WO1995034558A1 - Chondramides, production methods, compositions comprising chondramides and cultures for chondramide production - Google Patents

Chondramides, production methods, compositions comprising chondramides and cultures for chondramide production Download PDF

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Publication number
WO1995034558A1
WO1995034558A1 PCT/EP1995/002294 EP9502294W WO9534558A1 WO 1995034558 A1 WO1995034558 A1 WO 1995034558A1 EP 9502294 W EP9502294 W EP 9502294W WO 9534558 A1 WO9534558 A1 WO 9534558A1
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Prior art keywords
chondramide
methanol
chondramides
radical
silica gel
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PCT/EP1995/002294
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French (fr)
Inventor
Hans Reichenbach
Gerhard Höfle
Rolf Jansen
Brigitte Kunze
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Ciba-Geigy Ag
Gesellschaft für Biotechnologische Forschung mbH
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Priority to AU27921/95A priority Critical patent/AU2792195A/en
Publication of WO1995034558A1 publication Critical patent/WO1995034558A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to chondramides of the formula I
  • R 3 hydrogen, a C ⁇ _ 8 alkyl radical, an aryl radical, such as CgHg-, an alkaryl radical, a heterocyclic radical having an oxygen, sulfur or nitrogen atom, a formate radical, a hydrogen carbonate radical, an H 2 N-CO-O- radical, a C ⁇ alkoxy radical, an alkaryl- oxy radical, an al oxyalkyl radical, such as CH 3 -O-CH 2 - or CH 3 -0-CH(CH 3 )-, or an acyloxyalkyl radical, such as alkyl-CO-0-CH 2 -, and the pharmaceutically acceptable alkali metal or alkaline earth metal salts thereof.
  • the invention relates also to a chondramide having the empirical formula C 36 H 46 N 4 O 7 and having the following spectra: UV (methanol); - ⁇ [nm] (lg e):
  • the invention relates also to a chondramide having the empirical formula C 36 H 45 CIN 4 O 7 and having the following spectra: UV (methanol); ⁇ [nm] (lg e):
  • the invention relates also to a chondramide having the empirical formula C 35 H 44 N 4 O 6 and having the following spectra: UV (methanol); ⁇ [nm] (lg e):
  • the invention relates also to a chondramide having the empirical formula C 35 H 43 C1N 4 0 6 and having the following spectra: UV (methanol); ⁇ [nm] (lg e):
  • the invention relates also to chondramides that are producible produced as follows: (a) microorganism capable of producing a chondramide of the formula I wherein R 3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative of the tribe Sorangineae, more preferably a representative of the genus Chondromyces, more preferably a representative of the species Condromyces crocatus and most preferably the species Chondromyces crocatus DSM 8963 or Chondromyces crocatus DSM 10034 is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts, and (b) the chondramides are concentrated, separated and isolated in a manner known per se.
  • R 3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative
  • the invention relates also to chondramides that are producible as follows:
  • said microorganism capable of producing a chondramide of the formula I wherein R 3 is hydrogen is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances, such as vitamin B12, and mineral salts,
  • the cells are separated from the culture medium in the form of a moist biomass and extracted with an aliphatic ketone, such as acetone,
  • the chondramides can preferably be produced by in step (a) using vitamin B12 as growth-promoting substance and/or a medium based on single-cell protein, such as Probion, and/or culturing at approximately 30°C.
  • the chondramides can preferably be produced by in step (d) taking up in a methanol/water mixture (approximately 95:5).
  • the chondramides can preferably be produced by in step (f) chromatographing with an RP-silica gel (for example g and, for example, 100 Angstr ⁇ m/20 ⁇ m) and/or with a methanol/water mixture (approximately 65:35).
  • an RP-silica gel for example g and, for example, 100 Angstr ⁇ m/20 ⁇ m
  • a methanol/water mixture approximately 65:35
  • the chondramides can preferably be produced by in step(s) (gl) and/or (g2) chromato ⁇ graphing with petroleum ether/tert-butyl methyl ether/methanol (approximately 30:69.5:0.5).
  • the chondramides can preferably be produced by in step(s) (gl) and/or (g2) chromato ⁇ graphing with an RP-silica gel (Cig) and/or with methanol/water (approximately 60:40).
  • the invention relates also to a method of producing chondramides wherein
  • chondramide of the formula I wherein R 3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative of the tribe Sorangineae, more preferably a representative of the genus Chondromyces, more preferably a representative of the species Condromyces crocatus and most preferably the species Chondromyces crocatus DSM 8963 or Chondromyces crocatus DSM 10034is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts, and (b) the chondramides are concentrated, separated and isolated in a manner known per se.
  • R 3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative of the tribe Sorangineae, more preferably a representative of the genus Chondromyces, more preferably a representative
  • the invention relates also to a method of producing chondramides wherein:
  • said microorganism capable of producing a chondramide of the formula I wherein R 3 is hydrogen is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances, such as vitamin B12 and mineral salts,
  • the invention relates also to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of the above-mentioned chondramides together with a pharmaceutically acceptable carrier.
  • the invention relates especially to such a composition having anti-fungal and anti- neoplastic action, also against cell lines that are resistant to chemotherapeutic drugs.
  • the invention relates also to an antibiotic composition
  • an antibiotic composition comprising at least one of the above-mentioned chondramides together with a suitable carrier.
  • compositions according to the invention may comprise an effective amount, especially a prophylactically or therapeutically effective amount, of the active ingredient together with pharmaceutically acceptable carriers that are suitable for topical, enteral, for example oral or rectal, or parenteral administration and may be inorganic or organic and solid or liquid.
  • pharmaceutically acceptable carriers suitable for topical, enteral, for example oral or rectal, or parenteral administration and may be inorganic or organic and solid or liquid.
  • diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol, and/or lubricants, for example diatomaceous earth, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.
  • Tablets may also comprise binders, such as magnesium aluminium silicate, starches, such as corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxy- methylcellulose and/or polyvinylpyrrolidone, and, if desired, disintegrators, such as starches, agar, alginic acid or salts thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, colourings, flavourings and/or sweeteners.
