype of the organism. Microorganisms have the ability to adapt to their environment and therefore exhibit characteristic physiological flexibility. In the case of phenotypic adaptation in the process involved all the cells of the microorganism, and a different kind of change is not due to genetically and turn in the changed conditions (H. Stolp, Microbial ecology: organismus, habitats, activities. Cambridge University Press, Cambridge, UK, S. 180 (1988)).

Screening of mutants and/or variants that synthesize one or more proposed according to the invention compounds, carried out according to the following scheme:

- lyophilization fermentation environment;

extraction of freeze-dried using an organic solvent;

extraction of compounds from the culture filtrate using solid phases;

analysis by HPLC, thin layer chromatography or by testing biological efficiency.

The conditions of fermentation are important for Myxococcus virescens ST200611 (DSM 15898), as well as for mutants and/or variants.

The object of the present invention, then, is the use of microorganism Myxococcus virescens ST200611 (DSM 15898) or mutant and/or variant to obtain the compounds of formula (V), in particular the compounds of formula (IV), or its physiologically acceptable salts, as described above.

To detect inhibit the of cell proliferation using the test based on the determination of the intracellular concentration of ATP. You can use the well-known lines of tumor cells, such as ner-G2 and Colo205. In this test, the ATP content of metabolically active cells by reaction with luciferase serves as a measure of the number of living cells.

Compounds of formulas (II)-(IV) in the test used in a dose of 0.3 to 40 μm, and the dose is specified as the value TS, and (IIA) and (IIB) respectively mean the diastereoisomer of compounds of formula (II):

Table 1
Efficiency benhamed in the test for cell proliferation in the form of values TS in mcm
ConnectionHep-G2Colo 205
(II)1627
(IIA)1733
(IIB)610
(III)915
(IV)>4042
Benhamed E36 46
Benhamed F2733

The object of the invention, then, is the use of the compounds of formula (I) or its physiologically acceptable salts as a drug in human medicine or animal, in particular for the treatment and/or prevention of cancer. Preferably the invention relates to the use of compounds of formula (I) or physiologically acceptable salts for the treatment of breast cancer, colon cancer, stomach cancer, liver cancer, brain tumors, tumors of the ovary, cancer of the esophagus, kidney cancer, muscle cancer, especially carcinomas facial muscles, improve head and neck.

Further, the present invention relates to a medicinal product with a content of at least one of the compounds of formula (I) or a physiologically acceptable salt, and the compound or compounds of formula (I) can be entered as such in the form of a substance or preferably in a mixture with one or more conventional pharmaceutically suitable carriers or excipients.

Proposed according to the invention compounds are stable in the solid state and in solutions in the field of pH values from 2 to 9 and thus can be entered in the usual herbal composition.

Offer according to the image the structure of the medicinal product can be administered orally or parenterally, however, it is also, in principle, possible rectal application. Suitable solid or liquid galenovye forms of the compositions are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in ampoule form and also preparations with protagorean the release of biologically active ingredients, which are usually found pharmaceutically suitable carriers or excipients, as porofor, binder, means for coating, contributing to the swelling means giving lubricity tools or lubricants, flavorings, sweeteners or contributing to dissolved substances, such as magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.

If necessary, dosage units for oral administration can be microencapsulate to slow down or extend over a longer period of time of release, as, for example, by coating or the introduction of biologically active substances in the form of particles suitable for emery, waxes or the like.

Preferably the pharmaceutical drugs and get injected in the form of dosage units, with each unit as an active component contains a certain dose of one or more derivatives benhamed formula (I). In the case of solid dosage units such as tablets, capsules and suppositories, this dose can be up to about 500 mg, preferably, however, from about 0.1 to 200 mg, and in the case of injection solutions in ampoules up to about 200 mg, preferably, however, from about 0.5 to 100 mg per day.

Enter daily dose depends on body weight, age, sex and condition of the mammal. Depending on the circumstances, however, you can also enter a higher or lower daily doses. The introduction of the daily dose can be achieved by one-time injection in the form of an individual dosage unit or, however, as several smaller dosage units, as well as by repeated administration of divided doses at regular intervals.

Proposed according to the invention medicines get that one or more of the offer according to the invention compounds of formula (I), optionally together with one or more conventional carriers or excipients brought to a suitable form of administration.

Following the examples should serve for a more detailed explanation of the invention, without limiting in any way the scope of protection of the invention.

If not given any other instructions, the percentages relate to the weight, and the ratio of components in the mixture in the case of liquids apply to the scope.

