DE102004046142B4 - New selective active substances for the treatment of diseases caused by mycobacteria, process for their preparation and their use - Google Patents

Abstract

worin die Reste R1 bis R4 in den Ringen A und 8 unabhängig voneinander:
R1 Wasserstoff, eine verzweigte oder unverzveigte C₁-C₁₂-Alkylgruppe, eine substituierte oder nicht substituierte Aralkylgruppe ggf. ein oder mehrere Heteroatome enthaltend, (-C(O)-R_a)-gruppe oder (-C(S)-R_a)-gruppe, einer Cycloalkylgruppe bestehend aus 3 bis 10 Ringatomen, ggf. ein oder mehrere Heteroatome enthaltend, eine substituierte oder unsubstituierte Arylgruppe, ggf. ein oder mehrere Heteroatome enthaltend mit R_a bestehend aus Wasserstoff, einer verzweigten oder unverzweigten C₁-C₁₂-Alkylgruppe ggf. substituiert durch Carboxy-, Hydroxy und Aminogruppen;
R2 Wasserstoff oder ein doppelt gebundener Sauerstoff (Oxy);
R3 Wasserstoff, Hydroxy, ein doppelt gebundener Sauerstoff (Oxy), eine verzweigte oder unverzweigte C₁-C₁₂-Alkoxygruppe, (-C(O)-R_a)-gruppe, (-C(S)-R_a)-gruppe, (-OC(O)-R_a)-gruppe, (-OC(S)-R_a)-gruppe oder (-OC(O)O-R_a)-gruppe mit R_a wie oben definiert;
R4 Wasserstoff;
oder R1 und R2, sowie R3 und R4 bilden jeweils gemeinsam aber unabhängig voneinander eine Doppelbindung;
R5 Wasserstoff, eine verzweigte oder unverzweigte C₁-C₁₂-Alkylgruppe, eine substituierte oder nicht...Compounds of the general formula 1:

in which the radicals R 1 to R 4 in the rings A and 8 are independently of one another:
R _{1 is} hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group, a substituted or unsubstituted aralkyl group optionally containing one or more heteroatoms, (-C (O) -R _a ) -group or (-C (S) -R _a ) group, a cycloalkyl group consisting of 3 to 10 ring atoms, optionally containing one or more heteroatoms, a substituted or unsubstituted aryl group, optionally one or more heteroatoms containing with R _a consisting of hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group optionally substituted by carboxy, hydroxy and amino groups;
R2 is hydrogen or a doubly bonded oxygen (oxy);
R3 is hydrogen, hydroxy, a doubly bonded oxygen (oxy), a branched or unbranched C ₁ -C ₁₂ alkoxy group, (-C (O) -R _a ) group, (-C (S) -R _a ) group , (-OC (O) -R _a ) group, (-OC (S) -R _a ) group or (-OC (O) OR _a ) group with R _a as defined above;
R4 is hydrogen;
or R 1 and R 2, as well as R 3 and R 4, in each case together but independently of one another, form a double bond;
R5 is hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group, a substituted or not

Description

The The present invention relates to novel selective and non-toxic Antibiotics used to treat diseases caused by mycobacteria caused such. Tuberculosis and leprosy.

The Tuberculosis (TB) is among the infectious diseases worldwide the highest Death rate on. One third of the world population is with the pathogen Mycobacterium tuberculosis infects nearly three million people die annually at the infectious disease. Hunger and wars in Asia and Africa, but also the political and social upheavals in Eastern Europe with z.T. negative effects on health systems and their spread AIDS has dramatically increased the number of diseases. Emigration and worldwide tourism are also bringing the TB to the industrial nations back, in which until the eighties the disease already as largely overcome was. 1993 declared the World Health Organization made tuberculosis a global one Threat.

Furthermore there is a menacing increase in tuberculosis infections resistant mycobacteria. (B.R. Bloom, J.L. Murray, Tuberculosis: commentary on a reemergent killer, Science 257, 1992, 1055-1064).