  • binders such as magnesium aluminium silicate, starches, such as corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxy- methylcellulose and/or polyvinylpyrrolidone, and, if desired, disintegrators, such as starches, agar, alginic acid or salts thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, colourings, flavourings and/or sweeteners.
  • Such solutions are preferably isotonic aqueous solutions or suspensions, it being possible, for example in the case of lyophilised preparations that comprise the active ingredient on its own or together with a carrier, for example mannitol, for such solutions or suspensions to be made up before use.
  • the pharmaceutical compositions or preparations may be sterilised and/or comprise excipients, such as preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers.
  • compositions according to the invention which, if desired, may comprise further pharmacologically active substances, such as antibiotics, are produced in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes, and comprise, for example, approximately from 0.01 % to 90 %, in the case of lyophilisates up to 100 %, especially from approximately 0.1 % to approximately 50 %, more especially from 1 % to 30 %, active ingredient(s), a concentration of active ingredient of less than 1 % being suitable especially for topically administrable preparations.
  • the invention relates to Chondromyces crocatus DSM 8963 (mixed culture)as well as to Chondromyces crocatus DSM 10034or a strain derived therefrom provided that the derived strain is capable of producing a chondramide of the formula I wherein R 3 is hydrogen.
  • the vegetative cells of Cm c2 are cylindrical with broadly rounded ends,
  • yeast cells When yeast cells are present in the culture medium they are almost completely decomposed. After a few days fruiting bodies from 500 to 1000 ⁇ m in height may be formed: they consist of orange-coloured sporangioles, from 20 to 30 ⁇ m in diameter and from 30 to 45 ⁇ m in length, which are carried on a branched white stalk.
  • the interior of the sporangiols contains myxospores that are morphologically very similar to the vegetative cells but physiologically are dormant cells that are resistant to drying.
  • the strain grows, preferentially at 30°C, in small clumps of cells, both in shaken flasks at 160 rpm (100 ml of medium in a 250 ml Erlenmeyer flask or 400 ml of medium in a 1000 ml Erlenmeyer flask) and in bioreactors (tested up to a scale of 300 litres).
  • a suitable culture medium is, for example, Poll medium: Probion PS (single-cell protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; starch 0.3 %; MgSO 4 .7H 2 O 0.1 %; CaCl 2 .2H 2 O 0.05 %; 1 ml/litre standard trace element solution and 1 ml/litre standard vitamin solution (both Schlegel, Canale Mikrobiologie); pH 7.0.
  • the cultures are maintained at 30°C for 3 to 4 days.
  • the strain grows well also in culture media based on soybean meal. After separation of the accompanying bacterium described below, strain Cm c2 exhibits considerably poorer growth and lyses after a few cycles.
  • Strain Cm c2 can be preserved: for example by freezing vegetative cells from agar plates or liquid cultures in peptone solution at -80°C or in liquid nitrogen.
  • the pure strain, Cm c5 can be handled and cultivated in exactly the same manner and the same media.
  • the accompanying bacterium of Chondromyces crocatus, strain Cm c2 is gram-negative and rod-shaped. It can be separated from Cm c2 by plating of the mixed culture on complete culture media, for example nutrient agar. On those culture media it multiplies only slowly. In the liquid media mentioned under A.3.1. the bacterium multiplies approximately in parallel with strain Cm c2 but does not overgrow the latter.
  • An antibiotic activity against yeasts can be demonstrated in cell extracts of Cm c2.
  • the active substances are called chondramides.
  • No antibiotic activity can be demonstrated in cell extracts of the accompanying bacterium separated from the mixed culture. The same activity is observed in pure cultures of strain Cm c5.
  • chondramides For the qualitative detection of chondramides, cell masses of the production cultures are extracted with acetone. Aliquots of the concentrated extracts are tested against Candida albicans in an agar diffusion test. In a thin-layer chromatography test the different chondramides (A to D) are identified in comparison with isolated pure substance. Working-up, isolation and chemical characterisation are carried out in accordance with section B. A.6. Production conditions for chondramides:
  • Bioreactors 300 litre, supplied by Braun Melsungen, having two Intermag stirrers and a capacity of 200 litres.
  • Medium Probion PS (single-cell protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; MgSO 4 .7H 2 00.1 %; CaCl 2 .2H 2 O 0.05 %; standard trace element solution 1 ml/litre (Schlegel, dealt Mikrobiologie) and 0.23 mg/litre cyanocobalamin; pH 7.0.
  • 200 litres of medium are inoculated with 50 litres of culture from a well-grown culture from a seed fermentor.
  • the preferred cultivation temperature is 30°C.
  • the aeration rate is set at 33 standard litres/minute and the speed at 100 rpm. Because the medium produces a large amount of foam, 0.05 % of the anti-foam Tegosipon (Goldschmidt, Essen) is additionally added.
  • the p0 2 value which is 90 % saturation at the beginning of fermentation, decreases continuously to 60 % by the end of fermentation after 66 hours.
  • the pH value rises from 7.0 to 7.5 in the course of the fermentation.
  • Si-HPLC column: 20.5 mm internal diameter, 250 mm length, silica gel (Nucleosil 100, 7 ⁇ m, Macherey-Nagel, D ⁇ ren); eluant: petroleum ether/tert-butyl methyl ether/methanol 30:69.5:0.5; detection: UV absorption at 220 nm.
  • RP-HPLC column: 20.5 mm internal diameter, 250 mm length, RP-silica gel (Nucleosil C 18 , 7 ⁇ m, Macherey-Nagel, Diiren); eluant: methanol/water 60:40; detection: UV absorption at 220 nm.
  • the antibiotic activity is determined in an agar diffusion test with reference to the diameter of the inhibiting areola.
  • the chondramides inhibit the growth of some yeasts. At 20 ⁇ g/disc, chondramides are effective against:
  • the four chondramides act against mammalian cells in culture in the same concen ⁇ tration range and inhibit also the growth of cell lines that are resistant to other cytostatic drugs.
  • Tablets comprising 20 mg of active ingredient, that is to say one of the chondramides according to the invention, are produced in customary manner having the following composition:
  • the active ingredient is mixed with a portion of the wheat starch, with the lactose and the colloidal silicic acid and the mixture is then forced through a sieve, A further portion of the wheat starch is made into a paste with 5 times the amount of water on a water bath and the powder mixture is kneaded with the paste until a slightly plastic mass has been formed.
  • the plastic mass is pressed through a sieve of about 3 mm mesh size and dried and the resulting dry granules are again forced through a sieve.
  • the remainder of the wheat starch, the talc and the magnesium stearate are then mixed in and the mixture is compressed to form tablets each weighing 145 mg and having a breaking notch.

