CA1069449A - Process for the production of nocardicin c,d,e,f and g - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
New Nocardicins designated Nocardicin C. Nocardicin D, Nocardicin E, Nocardicin F and Nocardicin G are provided together with a process for their preparation, which comprises cultivating a Nocardicin C, D, E, F and/or G producing strain of the genus Nocardicin in an aqueous nutrient medium under aerobic conditions and recovering the Nocardicin C, D, E, F or G: suitable strains include Nocardia uniforms subsp. tsuyamanensis, for example Nocardia uniforms subsp. tsuyamanensis ATCC 21806 or a mutant thereof; the new Nocardicins posses specific antibiotic spec-trums, have low toxicity and are active against pathogenic bacteria.

Description

~L~G94~9 PROCESS FOR THE PRODUCTION
OF NOCARDICIN C, D, E, F ~ND G

This invention relates to a new process for the product-ion of new compounds, Nocardicin C, Dl E, F, and G.

More particularly, it relates to a new process for the production of new compounds, Nocardicin C, D, E, F and G by culturing a Nocardicin C, D, E, F and G - producing strain belonging to the genus Nocardia in a nutrient medium.

Accordingly, it is an object o~ this invention to provide new compounds, Nocardicin C, D, E, F and G.
. 15 The object compounds of this invention, Nocardicin C, D, E, F and G (hereinafter referred to as the present Nocardicins) are represented by the following formulae.

O
RlO ~ ~2- C- NH

N- ~H ~ OH
COOH

Nocardicin ~L . R2 :~:
: NH2 ~ :
C HOOC-CHCH2CH~- -CH- :-NH

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D ~ C~

N-OH ::

E ~H~ C~
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Nocardicin Rl R~
HO-N
F H- -C-G ~ " CH-Microorganism to be used in this invention is a strain belonging to the genus Nocardia which is capable of producing Nocardicin C, D, E, F and/or G~
~ 10 Among such a strain, preferred one is Nocardia uniformis subsp. tsuyamanensis, which was deposited on June 13, I972 with American Type Culture Collection (ATCC) located in 12301 Parklawn Drive Rockville, Maryland 20852, USA and assiqned the ATCC number 21806. This deposited Nocardia uniformis subsp.
tsuyamanensis ATCC 21806 is now available to the public and th~ details thereof, i.e. the microbiological characteristics, etc. are disclosed in~literatures, e.g. United States Patent No~ 3, 923; 377 and German O~fenlegungsschri~t~2242695~
.
; 20 It is to be understood that, for the pro~uction of the .
present Nocardicins, this invention is not limited to the use of ~specific) organism described~herein, which is given only -: -~ ~ for illustrative purpose. Further, this invention also includes - - . .

2~ the use of natural mutants as well as artifidalones which can be derived from the microoryanism as described herein in a -conventional manner such as radia~ion with X-rays or Ultra-violet, t~eatment with N'-nitro-N nitrosoguanidine, 2-amino- --purine or nitrogen mustards,~ and the like.
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The present Nocardicins are produced by culturing a Nocardicin C, D, E, F and/or G - producing strain belonging to the genus Nocardia such as Nocardia uniformis subsp.
tsuyamanensis in a nutrient medium containing assimilable -S carbon and nitrogen sources under submerged aerobic conditions.
Further, as a nutrient of the medium, there may be used any nutrient which can be utilized by the said microorganism for production of the present Nocardicins.

The preferred sources of carbon are carbohydrates such as glucose, sucrose, maltose, glycerin, starch and the like.
The preferred sources o~ nikrogen are organic nitrogen sources such as yeast extracts, peptone, gluten meal, cottonseed meal, soybean meal, corn meal, dried yeast, beeE extracts, casein hydrolysate, corn steep liquor, urea and the like, and inorga-nic nitrogen sources such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.) and the like. -I desired, there may be added to the medium, mineral salts such as calcium carbonate, sodium or potassium phosphate, magnesium chloride or sulfate and the like as a minor component.
Further, there may be added to the medium, one or more organic compounds such as tyrosine, glycine, serine, homoserine, p-hydroxyphenylglycine, d-aminobutyric acid, ~ diaminopropio-nic acid, N-acetyltyrosine, N-acetyltyrosineamide, p-hydroxy-phenylpyruvic acid, p-hydroxyphenylglycolic acid, p~-hydroxy-phenyIglyoxalic acid, shikimic acid, 2-amino-3-(4-hydroxy-phenyl)propionohydroxamic acid, 2-acetamido-3-(4-hydroxy-phenyl?prouionohydrazide~and the like. ~hese organic compounds , may work as a kind of precusor and may be useful for elevat-ing the productivity of the present Nocardicins.

In the fermentation process, submerged aerobic cultural conditions are preferably empoylyed for the production of the present Nocardicins in massive amounts. It is to be under-stood also that for the production in limited amounts, a shak-ing or surface culture in a flask or bottle can be employed.
Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production of the present Nocardicins. Accordingly, it is desirable f ir9t to produce a vegetative inoculum of the organism by inoculating a relat-ively ~mall quantity of culture medium with spores or myceliaof the organisman~ culturing them and to transfer the cultured vegetative inoculum aseptically to large tanks. The medium in which the vegetative inoculum is produced can be the substantially same as or different from medium utiliz d for the production of the present Nocardicins.

Agitation and aeration af the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or the similar mechanical agitation equipment, -by revolving or shaking the fermenter, by various pumpingequipment or by the passage of sterile air through the medium.

Aeration may be effected by passing sterile air throu~h the fermentation mixture. In the course of fermentation, especia~

.
lly when the culture medium is foamed remarkably, a defoaming agent such as plant ~ils ~e g. soybean oil, etc.), higher ',.

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alcohols (e.g. octadecanol, tetradecanol, etc.), silicones and the like, may be added to the medium.

The fermentation is usually conducted at a temperature about between 20C. and 40C., preferably about 30C., for a period of 50 hours to 100 hours.

