NO134219B - - Google Patents
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- Publication number
- NO134219B NO134219B NO932/71A NO93271A NO134219B NO 134219 B NO134219 B NO 134219B NO 932/71 A NO932/71 A NO 932/71A NO 93271 A NO93271 A NO 93271A NO 134219 B NO134219 B NO 134219B
- Authority
- NO
- Norway
- Prior art keywords
- antibiotic
- medium
- growth
- streptomyces
- fermentation
- Prior art date
Links
- 238000000034 method Methods 0.000 claims description 33
- 150000003839 salts Chemical class 0.000 claims description 26
- 241000187390 Amycolatopsis lactamdurans Species 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 16
- 235000015097 nutrients Nutrition 0.000 claims description 15
- 150000001408 amides Chemical class 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 12
- 241000187747 Streptomyces Species 0.000 claims description 11
- 150000002148 esters Chemical class 0.000 claims description 11
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- 238000000855 fermentation Methods 0.000 description 32
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/08—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin disubstituted in the 7 position
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cephalosporin Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
Foreliggende oppfinnelse angår en fremgangsmåte ved frem- The present invention relates to a method by producing
stilling av 7(3-(D-5-amino-5-carboxyvaleramido) -3-(carbamoyloxy-methyl)-7_methoxy-3-cefem-4-carboxylsyre (I), og salter, estere og amider derav. Forbindelsen med formel I er strukturelt beslektet med cefalosporinrekken av forbindelser, men mens cefalosporin C position of 7(3-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxy-methyl)-7_methoxy-3-cephem-4-carboxylic acid (I), and salts, esters and amides thereof. The compound of formula I is structurally related to the cephalosporin series of compounds, but while cephalosporin C
bare inneholder en D-5-amino-5-carboxyvaleramido-gruppe i 7-still- only contains a D-5-amino-5-carboxyvaleramido group in the 7-still-
ingen , har fremgangsmåteforbindelsen også en 7-methoxysubstituent, none , the process compound also has a 7-methoxy substituent,
og mens cefalosporin C er substituert med acetoxymethyl i 3_still- and while cephalosporin C is substituted with acetoxymethyl in 3_still-
ingen på ringen, har fremgangsmåteforbindelsen her en carbamoyloxy-methyl-gruppe. none on the ring, the process compound herein has a carbamoyloxy-methyl group.
Forbindelse I er her betegnet som "antibiotikum 842A". I Compound I is designated herein as "antibiotic 842A". IN
norsk patent 132.242 er omtalt fremstilling av og egenskaper for en forbindelse, "antibiotikum Al6886l", som senere er angitt å ha for- Norwegian patent 132,242 describes the preparation and properties of a compound, "antibiotic Al6886l", which is later stated to have
mel I ovenfor. Ifølge patent 132.242 fåes ved dyrkning av en ny mikroorganisme, Streptomyces clavuligerus NRRL 3585, en blanding av minst to nært beslektede antibiotika (Al6886l og AI6880II) flour I above. According to patent 132,242, when cultivating a new microorganism, Streptomyces clavuligerus NRRL 3585, a mixture of at least two closely related antibiotics (Al6886l and AI6880II) is obtained
og, om ønskes, kan antibiotikum Al6886l isoleres, eventuelt i form av et salt. Dyrkning av Streptomyces lactamdurans NRRL 3802 ifølge foreliggende oppfinnelse fører til dannelse av et enkelt, spesi- and, if desired, antibiotic Al6886l can be isolated, optionally in the form of a salt. Cultivation of Streptomyces lactamdurans NRRL 3802 according to the present invention leads to the formation of a single, specific
fikt distinkt antibiotikum 842A (eller Al6886l) hvorved man unngår de åpenbare ulemper som oppstår når man får en blanding av nært beslektede forbindelser som må skilles ved tidskrevende og dyre metoder for å få et rimelig rent antibiotikum Al6886l. Foreliggende fremgangsmåte er således enklere og billigere enn fremgangsmåten ifølge patent 132.242. obtained distinct antibiotic 842A (or Al6886l), thereby avoiding the obvious disadvantages of obtaining a mixture of closely related compounds which must be separated by time-consuming and expensive methods in order to obtain a reasonably pure antibiotic Al6886l. The present method is thus simpler and cheaper than the method according to patent 132,242.
Foreliggende oppfinnelse innbefatter også fremstillingen av The present invention also includes the production of
de farmakologisk godtagbare salter, estere og amider av forbind- the pharmacologically acceptable salts, esters and amides of compounds
elsen av formel I. Disse innbefatter organiske og uorganiske compounds of formula I. These include organic and inorganic
salter som f.eks. syreaddisjonssalter, metallsalter, kvartære salter salts such as acid addition salts, metal salts, quaternary salts
og aminsalter avledet av tertiære organiske nitrogenholdige baser. Passende estere og amidderivater innbefatter mono- og diesterne and amine salts derived from tertiary organic nitrogenous bases. Suitable esters and amide derivatives include the mono- and diesters
som dem som er avledet av alkanoler', cycloalkanoler, aromatiske alkoholer og aralkanoler, og mono- og di-amider som dem som er avledet av ammoniakk, la verealkylaminer', di-laverealkylaminer, aralkylaminer og heterocycliske aminer. Eksempler på disse derivater og metoder for fremstilling av dem er omtalt nedenfor. such as those derived from alkanols', cycloalkanols, aromatic alcohols and aralkanols, and mono- and di-amides such as those derived from ammonia, lower alkylamines', di-lower alkylamines, aralkylamines and heterocyclic amines. Examples of these derivatives and methods for producing them are discussed below.