Abstract

The invention relates to chondramides of formula (I), wherein R1 = OCH¿3? and R?2¿ = H or R1 = OCH¿3? and R?2¿ = Cl or R1 = H and R2 = H or R1 = H and R2 = Cl and R3 = hydrogen, a C¿1-8?alkyl radical, and aryl radical, such as C6H6-, an aralkyl radical, a heterocyclic radical having an oxygen, sulfur or nitrogen atom, a formate radical, a hydrogen carbonate radical, an H2N-CO-O- radical, a C1-8alkoxy radical, an alkaryloxy radical, an alkoxyalkyl radical, such as CH3-O-CH2- or CH3-O-CH(CH3)-, or an acyloxyalkyl radical, such as alkyl-CO-O-CH2-, and the pharmaceutically acceptable alkali metal or alkaline earth metal salts thereof. The invention relates also to a production method, to compositions comprising chondramides and to cultures of microorganisms capable of producing a chondramide.

Description

Chondramides, production methods, compositions comprising chondramides and cultures for chondramide production
The invention relates to chondramides of the formula I
Figure imgf000003_0001
wherein
Ri = OCH3 and R2 = H or
R1 = OCH3 and R2 = Cl or
R1 = H and R2 = H or
R1 = H and R2 = Cl and
R3 = hydrogen, a Cι_8alkyl radical, an aryl radical, such as CgHg-, an alkaryl radical, a heterocyclic radical having an oxygen, sulfur or nitrogen atom, a formate radical, a hydrogen carbonate radical, an H2N-CO-O- radical, a C^alkoxy radical, an alkaryl- oxy radical, an al oxyalkyl radical, such as CH3-O-CH2- or CH3-0-CH(CH3)-, or an acyloxyalkyl radical, such as alkyl-CO-0-CH2-, and the pharmaceutically acceptable alkali metal or alkaline earth metal salts thereof.
When produced from cultures of Chondromyces crocatus the chondramides are obtained with a free hydroxy group (R3 = OH). These naturally produced chondramides represent a preferred group within the compounds of the formula I. The introduction of a substituent R3 can be carried out in accordance with methods widely used for the derivatization of a hydroxy group. In this context reference may be made to Houben-Weyl, Methoden der Organischen Chemie, 4th Edition, G. Thieme-Verlag, Stuttgart and NY. The species Chondromyces crocatus DSM 8963 referred to below represents a mixed culture contaminated with small rod-shaped bacilli of an unknown accompanying bacterium. In contrast thereto the species Chondromyces crocatus DSM 10034 represents a pure culture of the same species. This can be confirmed by PCR sequencing technology. For production purposes one can use either the mixed or preferably the pure culture.
The invention relates also to a chondramide having the empirical formula C36H46N4O7 and having the following spectra: UV (methanol); -^ [nm] (lg e):
222 (4.56), 279 (3.79), 290 (3.67); IR; v [cm"1] : 3354, 1731, 1637, 1516, 1457, 1212.
The invention relates also to a chondramide having the empirical formula C36H45CIN4O7 and having the following spectra: UV (methanol); ^ [nm] (lg e):
222 (4.57), 279 (3.93), 290 (3.76); IR; v [cm"1] : 3346, 2931, 1731, 1638, 1517, 1452, 1212.
The invention relates also to a chondramide having the empirical formula C35H44N4O6 and having the following spectra: UV (methanol); ^^ [nm] (lg e):
222 (4.60), 279 (3.82), 290 (3.70); IR; v [cm"1] : 1727, 1637, 1516, 1457.
The invention relates also to a chondramide having the empirical formula C35H43C1N406 and having the following spectra: UV (methanol); ^ [nm] (lg e):
222 (4.52), 279 (3.87), 290 (3.73); IR; v [cm"1] : 3407, 1726, 1640, 1516, 1452.
The invention relates also to chondramides that are producible produced as follows: (a) microorganism capable of producing a chondramide of the formula I wherein R3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative of the tribe Sorangineae, more preferably a representative of the genus Chondromyces, more preferably a representative of the species Condromyces crocatus and most preferably the species Chondromyces crocatus DSM 8963 or Chondromyces crocatus DSM 10034 is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts, and (b) the chondramides are concentrated, separated and isolated in a manner known per se.
The invention relates also to chondramides that are producible as follows:
(a) said microorganism capable of producing a chondramide of the formula I wherein R3 is hydrogen is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances, such as vitamin B12, and mineral salts,
(b) the cells are separated from the culture medium in the form of a moist biomass and extracted with an aliphatic ketone, such as acetone,
(c) the aqueous acetone extract is freed of organic solvent and the aqueous phase that remains is extracted with ethyl acetate,
(d) the extract is concentrated, optionally after drying, and taken up in a methanol/- water mixture,
(e) the resulting liquid mixture is extracted with heptane and the methanolic phase is isolated,
(f) the isolated methanolic phase is concentrated and subjected to reversed-phase medium pressure chromatography:
RP-silica gel (RP = reversed phase); eluant: methanol/water mixture, then methanol - gradient to 100 % methanol; with the aid of UV detection (for example absorption at approximately 220 nm)
- a fraction containing predominantly a first chondramide (A) and
- a fraction containing predominantly three further chondramides (B, C and D) are obtained,
(gl) the fraction containing predominantly chondramide A
- is subjected to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol; and then - to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and chondramide A is isolated, (g2) the fraction containing predominantly chondramides B, C and D (g21 ) is subjected to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol;
- a fraction containing predominantly chondramides B and C and
- a fraction containing predominantly chondramide C are obtained,
(g22) the fractions obtained according to (g21) are each subjected to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and chondramides B and D, and C are isolated.
The chondramides can preferably be produced by in step (a) using vitamin B12 as growth-promoting substance and/or a medium based on single-cell protein, such as Probion, and/or culturing at approximately 30°C.
The chondramides can preferably be produced by in step (d) taking up in a methanol/water mixture (approximately 95:5).
The chondramides can preferably be produced by in step (f) chromatographing with an RP-silica gel (for example g and, for example, 100 Angstrδm/20 μm) and/or with a methanol/water mixture (approximately 65:35).
The chondramides can preferably be produced by in step(s) (gl) and/or (g2) chromato¬ graphing with petroleum ether/tert-butyl methyl ether/methanol (approximately 30:69.5:0.5).
The chondramides can preferably be produced by in step(s) (gl) and/or (g2) chromato¬ graphing with an RP-silica gel (Cig) and/or with methanol/water (approximately 60:40).
The invention relates also to a method of producing chondramides wherein
(a) a microorganism capable of producing a chondramide of the formula I wherein R3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative of the tribe Sorangineae, more preferably a representative of the genus Chondromyces, more preferably a representative of the species Condromyces crocatus and most preferably the species Chondromyces crocatus DSM 8963 or Chondromyces crocatus DSM 10034is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts, and (b) the chondramides are concentrated, separated and isolated in a manner known per se.
The invention relates also to a method of producing chondramides wherein:
