DE1227193B - Process for the production of the new antibiotics Verrucarin A and B and Roridin A. - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. CL:

C12d

German KL: 30 h -6

Number: 1227193

File number: S 78463IV a / 30 h

Filing date: March 13, 1962

Opened on: October 20, 1966

The present invention relates to a method for separating and isolating the new antibiotic effective components Verrucarin A, Verrucarin B and RoridinA from culture fluids and / or Mycelia of strains NRRL 3001 to 3005 of the fungal species Myrothecium verrucaria (Albertini et Schweinitz) Ditmar ex Fries and Myrothecium roridum Tode ex Fries. (The mushroom stems were at the Northern Utilization Research and Development Division, Peoria, I11./USA., deposited and received there the specified NRRL numbers.)

The isolation of the antibiotics from the culture fluid is carried out by precipitation, distribution, chromatographic Adsorption, partition chromatography or countercurrent distribution (fractional distribution).

Colonies of the fungus Myrothecium verrucaria and Myrothecium roridum were obtained from soil samples of different origins isolated. Morphologically correspond all these tribes correspond to the circumscriptions of the species, as they are, for example, in Gil man, J. C, A Manual of Soil Fungi, 1957, 2nd ed., The Iowa State College Press, Arnes. Iowa, USA., Given ,have been. The single ones. Isolations, in part Obtained from the same soil samples can differ both morphologically, e.g. by larger or smaller Willingness to sporulate, as well as physiological, e.g. B. in the claims to the nutrient medium for Production of antibiotics, differentiate.

Not only the culture fluid or the mycelium of strains of the fungus can be used as starting material Myrothecium verrucaria and Myrothecium roridum are used, but also the culture fluid mnd the mycelium of variants of these organisms, as they are e.g. B. granted by selecting sectors (Saltanten) will.

The cultivation takes place, for example, in dormant surface culture or submerged with shaking Mer while stirring with air or oxygen in holiday homes.

Strains of Myrothecium verrucaria and Myrothecium roridum can be found in a variety of nutrient media, the lie usual nutrients contain, breed. That means they utilize - glucose, starch, dextrin, lactose, Cane sugar, etc. as a carbon source, organic and inorganic nitrogen-containing compounds, such as Peptone, yeast or meat extracts, ammonium sulfate, ammonium nitrate, amino acids, etc. as nitrogen fuels, as well as the usual mineral salts and acid elements.

f It has been found that culture solutions of a number of strains of Myrothecium verrucaria can be used to produce processes for the production of the new antibiotics
Verrucarin A and B and Roridin A

Applicant:

SANDOZ A. G., Basel (Switzerland)

Representative:

Dr. W. Schalk, Dipl.-Ing. P. Wirth,
Dipl.-Ing. GEM Dannenberg
and Dr. V. Schmied-Kowarzik, patent attorneys,
Frankfurt / M., Große Eschenheimer Str. 39

Named as inventor:

Dr. Wolfgang Loeffler, Basel;

Dr. Hartmann Stähelin, Binningen;

Dr. Christian StolL_BaseI;

Dr. Christoph Tamm, Riehen;

Dr. Dorothea Wiesinger, Basel (Switzerland)

Claimed priority:

Switzerland of January 12, 1962 (360)

and Myrothecium roridum is able to isolate at least eight components in a pure, uniform and crystallized form. The presence and proportions of the components vary with the culture conditions. The process is characterized in that the active ingredients are extracted from the culture filtrate with chloroform, dichloromethane or ethyl acetate. The extracts obtained in this way are, after concentration to a smaller volume, ^{washed at 0} ° C. with sodium hydroxide solution and water, if the pH of the original culture liquid was less than 3.5. Extracts obtained from culture liquids with a pH above 3.5 are also washed with 2N hydrochloric acid. This allows inactive colored contaminants to be removed. After evaporation of the solvent, a yellow to brown colored oily crude product is obtained from which, under certain circumstances,

609 707/379

3 4

d. H. depending on the amount of oily ballast Myrothecium roridum present

substances by adding ether, a Rqhkristallisat _a ) strain NRRL 3004:

can be separated. After the analysis in the thin-film Roridin A

chromatogram mainly concerns substance A (not pure isolated)

VerrucarinA, which however still contains VerrucarinB and 5 _Κ λ ct „ _mm \ idtjt cmnc.

j .. ii -η -j -Α ι τ »ι -jj cc χτ. ·· ι ^ ") ötamm NKKL 3UU5:

eventueE contains Roridum B as accompanying substances, if Roridin A

working with cultures of M. verrucaria. Roridin B

When starting with cultures of M. roridum, Roridin C

the resulting crude crystallizate mainly from verrucarin A.

Roridin A, which is usually still Roridin B, Verru- to Verrucarin B

Contains carinA and verrucarin B as companions. Substance A (not purely isolated)

Separation into the uniform components takes place

by one or more subsequent careful cultures with the strains NRRL 3004

Adsorption chromatography on aluminum oxide and NRRL 3005 from M. roridum can be chromato-

or silica gel columns and by multiple _ umgraphic 15 yet another substance, which as

crystallize from mixtures of acetone — ether, substance A is designated. Substance A is so far

Methanol — ether, chloroform — ether — petroleum ether has not been isolated in pure form, but only lies

and dichloromethane ether, the purity being that in a mixture with Roiidin A before.

Fractions by thin layer chromatography The extraction of the mycelium of a culture batch

must be trolled. As a rule, it is possible to combine 20 of the strain NRRL 3001 of M. verrucaria with

the culture filtrates of M. verrucaria from those with aqueous methanol results after further purification

Chloroform and chloroform — methanol (99: 1) and the extract by partitioning between water and

(98: 2) eluted fractions of pure crystalline. Verrucarin C, ethyl acetate from the components soluble in ethyl acetate

D and E, Roridin A and B in varying amounts a crude product that can be found after further purification

to be separated off, while the main product VerrucarinA 25 of the silica gel acid in crude crystalline. VerrucarinA (nor

in this way usually cannot be obtained in pure form, containing verrucarin B) and separating roridin B.

but is still contaminated with verrucarin B. leaves.