Example 1

Store at a temperature of -135°C Myxococcus virescens ST200611 (DSM 15898)

Agar plate (1% fresh Baker's yeast, 1% CaCl2x 2H2O, 20 mm HEPES, 0,00005% cyanocobalamin, 1.5% agar, pH 7.2) inoculant strain of Myxococcus virescens ST200611 (DSM 15898) and incubated for about 7 days at a temperature of 30°C. the Cells of this superficial culture scraped with a sterile spatula with agar surface, resuspended in 0.5 ml Kapitonova environment (1% casitone (Difco), 0.15% Of MgSO4x 7H2O, pH=7.0) and stored at a temperature of -135°C.

Example 2

Getting preculture Myxococcus virescens ST200611 (DSM 15898) in Erlenmeyer flask

100 ml of Nutrient solution (1% fresh Baker's yeast, 1% CaCl2x 2H2O, 20 mm HEPES, 0,00005% cyanocobalamin, pH 7.2) in a sterile Erlenmeyer flask with a capacity of 300 ml inoculant using strain Myxococcus virescens ST200611 (DSM 15898) and incubated on a rotating machine for shaking for 4 days at a temperature of 30°C and with a speed of 180 revolutions per minute. 5 ml of this preculture then used to produce staple crops.

Example 3

The production of the liquid basis of the term culture Myxococcus virescens ST200611 (DSM 15898)

Sterile Erlenmeyer flask with a capacity of 300 ml with 100 ml of the following nutrient solution (0.5% yeast extract, 0.5% of casitone, 0,1% CaCl2x 2H2O, 0.2% of MgSO4x 7H2O, 0,00005% cyanocobalamin, pH=7,4) inoculant with 5 ml of preculture (see example 2) or grown on fresh agar plate culture (1% fresh Baker's yeast, 1% CaCl2x 2H2O, 20 mm HEPES, 0,00005% cyanocobalamin, pH 7.2, addition of 1.5% agar) and incubated in a machine for shaking at 30°C and a speed of 180 revolutions per minute. Maximum production proposed according to the invention benjaminov reach after 72-96 hours. For inoculation of the fermenter with a capacity of 10 l, 200 l enough 72-96 hours submerged culture (the amount of inoculum of approximately 5-10%) of the same nutrient solution as described in example 2.

Example 4

Getting the main liquid culture Myxococcus virescens ST200611 (DSM 15898) adding hydroxylysine for producing derived benhamed formula (IV)

Sterile Erlenmeyer flask with a capacity of 300 ml with 100 ml of the following nutrient solution (0.5% yeast extract, 0.5% of casitone, 0,1% CaCl2x 2H2O, 0.2% of MgSO4x7H2O, 0,00005% cyanocobalamin, and 1 mm hydroxylysine, pH=7,4) inoculant with 5 ml of preculture from example 2 or grown on the fresh and arovas plate culture (1% fresh Baker's yeast, 1% CaCl2x 2H2O, 20 mm HEPES, 0,00005% cyanocobalamin, pH 7.2, addition of 1.5% agar) and incubated in a machine for shaking at 30°C and a speed of 180 revolutions per minute. Maximum production derived benhamed formula (IV) reach after 72-96 hours. The analysis carried out by HPLC-MS. For inoculation of the fermenter with a capacity of 10 l, 200 l enough 72-96 hours submerged culture (the amount of inoculum of approximately 5-10%) of the same nutrient solution as described in example 2.

Example 5

Deriving benhamed in the fermenter

Fermenters with a capacity of 10 l and 30 l operate in the following conditions:

the inoculateapproximately 5%about 9%
the fermenter30 l10 l
nutrient medium:see example 2see example 2
incubation temperature:30°C30°C
speed stirrer:112 rpm150 rpm
aeration:8 l/min4 l/min
regulation of pH:from pH 7.8 to pH=7,5from pH 8.1 to pH=7,5
regulation of Rho2:nono

Regulation of pH is always performed using a 10%aqueous solution of KOH, 10%H2SO4. Due to repeated supplements Clerol FBA 265 (Cognis Deutschland GmbH), it is possible to suppress foaming. The maximum production is achieved after approximately 72-96 hours.

