CA1265758A - Antibiotics, and their production - Google Patents

Abstract

Abstract:
The invention provides novel compounds of the formula:

(I) wherein R is a hydrogen atom or a methyl group.
These compounds are useful as antibiotics.

Description

~s7~a Antibiotics, and their ~roduction __ _ _ _ ______ _____ ___ The present invention relates to antibiotics and to their production.
In the course of a search for new antibiotics, it has been found that a Nocardia species indexed as SC-4710 in the collection in the Research Laboratory of Sumitomo Chemical Company, Limited, Osaka, Japan, and on deposit with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ibaraki-ken, Japan under the deposition number FERM P-8233, produces antibiotics. When this microorganism i5 grown in a suitable nutrient medium, at least two different antibiotics, which can be represented by the following formula, are produced:

H~C
HO'~ , \ OH

! ~ OH O - CH3 H3C ~ / \~ ~ O ~ ~

wherein R is a hydrogen atom or a methyl group~ When R is a hydrogen atom, the antibiotic (I) is called "PC-766B".
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i7~5 !3 When R is a methyl group, the antibiotic ~I) is called "PC-766B"'.
Nocardia sp. SC-4710 was isolated from a soil sample collected at Shiga-ken, Japan and shows the following microbiological properties:
J 1) Morphological characteristics:-On an agar medium and in a liquid medium, the sub-strate mycelium elongates and branches well, and it is sometimes divided into bacillus-like or branched short rods. Good growth is observed on various media for classification. On the substrate mycelium, white to grayish white aerial mycelia are formed. ~erial mycelia are lightly wavy or spiral and irregularly branched. On observation of the grown culture by a scanning electron microscope, 3 to 10 spores in chain are seen on the aerial mycelium; the shape of the spores is ellipsoidal, the size is from 0.3 x 0.8 micron to 0.4 x 1.0 micron and the surfaces are smooth; no peculiar structures such as sporangium and sclerotium are observed.

2) Cultural characteristics:-Culture media were prepared according to the Sharing and Gottlieb's method [International Journal of Systematic Bacteriology, Vol. 16, page 313 (1966)], and the corresponding test was carried out. Observation was made after incubation for a period of three weeks at 27C.

The results are shown in Table 1 wherein the colors were determined in comparison to the standard colors in "Color Tone Mannual" edited by Japan Color Research Laboratory.

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PJ ~ Cr~ nu~ ~t P~ r~ ~ O t~ r~ P-~ ~ n ~ ~ ~ o ~ ~ r~~ PJ n~ r~ r~ Ul ~-I S OP~ O U~ Pl O ~ (D U~ r~ C
Pl ~n r~ _ n ~ ~ rD ~ rD ~
(D (D (D-- 3 H rt (DH ~ OH 01 ::~ `' P) H X ('G
1- 1 1(Dcn I I U~r~ t X
~ n ~ ~s r~
(D ~ P~It (D (DI t P~ ~ ~ ~3 ~ O PJ ~~ n ~Q~ n ~ ~
UJ ~~ ~ P~~ rt n t r~ IS rL tD

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--~ ~ C~
O ~ O~: O O ~ O ~ O ~ O ~ O ~ ~
PJ Q It O(D RJ ~I tt ~1~ O1', 0 1~ 0 n o 1~ (D (D O (D ~ tt ~ ~ (D ~
(D ~ ~I~ It ~ ~ ~ ~ rt P~ 0 P~ -rt U~ rt Y rl U~
(D ~tl- (D tl~ (D ~ ~ :5 ~3 0 H~
~ ~
(D :5 ~ ~ ~
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rt ~ 1-- rt rt rt ~
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C O ~t o ~ ~ C O ~ ~ ~ O ~ O (D O n o ~ tt 1~ (D 1~ tt 1~ t~ 1~ ~ 1~
1--rt l O lJ- 5 ~: U~ UJ rt 1~ ~ rl ~S tt C
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t~ ~ ~ U~ ~h P~ ~
O (D (D ~ - U) I ~ u, u~ u ~ C
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r~ ~D 1- rt ~D ~ ~3' ~3 (D ~
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~2~75~3 3) Physioloyical characteristics:-Observations were macle aecording to ~tandard methods. The results are shown in Table 2.
Table 2 Test Result Growth temperature 17 to 37C
Optimum temperature for growth 22 to 32C
Gelatin liquefaction Negative Starch hydrolysis Negative Nitrate reduetion Positive Hydrogen sulfide production Slightly positive ~iilk peptonization Slightly positive ~lilk coagulation Slightly positive Melanin-like pigment produetion Negative