From the culture filtrates of M. roridum, in addition to the classical methods of the characteristic analogous way, pure crystalline. Roridin A as the main sizing of the isolated crystall. Substances, the product, Roridin B and C as well as Verrucarin A and B 30 thin layer chromatography proves to be excellent diaas byproducts in pure crystal; Form won. Gnostic aid for checking the unity of the preceding with benzene eluted, both amorphous fractions and chromatography fractions can be oily constituents as well as crystallized substances. Separate one of the parts that are discarded. The particularly suitable embodiment is described in detail in Example 13 below with chloroform-methanol (98: 2), (95: 5), 35. So is z. B. the uniformity of (9: 1), (4: 1) and (1: 1) eluted fractions yield a verrucarin A and B without a chromatographic amorphous product. The separation of the crystal analysis method is very difficult to see,
Mixture of verrucarin A and B into the uniform The isolated substances can be characterized as follows with the help of the distribution:

Chromatography with water as the stationary phase 40 The antibiotic VerrucarinA forms after um- and water-saturated benzene-chloroform mixtures crystallize from mixtures of methanol-ether as the mobile phase. Diatomaceous earth serves as the carrier of the stationary phase or acetone-ether or chloroform-ether-petroleum. First of all, pure verrucarin B ether, colorless, rectangular flakes that do not melt below and in the later fractions, uniform verrucarin at 360 ° C. Carin A separate start at about 280 ⁰ C. 45 the crystals turn yellow and brown, since ZerTable I is based on the different settlements. They are insoluble in water, diethyl strains of M. verrucaria and M. roridum isolating ether, diisopropyl ether and in liquid saturated substances. Hydrocarbons (pentane, hexane, heptane, cyclo-

hexane), but soluble in lower ketones (acetone,

Table I ⁵ ° methyl ethyl ketone, etc.), lower alcohols (methanol, ethanol, propanol, etc.), in dioxane, in

Myrothecium verrucaria lower halogenated hydrocarbons pichloro-

^J methane, chloroform, carbon tetrachloride), in Pyn-

a) strain NRRL 3001: 4in. Benzene and in toluene. The specific rotation

VerrucarinA 55 values are: [α]? = + 206 ± 2 ° (chloroform) and

Verrucarin BM * - +208 ± 2 ° (dioxane). The elemental analyzes

Verrucaiin C gave the following values: C = 64.5, 64.7, 64.2% 5

Roridin B H-6.9, 7.0, 6.7% and O = 29.0, 28.4, 28.7%.

- The antibiotic contains neither N nor S nor

b) btamm NKKL 30Ü2: _6o halogen. The molecular weight determination according to

VerrucarinA _Rast (camphor) _{has a} molecular weight of 517

VerrucarmB ^ a _{thermoelectric} micro determination

Coridm A such of 527 results. That is consistent with that

c) strain NRRL 3003: result of the molecular weight determination from

Verrucarin A 65 of the radioactivity of the ¹⁴ C acetic anhydride

Verrucarin B constituted acetyl compound is calculated, viz

Verrucarin D 520. Based on these findings, the gross formula

Verrucarin EC ₂₇ H ₃₄ O ₉ (502.5) most likely. The UV * -

5 6

The absorption spectrum in ethanol shows a maximum or acetone-ether crystallizes the substance in color at 260 ηιμ, log = 4.25 (calculated on the molecular weight of loose prisms with a melting point of 223 to 224 ° C.
502.5). The IR spectrum of a solution of the anti-verrucarin D is also only formed in extremely low biotics in dichloromethane shows bands at (important amounts and not underlined under all culture conditions) 3540 , 2970, 1714 , 1635 , 1590 , 1475, 5. It crystallizes from ether-petroleum ether in 1410, 1385, 1213, 1190, 1186, 968, 878 and 820 cm " ^1. Needles with a melting point of 127 to 128 ° C.
IR spectrum I. In the solid state (suspension in the antibiotic Roridin A is according to Um-Nujol) bands can be seen at 3560-3580 . 2930, crystallize from acetone ether in colorless crystals-2570, 1709, 1673, 1631, 1587, 1457, 1377, 1275, 1213, len obtained either at 191 to 196 or 198 1195, 1123, 1103, 1085, 1053, 1032, 1023, 975, 967, io to 204 ⁰ C melt. They are readily soluble in the lower ^{887.832 cm "1.} Verrucarin A has no methoxyl ketones (acetone, methyl ethyl ketone), in lower groups, contains 5.5 ° / ₀ C methyl (according to Kuhn - alcohols (methanol, ethanol, Propanol), in dioxane, Ro th), 0.17 to 0.25% active hydrogen (Zer e- in lower halogenated hydrocarbons (Diw iti η ο ff), takes 3 moles of chloromethane, chloroform, carbon tetrachloride in the micro-hydrogenation) and H ₂ , gives pyridine with tetranitromethane in chloroform 15. The substance is sparingly soluble in water, does not turn yellow, discolours solutions of potassium diethyl ether, diisopropyl ether and hydrocarbons saturated in liquid permanganate in acetone and bromine in chloroform (pentane, hexane , Heptane, immediately. The reactions according to Tollens, Feh- cyclohexane, benzene and toluene) The specific ling, Molisch, Legal, Zimmermann rotation value is [cx] ff = + 129 ± 1 ° in chloro- and Liebermann are negative. Verru - * o form. The elementary analyzes have fol Low values carm A is therefore a lipoid-soluble neutral substance, results in: C = 65.1, 66.0, 6 * 5.4%; H = 7.4% and the antibiotic Verrucarin B shows very similar 0 = 27.4, 26.9, 26.9%. The molecular weight properties like VerrucarinA, with which it very determination after Rast (Camphor) results which like to crystallize out together. It crystallizes from values 459 and 503, the thermoelectric micro-acetone ether in needles, which is not below 360 ⁰ C as determination gives the value of 543. After this melt. The crystals begin to turn yellow and brown when the results for Roridin A come to the gross formulas 28O ⁰ C, which indicates a Zer- C ₂₂ H ₃₀ O ₇ (406.5) and C ₂₇ H ₃₈ O ₈ (490.6) and settlement indicates. They are insoluble in water, diethyl- C ₂₉ H ₄₀ _ ₄₂ O _e (533.6-535.6) in question,
ether, diisopropyl ether and saturated liquid. The UV absorption spectrum in ethanol shows hydrocarbons (pentane, hexane, heptane, cyclo- 30 maximum at 263 ηιμ, log = 4.27 (calculated on hexane), but soluble in lower ketones (acetone, molecular weight 533.6), in. The IR spectrum of a methyl ethyl ketone, etc.), lower alcohols (metal solution in dichloromethane contains bands at 3680th THANOL, ethanol, propanol, etc.), in dioxane, in 3570, 2970, 1721- 1724 . 1706 . 1637 . 1600 . 1180, 1123, lower halogenated hydrocarbons (dichloro-1100,1085,1035,1008, 970,927,843,813 and 650 cm- ¹ . Methane, chloroform, carbon tetrachloride), in pyrene 35 In the solid state (pressed in KBr) there are intendin, benzene and in toluene. The specific rotational bands at 3470 , 2970, 2860, 1742 . 1704 . Strikingly, 1634 values are strongly dependent on the nature of the 1594, 1482, 1418, 1335, 1170-1180, 1135, 1103, 1082, solvent. How do you find [«]? = ± 94 1037, 972, 815, 755 and 650 cm- ¹ . IR spectrum III. It ± 2 ° in chloroform; [a] f = + 101 ± 1 ° in dioxane there is a lipoid-soluble neutral substance.
and [a] o ³ = +147 ± 2 ° in benzene. After recrystallization from acetone analyzes, the elementary 4 ° Roridin B will have the following values: C = 65.4 chloroform — methanol in colorless needles or and 64.7%; H = 6.5 and 6.4% and O * = 28.1 YND leaflets of mp. 143 C ⁰ to 149 obtained. The 28.9%. Verrucarin B contains no N, no S and no crystals are readily soluble in lower ketones (acetone, halogen. The molecular weight determinations according to methyl ethyl ketone), lower alcohols (methanol, Rast (camphor) have values of 629 and 645 45 ethanol, propanol) , lower halogenated carbon and the thermoelectric micro-determinations of the hydrogen (dichloromethane, chloroform, tetra value of 490 result. On the basis of these findings chlorocarbon), dioxane, benzene and in pyridine, you get the gross formulas C ₂₇ H ₃₂ O ₈ (500, 5) and are sparingly soluble in water and in liquid saturated possibly C ₃₈ H ₃₈ O ₁₂ (662.7) and C ₃₆ H ₄₀ O ₁₂ (664.7) in hydrocarbons (pentane, hexane, heptane, cyclo- ■ · Question The UV absorption spectrum in ethanol 50 hexane). For specific rotation, the following values t have a maximum at 259 μm, log = 4.35 (calculated: [«]? = -123 ± 2 ° (chloroform) and molecular weight 490). The IR spectrum of a [a] f = -126 ± 2 ° (benzene). The elemental analyzes

