DE1909198A1 - Antibiotic A 204 and process for its preparation - Google Patents

Description

EIT Lilly and Company, Indianapolis , Indiana, V, St, Antibiotic A 204 and its method of manufacture

The invention relates to new antibiotic compounds "which are referred to herein as A 204 and A 204 II" and their salts, as well as a process for their preparation

Compound A 204 can be identified by the following information: It is a white crystalline solid. represents after recrystallization from Kthyläther at 96 - achisilst 98 ⁰ C, in esters, Benssol, halogenate hydrocarbon s, Dimethylformamidg DimethyIbuIfoxid; Ethers and ketones soluble, sparingly soluble in alcohols and very sparingly soluble in water; has acidic properties, a titratable group bit has a "pK'a value of 6 J " ®in BUO titration values a traitelte Eoleteilargtwioht τοη has about 900, 61 ₅ 74 $ carbon _for 9 _S 57 1> hydrogen ta "! 28j30 £ oxygen contains an aptxifise & en optical rotation value (a) ^ ⁵ = + 68.12 ° (G «1 /», Qm-pfol, in Hetnenoi) iiQöo when measured in the form of a chloroform lion, the following distinguishable bands ia lafrarotabsorptiosisapefetruai im iareieh from 2.0 to i5jO Mieron seigti 2-95? 3.43 * 5 ₃ 95; S _s & 7i 7.26; 7 _f 80f 8.52; 8.95s 9 »1? J 9 ₂ 34 * ₃ 9 _s 59i 9 ₃ O2i 10.0Ti 10, Mj 10 _s 51; 10.77-, 11 _a 40 and 11.80 micron * md koin oharakte-

having,

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The compound A 204 II can be identified more precisely by its mixed sodium-potassium salt, the white crystalline solid ₉ which in esters ₉ benzene ₍ halogenated hydrocarbons, dimethylformamide, dimethyl sulfoxide; ethers and ketones soluble, only slightly soluble in alcohols and in water very slightly soluble iat _e a titratable group with a pK'e value of _6; 3 in a 66 ti strength dimethylformamide-water solution _S contains a specific rotation (a) ^ ⁵ = +42 ° 3 ₅ ° (C = 1 $ Gew AoI in methanol) and, when measured in the form of a chloroforal solution, shows the following lower combined bands in the infrared absorption spectrum in the range from PO to 15 * 0 microns: 3 ₅ 2O; 3.45; 6 _; 26; 6-90; 7.30; 7c68; 7 ₆ 80; 8 _p 20; 8 _s 50; 8.80; 9.22; 9 ₉ 39; 9.59; 9.70; 9.82; 10; 08; 10p26; 10.48; 10 _s 83i 10 _; 96; 11,30; H _O 55 and 11 _f 78 microns,

The specific optical rotation value was determined from the crystalline free acidic compound after drying for about 1½ hours at low temperature in vacuo over anhydrous calcium chloride. The titration was carried out as eletetroiastri © cli @ titration in a 66 £ -igan Simethylformoiaid-VaaQer solution and for the Eleaentaranalyaen was dia in the vacuum at about 80 ^α ι
bindings used,

Vacuum at about 80 ⁰ G getisOekneten over phosphorus tntoside

W Antibioticua A 204 and A 204 II are produced by cultivating a strain of Strsptooycee albus with aerobic Bsdinguagen Ib a culture medium which contains aasimllbaren ICofeleaatoff _s Stickatoff and inorganic Salae.

The Orgenieaaa were for the first time from soil samples »ils sös Perry., Florida» V-St-A.- "came from following measured ieoilar'ss" Parts of the soil samples were suspended in sterile distilled water- The Suapenaionen were In Fora you Strc- Ifss

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coated on nutrient agar plates The inoculated nutrient agar plates were cubed several days at about 30 ⁰ C ^. * After the Inkubationedauer were colonies of A 204-producing organisms using a sterile platinum Bchlinge on slants überführt- The inoculated Schrägagarmedien were then _incubated larger quantities To obtain inoculum for the production of antibiotic A 204,

This antibiotic A 204-producing organism has been deposited without restriction in the permanent culture collection of the Northern Utilization Research and Development Division, Agricultural Research Service _v US, Department of Agriculture (fri!> · Northern Regional Research Laboratories) ^ Peoria, Illinois, and stands for Available to the public under cks Kulturnurnaer NRJiL 3384.

Because of the difficult Celts of the taxonomic classification \ ^ a organisms of group Streptomycee the classification of a newly discovered organism is always subject to some uncertainty, but it seems that the organism ,, the antibiotic A produces 204 "in its most important properties of the published description of Organisms Streptomyces albus, Streptomyces albidaflavus and Streptomyces flaveclue comes closest * On the basis of the description of the neotype strain of S-albus ATCC 3004 by Lyons and Pridhaa, fj Bacteriol 83 »370-380 (1962) J _V and the strain S-albue IMRU 3005 by Waksman, / "The Actinomyoetes, VoI, II" Classification, Identification, and Description of Genera and Specie, "Williams and Wilkins Co., Baltimore (196I) J, however, the culture is believed to be a strain of Salbus (Rossia Doria) Waksman and Henri ei, iet ..

