WO1989012038A1

WO1989012038A1 - New cyclites derived from streptomycetes, their preparation and use

Info

Publication number: WO1989012038A1
Application number: PCT/EP1989/000524
Authority: WO
Inventors: Susanne Grabley; Joachim Wink; Peter Hammann; Carlo Giani; Klaus HÜTTER; Axel Zeeck
Original assignee: Hoechst Aktiengesellschaft
Priority date: 1988-06-01
Filing date: 1989-05-13
Publication date: 1989-12-14
Also published as: EP0417152A1; JPH03504600A

Abstract

New cyclites with a growth-regulating action, in particular in dicotyledonous plants such as cress, can be isolated from a nutrient solution for streptomycetes.

Description

description

New cyclites from Streptomycetes, their production and their use

Cyclites are pseudosugars, i.e. Derivatives of saccharides in which the pyranoserine oxygen is replaced by a methylene group. These substances are potential glycosidase ether [H. Paulsen, Liebig's Analen der Chemie, p. 125 (1987)] or glyoxylase inhibitors [T.Takeushi, J. Antibiot. 28: 737 (1975)]. These substances are essentially synthesized by Streptomycetes in nature.

Umezawa et al. describe in The Journal of Antibiotics

[27, 579 (197-4)] a hydroxymethyltrihydroxycyclohexanone with a rotation [α] ² _D ⁰ of -168 °, which is excreted into the culture filtrate by Streptomyces filiplnensis. A compound with the same structural formula, but without specifying a rotation value, is described in Japanese patent application Sho-49 (= 1974) -54587. It is also mentioned there that this compound influences plant growth.

Keller-Schierlein et al. [Helvetica Chimica Acta 69, 1829 (1986) 7 have isolated a methyltrihydroxycyclohexanone with a rotation value of [α] ² _D ⁰ = -106 ° (in methanol) from Streptomyces albus, but no properties influencing the physiological activity of plants have been assigned to this product .

As described below, further cyclites with pharmacological and surprisingly good growth-regulating activity in plants could be isolated from Streptomycetes. The invention thus relates to:

1. The compound of general formula I

in the independently of each other

R ^{1 can be} an oxo group or a hydroxy group, R ^{2 can be} a methyl group or an acetoxymethyl group, R ³ and R ^{4 can be} hydrogen or together a double bond and R ^{5 can be} an oxo group or a hydroxyl group, except for the compounds in which

R ^{1 is} an oxo group, R ^{2 is} a methyl group,

R ³ and R ^{4 are} hydrogen and

R ^{5 is} a hydroxy group and has the rotation value [α] ² _D ⁰ = -106 ° (in methanol, c = 0.45).

2. A process for the preparation of the compound of general formula I characterized under 1, which is characterized in that a strain of the genus Streptomyces is cultivated in a nutrient solution until said compound accumulates in the nutrient solution.

3. The use of the compound of general formula I characterized under 1 as a growth-regulating substance. The invention is described in detail below, in particular in its preferred embodiments. The invention is further defined in the claims.

The compound of general formula I is produced by different St reptomycete strains, which were deposited with the German Collection of Microorganisms with the numbers DSM 4351, DSM 4354 and DSM 4353 under the conditions of the Budapest Treaty.

Streptomyces DSM 4351 synthesizes the compound of the formula

I. R ^{1 is} an oxo group, R ^{2 is} a methyl group, R ³ and

R ⁴ together are a double bond and R ^{5 is} a hydroxy group.

Streptomyces DSM 4354 produces the compound of the formula I in which R ^{1 is} a hydroxyl group, R ^{2 is} a methyl group or an acetoxymethyl group, R ³ and R ^{4 are} together a double bond and R ^{5 is} an oxo group.

Streptomyces DSM 4353 produces the compound of formula I in which R ^{1 is} an oxo group, R ^{2 is} a methyl group, R ³ and

R ^{4 is} hydrogen and R ^{5 is} a hydroxy group.

The streptomycete strains used according to the invention can be characterized as follows:

DSM 4351 DSM 4354 DSM 4353

Spore color: gray gray gray spore chain: straight to straight to narrow spiral wavy wavy

Spore surface: smooth smooth smooth melanin: + + + pigment formation:

Substrate mycelium: Endo: - - -

Exo: - - - Luftmycel: Endo - - -

Exo: - - - Sugar and sugar alcohol utilization: arabinose,

Xylose,

Rhamnose,

Raffinose,

Mannitol,

Inositol,

Fructose,

Sucrose

Instead of the strains mentioned, their mutants and variants can also be used, provided they synthesize the compound of the general formula I. Such mutants can be known in a manner known per se by physical means, for example radiation, such as with ultraviolet or X-rays, or chemical mutagens, for example ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) or 2-Hydroxy-4-methoxybenzophenone (MOB) can be generated.

