CA1209935A

CA1209935A - Biologically active ws 6049 substances, a process for the production thereof and their pharmaceutical compositions

Info

Publication number: CA1209935A
Application number: CA000427458A
Authority: CA
Inventors: Sumio Kiyoto; Motoaki Nishikawa; Hiroshi Imanaka; Masanobu Kohsaka; Hiroshi Terano; Morita Iwami
Original assignee: Fujisawa Pharmaceutical Co Ltd
Current assignee: Fujisawa Pharmaceutical Co Ltd
Priority date: 1982-05-24
Filing date: 1983-05-04
Publication date: 1986-08-19
Also published as: AU565322B2; SU1308200A3; ES8501440A1; AU1488683A; JPH0415236B2; EP0095154A1; ATE24933T1; DK232483A; DE3369158D1; JPH02257886A; KR900004066B1; KR840004783A; JPH0216756B2; US4578271A; JPS58212787A; EP0095154B1; ES522637A0; DK232483D0

Abstract

ABSTRACT OF THE DISCLOSURE

BIOLOGICALLY ACTIVE WS 6049 SUBSTANCES, A PROCESS FOR THE
PRODUCTION THEREOF AND THEIR PHARMACEUTICAL COMPOSITIONS

WS 6049 substances having antitumor activity are described.
The substances may be produced by culturing a strain belonging to the genus Actinomadura, which is capable of producing WS 6049 substances in an aqueous nutrient medium under aerobic conditions and recovering WS 6049-A substance and/or WS 6049-B substance.

Description

~ ~2~3~

This invention relates to new compounds having biological activities, hereinafter referred to as WS 6049 substances. More particula-rly, this invention relates to a new biologically active WS 6049-A sub-stance and WS 6049-B substance, which have antimicobial activities against various pathogenic microorganism and antitumor activities, to a process for the preparation thereof and to pharmaceutical compositions containing the same.
Accordingly, this invention seeks to provide new WS 6049 substances which are active against various pathogenic microorganisms and tumors, and useful for the therapeutical treatment of infectious diseases and of various cancers in human beings and animals.
This invention also seeks to provide a process for the production of WS 6049 substances by fermentatior Still further this invention seeks to provide a pharmaceutical composition containing, as an active ingredient, a WS 6049 substance.
In the preceding and following descriptions, it is to be noted that the wording "WS 6049 substances" is intended to include WS 6049-A substance and WS 6049-B
substance.
Characteristics of the WS 6049 substances are shown in the accompanying drawings in which:
Figure 1 is a 13C NMR spectrum (in CDC13) of WS 6049-A substance, Figure 2 is a lH NMR spectrum (in CDC13) of WS 6049-A substance, Figure 3 is a 13C NMR spectrum (in CDC13) of WS 6049-B substance; and Figure 4 is a H ~MR spectrum (in CDC13) of WS 6049-B substance.

~ I ~`J

~ ~2~$~3~i -WS 6049 substances of this invention can be produced by fermentation of WS 6049 substance-producing strain belonging to the genus Actinomadura, for example Actinomadura pulveraceus sp. nov~ No. 6049 or the like in a nutrient medium.
Particulars o~ microorganism used for producing WS 6049 substances and production thereof will be explained in the following.
T~ MI CROORGAN I SM
The microorganism which can be used for the product-ion of WS 6049 substances is a strain belonging to the genus Actinomadura, among which a strain of Actinomadura pulveraceus sp. nov. ~o. 6049 has been newly isolated from a soil sample collected at Wakayama City in Wakayama Prefecture, Japan.
A culture of the newly isolated living organism of Actinomadura ,pulveraceus sp. nov. No. 6049 has been de--posited with and added to a stoek eulture colleetion of the American Type Culture Colleetion under the Budapest Treaty and its deposit number is ATCC 39100 (deposit date:
April 12, 1982).
It is to be understood that, for the production of WS 6049 substanees, this invention is not limited to the use of the partieular organism as described herein, which is given for illustrative purpose only.
Further, this invention also ineludes the use of any mutants whieh are capable of produeing WS 6049 substanees, including natural mutants which are produced by natural -mutation of the organisms as well as artificial mutants which can b~ produced f~om the described organism by conventional means, such as X r ys, ul~ra-~iolet radia-tion, nitrogen mustard oils, etc. and genetic engineer-ing means Actinomadura pulver~ceus sp. nov. No. 6049 has the .
following morphological, cultural and physiological characteristics.
(1) Morphological characteristics:
The methods described by Shirling and Gottlieb were employed for observation of the morphological characteristics (Shirling, E.B. and D Gottlieb : Methods for characteriza-tion of Streptomyces species~ Int. J. Syst. Bacteriol 16 :
313-340, 1966)~ -Morphologlcal observations were made with light and electron microscopy on cultures grown at 30C for 21 days on yeast extract-malt extract agar, inorganic salts-starch agar and oatmeal agar The mature spores occurred in chains of 5 to 20 spores forming mainly hook and sometimes loose spirals. The spores were oval ana 0 8 - 1 0 X
1,2 - 1.4 ~ in size with warty surfaces.
t2) Cultural characteristics:
2S Cultural chaTacteristics were observed on ten kinds of meaia described by Shirling and Gottlieb (mentionsd hereinabove) and Waksman (Waksman~ S.A : The Actinomycetes.
Vol, 2. Classification, identification and description of genera and species~ The Williams and Wilkins Co "
Baltimore, 1961). The incubation was made at 30C for 21 days. The color names used in this study were based on Color Standard (Nihon Shikisai Co., Ltd )~ As shown in Table 1, olonies belonged to the blue color series when the strain was grown on yeast extract-malt extract agar, oatmeal agar and inorganic salts-starch agar Soluble ~Zg~3~