I * solution in dichloromethane shows bands at 2880, give the following values: C = 83.8%; H = 11.1%

1736. 1701 . 1670 . 1623 . 1584 . 1419, 1240, 1100, 1080, and O = 4.8%. The molecular weight determination

1038, 995, 965 and 877cm- ¹ . IR spectrum II. In 55 according to Rast (Camphei) the value is 318, the

In the solid state (suspension in Nujol), the thermoelectric micro-determination bands have a value of 426.

before at 2930, 2885, 1736. 1712 . 1675 . 1631 . 1590. With these findings, the gross formulas C ₂₁ H ₃₂ O

1460, 1378, 1263, 1200, 1104, 1087, 1038, 970, 892, (300.5) and C ₂₈ H ₄₆ O (398.6) are compatible.

825, 740 and 646 cm- ¹ . The compound does not contain any. The UV absorption spectrum in ethanol is empty. Methoxyl groups and no active hydrogen 60 IR spectrum (firmly pressed in KBr) are bands at

(Z erewiti η ο ff). In the case of microhydration, 3400-3450 . 2950, 2860, 1655, 1605, 1460, 1370, 1068,

it needs 3 moles of H ₂ (calculated on the molecular weight of 670) 1040, 985, 970, 833 and 804 cm- ¹ recognizable. In the

or 4 moles of H ₂ (calculated on the molecular weight of 490). The microhydration is 4 to 5 moles of H ₂ (calculated

contains 2.9% C-methyl (according to Kuhn-Roth). to molecular weight 318).

Like verrucarin A, verrucarin B is also a lipoid 65 The antibiotic verrucarin A is strong

soluble neutral substance. Inhibitory effect against various microorganisms

Verrucarin C is only formed in very small quantities. The growth-inhibiting effectiveness of

and not all cultural approaches. From methanol — ether Verrucarin A was made by the usual for this

Purpose used dilution method is determined. The lowest concentration of substance that shows a total or a strong partial growth-inhibiting effect in a suitable nutrient solution was determined for each germ. There were Testkonzentratipnen of 10 "* ^'3 to ΙΟ" ~ ⁴ uses ⁸ · ³ and 10 to 10 ~. ⁸ The results of the various determinations are compiled in the table below.

Test organism A. Saccharomyces
cerevisiae - lied
with w £
total the lowest
self-inhibition
partially concentration
end effect
Incubation duration
hours Candida albicans 5.8
5.8
5.3 6.3
6.3
5.8 6th
20th
40 Candida albicans
Strain dermat. Univ.
Clinic Zurich,
No. 1101 A 5.0 5.5 20th Candida tropicalis
. Strain dermat. Univ.
Clinic Zurich No. 169/61 5.5 6.0 20th Candida krusei
Strain inst. Trop. Med.
London No. 270 6.3
5.8
5.3 7.8
6.3
6.3 6th
20th
40 Torulopsis glabrata
CBS strain No. 138 6.3
6.3
6.3 6.8
6.8
6.8 6th
20th
40 Absidia lichtheimi
Strain CBS-Lendner 4.0 5.0 20th B ^< Aspergillus fumigatus 5.0 5.5 20th Blastomyces
brasiliensis 5.5 6.0 20th C < Trichoph. gypseum 5.5 6.0 20th Achorion quinckeanum
Strain dermat. Univ.
Clinic Zurich No. 964 4.3 5.3 3 Serratia marcescens 4.3 5.3
4.3
4.3 3
7th
14th Proteus mirabilis
Strain NCTC 5887 4.3
4.3 6th
20th Pseudomonas
aeruginosa 4.3
4.3 6th
20th 4.3
4.3 6th
20th

A = yeast and other fungi except B

B = skin fungi C = gram germs

Nutrient medium

Difco-Nutrient Broth (except. Sacch.Cerev. = Sabouraud
Malotse B bread; Torulopsis glabrata)
Sabouraud Maltose Agar
Difco-Nutrient Broth

