EP0368953A4

EP0368953A4 - Method and microorganism for the production of faeriefungin

Info

Publication number: EP0368953A4
Application number: EP19890904883
Authority: EP
Inventors: Alan R. Putnam; Saroj K. Mishra; Muraleedharan G. Nair
Original assignee: Michigan State University MSU
Current assignee: Michigan State University MSU
Priority date: 1988-04-05
Filing date: 1989-02-21
Publication date: 1991-09-11
Also published as: EP0368953A1; WO1989009770A1; JPH02503805A; JPH0699421B2; CA1337643C

Abstract

A pentaene macrolide composition, referred to as faeriefungin, and compounds contained therein is described. The composition is produced by Streptomyces griseus var. Autotrophicus var. nov. The composition is useful for inhibiting the growth of certain viruses, bacteria and fungi as well as nematodes and insects.

Description

METHOD AND MICROORGANISM FOR THE

PRODUCTION OF FAERIEFUNGIN

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a novel carbonyl pentaene macrolide composition, referred to as "faeriefungin", and the compounds contained therein as produced by a unique strain of Streptomyces which are antimicrobial, nematicidal and insecticidal. In particular, the present invention relates to faeriefungin produced by Streptomyces griseus var. autotrophicus var. nov ATCC 53668.

(2) Prior Art

The prior art has described carbonyl pentaene macrolide mixtures having the molecular formulae C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀ which are stereoisomers of the faeriefungin of the present invention. However, the prior art stereoisomeric forms have a limited ability to inhibit microorganisms, have significant cytotoxicity, and are unstable at room temperature.

The stereoisomer "mycoticin" from Streptomyces ruber has been described by Burke, R. et al in Journal of Investigative Dermatology 23: 163-168 (1954). Mycoticin was active in vitro against yeasts and molds, was rapidly inactivated and relatively highly cytotoxic. It produced hemolysis in blood agar medium. The molecular and structural formulae for mycoticin were accurately determined much later by Wassermann, et al, J. of American Chemical Society 89, 6 (1967) and Wassermann, et al., Chemical Communications, 1634 (1970) and detailed analytical data was presented. The structural formulae were as follows :

where R is H- or CH₃- and referred to as "A" and "B", respectively. It is uncertain whether the pentaene macrolides being produced in the Wassermann et al studies were the same as those of Burke et al because of differences in the data.

Flavofungin is described by Bekesi, Nature, Vol. 181: 908 (1958). It was inactive against bacteria. Schneider, et al, Russian Journal (1967) compare related pentaene macrolide compositions. The mycoticin (mycothicin) from Streptomyces ruber is indicated to be a 1 to 1 mixture of A and B. Flavofungin is described as a 9:1 mixture of A to B. Antiviral activity is described. Vetlugiha, L. A., et al, Russian Journal (1974) set forth further analytical data including chromatographic spectra. Bognar et al, Tetrahedron Letters 7 , 471-474 (1970) concludes that mycoticin and flavofungin contain the same compounds in different proportions. The ratio of A to B in flavofungin is set forth as 9:1. Bognar et al., J.C.S. Perkin - I 1848 (1972) describe flavofungin and mycoticin. Analytical data is presented. Summaries of the properties of flavofungin and mycoticin are set forth in Antibiotics Vol. II Koryyoski et al, American Society for Microbiology (1978) and in Kirk-Othmer 3 , pages 21-47 (1978). Objects It is an object of the present invention to provide a novel pentaene macrolide composition containing compounds having molecular formulae which are the same as flavofungin and mycoticin but with different stereoisomeric structures, stabilities and antimicrobial properties. Further it is an object of the present invention to provide a broad spectrum antimicrobial composition which inhibits certain virus, fungi and bacteria. Further still, it is an object of the present invention to provide an antimicrobial composition from a unique microbial strain. These and other objects will become increasingly apparent by reference to the following description and the drawings. In the Drawings

Figure 1 is a flow chart showing the method for isolation of faeriefungin using methanol (MeOH) or trichloromethane (CHCI₃). Figure 2 shows an x-ray diffraction pattern for the novel pentaene macrolide composition of the present invention, referred to as faeriefungin (FUNGIN.RD), wherein the ratio of A to B by weight is about 1 to 1. Figure 3 shows an x-ray diffraction pattern for the prior art mycoticin (MYCON.RD; MYC) wherein the ratio of A to B is 1 to 1. General Description

The present invention relates to a carbonyl pentaene macrolide compound from a composition referred to as faeriefungin having a structural formula

wherein R is selected from hydrogen (A) and methyl (B) and all double bonds are trans and having negative optical rotation and an x-ray diffraction pattern showing 28 crystalline peaks at a d-spacing between

1.5408 and 43.9165 Angstroms for a 1 to 1 molar ratio of A to B. The d values are shown in Table 4. Further the present invention relates to a carbonyl pentaene macrolide composition referred to as faeriefungin characterized by a melting point of 209-212ºC with partial decomposition, by a molecular formulae selected from the group consisting of C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀ and mixtures thereof and positive ion fast atom bombardment mass spectrometry indicated molecular ions of 651.4017 for C₃₆H₅₉O₁₀ and 665.4237 for C₃₇H₆₁O₁₀, a negative optical rotation and an x-ray diffraction pattern as shown in Figure 1 for a 1 to 1 molar mixture.