Despite the similarities, however, enough differences exist that the new organism described herein _s

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differ from all previously described strains of S. albue. That is why the organism NRRL 3384 became the newer Streptomyces albue classified "

The detailed description given herein focuses primarily on the new organism NRRL 3384 It is emphasized, however, that the production of antibiotic A 204 with the aid of mutants of this A 204-producing organism Also within the scope of the invention- Such mutants can be generated by known measures, for Example by treating the above-identified strain with X-rays, ultraviolet radiation or chemical ™ Agents such as nitrogen mustard or other mustard oils.

Pur the taxonomiflchen investigations of antibiotic A 204 erzeugenuen strain of S Albue were the methods _s recommended for the International Streptomyces Project and Shirling and Gottlieb / Methods for Characterization of Streptomycee Species, "Intern. Bull" Systematic Baeteriolg 313-340 (1966 ) were used together with additional tests. The determinations of etoffverwertung were carried out according to the method carried out by ii @ Pridhasj and Gottlieb, J-Bac- 56, 107 (1948) de- The Ergobnisae of the taxonoraisehen determinations are given in table form -HBS method (Kelly and Judd ₉ "The ISCC-NBS-HetaoS o £ Designating Colors and a Dictionary of Color of CoisBiarce Circ 553 Washington, DC) gsgebea Klanaera refer to the numbers given by Tresner and Backu3 (Tresner and Backus / V stem of Color Wheels for Streptomyces Taxonomy, "Appld, ffierofeiol 11: 335-338), Parb blocks according to Maerz and Paul (Maerz and

1950. Dictionary of Color. HcGrav-Hill, New York) are in square brackets

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- 5 - Table I.

Description of the culture NRRL 3384

observed feature

properties morphology

Cultural characteristics on: oalciumaalat

usually forms epiral sporophores aged 3-50 spores per chain 10-50; Spores ovoid bit elongated 1 »9yU χ 1« 3 »i smooth spore recognizable surfaces in electron microscope images»

Growth abundant, underside pale yellow- green / TOBlJ ', sporulation and aerial mycelium abundant, (7) pale yellow, 2ba ^ TlCl7; no soluble pigment; Medium slightly clarified

Tomato paste - oatmeal

profuse growth, grayish yellow underside ^ 1B27; Abundant sporulation and aerial mycelium (W) knows a; no soluble pigment.

iei ^J Mr. 2

(Yeast-Itele agar) abundant growth, grayish yellow underside ^ "1BiJ; sporulation and aerial mycelium abundant (Y) pale yellow, 2ba ^ 1CiJi no solubleβ pigment.

(HefoFaehl agar) moderate growth, white underside; Sporulation and aerial mycelium moderate (W) a; no soluble pigment

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Table I (Forteο)

observed characteristic properties

ICF No. 4

(Inorganic salts «starch · agar; abundant growth _B underside gray tichy yellow / TiB ^ s spurlation and aerial mycelium abundant (Y) pale yellow 2ba ß \ CiJi keia soluble pigment»

ICP Mr. 5

(Glycerine Asparagine Agar) abundant growth lower rope © pale yellow green / TOBiJs spoliation and air celium plentiful ä; no löaliciiea

Czapek -Agar gutee
Sporulation wiü (W) white

Uatereoit © Weißι ImS tmysel gut

Tyrosine »agar colors ssugeseite

> Spoj? Ulation rai myeel moderately (Y) felaßgeXfe no ISaliches pigment,

Emerson agar

Bennett agar abundant naüMaüwao gray tichig -; g @ llb lation and Lu £ tmj <s @ l (GY) yellow lee-g; no

very Colours

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Table I (Porte.)

observed characteristic properties

Nutrient agar

Effect on milk

Nitrate reduction Melanin = education

Peptone iron agar tryptone yeast extract

Gelatine liquefaction temperature requirements moderate growth _ς underside greyish-yellow ßV # 2J \ sporulation and aerial mycelium moderate (W) white a; no soluble pigments

no change after 14 days "

positive positive

negative negative

none in 14 days

Growth and sporulation moderate at 26 o; well at 30 ° i ** ege tatives growth at 37 ⁰ C; Trace white vegetative only at 43 -

No agreement

leaktioa ron substrate =

l a «f pH change

Reaction won soluble pigment pH Ä? I.derung Alls media up to ICP No. unaffected change on ICB No. 4 · Ton su white or brown färb ins at Zusatss 0 _r 05n WD or O ₉ Oju NaOHc

no soluble pigment

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Table I (Porte.)

observed characteristic properties

Carbon recovery

L-ArabiRoße 4-Saeeharose (-) D xylose O) D-pructose (ψ) Glucooe ψ lihamxioee + Caffinose -ΦΙ »inositol + D-mannitol ^ without carbon (control)

Explanation of symbols?

