CS274177B1 - 2-amino-2-hydroxymethyl-3,4-trans-epoxy-14-oxo-6-trans-eikosenic acid and method of its preparation - Google Patents

Abstract

The invention concerns microbiology with use in healthcare or agriculture. The new compound is 2-amino-2-hydroxymethyl-3,4-trans-epoxy-14-oxo-6-trans-eikosenic acid, which belongs in the group of amino acids with long aliphatic chains of general formula I. The acid is significant in its antibiotic effects against yeasts and filamentous fungi. The method of preparation is conducted by aerobic submersion cultivation of the productive strain of Melanconis flavovirens, preferably its mutants (CCF No 1,886), and the given acid is isolated from the acquired biomass by extraction and chromatographic procedures.

Description

The invention relates to 2-amino-2-hydroxymethyl-3,4-trans-apoxy-14-oxo-trans-eicosanoic acid and to a process for its preparation.

It is a further object of the invention to use the compound as an antibiotic. Acid aa is characterized by high antibiotic activity against yeast and filamentous fungi.

Znám and I know a small number of antibiotics and synthetic preparations used in the treatment of mycotic diseases in humans. Due to the current increasing number of these diseases and the increasing resistance of pathogens to the drugs used, there is a continuing need to look for other substances that have an antifungal effect.

Among the antifungal antibiotics used are, in particular, polyene-type antibiotics produced by straptomycates, such as nystatin (Házen EL and Brown R., Science 112: 423, 1950), amphotaricin B (Gold W. et al., Antibiotics Ann .: 579, 1955). (1956) or primaricin (Struyk A, P. et al., Antibiotlcs Ann .: 878, 1957/1958) of griaaofulvin produced by Panicilllum grieaofulvum culture (Oxford A, F. et al., Biocham., 33: 240, 1939) ) and finally the Czechoslovak mucidine (Mucidermin SPOFA) produced by the culture of Oudamansialla mucida (Cs. pat. 136 492).

Eyntatically prepared antifungal agents have also been used to treat fungal diseases, including imidazole compounds in recent years.

A disadvantage of some of these known substances is that, for example, many of them are not effective against yeast or their specific activity is low.

These drawbacks are overcome by 2-amino-2-hydroxymethyl-3,4-trans-apoxy-14-oxo-6-trans-aicosanoic acid of the general formula

O / \

NH,

CH ₃ - (CH ₂ ) ₅ -CO- (CH ₂ ) ₆ -CH ₂ CH-CH ₂ -CH-CH-C-COOH

CH ₂ OH which has been found to be highly antibiotic against yeasts and filamentous fungi.

For this reason, this acid can be used as an effective antibiotic.

2-Amino-2-hydroxymethyl-3,4-trana-apoxy-oxo-6-tran8-aicosanoic acid is prepared by aerobically cultivating the fungus Malanconia flavovirana on a liquid medium containing simple sugars or polysaccharides at a concentration of 1 to 10% wt. as a carbon source and a complex nitrogen source at temperatures ranging from 18 to 30 ° C, pH ranging from 4.0 to

6.5.

As a production organism, for example, a strain of Malanconis flavovirana deposited in ablrca CCF under No. 1,886 can be used. The acid according to the invention has antifungal activity, in particular against yeasts and filamentous fungi.

Detailed characteristics of the new antibiotic

2-Amino-2-hydroxymethyl-3,4-trans-apoxy-14-oxo-6-trancaicosanoic acid (hereinafter referred to as flavovirin) is a white crystalline compound having the following properties: mp 156-158 ° C, optical Rotation +13.5 ° + 1 ° (c0.66% pyr.), UV spectrum has no characteristic maximum, IR spectrum (KBr) has significant peaks at 3,415,

926, 2,854, 1709, 1,645, 1,499, 1,457, 1,254, 1,064, 972, 908 cm -1. See Fig. Lj ¹ H NMR spectrum of 13, see Fig. 2, C NMR, see Table 1. Elemental analysis for molecular formula C ₃₇ H N0 _{2 l 5} (MH 383) calculated: C 65.77%, H 9.72 %, N 3.65% found: C 65.67%, H 9.65%, N 3.72%. Positive reactions: and ninhydrin and dinitropanylhydrazinam. Solubility: flavovirin is insoluble in patrolstyr, disthylathr, benzene, partially soluble in chloroform, methanol, ethanol, n-butanol, dimethylformamide, dimethylsulfoxide, pyridine.