The present Nocardicins t as produced above, can be recovered from the cultured broth in a conventional manner, which are generally used for the recovery o~ fermentation products. The present Nocardicins in the cultured broth are present in mycelia (intracellularly) and/or out of mycelia (extracellularly).
~.'' '" .
As the first step, the cultured broth is divided into filtrate (supernatant) and filter cake by means of filtrat- ~
ion or centrifuge. ~;

Extraction of the present Nocardicins from the filter cake is conducted by treating sàid cake with an organic solvent in which the present Nocardicins can be soluble,for example, alcohols (e.g. methanol, ethanol, etc.), ketones (e.g. acetone etc.), aqueous alcohols ~e.g. aqueous methanol, aqueous ethanol, etc.) and the like.
- ~25 From thus obtained filtrate and/or extract, the present Nocardicins may be isolated and purified ~y conventional means~ ~ `
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.

S~6 ,9~9 As said conventional means, there may be exemplified~
treatment with adsorbents (e.g. activated charcoal, silicic acid, silica gel, alumina, etc.), anionic or cationic ex-change resins, or macroporous nonionic adsorption resins p?6~/'~
~e.y. Amherlite XAD-2, XAD-4,.XAD-7 and XAD-8 (trade ~e, B made by Rohm & Haas Co.), Diaion HP10, HP20, HP30, flP40 and HPS0 (trade ~e, made by Mitsubishi Chemical Industries Ltd.~, etc.]; e~traction with solvent; concentration under reduced pressure; lyophilization; ~H adjustment; crystallization;
recrystalliza-tion; and the like. rrhese means may be prefer~
ably employed independently or in combination thereof in optional order or repeatedly.

.
In the case of a crude material (e.g. culture filtrate, extracts from filter cake) containing all the Nocardicinsj iae.
Nocardicin A, Nocardicin B which is a geometric isomer of Nocardicin A at the hydroxyimino group, Nocardicin C, Nocardicin D, Nocardicin E, Nocardicin F and Nocardicin G, each of the Present Nocardicins can ~e separated from it, for example according to the separation method as illustrated in the following scheme.
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S tep a) Separ~tion of Nocardicin C ~nd G from other Nocardicin A/B,D,E and F;
When the said crude material is subjected to a column chroma-tography using a macroporous nonionic adsorption resin (e.g. Diaion HP 20), Nocardicln A,B,D,E and F are adsorbed to the resin. On the other hand, Nocardicin C and G are passed through the column.
Step b) Separation o~ Nocardicin C from Nocardicin G:
The passed solution, as obtained above, is then fractionated by subjecting to a column chromatography using cellulose with a suitable developing solvent ~e.g. n-butanol saturated with water, a mixture of n-butanol, acetic acid and water (20:1:6)] to give Fraction I containing Nocardicin C and Fraction II containing Nocardicin G.
Step c) Separation of Nocardicin D from other Nocardicin A,B,E and F:
The adsorbate of foregoing Step a), is eluted from the resin with a hydrophilic solvent (e.g. aqueous methanol). The eluate which contains Nocardicin A,B,D,E and F is then fractionated into three Fractions,i.e. F~action III containing Nocardicin E and F, 2~ Fraction IV containing ~ocardLcin D and Fraction V containing Nocardicin A and B, by a col~unn chromatography on cellulose with a suita~le developing solvent [e.g. n-butanol saturated with water, an upper layer of a mixture of ethyl acetate, n-butanol and water (5:5:2)3.
Step d) Separation of Nocardicin E from Nocardicin F:
The Fraction III, as obtained in the foregoing Step c) is further fractionated ~y acolumn~chromatography on silicic acid `
with a suitable developing solvent[e.g. a mixture of chloroform and ethyl acetate ( 4:1)r a mixture o~ chloroform and methanol (10:1)] to give Fraction VI containing Nocardicin E and Fraction ;;

VII containing Nocardicin F.

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S~6-3 ~ ~ ~9 ~9 S-tep e) Alternative me-thod for separa-tion of Nocardicin E
and Nocardicin F:
Nocardicin E and F can be also separated from the crude material by a column chromatography on silicic acid with the same suitable developing solvent as exemplified above, since Nocardicin A,B,C,D and G are strongly adsorhed to the silicic acid and can be hardly eluted with said solvent.

~ .
Each of Nocardicin C, D, E, F and G can be purified from the fractions, i.e. Fractions Il II, IV, UI and VII in a conventional manner.

The present Nocardicins, produced in the culture broth, can be isolated in the free form, and optionally in the form of their alkali metal salts by treating crude material containing the present Nocardicins with an alkali metal material (e.g. sodium or potassium hydroxide) during the isolation or purificatlon processes.

~0 The present Nocardicins obtained in their free form may be also converted to the salt with an inorganic or organic base (e.g. potassium or sodium hydroxide, ethanolamine, dicyclohexylamine, etc.) in a conventional manner~
-Further, the inorganic or organic base salt of the present Nocardicins may be easily converted to their free form by treating the said salt with an acid such as a m-ineral acid (e.g. hydrochloric acid,etc.J in a conventionaI

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s-7 3L~3369~9 Physico-chemical properties of the present Nocardicins are mentioned as follows.

Table 1. Physicochemical Properties o~ Nocardicin C and D
Nocardicin C ~ocardicin D
Appearance White crystals White crystals Amphoteri.c Amphoteric .