The effectiveness of verrucarin A, verrucarin B ascites tumor, sarcoma 180, sarcoma 37 of the mouse and RoridinA against animal tumors wuide on 55 and Yoshida sarcoma in the rat. The following experimental vaccine tumors of the mouse and the tables show the rat detected. Among the animal tumors that have proven to be susceptible to influence count honestly

show the effects of Verrucarin A, Verrucarin B and Roridin A on some animals Tumors:

Art

Dose once a day

duration

inhibition

Animals used for tolerance I died

Ehrlich's ascites tumor of the mouse

Sarcoma 180 - mouse

Sarcoma 37 - mouse

Yoshida sarcoma - rat

Verrucarin A 7 days · 94% 0.38 mg / kg 7 days 23 7o 0.125 mg / kg 7 days 43% 0.75 mg / kg 8 days 33% 1.0 mg / kg 14 days 35% 0.05 mg / kg

6th
6th
6th
6th
6th

9 Art dose
once a day duration inhibition 10 compatibility
animals used died

Verrucarm B

Ehrlich's ascites tumor of the mouse
Sarcoma 37 · ....-.

Sarcoma 37

1.0 mg / kg 6 days 84% 6th 0 3.0 mg / kg 7 days 95% 6th 1 1.0 mg / kg 7 days 23% 6th 0

Roridin A
0.5 mg / kg I 8 days

18%

Verrucarin A, Verrucarin B and Roridin A also have a pronounced effect in cell culture. The effect on cultures of chicken embryonic fibroblasts gave the following result:

After 6 hours of exposure to verrucarin A, the ED ₆₀ (concentration which interrupts the process in half of the mitoses) is 0.0025 y / ml. At this concentration, and also at a concentration 100 times higher, the cells that are not dividing are not morphologically changed. The dividing cells, however, disintegrate under the influence of Verrucarin A.

The effect of Verrucarin B is qualitatively the same as Verrucarin A, but somewhat weaker; ED ₆ O = O ₅ OIyZmI.

. The effect of Roridin A is qualitatively the same as that of Verrucarin A; ED ₅₀ = 0.003 y / ml.

The inhibition of the proliferation of tumor cells in vitro (cells of the mouse masocytoma P-815) resulted the following results:

In a suitable nutrient solution, these tumor cells multiply within 40 hours to 4 to 5 times the number of starting cells. The ED ₅₀ (concentration which inhibits this proliferation by 50%) of verrucarin A against these cells is 0.0006 y / ml (= 1: 1,600,000,000).

The ED ₅₀ of verrucarin B against these cells is 0.003 y / ml and of roridin A 0.001 y / ml.

The toxicity of Verrucarm A, Verrucarin B and Roridin A was examined in particular in the mouse. The acute toxicity DL ₅₀ was determined by administering the products by the intravenous route.

Verrucarin A DL ₆₀ i. ν 1.5 mg / kg

Verrucarin B DL ₆₀ i. ν 7 mg / kg

Roridin A DL ₆₀ iv 1 mg / kg

Verrucarin A is also used to treat blood disorders (such as myeloid or lymphatic diseases Leukemia) and diseases of the lymphatic system (e.g. lymphogranuloma Hodgkin, lymphosarcoma).

If dogs are treated intravenously with doses of 0.1 mg / kg of Verrucarin A once a day, so after multiple injections, there is a strong decrease in the number of leukocytes in the peripheral blood.

Examples

Dog A
Dog B
Dog C

Before treatment | After treatment
Leukocytes per cubic millimeter

8900

12 500

9,300

2 900

100

5,000

Verrucarin A, Verrucarin B and Roridin A can be used in the form of pharmaceutical preparations Find. These contain the compounds mentioned in a mixture with one for enteral, parenteral or local application of suitable organic or inorganic carrier material. Come for the same those substances in question that do not react with the new compounds, such as. B. gelatine, milk sugar, starch, Magnesium stearate, talc, vegetable oils, benzyl alcohols, rubber, polyalkylene glycols, petrolatum, cholesterol or other known excipients. the

As pharmaceutical preparations can e.g. B. as tablets, Dragees, powders, creams, suppositories or in liquid form as solutions, suspensions or Emulsions are present. If necessary, they are sterilized and / or contain auxiliary substances such as preservatives, Stabilizing, wetting or emulsifying agents. You can also find other therapeutically valuable ones Contain substances.

The invention is described in the following examples, without thereby limiting the Subject of the invention is intended. The temperatures are given in degrees Celsius. the Ratios for solvents always relate to volumes.

example 1

Manufacture and isolation of raw verrucarin A, verrucarin B and verrucarin E.

100 shake cultures (500 ml Erlenmeyer flasks with 100 ml nutrient solution each) with Richard's nutrient solution, modified according to G. Luz, Phytopathol. Z., 1934, 7, p. 589: 50.0 g glucose puriss., Ph. H. V, the following salts in analytically pure quality: 10.0 g ammonium nitrate, 5.0 g primary potassium phosphate, 2.5 g magnesium sulfate (-7H ₂ O), 0.02 g iron (III) chloride, demineralized water ad 1.01, are inoculated with a conidia suspension of the strain Myrothecium verrucaria NRRL 3003. After incubation for 9 days at 27 ° on a shaking machine with amplitude movement (frequency 100 movements per minute), the mycelium is sucked off and the clear culture nitrate is extracted twice with 11 ethyl acetate each time. The extracts are each washed once with 2N hydrochloric acid, 2N sodium hydroxide solution and water and evaporated in vacuo. 115 mg of brown-colored crude product results, which in the thin-layer chromatogram [carrier: silica gel; Mobile phase: chloroform-methanol (97: 3); Staining of the substance stains in iodine atmosphere (yellow-brown)] shows two stains, which correspond to Verrucarm A and B. For further purification, it is chromatographed on 6 g of silica gel. 12 ml of solvent per fraction are used for elution.