ν ~ positive

(-2-) «recovery w

(-) - Location fragliek

- = k8l?: <e Ye

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Ale culture medium ear production of antibiotic A 204 by breeding dee Organlsraue above described can be used vereehiedeneten media _β of the organism since, as is apparent Auss the ¥ above ®rwertungsteate It unterschiedlich® rgi®qM © @ ll®E to utilize vermag- For a wirteciiaftlleh® production _s maximal® build-ups of antibiotic and IeIeMe insulation $ © e Antibioticuina, however, some proportionally eiafeeii® sources are imminent to B © iopi © lm @ ise ®nthB.tt, en di © media »which are used for the production ά © @ AatlfetotlcmsB worteilfeaft are * ain © aseimiliarbare Qusll © fto löteleaetoff wi © Fructoe®, Starkes Glyseri ©, Si @ l © ese _s Bsxtrin or second ο the hevcr ^ ugm Qm © ll @ fiif Koiilsastoff is Cligaltisae @ @ ^ nth feiles gIuq Q &, ell® for

peptoa or Sojap®ptoa) _B Gb © © 1e Bevoffsiigta Quelles fü? S ^ ieiso ^ off ciisä Popton © “Bofel and AmlnQB'awsQgQmiBuliQο W® ä © a © sao ^ igaEAiQ c! L® dsü k ^ ltiiffQQiiiöB stagGscSst viskos k5aa © E _c

^J iiosptet- _{0 0} sulfate ßhlo £ ä {I ~ ₉ ^ ee feeao ^ = wa & öfeläeh ©! On "α

ilo fSr ola © ptiealeso ^T «ie @! iotiai3

iss OrganleeiiSg öqs ³ sias ¹ f ^ oimfetion, ν-α δ AatifeaotlisMH A 204 ^ ®stsQmuGt ι-ιίτύο opfo ^ offlieti sind, 3-jHea sbafifalls la a®m Knltm ^ mQuitim © Öelsh = euillQB, sate? . als ^ gmmr®iiiigimg & n in ^ ea BosoagieHsil®d © s I ^ diuas la Maagsa? οϊ, di ©

Do? aafSagliete pH ~ W @ rt d®a Kisltujpaediyms kana scnvmnkena Ee was jadoeh gsfimdan »iaß the Aafsfögs pH value dee mediuna 3, i © c; ksf ^: iiiig ETwisehasi €> ₉ ^ imü 7 _S 5 was observed as with other astiaehtycotan was not the pH value of the medium

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during the Wachotuina period dee Organieaua ,. _s is gradually is generated to the antibiotic and may include a waiting 7 ₉ 0 to 7 _S 6, or reached about "The End -pu- value depends wenigetene partly before the beginning = pM-W © 3 t of MediuffiB?; the buffers contained in the medium and the period of time during which the organism is allowed to grow v / i yd _{β ;}

Submerekulturen are the conditions of choice for the production of antibiotic A 204- To produce relatively klelnar amounts can be used in bottles Sehüttelkolbssikulturea and Oberfläshenkultüren ,, For Hero distribution of large quantities wäitl contrast, aerobic © -Suabers culture in sterile tanks preferably-Medium in &<sim<Bt®ril® tank canoe tait Elsser SpoFensiaspeaoiaa lao & uliert v / ground "V / egoin the WachstUES ^ ^ srziJ srungi, dis feei - fsrwendung a Sporeneuopeneion as liDcalua beobaehtat v / ill ₀ Uird j © Teh vegetative Sulturfora pre ^ agt «Isäea auf di®s® Weios the Wasfestiame" FersögerErig TsyEiaöea wi? d _e becomes a® wi ^ toiia ©? ©

dar FQrme ^ tatioasvor ^ ieh ^ iaHg © ^ slelt- It is therefore s saaäshet eie νog © ¹ SDtIvQs Xaocalua des Organiaau © dursh iJsoeiiiieffG'ä silier vc-yhaltenaiBisäßig klsiaen M © ag © KuI-tunssedluia-old of the SpI-tunssedluia

wecn a j'uigeo aktif®3 ^ egsfati ^ as is to transfer the vegetafi ·? © Xaceuiuia aseptiseh in big seals Bas Hsdlu @ ₈ in dam da® iregetative Inciculua is suckled »can be either the same or a different one Meiius ice that used on a large scale for the E ^ sougang of Antibiotic I 204.

The OygsLnisz.ua} of Antibiotieiia A 204 gVs © ugfe ₈ xf & © km% lsi a-far-an Tesperaturboreich voa 25 bla 31 ⁰ that an optimal production of A 204 ^{takes place at Tesapsrature 26 to 30 0} C 5 <> generally takes place © ine formation of the Astibioticuns innerliallb of about 48 "=? 2 after Inc-culate üca culture medium- As as at

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Merskulvurverfahren is common, v / ird through the culture medium sterile air blown »To achieve a beneficial Growth of the organism and an effective formation of antibiotic Λ 204 should contribute to the volume of air the tank production of A 204 0.2 to 0.4 parts by volume Air per minute and volume part of culture medium. Prevents 0.35 parts by volume of air per minute and part by volume Culture medium

