DE2903997A1 - METHOD FOR MANUFACTURING MONORDERS AND MONORDEN DERIVATIVES - Google Patents

Description

description

The invention relates to the production of monordes and monorde derivatives. Monorden is also known as Radicicol and is available using both names by RN Mirrington, E. Ritchie, CW Shoppee, WC Taylor in Tetrahedron Letters No. 7, p. 365, 1963, and F. McCapra, AI Scott in Tetrahydron Letters No. 15 , Page 869, 1964. The manufacture of monords using new species of the fungi Humicola gresea Traaen (fungi imperfecti, Minoliales) is also described in US Pat. No. 3,428,526.

The present invention provides a process for the production of monords, which is characterized in that Diheterospora chiamydosporia is cultured in a conventional nutrient medium and monodes are isolated from this culture medium in a conventional manner, for example by adsorption or extraction. A culture of the Diheterospora chlamydosporia which can be used according to the invention is available under registration no. NRRL 11178 on file with the United States Department of Agriculture (Northern Utilization Research and Development Division) in Peoria, Illinois, USA. The microorganism was also registered on January 3, 1979 under the registration no. ATCC 20537 deposited with the American Type Culture Collection. This deposit is publicly available. Diheterospora chlamydosporia is a commonly available mold

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with a flaky hyphal appearance (floccose hyphal morphology), which can be characterized as follows:

Aleurospores:

Phialospores:

Hyphae:

Cultural characteristics:

Spore structure remarkably large (10 to 25 .mu.m in diameter), muriform and subglobal (muriform and sub-globose) in outline. These The spore is initially a vesicle without partitions; when growing arise at right angles walls standing to each other, and finally a large spore with crossed walls (criss-cross walled spore).

Small, 1.5 to 2.5 µm, subglobal (sub-globose) to elliptical in outline. Transparent for microscopic specimens.

1 to 2 µm wide, transparent; and appear white in colonies.

White and flaky, after 3 days on potato dextrose medium or on Czapek's synthetic Culture medium about 4 cm in diameter.

Mutants of Diheterospora chlamydosporia which are obtained by irradiation with ultraviolet or X-rays or by chemical means, for example treatment of laboratory cultures with suitable chemicals, can also be used in the method according to the invention. D. chlamydosporia is grown on a variety of nutrient media containing conventional nutrients. For example, nutrients suitable for carbon heterotrophic organisms can be used;

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specific examples of the carbon source are glucose, Starch, dextrin, lactose and cane sugar; organic or inorganic nitrogen-containing nitrogen sources can be used as the nitrogen source Compounds can be used, specific examples being peptones, yeast and meat extracts (e.g. potato dextrose culture medium or trypticase soy agar), ammonium sulfate, ammonium nitrate and amino acids are; Usual mineral salts and trace elements are also suitable for use in the nutrient medium.

In one method of making monords, a liquid nutrient medium is inoculated with a culture of D. chlamydosporia (grown on potato dextrose medium or trypticase soybean medium). The nutrient medium is kept at a temperature of about 20 to 35 ° C. for a period of about 2 to 15 days. If the cultivation time is less than about 2 days, the yields of monordes are too low for practical purposes, and if the cultivation time exceeds 15 days, the process becomes unattractive in terms of time and energy consumption. Preferably the temperature used is about 24 to 30 ° C.

The cultivation can be carried out using a static surface culture or in an immersed culture with shaking or in fermenters with aeration with air or oxygen take place. Following the breeding period, the

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Broth filtered and the monorden isolated by conventional extraction or adsorption methods. A particularly suitable method for isolating monords is solvent extraction from the fermentation broth using methylene chloride as the solvent. However, other solvents such as benzene, butyl acetate, methyl acetate, chloroform or butanol can also be used. The extracts are then separated from the solvent, for example by evaporation or distillation, and the residue is purified by chromatography using adsorbents, for example activated aluminum oxide, silica gel or magnesium silicate, or by means of countercurrent distribution. In this procedure, Monorden is isolated which has the physical properties given in the literature: For example, melting point 195 ° C., UV spectrum maximum at 265 μm; Infrared bands at 3100, 2980, 1655, 1572, 1430, 1352, 1310, 1245, 1110, 1045, 983, 925, 845 cm " ¹ (KBr) and among other lines in the nuclear magnetic resonance spectrum at ($ 1.52 (doublet) and at J "6.68 (singlet) with an _{empirical formula of C 1} OH ₁₇ OgCl.