pigment was not produced, The cell-wall composition analysis was performed by the me~hods of Becker et al, ~Becker, B " M,P,Lechevalier, R,E,Gordon and H,A,Lecheralier:Rapid differentiation bet-ween Nocardia and Streptomyces by paper chromato~Taphy ofwhole-cell hydrolysates, Appl, Microbiol,12, 421-423,1964 and Yamaguchi (Yamaguchi, T,: Comparison of the cell-wall composition of morphologically distinct actinomycetes3 J,Bacteriol,89,444-453,1965), The cell wall of this strain No, 6049 contained meso-diaminopimelic acid, A
whole cell hydrolysate showed the presence cf glucose, mannose and madurose (3-o-methyl-D-galactose)~
(3~ Biological and physiological properties:
T~mperature range for growth and optimum temperature were determined on yeast extract-malt extract agar using temperature gradient incuba~or (Toyo Kagaku Sangyo Co " Ltd,), The pH range for growth and the optimum pH were determined by liquid cultivation in yeast extract-malt extract broth with shaking at 30C for 7 days, Gelatin li~uefaction was examined at 30DC for 14 days on gelatin medium. Starch hydrolysis was observed by the starch-iodine reaction after incubation on inorganic salts-starch agar plate at 30C
for 14 days, Milk peptonization was observed in skim-milk medium at 30C for 14 days, Melanoid pigment production was observed on tyrosine agar, peptone-yeast extract-iron agar and tryptone-yeast extract broth, As shown in Table 2, tempeTatUre range for growth was from 20~ to 41C with optimum from 30C to 35C, Starch hydrolysis, mîlk peptoni-zation, H2S production, urease activity, nitrate reduc~ion and melanine pToduction were negative, whereas gelatin liquefaction and milk coagulation were positive, U~ilization of carbon sources was examined according to the method of Pridham and Gottlieb (PTidham,T,G, and D, Gottlieb: The utilization of carbon compounds by some Actinomycetales as an aid for species determination~

33~

J,BacteTiol,56:107-114,1965), The results were deter-mined after 14 days incubation at 30C, As shown in Table 3, almost all carbon sources were doubtfully or not utilized, Only D-xylose, D-glucose and D-trehalose S were utilized, The aforementioned cell wall composition and whole cell sugar components indi~ate the strain No.
6049 is a species of the genus Actinomadura, A comparison of this oTganism was made with the published descriptions of Actinomadura species ~Nonomura,H, and Y.Ohara:Distribution of actinomycetes in soil, XI) Some new species of the genus Actinomadura Lechevalier e~
al" J,Ferment,Technol,49:904-912,1971, Goodfellow,M" G,Alderson and J,Lacey:Numerical taxonomy of Actinomaaura and related actinomycetes~ J.Gen,Microbiol, 11~:95-111,1979;
Tomita,K"Y,Hoshino,T,Sasahira and H,Kawaguchi: BBM-928, a new antitumor antibiotic complex 2, Taxonomic studies ~ on the producing organism~ J,An~ibiotics 33: 1098-1102, 1980~, Strain No, 6049 is consiaered to resemble Actinomadura verrucosospora, It was found however, that strain No, 6049 could be differen~iated from this species in the following points, As showr, in Table 4, utilization of D-fructose, L-arabinose, mannitol, sucrose and glycerin is different, 2S Differences are also observed in nitra~e Teduction and milk peptonization, Aerial mass color of strain No, 6049 belonged to the white color series on inorganic salts-starch agar, whereas that of A,verruco ospora belonged to the gray color series, Direct comparison of cultural characteristics between strain No, 6049 and A,verruCos-ospora was shown in Table 5.
As a result of above comparisons, strain No, 6049 is considered a new species of the ge~us Actinomadura, The name Ac~inomadura pulveraceus sp, nov, is proposed for strain No, 6049 referring to the powdery aerial mycelium ~$~3~

on yeast extract-malt extract agar, oatmeal agar and inorganic salts-starch agar, Table 1. Cultural characteris~ics of strain No. 6049 Reverse Soluble Medium Aerial Myc.e.l.ium .. .S.iae .Color . P.igme~t Oatmeal agar greenish white colorless none Yeast extract-malt extract agar pale blue pale yellow none Inorganic salts-starch agar white light red none Glucose asparagine agar none pink none Gl~cerin-asparagine agar trace pale pink none .. Sucrose-nitr~te agar none pale pink none Nutrient agar none light red none Potato-dextrose agar none . pale pink none Tyrosine agar grayish white pale pink . none Peptone-yeast extract-iron agar none pale pink none ~L2~ 3l~

Table 2. Physiological PToperties of Strain No. 6049 Temperature range for growth 20C - 41C
Optimum temperature 30G - 3SC
pH Range for growth 6 - 10 Optimum pH 7 - 8 NitTate reduction negati~e Starch hydrolysis positive Milk coagulation weakly positive Milk peptonization negative Gelatin li~uefaction positive Melanine production negative H2S produc*ion negati~e Urease . negative ~L2~35 Table 3. Carbon sources utilization of strain No.6049 C-sources none GlyceTin +
D-Xylose +
Sodium citrate Lactose D-Pructose Rhamnose Maltose +
Sodium succinate Inulin Inositol Raffinose D-Galactose L-Arabinose D-Glucose t Mannitol D-Mannose -Sucrose Cellulose D-Trehalose Salicin Chitin Sodium acetate + utilization + : doub~ful utilization - : not utilization g Table 4, Different points of carbon utilization and physiological pToperties between strain No~6049 and Actinomadura errUcososPOr~-No, 6049 A, errucosospora -D-Fructose L-Arabinose - +
Mannitol ~ +
Sucrose ~+
Glycerin ~ +~
Nitrate reduc~ion negative positive Milk peptonization negative positive I

-+ : good utilization ~ + : utilization + : d~ubt ~ utilization - : not utilization lZ~ 3~

Table 5. Direct comparison between strain No. 6049 .
and Actinomadura verrucosos~ora . ~
I Strain No~6049 Medium ~ . .
Growth¦ Aerial Mycelium Reverse side Soluble I ColoT ~
__ . . . .
1 abundant none pale yellow none

2 poor none colorless none

3 abundant blue pink none

4 abundant white pink none

5 moderate grayish white colorless none

6 abundant none colorless none

7 poor grayish white colorless none

8 moderate pale blue pale pink none abundant pinkish grey pink ~one ~ l Actinomadura verrucosospora Medium _ ....... _ . .......... _ - -1 Growth Aerial Mycelium Reverse side Soluble color Pi~ment . . _ _ . _ _ . .
. 1 moderate no~e pink none . 2 moderate none pink none . 3 poor none pink none 4 poor none pale pink none Z5 5 moderate grayish white pink none 6 abundant none pink none 7 poor grayish blue pink none 8 abundant pale blu sh pink none