The concentration of antibiotic activity in the culture medium can of samples of the culture medium for their inhibitory effect pm £ ias growth of organisms that are inhibited by the presence of r y isitibioticuia A 204 during the fermentation by Fv ^ f "ng easily overcome _β pursues the organisms Staphylococcus aureus , Sarcina lutei? ,, Bacillus aubtilis and Mycobacteri & m aviuss have proven to ^{be suitable for the 1} % bar. The test of the samples can be carried out using the AeT oekssHiten turbidometric method or the known agar bluffing method »

Ίί in general, in aerobic submere culture or in shake flask culture, a maximum of A 204 production of 2 to 3 days aaoh of isoculating the culture

Bio w £ b * enö the fermentation arseugte aatibiotic activity

aQT occur in. the antibiotic broth or in the i ^ ycel both. Those involved in the production of A? 04

n Isolation therefore ao selected to eiae iraxicale recovery of the antibiotic enable from each or baiäan sources "Thus, for example, the Peraentationabrühs _r as it is erlialten, filtered and the filtrate for recovery of the antibiotic activity that is not retained by (lea mycelial cake is extracted with a suitable solvent. Furthermore, the in the mycelium

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the antibiotic contained in the cake by thorough extraction obtained with a suitable solvent = in any case the antibiotic can be added to the extract using customary methods be won.

Chemical and physical analyzes of crystalline

A 204 show the presence of 4 + 5 methoxy groups esa ο

You? ApieL * ohrßsiatographie of antibiotic A 204 on Whatman paper Kr. I gave the following R _f -tfortes H ^. «O ₈ 14

in a Lueusigsmittelyeten aue water _t ^ ethanol »Aeetoia and Beaeol in a volume ratio of 72 s 24 ₉ 5 s 3 8 0 _B 5» K ^ s 0.87 in an "" oBungoffiitteleyetem from 10 $ ~ ± §®m water flgom n- propanol R * »0.33 in a Mswsgamittelsjsteii from ¥ qs®qf _b methanol nnü losigsäure in a ratio 'won 12 § 3 § 1 (withdraw solution is old NH ₄ OH to a pH of 1O ₈ 5 wad ia & ia e t S ^ P © ^

on pH 7sS tsiagasicsllt) · Eu? Beatiiaswsig de? WFefeiaeiisi asago = gebeüea Wss ^ t'S "iiiiräle daa Aatl ^ ioticiiia in astliaaelilsoSi ^ s ¹ M = aufget3Peg © a _e IMs ° eli ÄHflsgesi io ©

ίο osispft wai * ®a ₉ ι «ΐιΜ © ΐι Bloantog ^ aisao

Bti BünhsoSäicSitahroaiatagffßpSii © ^ © s & 204 ami? 5οώ lsi Atfejlscatat as LcO ^ agaEi'StGl ϊ © ΰ '7üs 7aalilin oier SshwofeleSMS ³ © © 1q IJq © Sau © i © al'S-Söl

Bas feti ^ i-uv ^ jQ ^ g ^^ a AiitibiöiiieiBia SL 204 Iq ¹ S o £ a uqISoj? liner Fs «<kj ^ off , j which at 144 = 14§ ^Q Q oefeiailst? 3fää duroh BSatseasEslyss fceotiaatse Hslekialas'geViTiigia'ö v © a 960. Bas Natrlusiealz let la prayers ₉ Bqslso1 _ö ImIogenlerten Kuhleavaasaratoffea, DHP ₉ BHSO ₀

borrowed and little itialish in alcohols. Bar __

"250
Rotation value (a) jf des be :. Room temperature in

anhydrous Calciuinchlorid getroekaetea for about 15 hours

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crystalline sodium salses of antibiotic A 204 is + 54 ₉ 95 ° (C * 2 #p wt. AoI. in methanol),

The infrared absorption spectrum of the sodium salt of antibiotic A 204 in chloroform is in Pig. 2 of the accompanying drawings: “In the infrared spectrum, you can distinguish _{between the following bands in the range of 2 s} 0 to 1 ^ 0 microß? 3 _f 1-3j2 (broad) i 3 ₀ 44; 6 _B 26; 6 _B 87; 7 _P 29i 7 _B 67; 7.79; 8 _P 45; 8.96; 9 _t H; 9 * 18; 9 _β 36ϊ 9d56; 9 ₀ 82 | 10.05; 10.26; 10.54; 10 * 95; and 11 _B 77 micron,

Since the molecular weight of the hydrated crystalline silver salts of antibiotic A 204, determined by X-ray dialysis, is about 1099%. Crystals of the silver salt lake are colorless, however, no! ? Light and Rönt.gensti ahleß © mpfindlich and after two days long irradiating in air dark brown- The by flotation in aqueous solution ZnGi ₂ = measured density of the Silberealzes is ren 1 ₀ 293 g /. ^ - di © ingenious Ex öitikiilonßPegel is hkls h * k - Zm, _t something on the monoclinic space group C2 without Syitunetrie ^ entrum hiaweiat.- There are 4 aeymiaoteieehe monoclinic units and 1 molecule per aeymm © triscs! ie unit in an Eleraentaraell © with the following dimensions

a * 25.-977 A ₀ b β 14 _S 517 A? O = U ^ 'B a, B ^ - 9I ₁ 94 A.