4) Utilization of earbon sourees The utilization of carbon sourees on the Pridham-Gottlieb agar medium is shown in Table 3 wherein the marks "+" and "++" indicate , respeetively, good and better utilization and the mark "-" indieates no utilization.
Carbon souree Result None L-Arabinose D-Xylose D-Glucose +~
D~Fructose ~ +~
Sucrose Inositol ~+
L-Rhamnose ::

- 6 ~ ~ 6 S7 ~ 8 Rha~finose D-Mannitol -~

5) sacterial composition:-Diaminopimelic acid in the bacterial cell is in the meso form, and arabinose and galactose were detected as the saccharides. By the analysis of the phopholipids according to Lechevalier's method [Biochemical Systematics and Ecology, Vol. 5, page 249 ~1977)], phosphatidylinositol, phosphatidylinositol dimannoside, diphosphatidylglycerol and phosphatidylethanolamine were detected, but phosphatidyl-choline was not detected. In the analysis of micolic acid according to Lechevalier's method [Canadian Journal o~
Microbiology, Vol. 19, page 965 (1973)] and Minikin's ~Journal of Chromatography, Vol. 188, page 221 (1980)], the spot for nocardomicolic acid methyl ester was detected.
From the above obs~rvation results, the charac-teristics of the mieroorganism SC-4710 are summarized as fvllows: the eell wall type is Type IV, and the phospho-lipid type is Type PII. Regarding the culture propertie~, the substratum myeelium shows brown to pale yellow, and the aerial myeelium shows white to grayish white. Morphologic-ally, spores are formed in a ehain on the aerial myeelium, and their sur~aces are smooth. Based on these eharaeteris-ties, the mieroorganism SC-4710 has been elassified to oeardia and named Nocardia ~ SC-4710 (FERM P-~233;
eorresponding to FERM BP-1203 used as the International Deposition ~umber under the Budapest Treaty).

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The antibiotics (I) of the invention are produced during cultivation oE a stanclard strain of r~_cardla sp.
SC-4710 or a natural or artificial variant or mutant thereof in an aqueous nutrient medium. The composition of this nutrient medium may be varied over a very wide range.
Essentially what is required is a carbon source, a nitrogen source and trace inorganic elements. Examples of suitable carbon sources are glucose, maltose, starch, dextrin, glycerol, molasses, etc. Examples of suitable nitrogen sources are soybean meal, corn steep liquor, cotton seed flour, peptone, meat extract, yeast extract, casein hydrolyzate, ammonium salts, nitrates~ etc. Examples of suitable sources of inorganic elements are mineral salts, e.~. chlorides, carbonates and phosphates of magnesium, potassium, calcium, sodium, iron, manganese, etc.

Cultivation is usually carried out under aerobic conditions, and preferably under aeration and agitation.
The temperature required for the cultivation may be appropriately decided within the range in which the microorganism grows and the antibiotics ~I) are produced.

A preferred temperature range is from about 25 to 30C.
The p~ is normally from about 7 to 8. The antibiotic potency accumulated in the nutrient medium reaches usually the highest ~ithin a cultivation period of about 80 to 120 hours.

For recovery of the antibiotics (I) from the fermentation broth a~ter the cultivation, any conventional procedure may be adopted. For example, a procedure ~` .
.. . . . .

: ~ ;`'" "' ,, ' - B -utiLizing diEEerences ;n solubility between the clesired substance and the impurities, a procedure utilizirlg differences in adsorption a~finity onto various adsorbents e.g. activated carbon, macroporous non-ionic resins, silica gel and alumina, a procedure using ion exchan~e resins for the removal of impurities, etc. may be adopted either alone or in combination.
A typical example of the separation and purifica-tion of the antibiotics (I) from the ~ermentation broth after cultivation is as follows.
The fermentation broth may be separated into the bacterial body and the supernatant or filtrate by centri-fugation or filtration. The bacterial body can then be extracted with acetone or methanol and the extract extrac-ted with a suitable organic solvent for the antibiotics(I), e.g. ethyl acetate or n-butanol. Alternatively, the supernatant or filtrate may be extracted with an organic solvent which is not miscible with water and can dissolve the antibiotics (I) therein, e.g. ethyl acetate, n-butanol or methylisobutylketone The organic solvent extracts comprising the antibiotics ~I) are combined together, and a per se conventional procedure for purification of a fat-soluble substance is applied thereto to recover the antibiotics (I). For example, the ethyl acetate extract may be washed with water and concentrated under reduced pressure, ~ollowed by addition of n-hexane or the like to precipitate the active component. 1~he precipltate can then be collected by centriEugation or filtration to give : ,~