(a) said microorganism capable of producing a chondramide of the formula I wherein R3 is hydrogen is cultured aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances, such as vitamin B12 and mineral salts,
(b) the cells are separated from the culture medium in the form of a moist biomass and extracted with acetone,
(c) the aqueous acetone extract is freed of organic solvent and the aqueous phase that remains is extracted with ethyl acetate,
(d) the extract is dried, concentrated and taken up in a methanol/water mixture,
(e) the resulting liquid mixture is extracted with heptane and the methanolic phase is isolated,
(f) the isolated methanolic phase is concentrated and subjected to reversed-phase medium pressure chromatography:
RP-silica gel (RP = reversed phase); eluant: methanol/water mixture; with the aid of UV detection (absorption at approximately 220 nm)
- a fraction containing predominantly a first chondramide (A) and
- a fraction containing predominantly three further chondramides (B, C and D) are obtained,
(gl) the fraction containing predominantly chondramide A
- is subjected to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol; and then - to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and chondramide A is isolated, (g2) the fraction containing predominantly chondramides B, C and D (g21) is subjected to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol;
- a fraction containing predominantly chondramides B and C and
- a fraction containing predominantly chondramide C are obtained,
(g22) the fractions obtained are each subjected to reversed-phase HPLC chromato¬ graphy: RP-silica gel; eluant: methanol/water; and chondramides B and D, and C are isolated.
The invention relates also to a pharmaceutical composition comprising at least one of the above-mentioned chondramides together with a pharmaceutically acceptable carrier.
The invention relates especially to such a composition having anti-fungal and anti- neoplastic action, also against cell lines that are resistant to chemotherapeutic drugs.
The invention relates also to an antibiotic composition comprising at least one of the above-mentioned chondramides together with a suitable carrier.
Compositions according to the invention may comprise an effective amount, especially a prophylactically or therapeutically effective amount, of the active ingredient together with pharmaceutically acceptable carriers that are suitable for topical, enteral, for example oral or rectal, or parenteral administration and may be inorganic or organic and solid or liquid. For oral administration there are used especially tablets or gelatin capsules comprising the active ingredient together with diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol, and/or lubricants, for example diatomaceous earth, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Tablets may also comprise binders, such as magnesium aluminium silicate, starches, such as corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxy- methylcellulose and/or polyvinylpyrrolidone, and, if desired, disintegrators, such as starches, agar, alginic acid or salts thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, colourings, flavourings and/or sweeteners. The pharmaceutically or pharmacologically active compounds of the present invention can also be used in the form of parenterally administrable preparations or in the form of infusion solutions. Such solutions are preferably isotonic aqueous solutions or suspensions, it being possible, for example in the case of lyophilised preparations that comprise the active ingredient on its own or together with a carrier, for example mannitol, for such solutions or suspensions to be made up before use. The pharmaceutical compositions or preparations may be sterilised and/or comprise excipients, such as preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical compositions according to the invention, which, if desired, may comprise further pharmacologically active substances, such as antibiotics, are produced in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes, and comprise, for example, approximately from 0.01 % to 90 %, in the case of lyophilisates up to 100 %, especially from approximately 0.1 % to approximately 50 %, more especially from 1 % to 30 %, active ingredient(s), a concentration of active ingredient of less than 1 % being suitable especially for topically administrable preparations.
Finally, the invention relates to Chondromyces crocatus DSM 8963 (mixed culture)as well as to Chondromyces crocatus DSM 10034or a strain derived therefrom provided that the derived strain is capable of producing a chondramide of the formula I wherein R3 is hydrogen.
The invention is described in greater detail below, especially by means of experimental data.
A. Production conditions
Though the following protocol refers specifically to the mixed culture of Chondromyces crocatus, strain Cm c2 it also applies to the any pure cultures of Chondromyces crocatus. Moreover, the skilled artisan can easily adapt this protocol for each other microorganism suitable for the production of a chondramide. A.1. Production culture:
The bacterium Chondromyces crocatus, strain Cm c2, tribe Sorangineae, order Myxococcales, contaminated with small rod-shaped bacilli of an unknown accompanying bacterium.
Further strains of Chondromyces crocatus have also been found to grow well in the absence of the symbiotic bacterium, i. e., completely pure culture. These strains still produce chondramides, e.g., the pure strain Cm c5 (DSM 10034) isolated at the GBF from a sample collected in Brazil.
A.2. Origin of the production mixed culture:
Isolated in June 1985 at the GBF (Gesellschaft fur Biotechnologische Forschung mbH) from a soil sample from Madeira.
A.3. Description of the production mixed culture:
A.3.1. The vegetative cells of Cm c2 are cylindrical with broadly rounded ends,
0.8 x 6 to 8 μm. On solid culture media the organism grows well in the presence of the accompanying bacterium on yeast agar, for example VY/2 agar (baker's yeast 0.5 %, based on fresh weight; CaCl2«2H2O 0.1 %, cyanocobalamin 0.5 mg/1, agar 1.5 %; pH 7.2). No growth is observed on pure glucose/mineral salt media. Proteins, for example casein or single-cell proteins, are hydrolysed. Chitin is not attacked. The strain grows preferentially at 30°C, in a neutral pH range and aerobically. On suitable culture media the colony gradually spreads over the culture plate. Short, wide, radial channels are formed on the surface. When yeast cells are present in the culture medium they are almost completely decomposed. After a few days fruiting bodies from 500 to 1000 μm in height may be formed: they consist of orange-coloured sporangioles, from 20 to 30 μm in diameter and from 30 to 45 μm in length, which are carried on a branched white stalk. The interior of the sporangiols contains myxospores that are morphologically very similar to the vegetative cells but physiologically are dormant cells that are resistant to drying.
In liquid media the strain grows, preferentially at 30°C, in small clumps of cells, both in shaken flasks at 160 rpm (100 ml of medium in a 250 ml Erlenmeyer flask or 400 ml of medium in a 1000 ml Erlenmeyer flask) and in bioreactors (tested up to a scale of 300 litres). A suitable culture medium is, for example, Poll medium: Probion PS (single-cell protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; starch 0.3 %; MgSO4.7H2O 0.1 %; CaCl2.2H2O 0.05 %; 1 ml/litre standard trace element solution and 1 ml/litre standard vitamin solution (both Schlegel, Allgemeine Mikrobiologie); pH 7.0. The cultures are maintained at 30°C for 3 to 4 days. The strain grows well also in culture media based on soybean meal. After separation of the accompanying bacterium described below, strain Cm c2 exhibits considerably poorer growth and lyses after a few cycles.