According to the invention can be produced in the manner described Monorden can be converted into new monord derivatives by alkylation with the following formula:

OR

RO

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in which R is an alkyl group having 2 to 8 carbon atoms and the two R groups are the same or different. Because of the synthetic methods used, the two R groups are common identical. The R groups can either be straight-chain or branched-chain and preferably each have 2 up to 4 carbon atoms. Examples of such derivatives are Diethoxymonorden, Dipropoxymonorden, Diisopropoxymonorden, Dibutoxymonorden, Diisobutoxymonorden and Dioctylmonorden.

Using monordes as a starting material, the new dialkoxy derivatives prepared by alkylation preferably according to the Williamson synthesis. In this procedure Monorden reacted with an alkyl halide of the formula R-Hal in the presence of an acid-binding agent. It can also be a Procedures similar to that described by Mirrington et al for the preparation of dimethoxy derivatives can be used. In detail, the derivatives according to the invention are made by reacting monordes with potassium carbonate and the appropriate alkyl iodide made in an organic material that is a solvent for the reactants but does not react with them. In general, the molar ratio of monordes to alkyl iodide is about 1 to 1.5. Suitable solvents include Acetone and other ketones such as heptanone, hexanone and methyl ethyl ketone. Solvents like alcohols or acids are used because of Possibility that these could be reactive not used. Ethers (e.g. diethyl ether and dioxane) can also be used

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will. The reaction is generally carried out for a period of about 2 to 10 hours at a temperature of about 30 to 120 C. The variables like time, temperature, pressure, ratio of reactants and solvents can be used to maximize the yield can be changed. It is not believed that any of these variables are more critical in terms of The meaning is that a certain precise value must be adhered to in order to get a reaction. It is believed, that reaction conditions are sufficient which correspond approximately to the above. When using an alkyl iodide (as above described) the progress of the reaction can optionally be recorded and followed colorimetrically.

After the reaction, the reaction mixture is worked up and the dialkoxy derivative is isolated by conventional methods. For example, the solvent (eg acetone) is removed by distillation and the dry residue is taken up in a less polar solvent (eg chloroform), whereby K ₃ CO ₃ in the residue is removed. The volume of the chloroform solution is reduced to an easily manageable amount (e.g. about 25 mg of product per ml) and the resulting solution is chromatographed on a suitable substrate, e.g., silica or alumina. The derivative can be eluted with chloroform or mixtures of chloroform / hexane. The eluate is monitored (continuous recording) in order to isolate the fraction from which the maximum

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male yield of the dialkoxy derivative is available.

It has been found that monomer derivatives prepared as described inhibit the growth of cancerous human nasopharynx (KB) cells in vitro. This is a recognized one Test for anti-tumor activity, see e.g. Cancer Chem. Rpts., 25, 52 (1962). The monorder derivatives are also active against lymphocytic leukemia cells in vitro (P. 388). This is also a recognized test for anti-tumor activity, see e.g. National Cancer Institute protocol, Drug Screening Section.

The monordene derivatives according to the invention are also nematocides.

The invention will be explained in more detail with the aid of the following examples.

example 1

Diheterospora chlamydosporia was kept on potato dextrose medium and a spore suspension (about 1 ml -

fi Q

10 to 10 spores per 100 ml) produced by adding to the inclined nutrient medium sterile distilled water was given. The suspension was used to inoculate 100 ml of medium into a 250 ml Erlenmeyer flask used. The medium had the following composition :

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Weight%

Glucose 4

Cottonseed flour 0.3

Corn flour 0.1

Soybean oil flour 0.1

K ₂ HPO ₄ 0.1

MgSO ₄ .7H ₂ O 0.1

FeSO ₄ .7H ₂ O 0.001

CaCO ₃ 1

NaCl 0.5

and demineralized water to make up to 100 ml. The water contained about one drop of an anti-foaming agent (Hodag FD-62 Antifoam - 10% aqueous emulsion of methylpolysiloxane).

Numerous other flasks were prepared in the same way and placed on a rotating shaker at 150 rpm. Maintained at 25 C for days. The flask contents were combined and filtered. 8 liters of filtered fermentation broth were extracted with methylene chloride (1: 4, vol / vol). This extraction was carried out three times and the obtained methylene extracts were combined with each other. CH ₂ Cl ₂ was then removed under reduced pressure. A syrup was obtained which was chromatographed on an 8 × 20 cm column of silica gel (0.063-0.2 mm) by elution chromatography using chloroform as the eluent. The monord formed

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white crystals with a melting point of 191.5 to 192 ° C (not corrected) and with the UV, IR and NMR (CDCl ₃ ) spectra described above. The yield of monorden was about 45 mg / l of the filtered fermentation broth.