Antibiotikum 84 2 A : Dette antibiotikum er 7|3 - (D-5-aiiiino-5 - carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cefem-4-carboxylsyre (I), og dens salter, estere og amider. Denne forbindelse har følgende planformel: Antibiotic 84 2 A : This antibiotic is 7|3 - (D-5-aiiiino-5 - carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid (I), and its salts, esters and amides. This connection has the following plan formula:
Dette produkt (I) dannes av en ny stamme av Actinomycet.es og en prøve av denne mikroorganisme, betegnet som MA-2908, er anbrakt i kultursamlingen til Merck & Co., Inc., Rahway, New Jersey. En prøve av denne kultur er også deponert i kultursamlingen til Northern Utilization Research and Development Branch of the U.S. Department of Agriculture, Peoria Illinois, og har fått kul-turnummer NRRL 3802. Foruten å være antibiotisk aktivt er dette produkt (I) også et mellomprodukt ved fremstillingen av de tilsvarende 3-hydroxy-, 3-acyloxy- og carbamoyloxy-derivater. Nedenfor vil produktet i, dvs. 73-(D-5-amino-5-carboxyvaleramido|-3-(carba-moyloxymethyl)-7-methoxy-3-cefem-4-carboxylsyre, bli betegnet som Antibiotikum 842A, eller ganske enkelt 842A. This product (I) is produced by a new strain of Actinomycet.es and a sample of this microorganism, designated MA-2908, is deposited in the culture collection of Merck & Co., Inc., Rahway, New Jersey. A sample of this culture is also deposited in the culture collection of the Northern Utilization Research and Development Branch of the U.S. Department of Agriculture, Peoria Illinois, and has been given the cult number NRRL 3802. Besides being antibiotically active, this product (I) is also an intermediate in the production of the corresponding 3-hydroxy-, 3-acyloxy- and carbamoyloxy derivatives. Below, the product i, i.e. 73-(D-5-amino-5-carboxyvaleramido|-3-(carba-moyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid) will be referred to as Antibiotic 842A, or simply 842A.
Akt ivitet Activity
En vesentlig vanskelighet i antimikrobiell terapi er tilbøye-ligheten hos de fleste antibiotika til enzymatisk nedbrytning. Penicillin G f.eks. er virksomt mot en lang rekke gram-posit ive og grai>-negat ive mikroorganismer, mén i nærvær av penicillinase ned-brytes det til en form som er ineffektiv mot de fleste patogener. A significant difficulty in antimicrobial therapy is the propensity of most antibiotics to enzymatic degradation. Penicillin G e.g. is effective against a wide range of gram-positive and gram-negative microorganisms, but in the presence of penicillinase it breaks down into a form that is ineffective against most pathogens.
En måte for å løse dette problem har vært utviklingen av nye antibiotika som inneholder "cefem"-ringegenskapene av cefalosporin C. Cefalosporin C har en. iboende resistens mot penicillinase og er aktiv mot både gram-negat ive og gram-positive bakterier, men det er bare moderat aktivt, og der finnes enzymer andre enn penicillinase som er effektive til å ødelegge dens aktivitet. Disse enzymer betegnes som cefalosporinaser. Fremgangsmåtepfoduktene (I) oppviser resistens ikke bare mot penicillinase, men også mot cefalosporinaser. De oppviser aktivitet mot både gram-negative og gram-positive bakterier, men graden av aktivitet og området av organismer mot hvilke de er virksomme, er ikke identisk. One way to solve this problem has been the development of new antibiotics that contain the "cephem" ring properties of cephalosporin C. Cephalosporin C has a. intrinsic resistance to penicillinase and is active against both gram-negative and gram-positive bacteria, but it is only moderately active, and there are enzymes other than penicillinase that are effective in destroying its activity. These enzymes are called cephalosporinases. The process products (I) show resistance not only to penicillinase, but also to cephalosporinases. They show activity against both gram-negative and gram-positive bacteria, but the degree of activity and the range of organisms against which they are effective are not identical.