609 707/379

II 12

Fractions 1 to 5 [eluted with benzene, chloroform biert. 11 Culture broth is, as described in Example 2, *

and chloroform, - methanol (99: 1)] yield 3.6 mg extracted with ethyl acetate. The extracts are made twice

amorphous material (discarded). Fractions 6 and each with 50 ml of -2N sodium hydroxide solution and — twice — each with

7 [26 mg, eluted with chloroform-methanol (99: 1)] - 50 ml of water and evaporated in vacuo.

result from ether 8 mg crude crystall. Verrucarin A 5 The result is .142 mg crude extract, which in the thin-layer

(still containing Verruearin B ') .. Fractions 9 _; chromatogram showing three fleeke, two of which

and 10 [eluted with chloroform-methanol (99: 1)]; Verrucarin A and Verrucarin B correspond. To the

result from ether '13 mg crystall. Verrucarin E. Im> further purification is carried out on 7 g silica gel

Thin-layer chromatogram shows verrucarin E in graphed, with 15 ml of solvent for elution

Serve one red i'o per fraction after about 10 minutes in the iodine atmosphere. Fractions 1 to 8 [eluted

purple spot. Fractions 16 to 24 [eluted with: with benzene, methylene chloride and methylene chloride-

Chloroform-methanol · (99: 1) and (98: 2)] give 'methanol (99: 1)] give 54 mg of amorphous' material

15 mg of amorphous material (discarded). (discarded). Fraction 9 [6.5 mg eluted with methylene

. · "" ■ - i .-. chloride — methanol (99: 1)] yields 0.5 mg from ether

. With so ie 1 2 * ⁵ ^ ^st · Verrucarin A (still containing Verrucarin B).

P The. Fractions 10 to 14 [eluted with methylene chloride-

Production and recovery of methanol (99: 1)] and 15 to 20 [eluted with methylene

of crude verrucarin A and verrucarin B chloride — methanol (98: 2)] yield 28 mg. amorphous

..- '■ ... material in which with the help of the thin-film

The strain. NRRL 3003 is in · 101 of the examples o _a chromatogram still further amounts of .Verrucarin

Game 1 described nutrient solution in a 14-1- A and B can be detected. . _:

Fermenter for 6 days at 450 revolutions each

Minute of the stirrer and with ventilation (101 air

per minute) at 27 ^? ~ / bred. 11 of the culture broth example 4

is filtered through a layer of kieselguhr and the 25th

2N hydrochloric acid, twice with 50 ml of 2N sodium hydroxide solution each time The strain NRRL 3001 is produced under the conditions

and washed twice with 50 ml of water each time and datftpft in a vacuum in Example 1, but in Weindling's nutrient solution. This results in 277 mg of brown- ₃₀ (cf. Example 3), incubated .; Then 890 ml of colored crude extract, which is filtered in thin-layer chromatography culture broth to remove the mycelium, shows three spots, which verrucarin A, B and E express the clear filtrate twice, each with 1.1 ethyl acetate. The entire amount is shaken on 14 g of silica gel, the extracts are chromatographed once with water and twice. <· For elution, 30 ml each with 2N sodium hydroxide solution and twice with water-based solvent per fraction are used. Wash fractions 1 to 11 ₃₅ and evaporate in vacuo. It. result "[eluted with benzene, methylene chloride and methylene 428 mg of yellow-colored crude product, which in the thin chloride-Methano.1 (99: 1)] result in 127 mg of amorphous layer chromatogram (execution as in example 1 material, in which according to thin layer chromatography described) two Spots shows that verrucarin A and verrucarin B are detectable. These correspond to verrucarin B. For further purification, fractions 12 and 13 [11 mg eluted with methylene ₄₀ , the crude product is chromatochloride-methanol (99: 1) on 25 g silica gel ] yield 3.5 mg graphed from ether (execution analogous to the description in the crystall. Vemicarin E. Fractions 14 to 22 [eluted in example 1). 3.5 mg of crude crystalline result. Vernimm methylene chloride-methanol (99: 1)] result in 30 mg carin A (still containing verrucarin B),
amorphous material. The material of the factions 6

to 11 and 14 to 17 and the mother liquors of Example 5

Fractions 12 and 13 are combined (125 mg)

and again analogously to 13 g of silica gel chromate production and detection

graphed. From the methylene chloride-methanol ^of verrucarin A and verrucarin B

(99.5: 0.5) _r fractions are eluted from ether. The strain NRRL 3001 is produced under the conditions

a total of 3 mg of crude crystalline. Verrucarin A, which is still _{incubated 50} gene from Example 1. The extraction and which contains some Verrucarin B are won. further work-up of 780 ml of culture broth takes place

exactly the same as described in example 1. It results

■ p. 1 α animal 44 mg raw ethyl acetate extract (brown colored),

^exs P * ^e in the thin-layer chromatogram (execution as

Production and recovery ^{55 described in Beis} P ^iel 1) shows two spots whose

of raw verrucarin A and verrucarin B running tracks verrucarin A and verrucarin B ent

speak.

The strain NRRL 3003 is produced under the conditions of Example 6

of Example 1 in Weindling's nutrient solution [R. ", ■ _,. . . _Λ

W e i η d 1 i η g, Phytopathol., 24, p. 1155, R. Weind- 60 Production and isolation of crude verrucarin A, 1 i η g and A. E m e r s ο η, loc. Cit., 26, p. 1068: 25.0 g verrucarin B and Roridm A

Glucose puriss., Ph. H.V., the following substances The strain NRRL 3002 is in a 7-1 fermenter

in analytically pure quality: 2.0 g ammonium tartrate, with 51 nutrient solution (Richard's nutrient solution [cf. 2.0 g primary potassium phosphate, 1.0 g magnesium example I]) with aeration and stirring (300 umsulphate (· 7 H ₂ O) , 1.0 mg iron sulfate (· 7 H ₂ O), 0.15 mg 6 ₅ rotations per minute) at 27 °. After copper sulphate (· 5 H ₂ O), 1.0 mg zinc sulphate (· 7 H ₂ O), filtering off the mycelium, 4.11 pale yellow colored 1.0 mg manganese sulphate (· H ₂ O), 0.1 mg sodium KulturfUtrat extracted three times with 51 ethyl acetate each time, manganate demineralized water ad 1.01], the combined extracts washed once with 11 water.