Further still the present invention relates to a composition which comprises Streptomyces griseus var. autotrophicus var. nov ATCC 53668 and a synthetic culture medium which maintains the Streptomyces griseus. The present invention also relates to a method for producing a carbonyl pentaene macrolide referred to as faeriefungin which comprises growing Streptomyces griseus var. autotrophicus var. nov ATCC 53668 mycelia in an aqueous growth medium containing sources of carbon, nitrogen and inorganic substances to produce the carbonyl pentaene macrolide having a negative optical rotation pattern and an x-ray diffraction pattern as shown in Figure 1. The present invention also relates to a method for inhibiting the growth of a microorganism which comprises exposing the microorganism to an effective amount of a carbonyl pentaene microlide compound from a composition referred to as faeriefungin having a structural formula:

wherein R is selected from hydrogen (A) and methyl (B) and all double bonds are trans and having a negative optical rotation and an x-ray diffraction pattern with peaks at a d-spacing between 1.5408 and 43.9165 Angstroms for a 1 to 1 molar ratio of A to B.

Further the present invention relates to a mixed carbonyl pentaene macrolide composition referred to as faeriefungin for inhibiting the growth of microorganisms characterized by a melting point of 209-212°C with partial decomposition, by molecular formulae of C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀ and mixtures thereof and fast atom bombardment mass spectrometry indicated molecular ions of 651.4017 for C₃₆H₅₉O₁₀ and 665.4237 for C₃₇H₆₁O₁₀, by a negative optical rotation and by an x-ray diffraction pattern as shown in Figure 1 in a carrier in an amount between about 1 and 100 micrograms per ml or per gram.

In the present specification, the word "carrier" means a pharmaceutically acceptable non-toxic substance that when mixed with the active ingredient or ingredients renders it suitable for administration. The expression preferably excludes water and low-molecular weight organic solvents commonly used in chemical synthesis, except in the presence of other phramaceutically necessary ingredients such as salts in correct quantitites to render the composition isotonic, buffers, surfactants, coloring and flavoring agents, and preservatives. Examples of suitable solid and liquid diluents and carriers are the following: water containing buffering agents which can be rendered isotonic by the addition of glucose or salts; non-toxic organic solvents; such as paraffins, vegetable oils; alcohols; glycόls; natural ground rock (for example kaolins, aluminas, talc or chalk); synthetic rock powders (for example highly dispersed silica or silicates); and sugars. Oral administration can be effected utilizing solid and liquid dosage unit forms such as powders, tablets, dragees, capsules, granulates, suspensions, solutions and the like. Where appropriate, dosage unit formulations for oral administration can be microencapsulated to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like.

Parenteral administration can be effected utilizing liquid dosage unit forms such as sterile solutions and suspensions intended for subcutaneous, intramuscular or intravenous injection. These are prepared by suspending or dissolving a measured amount of the compound in a nontoxic liquid vehicle suitable for injection such as an aqueous or oleaginous medium and sterilizing the suspension or solution. Stabilizers, preservatives and emulsifiers can also be added.

Generally the parenteral dosage will be from 0.01 to 50 mg/kg, preferably from 0.1 to 10 mg/kg, of body weight per day, and the oral dosage form will be from 0.1 to 500 mg/kg, preferably 0.5 to 100 mg/kg, of body weight per day.

Streptomyces griseus var. autotrophicus var. nov has been deposited under the Budapest Treaty with the American Type Culture Collection as ATCC 53668. This strain produces the composition referred to herein as "faeriefungin". SPECIFIC DESCRIPTION Example 1

Faeriefungin was isolated from the mycelium of Streptomyces griseus var. autotrophicus var. nov, ATCC 53668 (also known as MSU SM008). This strain was isolated from a soil sample collected from a fairy ring from a grass lawn. The composition was produced by growth in bacto peptone, glucose, Brer Rabbit-Green™ Label-molasses, 5:10:20 grams per liter in distilled water (A-9 medium) under the following conditions. Smaller batches of cultures were grown in two liter baffle-bottomed Erlenmeyer flasks containing 400 ml medium, placed on a rotary shaker at 100-200 rpm at 26ºC for 5 to 7 days. Larger batches were grown in a 130 liter fermentor containing 100 liter of A-9 medium, aerated at 100 L/minute and stirred at 100 rpm at 26ºC for 5 days. Streptomyces griseus var. autotrophicus var. nov produced approximately 1.0 g/L or more of faeriefungin over a 7-12 day period in A-9 medium. A. Microbiological Aspects:

1. Isolation and Growth. ATCC 53668 was isolated from a soil sample collected from the center of a fairy ring. The soil was suspended in sterile physiological saline and serial dilutions were plated on various isolation media. The colony of this strain was picked up from a Czapeck agar plate (sucrose 20.0g, NaNO₃ 3.0g, K₂HPO₄ 1.0g, MgSO₄·7H₂O 0.5g, KCl 0.5g, FeSO₄·7H₂O 0.01g, bacto agar 15.0g, distilled water 1 liter). The microorganism grows well at room temperature (25ºC) on most of the laboratory media. On YMG agar (yeast extract, malt extract, glucose, agar; 4:10:4:18 grams per liter in distilled water), it produced slightly wrinkled colonies that were yellowish orange with abundant aerial hyphae at the periphery.