Strain Cm c2 can be preserved: for example by freezing vegetative cells from agar plates or liquid cultures in peptone solution at -80°C or in liquid nitrogen. The pure strain, Cm c5 can be handled and cultivated in exactly the same manner and the same media.
A.3.2. The accompanying bacterium of Chondromyces crocatus, strain Cm c2, is gram-negative and rod-shaped. It can be separated from Cm c2 by plating of the mixed culture on complete culture media, for example nutrient agar. On those culture media it multiplies only slowly. In the liquid media mentioned under A.3.1. the bacterium multiplies approximately in parallel with strain Cm c2 but does not overgrow the latter.
A.4. Properties of the production cultures:
An antibiotic activity against yeasts (for example Candida albicans) can be demonstrated in cell extracts of Cm c2. The active substances are called chondramides. No antibiotic activity can be demonstrated in cell extracts of the accompanying bacterium separated from the mixed culture. The same activity is observed in pure cultures of strain Cm c5.
A.5. Detection of chondramides:
For the qualitative detection of chondramides, cell masses of the production cultures are extracted with acetone. Aliquots of the concentrated extracts are tested against Candida albicans in an agar diffusion test. In a thin-layer chromatography test the different chondramides (A to D) are identified in comparison with isolated pure substance. Working-up, isolation and chemical characterisation are carried out in accordance with section B. A.6. Production conditions for chondramides:
In shaken flasks the chondramides are formed during growth and attain the highest activity after 3 to 4 days at the end of the logarithmic phase up to the early stationary phase.
Example of fermentation in a bioreactor
Bioreactors (300 litre), supplied by Braun Melsungen, having two Intermag stirrers and a capacity of 200 litres. Medium: Probion PS (single-cell protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; MgSO4.7H200.1 %; CaCl2.2H2O 0.05 %; standard trace element solution 1 ml/litre (Schlegel, Allgemeine Mikrobiologie) and 0.23 mg/litre cyanocobalamin; pH 7.0. 200 litres of medium are inoculated with 50 litres of culture from a well-grown culture from a seed fermentor. The preferred cultivation temperature is 30°C. The aeration rate is set at 33 standard litres/minute and the speed at 100 rpm. Because the medium produces a large amount of foam, 0.05 % of the anti-foam Tegosipon (Goldschmidt, Essen) is additionally added. The p02 value, which is 90 % saturation at the beginning of fermentation, decreases continuously to 60 % by the end of fermentation after 66 hours. The pH value rises from 7.0 to 7.5 in the course of the fermentation.
B. Isolation and characterisation
1. Isolation
Approximately 2.8 kg of moist biomass are obtained by centrifugation of a 250 litre culture of the strain Cm c2 and are then extracted in succession with 9 litres and 6 litres of acetone. The combined acetone extracts are freed of solids by filtration and concentrated in vacua until the aqueous phase remains. The aqueous phase is extracted with ethyl acetate. The extract is dried with sodium sulfate and concentrated in vacuo. The residue (32 g) is taken up in 500 ml of methanol/water (95:5) and extracted with 800 ml of heptane. After concentration of the methanol phase in vacuo there remain 11.7 g of crude product which is separated in 3 portions by reversed-phase medium pressure chromato¬ graphy [glass column: 60 mm internal diameter, 530 mm length (Kronwald Separations- technik, Sinsheim) filled with RP-silica gel Eurosil Bioselect 100-20 C18, 15 to 25 μm; (Knauer, Berlin); eluant: methanol/water 65:35 for 140 min, then gradient to 80 % methanol in 90 min, 80 % methanol for 100 min and gradient to 100 % methanol in 60 min, flow = 36 ml/min; detection: UV absorption at 254 nm]. The peaks at tR = 63 min (fraction 1) and tR = 71 min (fraction 2) are combined without the preceding shoulder, concentrated in vacuo until the aqueous phase remains and extracted with dichloromethane.
The residue from fraction 1 (176 mg) is purified in two portions by Si-HPLC (see below) (flow = 13 ml/min). The fractions of the main peak at about tR = 15 min are combined and concentrated in vacuo to 60 mg of crude chondramide A. 46 mg of pure chondramide A (tR = 29 min) are isolated therefrom by means of RP-HPLC (see below) (flow =
13 ml/min). Fraction 2 (540 mg) is separated in 5 portions by Si-HPLC (see below) (flow = 14 ml/min) into fraction 2.1 (tR = 10 min, 120 mg) and fraction 2.2 (tR = 15.5 min, 171 mg). Fraction 2.1 is separated in two chromatography runs by RP-HPLC (see below) (flow = 13 ml/min) into 61 mg of chondramide B (tR = 36.5 min) and 14 mg of chondramide D (tR = 52 min).
Fraction 2.2 is separated in two chromatography runs by RP-HPLC (see below) (flow =
14 ml/min) into a further 35 mg of chondramide A (tR = 28 min) and 86 mg of chondramide C (tR = 39 min).
Si-HPLC: column: 20.5 mm internal diameter, 250 mm length, silica gel (Nucleosil 100, 7 μm, Macherey-Nagel, Dϋren); eluant: petroleum ether/tert-butyl methyl ether/methanol 30:69.5:0.5; detection: UV absorption at 220 nm.
RP-HPLC: column: 20.5 mm internal diameter, 250 mm length, RP-silica gel (Nucleosil C18, 7 μm, Macherey-Nagel, Diiren); eluant: methanol/water 60:40; detection: UV absorption at 220 nm.
2. Structures
Figure imgf000014_0001
chondramide A R1 = OCH3 R2 = H C36H46N407 (M = 646.8) chondramide B R1 = OCH3 R2 = C1 C36H45C1N407 (M = 681.2) chondramide C R^ H R2 = H CasH^Og (M = 616.7) chondramide D R! = H R2 = C1 C35H43C1N406 (M = 651.2)
3. Characterisation of chondramides
TLC: Aluminium sheets with 0.2 mm silica gel 60 F254 (Merck, Darmstadt); eluant: toluene/ethanol (9:1), chondramide A Rf = 0.19, chondramide B Rf = 0.25, chondramide C Rf = 0.20, chondramide A Rf = 0.26, and methylene chloride/methanol (9:1) all Rf = 0.46, UV quenching at 254 nm, staining with vanillin/sulfuric acid reagent, chondramides A and C violet, chondramides B and D light-blue after brief heating at 120°C.
HPLC analysis: Column 2 mm internal diameter, 125 mm length with preliminary column 11 mm (Macherey-Nagel, Dϋren) filled with RP-silica gel (Nucleosil 120-5 C18, 5 μm); eluant gradient with methanol/water, 65 % to 95 % methanol in 8 min, then 95 % methanol isocratically, flow = 0.3 ml/min, UV detection at 222 nm, chondramide A tR = 3.88 min; chondramide B tR = 4.27 min; chondramide C tR = 4.37 min; chondramide D tR = 4.81 min.
Specific rotation values fαl " p of chondramides in methanol
22 c
Chondramide [ ]D
[g/100 ml]
A + 2.1 2.0
B + 20.8 2.0
C + 40.2 2.4
D + 49.6 1.3
UV data of chondramides in methanol
Chondramide 222 279 290 Λmax [nm]
A 4.56 3.79 3.67 B 4.57 3.93 3.76 lg 6 C 4.60 3.82 3.70 D 4.52 3.87 3.73 data of chondramides f υ [cm'1] intensity)
Chondramide A Chondramide B Chondramide C Chondramide D lmg/lOOm KBr lmglOOmgKBr lmg/100mgKBr 0.5mg/100mg KBr
3354 s 3346 S 3337 m 3407 s
2931 m 2931 s 2932 m 2929 m
1731 s 1731 s 1727 s 1726 s
1637 s 1638 s 1637 s 1640 s
