USOO537,000A United States Patent (19) 11 Patent Number: 5,371,000 Hummel-Maquardt et al. 45) Date of Patent: Dec. 6, 1994 54 PROCESS FOR THE PRODUCTION OF tive Stimulus Properties...' J. Med. Chem, 1985, vol. ERGOLINE DERVATIVES 28, pp. 1252-1255. 75 Inventors: Heidi Hummel-Maquardt; Mario Primary Examiner-Mukund J. Shah Kennecke; Alfred Weber; Klaus Assistant Examiner-Y. N. Gupta Nickisch; Gregor Haffer, all of Attorney, Agent, or Firm-Millen, White, Zelano & Berlin, Germany Branigan 73) Assignee: Schering Aktiengesellschaft, Berlin, 57 ABSTRACT Germany A process for the production of ergoline derivatives of 21 Appl. No.: 930,692 general formula I is described 22, PCT Filed: Feb. 6, 1992 R3 (I) 86 PCT No.: PCT/DE92/00088 S 371. Date: Oct. 7, 1992 S 102(e) Date: Oct. 7, 1992 87, PCT Pub. No.: WO92/13857 PCT Pub. Date: Aug. 20, 1992 30 Foreign Application Priority Data Feb. 8, 1991 DE Germany ............................. 4104379 in which the bonds 51 int. Cl. ..................... C12P 17/18; C07D 457/14 . represent two single-bonds or a double bond and a 52 U.S.C. .................................... 435/119; 435/121; single bond. 435/886; 546/70 R1 means a hydrogen atom or an alkyl group with 1-6 58) Field of Search .................... 546/70,435/42, 119, carbon atoms 435/121,886 R2 symbolizes a hydrogen atom or an alkyl group with 56) References Cited 1-6 carbon atoms, PUBLICATIONS R3 represents a carboxyl group or a grouping of formula Ishii et al. Journal of Chemical Society, Perkin I, 902-905, 1980. Ishii, H. et al., "Studies on Lysergic Acid Diethylamide and Related Compounds . Chen. Pharm. Bull., with R4 and R5 meaning hydrogen or an alkyl radical 27:12, pp. 3029-3038, 1979. with 1-6 carbons atoms optionally substituted by a hy Chemical Abstracts, 59, No. 3, p. 3099(Yamano et al.), droxy group and R6 and R7 meaning an alkyl group 1962. containing 1-4 carbon atoms and Q meaning an oxygen Ishii et al., Chem. Pharm. Bull 27(7), vol. 27, pp. or sulfur atom. 1570-1575, 1979. Hoffman et al., “Synthesis and LSD-like Discrimina 3 Claims, No Drawings 5,371,000 1. 2 PROCESS FOR THE PRODUCTION OF R3 (IIE) ERGOLINE DERVATIVES 5 The invention relates to a process for the production of ergoline derivatives of general formula I Nr. O , (I) R-N 1. R2 in which . R, R2, and Rs have the above-mentioned mean 15 ing and R4 represents an alkyl radical with 2 to 6 car bons atoms (EP-B 0021206; EP-A 0351 352 and J. Med. Chem. 28, 1985, 1252–1255). R-N The process according to the invention makes it pos 20 sible to produce the ergoline derivatives of general in which the bonds formula I in a significantly simpler way and under very much more environmentally favorable conditions, than . represent two single bonds or a double bond and is possible by conventional processes (J. Med. Chem. a single bond. 28, 1985, 1252-1255). R1 means a hydrogenatom or an alkyl group with 1-6 25 It has already been mentioned that the process ac cording to the invention is performed with a bacterial carbon atoms culture of genus Streptomyces. In the hitherto per R2 symbolizes a hydrogen atom or an alkyl group formed studies microorganisms Streptomyces purpurasc with 1-6 carbon atoms, ens (DSM 40310) or Streptomyces roseochromogenes R3 represents a carboxyl group or a grouping of for 30 (IFO 3363 and IFO 3411) have proved to be suitable; but it is very probable that numerous other cultures of mula genus Streptomyces can be found that are suitable to perform the process according to the invention. The process according to the invention is performed 35 under the same fermentation conditions which are also with R4 and R5 meaning hydrogen or an alkyl radical used in the known microbiological conversions of sub strates with bacterial cultures. with 1-6 carbons atoms optionally substituted by a hy Under the culture conditions usually used for bacte droxy group and R5 and R7 meaning an alkyl group rial cultures-especially those of genus Strep containing 1-4 carbon atoms; and Q meaning an oxygen tomyces-submerged cultures are cultivated in a suit or sulfur atom, which is characterized in that an ergo able nutrient medium with aeration. Then the substrate is added to the cultures (dissolved in a suitable solvent line derivative of general formula II or in emulsified form) and fermented, until a maximum substrate conversion is achieved. 