The growth was powdery on N.Z. Amine-A (NZ amine-A 3 g in 1 liter distilled water) agar and leathery on nutrient agar (Difco, Detroit, Michigan). Older colonies developed cracks typical of Nocardia autotrophica. During the microscopic examination, the .aerial as well as substrate hyphae appeared straight with branchings at right angles. Spirals, sporangia, spore chain or endospores were not seen. The microorganism decomposed adenine, tyrosine, hypoxanthine, xanthine, and casein. It produced acid with adonitol, cellobiose, glucose, galactose, inositol, lactose, maltose, mannitol, melibiose, a-methyl-D-glucoside, raffinose, trehlose, and xylose. Acid production was not observed with arabinose, erythritol, melezitose, rhamnose, and sorbitol.

Although the colonial morphology of ATCC 53668 was similar to that of N. autotrophica, its physiological characteristics were closer to those of Streptomyces griseus. Consideration of these two major traits warranted recognition of this strain as a new variety of S. griseus. We adopted the nomenclature, S. griseus var. autotrophicus var. nov.

B. Isolation, Purification and Chemical Characterization:

The isolation and purification of faeriefungin is as set forth in Figure 1. As can be seen from Figure 1, the culture is grown in broth and then the cells are harvested. The cells are treated with methanol and the broth is treated with trichloromethane. The crystals (X'ls) of faeriefungin are separated by cooling from the methanol twice and then the filtrate is extracted with trichloromethane. The crystals can be combined. Thus as can be seen, faeriefungin is both intra- and extra-cellular.

1. Basic Physical Data: Melting Point: 209-212°C with partial decomposition. Solubility: Faeriefungin is sparingly soluble in methanol, fairly soluble in chloroform-methanol mixtures, partially soluble in water, completely soluble in DMSO at room temperature. Previously reported pentaene macrolides are soluble in methanol at the same amount and temperature at which faeriefungin was studied.

Stability: Pure faeriefungin is stable at room temperature and under laboratory artificial light conditions, whereas previously reported pentaene macrolides are unstable under those conditions.

2. Spectral Data: MeOH a. UV Spectra. λ Max (meOH) = 262, 363.5, no base shift by addition of

5% KOH in MeOH. UV spectra of peracetate of faeriefungin has the

MeOH following λ_Max values: 210, 261,

363.5; with previously studied acetates of pentaene macrolides, the max 363 . 5 resolved into 3 peaks.

Faeriefungin acetate did not. b. Mass Spectra: Faeriefungin (homologous mixtures of C₃₆H₅₈O₁₀ and C₃₇H₆₀H₁₀)

M⁺650, FAB, High Resolution, Peak

Matching, 651.4017 (C₃₆H₅₉O₁₀, M⁺ +

H).

M₊664, FAB, High Resolution, Peak

Matching, 665.4237 (C₃₇H₇₁O₁₀, M⁺ +

H). c . IR-Spectra: Max 3400 (OH, 1695 (lactone C=O),

1610 (C=C), 1570 (conjugated C=C) cm^-1 d. ¹³C-NMR: Natural abundance ¹³C-NMR indicated resonances for a lactone carbonyl, olefinic carbons, carbon bearing secondary hydroxyl groups, carbon attached to oxygen which is part of the lactone ring, methyl groups, methylene groups, and methyne groups. e. ¹H-NMR:

Faeriefungin contains five adjacent and one separate C=C bonds. All of the double bonds are trans, contains two secondary (2°) methyl groups and contains eight secondary (2°) -OH groups, which was confirmed by acetylation.

Faeriefungin was a mixture of 13, 15, 17, 19, 21, 23, 25, 27 octahydroxy-31-isopropyl-14, 30-dimethyl-hentriaconta -2,4,6,8,10, 28-hexen-31-olide (A) and 13, 15, 17, 21, 23, 25, 27 octahydroxy-14, 30-dimethyl-31-S-butyl hentriaconta-2,4,6,8,10,28-hexene-31-olide (B).

The stereochemistry of the -OH and alkyl substituents on the macrolide ring is not known. Faeriefungin is a 1:1 molar mixture of C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀.

Table 1 shows a comparison of the properties of faeriefungin, mycoticin and flavofungin.

Table 1 Faeriefungin Mycoticin Flavofungin Appearance pale yellow yellow yellow needles crystals crystals Stability stable, when stored Decomposes Decomposes at 25°C in solid form

Converts to new Unknown Unknown compound in dioxane, chloroform, methanol, dichloromethane, pyridine when kept in light. No change in the dark. m.p. 210-213 220-222 210 charring at 310°C (decomposes) (decomposes)

363.5 (965), 364 (948), 363(8%) 259.5 (71) 262 (79)

363.5 (ε=63413), -

259.5 (ε=4649.54)

IR (KBr) 3375, 3000, 2950 3400, 1695 3400, 2945,

2925, 2900, 2850, 1610, 1710, 1680,

1678, 1605, 1560, 1570 cm^-1* 1232, 1122,

1420, 1350, 1300 1020, 840cm^-1*

MF C₃₆H₅₈O₁₀ (50%) C₃₆H₅₈H₁₀ C₃₆H₅₈O₁₀ (50%) (90%)

C₃₇H₆₀O₁₀ (50%) C₃₇H₆₀O₁₀ C₃₇H₆₀O₁₀ (50%) (10%)

M. wt 650 and 664 650 and 664 650 and 664

Example 2 The specific rotations for the compositions produced by two Streptomyces strains, ATCC 3348 of Burke et al and Wassermann et al and ATCC 53668, in A-9 medium and the medium of Burke et al (1954) were determined. The [α]² _D ⁵ values were for a 0.2% solutions in pyridine and dioxane and 0.15% in MeOH. The results are shown in Table 2.