Example 6

The allocation of the derivatives benhamed formulas (II) and (III), and benjaminov E and F from grown with constant shaking cultures of Myxococcus virescens ST200611 (DSM 15898)

At the end of fermentation Myxococcus virescens ST200611 (DSM 15898) culture broth from example 3 (30 l culture broth) together with biomass lyophilizer and lyophilized extracted with methanol (2 x 5 l). The methanol extract was reduced in vacuum to a volume of 1.2 l and then applied to the prepared column, which is filled with approximately 15 l material CHP-20P (MCI®gel, 75-150 micron, Mitsubishi Chemical Corporation). Elute with 95%methanol. Collect the flow from the column (120 ml/min) and in vacuum to reduce the volume of 5 liters

Example 7

Preliminary separation of the derivatives benhamed formulas (II) and (III), and benjaminov E and F by chromatography with reversed phase C18

1.5 l Obtained according to example 6 solution contribute to column Phenomenex Luna®, 10 micron, C18(2) (size: 50 mm x 250 mm) with pre-column Luna®, 10 micron, C18(2) (size: 21.2 cm x 60 mm) and elute within 60 minutes using a gradient from 5% to 95% acetonitrile in water (0.1% ammonium acetate, pH=4,6 set using acetic acid). The flow rate of 150 ml/min, the volume fractions of 200 ml Benhamadi are in fractions 5-9, 10-11 and 12-14.

Example 8

Clearing derivatives benhamed formulas (II) and (III), and benjaminov E and F

Individual fractions from example 7 lyophilizer and again purified by HPLC using a column Phenomenex Luna®, 10 micron, C18(2) (size: 21 mm x 250 mm) with pre-column XTerra®Prep MS C18, 10 μm (Waters; size: 19 mm x 10 mm). Elute within 40 minutes using a gradient from 5% to 40% acetonitrile in water (with the addition of 0.1% ammonium acetate, pH 8,8 set with triethylamine). Flow from the column (50 ml/min) to collect fractions (fractions of 7.5 ml). Fraction 5-9 example 7 contains a compound of the formula (III), and after chromatography and lyophilization gives 86 mg benhamed E (purity >95%). Fraction 10-11 of example 7 after chromatography Yes the t 145 mg benhamed formula (II) (purity > 95%, mixture of diastereomers in the ratio of 70:30) and 5 mg benhamed F (purity >95%). From fractions 12-14 from example 7 receive 35 mg benhamed formula (III) (purity >95%) as a mixture of diastereoisomers in the ratio of 75:25. In the case of diastereoisomers it is, respectively, about the respective epimeric C-16.

Example 9

Selection hydroxybenzamide formula (IV) grown with constant shaking cultures of Myxococcus virescens ST200611 (DSM 15898)

At the end of fermentation Myxococcus virescens ST200611 (DSM 15898) culture broth from example 5 (10 l culture broth) together with biomass lyophilizer and lyophilized extracted with methanol (2 times in 3 years). Methanol extract reduced in vacuo to a total volume of 400 ml and then applied to the prepared column, which is filled with 0.6 l of material CHP-20P (MCI®gel, 75-150 micron, Mitsubishi Chemical Corporation). Elute with 5% to 95% methanol in water. Flow from the column (100 ml/min) collect within 60 minutes of the fractions (0.5 minutes per fraction). Containing desirable derived fractions (fraction 45-109) combine and reduce vacuum to a volume of 700 ml

Example 10

Pre-treatment hydroxybenzamide formula (IV) by chromatography with reversed phase C-18

The solution from example 9 then bring in column Phenomenex Luna®, 10 micron, C18(2) (size: 50 mm x 250 mm) with pre-column Luna® , 10 micron, C18(2) (size: 21.2 cm x 60 mm) and elute within 60 minutes using a gradient from 5% to 40% acetonitrile in water (0.1% ammonium acetate, pH=48,8 set with triethylamine). The flow rate of 150 ml/min, the volume fractions of 225 ml. Fraction 22 contains desirable derived benhamed.

Example 11

Cleaning hydroxybenzamide formula (IV)

Fraction 22 from example 10 lyophilizer and again purified by HPLC using a column Phenomenex Luna®, 10 micron, C18(2) (size: 21 mm x 250 mm) with pre-column Waters XTerra®Prep MS C18, 10 μm (size: 19 mm x 10 mm). Elute within 60 minutes using a gradient from 5% to 95% acetonitrile in water (with the addition of 0.1% ammonium acetate, pH=4,6 set using acetic acid). Flow from the column (50 ml/min) to collect fractions (fractions of 7.5 ml). Containing benhamed fraction (fraction 26-28) unite, absoluut and subjected to drying by freezing. You get a 7 mg benhamed formula (IV) as a mixture of diastereoisomers in the ratio of 75:25.