45 Suitable substrate solvents are, for example, metha (II) nol, ethanol, glycolmonomethylether, dimethylform amide or dimethyl sulfoxide or aqueous mineral acids such as phosphoric acid or sulfuric acid. The emulsifica tion of the substrate can be brought about, for example, 50 by injecting the latter in a micronized form or in a wa ter-miscible solvent (such as methanol, ethanol, ace tone, glycolmonomethylether, dimethylformamide or dimethyl sulfoxide) dissolved under strong turbulence 55 in (preferably decalcified) water which contains the usual emulsification auxiliary agents. Suitable emulsifi cation auxiliary agents are nonionogenic emulsifiers, in which such as, for example, ethylenoxy adducts or fatty acid . R, R2, R3 have the above-mentioned meaning, esters of polyglycols. As suitable emulsifiers there can is fermented with a bacterial culture of the genus Strep be mentioned as examples the commercially available tomyces. wetting agents Tegin (R), Tween (Rand Span (R). The optimal substrate concentration, substrate addi The ergoline derivatives of general formula I, as is tion period and fermentation time depends on the type known, are valuable intermediate products, which can of substrate and microorganism used and the fermenta be used, for example, for the synthesis of the pharmaco 65 tion conditions. These values have to be determined, as this is generally necessary in microbiological substrate logically effective ergoline derivatives of general for conversions, in the individual case by preliminary tests, mula III as they are familiar to one skilled in the art. 5,371,000 3 4 It is surprising to one skilled in the art that the ergo ine in a ratio of 75:25:0.1 was used as the mobile solvent. line derivatives of general formula II are selectively The fractions, which contain the desired product, were demethylated in the N-6 position under the conditions combined, evaporated to dryness and precipitated with of the process according to the invention, for one skilled ethylene chloride. 0.25 g of 9,10-didehydro-ergolinyl in the art would have had to expect that the substituents in the 8-position of these substrates would also be 8o-carboxylic acid amide was obtained. The structure cleaved. was confirmed by NMR and IR spectroscopy. Indeed from the tests of S. Yamatodaniet. al. (Takeda EXAMPLE 2 Kenkyusho Nempo 21, 88–94; ref: C. A. 59, 1963, 3099c) it is basically known that ergoline derivatives O The N-6 demethylation of lysergic acid amide/isoly can be demethylated in the N-6 position. But these tests sergic acid amide was performed with Streptomyces were performed with ergoline derivatives that have no roseochromognes IFO 3363. The cultivation, substrate cleavable groups in the 8-position (agroclavin and production, fermentation and working up took place as elymoclavin), the substrate concentrations used in these indicated in example 1. The yield was close to 0.3 g. tests are very low. Moreover secondary reactions, such 15 as a hydroxylation in the 8-position, were observed in EXAMPLE 3 these reactions so that this process seems to be unsuit able for industrial use. The N-6 demethylation of lysergic acid amide/isoly The following embodiments are used for a more de sergic acid amide was performed with Streptomyces tailed explanation of the process according to the inven 20 roseochromogenes IFO 3411. The cultivation, substrate tion. production, fermentation and working up took place as EXAMPLE 1. indicated in example 1. The yield was close to 0.3 g. a) Spores of Streptomyces purpurascens DSM 40310 EXAMPLE 4 were applied on agar plates of the following composi 25 The N-6 demethylation of lisuride took place with tion: 2% starch Streptomyces purpurascens DSM 40310. The cultivation, 0.4% yeast extract fermentation and working up was performed as de 2% agar scribed in example 1. - adjusted to pH 7.2 30 Production of the substrate: 1 g of lisuride was dis and incubated 7 days at 30° C. The strain formed red solved in 50 ml of 10% H3PO4 and sterilized by filtra dish to violet substrate mycelium and whitish spores. tion. b) The spores were elutriated from the plate with 1 ml The reaction took place with a yield of 0.4 g of D-3- of physiological NaCl solution and a 500 ml Erlenmeyer (9,10-didehydro-8a-ergolinyl)-1,1-diethylurea. The flask with 100 medium of the following composition is 35 structure was confirmed by NMR and IR spectroscopy. inoculated with it 0.75% soy meal EXAMPLE 5 0.1% NaCl 0.5% soluble starch The N-6 demethylation of lisuride was performed 0.65% tris(hydroxymethyl)-aminomethane with Streptomyces roseochromogenes IFO 3363. The cul 0.2 % K2HPO4 tivation, fermentation and working up took place as - adjusted to pH 6.2. described in example 1; the substrate production was The cultivation flask was incubated for 48 hours at 30 performed as described in example 4. The yield was C. and 180 rpm. close to 0.45 g. c) Production of the substrate: 10 g of lysergic acid 45 amide/isolysergic acid amide and 0.5 g of Tween 80 EXAMPLE 6 were ground for 1 hour under water cooling in 500 ml The N-6 demethylation of terguride took place with of deionized water in the ball mill with corundum Streptomyces purpurascens DSM 40310.