Table 2

Compound Pyridine Dioxane MeOH t=0 t=10 t=0 t=10 t=0 t=10

FF -70.5 -70.5 -40.5 -52.5 -34.0 -35.33 Mycoticin - - +63.5 +81.7 - -

(Burke et al)

MYC-N -74.5 -74.5 -58.0 -58.0 -44.66 -42.0

Flavofungin -85-90 - - - -47.51 -

(from literature) MYC -72.0 -73.5 -50.0 -51.5 -38.66 -38.66

(from literature) FF-II -46.0 -54.0 - - - -

t = time in minutes. FF = Faerifungin, produced by ATCC 53668 in A-9 medium (1 to 1 A to B).

MYC-N = Compound produced by ATCC 3348 in A-9 medium (9:1 A to B). MYC = Compound produced by ATCC 3348 in 'Burke's' medium (supposed to be mycoticin (1 to 1 A to B)).

FF-II = Compound produced by ATCC 53668 in 'Burke's' medium. Flavofungin = (9 to 1 A to B).

ATCC 3348 is Streptomyces ruber, the original mycoticin A and B producing organism. Burke's medium was the original medium used to produce mycoticin A and B, [α]² _D ⁵ (c=0.48%, dioxane) = +63.5, by ATCC 3348. Streptomyces ruber ATCC 3348 no longer produced mycoticin A and B in the original Burke's medium. ATCC strain 3348 produced a 90:10% composition of a polyene macrolide (MYC-N) in A-9 medium with molecular formulae (MF): C₃₆H₅₈O₁₀ (90%) and C₃₇H₆₀O₁₀ (10%). This was confirmed by C¹³-NMR and MS. Streptomyces griseus var. autotrophicus ATCC 53668, produced a 50:50 (1:1) mixture of faeriefungin (FF) with MF C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀. MYC (literature) had a specific rotation different from FF where the molar proportions of A to B were about equal; however, this was not considered to clearly differentiate FF from MYC.

Bognar et al confirmed the specific rotation change of mycoticin found by Burke et al in dioxane with time. The [α]_D2 ⁵ value in dioxane changed from +63.5 to +81.7° in 9.5 minutes and reached zero value at 15 minutes. The final rotation at t = ∞, was -18.2°.

Flavofungin did not show this effect, possibly because of the high proportion of A to B (9:1). Faeriefungin showed some change in rotation with different solvents, but retained the same negative value obtained after t = 10 minutes. It appears that flavofungin and mycoticin (MYC-N or MYC) differ only in the proportions of A to B. Faeriefungin (FF) appears to be a different stereoisomeric composition.

Example 3 Circular dichroism (CD) studies of octa-acetylmycoticin and -flavofungin in dioxane were made and compared to the CD values for faeriefungin in Table 3.

Table 3 ⁽¹⁾ Octa-acetyl Octa-acetyl Octa-acetyl flavofungin faeriefungin mycoticin λ Δε λ Δε λ Δε

208 +2.34 347 -11.3 210 +0.85

224 -2.34 363 -19.2 224 -2.04

260 -5.19 385 -11.9 260 -3.73

343 -2.51 faeriefungin 345 -1.86

371 -2.18 387 -10.2 362 -2.04

389 -2.51 366 - 9.7 380 -1.86 347 -10.2 (1) No CD transitions were observed in the 260 to 240 nm region for faeriefungin octa-acetate or faeriefungin. Table 3(a) Circular dichroism (CD) of faeriefungin and mycoticin-N in MeOH.

Faeriefungin Mycoticin-N λ Δε λ Δε 380 -14.6 377 -11.4 374 -16.3 372 -11.4 368 -11.8 370 -16.6 362 -13.5 365 -15.8 360 -13.5 360 -15.0 354 -10.1 355 -13.4 349 -10.1 348 - 7.1 344 - 9-6 339 - 3.9

The CD curves of the octa-acetate of mycoticin and flavofungin were similar in shape, but quantitatively different as shown in Table 3 (Bognar et al., and Brown et al., J. C. S. Perkin I, 1848, 1972). The octa-acetyl faeriefungin produced a CD curve different from mycoticin and flavofungin octa-acetates. It is different both quantitatively and in shape. Table 3(a) shows that faeriefungin and mycoticin N have different CD values. The results show that faeriefungin contains significantly different stereoisomers. The structures are different from those characterized by Schreiber, S. L. et al in Tetrahedron Letters 28, 6001-6004 (1987) and 28, 6005-6008 (1987) for mycoticin A and B produced in the manner of Wasserman et al (1967 and 1970). Schreiber, S. L. et al indicated that there were 64 possible stereoisomers. Example 4 The x-ray diffraction patterns for faeriefungin and mycoticin (MYC-N) were determined as shown in Figures 2 and 3. Faeriefungin had 28 discernible crystalline peaks whereas mycoticin (MYC-N) had 14 as shown in Tables 4 and 5. Only about 6 of the peaks are common. The results show that faeriefungin contains significantly different stereoisomers.