1516 s 1517 s 1516 s 1516 s
1457 s 1452 s 1457 s 1452 s
1212 s 1212 s 1268 m 1270 m
1104 m 1105 m 1206 m 1207 m
836 w 837 w 1088 m 1079 m
742 m 742 m 835 w 743 w 742 m
NMR data of chondramides
Η-NMR data of chondramides in [d6]DMSO [600 MHz]
Figure imgf000017_0001
Figure imgf000018_0001
NMR data of chondramides (continued)
Η-NMR data of chondramides in [d6]DMSO [600 MHz]
No. Chondramide A Chondramide B Chondramide C Chondramide D
δπ mult.J" δH mult . J κ mult . J δH mult.J" lO'-H 3.06 s 3.06 s _ — _ •
3'-NH 9.00 d 9.6 9.18 d 9.6 9.72 d 9.4 8.90 d
7' -OH 9.35 s br 9.33 s br 9.32 s br 9.35 s Os
2' '-H 5.48 dd 4.9, 11.1 5.54 dd 11.6, 4 3 5.50 dd 10.0, 5.3 5.56 dd
3"~Ha 2.97 dd 11.1, 14.5 2.97 dd 11.7, l4.8 2.98 dd 9.9, 1 4.7 3.00 dd
3"-Hb 2.64 dd 4.9, 14.5 2.48 m M_ 2.85 dd 5.3, 1 1.2.73 dd 14.6, •
5' '-H 6.99 d 1 -. - - - 7.04 d 2 - . - -
7"-H 7.26 d 8.1 7.18 d 8 7.28 d 8.2 7.21 d
8"-H 7.01 dd 7.2, 8.1 7.05 dd 8, 7 br 7.02 dd 7, 8, br 7.06 ddd
9' '-H 6.94 dd 7.2, 7.8 7.00 dd 7, 8 br 7.00 dd 1, 8, br 7.01 ddd
10' '-H 7.58 d 7.8 7.58 d 8 7.60 d 7.6 7.61 d
12' '-H 3.01 s 3.01 s 2.99 s - 3.00 s
NH' ' 10.72 d 1 11.52 s 10.74 d 2 11.55 s
2" ,-H 4.61 dq 7.0, 9.5 4.57 dq 7.0, 9.:$ 4.61 dq 8.8, 1 .1 4.56 dq
3" '-H 0.57 d 7.0 0.48 d 7.0 0.66 d 7.1 0.54 d
3' ' '-N 17.81 d 9.5 7.80 d 9.3 7.82 d 8.8 7.80 d
Wbelow DMSO signal
Figure imgf000018_0002
C-NMR data of chondramides in [d6]DMSO [150 MHz]
o. Chondramide A Chondramide B Chondramide C Chondramide D δc mult. δc mult. δc mult. °c mult.
172.89 s 172.86 s 173.17 s 173.11 s
37.35 d 37.29 d 37.59 d 37.53 d
44.85 t 45.02 t 44.76 t 44.91 t
133.05 s 133.07 s 133.18 s 133.18 s
127.06 d 127.01 d 127.07 d 127.11 d
36.91 d 36.92 d 36.66 d 36.68 d
77.32 d 77.39 d 77.39 d 76.79 d
17.62 q 17.53 q 17.24 q 17.67 q
18.57 q 18.54 q 18.78 q 18.75 q 0 15.60 q 15.52 q 15.75 q 15.67 q 1 17.72 q 17.64 q 17.65 q 17.27 q ' 171.55 s 171.71 s 171.35 s 171.49 s ' 81.67 d 81.48 d 41.86 t 41.67 t ' 53.54 d 53.69 d 49.90 d 50.09 d ' 130.25 s 130.34 s 132.57 s 132.62 s ' 128.77 d 128.81 d 127.07 d 127.11 d ' 114.77 d 114.82' d 115.04 d 115.09 d ' 156.48 s 156.49 s 156.25 s 156.31 s ' 114.77 d 114.82 d 115.04 d 115.09 d ' 128.77 d 128.81 d 127.07 d 127.11 d 0' 57.03 q 57.06 q - .- - -.- - ' ' 169.16 s 168.60 s 169.06 s 168.58 s ' ' 53.87 d 52.77 d 54.31 d 53.25 d " 25.52 t 24.50 t 25.18 t 24.18 t " 109.02 s 105.54 s 109.25 s 105.78 s ' ' 123.43 d 121.54 s 123.42 d 121.51 s " 136.05 s 134.39 s 134.08 s 134.42 s " 110.99 d 110.37 d 111.04 d 110.42 d " 120.69 d 121.45 d 120.73 d 121.45 d " 117.99 d 119.01 d 118.04 d 119.04 d 0' 118.56 d 118.60 d 118.52 d 118.53 d 1' 127.11 s 127.08 s 127.07 s 127.08 s 2' 29.81 q 30.26 q 29.92 q 30.33 q " 172.78 s 172.77 s 172.53 s 172.47 s ' ' 43.32 d 43.17 s 43.62 d 43.46 d " 17.72 q 17.64 q 17.82 q 17.56 q MS data of chondramides
Chondramide A
EI-MS (70 eV, 180°C): mlz (%) = 646 (12) [M+], 616 (1), 574 (1), 525 (3), 485 (3), 436 (1), 356 (11), 291 (42), 267 (9), 236 (5), 208 (7), 173 (19), 170 (100), 130 (26).- (+VFAB-MS (3-NBA): mlz (%) = 669 (100) [(M + Na)+], 647 (38) [(M + H)+].- High resolution: calc. 646.3367 found 646.3388 C36H46N4θ7
Chondramide B
EI-MS (70 eV, 190°C): mlz (%) = 682 (2.7), 680 (6.4), 645 (100), 615 (2), 559 (15),
544 (6), 517 (3), 485 (9), 470 (4), 461 (53), 430 (5), 356 (39), 325 (38), 267 (68), 236 (36),
208 (31), 207 (50), 204 (62), 185 (11), 171 (18), 164 (33), 150 (10), 137 (11), 122 (25).-
(+VFAB-MS (3-NBA : mlz (%) = 681 (59) [(35C1-M + H)+], 682 (16), 683 (15)
[(37C1-M + H)+], 645 (15) [(M-C1)+], 169 (100).-
(-)-FAB-MS (3-NBA): mlz (%) = 833 (10) [(M + 3-NBA - H)"], 679 (100) [(35C1-M - H)"],
680 (40), 681 (36) [(37C1-M - H)'].-
High resolution: calc. 680.2977 found 680.2971 C36H45 35ClN4O7
Chondramide C
EI-MS (70 eV, 190°C): mlz (%) = 616 (100), 577 (1), 487 (3), 446 (94), 415 (3), 350 (8), 267 (41), 236 (24), 235 (10), 208 (23), 186 (13), 173 (22), 170 (29), 162 (35), 130 (39).- High resolution: calc. 616.3261 found 616.3260 CssH^N^
Chondramide D
EI-MS (70 eV, 200°C): mlz (%) = 650 (0.5) [(M35C1)+], 615 (17), 487 (0.5), 431 (8),
400 (2), 349 (2), 267 (13), 236 (9), 208 (8), 207 (6), 185 (10), 171 (5), 149 (11), 145 (16),
129 (8), 111 (12), 109 (8).-
CI-MS (isobutane : mlz (%) = 651 (3.01) [(35C1-M + H)+], 652 (1.02) [(13C35C1-M + H)+],
653 (1.16) [(37C1-M + H)+], 152 (100).-
High resolution: calc. 650.2871 found 650.2857 C35H43ClN4O6 C. Biological characterisation of chondramides
Cl. Antimicrobial activity
The antibiotic activity is determined in an agar diffusion test with reference to the diameter of the inhibiting areola. The chondramides inhibit the growth of some yeasts. At 20 μg/disc, chondramides are effective against:
Test organism inhibiting zone (mm)
Chondramide
A B C D
Candida albicans 13 14 9 12 Candida utilis 0 trace 0 0 Hansenula anomala 12 13 7 12 Lipomyces lipofer 11 14 9 12 Schizosaccharomyces pombe 0 16 0 0 Torulopsis glabrata 9 14 9 11
C.2. Cytostatic activity
The four chondramides act against mammalian cells in culture in the same concen¬ tration range and inhibit also the growth of cell lines that are resistant to other cytostatic drugs.
D. Formulation example
Tablets comprising 20 mg of active ingredient, that is to say one of the chondramides according to the invention, are produced in customary manner having the following composition:
Composition Amount active ingredient 20 mg wheat starch 60 mg lactose 50 mg colloidal silicic acid 5 mg talc 9 mg magnesium stearate 1 mg total 145 mg For preparation, the active ingredient is mixed with a portion of the wheat starch, with the lactose and the colloidal silicic acid and the mixture is then forced through a sieve, A further portion of the wheat starch is made into a paste with 5 times the amount of water on a water bath and the powder mixture is kneaded with the paste until a slightly plastic mass has been formed.
The plastic mass is pressed through a sieve of about 3 mm mesh size and dried and the resulting dry granules are again forced through a sieve. The remainder of the wheat starch, the talc and the magnesium stearate are then mixed in and the mixture is compressed to form tablets each weighing 145 mg and having a breaking notch.
Deposits of microorganisms
Cm c2 (plus the accompanying bacterium / mixed culture) has been deposited on February 4, 1994 (4.2.94) in accordance with the Budapest Treaty under No. DSM 8963 at the Deutsche Sammlung von Mikroorganismen (DSM) (Mascheroder Weg lb, D-38124 Braunschweig [Germany]) and the pure culture of Cm c5 has been deposited on June 6, 1995 (6.6.95) under No. DSM 10034 with the same depository.
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
A. The indications made below relate to the microorganism referred to in the description on page 20 , line 10-16
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | [
Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM)
Address of depositary institution (including postal code and country)
Mascheroder Weg IB D-38124 Braunschweig Germany
Date of deposit Accession Number
04 February 1994 (04.02.94) DSM 8963
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet ~~
We request the Expert Solution where available
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the international Bureau later (specify the general nature of theindications e.g., "Accession Number of Deposit")
For International Bureau use only
I ] This sheet was received by the International Bureau on:
Authorized officer
Figure imgf000023_0001
Form PCT RO/134 (July 1992)