Extreme dangerous is the development of multidrug resistant mycobacteria that oppose the Standard tuberculostatic agents isoniazid, rifampicin, streptomycin, pyrazinamide and ethambutol have become insensitive. The proportion of Tubekuloseinfektionen, which were caused by multidrug-resistant pathogens is in some countries already higher than 20%. In Germany, the resistance to individual drugs increased since 1995 by 50%.

All in all is estimated that the known drugs for the treatment of tuberculosis and thus related diseases, the demands for adequate antibacterial effect and good compatibility do not meet what the search for new compounds, especially with a new mechanism of action, required.

Out the class of cyclodepsipeptides are some natural products such as e.g. Aurantimycin, verucopeptin and others with antibacterial activity known. However, these substances all have a strong cytotoxicity and are therefore for This indication has not been pursued. The substances of this invention are in contrast to the known cyclodepsipetides not cytotoxic.

Of the Invention is based on the object, suitable, readily soluble, selectively effective and well tolerated To provide drugs. The compounds should be effective Allow treatment of diseases, which are caused by infections with mycobacteria, such as e.g. Tuberculosis and leprosy.

Piperazic-containing Cyclodepsipeptides whose ring system consists of four amino acids and those with a longer one Alkyl side chain are substituted, show the desired Effects.

worin
die Reste R1 bis R4 in den Ringen A und 8 unabhängig voneinander
R1 Wasserstoff, eine verzweigte oder unverzweigte C₁-C₁₂-Alkylgruppe, eine substituierte oder nicht substituierte Aralkylgruppe ggf. ein oder mehrere Heteroatome enthaltend, (-C(O)-R_a)-gruppe oder (-C(S)-R_a)-gruppe, einer Cycloalkylgruppe bestehend aus 3 bis 10 Ringatomen, ggf. ein oder mehrere Heteroatome enthaltend, eine substituierte oder unsubstituierte Arylgruppe, ggf. ein oder mehrere Heteroatome enthaltend
mit R_a bestehend aus Wasserstoff, einer verzweigten oder unverzweigten C₁-C₁₂-Alkylgruppe ggf. substituiert durch Carboxy-, Hydroxy und Aminogruppen;
R2 Wasserstoff oder ein doppelt gebundener Sauerstoff (Oxy);
R3 Wasserstoff, Hydroxy, ein doppelt gebundener Sauerstoff (Oxy), eine verzweigte oder unverzweigte C₁-C₁₂-Alkoxygruppe, (-C(O)-R_a)-gruppe, (-C(S)-R_a)-gruppe, (-OC(O)-R_a)-gruppe, (-OC(S)-R_a)-gruppe oder (-OC(O)O-R_a)-gruppe mit R_a wie oben definiert;
R4 Wasserstoff;
oder R1 und R2, sowie R3 und R4 bilden jeweils gemeinsam aber unabhängig voneinander eine Doppelbindung;
R5 Wasserstoff, eine verzweigte oder unverzweigte C₁-C₁₂-Alkylgruppe, eine substituierte oder nicht substituierte Aralkylgruppe ggf. ein oder mehrere Heteroatome enthaltend, (-C(O)-R_a)-gruppe, (-C(S)-R_a)-gruppe, (-OC(O)-R_a)-gruppe, (-OC(S)-R_a)-gruppe oder (-OC(O)O-R_a)-gruppe mit R_a wie oben definiert sind.The compounds according to the invention have the general formula 1:

wherein
the radicals R1 to R4 in the rings A and 8 independently
R _{1 is} hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group, a substituted or unsubstituted aralkyl group optionally containing one or more heteroatoms, (-C (O) -R _a ) -group or (-C (S) -R _a ) group, a cycloalkyl group consisting of 3 to 10 ring atoms, optionally containing one or more heteroatoms, a substituted or unsubstituted aryl group, optionally containing one or more heteroatoms
with R _a consisting of hydrogen, a branched or unbranched C ₁ -C ₁₂ alkyl group optionally substituted by carboxy, hydroxy and amino groups;
R2 is hydrogen or a doubly bonded oxygen (oxy);
R3 is hydrogen, hydroxy, a doubly bonded oxygen (oxy), a branched or unbranched C ₁ -C ₁₂ alkoxy group, (-C (O) -R _a ) group, (-C (S) -R _a ) group , (-OC (O) -R _a ) group, (-OC (S) -R _a ) group or (-OC (O) OR _a ) group with R _a as defined above;
R4 is hydrogen;
or R 1 and R 2, as well as R 3 and R 4, in each case together but independently of one another, form a double bond;
R5 is hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group, a substituted or unsubstituted aralkyl group optionally containing one or more heteroatoms, (-C (O) -R _a ) -group, (-C (S) -R _a ) group, (-OC (O) -R _a ) group, (-OC (S) -R _a ) group or (-OC (O) OR _a ) group with R _a as defined above.

mit R⁶ Wasserstoff oder Hydroxy.Preferred in formula 1 are:
R 1 is hydrogen, (-C (O) -R _a ) -group with R _a as defined above, a cycloalkyl group consisting of 3 to 10 ring atoms, optionally containing one or more heteroatoms, a substituted or unsubstituted aryl group, optionally one or more Containing heteroatoms;
R2 hydrogen
R3 is hydrogen, hydroxy, (-C (O) -R _a ) -group, with R _a as defined above;
R4 is hydrogen;
or R ¹ and R ² , and R ³ and R ⁴ together but independently form a double bond;
R5

with R ^{6 is} hydrogen or hydroxy.

Of Further, the invention all stereoisomers of Lydiamycine included, both pure diastereomers and enantiomers, as also their mixtures.

Further are tautomeric forms of lydiamycins and their derivatives as well of the invention.

salts Lydiamycins, both internal salts, as well as by adding appropriate organic or inorganic acids or bases, are included in the invention, such as also the salts formed by quaternization of amino groups.

Farther The present invention relates to the pharmaceutical application of Lydiamycins or their derivatives for the control of diseases which caused by mycobacteria, e.g. Tuberculosis and Leprosy.

The lydiamycins and their derivatives can be synthesized by known chemical methods or isolated from the strain Streptomyces lydicus HKI 0343. For this purpose, the strain is grown in submucosal culture in a soybean-based medium. It is also possible to isolate precursors from this strain and to derivatize by known methods. The isolation of the lydiamycins is illustrated in the examples given. The detection and isolation methods include thin layer chromatography on silica gel with two solvent systems, four staining agents to visualize the metabolite spectrum (Grabley, S., Thiericke, R., Drug Discovery from Nature, Springer Verlag, Berlin). The most striking methanolic extracts are purified. The products are purified by means of HPLC on reversed phases (eg silica gel C18, acetonitrile / water) in aqueous systems with organic modifiers, as well as on Sephadex LH20. The detection is carried out both by light spectroscopy (eg by means of DAD) and by mass spectroscopy. It is apparent, with relevant experience, that lydiamycins and their derivatives of this invention can also be produced wholly as synthetically. Examples of methods include: "March's Advanced Organic Chemistry: Reaction, Mechanisms and Structure," 5 ^th ed., By Michael B. Smith, Jerry March, Wiley-Interscience, 2001, or "Classics in Total Synthesis: Targets, Strategies, Methods by KC Nicolaou, EJ Sorensen, John Wiley & Son Ltd, 1996 or in "The Art and Science of Total Synthesis at the Dawn of the Twenty-First Centruy", Nicolaou KC, Vourloumis D., Winssinger N., Baran PS, An wt. Chem. Int. Ed. Engl. 2000, 39 (1): 44-122. The strain has been deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) in Braunschweig and is registered under number DSM 16701.

Farther For example, the present invention is illustrated by the following examples explained.

example 1

Isolation and cultivation of the strain Streptomyces lydicus HKI 0343

Of the Strain Streptomyces lydicus HKI 0343 is from a soil sample of a wet meadow from the Thuringian Forest. He was following a standard dilution principle and by plating isolated.

Of the Strain S. lydicus HKI 0343 was based on a 16S rDNA analysis It is identified by a well-developed white aerial mycelium (in malt extract extract / yeast extract medium) with a yellow substrate mycelium and by the formation of a yellow pigment in the culture medium excreted is characterized.