Table 4 Summarized X-ray Diffraction Pattern for Faeriefungin

PEAKS

Listed DI file name FUNGIN.DI Raw data file name FUNGIN.RD Sample identification fungin Date of measurement 9-DEC-87 Generator settings 35 kV, 20 mA Step size, count time 0.020 deg, 1.00 s Angle range (2theta) 2.010 - 59.990 deg Al, A2 wavelengths 1.54056, 1.54435 Ang Range in d-spacing 1.5408 - 43.9165 Ang Monochromator used Yes Maximum peak counts 19404. cts, 19404. cps

Peak Angle Tip width Peak Backg D spac I/Imax Type Qual no (deg) (deg) (cts) (cts) (Ang) (%) A1 A2 0t

1 5.4325 0.12 71. 46. 16.2542 0.36 X X 0.93

2 6.4100 0.08 19404. 55. 13.7775 100.00 X X 31.62

3 6.9675 0.14 279. 66. 12.6763 1.44 X X 5.62

4 9.5950 0.12 734. 69. 9.2101 3.78 X X 10.23

5 10.7275 0.16 56. 72. 8.2402 0.29 X X 0.85

6 11.2600 0.14 681. 74. 7.8517 3.51 X X 11.75

7 12.0900 0.16 67. 77. 7.3144 0.35 X X 1.58

8 13.3125 0.12 34. 69. 6.6454 0.17 X X 1.29

9 14.8850 0.20 53. 64. 5.9467 0.27 X X 1.07

10 15.3300 0.16 55. 72. 5.7751 0.28 X X 0.87

11 15.8300 0.20 59. 72. 5.5938 0.31 X X 1.20

12 16.8775 0.20 1901. 94. 5.2489 9.80 X X 32.36

13 17.7950 0.20 142. 102. 4.9802 0.73 X X 1.55

14 18.1700 0.32 117. 102. 4.8783 0.60 X X 1.51

15 18.8125 0.10 146. 110. 4.7131 0.75 X X 1.29

16 19.8175 0.22 784. 130. 4.4763 4.04 X X 17.38

17 20.8150 0.16 172. 135. 4.2640 0.88 X X 1.05

18 21.1850 0.16 185. 142. 4.1903 0.95 X X 0.83

19 21.5100 0.12 156. 151. 4.1278 0.81 X X 1.02

20 22.5600 0.12 524. 204. 3.9380 2.70 X X 3.63

21 24.0625 0.20 1384. 204. 3.6954 7.13 X X 24.55

22 24.8900 0.24 94. 204. 3.5744 0.48 X X 1.66

23 28.2900 0.16 96. 193. 3.1520 0.49 X X 0.98

24 30.4700 0.16 62. 193. 2.9313 0.32 X X 0.83

25 34.0125 0.20 18. 161. 2.6337 0.10 X X 0.85 26 39.9900 0.96 16. 121. 2.2527 0.08 X X 0.83

27 43.0925 1.28 14. 128. 2.0974 0.07 X X 0.89

28 49.2900 0.96 10. 125. 1.8472 0.05 X X 0.81

28 peaks identified 28 crystalline 0 amorphous All listed

Table 5

PEAKS

Listed DI file name MYCON.DI Raw data file name MYCON.RD Sample identification mycon Date of measurement 9-DEC-87 Generator settings 35 kV, 20 mA Step size, count time 0.020 deg, 1.00 s Angle range (2theta) 2.010 - 59.990 deg A1, A2 wavelengs 1.54056, 1.54435 Ang Range in d-spacing 1.5408 - 43.9165 Ang Monochromator used Yes Maximum peak counts 1156. cts, 1156. cps

Peak Angle Tip width Peak Backg D spac l/lmax Type Qual no (deg) (deg) (cts) (cts) (Ang) (%) A1 A2 0t

1 6.4075 0.12 1156. 69. 13.7829 100.00 X X 4.07

2 6.9625 0.10 303. 74. 12.6854 26.19 X X 4.47 3 8.2575 0.16 106. 67. 10.6987 9.18 X X 1.82

4 10.3525 0.20 37. 62. 8.5378 3.22 X X 0.78

5 10.8200 0.28 77. 62. 8.1700 6.70 X X 1.62

6 12.1875 0.12 37. 79. 7.2562 3.22 X X 1.86

7 14.1025 0.24 21. 71. 6.2748 1.83 X X 0.78 8 16.7425 0.56 85. 81. 5.2909 7.32 X X 4.37

9 17.9225 0.24 50. 92. 4.9451 4.36 X X 0.91

10 19.9275 0.80 161. 137. 4.4518 13.95 X X 9.77

11 21.3850 0.24 61. 159. 4.1516 5.26 X X 1.07

12 22.8475 0.24 119. 177. 3.8891 10.28 X X 0.91 13 24.8600 0.48 10. 243. 3.5786 0.89 X X 1.48

14 26.0400 0.96 10. 213. 3.4190 0.89 X X 1.91

14 peaks identified 14 crystalline 0 amorphous All listed

Example 5

Table 6 shows comparisons of minimum inhibitory concentrations (CMIC) of faeriefungin and other antifungal agents in ug/ml. Table 6

Substance Organisms

Candida Aspergillus Microsporum Trichophyton albicans fumigatus cams rubrum Faeriefungin 5.5 3.2 3.2 3.2

Amphotericin B 3.2 80 16 16

Nystatin 3.2 >100 >100 >100

Pimaricin 5.5 16 16 16

Ketoconazole 0.5-500 1-500 5.5-80 5.5-80 Miconazole 1-50 8-500 16 80

Clotrimazole 1-50 1-500 16 80

5-flurocytosine*0.5-500 50-100 >500 >500

*This data is based upon the literature. The prior art antifungal agents are listed by their generic names.