Assimilable carbohydrates and sugar alcohols, such as glucose, lactose or mannitol, as well as carbohydrate-containing natural products, such as malt extract, are suitable as the preferred carbon source for aerobic fermentation. As preferred Nitrogen-containing nutrients come into consideration: amino acids, peptides and proteins and their breakdown products, such as peptones or tryptones, also meat extracts, ground seeds, for example from corn, wheat, beans, soybeans or the cotton plant, distillation residues of

Alcohol production, meat flours or yeast extracts, but also ammonium salts and nitrates. In addition, the nutrient solution can contain, for example, chlorides, carbonates, sulfates or phosphates of the alkali or alkaline earth metals, iron, zinc and manganese as additional inorganic salts.

The formation of the compound of general formula I proceeds particularly well in a nutrient solution, the oatmeal in concentrations of 0.5 to 6%. preferably 2 to 4%, soy flour in concentrations of 0.1 to 4%, preferably 0.5 to 2%. and contains trace elements, each based on the weight of the entire nutrient solution.

The fermentation is carried out aerobically, for example submerged with shaking or stirring in shake flasks or fermenters, optionally with the introduction of air or oxygen. The fermentation can take place in a temperature range from about 18 to 40 ° C., preferably at about 25 to 30 ° C., in particular at 28 to 30 ° C. The microorganism is cultivated under the conditions mentioned until the stationary phase is reached, about 60 to 120 hours, preferably 70 to 75 hours.

Cultivation is advantageously carried out in several stages, ie one or more precultures are first prepared in a liquid nutrient medium, which are then inoculated into the actual production medium, the main culture, for example in a volume ratio of 1:10. The preculture is obtained, for example, by inoculating a spilled mycelium in a nutrient solution and allowing it to grow for about 48 to 72 hours. The exported mycelium can be obtained by using the Let the strain grow on a solid or liquid culture medium, for example yeast-malt agar, for about 7 days.

The course of the fermentation can be monitored on the basis of the pH value of the culture or the mycelium volume, by thin-layer chromatography or by testing the biological activity.

The cyclites of the general formula I are isolated from the culture medium by known methods, taking into account the chemical, physical and biological properties of the products. The cyclites of the general formula I are present in the mycelium or in the culture broth. They can be extracted from the unfiltered culture broth with an organic solvent which is immiscible or only slightly miscible with water, such as chloroform or ethyl acetate. However, since they are only a small part of the mycelium, it is advantageous to separate the culture broth from the mycelium, for example by centrifuging or filtering, preferably with the addition of

Filter aids. The compound of general formula I can then be isolated from the supernatant or filtrate, expediently in the slightly acidic to neutral pH range, preferably at pH 6 to 7. For this purpose, it is possible to use organic solvents which are poorly or immiscible with water, in particular chlorinated hydrocarbon such as chloroform or methylene chloride or esters such as ethyl acetate or acetone. The extracts, if appropriate after evaporation and taking up in a polar organic solvent, are freed from strongly lipophilic constituents by precipitation with a non-polar solvent, advantageously a hydrocarbon such as petroleum ether, which can make up to about 80% of the total extract. The cyclites can be isolated from the residue by chromatography.

Instead of an extraction, the cyclites can also be obtained from the adsorber resins by adsorption onto commercially available resins Culture broth are isolated. It has also proven advantageous to dry the fermenter content mentioned, for example by spray drying or freeze drying.

The usual process steps can be used to isolate the pure cyclites, such as chromatography, gel filtration or precipitation from their solutions in suitable organic solvents. Chromatography on silica gel has proven particularly useful, a mixture of ethyl acetate and hexane in a volume ratio of, for example, 1: 2 being used as the eluent.

The cyclites of the general formula I are oily and readily soluble in methanol, acetone, DMSO, dioxane and chloroform, but little in water and not in alkanes. The substances are stable in the solid state and also as a solution in the pH range from 3 to 9, in particular from 5 to 8. The growth-regulating effect can be shown particularly well on dicotyledonous plants such as cress.