Optical [d]D4=-95(C=0.6, H2O) ~f~]20=-171(C=l, H2O) rotation Melting point 220-225C(decomp.) 230-235~C(decomp.) W spectrum Amax(~1%m)=227(434~, ~ max(Elcm)=226(395), 272(43), 277(sh, 37)nm 298(313)nm in C2H5OH-H2O
in H2O, 232(319), 243 (1:1), 246(305), 298 (sh, 289), 280(54), (300)nm in C2H5OH-292(sh, 44)nm in 0.1N Q.lN aq~ NaOH(l:l) . ..
aq. NaOH.
-~
IR spectrum ~ mU~l=3430,2920,2850, J nU~l=3430,3210,2900, 1745,1670,1610,1580, 2850,1735,1670,1655, 1560,1515,1465,1375, 1610,1600,1570,1510, :`
- - 1340,1310,1255,1180, 1465,1455,1420,1~08, :~.
-: 1170,1110,1040,97~, 1375,1340,1320,1280, 940,930,890,850,820, 1260,1f~0,1178,1140, 795,760,740,680cm 1 1120,1080,1050,1030, -1010,980,930,845cm 1 :

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Nocardicin C Nocardicin D
_ Color Positive: ninhydrin and Positive: ninhydrin reaction ferric chloride react- and ferric chloride ions, reactions~
N~gative: Ehrlich and Negative: Molisch test, Molisch tests, reaction reactions with Tollens with Tollens reagent. reagent and Dragendorff reagent Solubility EIighly soluble:aqueous Highly soluble:aqueous ; alkaline solution(e~g. alkaline solution(e.g.
aqueous ammonia, aqueous ammonia, aqueous sodium hydrox- aqueous sodium hydrox-ide solution3, ide solution), pyridine dimethyl sufoxide, Sparingly soluble:H20, Spo Sparingly soluble:H20, CH30H, C2HsOH
CH30H Insoluble:CH3COOC2H5, Insoluble:C~H50H, CH~Q3l C2H50C2 5 ' C~3CCH31CHCQ3~ :~

Table 2. Physicochemical Properties of Nocardicin E and F
~Nocardicin~E Nocardicin F
Appearance White crystals White crystals Weakly acidic Weakly acidic Elemental C56.95:H4.20:N10.48 C56.84:EI~.35:N10.23 - analysis Opt cal [~]2 =-192(~C=1, H20) ~ ¦~3~ =-181 ~C=1, X20) :' , .
- 11 - ' ' 1~6~9 Nocardicin E Nocardicin F
Melting 228-231C(decomp.) 230-231C(decomp.) point spectrum ~max(Elcm)=22~(sh,557~t Amax(Elcm)=224(516), 272(396)nm in CH30H 270(248)nm in CH30H, 248(719), 298(324)nm 247(720), 295(253)nm in CH3oH-lN aq. NaOH in CH30H-lN aq. NaOH
(9: 1) (9 ~

10spectrum ~ma~ =3380,3280,2920, ~ ma~ =3420r3300,3280,2840,1745,1675,1645, 2g50,29~0,1900,1745, 1610,1595,1540,1515, 1680,1655,1610,1595, 1510,1~60,1435,1375, 1550,1515,1465,1450, 13~0,1325,1310,1275, 1410,1380,1360,1310, 1260,1220,1175,1140, 1295,1280,1270,1250, 1115,1105,1055,1030, 1220,1180,1140,1120, 1005,945,935,910,855, 1110,1060,1030,1010, 845,825,755,735,730l 980,935,900,870,860, 700,685cm l .840,820,780,740,720, :: 630cm 1 .

Color Positive: ferric chlo~ Positive: ferric reaction ~:
ride-potassium chloride-potassium -~
ferricyanide reaction ferricyanide reaction :
Negative: Ehrlich ~est, Negative: Ehrlich test, : ~
:
ninhydrin reaction ninhydrin reaction : .
: :: : : Solubility Hlghly soluble: aqueous Highly soluble: aqueous alkaline solution~e.g. ~ al~aline solution, aqueous ammonia, pyridine, : . '' ' ~ ' ' ' ~ I2 -. .

s-lo 4~9 Nocardicin E Nocardicin F
Solubility aqueous sodium hydrox- dimethylsulfoxide ide solution), pyridine, Sparing soluble:CH3OH, dimethylsulfoxide, 2 5 Sparingly soluble:CH3OH, Insoluble: C~l3COCH3 C2H5H' CH3COOC2H5' CHC~3 Insoluble: CH3COCH3 CH3CC2HS ~ CHCQ3 1~ Table 3. Physicochemical Properties of Nocardicin G
Nocardicin G
Appearance white crystals Amphoteric '.
; 15 Elemental analysis C57.62; H5.14: N 10.39: H2O 2~81 .

Optical rotation 1~]D =-205(C=1, 1% aq. NaHCO

`~ Melting point 227 - 233C(decomp.) ~The Nocardicin G
:~ 20 gradually changes to red about at 2aooc and reddish black about at 225C) ,:, UV spectrum ~max~Elcm)~228(454j, 273(52), 278(sh, 45)nm in n.lN aq. HCQJ 248(562), 291(107)nm in 0.LN aq. NaOH.
.
IR spectrum ~ ~ nU~1=3250,3150,2900,2850,174~5,1690, 1610,1590,1560,1510,1460,137S t 1340,1295, 1270,1255,~1185,1170,1135,1100,1050,102 ~ 940,925,880,850,830,825,815,760,750 r720 : ~

s- ll -~ _ Nocardicin G
IR spectrum 680cm 1 Color reaction Positive: ninhydrin and ferric chloride reactions Negative: Ehrlich and ~lolisch tests, Fheling reaction, and reaction with Tollens reagent.
Solubility Highly soluble: dimethylsulfoxide, H20, Sparingly soluble: CH30H, C2H50H
Insoluble:CH3COcH3 CH3COOC2H5, CHCQ3 , :

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s-14 )6g'~ 3 As the result of further study, chemical structures of the present Nocardicins having the above physico-chemical properties have been identified and assigned as mentioned above.

The present Nocardicins and their s.alts possess spec.ific antibiotic spectrums, showing activity against pathogenic bacteria with low toxicity. Accordingly, the present Nocar-dicins and salts thereof may be useful in t.reatment of infect-ious diseases caused by .such bacteria in mammalsO

The pharmacological tests of some of the present Nocar- ;.
dicins i~e. antimicrobial and toxicity tests are mentioned as follows.