Example 6 In order to confirm the data from the literature, the cultures were grown in 500 ml baffled bottom Erlenmeyer flasks, each containing 100ml of the liquid A-9 medium. The inoculated flasks were placed on a rotary shaker at 200 r.p.m., 26°C. The pH and the antifungal activities were monitored daily up to a period of 14 days. The antifungal activities were determined by spreading evenly a thick suspension (200 × 10 cells/ml) of the spores or yeast-like cells of the test species on Emmons (neopeptone, glucose, bacto agar; 10:20:18 grams per liter in distilled water) agar in 100 mm Petri dishes and placing 25 ul of the culture broth on the surface. The test culture was incubated at 26°C for 2 days. A clear zone of inhibition was indicative of antifungal activity. Production of the antifungal compound could be detected after 3 days, peaked on 8th day and declined after 11th day. During this period, pH of the medium ranged between 6 and 8. Larger batches were grown in 2 L flasks containing 400 ml of A-9 medium. After 8 days of growth at 26°C (shaker speed: days 1-3, 100 r.p.m.; days 4-5, 150 r.p.m.; days 6-8, 200 r.p.m.) the culture broth was centrifuged and the cake was extracted in methanol as shown in Figure 1. The purified substance(s) was dissolved in DMSO and known amounts are bioassayed against Candida albicans, Aspergillus flavus and Fusarium species. Comparable amounts of nystatyin, pimaricin and amphotericin B also dissolved in DMSO were tested simultaneously. Faeriefungin was more active than a Amphotericin B against A. fumigatus, it was slightly less active than pimaricin, but better than nystatin. Against C. albicans, this compound had similar activity as amphotericin B. Against Fusarium species, Faeriefungin was better than any of the three antibiotics tested. Faeriefungin also has a low MIC against a broad spectrum of fungi.

Table 7 shows the minimum inhibitory concentrations (MIC) in ug/ml of faeriefungin, amphotericin B and nystatin against various bacteria and fungi.

Table 7

Organisms Faeriefungin Amphotericin B Nystatin

FUNGI :

Aspergillus fumigatus 3.2 80 80

A. flavus 3.2 80 80

Candida albicans 5.5 3.2 16

C. trophicalis 5.5 5.5 16

Microsporum canis 3.2 16 >100

Trichophyton rubrum 3.2 16 >100

Alternaria solani 3.2 >100 >100

Fusarium oxysporum 3.2 >100 >100

Fusarium moniliforme 3.2 >100 >100

Pythium ultimum 12.0 >100 >100

Phialophora graminicola 12.0 >100 >100

Leptosphaeria korrae 12.0 >100 >100

BACTERIA:

Escherichia coli not active not active not active

Proteus vulgaris not active not active not active

P. mirabilis not active not active not active

Pseudomonas aeruqmosa not active not active not active

Staphylococcus aureus 16.0 not active not active

S. saprophyticus 64.0 not active not active

Streptococcus bovis 16.0 not active not active

S. agalactiae 16.0 not active not active

S. faecalis 16.0 not active not active

S. pneumoniae 16.0 not active not active

S. pyogenes 16.0 not active not active

S. sanguis 16.0 not active not active

Streptococcus group C 16.0 not active not active

Streptococcus group D 16.0 not active not active

Neiseria gonorrhaeae 16-24 not active not active

N. meningitidis 32-64 >128 not active

N. cinerea 32-64 >128 not active

N. flava 64 >128 not active

N. mucosa 64-128 >128 not active

N. subflava not active not active not active

N. sicca 64 >128 not active

N. perflava not active not active not active

Haemophilus influenzae 64-128 >128 not active

Acenetobacter cales not active not active not active

Branhamella catarrhalis 64 >128 not active

Klebsiella pneumoniae not active not active not active

As can be seen faeriefungin is active against a number of strains of bacteria whereas the prior art compositions are not. Tables 8 and 9 show the reported activities for flavofungin and mycoticin.