Antibiotikum 842A kjennetegnes ved en øket aktivitet mot gram-negative mikroorganismer. Ulik cefalosporin C som har en relativt lav antibakteriell aktivitet, oppviser dette produkt en be-traktelig in vivo gram-negativ effekt med en styrke som i alminnelighet er større enn for cefalothin. Denne aktivitet innbefatter effektivitet in vivo mot Proteus morganii og en effektivitet mot de følgende gram-negative bakterier: Escherichia coli, Proteus vulgaris, Proteus mirabilis, Proteus morganii, Salmonella schottmuelleri, Klebsiella pneumoniae AD, Klebsiella pneumoniae B og Paracolobactrum arizoniae. Antibiotic 842A is characterized by an increased activity against gram-negative microorganisms. Unlike cephalosporin C, which has a relatively low antibacterial activity, this product exhibits a considerable in vivo gram-negative effect with a potency that is generally greater than that of cephalothin. This activity includes effectiveness in vivo against Proteus morganii and an effectiveness against the following Gram-negative bacteria: Escherichia coli, Proteus vulgaris, Proteus mirabilis, Proteus morganii, Salmonella schottmuelleri, Klebsiella pneumoniae AD, Klebsiella pneumoniae B and Paracolobactrum arizoniae.
I tillegg til en generelt øket gram-negativ effekt og en øket styrke sammenlignet med cefalothin og en større motstandsdyktighet overfor cefalosporinaser, er 842A kjennetegnet ved en lav grad av toksisitet og gir hurtig et blodspeil. Innen 6 timer efter administrasjon er ca. 100% eliminert i urinen. Dessuten er det mere resistent overfor enzymatisk nedbrytning enn cefalosporin C, og resistens overfor det utvikles langsomt, og det er baktericid. Gitt oralt beskytter det mot infeksjoner som skyldes Paracolobactrum arizoniae 3270, Proteus vulgaris 1810 og Salmonella schottmuelleri 3010, og administrert subkutant er det fra to til ti ganger mere effektivt enn cefalothin mot de samme infeksjoner. In addition to a generally increased gram-negative effect and an increased potency compared to cephalothin and a greater resistance to cephalosporinases, 842A is characterized by a low degree of toxicity and provides a rapid blood count. Within 6 hours after administration, approx. 100% eliminated in the urine. Moreover, it is more resistant to enzymatic degradation than cephalosporin C, and resistance to it develops slowly, and it is bactericidal. Given orally it protects against infections caused by Paracolobactrum arizoniae 3270, Proteus vulgaris 1810 and Salmonella schottmuelleri 3010, and administered subcutaneously it is from two to ten times more effective than cephalothin against the same infections.
Mikroorganismen The microorganism
Mikroorganismen som danner Antibiotikum 842A, er en tidligere ukjent stamme av Actinomycetes. Det opprinnelige isolat ble erholdt som en enkelt koloni fra jord på en agar-skråkultur og dyrket i et medium med følgende sammensetning: The microorganism that forms Antibiotic 842A is a previously unknown strain of Actinomycetes. The original isolate was obtained as a single colony from soil on an agar slant and grown in a medium with the following composition:
Efter flere dagers vekst viste det seg at ingen sporulering kunne påvises. After several days of growth, it turned out that no sporulation could be detected.
Taksonomi: Mikroorganismen (stamme MA-2908) som danner 842A, er identifisert som en ny stamme av Actinomycetes. Taksonomien anvendt ved denne bestemmelse er beskrevet i "Bergey<*>s Manual of Determinative Bacteriology", J. utgave og i "The Actinomycetes", Vol. 2, "Classification, Identification and Description of Genera and Species", S. A. Waksman (1961). Under anvendelse av denne metode viste det seg at kulturen tilhørte slekten Streptomyces, og den har mange av attributtene til den kjente art Streptomyces Taxonomy: The microorganism (strain MA-2908) producing 842A has been identified as a new phylum of Actinomycetes. The taxonomy used in this determination is described in "Bergey<*>'s Manual of Determinative Bacteriology", J. edition and in "The Actinomycetes", Vol. 2, "Classification, Identification and Description of Genera and Species", S. A. Waksman (1961 ). Using this method, the culture was found to belong to the genus Streptomyces, and it has many of the attributes of the known species Streptomyces
.fradiae. Biokjemisk er den et i det vesentlige fullstendig side-stykke til sistnevnte. Morfologisk er der imidlertid viktige forskjeller. Eksempelvis er farven av luftmyceliet av S. fradiae "seashell pink", mens kulturen MA-2908 vanligvis er "cream"-farvet. Dessuten viser den vegetative vekst i MA-2908-kulturen pig-mentforskjeller på de forskjellige medier som anvendes, og, som an-ført nedenfor, ble ingen sporulering påvist på standardtaksonomiske medier. På grunnlag av disse forskjeller er kulturen tildelt et nytt artsnavn: Streptomyces lactamdurans. Tabell I nedenfor be-skriver de biokjemiske attributter av Streptomyces lactamdurans-arten og av den kjente Streptomyces fradiae. Alle avlesningene i tabell I ble gjort efter 3 ukers inkubasjon ved 28°C hvor annet ikke er anført; pH av det anvendte medium i disse undersøkelser var tilnærmet nøytral, dvs. 6,8 til 7,2. De fysiologiske prøver ble utført ved utløpet av den syvende og enogtyvende dag. .fradiae. Biochemically, it is essentially a complete side piece to the latter. Morphologically, however, there are important differences. For example, the color of the aerial mycelium of S. fradiae is "seashell pink", while the culture MA-2908 is usually "cream" colored. Moreover, the vegetative growth in the MA-2908 culture shows pigment differences on the different media used, and, as stated below, no sporulation was detected on standard taxonomic media. On the basis of these differences, the culture has been assigned a new species name: Streptomyces lactamdurans. Table I below describes the biochemical attributes of the Streptomyces lactamdurans species and of the known Streptomyces fradiae. All the readings in Table I were made after 3 weeks of incubation at 28°C where otherwise not stated; The pH of the medium used in these investigations was approximately neutral, i.e. 6.8 to 7.2. The physiological tests were performed at the end of the seventh and twenty-first day.