~inimum Inhibitory Concentration ~M.I.C.) M.I.C. test was conducted by a usual serial agar dilution method, using heart infusion agar (or Mueller Hinton agar) which was incubated at 37C for 20 hours. M.I.C. value is expressed as the minimum concentration of the Nocardi.cin C, D, E or F (mcg/ml) which inhibits growth of the micro-organism. The results are shown in the following table 6.
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~ 18 -Acute Toxicity An aqueous sodi~ hydroxide solution (pH 7.4) of Nocardicin C was intravenously injected into each of five ICR-strain male mice weighing 18 - 22g (Dose: lg/kg), and S the observation was continued for one week after said admini-stration~as the result of which all the mice tested were normal for said period.

Further, each of aqueous sodium hydroxide solution (pH 7.4) of Nocardicin D, E, F or G was subcutaneously injected in each of five ICR-strain mice weighing 18 - 22g (Dose: 500mg/kg) and the observation was continued for one week after said administration, as ~he results of which all the mice tested were normal for said period.

The present Nocardicins and pharmaceutically acceptable salt thereof can be formulated for administration in any convenient way, analogously with known antibiotics, by admixture with a pharmaceutical carrier.

A pharmaceutically acceptable salt of the present Nocardicins may include salt with an inorganic or organic base such as sodium hydroxide, potassium hydroxide, calcium hydroxide~ ammonia, ethanolamine, triethylamine, dicyclo-hexylamine and the like.
. . .

Thus, the antimicrobial composition can be used inthe form of pharmaceutical preparation, for example~ in solid~ semisolid or liquid form~ which contains the active object compound in admixture with a pharmaceutical organic `:
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-- 19 -- , .
- `' , ' ` ~' , ".. ',' . . ` ' ' ` ' ,~ .`. ,. ' . ' ~ ., or lnorganlc carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with usual carriers for tablets, peletts, capsules, suppositories, solutions, emulsions, suspensions, and other form suitable for use.
The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, manni~ol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid foxm, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes. ~he antimicrobial compositions can also contain preserving or bacteriostatic agents thereby keeping the active ingredient in the desired preparations stable in activity. The present Nocardicins or salts thereof is in~]uded in the antimicrobial co~posit- -ion in an amount sufficient to produce the desired thera-peutic effect upon the bacterially infected process or condition.

For applying the present antimicrobial composition to human, it is preferably to apply in a form of intraven-ous or intramuscular injection. While the dosage or therapeutically effective quantity of the present Nocardicins or salt salt thereof varies from and also depends upon the ::
age and condition of each individual patient to be treated, a daily dose of about 2 - 50mg of the active ingredient/kg of human or an animal is generally given for treating ~iseases.
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~9~45a The present Nocardicins can be also used as intermedi-ates for preparing the other 3-acylamino~ -carboxy-4-hydroxybenzyl)-2-azetidinone, which is more improved in the antimicrobial activity against pathogenic Gram-positive S bacteria. For reference, with regard to some of the present Nocardicins, there is illustrated the production of 3-acylamino-l-(~-carboxy-4-hydroxybenzyl)-2-azetidinone from the present Nocardicins by the scheme as follows.

N~20 HOOC-I~l(CH2~20 ~ CH-C-HN ~
NH2 -IH ~ _OH
COOH

(Nocardicin C) ~ ¦ ~ N=C=S

HOOC-CH(CH2)20 ~ CIH- ~-HN ~

NHCSNH ~ IHCSNH~ ~ -OH

Hydrolysis ,.
~ 20 H2N ~ Acylation ; ~ N-~H ~ -OH - ~ acyl-HN ~ I :
OOH ~ N-CH ~ OH
COOH

3-acYlamino-l-(d-carboxy-4 Hydrolysis O hydroxybenzyl)-2-azetidinone~ :
: 25: HO ~ CH-C HN ~
NHCSNH- ~ ~ N-IH ~ OH

O ~ N=C=S
~ HO ~ CH- C - HN ~
: NH2 ~ ~ ~ N-CH ~ -OH
(Nocardi~in G) COOH
<

, , .
.' .:

'' ' ' " ' ' '. . ' ' ' ' . ' ' ., '.'' ` , . '' ' ., ' ',, . ' ', . ,,,, ' ' ~.,' '' ' '' . . '' ' . " ' ' ' ,' , ' The following examples are given for the purpose of illustrating this invention.
' Example 1 (Production of Nocardicin C) An aqueous medium (lOOml) containing 2 % of sucrose, 2 ~ o cottonseed meal, 1 % of dried yeast, 2.18 % of KH2PO4 and 1.43 ~ of Na2HPO4 12H2O was poured into each of twenty 500ml. shaking-flasks, and sterilized at 120 C for 20 min-~tes. A loopful oE a slant cultur~ of ~ocardia uniformis subsp. tsuyamanensis ATCC 2i806 was inoculated into each of the media, and then cultured at 30 C for 48 hours.

On the other hand, another aqueous medium (100 liters) containing 2 % of soluble starch, 1 % of peptone, 0.4 % of yeast extracts, 1 % of KH2PO4, 1 % of Na2HPO4 12H2O, 0.5 %
of MgS04-7H20, 0.1 % of ~-tyrosine and 0.1 % of glycine was adjusted to pH 6.0 with 6N aqueous sodium hydroxide solution.
20-Liters of the medium were poured into each of five :
30-liter jar fermenters, and sterilized at 120 C for minutes.

~o each of the media, there was added the seed culture, as prepared above, in an amount of 2 % by volume of the medium. The organism was grown at 30 C for 96 hours.
During the grow~h period, the broth was stirred at 250 r.p.m.
and sterile air was blown through the broth in the amount of 20 liters per minute.