Vfie -ψ Lei © other antibiotics contains the Asitlbioticua. A g®leg @ R!; Lioh several closely related components · The sweite

is not found at allaa Perment-atioaen ₉ and / orliandc-a iat _c . It lies in the usual way bia au des.- Clesaiatamsbeiite tos ».-» This second smaller ₀ is given here with A 204 II .. Eh? has so far only been used in the form of the gamified na-

Eß obtained it t-mrds found ₉ that dae

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missfete sodium-potassium salt of A 204 II similar activity as the antibiotic A 204 exhibits.

Several Elemontaranalyoen in the vacuum at about 80 ⁰ G
Crystalline mixed sodium-potassium salt of A 204 dried over phosphorus pentoxide gave the following mean values: 60.66 1 » C ₉ 8.76 # H and 27 ₉ 09 JtO,

Several atomic absorption analyzes show that the a mixed sodium-potassium sale in ¥ ®rechiedeneu niaoen which contains 1 mole of the mixed cation per. The mixed sodium-potassium sals from A shows no characteristic ultraviolet absorpti © E-

By post chromatography on the mixed N. Potassium salees from Paktor II on gravel © leäuFeg @ lglatt © ia Xthyl acetate and proof by spraying with an R ^ -value of 0.75 wasetiiEst ..

Dt® antibacterial compounds of the invention
IV-J ⁰ industrial purposes, pharmaceutical purposes ©,
Suitable for specific and veterinary purposes, the term "industrial" is used to distinguish between dor Aa = phrase in human and veterinary medicine, Di® ferbiadumgon are usually combined in a known manner with suitable carriers to produce the desired peaticides.

The antibiotic A 204 and its salts g

Effective against coecidiosis infections «wenra -si ©« lern futter

added by chicks. With Kengen voss 10 tola §0 g

Antibiotic A 204 or common salt © per Tonn® patter (9

45 g / ton) island infections are allowed to E-te5a © lia _p

and Ε- combats maxima effectively »f4it the same% agon is © ia ©

effective control of a Miachiaf © ktios dareli E »

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IS. tanella as well as an infection by 8 cocci Bpeciefj (Coceivac type D) achieved "

The compounds according to the invention also have insect disease and aoaricidal activity. They also have an inhibitory effect on the wa cha turn of microorganisms for bacteria sand fungi, which are common to animals and
Plant pathogen siado The antibiotics bind therefore in the form of disinfectant solutions to sterilize the unavailable soil “of stables and the like and to keep such areas free from bacteria and fungi
suitableο

Bie compounds of the invention are further ivrigen against ^Λ bean powdery mildew and dwarf virus Corn In KonzentratΙο & θώ ^ ^t; 16-400 ppa v / irksam and therefore to combat such plant viruses and mushrooms suitable _β

Burch the folgenien example, © ^ iS ⁵ O & ± invention above described siäher "·

Bi © feoeiiktioa clay Aatibiotieum A 204 la shake flask

feiiltwrsE is dii:? @ h the following © example explains:

aue 10 g Bssj "feriii _c 2 g with

aii% © söiilt sseaem 0as © iia ₉ 1 g beef extract, - 1 g Hs-fGosstrak ^5;? 20 agar Imae water s "is sxergefatellt up to a total volume of 1 t ₀ is lüGüuliert with spores of Streptomy-jes IERL 3384 and 4 ms 5 days» © ei 30 ⁰ C

Ground the Sohrägfculutren with sterile distilled water

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Waeoer docked and carefully scraped off? to remove the organisms, and to obtain an aqueous suspension thereof. 1 ml of the resulting new spore suspension is used to inceulate 100 ml of the vegetative medium in each case

The vegetative culture medium is prepared by mixing 15 g glucose, 15 g seybean meal, 5 g corn spring water solids, 5 g sodium chloride, 2 g calcium carbonate and tap water up to a total volume of 1 liter. The vegetative inoculum is shaken for 48 hours at 30 ⁰ C on a reciprocating shaker with a rash of 5 ca (2 ") at 108 rpm. The inoculum thus prepared is then used as follows for the production of antibiotic A 204»

JSs becomes a production medium with the following composition manufactured:

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Soybean flour 15 g / l Casein 1 g / l

HaNO ₃ 3 g / l

Glucoee syrup 20 gA CaCO ₃ 2p5

tap water

100 al dee production media are filled into 500 ml Erlenmeyer flasks, which are then ^{sterilized at 120 °} C. for 30 minutes. After cooling, each flask is inocullertp with a 5 ^ -igeη vegetative inoculum which is prepared as described above.

