CA1068225A - Naphtacene derivatives, their preparation and compounds thereof - Google Patents

Abstract

The subject of the present invention is the preparation of a new naphthacene derivative of formula (I) (I), its addition salts with pharmaceutically acceptable acids and the compositions which contain it. This new derivative designated by the number 32 999 RP is a basic compound of dark red color particularly useful for the treatment of tumors.

Description

i ~ 6 ~ zzs Ia pr ~ en ~ e invention relates to a new derivative of napht ~ c ~ ne of formulQ:

O OH
ll I CHOH-CH3 ~ 1 ~ O. I
HO O OH O ~ CH - CH2 - ICH _ ICH _ CH _ CH3 ~ H2 OH

~ es salts, its preparation and the ~ compositions containing it.
~ e product of formula (I), which will be designated by the - number 32 999 RP ~ has a very special interest by ~ uite of ~ on anti-tumor activity.
~ e 32 999 RP e ~ t a ba ~ ic compound of red color dark forming of ~ addition salts with the ~ pharmaceutical acids ~
mentally acceptable ~, - ~ e hydrochloride of 32 999 RP is soluble ~ reason about 100 mg / cm ~ dan ~ m ~ thanol, pyridine, dimethyl-formamide and esu ~ en ~ iron 10 mg / cm3 dan ~ n.butanol ~ aturé
of water ~ 20C ~ in ~ iron 5 mg / cm3 in ~ thanol, and practically insoluble (less than Ool mg / cm3) dsn ~ i'ac ~ tone ~ chloro ~ ormet methylene chloride ~ acetate ~ methyl ~ benzane ~ la m ~ thyli ~ obutylketone ~ dio ~ ne and hexane.
The 32 999 RP hydrochloride contains carbon hydrog ~ ne, oxygen ~ ne, nitrogen and chlorine. Its composition elementary e ~ t close to:
C% = 56 ~ 6 H% ~ 5.7 0% = 29.41 N% = 2.39 Cl% - 6 ~ 0 It is also c ~ rsotéri ~ é by the properties ~ s phy3ico-chemi ~ ues ~ ui ~ ntes:
- as ~ e ~ t: microori powder ~ talline ~ orange-red ~ point ae ~ usion: ~ ers 200C (with decomposition) . ~

1068; ~ Z5 - ~ ouvo`i ~: rotary: (determin ~ en ~ olution ~ 0,09 ~ in ~ thanol ~ 0.1% HCl 1 N) r ~] - + 208 + 32 ~ 8 ~ 9 ~ determination ~ termination ~ from an olution ~ 10 ~ 6 ~ mg / l dan ~ thanol ~ OJ1 ~ de HCl 1 N

_ .. __ I
Maximum absorption ~ tlon ~ 1 cm 233 nm 620 : `
. 254 nm 495 ._ ._ 292 nm 140 This specter is represented by FIG. 1 attached, - s ~ ctre ~ is ~ be: determination from a ~ solution ~
10 ~ 65 mg / l in ethanol with 0.1 ~ of 1N HCl.

.: - ---: ~ Maximum absorption at: 1 cm . I. I
: - 493 nm 272 ,. . I
512 nm 189 _. .__ 527 nm 190 .
Ce ~ pectre e ~ t ~ represented ~ by Figure 2 appended ~ e.
- ~: (determination ~ from tablets in . 20 mix with KBr) ; This ~ pectre e ~ t represented ~ ented by the ~ igure 3 attached ~ ur which we wore ~ absc ~ se ~, on the one hand the wavelengths ~
; - expressed in microns ~ ~ scale ~ up) and on the other hand : number of waves in cm 1 (lower scale) and ordinates on ~
den ~ ité ~ optical ~.
Dan ~ Table I below indicates the main 9 ~ 0 68 2 ZS

absorption bands in ~ ra-red for this product expressed in ~
wave number (cm 1):
3410 F 2680 th. 1580 th. 1955 F 915 th. 725 m 53Q f 3250 ~ p. 2600 th. 1510 th. 1165 ~ 885 m 708 m 485 m 3220 th. 1980 tf 1460 F1115 ~ 875 m 695 ~ p. 475 3050 ~ p. 1930 ep, 1440 F1062 F 845 ep. 665 th. 465 2970 ~ 1900 tf 1405 ~ 1050 ~ p ~ 840 ep, 640 ~ p. 450 , 2930 F 1800 tf 1370 th. 1030 th. 820 ~ 625 ~ p. 438 m 2900 ~ p. 1735 ~ P. 1315 ~ p.1010 F 810 th, 600 m 415 tf - lO 2860 th. 1690 th ~ 1285 F985 ~ 780 m 580 f 390 m 2820 th. 1635 th. 1255 m960 ~ p ~ 762 m 565 f 320 1600 tF 1235 ~ 930 m 750 m 540 f , ~ t ~ - ~ very strong f ~ weak F = strong tf = very very ~ weak m _ average ~ p. = shoulder ';
~ e 32.999 RP can be characterized by chromatography - ~ ur thin layer of ~ ilice gel using ~ ant as ~ ol ~ ant development of the methyl chloride ~ ne - formic acid mixture methanol (80-17-3 by volume) at a temperature of 24C. In this, sy ~ tame the hydrochloride of 32 999 RP has an R ~ pathin of 0.2.
~ 'hyaroly ~ e acid of 32.999 RP or one of se3 eel ~
allows to obtain the product of formula:

- OH (II) HO O OH OH
which has the lycone of 32.999 RPo Aglycone ~ u 32.999 RP e ~ t ~ oluble ~ rai ~ on de 20 ~ ,, lQ68ZZ5 mg / cm3 in pyridine ~ 5 mg / cm3 in methsnol ~ dioxane ~
chloroform ~ and chloride of m ~ thylane and ~ less than 0 ~ 1 mg / cm3 in acid, ethyl acetate, - hexane and water.
This product also has the ~ propri ~ t ~ phy ~ ico-chemical ~ sui ~ a ~ te ~:
powder ~ ~ orange-red microcrystalline ~
- Point de fu''sion: ~ er ~ 185C (with d ~ compo ~ ition) - anal ~ e'é ~ elementary: it is ~ close to:
C% = 61.5 ~ H% = ~, 31 0 ~ - 30.16 -, ou'vo ~ r rotary: (d ~ finished in solution ~ 0,1% dan ~ le :; dioxane) ~ 3 20 = ~ 249 ~ 32 , s ~ ec ~ '~ e' ult ~ a'- ~ olet: d ~ termination from solutions to - 50 and 5 ~ g / 1 dan ~ l ~ ethanol ~ 0.1 ~
., HCl 1N
. .

. -,, Maximum absorption at: E1 ~ o .,.
234 nm 770 . . . _ ~. ~.
. ~ 254 nm 640 293 nm 1_0 __ _ This spectrum is ~ represented ~ ent ~ by the flgure 4 annex ~ e ~ ur which the ~ curve ~ I and II correspond ~ respectively lay ~ de3 solutions ~ 50 and 5 mg / l ~
- s ~ e'dt ~ if ~ the: determinate ~ from ~ a 10 mg / l solution in ethanol at 0 ~ 1 ~ o of 1N HCl :

1 ~ 682Z5 M ~ imum of ab ~ orption ~ 1 - -.: ~ _. _ _ _ ~ `493 nm _370 _ 5 ~ 5 ~ _ 255 `528 nm 270 _ This spectrum is represented by the appended FIG. 5, ;; - infra-red s ~ pectre: (det ~ rmi ~ ation ~ from compiim ~ en mix with K ~ r) ~ Q spectrum is represented ~ ente by the ~ igure 6 ~ ur which - ~ we brought in ab ~ ci ~ ses ~ on the one hand the wavelength ~
expressed in micron ~ (~ upper ~ cheile) and, on the other hand, the ~
wave numbers e ~ awarded in cm ~ 1 (lower scale) and ordonn ~ e ~ le ~ den ~ it ~ optical ~.
Dan ~ table II below ~ we indicate the main ones ~
ab bands ~ infrared orption for this product expressed ~ in number of waves 9 (cm ~ 1):

3500 ~ p, 2680 th, 1412 ~ 1095 f 910 m 740 m 530 tr 3420 F 1980 tf 1370 ~ 1070 th. 885 m 710 m 505 th.
3360 ep. 1705 f 1315 th, ~ 1062 P 875 m 700 th, 485 m 3080 th, 1640 th, 1285 t ~ 1050 th, 865 f 680 tf 465 m 2975 m? 600 tf 1255 tF1030 m 840 m 665 tr 450 m 2,925 m 1,580 th. 1240 ~ p, 1020 m 815 ~ 625 th, 435 m 2910 ~ p. 1540 th, 1195 F1005 m 805 th, 595 f 385 tf 2890 th, 1458 P 1165 F975 m 785 th, 585 f 360 tr 2860 ~ p, t448 F 1130 m950 m 775 th. 570 f 320 tf 925 m 765 m 545 tF = very strong ~ = strong m = medium = low tf - tra ~ low epO = ~ payment ~ 32 9g9 RP aglycone can be characterized by ~ ilice gel minoe layer chromatography using comm ~ ~ development solvent the methylene chloride mixture -formic acid - methanol (80-17-3 in ~ olume) at a temperature of 24C. In this sy ~ teme the aglycone has an Rf close to 0.7.
According to the present invention, the 32,999 RP can be obtained according to one of the following processes:
1 by microbiological reduction of the antibiotic called carminomycin which answers the formula:

~ aoo (III) HO O OH O_ CH - CH2 - CH _ CH _ CH _ CH3 ~ 'NH2 O ~ I
~ Carminomycin and 8a prepsrstion by culture - from Actinomsdura carmina ~ a Sp. nov. have been described ~ dan ~ la French patent application 74.00349 which was published under num ~ ro 2 ~ 235,700. ~ the structure of carminomycin has been published by MG, ~ razhnikova et al, J. of Antibiotics, XXVII, no 4, 254-259 (1974).
~ ar ~ microbiological production of osrminomycin g ~ n ~ rslement in an aqueous medium by means of culture of microorganisms that have reached a suitable stage of development ment, 80it of cells isolated from these cultures, ie extracts obtained from these microorganisms ~.
~ microorganisms which can be used depending on the proc ~ die of the present invention little ~ ent belong to the category of streptomycete ~ or bacteria. Among the micro-organi ~ my which are particularly suitable can 8trecités Streptomyces lavendulae (A ~ CC 8664), Streptomyce ~ roseochro-. .

'' '-~ 1068ZZ5 mogenes (A ~ C ~ 13.400), Coryn ~ bacterium simplex (A ~ CC 6946) or Bacterium cyclooxydang (ATCC 12.673). ~ are better ~ re ~ ultat ~
are obtained ~ ~ from ~ orynebacterium ~ implex 1ATCC 6946), Io ulture of the microorganism producing the system enzyme capable of reducing carminomycin to 32,999 RP
can be done by any aerobic culture method in surface or in depth but the latter e ~ t ~ prefer for convenience ~. We use for this purpose seeding and fermentation techniques and the ~ different ~
type ~ of devices which ~ are in common use in industry fermentations.
the culture medium of the microorgani ~ me must contain e ~ substantially ~ carbon and assimilable nitrogen forces ~, mineral elements and growth factors ~ all ~ all oe ~
elements which can be supplied in the form of well-formed products - die ~ ini3 or by ~ me ~ complex angels ~ such ~ qulon in encounter in a biological product of various origins, As source ~ of carbon ~ s ~ imilable one can use ~ carbQne hydrates such as glucose or other ~ sub ~ tances - 20 csrbonées as ~ sugar ~ alcohol ~ or as certain ~ acids organic. Certain ~ animal ~ or vegetable oils ~ such as oil bacon or soybean oil can advantageously replace these sources o ~ rbon ~ e ~ or their ~ be anointed ~, l ~ es Mource ~ of nitrogen a ~ like ~ have extremely varied. It ~ can be very simple chemicals ;; like salt ~ min ~ raux or gold ~ ani ~ ues of ammonium, urea, certain ~
amino acids. They can also be brought by sub-~ complex tances ~ mainly containing nitrogen under ~ elm protein: casein, lactalbumin, gluten and their ~ hydrolyte ~ ats, flour ~ o ~, ar ~ chide, fish3, peptone, extracts meat, yeast, di ~ soluble tillersl ~, corn-steep.
Among the ~ minimal elements ~ added 8 ~ 9 ~ some may -have an ef ~ and buffer or neutralizer like the ~ pho ~ phates alkaline or alkaline earth. Others bring balance ionic necessary for the development of the microorgani ~ me like le3 chlorurec and the ~ ~ ulfates of m ~ alkaline or alkaline earth rates ~
~ es factor ~ of growth ~ have products ~ of nature ~ itaminic ~ such as ribo ~ lavine ~ l ~ aoide fol ~ eu, acid pantothenic.
;; ~ e pH of the fermentation medium at the start of the culture must be included between 6.0 and 7.8 and preferably between 6.5 and 7.5. IB optimal temperature for the ¢ ulture of mioroorgan-- ism is 29-31C but a satisfactory development is obtained at aes temperature ~ compri ~ es between 26 and 37C.
. ~ ta ~ ration of the culture medium can ~ arier between : wide enough values. However, it was found that aeration 0 ~ 3 to 3 liters of air per liter of broth per minute : particularly suitable ~
Of more ~, to obtain a culture po ~ s ~ dant a good reductive activity, it is pr ~ fable to stir the medium of oulture for example by means of an agitator whose rapidity of rotation can vary between 100 and 250 revolutions / minute.
Generally the culture of the microorganism reaches a degree of satisfactory development after a period of 24 48 hours.
~ r ductrioe activity essentially depends on the amount of cells formed ~ 8U during culture ~ Ave ¢ de ~
cell ~ i ~ olées ~ for example after centrifugation of the medium culture ~ the amount of 32,999 RP formed ~ from carmine-mycine is directly related to 18 cell concentration reaction medium; riohe ~ cells ~ cell ~ possadent a reducing power ~ higher ~ 8 reduction of carminomycin to 32,999 RP s ~ takes place by contacting an aqueous solution of carminomycin or 1 ~ 682Z5 . .
of one of 3es 3el ~ a ~ ec culture of the microorgani ~ me having ~ reached a degree of de ~ development and a louse ~ oir sufficient reducer ~ ' or with the cells i ~ olée9 of this ~ culture ~ or a ~ ec dege ~ enzymatic traits ~ obtained ~ from this ~ cell 9.
~ e ~ extract ~ in ~ ymatique ~ of ~ cell 9 little ~ ent be obtained after ~ destruction of the ~ cell wall ~ ~ oit by ~
~ chemical or biochemical method ~ ~ or by methods physical. Preferably, the destruction of cell walls is carried out from the culture of the microorganism or cell isolated from this culture in su ~ pension in the middle aqueous either by t them having an enzyme capable of lyzing the cell wall, such as lysozyme, ~ oit e ~ le ~ 90 nPttant action of ultrasound. ' ~ es enzymatic extracts are in solution in the liquid ~ urnant which is obtained after centri-~ ugation of the environment Generally the solution of e ~ traits enzymatic is used ~ a ~ s other subsequent treatment in the reduction phase. ' Generally the reduction takes place in an agitated environment '~ ~ a temperature between 23 and 37C, pr ~ preferably between 26 and 30C and it is flat after 1 ~ 4 ~ contact bear.
Ia reduction can take place ~ a p between 5 and 10 maie it is preferable ~ to operate at a p ~ ¢ ompris between 7 and ~ e mllieu reaction can ~ be acted ~; for example by an agitator whose ~ rotational speed can ~ range between 100 and 250 rpm / minute.
Ia reductlon can be performed ~ e ~ oit ~ ur carmino-mycine or one of 8e9 pure salts, ~ oit on an impure product ~
either on the acid iltrate of a culture having produced carminomycin ~ To get good results ~ nt ~ 9 the concentration carminomycin in the reducing medium is advantageously between 0 ~ 1 and ~ g / l 8U d ~ reduction goal _g_ : `10682ZS