Table 8

Inhibitory Cone. ug/ml

Fungi:

Aspergillus clavatus 15

Penicillium chrysogenum 8

Penicillium novum hybrid 8 Penicillium sp. (two strains) 10-20

Scopulariopsis sp. 15

Cephalosporium sp. 15

Monosporium apiospermum 15

Helminthosporium sp. 10 Tricholeccum roseum 15

Mastigocladium sp. 10

Yeast and Yeast-Like Fungi :

Candida albicans (three strains) 4-5

Candida krusez 6 Candida tropicalis 15

Saccharomyces cerevisiae (three strains) 3-20

Saccharomyces niger 2

Cryptococcus neoformans 2

Torula utilis 10 Hansenula anomala 10

Rhodotorula sp. 2

Torulopsis pulcherrima 2 Pathogenic Fungi:

Trichophyton mentagrophytes 20

Trichophyton tonsurans (two strains) 8-30

Trichophyton rubrum (two strains) 10 Trichophyton. gypseum (two strains) 20 Trichophyton sulfureium 8

Epidermophyton Kaufmann-Wolf (three strains ) 15-20

Epidermophyton inguinule 10 Microsporum canis 8 Microsporum gypseum 8

Achorion quinckeanum 8

Ceratinomyces 10

Phialophora verrucosa 20

Histaplasma capsulatum 10 Sporolrichum schenkii 10 Hormodeudrum compactum 8

Nocardia asteroides 20

Geotrichum sp. 20

Burke et al, the Journal of Investigative Dermatology, 23, 163 (1954).

Table 9

Inhibitory Concentration

Organism mcg/ml, No. Hours on Agar

Cryptococcus neoformans ATCC 10226 6-8 Candida albicans ATCC 10261 6-10 Blastomyces dermatitidis ATCC 10225 2-4 Histoplasma capsulatum ATCC 10220 1-2 Sporotrichum schenckii ATCC 10213 1-3 Hormodendrum pedrosoi ATCC 9475 7-10 Coccidioides immitis Conant 2150 2-3

Trichophyton mentagrophytes ATCC 9972 5-7 Trichophyton mentagrophytes Harvard 16 6-9 Trichophyton rubrum ATCC 9806 3-5 Microsporum audonini ATCC 10216 2-3 Microsporum canis ATCC 9084 4-7 Microsporum gypseum Harvard 22 8-.0 Bacillus subtilis >100 Escherichia coli >100 Staphylococcus aureus >100 Mycobacterium phlei >100

Streptococcus hemolyticus >100 Salmonella typhi >100 Leishmania donovani >100 Tryphanosoma tropica >100 Endamoeba histolytica >100

Table 10 shows a summary of the biological activity of faeriefungin, mycoticin and flavofungin.

Table 10 Faeriefungin Mycoticin Flavofungin Biological Antifungal Antifungal Antifungal Activity . Antibacterial Not anti- Not anti- bacterial bacterial

Antiviral Not anti- Antiviral viral Antiviral activity of carbonyl pentaene macrolides except faeriefungin has been studied on influenza A and B virus, variolavaccine virus and Rous sarcoma virus by M. A. Schneider et al (Schneider, et al., Russian Journal, 1967). It was reported that flavofungin was the most active against the above viruses.

Example 7 Table 11 shows the toxicity of the faeriefungin to human erythrocytes.

Table 11

Concentration Faeriefungin Amphotericin B

100 ug/ml Toxic Toxic

20 ug/ml Toxic Toxic 4 ug/ml Non-τtoxic Toxic

0.8 ug/ml Non-toxic Non-toxic

As can be seen, faeriefungin is non-toxic at low levels in vitro.

Example 8 Broths of Streptomyces ATCC 53668 containing faeriefungin also showed a highly toxic rapid response (greater than 50% kill within 2 hours) on mosquito (Aedes aegypti) larvae. However, pure mycoticin (MYC-N) was only moderately toxic to mosquito larvae (35% mortality at 100 ppm for 24 hours), possibly indicating that another component produced by ATCC 3348, may be the primary insecticidal moiety. Broths were moderately toxic (rapid to slow) to cultures of the nematode Panagrellus redivivus. The biosynthesis of mycoticins A and B has been studied using labelled acetate, propionate, and methionine by Wassermann, et al., Chem. Communication, 1634 (1970). The results indicated that the macrolide pentaene ring is biosynthesized through acetate. The alkyl side chain and two methyl groups substituents are biosynthesized from the propionate, i.e., C-14 and C-30 methyl substituents and the isopropyl (isobutyl) side chain at C-31 (Wassermann, et al., Chem. Communication, 1634 (1970)). No methionine uptake was reported. In the case of faeriefungin, experiments with ¹³C-labeled acetate indicated incorporation of acetate for the ring carbons. Propionate was biosynthesized for C-14 and C-30 methyls. There was no incorporation of either acetate nor propionate for the C-31 carbon and the alkyl side chain (isopropyl or iso-butyl) at C-31. The experiments confirmed that faeriefungin production is proceeding through a different biosynthetic pathway than the reported route for the production of mycoticins and that the compounds are different for this reason. The faeriefungin can be used to inhibit plant or animal pathogens. Various well known solid or liquid carriers for pharmaceutical, veterinary or plant use can be used. The composition can be applied topically very effectively. It is intended that the foregoing Examples are only illustrative of the present invention and that the invention be limited only by the hereinafter appended claims.

Claims

I CLAIM :

-1- A carbonyl pentaene macrolide compound from a composition referred to as faeriefungin having a structural formula

wherein R is selected from hydrogen (A) and methyl (B) and all double bonds are trans and having negative optical rotation and an x-ray diffraction pattern with 28 crystalline peaks at a d-spacing between 1.5408 and 43.9165 Angstroms for a 1 to 1 molar ratio of A to B.