The Laboratory cultivation is carried out in a medium of 0.4% glucose, 1% malt extract, 0.4% yeast extract and 2% agar or in a liquid medium of 2% soy flour and 2% mannitol without agar at a pH of 7.5. The incubation temperature is 28 ° C. The Preservation takes place in liquid Nitrogen with addition of 5% DMSO to the medium or by storage at -80 ° C with admixture of 50% glycerol to the culture medium.

Of the Strain Streptomyces lydicus HKI 0343 is number DSM 16701 at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) in Braunschweig.

Example 2

insulation of lydiamycin A-D

The Compounds of the present invention can be obtained from the culture broth of the strain HKI 0343 can be isolated by placing the strain in a soy liquid medium (20 g / l soy flour, 20 g / l mannitol) at a pH of 7.5 for 4 days 28 ° C in 500 ml Erlenmeyer flask is incubated. This culture becomes a seeding of a 300 l fermentor under the same conditions.

After sufficient product formation, the cultivation is terminated by separation of the cells. For this purpose, the biomass is separated from the culture broth by centrifugation or filtration. The substances of interest are recovered from the culture filtrate. The first step involves adsorption on an adsorbent gel (eg, Amberchrom 161c), followed by elution with methanol in accordance with Schmid, I. et al., Biomol. Screening, 1999, 4, p. 13-23. After separation of the solvents, the substances are purified by means of standard chromatographic procedures, as described below. The chemical structure is undoubtedly determined by means of mass spectroscopy, one- and multi-dimensional ¹ H and ¹³ C NMR spectroscopy in CDCl ₃ solution, as well as polarimetric. All solvents used were free from plasticizer or pA quality.

Lydiamycin A

• ESI-MS: m / z 686.8 [M + Na] ⁺ , 681.9 [M + NH ₄ ] ⁺ , 664.9 [M + H] ⁺ , 662.6 [M - H] ⁺ ; HREI-MS: m / z 686.3495 (C31H49NaN7O9 calc.686.3489);
• IR (film) νmax cm ^-1 : 3295, 2954, 2359, 2341, 2163, 2051, 1745, 1663, 1539, 1408, 1218, 793, 667, 629; ¹ H and ¹³ C NMR data see Table 1; α _D (0.23 g / 100 ml methanol) -19.8 °.

Lydiamycin B

• ESI-MS; m / z 702.8 [M + Na] ⁺ , 698.0 [M + NH ₄ ] ⁺ , 680.8 [M + H] ⁺ , 678.6 [M - H] ⁺ ; HREI-MS: m / z 702.3444 (C31H49NaN7O10 calc.702.3439);
• IR (film) ν max cm ^-1 : 3299, 2932, 2358, 2324, 2163, 2073, 1754, 1662, 1538, 1415, 1260, 1218, 1093, 773, 630; ¹ H and ¹³ C NMR data see Table 2; α _D (0.17 g / 100 ml methanol) -34.5 °.

Lydiamycin C

• ESI-MS; m / z 684.8 [M + Na] ⁺ , 662.9 [M + H] ⁺ , 679.9 [M + NH ₄ ] ⁺ , 660.6 [M - H] ⁺ ; HREI-MS: m / z 684.3325 (C31H47NaN7O9 calc.684.3333);
• IR (film) νmax cm ^-1 : 3301, 2954, 2359, 2341, 2164, 2049, 1746, 1718, 1666, 1627, 1539, 1416, 1261, 1218, 1048, 988, 960, 772, 630; ¹ H and ¹³ C NMR data see Table 3; α _D (0.20 g / 100 ml methanol) -16.2 °.