2 per culture a ~ rob ~ e of microorgani ~ my app ~ rte-n ~ nt to the genus ~ treptomycete and choi ~ i among Streptom ~ these coerul-eorubidus DS 8899 (NRR ~ 3046), Streptomyce ~ coeruleorubidus DS 31.723 (NRR ~ 3045), Streptomyce ~ coeruleorubidu ~ DS 7126 (NRR ~ 8098) ~ and Streptomyces bifurcu ~ DS 2 ~ .219 (NRRL 35 ~ 9) dan ~ of ~ nutrient media ~ f ~ con ~ enant awc ~ treptomyces, in pre ~ ence dlad ~ uvant ~ selected ~ from ~ am ~ thopt ~ rine, sulfanil-amide, ~ ulfathiazole, sulfapyridazine ~ 'barbital, '; phenobarbital, butobarbital and penthiobarbital.
~ a quantity of antibiotic 32,999 RP formed 8U court ~
of culture may vary depending on the composition of the ~ - culture medium, the amount of ad ~ u ~ ant added and the time of its addition, as will be shown below ~ s. ' ~ es microorgani ~ mes Streptomyces coeruleorubidus DS 8899 (NRR ~ 3046) and Streptomyce ~ coeruleorubidus DS 31 723 (NRR ~ 3045) ~ have die ~ declt ~ in the patent françsi ~ lj 380, 810 and its various corre9pondante ~ Streptomyces bifurcue DS 23 219 (NRR ~ 3539) is described in ~ le bre ~ and ~ French 1,593,235 and ~ es various correspondents.
Streptomyces coeruleorubidus DS 7126 is a strain new ~ which was ~ isolated ~ from ~ a plot of land.
A sample was deposited at the Northern Regional Research '~ US Department of Agriculture laboratory ~ Peoris ~ Ill, (United States) ~ where it was registered ~ or ~ the reference ~ NRR ~
; ~ 8098, `D ~ sampled of this microorgani ~ me little ~ ent to be obtained ; of this laboratory ~ ur re ~ erence to this document, ~ 'isolation of this strain was e ~ ectu sui ~ ant the general method which requires you to put a small amount of soil suspended in sterile distilled water, ~ di ~ uer the suspension ~ different concentration ~ and ~ tsler a little volume of each dilution on the ~ urface of the box ~ of P ~ sorting containing frozen nutrient medium ~ After an incubation of ~ 068ZZ5 some ~ ~ bear ~ 26a ~ which allows ~ microorganism ~ of ~ e to develop, the colony ~ we want to continue to pursue it the study are taken and transplanted for nutritious gums inclined ~ es in order to obtain more abundant cultures ~.
Streptomyce ~ DS 7126 is related to the species Stre ~ tom ~ these coeruleorubidus, described by ~ Fo GAUZE et al.
. (Problem ~ in the cla ~ eification of Antagoni ~ t ~ c Actinomycete ~ -The American In ~ titute o ~ Biologicai Science ~ Washington ~ 1959, p ~ 100) ~ which it po ~ sows the ~ main characters ~ It presents indeed a myc ~ aerial lium ~ porulated blue ~ blue green ~ be clear on the mineral medium I of Gauze ~ it develops a mycelium ~ aegative which takes on a reddish color and develops a pig-ment ~ soluble of the same color which diffuse in the g ~ lose ~ all ~
these properties being characteristics of the ~ e ~ Stre species ~ tom ~ this ~
coeruleorubidu ~. That’s why we have it ~ m ~ Streptomyce ~
coeruleorubi'dus ~ ~ ouche DS 7126.
Stre ~ tomyce ~ coeruleorubidu ~ DS 7l ~ for ~ e de ~ ~ pores spherical ~ short oval, measuring 0.7 ~ 0.9 ~ / 0.8 to 1 ~
It develops from ~ sporophore 9 simple 8 OR in cluster 8; ~ e 9 channels of ~ pores ~ that are enough ~ long ~ and can include ~ up to several dozen spores9 curl up forming spirals more or less open, usually 2 ~ 5 or 6 towers ~ pirated By ~ on mode of ~ porulation, Streptomyoea coeruleorubidus DS 71 ~ 6 is located in Section S ~ ira of the Pridham classification Streptomyces coeruleorubidu ~ DS 7126 grows well at 25C, slightly worse at ~ 7C, and not ~ 50C, Dan ~ ses cultures, carried out at 26C, it has the ~ character ~ bio-following chemicals:
- melanin production: positive - production of ll2S: po ~ itiYe - tyroaina3e: po3itive ~ 0682Z5 . ~
- the qué ~ action of the Latin g: po ~ iti ~ e, slow - use of cellulo ~ e: positive - production of nitrite ~ ~: n ~ negative on broth from nitrate nutritive nitrat ~, ~ positive on backgrounds ~ ynth ~ tick 9 - hydrolysis of ~ 'starch: positive, mod ~ rée - oulture ~ ur milk ~ cream ~: no coagulation nor of pepton ~ ation In Tables III ~ VI oi-après ~ are r ~ appeared the wild cows ~ cultural than present ~ stre ~ tomA ~ ce ~ coeruleorubi-,, due DS 7126 ~ a number of backgrounds usually useful for determining the morphological characteristics of the strains of streptomyces, cultures ~ ~ ur media g ~ losé ~ ~ both ~ e ~ made ~ on ae ~ gelo ~ e ~ inclined, These caraotareg ~ have eyes of cultures arrived ~ at a good stage of development ~ c ~ e ~ t- ~ -say about 3 weeks ~ 26C except ~ contrary indications.
Ie ~ references of the shuttering media used ~ - ~ have le9 : ~ ui ~ antes:
. ~ - R ~ f, A - "~ ediuL 1 with a mine ~ al source of nitrogen" -G ~. GAUZE et al - Problems in the Claa ~ ification of Antagonistic A ¢ tinomycetes_ p. 13 - The American 20. In ~ titute of Biological Science ~ Washington 6 ~ D ~ C ~
. 1959 _ R ~, B - l ~ he Aotinomycete ~ - S, A WAKSMAN, Chronica ; Botsnica Company ~ Waltham, ~ ass ~ U SA, 1950.-p. 194 - # 10 - Ref C - "Inorganic Salt ~ - Starch Agar" - TG
PRIDHAM et al. - Antibiotic ~ Annual ~ 1956-1957 -_ Re ~ D - The Actinomycetes - SA WAKSMAN ~ Chronica Botanica Company, Wsltham ~ Ma ~, UoS ~ A "1950 -p ~ 193 - n 3 j ~ ~, t 1 ~ 68225 Ref, E _ The Actinomycetes - S, A. V ~ KSMAN ~ Chron ~ ca Botanica ~ ompany, W ~ ltham, Mass., USA, 1950 -P. 193 - n1 D ~. F _ Corre ~ pond ~ la R ~ f, E - o ~ 3 ~ de ~ accharo3e ~ have replaced ~ 3 with 1.5% gluc09 ~
R ~ g G _ ~ he Actinomycete ~ - SA WAES ~ AN, Chronica Botanica Company ~ Walth ~ m, N ~ tss ~ l ~ US A, 1950 -p. 195 - no 18 Ref H _ Corre ~ pond 8 la Ré ~ G - or 3% of ~ acchsro ~ e 30nt re ~ placed by 1.5% gluco ~ e Ref. I _ Corresponds to R ~ f. G - where the ~ accharo3e e ~ t deleted and replaced ~ by small ~ bsndes of pap ~ er submerged filter ~ partly in the liquid Ref J - ~ nual of Method ~ for Pkre ~ ulture Study o ~
~ scteria Society o ~ America ~ ~ acteriologist ~ -Gene ~ a ~ N, Y, II50 ~ 18 D ~. K _ "~ ennettls Agar" - SA ~ AKS ~ AN - ~ he Actinomycete ~, vol. 2, p. 331 - n 30 - ~ he Williams snd Wilkin ~ Compan ~ ~ altimore, 1961 Ref. ~ - "Melanin formation medium" - SA, WAKSMAN -The Actinomycetes, vol. 2, p. 333 - # 42 - The William ~ and Wilkin ~ Company, ~ altimore, '1961 R ~ f. M _ ~ he Actinomycete ~ - SA WAKSMAN ~ Chronica ~ Natica Company ~ Waltham, Ma ~ s. ~ US A., 1950 -p. 197 - # 27 R ~ f, ~ - "Plain Gelatin"'- pr ~ ready ~ uivsnt the indi-cations from the "Msnual of Method ~ for Pure Culture Study of ~ cteria "- Society of American ~ acteriologiats, Geneva, NY ~ II50 -18 R ~ f. 0 - ~ creamed commercial powder, reconstituted ~ according to the manufacturer's 9 indications R ~ f ~ P _ Medium indicated ~ for research of the . ~ H2S duction by: H ~, D. ~ ER and F, DANGA -Joumal of Bacteriology, 76 ~ 239-244, 1958 ..

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- "iO68225 Ie Strç ~ tom ~ these DS 7126, by the blue coloring ~
blue green ~ tre of its mycelium has ~ nothing ~ porulated, belongs ~ the ~ rie ~ Coerule ~ cen ~ described by Gauze et al. Among the ~ strain ~
described in this 8eries ~ it pre ~ ente a myc ~ vegetative lium red dye such as S ~ cioerulerorubidus and S bicolor.
Toutefoi ~, it differs from S bicolor by the ~ a ~ ue, at - contr ~ ire of the latter ~ it uses cellulose ~ it does not clot not milk ~ and above all is likely to form pigment - soluble red diffusing in the gel ~ e on ~ aesthetic medium, property which it only shares with the species S coerulerorubidus.
This last property ~ anointed with the set of ~ e ~ characters - shows that it is connected ~ l ~ e3pèce S _coeruleorubidus.
- The capacity of Stre ~ tom ~ these coeruleorubidu ~ DS 7126 use various sources of carbon or nitrogen to ensure its development has been determined according to the principle of Prldham and Gottlieb method (J of Bact, ~ 6 ~ 107-114 ~ 1948) the degree of development has been observed on the basic medium .
indicated ~ by 9 sutors by replacing ~ oit glucose by ~
; different ¢ arbone souroes respectively tried, ie S04 (NH4) 2 by the ~ various sources of a ~ ote respectively e ~ sayées.
hes result ~ are indicated ~ in table VI
... .
following:

.
'' : 1068ZZ5 TA ~ LEAU VI
Car sources- Use Nitrogen sources Use bone tried tried tried D-Xylose positive NO ~ Na positive ~ -Arabinosis positive 2 positive ~ -Rhamnose positive S04 (NH4) 2 positive D-Glucose positive P04H (NH4) 2 positive D-Galactose positive Urea positive D- ~ ructose positive L-Asparagine po ~ itive 10 M-Mannose po ~ itive Glycocolle positive ~ positive actose Sarcosine negative Maltose positive D ~ -Alanine positive DL-Valine positive sucrose Trehalosis positive ~ - ~ ysine positive D ~ -methionine positive, slow cellobiose Dextrin positive ~ aurine negative : ~ Inulin negative ~ - ~ yrosine positive Positive starch Glycerol positive Erythritol negative Dulcitol negative D-Mannitol positive D-Sorbitol positive :. Inositol positive ; 5alicin positive.