_{The mycelium was mixed with CH 3} Cl ₂ in a ratio of 1: 1 (vol / vol) and stirred vigorously (in a Waring mixer) for 10 minutes. The solvent was separated and the procedure was repeated 3 times. The extracts were combined and the methylene chloride was removed by distillation. Chromatography was then carried out as described above. The yield of monordes was 60 mg / l.

Example 2 Diethoxymonorden

200 mg of monordes (0.0055 moles) were dissolved in 8.3 ml of dry acetone (distilled over potassium carbonate) dissolved. To this mixture, 0.7746 g (0.0055 mol) of anhydrous potassium carbonate and 0.652 ml (0.0082 mol) of ethyl iodide was added. The mixture was then refluxed on a 56 ° C water bath for 6 hours cooked. What was soluble in acetone was pipetted off and dried to give 357.2 mg of product. The dried material was extracted with chloroform to remove potassium carbonate. The chloroform soluble was 199.6 mg. Thin-layer chrome

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matograms of the extract were in chloroform / methanol 9: 1, Methanol and chloroform produced. It has been found that the best separation is obtained using chloroform became. Impure diethoxymonord (199.6 mg) was then added placed on a 134 cm silica gel column and eluted with chloroform. Diethoxymonorden was eluted and off as the main component Recrystallized methanol. Fine, pale yellow, needle-shaped crystals were obtained.

The yellow crystals identified as diethoxymonords had the properties given in Table I.

Example 3 Di-n-propoxymonorden

Di-n-propoxymonorden was prepared as described in Example 2 by dissolving 200 mg of monorden (0.0055 mol) in 8.3 ml of dry acetone. To this mixture were added 774.6 mg (0.0055 mol) of anhydrous potassium carbonate and then 0.796 ml (0.0082 mol) of 1-propyl iodide. The mixture was refluxed at 60 to 70 ° C. If necessary, acetone was added to keep the mixture in solution. After 6 hours, the acetone soluble was pipetted off and dried under reduced pressure in a rotary evaporator. To remove the potassium carbonate, extraction _{was then carried out with CHCl 3 as described above.} The in CHCl ₃ -

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Solubles were chromatographed on a Porasil A (Water's trade name for a high pressure ligand chromatography column) using CHCl ₃ : hexane (1: 1). The chloroform / hexane extracts were evaporated to dryness and the dried material was recrystallized from hexane. The product obtained had the properties given in Table I.

Example 4 Di i sopoxymonorden

200 mg of monorden (0.0055 mol) was dissolved in 8.3 ml of dry acetone (distilled over potassium carbonate) and it became 0.7746 g (0.0055 mol) anhydrous potassium carbonate added. Then 0.815 ml (0.0082 mol) of 2-propyl iodide were added. The mixture was then refluxed at 56 ° C. for 6 hours. If necessary, acetone was added to make the mixture in Hold solution. The acetone soluble was decanted and the acetone was removed under reduced pressure. The resulting Solids were extracted with chloroform. Diisopropoxymonorden was isolated by crystallization from methanol. The physical properties of diisopropoxy monodes are shown in Table I.

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Table I.

Di-ethoxymonorden Dipropoxymonorden Isopropoxy monorden Enamel 142-144 ° C 136-139 ° C Point 280 nm 280 nm 140-141 ⁰ C. UV peak 1750 1705 280 nm IR peaks 1690 1690 1705 (Nujol) 1670 1662 1688 1610 1608 1662 1595 159 3 1605 1460 1570 1590 1440 1468 1568 1425 1460 1468 1400 1425 146O 1388 1408 1422 1380 1380 1403 1355 1353 1379 1330 1332 1368 1305 1318 1350 1290 1300 1330 1280 1278 1315 1260 1260 1297 1250 1247 1272 1200 1230 1258 1180 1190 1240 1152 1145 1277 1140 1130 1190 1120 1115 1140 1112 1098 1125 1078 1070 1110 1032 1048 1092 950 1035 1068 930 1000 1042 870 985 1030 860 978 1007 825 950 994 800 920 980 740 903 972 720 860 962 830 943 820 912 745 900 720 890 856 825 818 740 715

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Example 5 Activity against tumor cells

Human nasopharyngeal tumor cells (also known as KB cells) were used. According to the procedure described in Cancer Chem. Rpts. 25, 52 (1962), the KB cells were cultured and inoculated with various amounts of diethoxymonord. The ED ^ q value of this compound was found to be 3.1 µg. In the test mode described, amounts of 4. µg / ml or less showed significant activity against tumor cells. The ED ₅ values for di-n-propoxymonorden and diisopropoxymonorden were 3.1 µg and 3.9 µg, respectively. Dimethoxymonorden, a known compound, has an ED ₅ of 1.9 µg.