Lydiamycin D

• ESI-MS; m / z 702.8 [M + Na] ⁺ , 680.9 [M + H] ⁺ , 697.8 [M + NH ₄ ] ⁺ , 678.6 [M - H] ⁺ ; HREI-MS: m / z 702.3453 (C31 H49NaN7O10 calc.702.3439);
• IR (film) ν max cm ^-1 : 3280, 2933, 2867, 2359, 2163, 1744, 1721, 1657, 1629, 1563, 1535, 1442, 1408, 1309, 1253, 1206, 1181, 1054, 1033, 1011, 912 , 869, 696; ¹ H and ¹³ C NMR data see Table 4; α _D (0.32 g / 100 ml methanol) -21.2 °.

Example 3

biological characterization

Tests on Growth inhibition of some mycobacteria

The test substances are dissolved in DMSO at a concentration of 1 mg / ml and dissolved with methanol Diluted to 0.4 mg / ml. From this solution of the test substances, dilution series are prepared in microtiter plates (Greiner, Germany) with Mueller-Hinton broth as culture solution, which after addition of the mycobacteria as inoculum a graded in half steps concentration of 100 ug / ml up to 0.05 ug / ml exhibit. The test organisms used are Mycobacterium smegmatis SG 987, M. vaccae IMET 10670 and M. aurum SB66.

The mycobacteria are inoculum in test broth (glycerol 1%, meat extract 0.5%, peptone pancreatic from casein 0.5%, NaCl 0.3%, distilled water, pH 7.0) 48 h at 32 ° C shaken. The inoculum is adjusted to McFarland Standard 0.5 (BioMerieux, Laupheim, Germany) and has a final microtiter plate concentration of 5 x 10 ⁵ CFU / ml.

The Microtiter plates become after inoculation of the dilution series the test substances with the respective mycobacterial strains 24h at 37 ° C incubated. Thereafter, with the help of a reading mirror is the lowest Concentration at which no visible growth occurs, found and given as minimum inhibitory concentration (MIC). This is the Growth in wells with test substance compared to growth in wells without test substance but with solvent as well as in the wells without test substance and without solvents, each carried as controls.

Result

Tests for cytotoxicity

L-929 and K-562

Adherently growing Cells of the L-929 mouse fibroblast line (DSMZ, ACC 2) and as a suspension culture growing cells of K-562 (DSMZ, ACC 10) cell line [cell culture medium: RPMI 1640 (GIBCO BRL) with additives of 25 μg / ml Gentamycin sulfate (Cambrex), 10% FCS, heat-inactivated (GIBCO BRL) and GlutaMax-1 (GIBCO BRL)] are available at various concentrations the test substances in microtiter plates (NUNC) and standard 3 days long incubated under physiological conditions.

In front the sowing in the microplates is a quality check of the Cell material by CASY1 - cytoanalysis. This allows the determination of distribution curves of cell diameters with which Help cell density (cell count / ml), total cell volume, mean Evaluate cell volume, cell volume distribution and cell size changes.

The aim is to determine the half-maximal antiproliferative efficacy (GI ₅₀ ) using the CASY1 cell analyzer (SCHÄRFE, Reutlingen) or absorbance measurement in the microplate reader ("Sunrise", TECAN, 660 nm) Both methods are advantageous for rapid information on substance effectiveness because they are sufficiently sensitive.

After the incubation period, all K-562 cells of each microtest plate well are diluted with the CASY1 cell analyzer (SCHÄRFE, Reutlingen) at a dilution of 1:50 in physiological saline (CA. SYTON, SHARPNESS) in the cell diameter range of 0-30 μm.

Based on an untreated control, dose-response curves are generated by means of an integrated evaluation program (CASYSTAT) from which, among other things, the GI ₅₀ values (growth inhibition) can be read off. The detection of the proliferation of the adherent L-929 cells is carried out by extinction measurement after previous glutaraldehyde fixation and methylene blue staining of the living cells. Unbound dye is removed by subsequent washing. After eluting the bound methylene blue with 0.2 ml of 0.33 molar HCl, the optical density (OD) of all wells is measured in the microplate reader. An evaluation program ("Magellan") makes it possible to determine the GI ₅₀ by offsetting the OD values of the respective substance concentrations against the OD mean value of all negative controls after blank value correction.

Result: At the maximum test concentration of 50 μg / ml, no effect was observed become.