`--19--~ ~ ~ 68 2 Z 5 Ie method of preparation of 32 999 RP consists essentially ~ cultivate stre strains ~ tomycete ~ cit ~ es above on a medium and in suitable conditions presence of a ~ sufficient amount ~ ante adjuvant and ~ ~ sparing the product form ~ during 1 ~ culture.
The culture of microorganisms3 can be carried out by any method of aerobic culture at the surface or at depth ~
May ~ the latter is ~ prefer for ~ rai ~ ons of convenience.
We use ~ this end the different type ~ of devices that are u3a ~ e common in the iron industry ~ entatio ~ s.
We can in particular adopt the walk ~ ui ~ ante for ~ - the conduct of 9 operations:
Producing strains ~ - Stock Culture on agar . l,.
Inoculum culture in shaken flask .,., ..;

Culture inoculum in fermenter ~. J, Production culture in fermenter ~ th fermentation medium must contain a carbon souroe and a nitrogen souroe a ~ slmilable ~ de ~
él ~ mente min ~ raux ~ in particular chloride ~ or ~ carbonate3, ~; and é ~ entionnement factor ~ of growth ~ tou ~ these elements can be brought ~ OU9 product form ~ well d ~ inis or by complex mixtures, such as we find in le3 produita biological origin3 various, As sources of assimilable carbon ~ one can use carbohydrates such as glucose, lactose, 9 dextrins, starch or other carbon substances ~ e3 comm ~
~ alcohol ucres ~ (glyc ~ rol) or as certain ~ organic acid3:
lactic, citric acids, Certain animal or vegetable oils lQ682ZS
t ~ comn ~ bacon oil or so oil a can replace advantageous ~ nt these different sources ~ carbonaceous ~ or their be attached ~.
Ie9 90urce9 suitable ~ nitrogen a ~ imilable ~ have extr ~ mem ~ nt varied ~. It ~ can be chemicals very simple like ~ el ~ minerals or organi ~ ues of ammonium ~
- urea, certain amino acids. They can also be brought by dee substance ~ complexes containing mainly - nitrogen sou ~ protein form: casein ~ lactalbumin ~ gluten and their hydrolysates ~ soy flour ~ peanut, poi ~ on ~
extracts of meat, yeast, soluble distillerst, corn-steep.
Among the ~ element ~ min ~ rauæ added, some ~ may have a buffering or neutralizing effect like pho ~ phate ~
slcalins or aloalino-earth or calcium carbonate and magnesium. Others provide the ionic balance necessary for development of microorganisms and the development of 32,999 ~ P like chlorides and sulfates of alkali and alkali metals-earthy Finally some act more specifically as an activa-of the ~ m ~ tabolic reactions ~ of 3 microorganisms, these ~ have ~ els of zinc, cobalt, iron, copper, mangane ~ e, ~ es ~ actor8 de croi ~ ance 30nt g ~ generally d ~ s pro natural products such as thiamine, 18 ribo, milk, the pantothic scene, ~ es ad ~ uvants can ~ be ~ out ~ s during ~
different stages of culture: inoculum culture in vial acts ~ e) production oulture in fermenter.
However, the best results are obtained when C8 ~ ad ~ uvants are added ~ s either at the start of the pro-duction ~ oit au cour ~ of this one, Among the adjuvants ~ which are particularly suitable well can be cited ~ s sulfanilamide, ~ ulfathia ~ ole and sulfapyrid ~ zine :
-.

~ 68225 I, a concentration of the adjuvant ~ sn ~ the medium of fermentation can vary in ~ wide ~ limits according to 18 composition of the culture medium and depending on the nature of the adjuvant. ' Generally, concentrations between C, 01 and 1 g per ~ - liter of broth are particularly suitable.
Ie of the fermentation medium at the start of the culture must be between 5.6 and 7.4 and preferably between 5 and 8 and 7.2, ~ at optimum temperature for ~ ermentation is included between 25 and 30C, but satisfactory production is obtained for temperatures between 23 and 3 ~ C ~ 'ventilation the fermentation can vary between from ~ va ~ eur ~ to ~ sez large. We found, however, that ~ ~ 3 ~ 3 liter ~ air vents by liter of broth and per minute particularly suitable good. Ie maximum yield in 32,999 RP is obtained after ~ 2 to 10 days of culture, this time depending mainly on the environment useful From the above, it can be seen that the conditions general ~ culture of: microorganisms in the presence of adju-can vary to a large extent and be adapted every special need

3 ~ from the culture media of a new microorganism ~ identified more ~ completely oi-after ~ belonging ~ nt to the genus Streptomyce ~ and designated ~ designated by the name Streptomyces atro ~ iolaceu ~ DS 8938. A ~ sample of this strain was depo ~ é ~ the United States Department of Agriculture ~ Peoria ~
Illinois, ~ or ~ NRR number ~ 8148 Dee sample ~ of this mioroorganism little ~ ent be obtained ~ from this laboratory ~ ur reference to the pre ~ ent document.
This organism, which was isolated from a sample of earth ~ has characteristics ~ which did not allow identify it with a space already described. It must therefore be considered a new species Isolation was done using the method general which is to put a small amount of soil in suspension in sterile distilled water, to dilute the above board at different concentrations, and spread a little vo-lume of each dilution on the surface of petri dishes holding an agar nutrient medium. After an incubation of a few ~ bears at 26C, which allows microorganisms to develop, the colonies that we want to isolate to continue the study are taken and subcultured on nutrient agars in order to obtain more abundant crops.
streptomyces atroviolaceus DS 8938 forms spores oval, measuring 0.4 to 0.6 ~ / 0.6 to 0.9 ~. It develops unbranched sporophores that arise on the mycelium air. Its spore chains are long, usually counting several dozen spores, and roll up describing several generally tight spiral towers. By its mode of sporulation, this strain is classified in the Spira Section of the Pridham classification.
Streptomyces atroviolaceus DS 8938 grows well at 25C and 30 C, less well at 37 C and not at 50 C. It forms the -.. ...
more often on its culture media a vegetative mycelium first yellow brown to red brown which quickly takes on a hue dark ranging from very dark brown to purple brown or blackish brown.
Sporulation only develops on a number of media limited, where it appears difficult and late, making then take a very light gray coloring in the aerial mycelium;
it should also be noted that on a certain number of media aerial mycelium may turn pale pink or pale purplish when dark purple soluble pigment is pro-is abundantly cultivated.
Streptomyces atroviolaceuS DS 8938 does not produce 10 ~ 8ZZS

pigment ~ black melanol soluble ~ ur gel ~ e special tyro ~ ine-Waksman yeast extract (Melanin formation medium); any-times it is likely to develop on many environments, au99i bien ~ ynthétiques gu'organique ~, de ~ pigment ~ soluble a ~ darkened ~ g ~ lose by coloring it in purplish brown, purple black, blackish brown or black.
In his oultures ef ~ ectue ~ ~ 26C ~ he pre ~ ente character ~ biochemical ~ ~ next ~:
- melsnine production: negative _ H2S production: negative - tyro ~ inase: n ~ gative - gelatin liquefaction: positive - use of cellulose: negative - production of nitrites from: weakly posited on of ~ nitrates synthetic media at early cultures - ~ starch hydrolysis: positiYe - oulture ~ ur milk: no coagulation nor peptoniaation pH aans appreciable change in 1 month ~

~ es cultural characters of Stre Ptom ~ ce 9 atroviolaceu ~ DS
8938 ~ have ra ~ aemblés dan ~ the table ~ YII to XI below. This ~ have those of cultures arrived at a good stage of development ~
c'e ~ t-dire d ~ en ~ iron troi9 ~ emainea ~ 26a ~ ~ auf indication ~
otherwise ~. These caraotares were ob ~ erv ~ on dee g ~ lo ~ e ~
nutriti ~ ea and de bouillona usually used for determine the ~ morphological characteristics of the strep strain ~
tomyces ~ le ~ cultures on medium g ~ lo ~ és being carried out aur of inclined plates. A number of culture media employees were prepared according to ~ the formulas indicated ~ in "The Actino ~ ycetes" ~ S ~. WAKS ~ AN, p. 193-197 ~ Chronica Botsnica Company, Walthamt ~ aa ~., US, A, 1950 ~ in this caq it ~ aont indicated ~ by the letter W with the number assigned to them ~

1 ~ 68; ~ ZS

in "The Actinomy ¢ ete3", I ~ e9 references or constitutions of other places 90nt 1e9 gui ~ ante9:
R ~ f, A - "Yea ~ t Extract Agar" - ~ .G. PRIDHAM et al. -Anti ~ iotic ~ Annual ~ 1956; -1957 ~ p. 950 R ~ r, ~ emlett's Agar "S, A, WAK ~ MAN - The Actinomy ~
ce ~ es, vol.2, p. 331- n 30 - ~ he Williams and WiIkin ~
Company ~ Baltimore ~ t961 Ref. C - Formula W-2 ~ added with 2% of g ~ lose R ~ f, D - "Hickey and Tre ~ ner '~ Agar" - ~, G. PRIDHAM and collar ~
10 - Antibiotics annual, 1956-1957, p. 950 R ~ f. ~ omato Paste Oatmeal Agar "- T, G, PRIDHAM et al.
_ Antibiotios Annual, 1956-1957 ~ p. 950 Ref, ~ _ "Melanin formation ~ edium" - SA ~ l ~ AKSDL9N - 'The Actinomycete ~, vol. 2, p. 333, n42 - ~ he ~ Nilliam3 and Wilkins Company ~ Baltimore, 1961 Ref. G. _W. ~, GRU ~ D ~ and col, - Antibiotics and Chem, 2, 401, 1952 ~ é ~; H _ "Inorganic Salt3 - Starch Agsr" - TG, PRIDHAM
and col. - Antibiotios An ~ ual 1956-1957, p. 951 Ref. I _ Corresponds to formula W-1 ~ o ~ 3% saccharo ~ e ~ have been replaced by 1 ~ 5% glucose Ref.l J _ Corre ~ pond ~ formula W-1 ~ o ~ 3% saccharo ~ e are replaced with 1 ~ 5% glyoerol Ref. K - Corre ~ pond to formula W-18, where 3% of ~ aocharo ~ e ~ have been replaced by 1J5% glucose Ref. ~ _ Corresponds ~ la ~ ormule W-18, o ~ le ~ accharo ~ e e ~ t deleted and replaced with small ~ pspier strips filter partially submerged in the liquid Ref. M - ~ Manual of Methods for Pure Culture Study of Baoteris "- Society of American Bacteriologists - Gene ~
N, Y. II50 -18 Ref ~ N - "Plain gelatin" - prepared ~ ~ following the indications ~ ~ `,. , ., ~ 68225 du ~ anual of Method ~ fsr Pure Culture Study of .Bacteria "
_ Sooiety of A ~ nerican: Bacteriologi ~ ts - Geneva ~ ~, Y. II5o Ref. ' P - Iait ~ cr ~ born in commercial powder - reconstituted ~
~ according to ~ manufacturer's instructions Ré ~, 'Q - ~ ilieu ~ ndiqu ~ for 18 research of the pro-duction of H2S by: H, D. ~ PES ~ ER e ~ F ~, DANGA - Journal of ~ acteriology,? 6, 239-244, 1958.

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iO68ZZ5 Strept'o ~ ces a'tr ~ io ~ a'c'e ~ DS-8938 presents ~ en emble of characters which do not coincide with any of the eyes of ~ ouche ~ die ~ described ~, and that is why we must con-~ think of it as something new.
By considering the e ~ pace ~ whose description is given dan ~ "The Aotinomycetes" (vol, 2, SA WAK ~ N, ~ he William ~ and Wilkins Company ~ Baltimore ~ 1961) plus ~ ue dan ~ le Bergey '9 ~ anusl o ~ Determinative ~ acteriology (7th edition, The Williams and Wilkin ~ Company, Baltimore ~ 1957) l ~ espace ~ la ~ uelle on can please ~ gladly compare it e ~ t S't ~ e ~ t ~ m ~ cé ~ ~ iola ~ 'è ~ hi ~ er ~ as given ~ as last, it does not produce pigment ~ black melanol soluble, 'mai9 gives by cons ~ r gélo3e Czapek synthetic with saccharo ~ e (sucroee nitrate agar) un soluble pigment brown ~ iolet of ~ enant black very quickly;
S. 'at ~' o ~ i ~ lta'c'eu's DS 8g ~ 8 ~ elm elsewhere ~ ~ ur many pigment backgrounds ~ soluble ~ Yiolaceous brown ~ black violet, ranging even to black in a certsin number of cases` ~ Of plu9 ~
like ~ v ~ blaceo'ni ~ er '~ atrov'i'olac'eu ~ iDS 8 ~ presents a myc ~ lium aerial sporulated gray tint and ~ es spore chains wind in a spiral ~ out ~ ois, S, 3tro ~ iola'ceu ~ 'DS 8 ~ 8 and S ~ vi'ollaic ~ 'o ~ i ~ e ~ doi ~ ent be con ~ id ~ ré ~ like two spaces3 different ~. In fact, despite the analogies which have just been oited ~ it should be noted that S. ~ iolaoeoni ~ er does not give pa ~ of pig-mentally soluble on potato, develops a vegetative mycelium . gri ~ sur g ~ latin, and ~ urtout shows by aging a black-: ci ~ ement of its myc ~ lium ~ érien which makes it include dan ~ le group of S.'h, ~ ro ~ co ~ lcu ~ ', while S ~ a't'ro ~ i! olaceu ~ DS 8 ~ 8 gives a potato pigment ~ brown oily ~ purplish brown : fonc ~, develops on gelatin a myc ~ lium v ~ g ~ tative brown ~ tre violac ~, and even ~ dsn ~ these tra tra ~ ~ gée ~ ne pré ~ ente jamai ~
blackening of its aerial mycelium which allows bring closer to the group of S. hydro ~ copicu ~. We must also note that ~ in p ~ rticulier ~ ur g ~ lo ~ e synth ~ tique de ~ zapek au 8 ~ ccharose (sucrose nitrate agar), pigm ~ nt ~ soluble ~ labore by S, violaceoni ~ er takes ~ u beginning of the culture a bluish tone ~
that does not present pa ~ the pigment ~ o ~ uble ~ worked p ~ r ~ I_e ~
~ 9 ~ a ~ which ~ for sn part, shows a ~ nt de ~ 'obscurc ~ r a purple-purple and not bluish-purple coloration.
-. The capacity of S, atroviolaceus DS 8938 ~ useful iverse ~ ~ ourcas of carbon or ~ zote to measure sound development has been determined according to the principle of the method de Pridham and Gottlieb (~. of Bact, ~, 107-114, 1948) ~ le : degree of development was ~ observed ~ on the middle of baee indicated ~ by the authors ~ by replacing either the gluco ~ e by the various ~ ~ ource ~ of carbon respect ~ vement essay ~ es ~ eoit S04 (NH4) 2 by the various ~ source ~ dlazote respectively ~; es ~ ay ~ ea. The results are indicated in the following table:

-3 ~
"