Claims

What is claimed is:
1. A chondramide of the formula I
Figure imgf000024_0001
wherein
R1=OCH3andR2 = Hor
R1 = OCH3 and R2 = Cl or
R1=HandR2 = Hor
R1=HandR2 = Cland
R3 = hydrogen, a Cι.8alkyl radical, an aryl radical, an aralkyl radical, a heterocyclic radical having an oxygen, sulfur or nitrogen atom, a formate radical, a hydrogen carbonate radical, an H2N-CO-0- radical, a Cμgalkoxy radical, an alkaryloxy radical, an alkoxyalkyl radical or an acyloxyalkyl radical, or a pharmaceutically acceptable alkali metal or alkaline earth metal salt thereof.
2. A chondramide of the formula I according to claim 1, wherein R1=OCH3andR2 = Hor R1'=OCH3andR2 = Clor R1=HandR2 = Hor R1=HandR2 = Cland R3 = hydrogen.
3. A chondramide having the empirical formula C36H46N407 and having the following spectra:
UV (methanol); λ^ [nm] (lg e): 222 (4.56), 279 (3.79), 290 (3.67); IR; v [cm"1] : 3354, 1731, 1637, 1516, 1457, 1212.
4. A chondramide having the empirical formula C36H45C1N407 and having the following spectra: UV (methanol); λ^ [nm] (lg €): 222 (4.57), 279 (3.93), 290 (3.76); IR; v [cm ] : 3346, 2931, 1731, 1638, 1517, 1452, 1212.
5. A chondramide having the empirical formula C35H44N406 and having the following spectra: UV (methanol); λ^ [nm] (lg e): 222 (4.60), 279 (3.82), 290 (3.70); IR; v [cm'1] : 1727, 1637, 1516, 1457.
6. A chondramide having the empirical formula C35H43C1N406 and having the following spectra: UV (methanol); λmax [nm] (lg e): 222 (4.52), 279 (3.87), 290 (3.73); IR; v [cm"1] : 3407, 1726, 1640, 1516, 1452.
7. A chondramide producible by a process comprising the following steps:
(a) culturing Chondromyces crocatus DSM 8963 (mixed culture) or Chondromyces crocatus DSM 10034 (pure culture) aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts, and
(b) concentrating the chondramides, separating and isolating them in a manner known per se.
8. A chondramide according to claim 7, producible by a process comprising the following steps:
(a) culturing Chondromyces crocatus DSM 8963 (mixed culture) or Chondromyces crocatus DSM 10034 (pure culture) aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts,
(b) separating the cells from the culture medium in the form of a moist biomass and extracting them with an aliphatic ketone,
(c) deleting the solvent from the aqueous extract and extracting the aqueous phase that remains with ethyl acetate,
(d) concentrating the extract, optionally after drying, and taking it up in a methanol/- water mixture,
(e) extracting the resulting liquid mixture with heptane and isolating the methanolic phase,
(f) concentrating the isolated methanolic phase and subjecting it to reversed-phase medium pressure chromatography: RP-silica gel (RP = reversed-phase); eluant: methanol/water mixture, then methanol - gradient to 100 % methanol; with the aid of UV detection,
- collecting a fraction containing predominantly a first chondramide (A) and
- collecting a fraction containing predominantly three further chondramides (B, C and D),
(gl) subjecting the fraction containing predominantly chondramide A to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol; and then
- to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and isolating chondramide A, (g2) subjecting the fraction containing predominantly chondramides B, C and D (g21) to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol;
- collecting a fraction containing predominantly chondramides B and C and
- a fraction containing predominantly chondramide C are obtained,
(g22) subjecting each of the fractions obtained according to (g21) to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and isolating chondramides B and D, and C.
9. A chondramide according to either claim 7 or claim 8, wherein in step (a) there is used vitamin B 12 as growth-promoting substance and/or a medium based on single-cell protein and/or cultivation is carried out at approximately 30°C.
10. A chondramide producible either in accordance to claim 8 or claim 9 wherein the extract of step (d) is taken up in a methanol/water mixture (approximately 95:5).
11. A chondramide producible in accordance to any one of claims 8 to 10 wherein the isolated methanolic phase of step (f) is chromatographed with an RP-silica gel and/or with a methanol/water mixture (approximately 65:35).
12. A chondramide producible in accordance to any one of claims 8 to 11 wherein the fraction obtained in step(s) (gl) and/or (g2) is chromatographed with petroleum ether/- tert-butyl methyl ether/methanol (approximately 30:69.5:0.5).
13. A chondramide producible in accordance to any one of claims 8 to 12 wherein the fraction obtained in step(s) (gl) and/or (g2) is chromatographed with an RP-silica gel (C18) and/or with methanol/water (approximately 60:40).
14. A method of producing chondramides comprising the following steps:
(a) culturing a microorganism capable of producing a chondramide of the formula I wherein R3 is hydrogen, preferably a representative of the order Myxococcales, more preferably a representative of the family Myxococcaceae, more preferably a representative of the tribe Sorangineae, more preferably a representative of the genus Chondromyces, more preferably a representative of the species Condromyces crocatus and most preferably the species Chondromyces crocatus DSM 8963 or Chondromyces crocatus DSM 10034 aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts, and
(b) concentrating the chondramides, separating and isolating them in a manner known per se.
15. A method of producing chondramides according to claim 14 comprising the following steps:
(a) culturing said microorganism aerobically in a medium containing carbon sources, nitrogen sources, growth-promoting substances and mineral salts,
(b) separating the cells from the culture medium in the form of a moist biomass and extracting them with an aliphatic ketone,
(c) deleting the solvent from the aqueous extract and extracting the aqueous phase that remains with ethyl acetate,
(d) concentrating the extract, optionally after drying, and taking it up in a methanol/- water mixture,
(e) extracting the resulting liquid mixture with heptane and isolating the methanolic phase,
(f) concentrating the isolated methanolic phase and subjecting it to reversed-phase medium pressure chromatography: RP-silica gel (RP = reversed-phase); eluant: methanol/water mixture, then methanol - gradient to 100 % methanol; with the aid of UV detection,
- collecting a fraction containing predominantly a first chondramide (A) and
- collecting a fraction containing predominantly three further chondramides (B, C and D),
(gl) subjecting the fraction containing predominantly chondramide A to HPLC chromatography: silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol; and then
- to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and isolating chondramide A, (g2) subjecting the fraction containing predominantly chondramides B, C and D (g21 ) to HPLC chrom atography : silica gel; eluant: petroleum ether/tert-butyl methyl ether/methanol;
- collecting a fraction containing predominantly chondramides B and C and
- a fraction containing predominantly chondramide C are obtained,
(g22) subjecting each of the fractions obtained according to (g21) to reversed-phase HPLC chromatography: RP-silica gel; eluant: methanol/water; and isolating chondramides B and D, and C.
16. A pharmaceutical composition comprising at least one chondramide according to any one of claims 1 to 12 together with a pharmaceutically acceptable carrier.
17. A pharmaceutical composition according to claim 15 having anti-fungal and anti- neoplastic action, also against cell lines that are resistant to chemotherapeutic drugs.
18. An antibiotic composition comprising a chondramide according to any one of claims 1 to 13 together with a suitable carrier.
19. Microorganism capable of producing a chondramide of the formula I
Figure imgf000029_0001
wherein
R1 = OCH3 and R2 = H or R1 = OCH3 and R2 = Cl or Rx = H and R2 = H or R1 = H and R2 = Cl and R3 = hydrogen.
20. Microorganism according to claim 19 selected from the group consisting of a representative of the order Myxococcales, a representative of the family Myxococcaceae, a representative of the tribe Sorangineae, a representative of the genus Chondromyces, a representative of the species Condromyces crocatus and the species Chondromyces crocatus DSM 8963 or Chondromyces crocatus DSM 10034 or a strain derived therefrom which is still capable of producing a chondramide.
PCT/EP1995/002294 1994-06-16 1995-06-13 Chondramides, production methods, compositions comprising chondramides and cultures for chondramide production WO1995034558A1 (en)