Example 12

Characterization of the compounds of formula (II)

Total formula: C18H32N2O6

Molecular weight: 372,47

Mixture of diastereomers: 75:25

Table 2
Chem is ical shifts in NMR benhamed formula (II),
mixture of diastereomers, C=3 mg/ml in DMSO, 300 K
Position1N13
1-173,99
2to $ 7.91-
33,19/3,0640,56
41,74/1,2028,75
51,87/1,6427,55
61,87/1,3630,72
74,3951,27
87,78-
9-169,61
103,69the point 81.60
10-OMe3,2557,32
11to 3.5870,72
11-HE4,46-
123,3372,80
12-HE4,36-
133,9772,46
13-HE4,56-
14lower than the 5.37129,05
155,48136,57
161,9937,41
16-IU0,9319,90
171,2629,15
180,8111,58

Example 13

Characterization of the compounds of formula (III)

Total formula: C19H34N2O6

Molecular weight: 386,49

Mixture of diastereomers: 75:25

Table 3
Chemical the deeds in NMR benhamed formula (III),
mixture of diastereomers, C=3 mg/ml in DMSO, 300 K
Position1N13
1-171,95
22,9135,28
33,61/3,2149,20
41,71/1,3126,13
51,82/1,6727,11
61,84/1,3230,80
74,5551,14
8to 7.84-
9-169,54
103,6981,55
10-OMe3,2557,28
113,5770,69
11-HEof 4.45-
123,3372,77
12-HE4,37-
133,9772,47
13-HE4,56-
14lower than the 5.37129,04
155,48136,58
161,9937,41
16-IU0,9319,89
171,2629,15
180,8111,58

Example 14

Characterization of the compounds of formula (IV)

Total formula: C18H32N2O7

Molecular weight: 388,46

Mixture of diastereomers: 75:25

Table 4
Chemical with the Whigs in NMR benhamed formula (IV),
mixture of diastereomers, with a=3.1 mg/ml in DMSO, 300 K
Position1N13
1-173,45
2at 7.55-
33,35/3,0245,05
43,7463,48
4-HE4,60-
51,81/1,7534,28
61,68/1,6424,25
74,3251,18
8to 7.77-
9-169,63
103,7081,59
10-OMe3,2657,30
11to 3.5870,73
11-HE4,47-
123,3472,82
12-HEof 4.38-
133,9772,47
13-HE4,56-
14lower than the 5.37129,06
155,48136,57
161,9937,41
16-IU0,9319,90
171,2629,15
180,8111,58

Example 15

Description benhamed E

Total formula: C17H30N2O6

Molecular weight: 358,44

Table 5
Chemical shifts in NMR benhamed E, C=3 mg/ml
in DMSO, 300 K
Position1N13
1-174,01
2to $ 7.91-
33,19/3,0640,56
41,74/1,2028,75
51,87/1,6427,56
61,87/1,3630,72
74,3951,27
87,78-
9-169,60
103,6981,61
10-OMe3,2557,30
11of 3.56 70,74
11-HE4,49-
123,3372,78
12-HEof 4.38-
133,9672,38
13-HE4,57-
145,38127,68
155,58137,85
162,2430,08
170,9522,27
180,9522,17

Example 16

Description benhamed F

Total formula: C18H32N2O6

Molecular weight: 372,47

Table 6
Chemical shifts in NMR benhamed F=3 mg/ml
in DMSO, 300 K
Position1-
22,91
33,62/3,21
41,69/1,33
51,84/1,69
61,84/1,33
74,56
8to 7.84
9-
103,70
10-OMe3,25
113,57
123,33
133,97
145,38
155,59
162,25
170,95
180,95

Example 17

Separation of diastereomers of compounds of formula (II)

Separation of a mixture of diastereoisomers of the compounds of formula (II) of example 8 is carried out on a column with a chiral phase (AD/H, Daicel, 20 mm x 200 mm, flow 0.5 ml, eluting agent: acetonitrile:methanol=4:1+0.1% ammonium acetate). Optical purity control on the analytical column AD/H (Diacel) (4.6 mm x 250 mm; 30°C; eluting agent: acetonitrile:methanol=4:1+0.1% ammonium acetate, flow 0.75 ml; output peak 1:9.9 minutes, the output peak 2:10.9 minutes).