`1061 ~ 225 . . .
Carbon sources Use Nitrogen sources Use tried tried tried . , D-Ribose negative N03Na positive D-Xylose negative ~ 02Na positive L-Arabino ~ e negative S04 (NH4) 2 positive L-Rhamnose negative P04H (NH4) 2 positive D-Glucose positive Adenine positive D-Galactose positive Adenosine positive D-Fructose positive, the negative uracile D-Mannose positive Urea positive ~ -Sorbose negative L-Asparagine positive ~ positive actose Glycocolle positive Maltose positive Sarcosine positive, len ~
. DL-Alanine positive sucrose . DL-Valine positive thehalosis Cellobiose positive Acid D ~ -æ ~ ti ~ e positive :. Raffinosis positive L-gluta ~ ic acid positive ~ - Dextrin positive L-Arginine positive : Inulin negative L-Lysine positive DL-Serine positive starch Glycogen positive DL- ~ hreonine positive D ~ -methionine positive glycerol Erythritol po ~ itive ~ negative aurine Adonitol positive D ~ - ~ hénylalanine positive Dulcitlonnegative L- ~ yrosine positive D-Mannitolpositive DL-Proline positive . D-Sorbitolegative L-Histidine positive Inositolpositive L- ~ ryptophane positive, ~ nte Salicin negative 3eta ~ ne negative - ~ th ~ pr ~ psrstion procedure of 32999 RP con3iste basically ~ cultivate Streptomyces atroviolaceus DS
8938_Sto ~ k or se: ~ mutants producers on a medium and d ~ n ~ des condition ~ appro ~ ri ~ ea and ~ a ~ adorn the product ~ orm ~ au cour ~
- ~ culture, The culture of Streptomyce ~ stro ~ iolaceu ~ DS 8938 ~ Stock : ~ can ~ be carried out by any method of culture ~ urface or deep ~ but the latter is ~ pre ~ er For convenience, we use this purpose for different types of devices that are in common use in the fermentation industry, We can in particular adopt the mar ~ he sui ~ ante for the conduct of operations ~:
Streptomyces at oviolaceus DS 8938- ~ tock Culture on agar '' l Culture in shaken flask , ~
Culture inoculum in fermenter Fermenter production culture he medium of fermentation must oontent e ~ sentielle-a ~ aroe of arbone and a ~ assimilable nitrogen oce ~
mineral elements, in particular chlorides or carbonates ~ and é ~ entirely growth factors, all these elements ~ can be brought ~ in ~ elm of good products : defined or by complex mixtures, such as meeting dan ~ organic ~ products of origin 9 di ~ erses, As ~ ources of carbon has ~ similar ~ we can use carbohydrates such as glucose, lactose9 dextr $ nes, starch or other carbon ~ es like ~
sugar alcohols (glycerol) or comm ~ certain organic acids:

-35 ~

~ 0682Z5 lactic, citric acids9 Certain animal or vegetable oils such as bacon oil or ~ oja oil can replace place svantageu ~ ement these different ~ ~ ource ~ carbonaceous ~ or their being assistant The assimilable nitrogen forces are very varied ~ e ~. It ~ can be ~ ub ~ tances ¢ himiques tr ~ s ~ implee like ~ mineral or organic salts ~ ammonium, urea ~
- ~ certain amino acids ~. They can also be brought ~ by complex substances containing mainly nitrogen 90U9 protein form, casein, lactalbumin, gluten and their ~
hydrolyzate ~, soybean, peanut, pea 30n extracts, extracts meat, yeast, soluble distillers, corn steep.
Among the ~ added mineral elements, some ~ can have a buffering or neutralizing effect, such as phosphates slcalin ~ or alkaline earth or calcium carbonates and magnesium. Others bring the necessary ionic balance to development of Streptomyces atroviolaceus DS 8938 and ~ ela-boration of 32999 RPI like chlorides and sulfste ~ of metals alkaline or alkaline earth. Some acted ~ felt more ~ pécisle-ment as stimulators of the metabolic reactions of Streptom ~ cesatrovio aceu ~ DS 8938 ~ these are the salts ~ of zinc, of cob ~ lt ~ of iron, copper, mangana ~ e ~
~ es ~ actor ~ of growth ~ sance are products of nature vitamins such as riboflavin, foligue acid, and acid pantothenic or thiamine, The fermentation pH at the start of the culture must be Compr ~ s between 5.8 and 7.8. ~ at optimum temperature for fermentation is between 25 and 30C, but a production sati ~ doing e ~ t obtained for ~ tempérsturee between 23 and 33C, fermentation aeration can vary between values as ~ ez large ~. However, it was found that from 9 0.3 ~ 3 liter ~ air vents per liter of broth and psr r ~ ~
~ 36-.f '; , :

`106 ~ 3Z25 minute are particularly well suited. The maximum yield in 32999 RP is obtained after ~ 2 ~ 8 days of culture ~ this temp ~
d ~ during e ~ sentiellem ~ nt of the medium utili3 ~.
D ~ apras which preoads, we can see that the conditions general of the culture of Streptom, yce ~ atro ~ iolaceus DS 8938 for the production of 32999 RP can vsrier in a wide measure and be adapted a each neces ~ it ~ part ¢ uli ~ re.
Ie 32999 RP can be separated from ~ on medium according to the ~ usual ~ method of isolation of ~ a ~ tibiotic ~
belonging to this family, For example ~ the ~ 2999 RP can be extracted from the medium ; red ~ ction or fermentation at a pH voi ~ in of 9 using of an organic solvent such as chloroform, chloride of methylene, n butanol or a mixture of these ~ olvants in pro-suitable portions ~.
~ e mo ~ t fermentation can ~ us ~ i be filtered into presence of an ~ iltration adjuvant ~ a pH compri ~ between 1.5 and 9. It is advantageous to carry out this operation in the middle acid ~ and particularly by acidifying ~ a pH between 1.5 and 2 using oxalic acid It is also possible perform filtration at a pH between 2 and 8 in pr ~ -~ presence of an aliphatic alcohol ccntenant 1 to 3 atom ~ d0 oarbone ~
~ e 32999 RP can ~ be extracted from the filtrate, after oonoentration, at using an organic solvent such as chlorofor ~ e, ohloride methylene ~ n.butanol or a mixture of these solvent ~ in ~ suitable proportions 9 -~ e 32999 RP can be isolated from organic extracts, apra ~ washing ~ and sucking ~ sive extractions, by pr ~ precipitation after concentration of extracts ~ a low volume or by addition in a ~ olvant ~ i ~ target dan ~ which the 32999 RP is practically ; in ~ soluble ~ such as hexane ~ after 3 tran ~ possible formation of the antibiotic into an addition salt with an acid such as :
~ 37--~ 068Z25 "hydrochloride.
Ie 32999 RP can 8tre possibly purified by the ~
various operation ~ cla ~ iqae ~ of purification such ~ ue the crystalli ~ ation or chromstography on di ~ ers ad ~ orbitants, ~ e 32999 RP and its ~ el ~ non toxi ~ ue ~, that is ~ t- ~ say acceptable ~ phsrmaceuti ~ uement ~ 9th 90nt shown ~ particularly active on tumor ~ graftable ~ mouse ~ ~ de3 dose3 between 0.125 and 0.250 mg / kg i ~ p. ~ ur leukemia L 1210, P 388 leukemia and leucossrcomato ~ e.
The toxicity of 32,999 RP hydrochloride has been studied : mainly in the ~ ouri ~. Its 50% tax (D ~ 50) has been determined (treatment for 4 ~ consecutive bears ~ a) by route intraperitoneally and it is close to 0.4 mg / kg.
~ es example 8 sui ~ ants, given ~ without limitation, show how the invention can be practiced. Dan ~
what follows ~ a) the identi ~ ic ~ tion of ~ 2,999 RP was performed by chromatography - ~ on ~ ouche ~ thin gel of ~ ilice by comparing the ~ Rf of products o ~ held ~ ~ that of 18 carminomycin or 32 999 RP
in the same solvent system, . b ~ the production of 32,999 RP is determined from chromatograms on thin couohe, 30it after ~ inten ~ it ~
compared ~ of ~ matching spots of e ~ trait and oelle of pure 32,999 RP taken as reference, or by comparison surfaces ~ peaks enregi ~ very ~ to the plate reader "Chromo ~ ¢ an" or finally by dosing oolorimétriement only 32,999 RP eluted from the chromatographic support at the stain of the product sought ~, ~: Example ~ 1 -A culture medium A is prepared, the composition of which in ~ / l is the following:
Arm yeast autolysate ~ erie powder .., 15 ~ 68Zz5 Pho ~ manopotassic phate ... 2.5 Sodium dipota phosphate.,. 2 ~ 5 Gluco ~ e anhydrous Ø 15 An ~ evolution of the first 3 components ~ dan ~ 1000 cm3 distilled water and adjusted to pH 6 ~ 9 by adding sodium hydroxide normal ~ t is repaired ~ ie in ~ 00 cm ~ ~ rai ~ on 50 cm3 fiQles by ~ iole. These ~ 30nt sterilized ioles ~ ~ 122C for 20 minutes later cooled. We complate the environment by having sterile in each 1.5 cm3 vial of a ~ sterile ~ solution glucose ~ 500 g / l. The pH of the medium is then 7.050 We have ~ emence 80 of these vials by having ~ outing in each 2.5 cm3 flask of an inoculum culture of the Corynebacterium strain simplex (A ~ CC 6946) ~ the oulture is developed ~ e ~ ur agitated table 220 rpm in a ~ 30C enclosure. After ~ 48 hours the culture e3t well developed and its pH is 7.70. We then have a ~ ol ~ 10 mg / cm3 aqueous solution of carminomycin hydrochloride ; rai ~ on of 2.5 cm3 per ~ iole. ~ a pha ~ e of reductlon is for-~ monitoring ~ ur agitated table ~ 220 rpm in an enclosure at 26C.
After 48 hours the reduction is complete and the pH of the culture is 8.5, ~ e content of 80 ~ ioles is met. We homogeneously removes 50 cm3 of the culture qul e ~ t treated ~
per 25 cm3 of a borate buffer so as to bring the pH to 9.0. We then extracted with 2 faiths 50 cm3 of a methyl chloride mixture normal ne-butanol (70-30 volu ~ e). ~ es extract ~ organic ~
are dried on anhydrous sodium sulfate.
~ a production of 32,999 RP e ~ td ~ terminated by chroma-tography of ~ organic extracts ~ ur thin layer of silica gel.
~ es volumes of ~ extracts deposited ~ are 10 ~ 20 ~ l. The plates 50nt developed with the following mixture:
m ~ thylene chloride - formic acid - m ~ thanol (80-17-3 in volume) to obtain good separation of carminomycin and 32,999 RP. ~ es R ~ product ~ obtained3 ~ have compared ~ its those of the reference products ~ chromatographi ~ in the same ~ ~
conditions.
~ quantitative evaluation of the production of 32,999 RP
- shows that the oulture of Corynebacterium ~ imple ~ (ATCC 6946) : reduces carminomycin to ~ 2,999 RP with a yield of 80%
~ ur csrm ~ nomycin mi ~ e implemented.
Example ~ -Qn ready and we left in ~ 300 cm ~ iole ~ a culture medium A pr ~ by ~ as dan ~ llexample 1.
We prepare and distribute also in a vial 9 of 300 cm3 a m ~ place of culture ~ whose composition is as follows:
Peptone .. 0 10 g Meat extract ................................ ..... 5 g Gluco ~ e ... 10 g distilled water q. ~ .p. ................................. 1000 cm3 the pH is ~ usté ~ 7.2 by addition of normal sodium hydroxide;
mdlieu e ~ t st ~ rili ~ é ~ 122C for 20 minutes.
Qn ~ emence a flask containing 50 cm3 of medium A
with a pH of 7.05 and a flask containing 50 cm ~ of medium ~ with a pH of 6.5 with, per vial, 2.5 cm3 of a shutter inoculum of the Corynebacterium simplex strain (ATCC 6946), ~ e ~ oultures are d ~ veloppée ~ ~ ur a table acts ~ e 220 rpm in a ~ 30C enclosure. After a growing time appropriate (48 hours for medium A and 72 hours for medium ~) ~ an ~ aqueous solution at 10 mg / cm3 of hydrochloride carminomycin is then added ~ 2.5 cm3 per vial to obtain a concentration of 0.5 g / l of carminomycin.
After these addition ~ cultures ~ A and B are incubated during 2 and 4 ~ our ~, re ~ pectively, ~ ur lme agitated table 220 rpm in a chamber ~ 26C ~ vials ~ 80nt milked ~ e ~
d ~ ns the 9 condition ~ described ~ Example 1 ~ 068ZZ5 ~ es yields in 32,999 RP, compared to carmine-mycine put into play, are the following 8 ~:

Middle Re ~ dem ~ nt At 80 ~ Exe ~ the 3 -...
We prepare ~ in ~ iole of 300 cm3 in oondit ~ ons described ~ example 1 of cultures on medium A of the ~ ouche Corynebacterium ~ implex (A ~ CC 6946). ~ 90nt cultures developed on an agitated table ~ 220 rpm in an enclosure :. at 30C.
After 30 hours of incubation, 5 vials are combined (250 ~ m3 of must) and the ~ cell ~ pr ~ ente ~ ~ harvested by centrifugation. The cell harvest in its entirety is resuspended in a ~ 300 c ~ 3 ole container 50 cm3 of a phosphate buffer M / 15 ~ pH 8.0, We also prepare in the same way a sutre ~ u ~ cell penaion in using for the recovery of ~ cells, 50 cm3 of a buffer phosphate M / 15 ~ pH 5 ~ 0, We have ~ everything in ~ uite dan ~ each vial 295 cm3 of ~ an aqueous solution ~ 4 mg ~ om3 of hydrochloride ~ of carminomyoine to obtain a concentration of 0 to 2 g / l in carminomycin.
~ e ~ vials are incubated for 48 hours in the conditions described ~ ~ Example 1 and are treated ~ as dan3 e ~ example 1.
~ e ~ antibiotic yields 32,999 RP compared carminomycin mi3e in play, are the following:
Yield.
8.0 90%.

5.0 60%

...
.