Morfologi: Sporoforer ble'■ ikke påvist når kulturen ble dyrket på mediene angitt i beskrivelsen av kulturegenskapene skjont gjentatte observasjoner ble gjort inntil 8 uker. Farvede mikro-skopiske preparater viste imidlertid lange filamenter, hvorav mange var segmentert i underenheter av forskjellige størrelser, Morphology: Sporophores were not detected when the culture was grown on the media indicated in the description of the culture characteristics, although repeated observations were made up to 8 weeks. However, stained microscopic preparations showed long filaments, many of which were segmented into subunits of different sizes,
i alminnelighet bredt formet og ca. 0,9 x 1,7 micron i storrelse. generally broadly shaped and approx. 0.9 x 1.7 micron in size.
Luftmyceliet var'kort, rett, lite forgrenet. Det synes å være av samme storrelse som det vegetative mycelium, 0,9 M> bredt. Det er lett, pudret og avskrape°s lett. The aerial mycelium was short, straight, little branched. It appears to be of the same size as the vegetative mycelium, 0.9 M> wide. It is light, powdery and scrapes off easily.
Det vegetative mycelium er gram-positivt, ikke syrefast. Det klynger seg til, og i noen medier, er det innleiret i agaren. Det er noen oppbrytning i staver ved rystekolbedyrkning, men dette er ikke utstrakt. Vegetativt mycelium fra rystekolber og stasjonære kolber (podningsmedium, ^—6 dager, 28°C) viste noen "knopper" og korte, fortykkede, nesten klubbeformige segmenter på myceliet, men disse var ikke tallrike q<y>g deres eventuelle betydning er ukjent. The vegetative mycelium is gram positive, not acid fast. It clings to, and in some media, is embedded in the agar. There is some breaking up of rods in shaking flask cultivation, but this is not extensive. Vegetative mycelium from shake flasks and stationary flasks (inoculum medium, ^—6 days, 28°C) showed some "buds" and short, thickened, almost club-shaped segments on the mycelium, but these were not numerous q<y>g their possible significance is unknown .
Tomatpasta- havremel- agar Tomato paste - oatmeal - agar
Vegetativ vekst - bakside, "orange" matt, tort utseer.de, rynket. Vegetative growth - reverse side, "orange" matte, tort utseer.de, wrinkled.
Luftmycelium - spredt, "cream". Aerial mycelium - spread, "cream".
Intet opploselig pigment. No soluble pigment.
Czapek- Dox- agar Czapek-Dox-agar
Vegetativ vekst - matt, "deep cream" Vegetative growth - matte, "deep cream"
Luftmycelium - pudret, '"creamish white" Aerial mycelium - powdered, '"creamish white"
Intet opploselig pigment No soluble pigment
Glycerol- asparagin- agar Glycerol- asparagine- agar
Vegetativ vekst - matt, bakside - "golden yellow" til "orange" Luftmycelium - pudret, "cream" med "pale peach tones" Opploselig pigment - "pale amber" Vegetative growth - matte, reverse side - "golden yellow" to "orange" Aerial mycelium - powdered, "cream" with "pale peach tones" Soluble pigment - "pale amber"
Eggalbumin- agar Egg albumin agar
Vegetativ vekst - matt, "cream" til "yellow" Vegetative growth - dull, "cream" to "yellow"
Luftmycelium - pudret, "cream" Aerial mycelium - powdered, "cream"
Intet opploselig pigment No soluble pigment
Caleiummaleat- agar Calcium Maleate Agar
Vegetativ vekst - matt bakside - "yellow" kantet mec. "orange" Vegetative growth - matte back - "yellow" edged mec. "orange"
Luftmycelium - pudret, "white" til "cream" kantet med "peach" Intet opploselig pigment Aerial mycelium - powdered, "white" to "cream" edged with "peach" No soluble pigment
Næringstyrosin- agar Nutrient tyrosine agar
Vegetativ vekst - matt, "tan" til "orange" Vegetative growth - dull, "tan" to "orange"
Luftmycelium - spredt, "cream" med "white" Aerial mycelium - scattered, "cream" with "white"
Intet opploselig pigment No soluble pigment
Tyrosinkrystaller spaltet Tyrosine crystals cleaved
Melas se- gjærhydrolysat- agar Molasses sticky yeast hydrolyzate agar
Vegetativ vekst - matt bakside - "orange" Vegetative growth - matte back - "orange"
Luftmycelium - pudret, "creamish white" Aerial mycelium - powdered, "creamish white"
Intet opploselig pigment. No soluble pigment.