The Produktionekolben are shaken for 48 hours at 30 ⁰ C on a rotary shaker with 250 UPK "The pH of the medium prior to inoculation is in a range of from 6.5 to 7.5- The pH at the end of Permentationecyclue is between 7.0 and 7.6. The antibiotic activity occurs both in the broth and in the mycelium. This activity is demonstrated by testing the broth and the mycelium against Bacillus Qubtilie according to the known agar diffusion method with agar discs or pans «

All of the broth (25 liters) obtained in this way is filtered in vacuo with diatomaceous earth as a filter aid. The mycelial cell is extracted three times with half the volume 50 36 ig in aqueous methanol. The three myeel extracts are combined and concentrated in vacuo to remove the methanol. The filtered broth and the aqueous concentrate of the mycelium extracts are combined, the pH of the mixture is adjusted to 3 with hydrochloric acid and the solution is extracted twice with half a volume of ethyl acetate * The ethyl acetate extracts are combined and concentrated to dryness _? and the residue is redissolved in chloroform (10 ml / g pesticide). The chloroform solution will

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through a 30 χ 102 co (12 χ 40 ") column of Pitfcurgh coal passed, which contains 2 g of charcoal per g of solid Then is the column is eluted with 5 to 10 times the amount of the original volume of chloroform.

The chloroforntluate fractions are combined, evaporated to dryness in a vacuum "dissolved in a small amount of existing methanol *, cooled and the resulting crystals filtered off." mg received. The crystals are by NMR analysis, IR Analyee, Dünnechiohtchromatographi "and Fapierchromatographie as described above identified as antibiotic A 204, and have a melting point of 96-98 ⁰ C.

Example 2 Production of antibiotic A 204

Antibiotic A 204 is according to the procedure of Example 1, . but generated using a production medium with the following composition!

glucose 15th β Soybean meal 15th G Maisquellwaseer-
foot fabrics 5 G NaCl 5 G CaCO, 2 β tap water
until 1 1

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Dae antibiotic generated is identified ale A 204 and has a melting point of 96-98 ⁰ C.

Antibiotic A 204 vd rd produced according to the procedure of Example 1 ₃ but using a production medium with the following composition:

Qlucobe 20 g (OT ₄ ) ₂ S0 ₄ 5 g CaCO ₅ 8 g KCl 4 g KH ₂ PO ₄ 0.4 g Soybean meal 5 g Tap water except for 1 1

Antibiotic produced by Bac is identified as A 204 and has a melting point of Tone 96 - 98 ⁰ C «

Example 4

Antibiotic A 204 is according to the procedure of Example 1, However, using a production medium with the following Composition produces:

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Glucose 10 g

Edible molasses 20 g

Peptcm 5 g

CaCQ ₅ 2 g

Tap water up to 1 g

The antibiotic produced is identified as A 204 and has a melting point of 96 · 98 ^° C.

At spie 1 5 production of antibiotic A 204 on a semi- independent scale

The production of antibiotic A 204 by subtle fermentation on a semi-organic scale is illustrated by the following procedure?

A 250 ml flask ₀ of 50 ml of a medium suitable for a vegetative tux a? S 10 g / l dextrose, 25 g / l starch _t 15 g / l Soyafutfcersohrots "» 0 g / l Ma 2 g / l Of'OO « in tap water containing _ff and _6? 5 eingeßtellt (Haoh) = is inoeuJiert with eiasr _Suspsßaion? AUO after surrounded in example method of Elnor Hähragarkultur 10 g ^ O ~ g H9fe- Extrakf "3 β NaCl, 0.01 g of 0 _r 25 g HgSO ^ - 7 H., 0 i? Ad 20 g Meerin 1 1 deioni3i8rtem V ^ asoer received words ο Das

Medium v / ird at «» ΐν / a 30. ⁰ C 48 hours on siner rotisreadta

Schüfe tel device with an arching v & m 5 effl (2 inch) passage se y "operated at 250 rpm" is incubated, 16 ml of the vegetative culture thus obtained

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= 21 -

used to inoculate a 1 l flask containing 200 ml of the medium described above. The inoeulierte medium is at about 30 ⁰ C for 30 hours on a rotary shaker with. A curve length of 5 cm (2 inch) diameter, which is operated at 250 rpm - 200 ml of the vegetative culture thus obtained are used to inoculate a 40 1 permentera, the 25 1 of a sterilized aqueous permeation medium from O ₉ 2 g / l antifoam agent (Dow Coral ag Antifoam): 25 g / l destrose, 15 g / l soya feed pots, 3 g / l dark molasses, .1 g / l casein and 2.5 g / l CaCO in tap water "operated at a temperature of 30 ⁰ C for 30 hours Y / ährenä the fermentation is stirred with a speed of 420 rpm =. Me Be ^ aeration during the fermentation takes place at a speed of Geschwin 0 _c 4 m / m medium and minute. The isolation of the antibiotic is carried out as in Example 1. The antibiotic produced is identified as A 204 and has a melting point of 96 98 ⁰ Co

Production, ren salts of the antiblotic A 204 aua broth and Μ, γοβΐ

The alkali metal, ammonium and amine salts of antibiotic A 204 are prepared from the broth and the Myeel according to the procedure of Example 7. By adjusting the pH of the Miachuag from filtered broth and aqueous concentrate of the Mjcelextrakta with the alkali metal, ammonium or in question Amine hydroxide adjusts to at least pH 8.5, extracted twice with half the volume of ethyl acetate and then as proceed as described above.