~ ~ ~ 8 2 Z 5 Example ~ the ~ -We prepare and distribute in a 300 cm3 flask a culture medium A whose pH is 6.8 and which is prepared as described ~ Example 1, ~ 50 cm3 per vial.
Gn seeding more ~ ieur ~ flask ~ whose pH is 6.8 by adding each of them ~ 5.5 cm3 of an inoculum culture of the strain ~ acillu ~ cyolooxydan ~ (A ~ CC 12.673). Culture is developed for 48 hours on a table at 220 rpm dan ~ an enclosure ~ ~ oC ~ `
The reduction of carminomycin is carried out in using:
- or the entire culture, the pE of which is 7.65 - either a ~ cell uspension in ~ a pH 8.0 pre-buffer psr ~ e in the ~ conditions described 9 ~ Example 3.
Gn adds in each ~ iole 1 cm3 of a ~ olution aqueous ~ 10 mg / cm3 of carminomycin to obtain a concentration 0.2 g / l.
~ the reduction phase is continued ~ ie for 4 days : ~ ur a stirred table ~ 220 rpm in a chamber ~ 26C. ~ es vials are treated ~ in the ~ conaitions described ~ ~ e ~ example 1.
~ es yields in 32,999 RP compared to carminomycin mi30 in ~ eu are the ~ ui ~ ants:

Reducing medium Yield . I
: Whole culture 94%
. . _. _ Isolated cells 75%.

Example 5 -We prepare dan ~ the ~ conditions described ~ example 4 cultures on medium A of the strain ~ acillus c ~ clooxydans (A ~ CC 12.673). Whole cultures ~ -incubated for 48 hours and whose pH is ~ 7.65, carminomycin is added as:

`` -42-: ~.

. , r "- 1068225 a) of an aqueous solution of 10 mg / cm of the pure product, 1 cm3 rai ~ per ~ iole (0.2 g / 1 carminimycin) b) of an aqueous solution ~ 10 mg / cm3 of a product crude which contains 16% carminomycin, ~ rai ~ on of 4 cm3 per vial (0.13 g / l carminomycin).
~ es flask ~ ~ then incubated ~ pend ~ nt 4 day ~ in the conditions described in Example 1 and treated in the same way m ~ niare. ~ e ~ yield ~ in 32,999 RP, compared to carmine-mycine mi ~ e at stake are the 8 8 following:

Product Yield: ~
. _ ~ _ : _armin pure mycine_ _ 94 ~ _ crude carminomycin _ 92% _ : ~ Example ~ on 6 -A culture medium C is prepared, the composition of which in g / l eat the following:
Flour of ~ oja ... 30 Soybean oil (d = 0.92) ~ 4.6 Ph08phate monopota ~ sique ...
Calcium carbonate, ~, 2.5 Glucoae anhydr ~ ... 50 The ~ constituents of the environment ~ except glucose are di ~ sou ~ or mi3 en ~ uspen ~ ion dsns 900 cm3 di ~ tilled water ~ A preparation, a ~ ust ~ e ~ a pH of 7.20 by addition of ~ oude N, e8t distributed in vial ~ of 300 cm3 ~ rai ~ on of 45 cm3 per vial.
Ce9 vials ~ sterilized ~ ~ 122C for 20 minutes ~ and re-coldness ~ We complate the environment by having ~ outing ~ very dana each 5 cm 3 vial of sterile solution of gluco ~ 500 g / l.
The pH of the medium ready for sowing is 7.0 ~
A 300 cm3 flask containing 50 cm3 of previous medium whose fold is 7.0 with 2.5 cm3 of a culture : ~) 68225 inoculum of ~ Streptomyces ouche ~ oseochromogene ~ (A ~ CC 13.400).
ha culture is developed for 48 hours ~ ~ ur a stirred table ~ 220 rpm in ~ an enclosure ~ 30C, We then harvest the ~
- cells produced 8 by this culture. ~ a reduction in car-minomycin en ~ ntibiotique 32.999 RP is performed using a ~ u ~ pension of cells dan ~ 50 cm3 of a phosphate buffer M / 15 at pH 8.0 containing 1 c ~ 3 of an aqueous solution ~ e ~ 10 mg / cm3 carminomycin hydrochloride to achieve a concentration 0.2 g / l. ~ a reduction phase ~ e continued for 48 hours on a table acts ~ ~ 22 ~ rpm in ~ an enclosure 26C. ~ a suspen ~ cell ion e ~ t braitée in the conditions ~
described ~ example 1. ~ e yield in 32,999 RP psr report the carminomyc ~ ne involved is 57%.
Example 7 -We prepare and distribute in 300 cm3 flasks a culture medium C prepared ~ under the conditions described 8 Example 6.
There are ~ th ~ of ~ vials containing 50 cm3 of medium, whose pH is 6.90, with 2.5 cm3 of an inoculum culture of la ~ ouche Streptomyces lavendulae (A ~ CC 8664). ~ a culture is developed for 40 hours on a stirred table ~ 220 rpm dan ~
an enclosure at 26C. We add alore dan ~ cha ~ ue flask containing the entire shutter, whose pH is 7.50, 1 cm3 of a solution aqueous at 10 mg / cm3 of carminomycin hydrochloride to obtain a concentration of 0.2 g / l. The reduction phase is for-followed for 4 ~ bears dan ~ the ~ same shaking conditions and of temperature. ~ the vial ~ are treated ~ e ~ in the conditions described in Example 1. ~ e yield in 32,999 RP, compared carminomycin involved, was 61%.
~ o Example ~ on 8 -A - ermentati ~ o ~

~ IL0682 ~ 5 We load dan ~ u ~ e fermenter of 170 liter ~
Peptone ... 600 g Meat extract .. ~ 600 g ~ yeast trait ... 600 g . Gluco3e monohydrate ,,, 600 g - City water q. g. p. ,,. t 10 liter 9 Apra ~ please ~ ust ~ pH ~ 6.8 with 40 cm3 of ~ oude 10 N on a ~ all:
Calcium carbonate.,. 300 g - 10 the pH of the medium is then close to 6.8 on ~ térili ~ e the medium by bubbling steam ~ 122C for 40 minutes;
after cooling ~ due to steam conden ~ ation at during ~ t ~ rilisation, the volume of bolt is 120 liters and the pH of 8.3. Ic pH is a ~ ust ~ at 7.0 by addition of 90 cm3 . hydrochloric acid 12 ~.
- We seed with 250 cm3 of an inooulum culture in iole erlenmeyer agitated from the souohe Corynebac ~ erium ~ implex (ATCC 6946), the oulture is developed ~ 30C for 32 hours 9 shaking and sererating with air ~ terile it is alor ~
suitable for the sowing of the productive culture.
~ a producing culture is carried out in a fermenter of 800 liters ~ loaded with the following ~ ~ ubstanoe ~:
Brewer's yeast autolysate, powder. 6 kg Phosph ~ te monopotas ~ ique ... 1 kg Phosphste dipotas ~ ique ... 1 kg Eau de vllle qgP. ~ - 355 liters After having ~ a ~ u ~ t ~ pH ~ 7.0 by adding 460 cm3 of ~ oude 10 N, the medium is sterilized by steam bubbling - at 122C for 40 minutes, After cooling, due to steam condensation during sterilization, broth volume is 385 liters; it is completed at 400 liter ~ by adding 15 liters of a sterile aqueous solution .

~ 0 6 82 2 5 containing:
Gluco3e monohydrate ~ .. 6 kg ~ e pH of the medium is ~ 6 ~ 8, We ~ em ~ nce with 40 liter ~ of inoculum shutter in ~ 170 liter ermenter ~
described ¢ above. The culture was developed at 30C by shaking with a rotating fan ~ S60 rpm and airing at ~ ec a volume of desire 20 m ~ / h. After 30 hours ~ culture is suitable for carrying out the biochemical transformation of carminomycin in 32,999 RP
We introduce into the culture 4 liter ~ of a ~ olution aqueu3e containing:
Carminomycin hydrochloride. 40 g ~ incubation and continued for 17 hours ~ at 26C ~
by shaking and aerating under the conditions described above ~ us.
~ ac ~ rminomycin was reduced to 32,999 RP with a yield see ~ in of 100 ~.
~ - xtractidn At 420 liters ~ of wort obtained as shown in example 8 A, 42 liters of n.butanol and 2.9 liters of ~ oude are added

6 N, ~ e active principle e3t extr ~ it against the current by n.butanol (60% by volume) ~ ur two centrifuges ~ e ~ ~ extract ~
whose volume is ~ t of 330 liters ~ e ~ t oonc ~ ntré ~ OU9 pre ~ ion r ~ pick (5 ~ 10 mm of mercury) at 40C ~ u3qu ~ a ~ olume of 20 liter 9.
With the concentrated extract we have 20 liters of chloride methylene and lsve this m ~ lange per 10 liter ~ water smen ~ s to pH 10 by addition of soda. ~ wash water, which contains 32 ~ 999 RP, 2 liver psr 10 liters are extracted from the ohloride mixture of m ~ thylene - n.butanol (80-20 by volume) ~ e ~ troi ~ organic extracts are combined ~ pUi9 con-centered under pre ~ s ~ on r ~ duite (5 ~ 10 mm of mercury) ju ~ qu ~ a volume of 15 liters. We add ~ while stirring 0.2 liter of : 1 ~) 6 ~ 225 mixture n, butanol - hydrochloric acid 11 N (90-10 by volume) pui ~ concentrated under reduced pres ~ ion (5 ~ 10 mm of mercury) up to a volume of 5 liters ~
~ sntibiotique 32.999 RP ~ u9 hydrochloride form was precipitated by the addition of the concentrate to 35 liters of hexane.
Ie pr ~ cipit ~ e ~ t e3soré, washed et ~ éch ~. 52 g of crude hydrochloride of 32,999 RP.
C - Purification ~
~ ...
a) At a ~ uspenqion of 50 g of raw hydrochloride 32.ggg RP, obtained as indicated ~ l ~ exe ~ ple 8 B, dsns 130 cm3 dleau, we have ~ 520 cm3 of dioxane, Ia ~ naked solution is filtered, ~ e hydrochloride of 32,999 RP e ~ t crystallized by addition of 4 liters of dio ~ aNne ~ ~ a temperature close to 20C, in the filtrate. IeY cri ~ rate 90nt e ~ qsorés, l ~ é ~ by dioxane and ~ ch ~ s, We thus obtain 27 ~ hydrochloride 32.999 RP semi purified, b) 4.2 g of crude hydrochloride obtained as the example 8 B are mi ~ en ~ olution in 0.2 liter of water, ~ e pH is adjusted to 4.5 by adding ~ or N 10.
antibiotic content in this ~ eolution e ~ t ~ fixed ~ a column containing 14 liters of Amberl ~ te IRC 50 (brand of trade) 90U9 aide form, the column is washed with 1 liter of water, 2 liters of methanol a ~ ueux à 50 ~ o, 2 liter ~ of methanol aqueous at 90 ~ and finally with 3 liters of aqueous me-thanol ~ 90 ~ o-holding 1% of sodium chloride, the eluate is concentrated reduced pressure (5 to 10 mm of mercury) ~ 40C ju ~ qu ~ a volume 0.5 liter. Ie concentrate, the pH of which has been adjusted to 10 by addition of ~ oude 11 N, is removed by 2 times ~ 0.5 liter of ohloroform and twice 0.5 liter of chloroform mixture -n.butanol (80-20 in ~ olume), le9 organic extracts combined ~ have concentrated ~ s 90U ~ reduced pressure (5 ~ 10 mm of mercury) / ~, ' ~ '-47-~ `106822S
`40C. During the ¢ oncentration we ~ add 10 cm3 of n, butanol .
; containing 1% (by volume) of 11 N hydrochloric acid and 5% ten ~ olume) dleau. Ie concentrate, whose volume is brought ~ ~ ~ 0 cm3 ~ is 8 slowly added ~ ment ~ 500 cm3 of hexane. ~ e precipitates ~
obtained e ~ te ~ sor ~, la ~ é and dried. 1.26 g of hydrochloride of 32,999 RP ~ em ~ -puriri ~.
- ~ the semi-purified product (27 g) obtained ~ according to a) is put in solution in ~ 300 cm3 of water, ~ a solution obtained is filtered, ~ e hydrochloride of 32,999 RP is crist ~ llized by addition to the filtrate of 2.7 liters ~ of ac ~ tone, ~ a temper-ture voi ~ ine de 20C ~ ~ es cristau ~ are e ~ sorés ~ washed ~ and dried ~ Cn thus obtains 17 g of hydrochloride of 32,999 RP pure.
Example i9 -Centrifugal sterile centrifugation at a similar temperature from 0C ~ 50 cm3 of a Corynebacterium simplex culture (A ~ CC 6946) developed for 30 hours under the conditions ~
described in Example 8 A. After ~ entrifugation the cells are resuspended in 50 om3 of M / 15 phosphate buffer at pH 7 ~ 5, ~ the cells ~ then have ~ omitted ~ the action of ultra-~ ons (25 kHz) for 15 minutes at a similar temperature from oa. After ~ centrifugation we have ~ oute li ~ uid ~ urnageant 10 mg carminomycin hydrochloride, ~ a ~ olu ~ ion ain ~ i obtained e ~ t mi ~ e to incubate dan ~ the conditions ~ ~ described in Example 1. After 40 hours, the carminomycin is reduced to 32,999 RP with a yield close to 60%.
S ~ emPle 10 -Cn cen ~ riruge sterile ~ a temperature vo $ 3ine of 0C 50 cm3 of a culture of Coryn ~ bacterium ~ implex (A ~ CC
6946) developed for 30 hours under the conditions which are described in Example 8 A. Apr ~ centrifugation ~ the ~ cells ~ have resumed in ~ uspen ~ ion in 50 cm3 of tri buffer ~ (hydroxy-"` 1 ~ 16 ~ 3Z2S