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AU27921/95A AU2792195A (en) 1994-06-16 1995-06-13 Chondramides, production methods, compositions comprising chondramides and cultures for chondramide production

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DE19944421113 DE4421113A1 (en) 1994-06-16 1994-06-16 Chondramide, extraction process, means with chondrams and mixed culture for chondramide production
DEP4421113.9 1994-06-16

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CN102369211A (en) * 2009-02-13 2012-03-07 诺瓦提斯公司 Nucleic acid molecule of a biosynthetic cluster encoding non ribosomal peptide synthases and uses thereof
US8415305B2 (en) 2007-08-17 2013-04-09 Novartis Ag Use of cyclic depsipeptides to inhibit kallikrein 7
US8614289B2 (en) 2011-04-20 2013-12-24 Novartis Ag Processes for the manufacture of macrocyclic depsipeptides and new intermediates
US8680054B2 (en) 2011-04-20 2014-03-25 Novartis Ag Suspension type topical formulations comprising cyclic depsipeptide
US8987413B2 (en) 2012-10-09 2015-03-24 Novartis Ag Aldehyde acetal based processes for the manufacture of macrocyclic depsipeptides and new intermediates
US9067978B2 (en) 2012-10-09 2015-06-30 Novartis Ag Solution phase processes for the manufacture of macrocyclic depsipeptides and new intermediates

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Title
T. M. ZABRISKIE ET AL: "Jaspamide, a modified peptide from a Jaspis Sponge, with insecticidal and antifungal activity", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY., vol. 108, no. 11, 1986, GASTON, PA US, pages 3123 - 3124 *

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US9127044B2 (en) 2007-08-17 2015-09-08 Novartis Ag Cyclic depsipeptides
JP2010536731A (en) * 2007-08-17 2010-12-02 ノバルティス アーゲー Cyclic depsipeptides
US8178650B2 (en) 2007-08-17 2012-05-15 Novartis Ag Cyclic depsipeptides
EA017735B1 (en) * 2007-08-17 2013-02-28 Новартис Аг Cyclic depsipeptides
US8415305B2 (en) 2007-08-17 2013-04-09 Novartis Ag Use of cyclic depsipeptides to inhibit kallikrein 7
CN103298827A (en) * 2007-08-17 2013-09-11 诺瓦提斯公司 Cyclic depsipeptides
WO2009024527A1 (en) * 2007-08-17 2009-02-26 Novartis Ag Cyclic depsipeptides
CN103298827B (en) * 2007-08-17 2017-05-31 诺华股份有限公司 Cyclic ester peptide
CN102369211A (en) * 2009-02-13 2012-03-07 诺瓦提斯公司 Nucleic acid molecule of a biosynthetic cluster encoding non ribosomal peptide synthases and uses thereof
JP2012517801A (en) * 2009-02-13 2012-08-09 ノバルティス アーゲー Biosynthetic cluster nucleic acid molecules encoding non-ribosomal peptide synthases and uses thereof
US8614289B2 (en) 2011-04-20 2013-12-24 Novartis Ag Processes for the manufacture of macrocyclic depsipeptides and new intermediates
US9114110B2 (en) 2011-04-20 2015-08-25 Novartis Ag Suspension type topical formulations comprising cyclic depdipeptide
US9278997B2 (en) 2011-04-20 2016-03-08 Novartis Ag Processes for the manufacture of macrocyclic depsipeptides and new intermediates
US8680054B2 (en) 2011-04-20 2014-03-25 Novartis Ag Suspension type topical formulations comprising cyclic depsipeptide
US9067978B2 (en) 2012-10-09 2015-06-30 Novartis Ag Solution phase processes for the manufacture of macrocyclic depsipeptides and new intermediates
US8987413B2 (en) 2012-10-09 2015-03-24 Novartis Ag Aldehyde acetal based processes for the manufacture of macrocyclic depsipeptides and new intermediates
US9493512B2 (en) 2012-10-09 2016-11-15 Novartis Ag Solution phase processes for the manufacture of macrocyclic depsipeptides and new intermediates

Also Published As

Publication number Publication date
AU2792195A (en) 1996-01-05
DE4421113A1 (en) 1995-12-21

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