1,74/1,20
Table 7
Chemical shifts in NMR diastereomers benhamed
formula (II), C=3 mg/ml in DMSO, 300 K
1N(A)1H(In)13(A)13With(In)
1--173,99173,99
2to $ 7.91to $ 7.91--
33,19/3,063,19/3,0640,5640,56
41,74/1,2028,7528,75
51,87/1,641,87/1,6427,5527,55
61,87/1/361,87/1/3630,7230,72
74,394,3951,2751,27
87,787,78--
9--169,61169,61
103,693,69the point 81.60the point 81.60
10-OMe3,253,2557,3257,32
11to 3.58to 3.5870,7270,77
11-OH4,464,47--
123,333,3372,8072,85
12-OH4,364,36--
133,973,9772,4672,37
13-OH4,564,56--
14lower than the 5.375,38129,05129,01
155,485,49136,57136,44
161,991,9937,4137,28
16-Me0,930,929,90 19,86
171,261,2629,15depreciation is 29.06
180,810,8211,5811,48

Example 18

Determination of cell proliferation in the case of different lines of tumor cells

To determine cell proliferation used lines of tumor cells ner-G2 (ATCC no HB-8065) and COLO 205 (ATCC no CCL-222). Cell lines were sown on medium for cultivation of cells in 1000 cells per well [Hep-G2], respectively 3500 cells per well [Colo205] and incubated for 4 hours at 37°C and in an atmosphere with 5% CO2.

Environment for Hep-G2: modified by way of Dulbecco medium Needle/Ham's F12-Mix (Gibco); NEAA (10%; non-essential amino acids, Gibco), sodium pyruvate (1%, Gibco), L-glutamine (1%, Gibco), fetal calf serum (5%; PAA).

Environment for COLO 205: RPMI 1640 (Gibco), L-glutamine (1%, Gibco), HEPES (1%, Gibco), fetal calf serum (10%, PAA).

After 4 hours was added in different dilutions of the compounds of formula (II), (III), (IV) and benhamadi E and F, dissolved in DMSO/medium for cell culture, and incubated 72 hours at 37°C and in an atmosphere with 5% CO2. The definition is the obsession of intracellular ATP was carried out using the test CellTiterGlo reagent (Promega).

The test results on cell proliferation are shown in table 1.

1. The compound of formula (I)

and
R1means H or (C1-C6)-alkyl;
R2means H or HE; and
R3means N;
or its physiologically acceptable salt.

2. The compound according to claim 1, and R1means H or methyl.

3. The compound according to claim 2, and R1means H or methyl, R2means H or HE and R3means N.

4. The compound according to claim 1, which represents a compound of formula (II)

5. The compound according to claim 1, which represents a compound of formula (III)

6. The compound according to claim 1, which represents a compound of formula (IV)

7. The use of the compounds of formula (I) or its physiologically acceptable salts according to any one of claims 1 to 6 to obtain drugs for inhibition of cell proliferation.

8. The use of the compounds of formula (I) or its physiologically acceptable salts according to any one of claims 1 to 6 to obtain drugs for the treatment and/or prevention of cancer.

9. Drug for inhibiting the proliferation of cells containing at least one compound of formula (I) or a physiologically acceptable salt according to any one of claims 1 to 6.

10. When persons obtain the compounds of formula (V)

and
R1means H or (C1-C6)-alkyl;
R2means H or HE;
R3means N; and
R4means methyl or ethyl;
or physiologically acceptable salts of the compounds of formula (V),
characterized in that

1. strain Myxococcus virescens ST200611 (DCM 15898) in suitable conditions are fermented in a culture medium until the accumulation in the culture medium one or more of the compounds of the formula (V);

2. the compound of formula (V) was isolated from the culture medium; and

3. the compound of formula (V), if necessary, derivatizing and/or, if necessary, converted into a physiologically acceptable salt.

11. The method according to claim 10, and R4means ethyl.

12. The method according to any of PP or 11, and independently from other R1means H or methyl, R3means N, and R4means ethyl.

13. The method according to item 12, and R2IT means, and the culture medium contains hydroxylysine.

14. The microorganism strain Myxococcus virescens ST200611 (DSM 15898) to obtain the compounds of formula (V).

15. The use of a microorganism strain Myxococcus virescens ST200611 (DSM 15898) to obtain the compounds of formula (V)

and
R1means H or (C1-C6)-alkyl;
R2means H or HE;
R3means N; and
R4means methyl is whether ethyl;
or its physiologically acceptable salt.



 

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