Examples:

1. Fermentation of the producer strains DSM 4351, DSM 4354 and DSM 4353

a) Preparation of a spore suspension of a producer strain: 100 ml nutrient solution (4 g yeast extract, 10 g malt extract, 4 g glucose, 1 1 tap water, pH before sterilization 7.3) in a 500 ml Erlenmeyer flask are inoculated with the strain and 72 Incubated for hours at 27 ° C and 120 rpm on the rotating shaker. Subsequently, 20 ml of culture liquid in a 500 ml Erlenmeyer flask with the nutrient medium of the above composition, to which 20 g of agar / 1 was added for solidification, are uniform distributed and decanted. The cultures are incubated at 27 ° C for 10 to 14 days. The spores of a flask formed after this time are washed off with 500 ml of deionized water, which contains a drop of a commercially available nonionic surfactant, used immediately or kept at -22 ° C.

b) Production of a culture or preculture of a producer strain in the Erlenmeyer flask:

A 500 ml Erlenmeyer flask with 100 ml of a nutrient solution of the composition 2% meat meal, 10% malt extract, 1% calcium carbonate and water ad 100% (pH 7.2 before autoclaving) is inoculated with a culture drawn on a slant tube or with 0.2 ml spore suspension and incubated on a shaker at 120 rpm and 27 ° C. The maximum production of the desired substance is reached after 72 hours. A 48 hour old submerged culture (5%) from the same nutrient solution is sufficient to inoculate 10 and 100 1 fermenters.

c) Preparation of the Cyclites:

A 10 1 fermenter is operated under the following conditions:

Culture medium: 2% oatmeal

0.5% soy flour 0.25% trace elements

Trace elements: 3 g / 1 CaCl.2H ₂ O

1 g / 1 FeC ₆ O ₇ H

0.2 g / 1 MnSO ₄

0.1 g / 1 ZnCl ₂

0.025 g / 1 CuSO ₄ .5H ₂ O

0.02 g / 1 Na ₂ B ₄ O ₇ .10H ₂ O

0.004 g / 1 CoCl ₂

0.01 g / 1 Na ₂ MoO ₄ .2H ₂ O

pH 7.2 Incubation time: 72 hours

Incubation temperature: 30 ° C

Stirrer speed: 250 rpm

Ventilation: 4 1 air / min.

The foam development can be suppressed by repeatedly adding a few drops of ethanolic polyol solution. The production maximum is reached after approx. 70 hours (pH = 5.3). The yields are approx. 20 mg / 1.

2. Isolation of the cyclite

After fermentation of the producer strains, the culture broth is filtered with the addition of 2% Celite as a filter aid. The mycelium is extracted with acetone, the organic phase is evaporated and the aqueous residue is extracted with ethyl acetate. The culture filtrate is dried and dissolved in MeOH. The crude product is chromatographed on a silica gel column (silica gel 60, Macherey-Nagel) with ethyl acetate / hexane (1: 2; v: v).

3. Characterization of the cyclites

a) Characterization of the compound:

Thin layer chromatography: silica gel 60, F ₂₅₄

- chloroform-methanol (9: 1; v: v): Rf 0.30

- n-butanol-acetic acid-water, upper phase (4: 1: 5; v: v: v) Rf 0.47

EI-MS (70 eV): m / e = 160 (M + 2H); 140 (MH ₂ O) corresponding to C ₇ H ₁₀ O ₄ (158.16) IR (KBr): 3200-3600, 1715, 1680, 1640 cm ^-1

UV (methanol): λ _max (log ε) = 242 (3.47) nm ¹ H-NMR (200 MHz, d ₆ -DSMO): δ = 1.70 (t, 3H, J ≈ 1 Hz, 2- CH ₃ ); 3.71 (ddd, 1H, J = 8.0 / 4.5 / ~ 5 Hz, 4-H); 4.23 (dd, 1H, J = 8.0 / 5.0 Hz); 4.34 (broad, 1H); 4.91 (d, 1H, J ≈ 4 Hz, exchanges, OH); 5.04 (d, 1H, J ≈ 5 Hz, exchanges, 4-OH); 5.37 (d, 1H, J ≈ 5.0 Hz, exchanges, OH); 6.65 (d, broad, 1H, J = 4.5 Hz, 3-H) ppm.

¹³ C NMR (50.3 MHz, d ₆ -DMSO): δ = 14.8 q (2-CH ₃ ); 67.6 d; 75.3 d; 76.4 d; 131.8 s (C-2); 145.3 d (C-3); 199.1 s (C-1) ppm.

[a] ² _D ⁰ (c = 1, methanol) -132 °

Characterization of the connection

Thin layer chromatography: silica gel 60, F ₂₅₄

Chloroform-methanol (9: 1, v: v): Rf 0.30

- n-butanol-acetic acid-water, upper phase (4: 1: 5; v: v: v): Rf 0.50

EI-MS (70 eV): m / e = 160 (M ⁺ , 9%); 124 (11%); 100 (6%), 88 (36%); 87 (57%); 73 (100%) corresponding to C ₇ H ₁₂ O ₄ (160.17)

IR (film): 3200-3500, 1720 cm ^-1 ¹ H NMR (200 MHz, CD ₃ OD): δ = 1.03 (d, 3H, J = 6.5 Hz, 2-CH ₃ ); 1.42 (dt, 1H, J = 13.8 / 13.7 / 2.2 Hz, 3-H _a ); 2.12 (ddd, 1H, J = 13.8 / 5.7 / 3.7 Hz, 3-H _b ); 2.93 (ddq, 1H, J = 13.7 / 6.5 / 5.7 Hz, 2-H); 3.48 (dd, 1H, J = 10/3 Hz, 5-H); 4.12 (ddd, 1H, J = 3/3/3 Hz, 4-H); 4.43 (d, 1H, J = 10 Hz, 6-H) ppm.