9 abundant pale bluish pink none _ ~ pink _ _ _ _ , 1 : Peptone-yeast extrac~-iron agar, 2 : Nutrient agar, 3 : Potato-dextrose agar, 4 : Sucrose-nitrate agar, 5 : Tyrosine agar, 6 : Yeast extract-malt extract agar, 7 : Oatmeal agar~ 8 : Inorganic salts-starch agar, 9 : Glucose-asparagine agar The WS 6049 substances of this invention are produced when a WS 6049 substance-producing strain belonging to the genus Actinomadura (e.g. Actinomadura pulveraceus sp. nov. No 6049) is gTOWn in a nutrient medium contain-ing sources of assimilable carbon and nitrogen under aerobic conditions (e.g. shaking culture, submerged culture, etc ) The preferred sources of carbon in the nutrient medium are carbohydrates such as xylose, glucose~ sucrose) starch ~ and the like.
The preferred sources of nitrogen are yeast extract, peptone~ gluten meal, cot~on seed flour, soybean meal, corn steep liquor, dried yeast, wheat germ, etc.S as well 1~ as inorganic and organic nitrogen compounds such as ammonium salts (eOg. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.~, ure~, amino acid and the like The carbon and nitrogen sources, though ad~antageously employed in combination, need not be used in th~ir pure form because less pure materials, which contain traces of growth factors and considerable q~antities of mineral nutrients, are also suitable for use.
When desired, there may be added to the medium mineral salts such as calcium carbonate, sodium or potassium phosphateS sodium or potassium chloride, magnesium salts, copper salts and the like.
If necessary, especially when the culture medium foams seriously a defoaming agent, such as liquid parafin, fatty oil, plant oil, mineral oil or silicone may be added.
As in ~he case of the preferred methods used for the production of other an~ibiotics in massive amounts, submerged aerobic cul~ural conditions are prefe~red for the production of WS 6049 substances in massi~e amounts.

~2~

For the production in smal:L amounts, a shaking or surface culture in a flask or bottle is employed.
Furthermore, when the growth is caTried out in large tanks, it is p~eferable to use the vegetative form of the organism for inoculation in the production ~anks in order to avoid growth lag in the process of production of ~he WS 6049 substances. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inorulating a relatively small quantity of culture medium with spores OT mycelia of the organism and culturing said inoculated medium, and then to transfer the cultured vegetative inoculum aseptically to large tanks. The m~dium, in which the vegetative inoculum is produced, is substantially the s~me as OT different from the medium u*ilized for the production of ws 6049 substances Agitation and a0Tation of the culture mixture may be accomplished in a variety of ways Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor~ by various pumping e~uipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixtuTe.
The fermentation is usually conducted at a tem-perature between about 20C and 40C, preferably 25 -30C~ for a period of about 50 hours to 100 hours.
When the fermentation is completed, the cultu~e broth is then subjected for recovery of WS 6049 sub-stances to various procedures conventionally used for recove~y and purification of antibiotics, for instance, solven~ extraction ~ith an appropria~e sol~ent OT a mixture of such solvents, chromatography, or recrystal-lization from an appropriate sol~ent OT ~ mixture of such solvent.
According to this invention9 in general, . . .

ws 6049 substances are found mainly in the cultured mycelial Accordingly, the cul~ure broth is separated by means of filtration or centrifuging to provide the mycelium cake, and then WS 6049 substances are recovered from said resulting mycelium cake by means of extraction using an appropriate organic solvent such as acetone, ethyl acetate or the like~ or a mixture of such solvents.
The extract is treated by a conventional manner to provide WS 6049 substances, fo~ example, the extract is concen~rated by evapora~ion or distillation to a smaller amount and the resulting residue containing active mateTials~ i.e. WS 6049 substances are puri-fied by conventional purification procedures, for example, chromatography or recrystallization from an appropriate solvent or a mixture of such solvents, WS 6049-A subs~ance and WS 6049-B substance can be separated by-dissolving the materials containing both product produced by fermentation in an appropriate organic solYent such as chloroform or the like a~d then b~ chromatographing the solution~ for example, on silica gel in a column with an appropriate organic solvent o~
a mixture of such solvents such as chloroform, acetone or the like and each of WS 6049-A substance and WS 6049-B substance thus separated can be further pu~i-fied by a conventional method, for example, high per-formance liquid chromatography.

SUBSTANCES
The WS 6049 substances i e. WS 6049-A substance and WS 6049-B substance as obtained ~ccording to the aforementioned process have the following physical and chemical properties _ _ .

1) Form and color:
Colorless powder 2) Color reaction: -Positive: Dragendorff reaction, Ehslich's reac~ion and cerium sulfate reaction Negative: ninhydrin reaction 3) Solubility:
Soluble: methanol, acetone, chloroform ~ Slightly soluble: diethyl eth~r Insoluble: hexane, water 4) Melting point:
150~C ~dec ) . 5~ Specific rotation:
~]25 = -208 ~c=l.0~ CHC13) . 6) Ultraviolet absorption spectrum:
~ CH30H s 252 nm tElC~ = 560) = 280 nm (El~m = 370) ~ 320 nm (ElCm = 220) ~ CH3Off~HCl = 252, 280, 320 nm max H3oH~NaoH = 250J 280l ca310(5h.)nm max 7) Infrared absorption spectrum:
v CHC13 = 3450~ 3350, 3250, 2960, 292~, max 1725, 1675, 1610, 1595~ 1520~
14.65, 1450l 1405, 1370, 13~0, 1310, 1250, 1180, 1155~ 1115, 1075, 1020, ~85, 955, 905, 850 cm~l ~z~
- lS -8~ Elementary analysis:
C: 52,03~, H: 5.71%, N: 4.15%1 S: 9,86%
9) Thin layer chromatography:
Stationary phase Developing Rf value solvent . . - . _ Chloroform: 0,59 methanol Silica gel sheet (lO:l)(v/v) Chlo~oform: 0,33 acetone (l:l)(v/v)

10) Molecular weight:
FD Mass: m/z 1333 (M +Na) FABQ MS: m/z 1311 (M +1) Gel permeation chromatography : 1100 to 1200 113 13C Nuclear magnetic resonance Spectrum (CDC13):
~ (ppm) : 13~9J 14,6, 16.7, 17.6, 13.9, 22.89 29.1, 34.0, 35.2, 39.6~ 42 3, 52.7, 55.8~
56.1, 56.2, 56.2, ~0,3, 61 7, 64.7, 66 7, 68,4, 69.0, 6g.3, 69.8, 70.5, 72.0, 75.9, 76.1, 77.2, 77 3, 83 2, 86,3, 88,5, 90.7, 97.3, 98 6, 98 9, 99.6, 99,7, ~03.9, 107.8, 112.7, 123.2, 1~5.0, 130~0, 131,1~ 136.8, 144.2, 146.6, ~54.0~ 154.6, 160.~9 166.6, 192. 4, as shown in Figure 1 of the accompanying draw~ng.