' ' ~ 22 -~;9~9 The resultant cultured broth (80 liters) was filtered with an aid of diatomaceous earth (8 Kg) to give filter cake and filtrate. To the filter cake, there was added 70 %
aqueous ethanol ~20 liters), and then the mixture was stirred for an hour and filtered. This extrac-tion operation was re~eated twice. The combined extracts (40 liters) were con-centrated to a volume of 2 liters under reduced pressure~

On the other hand, the culture filtrate, as prepared above, was concentrated to a volume of 10 liters. To the concentrate, there was added methanol (50 liters) with stirring, whereafter the mixture was allowed to stand for an hour and th~n filtered. The filtrate was concentrated to a volume of 2 liters under reduced pressure.

The combined concentrate was adjusted to pH 3.0 with 6N -hydrochloric acid and extracted twice with ethyl acetate ~ -(4 liters). The aqueous layer was concentrated to a volume of 1.8 liters under reduced pressure. The concentrate was adjusted o pH 4.0 with lN aqueous sodium hydroxide solution and then passed through a column packed with Diaion HP20 (4 liters) to give a passed solution. Subsequently, the column was washed with water to give washings. The passed solution and the washings were combined and the aqueous solution (6 liters~ was adjusted to pH 8.0 with 6N aqueous sodium hydroxide solution. To ~he solution, there was added an activated charcoal (120 g), whereafter the mixture was ; stirred for 10 minutes and then filtered. The activated charcoal was washed with water (1 liter) and then 80 %
methanol (1 liter) w~s added thereto.

,.
,:

The mixture ~as stirred for 10 minutes and filtered. The elution operation was repeated once more. The combined eluate (2 liters) was concentrated to a volume of 10 ml. To the concentrate, there was added methanol (200 ml~ with stirring and insoluble materials were filtered off from the mixture. ~he filtrate was concentrated to a volume of 10 ml.
~he concentrate was s~bjected to a column chromatography on cellulose ~developing solvent : upper layer of a mixture of n-butanol : acetic acid : water (100:5:30)l. The eluate (500 ml~ containing the object compound was collected. To the eluate, there was added n-hexane (500 ml), and the mixture was stirred and then the aqueous la~er was separated. To the aqueous layer, there was added ethyl acetate (lO0 ml), and the mixture was stirred. The aqueous layer was separated and -concentra~ed to a volume ~f 20 ml. To the concentrate, there was added acetone (40 ml~, and the mixture was allowed to stand in a refrigerator (5 C) to give crystals. The mixture was filtered and the obtained crystals were dried to give Nocardicin C (26 mg) in the form of crude crystals. This crude crystals were dissolved in water (4 ml) and to the solution, there was added acetone (8 ml). The mixture was allowed to stand to give crystals, which were separated and dried to give Nocardicin C (15 mg) in the form of white crystals. `
Example 2 (Production of Nocardicin D~
An aqueous medium (lO0 ml) containing 2 % of sucrose, 2 % of cottonseed meal,~ l % of dried yeast, 2.18 % of K~I2PO4 and 1.43 % of Na2HP04 12H2O~was poured into each of ~wenty 500-ml. shaking flasks, and sterilized at 120 C for 20 minutes.

- 2~ -__ __ _ _ __ _ ,____ _~, _ ,, _ __, . __ ~__ _ _ _ . ,.. _.. ~.. _.~. . _ .. _ ~_ ~.. ~_ . ~.. _.. A~ .. ~_~.

A loopful of slant culture of Nocardia uni~ormis subsp.
tsuyamanensis ATCC 21806 was inoculated into each of the media, and cultured at 30 C for 48 hours.

On the other hand, an aqueous medium (100 liters) containing 2 % of soluble starch, l % of peptone, 0.4 % of yeast extracts, 1 % of K~PO4, 1 % of Na2HP04 12H20, 0.5 % of MgSO4-7H20, 0.1 % of L-tyrosine and 0.1 ~ of glycine was adjusted to pH 6.0 with 6N aqueous sodium hydroxide solution, and 20 liters of the medium were poured into each of five 30-liter jar fermenters and sterilized at 120 C for 20 minutes.

To each of the media, there was added the seed cul~ure, as prepared above, in an amount of 2 % by volume of the medi~. The organism was grown at 30 C for 96 hours.
During the growth period, the broth was stirred at 250 r.p.m.
and sterile air was passed into the broth in the amount of 20 liters per minute.
To the resultant cultured broth (80 liters), there was added diatomaceous earth ~8 Kg~, and then the mixture was filtered to give filtrate and filter cake. The filtrate (70 liters) was concentrated under reduced pressure to a volume of 7 liters. To the ~oncentrate, there was added methanol (90 liters) with stirringj and then the mixture was allowed to stand for an hour and fiItered to remove precipi-tates. The filtrate (80 iiters) was concentrated t5 a volume . .
of 5 liters. To the concentrate~ there was added water ~15 liters) and actlvated charcoaI ~1 Kg). ~ ;

.
.

~ 2 5 ~

~9~'~9 The mixture was stirred for 10 minutes and filtered to collect the activated charcoal. To the activated charcoal, there was added 80 ~ aqueous methanol (10 liters), and then the mixture was adjusted to pH 8.0 with 5 % aqueous ammonia, stirred for 10 minutes and then filtered. This elution was repeated once more. The combined filtrates were concentrated under reduced pressure to a volume of 2 liters. The concentrate was ad-justed to pH 4~0 with 6N hydrochloric acid and passed through a column packed with Diaion HP 20 (4 liters~. This column was washed with water (4 liters), whereafter the object compound was eluted with 50 ~ aqueous ethanol. The eluate (1.5 liters) containing the object compound were collected and concentrated under reduced pressure to a volume of 100 ml. The concentrate was subjected to a column chromatography on cellulose (developing soluvent : n-butanol saturated with water).
The eluate (200 ml) containing the object compound was collected and concentrated under reduced pressure to a volume o~ 10 ml. The concentrate was further subjected to a column ~ -chromatography on cellulose [developing solvent : upper layer of a mixture of ethylacetate : n-butanol : water (5:5:2)~. The eluate (100 ml) containing the object compound was concentrated under reduced pressure to a volume of 10 ml to give precipitates, which were separated by filtration and then dried. The precipitates were suspended in water (5 ml), whereafter the suspension was adjusted to pH 8.0 with lN
aqueous sodium hydroxide solution to dissolve the precipitates.
The solution was adjusted to pH 3.0 with lN hydrochloric acid and allowed to stand overnight in a refrigerator (5 C) to g~ve crystals, which were separated by filtration, and then dried to give Nocardicin D (50 mg) ln the form o~ white crystals.