Wæringsagar Weathering agar
Vegetativ vekst - matt til "golden yellow" Vegetative growth - dull to "golden yellow"
Luftmycelium - pudret, "cream" Aerial mycelium - powdered, "cream"
Intet opploselig pigment No soluble pigment
Lakmusmelk Litmus milk
Spredt vekstring - 10 vegetative vekster - intet luftmycelium Scattered growth ring - 10 vegetative growths - no aerial mycelium
Peptonisering: alkalisk reaksjon; Peptonization: alkaline reaction;
pH 7,3 - 7, h (kontroll pH - 6,7) pH 7.3 - 7, h (control pH - 6.7)
Skummet melk Skimmed milk
Spredt vekstring - "tan" til "orange" Vegetativ vekst - intet luftmycelium "Light tan" opploselig pigment Peptonisering - alkalisk reaksjon Scattered growth ring - "tan" to "orange" Vegetative growth - no aerial mycelium "Light tan" soluble pigment Peptonization - alkaline reaction
pH 7,2 (kontroll pH - 6,6) pH 7.2 (control pH - 6.6)
Skummet melk- agar Skimmed milk agar
Vegetativ vekst - matt, "orange" Luftmycelium - moderat, ".cream" til "pale coral" "Light tan" opploselig pigment Hydrolyse av casein Vegetative growth - dull, "orange" Aerial mycelium - moderate, ".cream" to "pale coral" "Light tan" soluble pigment Hydrolysis of casein
Gelatinstikk Gelatin stick
Spredt "cream" til "orange" kolbevegetativ.vekst suspendert i hele roret Spread "cream" to "orange" cob vegetative. growth suspended throughout the rudder
Intet opploselig pigment No soluble pigment
Fullstendig forvæskning Complete liquefaction
Næringsgelatin- agar Nutritional gelatin agar
Vegetativ.-vekst - matt, "orange" Luftmycelium - spredt, pudret "cream" Intet opploselig pigment Vegetative growth - dull, "orange" Aerial mycelium - scattered, powdered "cream" No soluble pigment
Forvæskning av gelatin Liquification of gelatin
Nærings- stivelse- agar Nutrient starch agar
Vegetativ vekst - matt, "orange" Luftmycelium - spredt, pudret, "pinkish cream" Intet opploselig pigment Vegetative growth - dull, "orange" Aerial mycelium - scattered, powdery, "pinkish cream" No soluble pigment
Moderat hydrolyse av stivelse Moderate hydrolysis of starch
Syntetisk stivelse- agar Synthetic starch agar
Vegetativ vekst, - matt, "bakside - "cream" kantet med "orange" Vegetative growth, - matte, "backside - "cream" edged with "orange"
Luftmycelium - pudret, "white" kantet med "peach" Intet opploselig pigment Aerial mycelium - powdered, "white" edged with "peach" No soluble pigment
Moderat hydrolyse av stivelse Moderate hydrolysis of starch
Loeffler' s blodserum- agar Loeffler's blood serum agar
Vegetativ vekst - "cream"-farvet til "orange" Vegetative growth - "cream" colored to "orange"
Luftmycelium - intet Aerial mycelium - nothing
Intet opploselig pigment No soluble pigment
Ingen forvæskning No dilution
Pepton-. jern- gjærekstrakt- agar Peptone-. iron - yeast extract - agar
Vegetativ vekst - "cream" Vegetative growth - "cream"
Luftmycelium - spredt - "whitish" Aerial mycelium - scattered - "whitish"
Intet opploselig pigment No soluble pigment
Mikroaerofil vekst Microaerophilic growth
(Gjærekstrakt-dextrose-stikk - <1>+0 mm dypt stikk) (Yeast extract dextrose puncture - <1>+0 mm deep puncture)
God overflatevekst og langs ovre l/ h av stikklinjen. Good surface growth and along the upper l/h of the puncture line.
Temperatur - Gjærekstrakt- dextrose- skråkulturer Temperature - Yeast extract - dextrose - slant cultures
God vekst ved 28°C Good growth at 28°C
Spredt vekst ved 37°C Scattered growth at 37°C
Ingen vekst ved 50°C No growth at 50°C
Gjærekstrakt - dextrose- agar Yeast extract - dextrose agar
Vegetativ vekst - matt "golden yellow" Vegetative growth - matte "golden yellow"
Luftmycelium - pudret, "cream" til "pale flesh pink" Aerial mycelium - powdered, "cream" to "pale flesh pink"
Intet opploselig pigment No soluble pigment
Potetplugg Potato plug
Vegetativ vekst - torr, matt, "cream" til "orange" Luftmycelium - spredt, "creamish" Vegetative growth - dry, dull, "cream" to "orange" Aerial mycelium - spreading, "creamish"
Intet opploselig pigment No soluble pigment
Reduksjon av nitrater til nitriter - negativ Reduction of nitrates to nitrites - negative
Alle avlesninger ble tatt efter 3 ukers inkubering ved 28°C hvor annet ikke er angitt. Fysiologiske prover ble utfort på 7 All readings were taken after 3 weeks of incubation at 28°C where otherwise not stated. Physiological tests were carried out on 7
og 21 dag. and 21 days.