~ H Nuclear magnetic resonance spectrum (CDC13):
~ (ppm~ : 8.57 (lH, s), 7.48 (lH, s), 6 6 (lH,d,d), 6.2 ~lH, d, J=1.6HZ~, 6.18 (lH, br.s.), 5,93 (lH, d, JZ9.6HZ), 5,83 ~lH, d,d, J=9,6, 1,6HZ), 517 ~lH, d, J=2Hz), 5,5 (lH, m), 5.48 (lH, d, J=2.3Hz), 5,43 (lH, br.s), S~3~-4.97 (lH! d), 4 7 (2H, m), 4 56 (lHI d9 J-2 3Hz), 4 23 (lH9 s), 4 2 tO 3 6 (lQ to 14H), 3 97 (3H, s), 3.88 (3H, s), 3 79 (3H, s), 3.5 (~H, m), 3,43 (3H, s), 2.77 (lH, s), 2 7 (2H, m), 2 52 (3H, 5), 2.5 (1~
2 4 to 2.25 (3H, m), 2.17 ~lH, s), 2.12 (3H, s), 2,07 (lH, m), 1.77 (2H~ s), 1.5 (2H, m), 1 41 (3H, d, J=6Hz), 1.35 (3H, d, 3=6Hz), 1 32 (3H, d; J=6Hz), 1 2 (4H, m), as shown in Figure 2 of the accompanying drawing, 1) Eorm and color:
~ CO1OT1eSS powder 2) Color reaction:
Positive: Dragendorff reaction, Ehrlich's reaction and cerium sulfate reaction Negati~e: ninhydrin reaction 3) Solubility:
Soluble: methanol, acetone, chloroform Slightly soluble: diethyl ether Insoluble: hexane 4) Melting point:
2~ 145C ~dec.), 5) Specific ~otation:
~a~25 Z -201(C=1.0~ CHC13) 6~ Ultra~iolet absorption spectrum:

~ CH30H = 253 nm (El~ = 620) max = 280 nm (El~m = 450) = 320 nm (ElCm = 2 50) ~Z~3~

A C~3OH+HCl = 253, 280, 320 nm max ~ CH3OH+NaOH = 250, 282, 310 (sh.) nm 7) Infrared absorption spectrum:
C C13 = 3450, 3350, 3250, 2990, max 2920, 1725, 1675, 1610, 1595, 1520, 1465, 14~0, ~405, 1370, 1350, 1310, 1250, 1180, - 1155, 1115, 1070, 1020, 985, ~55 905, 880, 850 cm 1 8) Elementary analysis:
C: 51.58%, H: 5,75%, N: 4.27%, S: 9.80%
lS ~ 9) Thin layer chromatography:
S~ationary phase Dev~loping Rf value sol~ent - . chloroform:
methanol(lO:l) 0,53 Silica gel sheet (v/~) ..
chloroform: 0,18 acetone~
~v/v) 10) Molecular weight:
Gel permea~ion chromatography: llO0 to 1200 ~ 3C Nuclear magn~tic resonance spec~rum tCDCl~
~ppm) : 13.8, 16.7; 17,6, 19,8, 22.7, Z9.1 33,9, 34,1, 35.2, 39.5~ 52.8, 55.8, 56.1, 56,3, 6~.~, 61.5, ~4.~, 66.7, 68,4, 69.0, 69,3l 6g.8, 70.4l 71.91 7S.9, 76,2, 76.7, 77.~, 77,3, 77.6, 83.2, 86,6, 88,3, 90~7, ~7.3, ~8,6, 99.0, 99.6, 99.7, 103.9, 107.8, 112.7, 123,2, 124.9, 12g.8, 131,0, 136.8, 144.2, 146,3~ 154.0, 154.6, 160,9, 166,6, 19Z.4, as . '~-.. , ., . _ .. . .

3~

shown in Figure 3 of the accompanying drawing, 12)1H Nuclear magnetic TeSOnanCe spectrum (CDC13):
~ ~ppm) : 11.75 (lH, s), 8,57 (lH, s), 7,48 (lH, s~, 6,6 ~lH, d,d,)~ 6,24 (lH, d~ J=1,3Hz), 6,18 (lHJ br,s~, 5,93 ~lH, d, J=9,2Hz), 5.83 (lH, d,d, J=9Hz and 1.3Hz)l 5,70 (lH, brld), 5,5 (lH, m), S,47 (lH, d, J=2,3Hz), 5,42 (lH, br.s), 4,98 (lH, d, J=9Hz~, 4,70 to 4,6 (2H, m), 4,56 (lH, d, J=2,3Hz)~ 4,24 LO (lH, s), 4,15 to 3,4 (12 to 18H), 3,97 (3H, s), - 3,88 (3H, s), 3,79 (3H, s), 3,42 (3H, s), 2,52 (3H, s), 2,6 to 2,4 t2H, m), 2,4 to 2,2 (7 to 8H), 2,12 (3H, s), 2,2 to 2,0 (2H, m), 1,5 ~2H9 m), 1,4 (3H, d, J-6Hz), 1,35 (3H, d, J=6Hz), 1,33 (3H, d, J=6Hz), as shown } in Figure 4 of the accompanying drawing.

Biological properties of WS 6049 substances are explained in the following, 1) Antitumor ac~ivities of WS 6049 subs~ances:
The antitumor activities of WS 6049 substances were determined in experimental tumor system in mice, Lymphocytic leukemia P-388 was implanted intra-peritoneally into BDFl mice at an inoculum size of lx10~
cells per mouse, Twenty-~our hours after the implantation of ~umor cells, graded doses of the antibiotics were administered to mice intrape~itoneally, Treatments were given on day 1,2,3 and 4~
The wS 6049 subs~ances were suspended in physiological saline solution (o.9% saline).
Control animals received intraperitoneal doses of physiological saline solution. The injection volume was 0~2 ml in all experiments.

3~

The therapeutic responses measured was mean survival time, and ~esults were expressed as T/C~
(survival time of treated group/survival time of con~rsl group x 100), Toxicity was measured as weight loss between Days 0 and 4 after tumor inoculation.
Th~ result is presented in Table 6 and Table 7.
The WS 6049-A and WS 6049-B were quite active against the leukemia P-388. Doses between 0.025-12,0 ~g/kg on the schedule resulted in significant increase in life spzn in mice.