- 26 ~
. . ~
, .

1C1 6~

The filter cake containing the mycerlia, obtained above, was extracted twice with 70 ~ aqueous ethanol (20 liters).
The combined extracts were concentrated under reduced pressure ot a volume of 2 liters. The concentrate was 5 adjusted to pH 3.0 with 6N hydrochloric acid and extracted three times with ethyl acetate (2 liters) to remove impurities.
The aqueous layer was separated and concentrated under reduced pressure. The concentrate was adjusted to pH 4.0 with 6N aqueous sodium hydroxide solution and passed through a column packed with Diaion HP20 (2 liters). ~he column was washed with water, whereafter ~he object compound was eluted with 50 ~ ethanol ~1.5 liters). The eluate 51.5 liters) was concentrated under reduced pressure to a volume of 100 ml.
The residue obtained was subjected to a column chromatography on cellulose (developing solvent : n-butanol saturated with water). The eluate containing the object compound was collected and concentrated under reduced pressure. The residue obtained was subjected to a column chromatography on cellulose -[developing solvent : upper layer of mixture of ethyl acetate:
n-butano1 : water (5:5:2)]. The eluate containing ~he object compound was concentrated under reduced pressure to a volume of 10 ml to give precipitates, which were separated by filtration and then dried. The precipitates were suspended in water (2 ml) and adjusted to pH 8.0 with lN aqueous sodium hydroxide solution to dissolve the precipitates. Subsequently, the solution was adjusted to pH 3O0 with lN hydrochloric acid and allowed to stand overnight in a refrigerator (5 C) to give crystals, which were separated by ~iltratisn and dried --to give Nocardicin D (10 mg~ in the form of white crystals~
3~

' ;-; ' .

- - 27 - ~

~94~

Exa~ple 3 (Production ol Nocardicin E) An aqueous medium (300 ml) containing 2 ~ of sucrose, 2 % of cottonseed meal, 1 % of dried yeast, 2~18 ~ of KH2PO4 and 1.43 % of Na2HPO4 12H2O was poured into one liter Erlenmeyer flask and sterili~ed at 120 C for 20 minutes.
A loopful of slan$ culture of Nocardia uniformis subsp.
tsuyamanensis ATCC 21806 was inoculated into the medium and cultured at 30 C or 54 hours.

Into a 500-liter fermenter! there was placed the same medium (150 liters) as ment oned above. The fermentation medium was sterilized at 120 C for 20 minutes and then inoculated with the whole volume of the vegetative inoculum -prepared above, and cultured at 30 C for 42 hours.
On the other hand, an aqueous medium (3000 liters) containing 2 % of soluble starch, 1 % of peptone, 0.4 % of yeast extracts, 1 % of KH2PO4, 1 % of Na~HPO~ 12H2O, o.5 % of MgSO~ 7H2O, o~l ~ of L-tyrosine and 0.1 % of glycine was pured into 4000-liter fermenter and sterilized at 120 C for 20 minutes. The whole volume of the cultured broth, as prepared above, was inoculated into the medium.

The organism was grown in the fermentation medium at 30 C, for 119 hours. During the growth period, the broth was stirred at 230 r.p.m. and stexile air was passed through the broth in a ratio of 3000 liters per minute. After the culture was completed, the cultured broth was filtered with the aid of diatomaceus earth (180 Kg). To a part of the filtrate ~1500 liters), there was added and activated - - 28 - ~

.
- ~ ,' ' . ~ ' ' ' ' . .

~9~9 charcoal (45 Kg), whereafter the mixture was stirred for 30 minutes and filtered to separate the activated charcoal.
To the activated charcoal was added a mixture of acetone and water (7:3) (200 liters), whereafter the resultant mixture was adjusted to pH 8.0 with an aqueous ammonia and stirred for 60 minutes. The eluate (500 liters) thus obtained was concentrated to a volume of 50 liters. The concentrate was adjusted to pH 4.0 with 6N hydrochloric acid. Subsequently, to the concentrate, there was added n-butanol (60 liters), whereafter the mixture was ~tirred ~or 30 minutes. The n-butanol layer (50 liters) was separated and concentrated under reduced pressure to a volume of 10 liters.
The concentrate was adjusted to pH 8.0 with 5 ~ aqueous ammonia and stirred for 30 minutes. The aqueous layer tl5 liters) was separated and concentrated under reduced pressure to a volume of 1.3 litersO The concentrate was adjusted to pH 4.0 with 6N hydrochloric acid, mixed with diatomaceous earth (1.3 Kg) and then dried. The powder was washed with chloroform (3 liters) and the object compound was eluted with ~ ;
ethyl acetate. The eluate (5 liters) was concentrated under reduced pressure to a volume of 50 ml. The concentrate was mixed with diatomaceous earth ~50 g) and dried. The powder was subjected to a column chromatography on silicic acid ;
(developing solvent : ethyl acetate). The eluate ~600 ml) was concentrated under reduced~pressure to a volume of 10 ml.
The concentrate was mixed with diatomaceous earth (lO~g) and dried. The powder was subjected to a column ehromatography on silicic acid [developing solvent : a mixture o chloroform : methanol (100:7)]. The eluate ~200 ml) was :
~ 30 eoncentrated under reduced pressure to a volume of 3 ml. ~
.
,. :.:;: .:

- . - . . .. - . . . . . ~ -Subsequently, the concentrate was mixed with diatomaceous earth (3 g) and dried. The powder was subjected to a column-chromatography on silicic acid [developing solvent : a mixture of chloroform : ethyl acetate (1:4)]. The eluate (50 ml3 was evaporated to dryness under reduced pressure. The residue thus abtained was dissolved in methanol ~1 ml). To the solution, there was added chloroform (5 ml) and allowed to stand overnight at 4 C to give crystals ~230 mg), which were recrystallized from a mixture of me~hanol and chloroform ~1:5) (12 ml) to give Nocardicin E (190 mg) in the form of .. .
white crystals.