909851/1797

22nd

The acid and its salts can also be made from a number of Ketones, esters, alcohols and benzene hydrocarbons Mixtures imcrystallized or according to other known Methods, e.g. by column chromatography, are purified the.

Example 7 Preparation of the Watriua salt of Antibiotic A 204

A solution of 100 mg of the clay obtained according to Example "1 Antibioticume A 204 in 1 ml of acetone is treated with a drop of 5 η IfαOH and 1 ml of water. The mixture is clarified by gentle heating and at -15 ⁰ C ₉ bie stehengelaaaen crystallization takes place. The crystals are filtered off and urakriatallisiert from aqueous alcohol to 72 mg of the sodium salt of antibiotic A 204 with melting point ^{144-145 0} G,

Example 8

Production of the potassium salet from antibiotic A 204

The potash oil is produced according to the procedure of Example 7 using 1 drop of 5 η KOH anateile of 5 η NaOH obtained,

Example 9

Prod RECOVERY of S ilbersalges of Antibi otic A 204

670 mg of the sodium salt of antibiotic A 204 are ia 40 ral of methanol dissolved, with a solution of 270 mg of 8-alpha-nitrate mixed in 2 al water and at room temperature Left in the dark for 3 hours. The solution will be sur

909851/1797

Evaporated to dryness and washed with water to remove excess silver nitrate. The RUeketand is dissolved in 10 ml of methanol while warming, filtered hot through a ßinterglöatyichter and left to stand for 16 hours at 50 ⁰ C. Large white rectangular crystals of the silver salt of antibiotic A 204 are obtained, which after recrystallization from aqueous acetone 450 mg of the silver salt of antibiotic A 204 supply- The molecular weight of the salt was determined by X-ray analysis to be about 1099.

Example 10

! should contain antibiotics A 2 04, II

100 g of mixed sodium-potassium salt of antibiotic A 204, which is obtained as described in Example 6, are dissolved in 500 ml of a mixture of benzene and ethyl acetate (7 t 3). The solution is chromatographed on a column containing 2000 g of silica gel (Grace-Davieon Chemical Co, Grade 62). A mixture of benzene and ethyl acetate (7: 3) is used for elution. The fractions are eluted by thin-layer chromatography on silica gel using ethyl acetate as the developing solution and detection by spraying with H ₂ PO. followed- A 204 is eluted initially. After about 32 l of solvent have been collected, the solvent is switched to a 1: Ml@eh.ang of bensol and ethyl acetate. The elution is continued until the thin-layer chromatogram on the silicic acid gel plates no longer shows any further stains.

Until fractions _t contain high concentrations of A 204 II, the combined are evaporated to dryness and recrystallized in an acetone Waeeer solution. Crystals obtained here (3 g)

909851/1797

BATH ORIGINAL

are again chromatographed on a column containing 71 g of silica gel with a mixture of benzene and ethyl acetate (7: 3). evaporated to dryness and from a Aoeton-water solution to 788 mg of crystalline a 204 XI of melting point 177 - 179 ⁰ C recrystallized which activity units 1550 a / mg is the 204th

Beis piel 11

The insecticidal and acaricidal activity of antibiotic A 204 is given in Table II «

In Table II the following rating scale is used «

j> Kill rating

0-10 0

11 -20 1

21 - 30 2

31-40 3

41-50 4

51-60 5

61-70 6

71 - 80 7

81-90 8

91 -100 9

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Eabelle II

co ο to co

effectiveness insect of antibiotic A 204 as Insecticide 100 50 25th ge f2s isfet ge ¹ ladybug valuation «- Baiaawol "Iswfete Hie rve Route 1 000 500 250 8.5 3 ₀ 5 3 HelGnenlaus stomach 3 4th 0 «-» - Mageii 8th 9 3 T- Contact 3.5 2 1 8th 7th 6th W © 1 saai I c iiwa a se Syataw 7th 0 0 3 1 2 Contact 9 ? 7th IContact 9 9 4th

CD CX)

Example 12

The inhibitory concentrations were determined by the agar dilution test tea teat - With the agar thinning tea, the tea gate is · = ganismue in strips on a series of agar plates that are Contain different concentrations of Antibiotic A 204, applied to the minimum concentration of antibiotic A 204 in mcg / ml in the agar substrate su determine the das The growth of the organism has to be done by the board of directors for a period of time.

909851/1791 BAD ORfGfHAt

TabeljLjBLjLII

Minimum inhibitory concentration of tea organism (Stamn) mcg / ml 24 hours

Staphylococcus aureus (5055) 6 _; 25th

Bacillus subtiJLis (X12-1) 6.25

Hycobacterium aviun (X-85) 1.56

Streptococcus faecalis 1.56

Lactobacillus easel. 1.56

Leuconostoc citrovorun 0.39

Trichophyton mentagrophytee 25? 00

Xanthofflonae phaseoli 100.00

Botrytia clnerea 100.00

Colletotriohum piei 100.00

Helminthoeporum eatirum 10O ₁ , 00

Pullularia ep. 100.00 H. gruberi (HB-I) in

₂
Tissue culture 100.00

Dnο sodium sais of the antibiotiecbsη free acid A 204 shows a comparable activity in vitro.