'~

r~ ~
- g a crude prod~ct com~rising the antibiotics (I).
For purification/ the above crude product may be subjected to column chromatography using a carrier having a molecular sieve effect, e.g. Sephadex LH-20 (trade mark, manu~actured by Pharmacia, Sweden) and a developing solvent e.g. alcohols (e.g. methanol), halogenated hydrocarbons (e.g. chloroform), and their mixtures. For further purif-ication, the resulting product may be subjected to adsorp-tive chromatography, for example using a carrier generally used for adsorptive separation of antibiotics (e.g. adsorp-tive resins, silica gel, alumina) as an adsorbent. When silica gel is used as the adsorbent, chromatographic separa-tion may be effected by changing the mixing proportion of a polar solvent, e.g. an alkanol (e.g. methanol), and a non-polar solvent, e.g. halogenated alkanes (e.g. chloroform).
In order to isolate PC-766B and PC-766B~ from the above purified product~ chromatography using the above-mentioned adsorbent may be repeatedly applied thereto.
When~ for example, silica gel is used as the adsorbent, the silica gel on which the antibiotics (I) are adsorbed may be developed with a solvent system consistin9 of benzene and e~hyl acetate, whereby PC-766B' and PC-766B are eluted in this order. After assaying the purity by TLC (thin layer chromatography) or HPLC ~high performance liquid chromatog-raphy), the eluted fractions may be concentrated underreduced pressure to dryness so that PC-766B and PC-776B' can be respectively obtained as powdery crystals.

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PLC~766B and PC-766B' can both be manu~actured by cultivation of _ cardia ~ SC~q710. PC~766B' may also be manu~actured by methylation of PC-766B under appropriate conditions. This methylation can be effected, for example, by maintaining a solution oE PC-766B in methanol at room temperature (e.g. 20 to 25C).
The chemical structures of the antibiotics (I) were determined by spectroscopic investigation. Some character-istic physico-chemical properties of PC-766B and PC-766B' are shown below, in which reEerence is made to the accompany-ing drawings, wherein:
Figure 1 is a W absorption spectrum (in methanol) of PC-766B;

Figure 2 is an Infra Red spectrum of PC-766B;
Figure 3 is an H-NMR spectrum of PC-766B;

Figure 4 is a C-NMR spectrum of PC-766B; and Figure 5 is an ~-NMR spectrum of PC-766B'.
1. PC-766B

Appearance: colorless amorphous powder.
Elemental analysis: C, 61.29 ~; H, 8.11 %.

Molecular weight; 776.
Mass spectrographic analysis: FD, F~B-MS; m/z 799 (M -~Na), m/z 815 (M +K).

Molecular formula: Cg3H68l2.
Melting point: 132-134C.

Speci~ic ro~ation: [~]D = ~17 7a (C = 0.61, methanol).

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- lOa -Solubility: soluble in methanol, chrloro~orm and ethyl. acetate; insoluble in n-hexane and water.
Color reaction: positive in I2 absorption and anisaldehyde reaction; negative in ninhydrin reaction.
TLC (silica gel): chloroform-methanol (15 : 1 by volume), Rf = Oal4; benzene-ethyl acetate (1 : 4 by volume), Rf = 0.23.
UV absorption spectrum (in methanol): as shown in :` ~ , .
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Fig. 1 of the accompanying drawings.
IR absorption spectrum (in l<sr tablet): as shown in Fig. 2~
H-NMR spectrum (200 MHz, in CDC13, T~IS standard):
as shown in Fig. 3.
13c_N~1R spectrum (50.3 MHz, in CDC13, TkiS
standard): as shown in Fig. 4.
2. PC-766s' Appearance: colorless amorphous powder.
Molecular weight: 790.
Molecular formula: C44H70O12.
Solubility: soluble in methanol, chloroform and ethyl acetate; insoluble in n-hexane and water.
Color reaction: positive in I2 absorption and anisaldehyde reaction; negative in ninhydrin reaction.
TLC (silica gel): chloroform-methanol (15 : 1 by volume), Rf = 0.18; benzene-ethyl acetate ~1 : 4 by volume), = 0.35.
H-NMR spectrum (90 MHz, in CDC13, TMS standard):
as shown in Fig. 5.
The antibiotics (I) exhibit significant anti-microbial potency against various microorganisms. The minimum inhibitory concentrations of PC-765B against various microorgnisms as measured by the agar dilution method are shown in Table 4. PC~766B' also shows similar antimicrobial potency.