13 14

see and in a vacuum to a volume of 500 ml Example8

constricted. The concentrated solution is used on this

0 ° three times with 100 ml of 2N sodium hydroxide solution each time and three times production and isolation of raw Verrucarm A, Washed with 100 ml of water each and in a vacuum Verrucarm B and Rondin A

evaporated. This results in 680 mg of yellow colored 5 The strain NRRL 3005 (Saltante from strain Raw product. For further purification, this NRRL 3004) is described in Example 7 chromatographed on 30 g of silica gel. Nutrient solution is used for elution in a 50 liter fermenter with stirring 60 ml of solvent per fraction. Fractions 1 (1450 revolutions per minute) and aeration (501 up to 10 (eluted with benzene and chloroform) deliver per minute) and at an overpressure of 0.4 Atmo-318 mg of oil (discarded). Fraction 11 [81 mg, eluted in cultured spheres. The culture broth (501) is then added with chloroform-methanol (99: 1)] gives filtered from ether after addition of kieselguhr as a filter aid, the 34 mg crystall. Verrucarin A, which after the thin-clear yellow-colored culture filtrate is six times with 401 layer chromatogram (description in Example 1), ethyl acetate extracted and the extracts uniform with water behaves. Wash crystalline from the mother liquor. The mycelium-diatomaceous earth mixture is after ize from ether another 11 mg mixture, consisting of 15 addition of water and homogenized three times with each Verrucarin A and Roridin A. The fractions 12 101 ethyl acetate stirred. The after filtering and to 16 [152 mg, eluted with chloroform-methanol washing with water extracts are obtained with (99: 1)] result from ether 61 mg of crystal mixture, which combines the ethyl acetate extracts of the culture filtrate and according to the thin-layer chromatogram, concentrated to a volume of about 51, mainly in vacuo, consists of verrucarin A, but still verrucarin B ao after washing with 11 2N sodium hydroxide solution and twice and Roridin A contains. The combined mother liquors with 0.51 each of 2N sodium hydroxide solution and water is im of fractions 11 to 16 (125 mg) still contain about vacuum evaporated to dryness. The resulting 30 mg verrucarin A and 30 mg roridin A. The residue (206 g) yields after addition of ether Fractions 17 to 23 [eluted with chloroform - 78 g of crude crystallizate, which after the thin-layer chromethanol (99: 1) (98: 2) and (1: 1)] result in 79 mg 25 matogramm (execution as described in example 1 amorphous Material (discarded). ben) consists mainly of Roridin A, but still

Roridin C, Verrucarin A and Verrucarin B as secondary

_n . -i _{contains 7} products. 128.5 g of mother liquor remain

^ßeisp '' residue I. The raw crystallizate (78g) is in metha-

Production and isolation of Roridin A ³ ° ™ 1 taken up and nitrated. The insoluble in methanol

borrowed portions (2.5 g) represent an artificial material

, The strain NRRL 3004, Myrothecium roridum, which is predominantly enriched with roridin C, is found in 51 nutrient solution (20 g glucose puriss., Ph. H. V, but still contains verrucarin A as an impurity. 2.0 g malt extract, Switzerland. Ferment AG, 2.0 g yeast The portions soluble in methanol are emgeextract Difco, 2.0 g peptone Cudahay, 2.0 g primary 35, and the residue from methanol-ether kdium phosphate, 2.0 g magnesium sulfate (-7 H ₂ O), 0.2 g fractionally recrystallized. Firstly, iron sulfate (· 7 H ₂ O) results in a fermenter under 33.9 g of pure crystalline Roridin A, secondly, 26.0 g of crystalline aeration (5 liters per minute) and stirring (300 um- Roridin A, which still cultivates some Roridin C, verrucarin A revolutions per minute). After filtering off and containing substance A, and thirdly 15.6 gmother of the mycelium, the yellow-colored clear solution of 4 ° caustic residue II extracted with mother pH 6 three times with ethyl acetate each time. The alkali residue I combined (144 g) and un d in 520 ml of combined extracts are dissolved once with 1 liter of water, chloroform-benzene (3: 1) mixture. 260 ml of the washed, to a volume of 700 ml in a vacuum water solution (corresponding to 72 g of mother liquor and concentrated at 0 ° three times with 80 ml of 2N sodium hydroxide each time) are applied to 3.77 kg of silica gel analogously to the description and twice with each 80 ml of water washed 45 exercise in Example 1 chromatographed. From the with and evaporated. 5.7 g of yellow-colored chloroform-methanol (95: 5) eluted fractions of crude product result, which is 105 mg of crystalline from acetone-ether. 1.2 g of pure Roridin A with a melting point of 190 ° to 195 ° are obtained by crystallization with ether. (Obtained in the thin-crystal Verrucarin A,
Layer chromatogram uniform.) The mother liquors

(5.6 g) are chromatographed on 250 g of silica gel. 50 Example9
250 ml of solvent per fraction are used for elution. ττ ^ « _JT i- _n -j · λ τ. -j- τ. Fractions 1 to 10 [eluted with benzene, chloro production and isolation of Rondm A, Rondm B, form and chloroform-methanol (99.5: 0.5)] give ^{Rondm C} 'Verrucarin A and Verrucarm C.
2.32 g of oily product (discarded). Fractions 11 strain Myrothecium roridum NRRL 3005 is supplied in up to 21 [2.45 g, eluted with chloroform-methanol 55 in a 10-1 fermenter with 101 air per minute at (99: 1)] from chloroform-methanol (97: 3) 0.4 atü and a temperature of 27 ° in the following 1.2 g of crude crystalline. Roridin A, which after the thin nutrient solution incubated: 30.0 g glucose puriss., Ph. HV, layer chromatogram still contains some verrucarin A and 1.0 g yeast extract Difco, 4.0 g ammonium nitrate, an unidentified substance A. After 2.72 g of primary potassium phosphate, 1.23 g of magnesium crystallization from acetone-ether-petroleum ether, 60 sulfate (-7H ₂ O), 0.028 g iron (II) sulfate (-7H ₂ O), 428 mg crystall. Roridin A from m.p. 191 to 196 °, 0.003 g zinc sulfate (· 7 H ₂ O). The culture broth (101) 166 mg crystall. Roridin A, which still contains traces of the sub- is filtered through a layer of kieselguhr and contains punch A, and 218 mg of a crystal mixture, the clear yellow-colored culture filtrate is extracted three times with 101 each of Roridin A and substance A in the ratio of ethyl acetate. The washed with 11 water (1: 1) obtained. 65 extracts are completely evaporated in vacuo. Roridin A is recrystallized from the residue is dissolved in 1 liter of chloroform and the acetone-ether is obtained in colorless prisms with a melting point of 191 solution three times with 100 ml of 2N sodium hydroxide solution each time and up to 196 or 198 to 204 °. washed four times with 100 ml of water each time. After one-