HeLa

The aim is the colorimetric determination of the CC ₅₀ (Cytotoxic Concentration) as a measure of the half-maximal cytotoxic efficacy.

For this purpose, 1 × 10 ⁴ cells of HeLa (DSMZ, ACC 57) are seeded with 0.2 ml culture medium RPMI 1640 per microtest plate cavity. The microtiter plates are incubated without test substance by default (at 37 ° C, 5% CO and about 95% relative humidity) for 48 hours under physiological conditions to produce sub-confluent monolayers.

After that become the monolayer with dilution stages the test substances and 72 h under physiological Conditions incubated.

At the end of the incubation period, after glutaraldehyde fixation and methylene blue staining, the absorbance of all wells of the microtiter plate at 660 nm is measured with a microplate reader (Sunrise, TECAN) and the CC _{50 determined} using the "Magellan" evaluation program.

Of the Test is relatively low sensitive, because the preincubation of the HeLa cells already leads to the formation of a subconfluent monolayer, so that not cell proliferation, but cytolysis in the subsequent incubation with the test substance for the rating is crucial.

Result: At the maximum test concentration of 50 μg / ml, no effect was observed become.

The The present invention demonstrates the potential of lydiamycins for growth inhibition of mycobacteria. At the same time, no cytotoxicity could be confirmed become. Consequently, the present invention is in the field of Pharmacy and medicine of great Use.

attachment

Table 1. ¹ HNMR (300MHz in CD ₃ Cl) and ¹³ CNMR (75MHz in CD ₃ Cl) data from lydiamycin A

Table 2. ¹ HNMR (300MHz in CD ₃ Cl) and ¹³ CNMR (75MHz in CD ₃ Cl) data from lydiamycin B

Table 3. ¹ HNMR (300MHz in CD ₃ Cl) and ¹³ CNMR (75MHz in CD ₃ Cl) data from lydiamycin C

Table 4. ¹ HNMR (300MHz in CD ₃ Cl) and ¹³ CNMR (75MHz in CD ₃ Cl) data from lydiamycin D

Claims (5)

Compounds of the general formula 1:

in which the radicals R _{1 to} R 4 in the rings A and 8 are independently of one another: R _{1 is} hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group, a substituted or unsubstituted aralkyl group optionally containing one or more heteroatoms, (-C (O) R _a ) group or (-C (S) -R _a ) group, a cycloalkyl group consisting of 3 to 10 ring atoms, optionally containing one or more heteroatoms, a substituted or unsubstituted aryl group, optionally containing one or more heteroatoms with R _a consisting of hydrogen, a branched or unbranched C ₁ -C ₁₂ alkyl group optionally substituted by carboxy, hydroxy and amino groups; R2 is hydrogen or a doubly bonded oxygen (oxy); R3 is hydrogen, hydroxy, a doubly bonded oxygen (oxy), a branched or unbranched C ₁ -C ₁₂ alkoxy group, (-C (O) -R _a ) group, (-C (S) -R _a ) group , (-OC (O) -R _a ) group, (-OC (S) -R _a ) group or (-OC (O) OR _a ) group with R _a as defined above; R4 is hydrogen; or R 1 and R 2, as well as R 3 and R 4, in each case together but independently of one another, form a double bond; R5 is hydrogen, a branched or unbranched C ₁ -C ₁₂ -alkyl group, a substituted or unsubstituted aralkyl group optionally containing one or more heteroatoms, (-C (O) -R _a ) -group, (-C (S) -R _a ) group, (-OC (O) -R _a ) group, (-OC (S) -R _a ) group or (-OC (O) OR _a ) group with R _a as defined above. Process for the preparation of the compound according to claim 1, characterized in that Streptomyces lydicus in a nutrient medium cultured until the compound of Formula 1 accumulates and out this is isolated. Method according to claim 2, characterized in that that Streptomyces lydicus DSM 16701 in a nutrient medium is cultivated. Use of the substance according to claim 1 as a biolgic effective substance. Use of the substance according to claim 1 for pharmaceutical Application for control of diseases caused by mycobacteria.