Table 6. P-388 mouse leukemia sDFl mouse(,~, 7 week) tumor site i,p. ~lx106 cells/mouse9day 0) drug route i.p, (day 1,2,3 and 4) five mice per group Dose ~eigh~ change Mean survival .
Drug (~g/kg/day) (g~ T/C ~
. _ Day 0- Day 4 time ~days) _ 20~S 6049-A 25 ~g/kg/day -3,1 7,6 ~3xic 12 -2.0 16.4 173 (1/5)~
. . . _ 6 +0,1 21.5 226 3 ~0,2 21.5 226 (l/S)*
1.5 - - -+0,2~ 22.5 237 0.8 +0.3 _ ~2~0_ 232 _ _ o~ +n.2 21,6 227 . . ... ,_ 1, 0,2 ~0,2 20.0 _ 211 0.1 +0.2 18.6 lg6 0.05 +0.1 16.6 175 .. . , . . .
0,025 +0,6 12.3 129 ' -. . _ _ . .
Contral _ ~_ . +1.0 9.5 100 I

* Numbers of survivor at Day 30 To~al mice . l~q~93,~ .

Table 7. P-388 mouse leulcemia BDFl mouse (~, 7 week) 6 tumor si~e i,p, (lxlO ) drug route i,p, (day 1,2,3 & 4) five mice per group . . Wei ght Mean survival T/ C
Drug Dose change(g) time (days) Day 0-Day 4 _ ~S 6049-B10 ~g/kg/day~ -1.7 8,9 72 ~ . .
3,3 ~ -0,4 26,4 215 1.l -1,2 20,7 168 . 0,37 ,0.3 23.3 18~
0,l2 0 21.9 178 0,04 IO,S 24,8 202 0.013 +0,6 17,7 144 . _ . ... . ~ - . , Control vehlcle ~0.6 12, 3 100 . mouse/day _ 2) Antimicrobial acti~ities of WS 6049-A and WS 6~49-~
Antimicrobial activities of WS 6049-A and WS 6049-B were determined by a serial broth dilution method in bouillon medium for bacteria and in Sabouraud medium for fungi and yeast, Minimum inhabitory con-centrations ~MIC) were expressed in terms of ~g/ml after o~ernight incubation at 37C for bacteria and 48-72 hours incubation at 28~ for fungi and yeasts.
The antimicrobial spectra of WS 6049^A and WS 6049-B
are shown in Table 8.

3~

From the results, the antibiotics, WS 6049 substances have a broad antimicrobial activi~y and may be a.mong the most potent antibiotics ever dis-covered, Table 8. Antimicrobial spectra of WS 6049-A and ~icroorganlsm _ MIC (~g _ ~_ . = .
Staphylococcus aureus 0.0001-0.0002 0,OUOl-0.0002 Bacillus subtilis 0,001 0,001 t Escherichia coli 0.6 0,6 ... Pseudomonas aeruginosa 0,6 0.6 Proteus vulgaris 1.2 1,2 Candida albicans 5.0 5.0 Aspergillus oryzae 0,6 0,6 Penicillium chrysogenum 0.3 0,3 Aureobasidium sp. 0.15 0.1 _ . . .
3) Acute toxicity.of WS 6449-A and WS 6049-B
A~ute toxicity of WS 6049-A and B in ddY mice by intraperitoneal injection are both 0,05 mg/kg.

The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparat-ion, for example$ in solid, semisolid or liquid form, which contains WS 6049 substances 9 as an active ingredient, in admixture w~th an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets~ capsules~ suppositoTies, solutions, emulsions, S suspensions, and any other form suitable for use.
The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, kPTatin, colloidal silica, pctato starch, urea and other carriers suitable for use in manufacturing preparations, in - solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an - 15 amount sufficient to produce the desired antimicrobial effect upon the process or condition of diseases, For applying this composition to humans, it is preferably to apply it by intravenous~ intramuscular or oral administration, While the dosage or thera-Z0 peutically effective amount of the object compound of this invention varies from and also depends upon ~he age and condition of each individual patient to be treated~ a daily dose of about 0.01 - 50 ~g of the active ingredient/kg of a human being or an animal is generally given for trea~ing diseases, and an average single dose of about 0.1 ~g, 1 ~g, 10 ~g, 50 ~g, 100 ~g and 200 ~g is generally administered.

The following examples are given for the purpose of illustrating the present invention.

Example 1 ~n aqueous medium (160 ml) containing 2~ starch, 0 5~ glucose, 1~ cotton seed flour9 1% dried yeast~
0.5~ corn steep liquor and 0.2% CaCO3 (pH 7.0) was poured into each of three S00 ml Erlenmeyer flasks and sterilized at 120~C for 30 minutes, A loopful of slant culture of Actinomadura E~ sp, nov, ~, No, 6049 (ATCC 39100) was inoculated to each of the medium and cultured at 30C on a rotary shaker wi~h 3-inch throw at 220 rpm for 4 days, The resultant culture was inoculated to an aqueous medium (20 liters) containing 4% sucrose, 0,5~ dried yeast, 0.1% K2HPO4, 0,1% MgSO4~7H2O, 0,1~ NaCl, 0.2% ~NH4)2SO4, 0-2% CaCO3, 0,0001% FeSO4~7H2O, 0,0001% MnC12-4H2O, 0,0001% ZnSO4~7H2O
- and O,OOOOS~ of NaI in a 30 liter jar-fermentor, which had been sterilized at 120C for 30 minutes, and cultured at 30C for 4 days under aeration of 20 litersJminute and agitation of 300 r,p,m, The cultured broth thus obtained was filtered with an aid of diatomaseous earth (1 kg), To the mycelia obtained were added 10 liters of ethyl acetate and stirred for 10 minu~es, This extraction procedure was carried out twice and the extracts were combined, The extracts were washed with 10 liters of 1~ sodium bicarbonate and lO liters of 10% sodium chloride~
Then the extracts were concentrated in acuo to a volume of one liter, After dehydration with anhydrous sodium sulfate, the ~thyl acetate solution was further concentrated in vacuo and the oily materials obtained were applied to a column chromatography using silica gel (70 ml), The column was w~shed with 200 ml of chloroform and eluted with a mixture of chlo~oform-methanol ~40:1), Fractio~s con~aining active materials (S00 ml~ were concentrated in vacuo to give a crude powder (140 mg).
The powder was dissolved into 2 ml of chloroform and subjeoted to a column of silica gel (2U ml). The column was washed with a mixture of chloroform-acetone ~8:1), and WS 6049-A was eluted with a mixture of chloroform-acetone (4:1). WS 6049-B was eluted with a mixture of chloroform-acetone (2:1).
Each of fractions containing WS 6049-A and WS 6049-B was concentrated in vacuo to give a crude powder of WS 6049-A (17 mg) and WS 6049-B (15 mg), respectively. They were separately subjected to high performance liquid chromatography (HPLC)~ HPLC was carried out using a Waters Model 6000 A pump with a Waters Model U6K injector. Chromatography was moni-10 tored by a UV detector, Waters Model 440 at 254 nm. A
steel column (7.9 mm inside diameter, 300 mm length) packed with a ~ Porasil ("~ Porasil" is a trade mark of Water Associates Inc.) was used at a flow rate of 3 ml/minutes. Mobile phase used was a mixture of hexane, chloroform and methanol (25:10:2). HPLC under the above-mentioned conditions gave fraction A
(retention time: 18 min.) and fraction B (retention time: 26 min.). Twelve mg of colorless powder of WS 6049-A from Fraction A and 8 mg of colorless powder of WS 6049-B from fraction B were obtained.
The term "such as", as employed herein, shall be considered to mean "for example", and shall not be construed as limiting unless the context clearly indicates otherwise.