15 16

evaporating the washed chloroform solution, 80 g of aluminum oxide are chromatographed. For elution

12.0 g of yellow-colored crude product are obtained which are used to serve 100 ml of solvent per fraction. the

Further separation on 250 g of silica gel gives chromatographic fractions 1 to 11 (eluted with chloroform)

is graphed. Traces of amorphous material (discarded). The frac-

Chromatography: 500 ml each of 5 tions 12 to 21 [eluted with chloroform-methanol solvent per fraction are used for elution. Fractions 1 and 2 (99.5: 0.5)] yield from ether 200 mg of crystal mixture (eluted with benzene) containing 7.88 g of oil (discarded). of Roridin A and Roridin C. Fractions 22 to 6 (482 mg, eluted with chloro-51 [eluted with chloroform-methanol (99: 1)] yield form) give 23 mg from ether 432 from ether or petroleum ether mg uniform crystall. Roridin A. The krist. Roridin B of melting point 140 ° to 147 °, which is uniform after 10 fractions 52 to 67 [eluted with chloroform-methade thin-layer chromatogram. The nol (95: 5), (9: 1) and 4: 1)] give 46 mg of amorphous fractions 7 and 8 [eluted with chloroform-methanol material (discarded).
(99: 1)] yield 101 mg of amorphous material (discarded). Fractions 9 and 10 [1.48 g, eluted with Example 10
Chloroform-methanol (99: 1)] yield from ether 15 _,,. 646 mg of crystals with a melting point of 122 to 143 °. After reconstitution and isolation of crude Verrucarm A crystallize from acetone — ether, 400 mg ^{and B as well as} Verrucarm C and Roridin B result
Crystals with a melting point of 130 to 138 °, consisting mainly of Roridin A and verrucarin A and surface culture in a 500 ml Erlenmeyer flask with Roridin C each Nutrient solution in that described in Example 7 follows in chromatography II (see below). by adding 3.0 g of ammonium chloride per liter

Fraction 11 [305 mg, eluted with chloroform-Me supplemented nutrient medium, incubated at 27 °. The culture

ethanol (99: 1)] yields 106 mg of crystalline from ether. Roridin A, liquid is filtered off from the mycelium. By Extra-

which still contains traces of accompanying substances. The tion of 301 culture broth with ethyl acetate are 14 g

further purification is obtained through the chromatography 25 crude extract. 12.7 g of this extract will be

phie ΠΙ, which the mother liquor dissolved by the chromato- in 1.51 chloroform and the solution at 0 ° three-

graph II. Fractions 12 to 20 [eluted with chloromal with 150 ml of 2N sodium hydroxide solution each time and twice with

form — methanol (99: 1), (98: 2) and (1: 1)] provide 150 ml of water, washed and evaporated. It results

no crystals. The further purification of the fractions animals 9.5 g of neutral crude product, which on 250 g of silica

12 and 13 (330 mg) takes place in the chromatography II. 30 gel is chromatographed.
Chromatographies: The mother liquor residues are used for elution in each case with 400ml of the solvent

of fractions 9, 10 and 11 as well as fractions 12 tels per fraction. Fractions 1 to 10 [eluted with

and 13, a total of 1.39 g of material, are combined chloroform and chloroform-methanol (99.5: 0.5)]

and partition chromatography with 700 g give 1.46 g of amorphous material (discarded), the

Kieselguhr — water (1: 1) as the stationary phase and with 35 fractions 11 to 20 [4.324 g, eluted with chloroform—

the following water-saturated solvents (200 ml methanol (99: 1)] 200 mg crude crystalline verrucarin A,

per fraction) as a mobile phase. Which still contains Verrucarin B. The fractions 21 bis

Fractions 1 to 19 [eluted with benzene and benzene- 30 [eluted with chloroform-methanol (98: 2, (9: 1)]

Chloroform (95: 5)] provide 269 mg of amorphous material yield 4.21 g of amorphous material (discarded). the

(discarded). Fractions 20 to 25 [91 mg, 40 mother liquor residues from fractions 11 to 20 eluted

with benzene- ^ chloroform (95: 5)] obtained from ether (3.56 g) are neutral for further purification

13 mg pure crystall. Verrucarin B. The fractions 26 aluminum oxide of activity level II chromatograbis 30 [eluted with benzene-chloroform (95: 5)] eluted. 400 ml of solvent per give 62 mg of amorphous material (discarded) are used for elution. The parliamentary group. Fraction 1 [1.54 g, eluted with chloroform - fractions 31 to 38 [130 mg, eluted with benzene - 45 benzene (4: 1)] gives 25 mg of crude crystalline from ether. Verru-chloroform (95: 5)] yield 12 mg of pure carin A (still containing verrucarin B) from ether. The Frakkrist. Verrucarin A. From the mother liquors from tionen 2 to 19 [eluted with chloroform-benzene (4: 1), ether 20 mg pure crystalline. Roridin C of m.p. 117 chloroform, chloroform-methanol (99: 1), (98: 2) to 122 ^o . The fractions 39 to 43 [60 mg, eluted and (95: 5)] give 1.54 g of amorphous material (with benzene-chloroform ( 95: 5)] result thrown from ether 50). Fraction 20 [61 mg, eluted with chloroform - after fractional crystallization 28 mg of pure crystalline. Methanol (3: 1)] yields 8 mg verrucarin A and 17 mg pure crystalline from methanol-ether. Roridin C. The crystals have a mp _>: 226 to 228 °. According to recrystalline mother liquors, verrucarin B and roridin C.
Chloroform (95: 5), (9: 1) and (4: 1)] provide after 55 extraction of the mycelium: The dried mycelium is fractional crystallization 48 mg of pure crystalline. Verru three times with 21% methanol and 200 ml water 45 Micarin A. The mother liquors contain Verrucarin B and are extracted at 40 to 50 °, filtered and incorporated. Fractions 76 to 125 [eluted with steam. The residue (12.8 g) is then partitioned between zol-chloroform (1: 1) and chloroform] to give 500 ml of water and 1 liter of ethyl acetate and the aqueous 526 mg of amorphous material (discarded). The friable phase was eluted three more times with 11 ethyl acetate each time, 126 to 133 [350 mg, eluted with chloroform - shaken. The evaporated ethyl acetate extracts give ethyl acetate (9: 1)] yeasts from ether 305 mg of pure after evaporation 5.20 g of brown oil, which is crystallized as above. Roridin A from m.p. 194 to 202 °. The fraction described, 120 g of silica gel is chromatographed, ions 134 to 159 [eluted with chloroform-vinegar- From the esters eluting with chloroform-methanol (99.5: 0.5) (9: 1), (4: 1) and Ethyl acetate)] provide 278 mg of 65 th fractions (157 mg), 41 mg of amorphous material are obtained from ether (discarded). pure crystal Roridin B in needles from m.p. 147 to

Chromatography III: 800 mg of crystal mixture of 152 ° obtained. From later with chloroform metha-

Fractions 9 to 11 of chromatography I will crystallize out on nol (99: 1) eluted fractions

Ether 16 mg raw verrucarin A (still vernicarin B containing).