methyl) aminom ~ thane / HCl M / 5 ~ p ~ 8.1 and tran ~ féré ~ in a Erlenmeyer flask of 300 cm3 we have ~ then alor ~ 25 mg of ED ~ A
(acid ~ thyl ~ ne diamine tétraac ~ ti ~ eu), 25 mg of C ~ A ~ (bromide of c ~ tyltrimethylammonium) and 200 mg of ly ~ ozyme ~ then the vial is placed at 37C for 1 hour, ~ on a roasting table ~
220 rpm. Apra ~ centrifugation, add to li ~ uide ~ ur-swimming 5 mg carminomycin hydrochloride. ~ a solution thus obtained e ~ t mi ~ e ~ incubate dan ~ the ~ condition ~ which ~ have described ~ in e ~ example 1. After 40 hours, carminomycin e ~ t reduced to 32,999 RP with a yield close to 30 ~ 0.
Example 1 ~ -0 ~ 5 g of pure hydrochloride of 32,999 RP ~ have hydrolyzed ~
by dissolving in 50 cm3 of water add ~ tionn ~ e of 10 cm3 hydrochloric acid 11 N and heating ~ age ~ boiling for 2 hours. A Cree ~ tallline aglycone precipitate is being formed hydrolyte ~ e. the precipitate is drained, the ~ and ~ ch ~. Cn thus obtains ~ i 0.3 g of pure aglycone ~
Example ~ e ~ -- ~ We prepare a blanking medium A having the composition ~ ui ~ ante:
- flour of so ~ s. ,, 8 g - baking yeast in pa ~ n,., 40 g - pure lactose .0l.58 g - sodium chloride ... 2 g _ monopotassi phosphate ~ Ue ... 1 g _ magnesium sulfate heptahydrate ~, ..... 0.1 g - ~ ferrous heptahydrate ulfate. ~ .0 ~ 1 g _ zinc sulfate heptahydrate ~.,. 0 ~ 1 g - distilled water ~ e ~ 75C ,,, 800 cm3 - 30 ~ e pH of the ~ uspension s ~ t adju ~ té ~ 6.5 by addition of 1 cm3 soda 10 N, then we add - glyc ~ rine (d - 1 ~ 26). ~ ol 19 g : - calcium carbonate ,,. 2 g ~ - complete distilled water ~ m ~ nt ~. . ', 1000 cm3 ~ - This millet ~ had e ~ tr ~ gone ~ nf ~ olea of 300 cm3 ~ rai ~ on of 50 cm3 per vial. Ie ~ vials ~ have ~ been ~ used ~ ~ 122C for 20 minutes, after cooling the pH of the medium is 5.9, prepare ~ by the way ~ a ster solution ~ the sulfan ~ lEmide ~
10 mg / cm3 and ~ pH 9.0 per di ~ olution of sulfanilamide in a mixture of 5 N sodium hydroxide and distilled water PU9 filtration 0.45 ~ "nMillipore membrane.
~ e 3 vial 9 containing the medium ~ terile A are re-part ~ in 6 lot ~ of 2 vial ~ ch ~ cun.
~ e ~ vials of batch Al do not receive a ~ aition complementary.
~ e ~ vial ~ of the 5 other lots receive the addition ~
complementary ~ sui ~ ante ~:
- batch A2: 1 cm3 of the solution of ~ ulfanilamide (0.2 g / l) - batch A3: 2 cm3 of the 301ution of sulfsnilamide (0 ~ 4 g / l) - batch A4 s 3 ¢ m3 of the sulfanilamide solution (0 ~ 6 g / l) - batch A5 s 4 cm3 of the solution of sul ~ anllamide (0.8 g / l) - batch A6 s 1 cm3 of the ~ evolution of ~ ulfanilamide (0 ~ 2 g / l) ~ es ficle ~ of lot A1 and the ~ ~ iole3 qul received a addition of ~ ulfanilamide are in ~ emenc ~ e ~, at rai30n of 2.5 cm3 per vial, with an inoculum culture of Streptomyces coeruloeru-bidu3 DS 7.126 (NRR ~ 8098) frozen for 72 hours.
Ie9 cultures are developed on an agitated table 220 turns ~ / min in a ~ 26C enclosure, ~ vials of lot A6 receive apra ~ 48 and 72 hours of incubation 2 nou ~ they addi-tion ~ of the sulfanilamide solution (total concentration sulfanilamide 0.6 g / l). Apra ~ each of these additions ll ~ ncu-bation d ~ ~ iole ~ e ~ t resumed in the same conditions ~ d ~ agitated-106 ~ Z25 tion and temperature.
After 7 ~ incubation bear in the conditions indicated ~ e ~ of ~ su ~, we tr ~ ite ~ each faith ~ a vial of each of ~ lots to make the 32,999 RP, ~ e treatment is carried out by a ~ outing in each flask 4 gd ~ oxaliq acid ~ e (~ e which amane the fold of the must ~ 1.5) and 50 om3 of absolute ethanol.
- ~ a vial containing this mixture e ~ t tightly sealed ~ only e ~~ placed ~ e for 1 hour on a tab ~ e acts ~ e ~ at 220 rpm.
~ e mixture e ~ t ~ filtered uite ~ ur paper and the filtrate di3-0 till ~ 80U9 pres ~ ion reduced ~ reduced ~ 50C ~ only ~ half ~ of ~ on ~ olume init ~ al (50 cm3) ~ the concentrate is up to ~ pH ~ 4.5 by addition of 5 N sodium hydroxide pUi9 we have ~ out ~ u ¢ successively 50 cm3 methylane chloride n-butanol mixture (80-20 by volume3 ~
and 25 cm3 of 0.5 M borate buffer ~ pH 9.0. After shaking in ampoule ~ the sol phase is collected by decantation ~ colored ant and the treatment is repeated on the ~ water ~ -m ~ ree aqueu ~ es which are - ~ drawn ~ by 50 cm3 of m ~ em ~ lange ~ olvant. Ies phase ~ org2ni-ques are combined ~ and dried on ~ ulfatb of ~ anhydrous odium. ~ e volume of the organic pha ~ e is finally a ~ u ~ t ~ ~ 100 em3 per addition of the same mixture ~ olvant.
To determine the production of 32,999 RP we chroma-tograph these e ~ lines on thin layer, production has been evaluated qualitatively in resting on silica gel plates (Mbrcl ~ 60 - 3 glass holder - without indication of fluorescence) 100 ~ 1 of the extract and 2.510 ~ g of a reference of 32,999 pure RP. ~ th plate ~ ~ have developed ~ with the chloride mixture of m ~ thylane - ~ thanol -acetic acid - water (80/20/10/4 in volume). The presence possible of 32.999 RP dan ~ the extralt3 is mi ~ e in evidence, after ~ development ~ by examining the 3 chromatogram ~ ~ oit the light of the ~ our be in light ~ re ultra- ~ iolette ~ 254 nm, 2) - ~ a production of 32,999 RP e ~ t m ~ ur ~ e quantita ~ trademark -51-.

by depositing uricagel plates [Merck 60 F
254 - glass holder with ~ fluorescence indicator] of volume3 of extracts ~ mined dlaprè ~ qualitative chromatography understood to be 10 and 100 ~ l ~ We deposit ~ e ~ also ~ ur this ~ plate ~
a gsm (0.5 ~ 1.5 ~ g) of 32,999 pure RP - ~ the plates ~ have developed with the mixture ~ olvant described above ~ u ~ After ~
development these pla ~ ues ~ have pas3ées to the plate reader I'Chromo ~ c ~ n'l ~ ui records the ~ peak ~ o corresponding to 32.999 RP
: contained in the extracts ~. Cn calculates the ~ surface ~ of these pic9 and those corresponding to the range ~ t810n of 32,999 pure RP
and thus the production of 32,999 RP of the cultures is determined.
With ~ the ~ culture ~ treated ~ ~ Yec the ~ ul ~ anilamide we have obtained with this do3age method the following results:
. ' .. _,, :. ~ ot ~ Sulfanilamide (g / l) 32,999 RP (mg / l) ; A1 OO
: A2 0.2 O
. 3 ~ 4 26 4 0.6 42 0.8 33 A6 0 ~ 6 (0.2 x 3) 5 _ ~

Exem'Plei '1'3' We set out in a vial ~ of 300 cm3 ar ~ i ~ on of 50 cm3 per flask, a medium A identical to 3 that described in Example 12.
We also prepare a ~ olution of sulfanilamide 10 mg / cm3 dan3 le ~ conditions described in dan3 e ~ ample 12.
~ e ~ vial3 containing the sterile medium A are distributed in 4 lots A1, Al1 ~ A ~ A ~ 5 of 2 ~ ioles each and we have ~ out dan3 each vial of ~:
- - lots A5 and A'5: 4 cm3 of the sulfanilamide eolution (0 ~ 8 ~) lee vial ~ of ~ lot ~ A1 and A'1 ~ ubis ~ having no addition.
trademark '1068225 We inoculate ensui ~ e all the vials:
~ - lots A1 and A5 with 2.5 cm3 per culture flask - Streptomyces coeruleorubidu inoculum ~ DS 8899 (NRR ~ 3046) ~ 72 hour age ~
- of ~ lot ~ A ~ l and A'5 with 2.5 cm3 per vial of an ino- culture culum ae Streptomyces coeruleorubidus DS 31.723 (NRR ~ 3045) - 72 hours old ~.
~ e ~ cultures ~ have grown ~ ur agitated table ~ 220 laps ~ min in an enclosure ~ 26C. ~ es flole ~ incubated 7 days ~
d ~ n ~ these conditions ~ ~ have tra ~ tée ~ to dose the 32.999 RP as described in Example 12.
~ the following ~ production ~ were obtained 9:

: ~ ot ~ Sulftng / l) mide Production of 32,999 RP (in mg / l) Streptomyce ~ ~ $ 8 ~ 99 Streptomyce ~ DS31.723 . .. _ _ _ A1 A '1 0 0 A5 A '5_ 0, 8 _. _ 25 .. - ._ Example ~ le ~ ~ 4 '-We distribute in vials of 300 cm3 ~ reason of 50 cm3 ~.
per vial, medium A identical to that described in e ~ example 12.
A solution of ~ ulfsnilamidQ is also prepared 10 mg / cm3 in the 9 conditions described in example 120 ~ e ~ vials containing the medium ~ terile A ~ are distributed ~
in 4 batches of 2 vials each and we have ~ in each vial of:
- batch A2: 1 cm3 of the ~ sulfanilamide solution (0.2 g / l) - batch A3: 2 cm3 of the ~ ulfanilamide solution (0 ~ 4 g / l) - batch A4: ~ cm3 of the solutlon of ~ ulfanilamide (0.6 g / l) the ~ ~ ioles of the batch Al not subjected ~ ant any addition After this ~ addition ~ all ~ the ~ vial ~ of batch ~ A1 ~ A2 ~ A3 and A4 with 2.5 cm3 per flask of an ino- culture culum of Streptomyoe3 bifurcus DS 23.219 (NRR ~ 3539) ~ age 72 1 ~ 682ZS
hours.
~ cultures are grown ~ on a hectic table at 220 rpm in an enclosure at 26C. ~ the incubated vials

7 days in these conditions are processed to dose 32.999 RP as described in example 12. ~ the following productions have been obtained:

~ ots Sulfanilamid e (g / l) 32.999 RP (mg / l) ,. . . ._._. , .. ______ _. . .. _ A2 0.2 36 . A3 0.4 28 A4 0.6 13.

Example 15 -A culture medium B having the composition is prepared next:
- soy flour ... 30 g - soluble distillers ... 5 g - partially hydrolyzed starch "Fox head" (mark of - 5 g trade ) - soybean oil (d = 0.92) .., 27.5 g - sodium chloride ... 10 g - distilled water .. 1000 cm3 This medium, the pH of which is adjusted to 7.20 by addition of soda N, is distributed in flasks of 300 cm3 at a rate of 50 cm3 per vial and is sterilized at 122C for 20 minutes. After cooling the pE ~ is 6.60.
An aqueous solution of sulfanilamide is prepared identical to that described in Example 12.
~ the vials containing sterile medium B are distributed tied in 2 lots B1 and B2 of 2 vials each. ~ the vials of the batch B1 do not receive any other additional addition O We add 106 ~ 3225 in each vial of:
- lot B2: 2 cm3 of the sulfanilalDide solution (0.4 g / l).
`` After these additions we sow all the vials of the batches B1 and B2 with 2.5 cm3 of an inoculum culture of Streptomyces coeruleorubidus DS 31,723 (NRR ~ 3045) aged 72 hours. ~ es cultures are grown on a table stirred at 220 rpm in an enclosure at 26C. ~ the vials incubated for 7 days in these conditions are treated to dose the 32.999 RP as - described in Example 12, ~ the following productions3 have been obtained:

~ fan ~ amide (g / l ~ 32.999 : -, ~
Example 16 -We distribute in vials of 300 cm3 at a rate of 50 cm3 per flask, medium A identical to that described in the example 12.
A solution of sulfathiazole is also prepared at 10 mg / cm3 under the conditions described in Example 12 for preparing the sulfanilamide solution.
~ the vials containing the ~ sterile dilute A are repaired ties in 4 batches of 2 vials each and are made in each vial the following additions:
- lo ~ A7: 0.25 cm3 of the sulfathiazole solution (0.05 g / l) - batch A8: 1 cm3 of the sulfathiazole solution (0.2 g / l) - batch Ag: 0.5 cm 3 of the sulfathiazole solution (0.1 g / l) the vials in lot A1 not undergoing any addition.
Each vial of lots A1, A7 is then seeded, A8 and Ag with 2.5 cm3 of an inoculum culture of Streptomyces coeruleorubidus DS 7.126 (NRR ~ 8098) aged 72 hours. ~ es cultures are grown on a table stirred at 220 rpm in an enclosure at 26C. ~ the vials of batch Ag receive after ~
48 and 72 hours of incubation 2 new additions of 0.5 cm3 sulfathiazole solution (total sulfa- concentration 'thiazole 0.3 g / l). After each of these additions the incubation vials is repeated ~ e danæ the same co ~ stirring editions and - temperature.
The vials are incubated for 7 days and treated for dose 32,999 RP as described in example 12. ~ es produc-The following have been obtained:
. . .