~,~

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing at least one WS 6049 substance selected from WS 6049-A substance and WS 6049-B substance, wherein the WS 6049-A substance has the following properties:
1) Form and color:
Colorless powder 2) Color reaction:
Positive: Dragendorff reaction, Ehrlich's reaction and cerium sulfate reaction Negative: ninhydrin reaction 3) Solubility:
Soluble: methanol, acetone, chloroform Slightly soluble: diethyl ether Insoluble: hexane, water 4) Melting point:
150°C (dec.) 5) Specific rotation:
[.alpha.]? = -208° (C=1,0, CHC13) 6) Ultraviolet absorption spectrum:
= 252 nm ( = 560) = 280 nm ( = 370) = 320 nm ( = 220) = 252, 280, 320 nm = 250, 280, ca310(sh.)nm 7) Infrared absorption spectrum:
CHCl3 = 3450, 3350, 3250, 2960, 2920, max 1725, 1675, 1610, 1595, 1520, 1465, 1450, 1405, 1370, 1350, 1310, 1250, 1180, 1155, 1115, 1075, 1020, 985, 955, 905, 850 cm-1 8) Elementary analysis:
C: 52.03%, H:5.71%, N:4.15%, S:9.86%
9) Thin layer chromatography:

10) Molecular weight:
FD Mass: m/z 1333 (M+ + Na) FABQ MS: m/z 1311 (M+ + 1) Gel permeation chromatography: 1100-1200 11) 13C Nuclear magnetic resonance spectrum (CDCl3):
.delta.(ppm): 13.9 14.6, 16.7, 17.6, 19.9, 22.8, 29.1, 34.0, 35.2, 39.6, 42.3, 52.7, 55.8, 56.1, 56.2, 56.2, 60.3, 61.7, 64.7, 66.7, 68.4, 69.0, 69.3, 69.8, 70.5, 72.0, 75.9, 76.1, 77.2, 77.3, 83.2, 86.3, 88.5, 90.7, 97.3, 98.6, 98.9, 99.6, 99.7, 103.9, 107.8, 112.7, 123.2, 125.0, 130.0, 131.1, 136.8, 144.2, 146.6, 154.0, 154.6, 160.9, 166.6, 192.4, as shown in Figure 1 of the accompany-ing drawing, 12) 1H Nuclear magnetic resonance spectrum (CDCl3):

.delta.(ppm) : 8.57 (1H, s), 7.48 (1H, s), 6.6 (1H, d.d), 6.2 (1H, d, J=1.6Hz), 6.18 (1H, br.s.), 5.93 (1H, d, J=9.6Hz), 5.83 (1H, d.d J=9.6, 1.6Hz), 5.7 (1H, d, J=2Hz), 5.5 (1H, m), 5.48 (1H, d, J=2.3Hz), 5.43 (1H, br.s), 4.97 (1H, d), 4.7 (2H, m), 4.56 (1H, d, J=2.3Hz), 4.23 (1H, s), 4.2 to 3.6 (10 to 14H), 3.97 (3H, s), 3.88 (3H, s), 3.79 (3H, s), 3.5 (2H, m), 3.43 (3H, s), 2.77 (1H, s), 2.7 (2H, m), 2.52 (3H, s), 2.5 (1H, m), 2.4 to 2.25 (3H, m), 2.17 (1H, s), 2.12 (3H, s), 2.07 (1H, m), 1.77 (2H, s), 1.5 (2H, m), 1.41 (3H, d, J=6Hz), 1.35 (3H, d, J=6Hz), 1.32 (3H, d, J=6Hz), 1.2 (4H, m), as shown in Figure 2 of the accompanying drawing, and the WS 6049-B substance has the following properties:
1) Form and color:
Colorless powder 2) Color reaction:
Positive: Dragendorff reaction, Ehrlich's reaction and cerium sulfate reaction Negative: ninhydrin reaction 3) Solubility:
Soluble: methanol, acetone, chloroform Slightly soluble: diethyl ether Insoluble: hexane 4) Melting point:
145°C (dec.).
5) Specific rotation:
[a]? = -201°(c=1.0, CHCl3) 6) Ultraviolet absorption spectrum:
= 253 nm = 280 nm = 320 nm = 253, 280, 320 nm = 250, 282, 310 (sh.) nm 7) Infrared absorption spectrum: .
CHC13 = 3450, 3350, 3250, 2990, 2920, v max 1725, 1675, 1610, 1595, 1520, 1465, 1450, 1405, 1370, 1350, 1310, 1250, 1180, 1155, 1115, 1070, 1020, 985, 955, 905, 880, 850 cm-1 8) Elementary analysis:
C: 51.58%, H:5.75%, N:4.27%, S:9.80%
9) Thin layer chromatography:

10) Molecular weight:
Gel permeation chromatography: 1100 to 1200 11) 13C Nuclear magnetic resonance spectrum (CDCl3):
.delta. (ppm) : 13.8, 16.7, 17,6, 19.8, 22,7, 29.1, 33.9, 34.1, 35.2, 39.5, 52.8, 55.8, 56.1, 56.3, 61.0, 61.5, 64.6, 66.7, 68.4, 69.0, 69.3, 69.8, 70.4, 71.9, 75.9, 76.2, 76.7l 77.2, 77.3, 77.6, 83.2, 86.6, 88.3, 90.7, 97.3, 98.6, 99.0, 99.6, 99.7, 103.9, 107.8, 112.7, 123.2, 124.9, 129.8, 131.0, 136.8, 144.2, 146.3, 154.0, 154.6, 160.9, 166.6, 192.4, as shown in Figure 3 of the accompany-ing drawing, 12) H Nuclear magnetic resonance spectrum (CDCl3) :
.delta.(pp~) : 11.75 (1H, s), 8.57 (1H, s), 7.48 (1H, s), 6.6 (1H, d.d.), 6.24 (1H, d, J=1.3Hz), 6.18 (1H, br s), 5.93 (1H, d, J=9.2Hz), 5.83 (1H, d.d, J=9Hz and 1.3Hz), 5.70 (1H, br.d), 5.5 (1H, m), 5.47 (1H, d, J=2.3Hz), 5.42 (1H, br.s), 4.98 (1H, d, J=9Hz), 4.7 to 4.6 (2H, m), 4.56 (1H, d, J=2.3Hz), 4.24 (1H, s), 4.15 to 3.4 (1H to 18H), 3.97 (3H, s), 3.88 (3H, s), 3.79 (3H, s), 3.42 (3H, s), 2.52 (3H, s), 2.6 to 2.4 (2H, m), 2.4 to 2.2 (7 to 8H), 2.12 (3H, s), Z.2 to 2.0 (2H, m), 1.5 (2H, m), 1.4 (3H, d, J=6Hz), 1.35 (3H, d, J=6Hz), 1.33 (3H, d, J=6Hz), as shown in Figure 4 of the accompanying drawing, which comprises culturing a strain belonging to the genus Actinomadura, which is capable of producing said WS 6049 substances in an aqueous nutrient medium under aerobic conditions and recovering said WS 6049 substances from said medium.

2. A process for preparing WS 6049 substances according to claim 1, wherein said strain belonging to the genus Actinomadura is Actinomadura pulveraceus.

3. A process for preparing WS 6049 substances according to claim 2, wherein said strain belonging to the genus Actinomadura is Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100).

4. A process for preparing WS 6049-A substance, wherein the WS 6049-A substance has the following properties:

1) Form and color:
Colorless powder 2) Color reaction:
Positive: Dragendorff reaction, Ehrlich's reaction and cerium sulfate reaction Negative: ninhydrin reaction 3) Solubility:
Soluble: methanol, acetone, chloroform Slightly soluble: diethyl ether Insoluble: hexane, water 4) Melting point:
150°C (dec.) 5) Specific rotation:
[.alpha.]? = -208° (C=1.0, CHCl3) 6) Ultraviolet absorption spectrum:

= 252 nm ) = 280 nm = 320 nm = 252, 280, 320 nm = 250, 280, ca310(sh.)nm 7) Infrared absorption spectrum:
= 3450, 3350, 3250, 2960, 2920, 1725, 1675, 1610, 1595, 1520, 1465, 1450, 1405, 1370, 1350, 1310, 1250 1180, 1155, 1115, 1075, 1020, 985, 955, 905, 850 cm-1 8) Elementary analysis:

C: 52.03%, H:5.71%, N:4.15%, S:9 86%
9) Thin layer chromatography:

10) Molecular weight:
FD Mass: m/z 1333 (M+ + Na) FABQ MS: m/z 1311 (M+ + 1) Gel permeation chromatography: 1100-1200 11) 13C Nuclear magnetic resonance spectrum (CDCl3):
.delta.(ppm): 13.9 14.6, 16.7, 17.6, 19.9, 22.8, 29.1, 34.0, 35.2, 39.6, 42.3, 52.7, 55.8, 56.1, 56.2, 56.2, 60.3, 61.7, 64.7, 66.7, 68.4, 69.0, 69.3, 69.8, 70.5, 72.0, 75.9, 76.1, 77.2, 77.3, 83.2, 86.3, 88.5, 90.7, 97.3, 98.6, 98.9, 99.6, 99.7, 103.9, 107.8, 112.7, 123.2, 125.0, 130.0, 131.1, 136.8, 144.2, 146.6, 154.0, 154.6, 160.9, 166.6, 192.4, as shown in Figure 1 of the accompany-ing drawing.
12) 1H Nuclear magnetic resonance spectrum (CDCl3):
.delta.(ppm) : 8.57 (1H, s), 7.48 (1H, s), 6.6 (1H, d.d), 6.2 (1H, d, J=1.6Hz), 6.18 (1H, br.s.), 5.93 (1H, d, J=9.6Hz), 5.83 (1H, d.d J=9.6, 1.6Hz), 5.7 (1H, d, J=2Hz), 5.5 (1H, m), 5.48 (1H, d, J=2.3Hz), 5.43 (1H, br.s), 4.97 (1H, d), 4.7 (2H, m), 4.56 (1H, d, J=2.3Hz), 4.23 (1H, s), 4.2 to 3.6 (10 to 14H), 3.97 (3H, s), 3.88 (3H, s), 3.79 (3H, s), 3.5 (2H, m), 3.43 (3H, s), 2.77 (1H, s), 2.7 (2H, m), 2.52 (3H, s), 2.5 (1H, m), 2.4 to 2.25 (3H, m), 2.17 (1H, s), 2.12 (3H, s), 2.07 (1H, m), 1.77 (2H, s), 1.5 (2H, m), 1.41 (3H, d, J=6Hz), 1.35 (3H, d, J=6Hz), 1.32 (3H, d, J=6Hz), 1.2 (4H, m), as shown in Figure 2 of the accompanying drawing, which comprises culturing a strain belonging to the genus Actinomadura, which is capable of producing WS 6049-A
substance in an aqueous nutrient medium under aerobic conditions and recovering WS 6049-A substance from said medium.

5. A process for preparing WS 6049-A substance according to claim 4, wherein said strain belonging to the genus Actinomadura is Actinomadura pulveraceus.

6. A process for preparing WS 6049-A substance according to claim 5, wherein said strain belonging to the genus Actinomadura is Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100).