-2-

A carbonyl pentaene macrolide composition referred to as faeriefungin characterized by a melting point of 209-212°C with partial decomposition, by a molecular formulae selected from the group consisting of C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀ and mixtures thereof and positive ion fast atom bombardment mass spectrometry indicated molecular ions of 651.4017 for C₃₆H₅₉O₁₀ and 665.4237 for C₃₇H₆₁O₁₀, a negative optical rotation and an x-ray diffraction pattern as shown in Figure 1 for a 1 to 1 molar mixture.

-3-

The composition of Claim 2 as the 1 to 1 molar mixture having ultraviolet absorption spectral peaks in methanol at 262 nm and 363.5 nm. -4- A carbonyl pentaene macrolide produced by Streptomyces griseus var. autotrophicus var. nov. ATCC 53668 having a negative optical rotation and x-ray diffraction pattern as shown in Figure 1.

-5- A biologically pure culture referred to as Streptomyces griseus var. autotrophicus var. nov. ATCC 53668 and producing an antimicrobial referred to as faeriefungin in a recoverable quantity upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.

-6- A composition which comprises:

(a) Streptomyces griseus var. autotrophicus var. nov. ATCC 53668; and

(b) a synthetic culture medium which maintains the Streptomyces griseus.

-7-

A method for producing a carbonyl pentaene macrolide referred to as faeriefungin which comprises: growing Streptomyces griseus var. autotrophicus var. nov. ATCC 53668 mycelia in an aqueous growth medium containing sources of carbon, nitrogen and inorganic substances to produce the carbonyl pentaene macrolide having a negative optical rotation pattern and an x-ray diffraction pattern as shown in Figure 1.

-8- The method of Claim 7 wherein the growth medium contains ingredients from cane sugar molasses. -9-

The method of Claim 7 wherein the carbonyl pentaene macrolide is extracted from the mycelia and the growth medium with an organic solvent for the carbonyl pentaene macrolide and then the carbonyl pentaene macrolide is removed from the organic solvent.

-10- The method of Claim 9 wherein the macrolide is extracted from the mycelia with methanol and from the growth medium with trichloromethane.

-11- The method of Claim 9 wherein the growth medium is centrifuged to produce a filtrate which is extracted with a solvent to produce the macrolide and a cake which is homogenized with the methanol and then is extracted with the methanol and cooled to produce the carbonyl pentaene macrolide which is then removed from the methanol.

-12- The method of Claim 9 wherein the filtrate is extracted with trichloromethane.

-13- The method of Claim 12 wherein the carbonyl pentaene macrolide is removed from the methanol as yellow crystals.

-14- The method of Claim 9 wherein the carbonyl pentaene macrolide is crystalized by cooling the solvent. -15-

A method for inhibiting the growth of certain microorganisms which comprises exposing the microorganisms to an effective amount of a carbonyl pentaene microlide compound from a composition referred to as faeriefungin having a structural formula:

wherein R is selected from hydrogen (A) and methyl (B) and all double bonds are trans and having a negative optical rotation and an x-ray diffraction pattern with 28 crystalline peaks at a d-spacing between 1.5408 and 43.9165 Angstroms, for a 1 to 1 molar mixture of A to B.

-16- A method for inhibiting the growth of a microorganism which comprises exposing the microorganism to an effective amount of a composition referred to as faeriefungin characterized by a melting point of 209-212°C with partial decomposition, by a molecular formulae selected from the group consisting of C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀ and mixtures thereof and positive ion fast atom bombardment mass spectrometry indicated molecular ions of 651.4017 for C₃₆H₅₉O₁₀ and 665.4237 for C₃₇H₆₁O₁₀, a negative optical rotation and an x-ray diffraction pattern as shown in Figure 1 for a 1 to 1 molar mixture. -17- A method for inhibiting growth of a microorganism which comprises exposing the microorganism to an effective amount of a carbonyl pentaene macrolide from Streptomyces griseus var. autotrophicus var. nov. ATCC 53668 having a negative optical rotation and an x-ray diffraction pattern as shown in Figure 1.

-18- The method of Claim 17 wherein the exposure is by applying the carbonyl pentaene macrolide topically.

-19- The method of Claim 18 wherein the carbonyl pentaene macrolide is admixed with a carrier.

-20-

The method of Claim 17 wherein the microorganism is exposed to the pentaene macrolide with a carrier in an amount between about 1 and 100 micrograms per ml or per gram.

-21- The method of Claim 17 wherein the microorganism is selected from the group consisting of a fungus, bacterium and virus.

-22-

The method of Claim 17 wherein the microorganism is a plant pathogen.

-23-

The method of Claim 17 wherein the microorganism is an animal pathogen. -24- A mixed carbonyl pentaene macrolide composition referred to as faeriefungin for inhibiting the growth of microorganisms characterized by a melting point of 209-212°C with partial decomposition, by molecular formulae of C₃₆H₅₈O₁₀ and C₃₇H₆₀O₁₀ and mixtures thereof and fast atoms bombardment mass spectrometry indicated molecular ions of 651.4017 for C₃₆H₅₉O₁₀ and 665.4237 for C₃₇H₆₁O₁₀. by a negative optical rotation and by an x-ray diffraction pattern as shown in Figure 1 in a 1 to 1 molar mixture in a carrier in an amount between about 1 and 100 micrograms per ml or per gram.

-25- The carbonyl pentaene macrolide of Claim 24 as the 1 to 1 molar mixture having ultraviolet absorption spectral peaks in method at 262 nm and 363.5 nm.