A ~ Sulfathiazole (g / l) 32,999 RP (mg / l) A70.05 O
A80.2 51 Ag_ _ ~ 64 Example ~ 17th -We distribute in vials of 300 cm ~ at a rate of 50 cm3 per flask medium A identical to that described in example 12.
A sulfathiazole solution is also prepared at 10 mg / cm3 under the conditions described in Example 16.
The vials containing sterile medium A are distributed tied in 4 lots of 2 vials each. The vials in lot A1 do not receive no further additions. We perform in each of the other vials the following additions:
- batch A10: 0.5 cm3 of the sulfathiazole solution (0.1 g / l) - batch A11: 2 cm3 of the sulfathiazole solution (0.4 g / l) - batch A12: 2.5 cm3 of the sulfathiaæole solution (0.5 g / l) After these additions, all the vials are inoculated .
lots Al, Alo, Al, l and A12 with 2.5 cm3 of an inocu-- lum of Streptomyces coeruleorubidus DS 31.723 (NRRL 3045) aged 72 hours. The cultures are grown on a stirred table at 220 rpm in an enclosure at 26C.
The vials incubated for 7 days under these conditions are processed to dose 32,999 RP as described in Example 12.
The following productions were obtained:

_. . _ ~. ~ Lots Sulfathiazole (g / l) 32.99 ~ RP (mg / l) - 10. . ._ . ': Al OO
Al o O 1 1 ~ O
All 0.4 9 .. Al 2 ~ ~ I
Example 1 ~ -We distribute in vials of 300 cm3 at a rate of 50 cm3 per flask, medium A identical to that described in Example 12.
A sulfathiazole solution is also prepared at 10 mg / cm3 under the conditions described in Example 16.
The vials containing sterile medium A are distributed in 4 batches of 2 vials each and carry out in each Eiole the following additions:
- batch Alo: 0.5 em3 of the sulfathiazole solution (0.1 g / l) - lct A8: 1 cm3 of the solution of sulfathiazol ~ e (0.2 g / l) - batch All: 2 cm3 of the sulfathiazole solution (0.4 g / l) the vials of batch Al not undergoing any addition.
After these additions, each vial is inoculated with lots Al, Alo, A8 and All with 2.5 cm3 of an inoculum culture of Streptomyces bifurcus DS 23.219 (NRRL 3539) aged 72 hours.
30 The eultures are developed on a table stirred at 220 rpm in an enclosure at 26C.

.

. ..
-10682 ~ 5 ~ the vials incubated for 7 days under these conditions are processed to dose 32,999 RP as described in Example 12.
~ The following productions have been obtained:

Dots Sulfathiazole (g / l) 32.999 '' ~ mg / l) AT? OO
0.1 18 A8 0.2 22 _ _ 0.4 5 Example 19 -A culture medium C is prepared containing:
- beans in grain ... 40 g - soluble distillers ... 10 g - glucose ... 15-g - soybean oil (d = 0.92) ... 18.5 g - cobalt chloride hexahydrate ... 0.02 g which is obtained as follows:
~ es beans ~ near autoclave cooking with 500 cm3 of distilled water for 45 minutes at 122C are reduced to mash potatoes.
Then add the di ~ tiller ~ 'soluble ~, the oil of soy, cobalt chloride and distilled water to get a volume of 900 cm3.
The pH of the mixture is a; just 7.2 by addition of soda 5 N. ~ e mixture is distributed in 300 cm3 flasks at a rate of 45 cm3 per vial. ~ the vials are sterilized at 122C during for 20 minutes. After cooling, sterile add ~ ns each 5 cm3 vial of sterile 15% glucose solution ~ e pH of this sterile medium ready for seeding is 6.70 F ' 1 ~ 1682Z5 An aqueous solution of sul ~ a- is also prepared thiazole identical to that described in Example 16, The vials containing sterile medium C are distributed tied in 2 lots C1 and C2 of 2 vials each. ~ the vials of the batch C1 do not receive any additional addition. We add in each vial of batch C ~ 0.5 cm3 of the sul ~ athiazole solution (0.1 g / l).
After this addition, each vial of batches C1 and C2 with 2.5 cm3 of an inoculum culture of Strepto-myces bifurcus DS 23.219 (NRR1 3539) 72 hours old. ~ es cultures are grown on a table stirred at 220 rpm in an enclosure at 26C. ~ the vials incubated for 10 days in these conditions are processed to dose the 32,999 RP co ~ me described in Example 12.
~ The following productions have been obtained:

~ ots Sulfathiazole (g / l) ~ ^, 999 RP (~
C1. __ ...
C2 --- ~ - - - 16 - Example 20 -We distribute in vials of 300 cm3 at a rate of 50 om ~
per flask, a medium A identical to that described in Example 12.
We also prepare a solution of ~ ulfapyrida-zine at 10 mg / cm3 by dissolving the product in a mixture of water distilled and hydrochloric acid 5 N ~ The solution whose pH is 2.0 is sterilized by filtration on a "Millipore" membrane 0.45 ~ "(trademark), ~ The vials containing the sterile medium A are distributed - in 3 lots of 2 vials each and sterile is carried out ~ ent in - each of the vials the following additions:

, ...... .

106 ~ 3Z25 - batch A13: 0.5 cm3 of the sulfapyridaein solution (0.1 g / l) - batch A14: 1 cm3 of the sulfapyridazine solution (o, ~ g / l) the vials in lot A1 do not undergo any addition.
Then each vial of lots A1, A1 ~ and A14 with 2.5 c ~ 3 of an inoculum culture of Streptomyce ~ coerule-orubidus DS 7.126 (NRR ~ 8098) ~ 72 hours old.
J. cultures are incubated for 7 days with shaking tion under the conditions described in Example 12. ~ es results dosages carried out under the conditions described in the example 12 are:

. ~ ots Sulfapyridazine (g / l) 32.999 RP (mg / l ~
___.

A13 0.1 38 _ _ 0.2 _ Example 21 -We distribute in vials of 300 cm3 at a rate of 50 cm3 per flask a medium A identical to that declt in the example 12.
We also prepare a solution of a ~ ethopterin at 20 mg / cm3 by dissolving the product in a mixture of water ~
5 ml of soda and soda. The solution with a pH of 10.0 is sterilized by filtration on a "Millipore 0, ~ 5 ~ u" membrane.
~ The vials containing the sterile medium A are distributed in 4 lots A1 ~ A1s ~ A16 and A17 of 2 vials G each I. esfioles of lot A1 do not receive any additional addition.
In each of the flasks of the other 3 batches, the following additions:
- batch A1 ~: 0.25 cm3 of the amethopterin solution (0.1 g / l) - batch A16: 0.6 cm3 of the solution of a ~ ethopterin (0.24 g / l) "- 60 -~) 68 ~ 25 - batch A17: 1 cm3 of the amethopterin solution (0 ~ 4 g / l) After these additions, each vial of lots A1, A15, A16 and A17 with 2.5 cm3 of an inoculum culture of Strepto ~ yces coeruleorubidus DS 7.126 (NRR ~ 8098) aged 72 - hours. ~ cultures are incubated for 7 days with shaking tion dan ~ the conditions described8 in Example 12.
~ The results of the 32,999 RP assay performed in the usual conditions are:

~ ots Amethopterin (g / l) ¦ 32,999 RP (mg / l) :,. ~.

A15 0.1 7, A16 0.24 11 A17 0.4 ~ ~ 1 - ~ Example 2? -We distribute in vials of 300 cm3 at a rate of 50 cm3 ~ Per flask, medium A identical to that described in Example 12.
A solution of barbital is also prepared at 50 mg / cm3 by dissolving the product in a mixture of distilled water 5N sodium hydroxide solution. The solution with a pH of 11.0 is sterilized by filtration on a "Millipore 0.45 / u" membrane.
The vials containing sterile medium A are distributed in 4 batches Al, A18, A ~ g and A20 of 2 vials each and carry out -; in each vial of lots A18 and A19 the following additions:
- batch A18: 0.5 cm3 of the barbital solution (0.5 g / l) - batch A19: 2 cm3 of the barbital solution (2 g / l) the vials in lot A1 not undergoing any addition and the vials of lot A20 receiving, under the conditions indicated below, an addition during the culture.
Each vial of lots A1, A18 is then inoculated '' ~ 61 -~ 6 ~ $ 322 A19 and A20 with 2.5 cm3 of an inoculum culture of Streptomyces coeruleorubidus DS 7.126 (NRR ~ 8098) aged 72 hours.
The vials are incubated in an enclosure at 26C on table stirred at 220 rpm. After 24 hours of incubation, add 1.5 cm3 of the solution to each flask in batch A20 barbital (1.5 g / l) and the incubation of these vials is continued under the same conditions of agitation and temperature.
After ~ 7, ~ day of incubation the vials are processed under the conditions described in example 12. ~ es results following were obtained:

~ barbital ote (g / l) 3,999 RP ~) Alg 2 ~ 5 23 A20 1.5 _ _ By operating as above, using a medium of crop A divided into lots A1, A21, A22, A23 and 24 each, lots A2l9 A22, A23 and A24 receiving additions 0.5 g / l sodium phenobarbital, 0.5 g / 1 butobarbital sodium (sodium salt of 5-butyl acid ~ 5-thyl acid barbiturate), 0.5 g / l and 2 g / l penthiobarbital (5-ethyl acid (1-methyl-butyl) -5 thiobarbiturate), and continuing the incu-feeding for 9 days, the following results are obtained:

6 ~ 2 2 5 ~ ots Adjuvants (g / l) 32 999 RP (mg / l) '_. .

A21 phenobarbital 0 ~ 5 9 A22 butobarbital 0.5 12 A2 ~ penthiobarbital 0.5 12 penthiobarbital 2 22 Example 23 -a) ~ ermentation -.
Charged in a 75 liter fermenter:
- soaa flour., 1500 g - soluble distillers ... 250 g - partially hydrolyzed starch., 1750 g - soybean oil ... 750 cm3 . . .
- sodium chloride.,. 500 g - city water qs ... 45 liters - ~ e pH is adjusted to 7.50 by addition of 40 cm3 of sodium hydroxide 10 N. The medium is sterilized by bubbling steam at 122C
- for 40 minutes. After cooling, due to the conden-sation of steam during sterilization, the volume of broth is 50 liters; the pH is 6.70. We seed then with 200 cm3 of an agitated Erlenmeyer culture of the Streptomyces coeruleorubidus DS 7.126 (NRR ~ 8098). ~ a culture ~
is developed at 30C for 25 hours while shaking and aerating with third air; it is then suitable for sowing cement of the productive culture.
~ a productive culture is carried out in a fermenter of 800 liters loaded with the following substances:
`- soy flour ........................................ 3.2 kg - baker's yeast cream ........................ 24 kg , 1C) 682ZS
- lactose ... 23.2 kg - sodium chloride ... 098 kg - monopotassium phosphate ... 0.4 kg - magnesium sulfate heptahydrate ... 0.04 kg - ferrous sulfate heptahydrate, ... 0.04 kg - zinc sulfate heptahydrate ... 0.04 kg - anti-foam - 30 cm3 - city water supplement to 200 liters After adjusting the pH to 6.50 by adding 210 cm3 of 10 N soda, add:
- glycerin ... 7.6 kg - calcium carbonate ... 0.800 kg - city water supplement to ... 360 liters ~ e pH of the medium is then equal to 6.45. We sterilize - broth by bubbling steam at 122C for 40 minutes;
after cooling, due to condensation of steam during sterilization, the broth volume is 400 liters; the pH of the medium is 6.25.
10 liters of a sterile aqueous solution are then added.
rile containing:
- sulfanilamide ... 308 g ~ e fold of the broth becomes equal to 6.80. We are ~ emence ,, ~., with 40 liters of inoculurn culture in 75 liter fermenter described previously. ~ a culture is maintained at 27C while shaking with a turbine rotating at 205 rpm and aerating with a sterile air volume of 20 m3 / h. After 24 hours of culture S liters of sterile aqueous solution containing:
- sul ~ anilamide. ,, 44 g and we continue the: ~ ermentation. After 140 hours of culture, the pH of the broth is 6.75 and its volume of 350 liters; the must content in 32,999 RP is 35 mg / l.
-.,. ~.

lV68Z25 b) Extraction To 350 liters of must obtained previously ~ ment is added 350 liters of methanol and 6N sodium hydroxide to bring the pH up at 7.5 The mixture is stirred for one hour. After addition of 25 kg of filter aid the suspension is filtered on a filter hurry. The filtrate is collected. ~ e cake is washed by 100 liters of 50% aqueous methanol. ~ e ~ iltrat and the ~ age - are combined and concentrated under reduced pressure to a volume of 250 liters. After alkalization by addition of 220 cm3 of sodium hydroxide 6 N the antibiotic contained in this concentrate is extracted with 2 times 150 liters of the ethylene chloride mixture ne - butanol (80-20 by volume ~ es 238 liters of extracts '' organic are concentrated under reduced pressure until a volume of 30 liters. Then added to the concentrate, 100 cm ~
of n-butanol saturated with 11N hydrochloric acid.
up to a volume of 3 liters, we slowly add the solution in 21 liters of hexane. ~ e precipitate obtained is - ~ wrung, the ~ é and dried. This gives ~ 37 g of hydrochloride from ~ 2,999 gross RP.
c) Purification -1) 112 g of crude hydrochloride of 32,999 RP obtained co ~ me indicated previously are dissolved in 1 liter of water at 50C. ~ a solution, the pH of which is 2.9, is filtered through sintered glass.
~ the colored impurities contained in this filtrate are extracted twice 500 cm3 then once 1 liter of chloroform. After addition of 75 g of sodium chloride to the aqueous phase on extracts the antibiotic with 3 times 0.5 liters of n-butanol. 1st pH of the aqueous phase is adjusted to 9.5 by addition of sodium hydroxide concentrated and the remaining antibiotic is extracted per 0.5 liter of n-butanol.
~ butanolic extracts containing the antibiotic are combined and concentrated under reduced pressure to a ",.

1 liter yolume. We then have ~ 1 liter of chloroform ~
this concentrate. ~ a ~ olution is washed ~ e by 2 times 500 cm3 of water alkalized ~ pH 9.5. The 80nt wash water extracted by 2 times 500 cm3 of the mixture ~ chloro mixture ~ elm - n-butanol (80 ~ 20 in volume) ~ pH 9 ~ 5. The organic extracts are gathered, then concentrated ~ ~ or reduced pre ~ sion. During the concentration we add ~ 00 cm3 of the n-butanol - water - hydrochloric acid mixture 11 N (94-5-1 by volume). ~ a concentration is continued ~ until a volume of 150 cm3 is obtained then the solution e ~ t a ~ out ~ e slowly ~ 2 liters of hexane. ~ e precipitate obtained is sssoré, washed and dried ~. 30 g of a product are thus obtained.
holding hydrochloride of 32,999 RP.
2) ~ 3 g of product thus obtained are dissolved in 26 cm 3 of the water - dioxane mixture (20- ~ 0 by volume) ~ 35C. ~ 'antibio-tick crystallizes by slow addition of 624 cm3 of dioxaneO
After 3 hours of stirring, the crystals are wrung out, washed and æ ~ ché ~. 2 g of a crystallized product are thus obtained ~ containing - ~ 320999 RP hydrochloride.
3) 4.47 gd ~ 'a criætallized product obtained as indicated before-cédem ~ ent are dissolved in 40 cm3 of water. ~ a solution e ~ t filtered. ~ e antibiotic hydrochloride 32. ~ 99 RP con-held in the filtrate crystallizes by adding 405 cm3 of acetate tone while shaking. ~ es cri ~ rates obtained are e ~ sor ~ s, la ~ és and dried. 2 ~ 47 g of a product containing 32.999 RP hydrochloride crystallizedO
4) 2.84 g of product obtained as indicated below from ~ su ~ are put dissolved in 2894 cm3 of the methanol - acetone mixture (50-50 in volume). The solution 16 ~ 9 cm3 of the acetone mixture is added.
water (84-16 by volume). By heating ~ age ~ 30C then cooling 3o the hydrochloride of 32.999 RP crystallizes. ~ crystals obtained ~
are wrung ~ s, washed and dried. This gives 2.33 g of semi-puri hydrochloride ~ i ~ of 32,999 RP crystallized ~

.