Stability: the compound is stable in solution over a pH range of 2 to 10 when heated to 100 ° C for 15 min. Its activity is halved, when stored at 4 ° C the antibiotic activity has not changed over several months.

Antibiotic activity is pronounced against yeast and some filamentous fungi, has been found against bacteria; see Table 2.

Table 1

C NMR Spectrum of Flavovirin (50.31 MHz, in haxadautarodimathylaulfoxide)

Carbon no. <ý ~ (ppm) 1 168.11 2 63.60 3 58.08® 4 53.66® 5 34.33 6 133.09 7 124.62 8 32.21 9 31.27 10 28,89 ^p 11 28,64 ^p 12 23.39 13 41.98 14 210.75 15 Dec 41.98 16 23.39 17 28; 58 ^p 18 28,45 ^p 19 Dec 22.16 20 May 14.09 21 62.16

and; b Assigning signals marked with the same letter may be confused

CS 274 177 Bl

Table 2

Biological spectrum of flavovirin and its comparison with myriocin

Testing organism BALL flavovirin ^§ (yUg / mL) myrlocin Candida albicans 0.39 1.56 C. pssudotropicalis 0.39 3.12 C. utilis 0.39 0.78 Cytospora leucostoma 0.78 0.78 Fuearium oxysporum 7200 6/25 Hansenula anomala 0/78 0.78 Chaetomium globosum 0.78 1.56 Kloecksra apiculata 0; 10 0.78 Metschnikowia pulchsrrlma 0.78 3.12 Penicillium stipitatum 200 7200 Pichia fermentans 0/10 1.56 Saccharomyces cerevisiae 0/39 1.56 S. cerevisiae var. ellips. 0.39 1.56 S. lipolytica 0.39 0.78 Verticillium cinnabarinum 25 7200 Bacillus subtilis 7 200 7 200 Escherichia coli 7200 z200

§ MIC - minimum inhibitory concentration

The tests were performed by the plate diffusion method.

Method of preparation of an antibiotic

The production organism is a strain of Melanconis flavovirens deposited in the CCF collection under No. 1886 (Author's Certificate No. 234 407). The culture is stored on sloping wort agar at 4 ° C; revaccinated after 6 months. The following are examples of the production of an acid (flavovirin) according to the invention.

Example 1

Flavovirin fermentation occurs either in shake flasks; or in a fermentation tank using soil having the following composition: glucose 3% by weight, maize steeping water (50% by weight of dry matter) 1.5% by weight, MgSO 4 .7H 4 O 0.15% by weight, pH 5, 5 (KOH adjusted before atherilization), optimal culture temperature 24 ° C. The inoculum is 5% by volume of submerged culture. The maximum production is between 12 and 14 days of cultivation, when the culture contains 20 to 35 mg of flavovirin in 1 L,

The grown submerged culture is separated into medium and mycelium by filtration. The mycelium is extracted with methanol. The concentrated methanolic extract was combined with the medium and further purified by chromatography on an amberlite XAD 2 column (water, methanol, up to 80% v / v conc. Methanol). The active fraction after evaporation of methanol is extracted with chloroform. After evaporation of the chloroform, the sample is purified by chromatography on a polyamide column (water: ethanol, up to 96% by volume of conc. Ethanol). Adsorption chromatography on a silica gel column (chloroform / methanol up to 80% v / v methanol) follows.

Since the antibiotic complex contains, in addition to flavovirin, also the antibiotic myriocin (thermozymocidin) (Aragozzini F. et al., Tetrahedron 28: 5493, 1972, U.S. Pat. No. 3,758,529, Begli 3. F. et al., 3. Org. 38: 1253 (1973), both compounds need to be separated. Flavovirin is isolated from the mixture by precipitation in ethyl acetate and by chromatography on silica gel (ieopropanol: HCOOH: HgO, 97: 1.5):

: 1.5) and analytical purity is achieved by repeated crystallization from ethanol: water (8: 2).