Example 4 (Production of Nocardicin F) An aqueous medium (100 ml) containing 2 ~6 of sucrose, 2 ~ of cottonseed meal, 1 % of dried yeast, 2.18 ~ of KH2PO4, 1,43 % of Na2HPO4 12H2O was poured into each of twenty four 500-ml. Sakaguchi 1asks and sterilized at 120 C for 20 minutes. A loopful of slant cultuxe of Nocardia uniformis ~ub~p. tsuyamanensis ATCC 21806 and the organism was cultured at 30 C for 48 hours.

On the othPr hand, an aqueous medium (20 liters) containing 2 % of soluble starch, 1 % of peptonel 0.4 ~ of yeast 2PO~I 1 % of Na2HPO4 12H2O, 0.5 ~ of MgSO
7H2O, 0.1 ~ of L-tyros~ine and 0~1 % of glycine was poured into each of six 30-liter ~ar fermen~er and sterilized;at 120 C
for 20 minutes. Subsequently, the cultured broth, as obtained - above, ~as inoculated into each of the media in a ratio of 2 by volume of the medium. The organism was grown in the medium at 30 C for 96 hours.

- ' ' ..

', 4g~

During the growth period, the broth was stirred at 250 r.p.m.
and sterile air was passed through the broth in a ratio of 20 liters per minutes. After the culture was completed, the cultured broth (lO0 liters) was filtered with the aid of diatomaceous earth (10 Kg). To the filtrate (90 liters), there was added an activated charcoal (1.5 Kg), whereafter the mixture was stirred for lO minutes and then filtered.
The activated charcoal was washed twice with water (10 liters) and the object compound was eluted twice with 80 % aqueous methanol (30 liters and 20 liters). The eluate (50 liters~
was concentrated under reduced pressure tb a volume of 6 liters. The concentrate was adjusted to pH 4.0 with 6N
hydrochloric acid and passed through a culumn packed with Diaion HP20 (3 liters). After the column was washed with water (l liter), the object compound was eluted with 40 %
ethanol (8 liters). The fractions (4 liters) containing the object compound was collected and concentrated under reduced pressure to a colume of lO0 ml. The concentrate was mixed with powdery cellulose (300 g), whereafter the mixture was dried under reduced pressure. The dried powder was packed into a column and elution was carried out with acetone (6 liters) from the column. The eluate was concentrated under reduced pressure to a volume of 50 ml. The concentrate was mixed with diatomaceus earth (50 g) and the mixture was dried.
The dried mixture was subjected to a colu~n packed with silicic acid ~developing solvent : a mixture of chloroform and methanol (lO~ . The fractions containing the object compound were collected and evaporated to dryness under reduced pressure.
The residue thus obtained was dissolved in methanol ~20 ml).
;~ 30 To the solution, there was add water (40 ml)~ whereafter the .~
~'.'.' ' E-ll the mixture was allowed to stand overnight at 4 C to give precipitatès ~560 mg), which were separated by filtration and dissolved in methanol (10 ml). To the solution, there ~-was added water ~20 ml), whereafter the mixture was allowed to stand overnight at 4 C to give crystals, ~hich were separated and dried to give Nocardicin F (300 mg) in the form of white crystals.

Example 5 ~Production of Nocardicin G) An aqueous medium (100 ml) containing 2 ~ of sucrose~
2 % of cottonseed meal, 1 ~ of dried yeast, 2.18 ~ of K~2PO4 and 1.43 % of Na2HPO4 12H2O was poured into each of eight 500-ml shaking flasks, and sterilized at 120 C for 20 minutes.
A loopful of slant culture of Nocardia uniformis subsp.
tsuyamanensis ATCC 21806 was inoculated into each of the media, and the organism was cultured at 30 C for 48 hours~

On the other hand, another aqueous medium (40 liters) containing 2 % of soluble starch, 2 % of cottonseed meal, 2 ~
of dried yeast, 2.18 % o XH2PO4, 1-43 ~ of Na2~PO4 12H2O ;~-0.5 % of MgSO4 7H2O, 0.1 % of L-tyrosine and 0.1 % of glycine was adjusted to pH 6.0 with 6N aqueous sodium hydroxide solution, whereafter 20 liters of the medium was poured into two 30-liter jar fermenter and sterilized at 120 C for 20 minutes. To each of the media, there was added the seed culture, prepared above, in an amount of 2 % by volume of the medium.
The organism was grown in the medium at 30 ~C for 96 hours.
During the growth period, the broth was stirred at 300 r.p.m.
and sterile air was passed through the broth at a rate of 20 liters per minute.

:' ,.

.

-hO69~9 Subsequently, the cultured broth (35 liters) was filtered with a aid of diatomaceous e~rth (3.5 Kg). The filtrate (30 liters) was concentrated under reduced pressure to a volume of 3 liters. To the concentrate, there was added with stirring methanol (15 liters), whereafter the mixture was allowed to stand for an hour to give precipitates, which were removed by filtration. The filtrate (17 liters) was concentrated under reduced pressure to a volume of one liter.
~All operations as mentioned hereinafter, were carried out 1~ in low-temperature (4 ~C) room.]
The concentrate was adjusted to p~ 2.0 with 6N hydrochloric acid. To the concentrate, there was added ethyl acetate -(2 liters), whereafter the mixture was stirred for ten minutes and the aqueous layer was separated. To the aqueous layer, there was added n-butanol (1.5 liters) and sodium chloride (250 g), whereafter the mixture was stirred for 10 minutes and the n-butanol layer was separated. This extraction was repeated once more. To the combined n-butanol layer (3 liters), there was added n-hexane (3 liters), whereafter the mixture was stirred for 10 minutes and the aqueous layer (400 ml) was separated. The aqueous layer was adjusted to pH 7.0 with lN aquPous sodium hydroxide solution and concentrated under -~
reduced pressure to a volume of 3Q0 ml. To the concentrate, there was added water (5 liters) and sodium chloride (50 g), whereafter the mixture was adjusted to pH 4.0 with lN hydrochloric acid. The mixture was passed throuyh a column packed with Diaion HP20 (2 liters). Subsequently, the object compound was eluted with water (20 liters) from the .
column. The eluate containing the object compound was collected.
To the eluate, there was added Na2HPO4 12H2O (60 g)~ whereafter ,'' ;.',:

...