Example 13

Da3 mixed sodium-potassium salt of A 204 II shows the in Tubelle IV stated major effect on the growth of a Set of microorganisms. The inhibitory concentrations were determined as described above *

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Table IV

Test organism Minimal Henun concentration

mcg / ml

staphylococcus aureue 12.50

Bscillue eubtilis 3 »12 Hyoobacterium avium 12.50 Streptococcus faecalia 3 »12 Lactobacillue easel 6.25 Leuconoetoc oltrovorum 6.25 Alternaria eolani 10O ₉ OO Botyryti · oinerea 50.00 Colletotrichunt pi ·! 50.00 Helfflinthoeporiuta eatirium 50 “00

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Claims (1)

Patent claims 1,. Antibiotic A 204 "A 204 II. Mixtures and their alkali earth metal and ₉ basic nitrogen salts. 2 «Antibiotic according to claim 1 ₉ characterized by the following feature white crystalline solid, which ^{melts after crystallization from ethyl ether at 96-98 0} C, soluble in esters, benzene, halogenated hydrocarbons, dine thy1-formamide, dimethyl sulfoxide ₉ ether and ketoses» little soluble soluble in alcohols and very little soluble in water; acidic compounds with a titratable group have a pK'a value of 6.11, determined by titration, of about 900; by going! dly © ß1 _e $ 74> C, 9e37 $> H and 2S ₉ 38 $ O ₀ sp @ sifie @ li @ r optical rotation (a) jj ⁵ ° "-6 68 ₈ 12 ° (0« 1 $ > ₉ Sew./?tons! »In methanol), when measured in a GhlorofosmUJaung is lüfrayötabsorptionsspektrum in the range vcra 2 _p 0 to 15 ₅ O microo the following distinguishable bands? $ 2.95 3.45 | 5 ₉ 95l 6 * 87? 7 "26; 7ρ80 x, 8.52; 8 _r 95; 9.17; _B 9 ₉ 34; 9-59; 9 _r S2; 10.07s 10 ₉ 24j 10.51; 10.77; 40% to 11.80 microns and no characteristic ultra-low oil absorption spectrum. 'S antibiotic according to claim I ₉ in Ροϊόι of the mixed Natriussä Kalium-Sa Iss aj gekanEizeiehiiet by the following characteristics? white crystalline solids soluble in esters, benzene, halogenated carbohydrates, Biaisthylfoffmamidj dimethyl, sulfo2cld ₉ ethers and KstoaeB? v / enig IBsIIeIi in alcohols and a © hr vigfiiglöslieSi la Waese ^ i aia @ tits-iorfea ^ e group with pIC'a ~ 6j3 in a $ 66 - £ g <gn Dimetliylforaaiaid-V / asaer ^ solution, spaeifischer Rotation value (e) ^ ~ * 42 ₉ 3 ^ü (C = « i> Gewo / Volo in methanol) and when measured as a normal form solution in the infrared absorption spectrum in the Bsi'Qieh vqu 2 ₉ Q to 15? 0 909851/1797 BAD ORfGlNAU Micron has the following distinguishable bands: 3? 2O; 3.45; 6.26; 6.9Oj 7.30; 7.68; 7.80; 8.20; 8.50; 8.80; 9.22; 9.39; 9.59; 9.70; 9 * 82; 10.08; 10.26; 10.48; 10.83; 10.96; 11.30; 11.55 and 11.78 microns. Process for the production of an antibiotic, characterized in that a strain of Streptomycee albus which produces antibiotic A 204 is grown in sinoa culture medium which contains assimilable sources of carbon, nitrogen, and inorganic salts under extreme aerobic conditions until a considerable amount of A 204 rom the organism in which Kulturnediu® is produced, Ancestors according to claim 4? thereby g @ taeßasoiohnet _B <3®§ StreptorayeeB albus HRRL 3384 used. 6s method according to claim 4 or 5? that the Kulturaiediun ran at ein®? Temperature "roa etua 25 ■ = 37 ° C and holding the organism MTWA 24-72 _o StwaäQa long aüchtet- ? - ■ method according to claim 4 or 5 S ₉ characterized gsteaaseieiänet _s 204 that wins the antibiotic A awa the culture medium. 8 _r method according to claim. 4- 5 or β _β thereby gel® aase ie te = net, that one wins the alkali metal , E ti alkali eh® ta Ii = baaisshen Stickatoffsals © τοπ Antibioticum A 204 Eulturmediua- 9- method according to claim 4 _; 5 _S ¹ B or I ₀ JDoes that antibiotics @? JiE Δ 2 A 204 II through conversion of a @ s AntiblotiaumB A 204 is Sals des Antibioticuns A 204 ₃ A ^ sorptioa des A 204 ψ ß a solution of the Sslssß an a g 909851/1797 BATH ORIGINAL Harts and selective blurring of A 204 II and of A 204o daa free iron A 204 II 1st, from the Harz as separate substances wins. 909851/1797