De morfologiske forskjeller mellom Streptomyces lactamdurans og Streptomyces fradiae er angitt i Tabell II. Observasjonene ble utfort på mediene angitt i Tabell II ved vekstintervaller på en uke, tre uker og åtte uker. Luftmyceliet av S. lactamdurans er kort og rett med liten forgrening. Det. synes å være. omtrent av samme storrelse som det vegetative mjicelium, dvs. 0,9 j.. Um bredt. Det er lett, pudret og avskrapes - lett. Det vegetative mycelium er gram-positivt, det er ikke syrefast. Det klynger seg til og er i noen medier innleiret i agaren. Der .er noen fragmentering i staver ved rystekolbedyrkning, men den er ikke vidtgående. Vegetativt mycelium er fra rystekolber og stasjonære kolber (podningsmedium h til 6 dager, 28°C) viste noen "knopper" og korte, fortykkede, nesten klubbeformige segmenter på myceliet, men disse var ikke tallrike. Alle avlesningene i Tabell II ble tatt efter 3 ukers inkubering ved 28°C hvor annet ikke er angitt. pH av mediene anvendt ved disse undersøkelser var omtrent noytral, dvs. 6,8 til 7,2. De fysiologiske prover ble utfort ved utlopet av den 7, og 21. dag. Farvene anvendt i beskrivelsen er i overensstemmelse med definisjonene i "Color Harmony Manual", h. utgave, 1958; Container Corporation of America. The morphological differences between Streptomyces lactamdurans and Streptomyces fradiae are indicated in Table II. The observations were carried out on the media indicated in Table II at growth intervals of one week, three weeks and eight weeks. The aerial mycelium of S. lactamdurans is short and straight with little branching. The. seems to be. about the same size as the vegetative mjicelium, i.e. 0.9 j.. Um wide. It's light, powdery and scrapes off - easily. The vegetative mycelium is gram-positive, it is not acid-fast. It clings to and in some media is embedded in the agar. There is some fragmentation in staves during shaking flask cultivation, but it is not extensive. Vegetative mycelium from shake flasks and stationary flasks (inoculum medium h to 6 days, 28°C) showed some "buds" and short, thickened, almost club-shaped segments on the mycelium, but these were not numerous. All the readings in Table II were taken after 3 weeks of incubation at 28°C where otherwise not stated. The pH of the media used in these investigations was approximately neutral, i.e. 6.8 to 7.2. The physiological tests were carried out at the end of the 7th and 21st day. The colors used in the description are in accordance with the definitions in the "Color Harmony Manual", first edition, 1958; Container Corporation of America.
CarbonutnytteIse: Streptomyces lactamdurans (MA-2908) ble også undersøkt på dens evne til å utnytte eller assimilere forskjellige carbonkilder ved å dyrke mikroorganismen i et primært syntetisk medium (T. G. Pridham and D. Gottlieb, 1948) som inneholdt 1% av carbohydratet ved 28°C i 3 uker. Tabell III viser ut-nyttelsen eller assimileringen av disse carbonkilder av Streptomyces lactamdurans (MA-2908)• Forklaringen av symbolene i Tabell III er som følger: + indikerer god vekst, ± indikerer dårlig vekst og - indikerer ingen vekst på den spesielle carbonkllde. Carbon utilization: Streptomyces lactamdurans (MA-2908) was also examined for its ability to utilize or assimilate different carbon sources by growing the microorganism in a primary synthetic medium (T. G. Pridham and D. Gottlieb, 1948) containing 1% of the carbohydrate at 28° C for 3 weeks. Table III shows the utilization or assimilation of these carbon sources by Streptomyces lactamdurans (MA-2908) • The explanation of the symbols in Table III is as follows: + indicates good growth, ± indicates poor growth and - indicates no growth on the particular carbon source.
Egenskapene angitt i Tabell I, II og III ble anvendt til å henføre Streptomyces lactamdurans (MA-2908) til en artskiassifika-sjon ved hjelp av nøklene angitt i "Bergey<*>s Manual of Determinative Bacteriology", 7. utgave, side 694-829 (1957) og i "The Actinomycetes" , Vol. 2: side 61-292 (196l). En sammenligning av de detaljerte egenskaper for Streptomyces lactamdurans med kjente arter viser at organismen biokjemisk svarer til Streptomyces fradiae. Som vist ovenfor, er der imidlertid viktige morfologiske forskjeller som f.eks. ved farven av luftmyceliet av S. fradiae som er "seashell pink" sammenlignet med "cream"-farven hos M^-2908-Dessuten mens den vegetative vekst hos S. fradiae oppviser pigment-forskjeller på de forskjellige medier, ble ingen sporulering iakttatt hos MA-2908- Pa basis av disse forskjeller og egenskapene beskrevet i foregående tabeller ble mikroorganismen som fremstiller antibiotikum 842A (MA-2908) gitt det nye artsnavn Streptomyces lactamdurans. The characteristics listed in Tables I, II and III were used to assign Streptomyces lactamdurans (MA-2908) to a species classification using the keys given in "Bergey<*>'s Manual of Determinative Bacteriology", 7th edition, page 694 -829 (1957) and in "The Actinomycetes", Vol. 2: pages 61-292 (196l). A comparison of the detailed characteristics of Streptomyces lactamdurans with known species shows that the organism corresponds biochemically to Streptomyces fradiae. As shown above, there are, however, important morphological differences such as e.g. by the color of the aerial mycelium of S. fradiae which is "seashell pink" compared to the "cream" color of M^-2908- Furthermore, while the vegetative growth of S. fradiae shows pigment differences on the different media, no sporulation was observed in MA-2908- On the basis of these differences and the characteristics described in the preceding tables, the microorganism that produces antibiotic 842A (MA-2908) was given the new species name Streptomyces lactamdurans.