7. A process for preparing WS 6049-B substance, wherein the WS 6049-B substance has the following properties:

1) Form and color:
Colorless powder 2) Color reaction:
Positive: Dragendorff reaction, Ehrlich's reaction and cerium sulfate reaction Negative: ninhydrin reaction 3) Solubility:
Soluble: methanol, acetone, chloroform Slightly soluble: diethyl ether Insoluble: hexane 4) Melting point:
145°C (dec.).

5) Specific rotation:
[.alpha.]? = 201°(c=1.0, CHCl3) 6) Ultraviolet absorption spectrum:
= 253 nm = 280 nm = 320 nm = 253, 280, 320 nm = 250, 282, 310 (sh.) nm 7) Infrared absorption spectrum:
= 3450, 3350, 3250, 2990, 2920, 1725, 1675, 1610, 1595, 1520, 1465, 1450, 1405, 1370, 1350, 1310, 1250, 1180, 1155, 1115, 1070, 1020, 985, 955, 905, 880, 850 cm-1 8) Elementary analysis:
C: 51.58%, H:5.75%, N:4.27%, S:9.80%
9) Thin layer chromatography:

10) Molecular weight:
Gel permeation chromatography: 1100 to 1200 11) 13C Nuclear magnetic resonance Spectrum (CDCl3):
.delta.(ppm) : 13.8, 16.7, 17.6, 19.8, 22,7, 29.1, 33.9, 34.1, 35.2, 39.5, 52.8, 55.8, 56.1, 56.3, 61.0, 61.5, 64.6, 66.7, 68.4, 69.0, 69.3, 69.8, 70.4, 71.9, 75.9, 76.2, 76.7, 77.2, 77.3, 77.6, 83.2, 86.6, 88.3, 90.7, 97.3, 98.6, 99.0, 99.6, 99.7, 103.9, 107.8, 112.7, 123.2, 124.9, 129.8, 131.0, 136.8, 144.2, 146.3, 154.0, 154.6, 160.9, 166.6, 192.4, as shown in Figure 3 of the accompany-ing drawing, 12) H Nuclear magnetic resonance spectrum (CDCl3) :
.delta. (ppm) : 11.75 (1H, s), 8.57 (1H, s), 7.48 (1H, s), 6.6 (1H, d.d.), 6.24 (1H, d, J=1.3Hz), 6.18 (1H, br.s), 5.93 (1H, d, J=9.2Hz), 5.83 (1H, d.d, J=9Hz and 1.3Hz), 5.70 (1H, br.d), 5.5 (1H, m), 5.47 (1H, d, J=2.3Hz), 5.42 (1H, br.s), 4.98 (1H, d, J=9Hz), 4.7 to 4.6 (2H, m), 4.56 (1H, d, J=2.3Hz), 4.24 (1H, s), 4.15 to 3.4 (12 to 18H), 3.97 (3H, s), 3.88 (3H, s), 3.79 (3H, s), 3 42 (3H, s), 2.52 (3H, s), 2.6 to 2.4 (2H, m), 2.4 to 2.2 (7 to 8H), 2.12 (3H, s), 2.2 to 2.0 (2H, m), 1.5 (2H, m), 1.4 (3H, d, J=6Hz), 1.35 (3H, d, J=6Hz), 1.33 (3H, d, J=6Hz), as shown in Figure 4 of the accompanying drawing, which comprises culturing a strain belonging to the genus Actinomadura, which is capable of producing WS 6049-B
substance in an aqueous nutrient medium under aerobic conditions and recovering WS 6049-B substance from said medium.

8. A process for preparing WS 6049-B substance according to claim 7, wherein said strain belonging to the genus Actinomadura is Actinomadura pulveraceus.

9. A process for preparing WS 6049-B substance according to claim 8, wherein said strain belonging to the genus Actinomadura is Actinomadura pulveraceus sp. nov. No. 6049 (ATCC
39100).

10. A process according to claim 1 wherein said step of recovering comprises isolating WS 6049-A from the recovered WS 6049 substance.

11. A process according to claim 1 wherein said step of recovering comprises isolating WS 6049-B from the recovered WS 6049 substance.

12. A process according to claim 1 wherein said step of recovering comprises recovering a mixture of WS 6049-A
and WS 6049-B and isolating WS 6049-A from said mixture.

13. A process according to claim 1 wherein said step of recovering comprises recovering a mixture of WS 6049-A
and WS 6049-B and isolating WS 6049-B from said mixture.

14. A process according to claim 1 wherein said step of recovering comprises recovering a mixture of WS 6049-A
and WS 6049-B.

15. A WS 6049 substance selected from WS 6049-A
substance and WS 6049-B substance, as defined in claim 1, whenever prepared by the process of claim 1, or by an obvious fermentative and/or chemical equivalent thereof.

16. A WS 6049 substance selected from WS 6049-A sub-stance and WS 6049-B substance, as defined in claim 1, whenever prepared by the process of claim 2, or by an obvious fermentative and/or chemical equivalent thereof.

17. A WS 6049 substance selected from WS 6049-A
substance and WS 6049-B substance, as defined in claim 1, whenever prepared by the process of claim 3, or by an obvious fermentative and/or chemical equivalent thereof.

18. A WS 6049-A substance, as defined in claim 4, whenever prepared by the process of claim 4, or by an obvious fermentative and/or chemical equivalent thereof.

19. A WS 6049-A substance, as defined in claim 4, whenever prepared by the process of claim 5, or by an obvious fermentative and/or chemical equivalent thereof.

20. A WS 6049-A substance, as defined in claim 4, whenever prepared by the process of claim 6, or by an obvious fermentative and/or chemical equivalent thereof.

21. A WS 6049-B substance, as defined in claim 7, whenever prepared by the process of claim 7, or by an obvious fermentative and/or chemical equivalent thereof.

22. A WS 6049-B substance, as defined in claim 7, whenever prepared by the process of claim 8, or by an obvious fermentative and/or chemical equivalent thereof.

23. A WS 6049-B substance, as defined in claim 7, whenever prepared by the process of claim 9, or by an obvious fermentative and/or chemical equivalent thereof.

24. WS 6049-A, as defined in claim 1, whenever prepared by the process of claim 10 or 12 or by an obvious fermentative and/or chemical equivalent thereof.

25. WS 6049-B, as defined in claim 1, whenever prepared by the process of claim 11 or 13 or by an obvious fermentative and/or chemical equivalent thereof.

26. A WS 6049 substance comprising a mixture of WS 6049-A and WS 6049-B, whenever prepared by the process of claim 14 or by an obvious fermentative and/or chemical equivalent.