¹³ C NMR (50.3 MHz, CDC1 ₃ ): δ = 13.2 q (CH ₃ ); 36.4 t (C-3); 36.7 d (C-2); 68.3 d; 77.1 d; 78.2 d; 210.5 s (C-1) ppm.

[α] ² _D ⁰ (c = 0.497, methanol) + 93 °

Characterization of the connection

Thin layer chromatography: silica gel 60, F ₂₅₄

Chloroform-methanol (9: 1, v: v): Rf 0.22

- n-butanol-acetic acid-water, upper phase (4: 1: 5; v: v: v): Rf 0.64

EI-MS (70 eV): m / e = 156 (C ₇ H ₈ O ₄ , M ⁺ -C ₂ H ₂ OH ₂ O); 138 (22%); 96 (66%); 68 (36%); 43 (100%).

IR (film): 3360, 2920, 2850, 1735, 1680 cm ^-1

¹ H NMR (200 MHz, CD ₃ OD): δ = 2.15 (s, 3H, 9-H ₃ ); 3.67 (dd, 1H, J = 8.4 / 11 Hz, 5-H); 4.08 (d, 1H, J = 11 Hz); 4.45 (d, broad, 1H, J = 8.4 / 1H _z ); 4.98 (m, 2H, 7-H _2); 6.04 (dd, 1H, J = 1.8 / 1 Hz, 2-H) ppm.

13 C NMR (50.3 MHz, CD ₃ OD): δ = 20.6 q; 63.7 t; 73.4 d; 78.0 d; 79.3 d; 122.5 d; 161.5 s; 172.0 s; 199.2 s ppm. [α] ²⁰ (c = 1, methanol) -34 ° D

Characterization of the connection

Thin layer chromatography: silica gel 60 F ₂₅₄

Chloroform-methanol (9: 1, v: v): Rf 0.17

- n-butanol-acetic acid-water, upper phase (4: 1: 5; v: v: v) Rf 0.54

EI-MS (70 eV): m / e = 140 (C ₇ H ₈ O ₃ , MH ₂ O); 98 (100%).

IR (film): 3460, 2930, 2850, 1670 cm ^-1

¹ H NMR (200 MHz, CD ₃ OD): δ = 2.09 (s, broad, 3H, CH ₃ ); 3.58 (dd, 1H, J = 11.2 / 8.8 Hz, 5-H); 4.01 (d, 1H, J = 11.2 Hz); 4.25 (d, 1H, J = 8.8 Hz); 5.96 (s, broad, 1H, 2-H) ppm

¹³ C NMR (50.3 MHz, CD ₃ OD): δ = 20.1 q; 75.2 d; 78.1 d; 79.2 d; 125.7 d; 165.7 s; 199.4 s ppm.

[α] ² _D ⁰ (c = 1, methanol) -48 °

Claims

Claims:

1 . Compound of the general formula I,

in the independently of each other

R ^{1 is} an oxo group or a hydroxyl group, R ^{2 is} a methyl group or an acetoxymethyl group, R ³ and R ^{4 are} hydrogen or together a double bond and

R ^{5 are} an oxo group or a hydroxy group, except for the compound in which

R ^{1 is} an oxo group, R ^{2 is} a methyl group, R ³ and R ^{4 are} hydrogen and

R ^{5 is} a hydroxyl group and has the rotation value [α] 20 -106 ° D

(Methanol, c = 0.45).

2. A process for the preparation of the compound according to claim 1, characterized in that the streptomycete strains

DSM 4351, DSM 4354 and DSM 4353 are cultivated until the compound mentioned accumulates in the nutrient solution.

3. The method according to claim 2, characterized in that the nutrient solution contains oatmeal in concentrations of 0.5 to 6% and soy flour in concentrations of 0.1 to 4%, each based on the weight of the entire nutrient solution.

4. The method according to claim 3, characterized in that the nutrient solution contains 2 to 4% oatmeal and 0.5 to 2% soy flour.

5. The method according to one or more of claims 2 to 4, characterized in that the cultivation takes place in a temperature range of 18 to 40 ° C.

6. The use of the compound of claim 1 as a growth regulating substance.

7. Streptomyces DSM 4351, DSM 4354 and DSM 4353 and their mutants and variants, provided that they produce the compound according to claim 1.