Antibiotikum A16886 kan ifølge norsk patent 132.242 fremstilles ved dyrkning av en organisme isolert fra jordprøver fra Syd-Amerika. According to Norwegian patent 132,242, antibiotic A16886 can be produced by cultivating an organism isolated from soil samples from South America.
Organismen ble isolert fra nevnte jordprøver ved å suspendere deler av jordprøvene i sterilt destillert vann og ved å stryke sus-pensjonene ut på næringsagar. De podede næringsagarplater ble inkubert ved 25-35°C i flere døgn. Ved slutten av inkubasjons-perioden ble kolonier av den antibiotika Al6886-produserende organisme ved hjelp av en steril platinaløkke overført til agarskrå-kulturer. Agarskråkulturene ble så inkubert for tilveiebringelse av egnede mengder inokulat for fremstilling av antibiotikum A16886. The organism was isolated from said soil samples by suspending parts of the soil samples in sterile distilled water and by streaking the suspensions onto nutrient agar. The inoculated nutrient agar plates were incubated at 25-35°C for several days. At the end of the incubation period, colonies of the antibiotic Al6886-producing organism were transferred to agar slant cultures by means of a sterile platinum loop. The agar slant cultures were then incubated to provide suitable amounts of inoculum for the production of antibiotic A16886.
Actinomyceten brukt i henhold til patent 132.242 for fremstilling av antibiotikum Al688°, angies å ha vært vanskelig å klassifisere i slekten Streptomyces på grunn av dens atypiske spore-morfologi. Imidlertid indikerer data for cellevegganalyser at kulturen bør betraktes som en art i Streptomyces-slekten. Følgelig ble organismen betraktet som en ny art og ble gitt navnet Streptomyces clavuligerus. The actinomycete used according to patent 132,242 for the preparation of antibiotic Al688° is stated to have been difficult to classify in the genus Streptomyces because of its atypical spore morphology. However, cell wall assay data indicate that the culture should be considered a species in the Streptomyces genus. Consequently, the organism was considered a new species and was given the name Streptomyces clavuligerus.
Denne organisme produserer karakteristisk et utstrakt nett-verk av korte,'; flerakset-forgrenede luft-hyfer som eventuelt deler seg i sporer. Korte kløverformede sidegrener dannes, og som vanligvis produserer fra 1 til 4 sporer hver. Det dannes ikke noe substrat-conidia. Elektronmikrofotografier viser glattveggede sporer. Celleveggpreparater inneholder den L,L-isomere av diamin-pimelinsyre og glycin i tillegg til hovedbestanddelene, asparagin-syre, glutaminsyre og alanin. Sporer er grå i mengder og pri-mærmycelium er blekgult til gulbrunt. Intet løselig pigment pro-duseres . Kulturen har et optimalt temperaturområde mellom 26° og 30°C. Ingen vekst opptrer ved 37°C. Morfologisk ligner denne kultur visse stammer av Thermomonospora og Micromonospora. This organism characteristically produces an extensive network of short,'; multiaxially branched aerial hyphae that eventually divide into spores. Short clover-shaped lateral branches are formed, which usually produce from 1 to 4 spores each. No substrate conidia are formed. Electron micrographs show smooth-walled spores. Cell wall preparations contain the L,L isomers of diamine-pimelic acid and glycine in addition to the main constituents, aspartic acid, glutamic acid and alanine. Spores are gray in quantity and the primary mycelium is pale yellow to yellowish brown. No soluble pigment is produced. The culture has an optimal temperature range between 26° and 30°C. No growth occurs at 37°C. Morphologically, this culture resembles certain strains of Thermomonospora and Micromonospora.
Den nye organisme som er istand til å produsere antibiotikum A16886, er blitt permanent deponert uten restriksjoner med hensyn til tilgjengelighet i kultursamlingen i The Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture (tidligere: Northern Regional Research Laboratories), Peoria, Illinois 6l6o4, og er tilgjengelig for publikum under kultur nr. NRRL 3585. The new organism capable of producing antibiotic A16886 has been permanently deposited without restriction as to availability in the culture collection of The Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture (formerly: Northern Regional Research Laboratories), Peoria, Illinois 6l6o4, and is available to the public under culture No. NRRL 3585.