: 33 . ,, , ~ . . ~ . , . ~

the mixture was adjusted to pH 9.0 with lN aquenous sodium hydroxide solution and passed through a column packed with Diaion HP20 (700 ml). The column was washed with 1 % aqueous secondary sodium phosphate solution (1 liter). Subsequently, the object compound was eluted with water (3 liters). The eluate t2 liters) containing the object compound was collected, adjusted to pH 8.0 with lN hydrochloric acid and then passed through a column packed with activated charcoal (300 g).

The column was washed with water (1 liter). Subsequently, the object compound was eluted with 80 % aqueous methanol (2 liters). The eluate was concentrated under reduced pressure to a volume of 50 ml. The concentrate was lyophili~ed mixed with a small amount of cellulose and then subjected to a column chromatography on cellulose. The column was developed with n-butanol saturated with water. The eluate (400 ml) containing the object compound was collected. To the eluate, there was added n-hexane (400 ml), whereafter the mixture was stirred for 10 minutes and the aqueo~s layer (60 ml) was separated. The aqueous layer was adjusted to pH 5.0 with lN
aqueous sodium hydroxide solution and concentrated under reduced pressure to a volume of 30 ml. The concentrate was allowed to stan~ overnight to give crystals, which were separated, washed with a small volume of water, dried and then recrystallized to ~ive Nocardicin G t150 mg) in the form ~5 of white crystals.

~' '~

, ~, ~ ~ , , . , : . . .

Claims (29)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for the production of a Nocardicin selected from the group consisting of Nocardicin C, Nocardicin D, Nocardicin E, Nocardicin F and Nocardicin G in which Nocardicin C has the following chemical structure:

Nocardicin D has the following chemical structure:

Nocardicin E has the following chemical structure:

Nocardicin F has the following chemical structure:

Nocardicin G has the following chemical structure:

which comprises cultivating a strain of Nocardia uniformis effective to product at least one Nocardicin of said group, in an aqueous nutrient medium under aerobic conditions and recovering the at least one Nocardicin,

2. A process according to claim 1, in which the strain is a strain of Nocardia uniformis subsp. tsuyamanensis.

3. A process according to claim 2, in which the strain is Nocardia uniformis subsp. tsuyamanensis ATCC 21806 or a mutant thereof.

4. A process according to claim 1, in which a mixture of Nocardicin C, D, E, F and G is produced, including a step of separating the mixture and recovering the individual Nocardicins.

5. A process according to claim 2, in which a mixture of Nocardicin C, D, E, F and G is produced, including a step of separating the mixture and recovering the individual Nocardicins.

6. A process according to claim 3, in which a mixture of Nocardicin C, D, E, F and G is produced, including a step of separating the mixture and recovering the individual Nocardicins.

7. A process according to claim 1, in which Nocardicin C is recovered from the cultured broth.

8. A process according to claim 2, in which Nocardicin C is recovered from the cultured broth.

9. A process according to claim 3, in which Nocardicin C is recovered from the cultured broth.

10. A process according to claim 1, in which Nocardicin D is recovered from the cultured broth.

11. A process according to claim 2, in which Nocardicin D is recovered from the cultured broth.

12. A process according to claim 3, in which Nocardicin D is recovered from the cultured broth.

13. A process according to claim 1, in which Nocardicin E is recovered from the cultured broth.

14. A process according to claim 2, in which Nocardicin E is recovered from the cultured broth.

15. A process according to claim 3, in which Nocardicin E is recovered from the cultured broth.

16. A process according to claim 1, in which Nocardicin F is recovered from the cultured broth.

17. A process according to claim 2, in which Nocardicin F is recovered from the cultured broth.

18. A process according to claim 3, in which Nocardicin F is recovered from the cultured broth.

19. A process according to claim 1, in which Nocardicin G is recovered from the cultured broth.

20. A process according to claim 2, in which Nocardicin G is recovered from the cultured broth.

21. A process according to claim 3, in which Nocardicin G is recovered from the cultured broth.

22. A Nocardicin selected from the group consisting of Nocardicin C, Nocardicin D, Nocardicin E, Nocardicin F and Nocardicin G, as defined in claim 1, whenever prepared by the process of claim 1, 2 or 3, or by an obvious equivalent.

23. A Nocardicin selected from the group consisting of Nocardicin C, Nocardicin D, Nocardicin E, Nocardicin F and Nocardicin G, as defined in claim 1, whenever prepared by the process of claim 4, or by an obvious equivalent.

24. A Nocardicin selected from the group consisting of Nocardicin C, Nocardicin D, Nocardicin E, Nocardicin F and Nocardicin G, as defined in claim 1, whenever prepared by the process of claim 5 or 6, or by an obvious equivalent.

25. Nocardicin C, whenever prepared by the process of claim 7, 8 or 9, or by an obvious equivalent.

26. Nocardicin D, whenever prepared by the process of claim 10, 11 or 12, or by an obvious equivalent.

27. Nocardicin E, whenever prepared by the process of claim 13, 14 or 15, or by an obvious equivalent.

28. Nocardicin F, whenever prepared by the process of claim 16, 17 or 18, or by an obvious equivalent.

29. Nocardicin G, whenever prepared by the process of claim 19, 20 or 21, or by an obvious equivalent.