:.

5) 1.85 g of hydrochloride se ~ i-purified obtained com ~ e indicated previously are dissolved in 400 cm ~ of water at pH 5.5.
Colored impurities are extracted from this solution by 10 times 400 cm ~ of the n-butanol - chloroform mixture (20-80 in volume). By concentration of the aqueous phase during which we add n-butanol we get a butanolic phase of 75 cm3 which is added slowly in 1.5 liters of hexane. ~ e precipitate obtained is drained, washed and dried. We thus obtain `1.4 g of pure 32,999 RP hydrochloride which is identical to product described in Example 8.

., A - ~ ERMEN ~ A ~ ION
Charged in a 170 liter fermenter: -- corn-steep (50% dry extract) .............................. 2400 g - sucrose ................................................ ..... 3600 g - ammonium sul ~ ate ........................................... .. 2 ~ 0 g - calciurn carbonate ........, ............ 900 g - - city water qs .......... ~ .......... 110 liters ~ e medium, whose pH is equal to 6.0, is sterilized by bubbling steam at 122C for 40 minutes. After cooling, due to steam condensation during from sterilization, the volume of the broth is 120 liters;
the pH is 6.65. Inoculated with 250 cm3 of a culture in an Erlenmeyer flask with Streptomyces atroviolaceus DS 89 ~ 8. ~ a culture is developed at 26C for 33 hours by shaking and aerating with sterile air; it is then suitable for the sowing of the productive culture.
~ a producing culture is carried out in a fermenter of 800 liters loaded with the following substances:
- flour ae 90 ja .......................................... 22 kg - soluble distillers .................................... 2,750 kg - starch (partially hydrolyzed) ....................... 16,500 kg ,.

- 10 ~ i8225 - so ja oil ....... ~ ............ 2,750 liters - sodium chloride ................. 5,500 kg - city water qs ................ 510 liters ~ e pH of the medium is adjusted to 7 ~ 5 by addition of . ~.
500 cm3 of ~ or 10N, then the medium is sterilized by bubbling steam at 122C for 40 minutes. After cooling, due to condensation of steam during sterilization tion, the volume of the broth is 550 liters. The pH of the medium is 6.60. Inoculum culture is carried out with 55 liters in a 170-liter fermenter described above. IJa culture is developed for 117 hours at 26C by stirring with a turbine rotating at 205 rpm and ventilating with a volume of air - sterile 15 m3 / hour. ~ e pH, at the end of culture, is equal to - 7.60.
B - EXTRACTION
To 560 liters of must obtained as described, add 28 kg of oxalic acid. After stirring for 1 hour at 40C and addition of 25 kg of filter aid, the must - acidified is filtered on a filter press. ~ e mycelial cake is washed with 120 liters of water at 50C, containing 3 kg of oxali-than. ~ e filtrate and washing combined, whose pH is equal to 1, are cooled to 10C. ~ e pH is adjusted to 4.5 by addition of 50 liters of 6N soda. ~ 'antibiotic present in this solution tion is fixed by passage 9ur a column containing 25 liters of AM ~ ERLI ~ E IRC 50 (trademark) in H ~ form. ~ has resi-is washed successively ~ ent with water, until the e ~ fluent colorless, then per 100 liters of methanol-water mixture (50-50 by volume) and per 100 liters of a methanol-water mixture (90-10 by volume). Finally the antibiotic is eluted by 250 liters a methanol-water mixture (90-10 by volume) containing 1%
(weight / volume) of sodium chloride. ~ 'eluate is concentrated -; - 1 SOU9 reduced pressure (5 to 10 mm of mercury) at 40C up to a . ~.

volume of 20 liters. The concentrate, whose pH is adjusted to 9 ~ ^ - by addition of 11N sodium hydroxide ~ is extracted once 20 liters, then 2 times 10 liters of chloroform. We get a total of 35 liters of extracts which are concentrated under reduced pressure (5 to 10 mm of mercury) at 40C up to a volume of 2 liters ~.
~ a remaining solution e ~ t acidified by addition of 50 cm3 of n.butanol saturated with 11N hydrochloric acid and concentrated to again up to a volume of 1 liter. ~ a solution is paid slowly in 7 liter ~ of hexane, ~ e precip ~ té which forms is wrung, washed and dried. 13 g of hydrochloride are thus obtained.
te antibiotic 32999 RP raw.
C - PURI ~ ICA ~ ION
a) the antibiotic is purified by distribution against the current in 3 ampoules of 2 liters A, B, C.
A mixture of methylene chloride-n.butanol- is prepared.
tarnpon phosphate M / 15 at pH 7.38 (8-2-10 by volume). When the mixture is in equilibrium, the two phases are separated.
In bulb A, 500 cm3 of an acid mixture are poured phosphoric (85% by weight) - water (1-125 by volume) and 500 cm3 of heavy organic phase. It is dissolved in this mixture of phases 13 g of crude antibiotic hydrochloride obtained as ; indicated paragraph B. ~ nearly 15 minutes of agitation and eliminated-tion of an insoluble matter by filtration, the pH 7.5 is adjusted by addition of soda N.
In bulbs B and C, 500 cm3 of phase are loaded light aqueous, the pH being respectively adjusted to 7 in bulb B and 6.5 in bulb C, with phospho-85% risk.
~ n starting from bulb A to pass through the bulbs B then C, a countercurrent distribution of 5 is carried out transfers, using the heavy phase (solvent) as phase mobile and light (aqueous) phases at pH 7.5 - 7 and 6.5 .
as stationary phases, implementing each time 500 cm3 of mobile phase and keeping the pH of the stationary phases.
The antibiotic contained in the aqueous phase of bulb C is extracted at pH 9.5 with twice 500 cm3 of the mixture ge chloroform-n butanol (8-2 by volume). ~ organic extracts that all are concentrated under reduced pressure (5 to 10 mm mercury) ~ 40C. During the concentration we add 30 cm3 n.butanol containing 6 ~ (by volume) hydrochloric acid 2N. ~ e concentrate is brought to a v ~ lume of 5 cm3, then slowly poured into 500 cm3 of hexane. ~ e precipitate which form is wrung, washed and dried. 275 mg of ; semi-purified 32999 RP hydrochloride.
b) 80 Mg of the hydrochloride obtained as indicated above are dissolved in a mixture of 1 cm3 of water and 1 cm3 of methanol. This solution is also distributed in the form a linear deposit on 4 analytical chromatography plates in a thin layer of MERCK silica gel. ~ the plates are developed ~ coiled for 1 hour in a tank, the walls of which are 20 covered with filter paper, containing chloride mixture methylene-formic acid-methanol (80-17-3 by volume). After drying, the area containing 32999 RP is recovered by scraping floor of the ~ ilice. ~ a powder obtained is suspended in 40 cm3 of water, the antibiotic is extracted at pH 9.5 by two times 20 cm3 of chloroform. ~ the organic extracts gathered are concentrates 90US reduced pressure (5 to 10 mm of mercury) at 40C.
During the concentration, 1 cm 3 of n.butanol is added.
6% (by volume) of 2N hydrochloric acid. ~ e concentrate is brought up to a volume of 2 cm3, then slowly poured into 50 cm3 of precipitated hexane ~ e which forms is drained, washed and dried.
This gives 4 mg of pure 32999 RP hydrochloride in the form of an orange-red microcrystalline powder melting around 200C

~, ~.

`~ 06 ~ 3Z25 a ~ ec decompo ~ ition which is identical to the product obtained at Example 8.

. ~

Claims (4)

The embodiments of the invention, about which an exclusive right of property or privilege is claimed, are defined as follows: 1. Process for the preparation of a new substance basic antitumor, designated by the number 32 999 RP, the hydrochloride has the following characteristics:
- it is an orange-red microcrystalline powder soluble at about 100 mg / cm3 in methanol, the pyridine, dimethylformamide and water, 10mg / cm3 in the butanol saturated with water at 20 ° C, 5 mg / cm3 in ethanol and below 0.1 mg / cm3 in acetone, chloroform, chloride methylene, ethyl acetate, benzene, methylisobutyl-ketone, dioxane and hexane - its elementary composition is close to:
C% = 56.6 H% = 5.7 0% = 29.41 N% = 2.39 Cl% = 6.0 - its rotary power determined in 0.09% solution in 0.1% ethanol of 1N HCl is:
[.alpha.]? 0 = + 208 °? 32 °
- its ultraviolet spectrum and its visible spectrum determined from a solution of 10.65 mg / l in ethanol at 0.1% of 1N HCl have absorption maxima at 233 nm, 254nm, 292 nm, 493nm, 512nm and 527nm - its infrared spectrum presents absorption bands-tion to 3410, 3250, 3220, 3050, 2970, 2930, 2900, 2860, 2820, 2680, 2600, 1980, 1930, 1900, 1800, 1735, 1690, 1635, 1600, 1580, 1510, 1460, 1440, 1405, 1370, 1315, 1285, 1255, 1235, 1195, 1165, 1115, 1062, 1050, 1030, 1010, 985, 960, 930, 915, 885, 875, 845, 840, 820, 810, 780, 762, 750, 725, 708, 695, 665, 640, 625, 600, 580, 565, 540, 530, 485, 475, 465, 450, 438, 415, 390 and 320 cm-1 - in ascending thin layer chromatography of silica gel with the mixture as a developing solvent methylene chloride-formic acid-methanol (80-17-3 by volume), it has an Rf of 0.2 - it is active on leukemia L 1210, leukemia P 388 and leukosarcomatosis at doses between 0.125 and 0.250 mg / kg ip in mice, and whose formula is said substance being taken as free base or salt addition with a pharmaceutically acceptable acid, this pro-ceded being characterized in that one reduces microbiolo-carminomycin formula or one of its salts, or cultivates a microorganism chosen from:
Streptomyces coeruleorubidus DS 7126 (NRRL 8098) Streptomyces coeruleorubidus DS 31 723 (NRRL 3045) Streptomyces coeruleorubidus DS 8899 (NRRL 3046) and Streptomyces bifurcus DS 23 219 (NRRL 3539) in an aerated nutritious medium suitable for Streptomyces, in presence of adjuvants chosen from amethopterin, barbital, phenobarbital, butobarbital, penthiobarbital, sulfanilamide, sulfapyridazine and sulfathiazole then extracts the antibiotic from the culture medium, or cultivates Streptomyces atroviolaceus DS 8938 (NRRL 8148) or its mutants producers in an aerated nutrient medium containing sources of assimilable carbon and nitrogen and mineral salts to a initial pH of 5.8 to 7.8 and at a temperature of 23 ° to 33 ° C then extracts the antibiotic from the culture medium. 2. Method according to claim 1, characterized in what reduction is done by means of a culture of a microorganism chosen from Streptomyces lavendulae (ATCC 8664), Streptomyces roseochromogenes (ATCC 13.400) Corynebacterium simplex (ATCC 6946) or Bacteriurm cyclooxydans (ATCC 12.673), or cells isolated from this culture or extracts enzymes obtained from cells of these microorganisms. 3. The basic antitumor substance 32 999 RP of which the hydrochloride has the following characteristics:
- it is an orange-red microcrystalline powder soluble at approximately 100 mg / cm3 in methanol, pyri-dine, dimethylformamide and water, 10 mg / cm3 in butanol saturated with water at 20 ° C, 5 mg / cm3 in ethanol and less than 0.1 mg / cm3 in acetone, chloroform, methylene chloride, ethyl acetate, benzene, methyl isobutyl ketone, dioxane and hexane - its elementary composition is close to:
C% = 56.6 H% = 5.7 0% = 29.41 N% = 2.39 Cl% = 6.0 - its rotary power determined in 0.09% solution in 0.1% ethanol of 1N HCl is:
[.alpha.]? 2 = + 208 °? 32 °
- its ultraviolet spectrum and its visible spectrum determined from a solution of 10.65 mg / l in ethanol at 0.1% of 1N HCl have absorption maxima at 233 nm, 254 nm, 292 nm, 493 nm, 512 nm and 527 nm - its infrared spectrum presents absorption bands-tion to 3410, 3250, 3220, 3050, 2970, 2930, 2900, 2860, 2820, 2680, 2600, 1980, 1930, 1900, 1800, 1735, 1690, 1635, 1600, 1580, 1510, 1460, 1440, 1405, 1370, 1315, 1285, 1255, 1195, 1165, 1115, 1062, 1050, 1030, 1010, 985, 960, 930, 915, 885, 875, 845, 840, 820, 810, 780, 762, 750, 725, 708, 695, 665, 640, 625, 600, 580, 565, 540, 530, 485, 475, 465, 450, 438, 415, 390, and 320 cm-1 - in ascending thin layer chromatography of silica gel with the mixture as a developing solvent methylene chloride - formic acid methanol (80-17-3 in volume), it has an Rf of 0.2 - it is active on leukemia L 1210, leukemia P 388 and leukosarcomatosis at doses between 0.125 and 0.250 mg / kg ip in mice and whose formula is said substance being taken as free base or salt addition with a pharmaceutically acceptable acid, when prepared by a process according to claim 1 or its equivalents manifest slow chemicals. 4. Substance 32 999 RP, when prepared by the process of claim 2 or its chemical equivalents manifestos.