Example 6 Nematocidal activity

Suspensions of diethoxymonorden (DEM) in water were prepared by adding 2 mg DEM in 200 μg dimethyl sulfoxide (DMSO) have been resolved. The DMSO solution was then rapidly dispersed in 1.8 ml of deionized water to form a suspension from microcrystalline DEM. The DEM suspension was sequentially in three consecutive stages diluted and placed in standard 13 χ 100 mm test tubes. Each Tube contained 1 ml of diluted DEM suspension. The amounts of

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DEM used (in mg / ml) were 0.0005, 0.0015, 0.0045, 0.0135, 0.0405, 0.1100 and 0.3300 and 1.0.

1 ml of water containing about 20 nematodes in suspension was added to each tube and mixed with the DEM suspension. The nematodes had previously been obtained from a soil sample by immersing the soil sample in water and then concentrating the nematodes by filtration. After 16 hours, the viability of the nematodes was determined by microscopic examination. No movement was judged dead. As a control, 1 ml of water with about 20 nematodes suspended in it was mixed with an aqueous solution containing 100 μg of DMSO (the highest amount of DMSO present in the DEM suspensions). After 16 hours, the death rate for the control sample was approximately 20%. According to the method described by Reed and Muench (Am. J. Hygiene 27: 493-497, 1938), the amount of DEM leading to a 50% death rate (ie the “LC ₅₀ value”) was determined after 16 hours. For DEM the LC ₅₀ value was 0.2 mg / ml, which clearly shows that DEM is an effective nematocide.

In a corresponding manner, the LC _5Q value for diisopropoxymonorden (DPM) was determined to be 0.8 mg / ml, which shows that DPM is also an effective nematocide.

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Claims (12)

UEXKÜLL & SVOLdERG BESELERSTRA3SE 4 2000 HAMBURG 52 29039S7 PATENTANWÄLTE OR. J.-D. FRHR. by UEXKÜLL OR. ULRICH GRAF STOLBERG D1PL.-ING. JÜRGEN SUCHANTKE W.R. Grace & Co. Avenue of the Americas New York, N.Y. 10036 / V.St.A. (Prio: February 1, 1978 US 874 207 and 874 348 October 30, 1978 US 955 705-15493) Hamburg, January 30, 1979 Process for the production of monords and monord derivatives Patent claims 1. A process for the production of Monorden, characterized in that Diheterospora chlamydosporia or a mutant thereof is cultivated in a nutrient solution which contains a source of carbon, nitrogen and mineral salts, and Monorden is isolated from this nutrient solution. 2. The method? According to claim 1 , characterized in that the nutrient solution at a temperature of about 20 to 35 ° C helped. 3. The method according to claim 1 or 2, characterized in that cultivated for about 2 to 15 days. 909836/0528 4. The method according to claim 1 to 3, characterized in that one monordes by solvent extraction of the Separates nutrient solution. 5. The method according to claim 4, characterized in that monordes are isolated from the solvent extract. 6. The method according to claim 1 to 5, characterized in that the monorden produced is alkylated to give dialkoxy monorden of the formula OR 0 RO 0
Cl
in which R is an alkyl group having 2 to 8 carbon atoms, it being possible for the two R groups to be identical or different. 7. The method according to claim 6, characterized in that dialkoxymonords with the same R groups are produced. 8. The method according to claim 6 to 7, characterized in that dialkoxymonords with R groups are produced which each contain 2 to 4 carbon atoms. 9. A method according to claim 6, characterized in that Dialkoxymonorden prepared in which R ^is ethyl · 909836/0520 10. The method according to claim 6, characterized in that dialkoxymonords are produced in which R is a propyl radical is. 11. The method according to claim 6, characterized in that dialkoxymonords are produced in which R is an isopropyl radical is. 12. The method according to claim 6 to 11, characterized in that the monordes by reaction with an alkyl halide of the formula R-Hal alkylated in the presence of an acid-binding agent. 909836/0528