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Practical and presently preferred embodiments of this invention are illustratively shown in the following Examples wherein part~s) and percenta~es are by weight, unless o~er-wise indicated.
Example _1 A loopful of Nocardia sp. SC-4710 (FERM P-8233) cultured on a slant agar was inoculated into a seed culture medium, which was then subjectedto a shaking culture at 27C
~ for 5 days. The resulting seed culture was added to a ! 10 pre-culture medium in an amount of 2 % by volume, and a shaking culture was carried out at 27C for 5 days. The resulting pre-culture was added to a nutrient medium (50 liters) charged tQ a 90 liter volume jar fermenter in an amount of 2 % by volume, and cultivation was carried out at 27C for 4 days under an aeration amount of 25 liters per minute at an agitation rate of 200 r.p.m. All of the seed culture medium, the pre-culture medi.um and the nutrient medium comprised glucose (2.5 %), soybean meal (1.5 ~), yeast extract (0.2 ~) and calcium carbonate ~0.4 %) and were used after adjustment to pH 7.2 and sterili~ation at 121C
for 20 minutes.
Example 2 The fermentation broth after cultivation as in Example 1 ~50 liters) was centrifuged to separate it into the microbial body and the supernatant. The microbial body in a wet state was shaken with acetone t5 liters~ and filtered, and this operation was repeated twice. The acetone extracts (15 liters) were combined together and .~ il .. i .. ...

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~6s~sa concentrated under reduced pressure to make a volume of about 100 milliliters. 'I'he concentrate was shaken with a mixture of water (1 liter) and ethyl acetate (1 litee) to extract the active fraction. Separately, said supernatant was adjusted to pH 7.0 and shaken with an equal volume of ethyl acetate.
The ethyl acetate extract was washed with water (30 liters), combined with the extract comprising the active fraction and concentrated under reduced pressure. n-~exane was added to the concentrate. The precipitate was collected by filtration and dissolved in a mixture of chloroform and methanol (1 : 1 by volume) (100 ml). The resultant solution was placed on a column of Sephadex LH-20 ~trade mark, manufactured by Pharmacia) (500 ml) equilibrated with a mixture of chloroform and methanol (1 : 1 by volume) and eluted with a mixture of chloroform and methanol to produce fractions of 50 milli-liters each. The active ractions were concentrated to dry-ness under reduced pressure, and the residue was dissolved in chloroform (about 5~ ml). The resulting solution was added to a column of silica gel ("Kiesel Gel 60" trade mark, manufactured by E. Merck in W. Gemany, 63 to 200 microns) (500 ml), and the column was eluted with chloroform and then a mixture of chloroform and methanol (97 : 3 by volume) to produce Eractions of 50 milliliters each. The active frac-tions were concentrated under reduced pressure to dryness to give a crude product (640 mg) of the antibiotic substance "PC-766" comprising PC~766B as the major product with a trace amount of PC-766B'.

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Example_3 The crude product of the antibiotic substance PC-766 (640 mg) as obtained in ~xample 2 was purified by the use of an apparatus for HPLC ~"System 500" trade mark, manu-Eactured by Waters, U.S.A.) under the Eollowing operation conditions:
Column: Preppack-500 (trade mark) silica gel column ~manuactured by Waters), two columns);
volume);
Flow rate: 100 ml/minute;
Detection: UV absorption at 254 nm;
Sample injection: the crude product "PC 766" (640 mg) dissolved in chloroform (30 ml) and then injected;
Fraction volume: 50 ml.
Fraction Nos. 27 and 28 were concentrated under reduced pressure to dryness to give a mixture l2 mg) of PC-766B and PC-766B'. Fraction Nos. 29 to 47 were concentrated under reduced pressure to dryness to give PC-766B (580 mg).
Example 4 The antibiotic substance PC-766B (50 mg) as obtained in Example 3 was dissolved in methanol (100 ml) and allowed to stand at room temperature (20 to 25C) for methylation. After one week, the resultant solution was concentrated under reduced pressure, subjec~ed to streak-adsorption on a preparative thin layer chromatogram and developped with a mixture of benzene and ethyl acetate (1 :
4 by volume). Observation was made under irradiation with . .

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UV ra~s. The bands or PC-766B and PC-766B' were respec-tively scraped o~f, extracted with methanol and concentrated under reduced pressure to yive unreacted PC-766B (34 mg) and PC-766B' (3 mg). These products gave the physico-chemical properties and biological properties as hereinabove described.

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Claims (8)

Claims:

1. An antibiotic of the formula:

(I) wherein R is a hydrogen atom or a methyl group.

2. An antibiotic according to claim 1, wherein R
is a hydrogen atom.

3. An antibiotic according to claim 2, wherein R
is a methyl group.

4. A process for preparing an antibiotic of the formula:

(I) wherein R is a hydrogen atom or a methyl group, which comprises cultivating Nocardia sp. SC-4710 in a nutrient medium under aerobic conditions and recoverying the accumulated antibiotic from the resulting fermentation broth.

5. A process according to claim 4, wherein R is a hydrogen atom.

6. A process according to claim 4, wherein R is a methyl group.

7. A process for preparing an antibiotic of the formula:

(I) wherein R is a methyl group, which comprises methylating the corresponding antibiotic (I) wherein R is a hydrogen atom.

8. A biologically pure culture microorganism, Nocardia s.p. SC4710.