Example 11

Manufacture and isolation of raw Verrucarin A, Verrucarin B and Roridin B

The strain NRRL 3001 is in the nutrient solution described in Example 10 in a 50-1 fermenter (Example 7) with stirring (1450 revolutions per minute) and with constant aeration (0.81 per Minute per liter of nutrient solution) for 63 hours. After adding kieselguhr, the culture broth becomes with a total of 221 ethyl acetate extracted (separation of the phases in the centrifuge). After concentrating the extract in a vacuum to 41 is washed four times with 400 ml of 2N sodium hydroxide solution and three times with 300 ml of water each time and evaporated. 19.5 g of purified ethyl acetate extract result. Ether become through Crystallization 2.31 g of crude crystalline. Verrucarin A (still containing Verrucarin B) obtained. The mother liquors (17.2 g) are chromatographed on 850 g of silica gel. 1.71 are used to rewash the fractions Solvent per fraction. Fraction 1 (7.0 g, eluted with benzene) yields 19 mg of crystals from ether-petroleum ether from m.p. 237 to 239 ° (unidentified). Fractions 2 to 6 (4.15 g, eluted with chloroform) result from ether-petroleum ether 60 mg crystall. Roridin B in needles with a melting point of 139 to 143 °. Fractions 7 to 10 [eluted with chloroform-methanol (99: 1)] yield 1.09 g of amorphous material (discarded). Fractions 11 to 18 [3.25 g, eluted with chloroform- Methanol (98: 2)] yield 2.18 g of crude crystalline from ether. Verrucarin A (still containing Verrucarin B). The following fractions are discarded.

Example 12

Manufacture and isolation of verrucarin A,

Verrucarin B and Verrucarin D
The strain NRRL 3003 is incubated as a shaking culture, as in Example 1, in a medium containing 30 g of cane sugar, 2.5 g of ammonium chloride, 1.0 g of primary potassium phosphate, 0.5 g of magnesium sulfate (· 7 H ₂ O), Contains 0.5 g potassium chloride, 0.01 g iron sulfate (-7H ₂ O) and water. 900 ml of culture broth are extracted with 21 ethyl acetate. After washing with water, 2N sodium hydroxide solution and water and after evaporation in vacuo, the extract yields 243 mg of oily crude product which is chromatographed on 12 g of silica gel. From the fractions eluted with chloroform, 2 mg of pure verrucarin D in needles with a melting point of 127 ° to 128 ° are obtained from ether-petroleum ether. In the thin-layer chromatogram, verrucarin D migrates at about the same rate as verrucarin A. 38 mg of crude crystalline are obtained from the following fractions eluted with chloroform-methanol (99: 1). Obtained verrucarin A, which still contains verrucarin B.

Example 13

Separation of raw crystal Verrucarin A

(Containing verrucarin B) into pure verrucarin A.

and Verrucarin B

9.53 g crude crystalline Verrucarin A (containing verrucarin B) are subjected to partition chromatography 5.4 kg of kieselguhr water (1: 1) were subjected. The for Eluting solvents are saturated with water (1.21 per fraction). Fractions 1 to 20 [eluted with benzene and benzene-chloroform (95: 5)] yield 445 mg of oily material (discarded). Fractions 21 to 34 [1.20 g, eluted with benzene-chloroform (95: 5)] give 747 mg from acetone-ether pure crystal Verrucarin B (uniform in the thin-layer chromatogram). The fractions 35 to 37 [172 mg, eluted with benzene-chloroform (95: 5)] deliver Acetone-ether 126 mg crystal mixture of about 50% Verrucarin A and about 50% verrucarin B. Fractions 38 to 40 [eluted from benzene-chloroform (95: 5)] result in 406 mg of material, which consists mainly of verrucarin A (main product) and three to four others unidentified by-products. Fractions 41 to 84 [8.6 g, eluted with benzene-chloroform (9: 1), (8: 2), (3: 2), (7: 3) and chloroform)] yield 6.13 g of pure crystalline from acetone-ether. Verrucarin A (uniform in the thin-layer chromatogram).

Example 14
Thin layer chromatography of the verrucarines

and Roridine

Thin-layer chromatography is carried out according to known methods.

Rf values

substance D. Rf values
2) 3) Color reaction
with J ₂ colour
in UV light Verrucarin A 0.70
0.83
0.74
0.70 0.28
0.47
0.28
0.28 0.59
0.69
0.59
0.59
0.91 Yellow-brown
Yellow-brown
Yellow-brown
Yellow-brown
violet bright
bright
brightly lit.
dark
dark Verrucarin B 0.70
0.55 0.18
0.36 0.55
0.41
0.42 Yellow-brown
Greenish black
Yellow-brown dark
dark
dark Verrucarin C Verrucarin D Verrucarin E. Roridin A Roridin B Roridin C

1) Carrier: aluminum oxide

2) Carrier: silica gel

3) Carrier: silica gel

Flux: chloroform-methanol (98: 2) Flux: chloroform-methanol (98: 2) Solvent - chloroform - methanol (97: 3)

Claims (1)

Claim: Process for obtaining the new antibiotics Verrucarin A, Verrucarin B and / or Roridin A, characterized in that the strains NRRL 3001 to 3005 of the species Myrothecium grows in saprophytic culture and the antibiotics obtained from the culture filtrate isolated in a manner known per se. 1 sheet of drawings 609 707ß79 10.66 © Bundesdruckerei Berlin