Example 2

The fermentation proceeded as in Example 1. The grown 9-well culture was homogenized with twice the volume of n-butanol. After separating the mycelium by filtration, the butanolic phases are evaporated and the residue is further processed by shaking between chloroform and water. The procedure is the same as in Example 1.

Comparison of flavovirin 8 myrlocinam (thermozymocidin)

Since flavovirin is a close myriocin (tharmozymocidin) structure, both antibiotics have been compared in terms of physicochemical properties; in terms of biological activity.

The first difference is in production organisms. Myriocin is produced by the tarmophilic fungus Myriococcum albomycez Cooney and Emerson, which is a synonym for Thiolavia albomycos (Coonay and Emerson) Malloch and Caln, a production culture deposited in the NRRL Collection under No. 3,858. mushrooms; which is deposited in the ATCC under No. 20 349. By contrast, the production organism Melanconia flavovirena (Otth) Wahm. it belongs to the group of fungi Pyrenomycates and in addition to myriocin (thermozymocidin) it produces another antibiotic flavovirin.

The difference in physicochemical characteristics between flavovirinam and myrlocinam is evident from Table 3. The chemical affinity of flavovirin with myriocin was used to address the flavovirin structure due to the known absolute co-configuration of myriocin.

Table 3

Comparison of some physicochemical characteristics of flavovirin and myriocin (thermozymocidin)

FLAVOVIRIN MYRIOCIN Summary composition ^C 21 ^H 37 ^NO 5 ^C 21 ^H 39 ^NO 6 Melting point Mp 156-158 ° C M.p. 176-179 ° C Optical rotation / ° £ / q * 1 ° (pyridine) + 13.5 ° (c 0.66%) + 19.5 ° (c n 0.48%) Chromatographic mobility TLC (Rf) a) Silica gel (n-amyl acetate: EtOH: HCOOH: H ₂ 0-12: 6: 1: 1) 0.49 0.39 b) Cellulose - org. phase (n-amyl acetate: : acetone: CH ₂ COOH: H ₂ 0 - 7: 2: 1: 4) 0.54 0.29

CS 274 177 Bl

Another difference between the two is in biological activity. When using the plate diffusion method, the effect of flavovirin compared to myriocin in most cases is up to 15 times higher in Pichia fermentans, see Table 2. Corresponding differences were also found using the tube dilution method, comparing the minimum inhibitory concentrations (MIC) and minimal fungicidal concentration (MFC) against selected yeast species, see Table 4,

Table 4

Comparison of minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of flavovirin and myriocin on selected yeast species ^

Testing organism BALL flavovirin (/ Ug / mL) myriocin MFC flavovirin (/ Ug / mL) myriocin Candida pseudotropicalis o; 25 2; 5 0.75 7.5 C. albicans 0.20 1.0 1.0 5.0 Kloackara aplculata 0/10 0.5 0.5 2.0

§ Tests were performed using the tube scaling method

Claims (4)

The first 2-Amino-2-hydroxymethyl-3,4-trans-epoxy-14-oxo-6-Trane-eicosenoic Formula I 'O _O NH Λ / CH ₃ - (CH ₂ ) ₅ -CO- (CH ₂ ) ₆ -CHmCH-CH ₂ -CH-CH-CH-C-C00H CHgOH 2. A process according to claim 1, characterized in that the Melanconie flavovirens fungus is cultivated aerobically on a liquid medium containing simple sugars or polysaccharides in a concentration of 1 to 10% by weight. as a carbon source and a complex nitrogen source at a temperature in the range of 18 to 30 ° C, a pH in the range of 4, 0 to 6; after which the acid is recovered from the biomass obtained; column chromatography and repeated crystallization. 3. A method according to claim 2, wherein the cultivation is carried out submergedly. 4. The method of item 2, characterized by; by using the strain Melanconis flavovirens deposited in the CCF Collection under No. 1,886.