Egenskapene til Streptomyces clavuligerus NRRL 3585 er angitt i de følgende tabeller. Metodene anbefalt av International Streptomyces Project (Shirling et al., "Methods for Characterization of Streptomyces Species", "Intern. Bull Systemic Bacteriol." 16, 313-340 (1966)) for karakterisering av Streptomyces-arter har vært brukt sammen med visse supplerende prøver. Farvenavnene ble fast-satt i overensstemmelse med ISCC-NBS-metoden beskrevet av Kelly m.fl. i "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names" (U.S. Department of Commerce Circ. 553, Washington D.C. 1955). Tallene i parenteser refererer seg til Resner og Backus' farveserier (Tresner m.fl.: "System of Color Wheels for Streptomyces Taxonomy", "Appl. Microbiol." 11, 335-338 (1937)) og farvetabellbetegnelser er understreket. Maerz og Paul *s farvebe-tegnelser (Maerz m.fl.: "Dictionary of Color" (McGraw-Hill Book Co., Inc. New York 1950)) er angitt i klammer. Kulturer ble dyrket ved 30°C i l4 dager hvis ikke annet spesielt er angitt. The characteristics of Streptomyces clavuligerus NRRL 3585 are indicated in the following tables. The methods recommended by the International Streptomyces Project (Shirling et al., "Methods for Characterization of Streptomyces Species", "Intern. Bull Systemic Bacteriol." 16, 313-340 (1966)) for the characterization of Streptomyces species have been used with certain supplementary tests. The color names were determined in accordance with the ISCC-NBS method described by Kelly et al. in "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names" (U.S. Department of Commerce Circ. 553, Washington D.C. 1955). The numbers in brackets refer to Resner and Backus' color series (Tresner et al.: "System of Color Wheels for Streptomyces Taxonomy", "Appl. Microbiol." 11, 335-338 (1937)) and color table designations are underlined. Maerz and Paul *'s color descriptions (Maerz et al.: "Dictionary of Color" (McGraw-Hill Book Co., Inc. New York 1950)) are indicated in brackets. Cultures were grown at 30°C for 14 days unless otherwise specifically stated.
I tabell V er angitt resultatene av carbonutnyttelsesforsøk, utført på organismen NRRL 3585. I tabellen anvendes følgende symboler : Table V shows the results of carbon utilization experiments carried out on the organism NRRL 3585. The following symbols are used in the table:
+ = vekst og utnyttelse + = growth and utilization
= ingen vekst, ingen utnyttelse = no growth, no utilization
= sannsynligvis utnyttelse = likely exploitation
(-) = tvilsom utnyttelse (-) = questionable exploitation
Ved sammenligning av egenskapene for Streptomyce lactamdurans NRRL 3802 og de i patent 132.242 angitte egenskaper for Streptomyces clavuligerus NRRL 3585 fremgår det at det dreier seg om to forskjellige stammer av Streptomyces-slekten. When comparing the properties of Streptomyce lactamdurans NRRL 3802 and the properties stated in patent 132,242 for Streptomyces clavuligerus NRRL 3585, it appears that these are two different strains of the Streptomyces genus.
In vitro- og in vivo- undersøkelser In vitro and in vivo studies
Antibiotikum 842 A, in vitro: Bestemmelsen av de in vitro biologiske egenskaper ble utført ved skive-plateagar-diffusjons-metoden. Disse prøver ble utført ved å anbringe 7 mm skiver, vætet med antibiotikumoppløsningen, på overflaten av petriskåler inneholdende 5 ml "Difco" næringsagar og 0,2% gjærekstrakt podet med 5 eller 10 ml standard cellesuspensjon (OD = 0,22 ved 66o nm) pr. 150 ml Antibiotic 842 A, in vitro: The determination of the in vitro biological properties was carried out by the disc-plate agar diffusion method. These tests were performed by placing 7 mm discs, wetted with the antibiotic solution, on the surface of Petri dishes containing 5 ml of "Difco" nutrient agar and 0.2% yeast extract inoculated with 5 or 10 ml of standard cell suspension (OD = 0.22 at 66o nm) per 150 ml
medium og inkubert ved 25°C eller 37°C i 16 timer som angitt. Metoden og grunnlaget for disse prøver er beskrevet i publikasjonen "Cross Resistance Studies and Antibiotic Identification", Applied Micro-biology, Vol. 6: side 392-398 .(1958). De følgende tabeller viser resultatene av disse prøver på antibakterielt spektrum og kryss-resistens, og angir de anvendte forsøksorganismer og de anvendte bet ingelser. medium and incubated at 25°C or 37°C for 16 hours as indicated. The method and basis for these tests are described in the publication "Cross Resistance Studies and Antibiotic Identification", Applied Micro-biology, Vol. 6: pages 392-398 .(1958). The following tables show the results of these tests on antibacterial spectrum and cross-resistance, and indicate the test organisms used and the conditions used.
Antibiotikum 81+ 2A. in vivo: Når 8^2 A gis subkutant til mus Antibiotic 81+ 2A. in vivo: When 8^2 A is given subcutaneously to mice
er det i alminnelighet mere aktivt enn cefalothin og omtrent lik cefaloridin, ampicillin eller kloromycetin til å beskytte mot infek-sjon av gram-negative organismer. Det er bemerkelsesverdig ugiftig og utskilles hurtig i urinen med ca. 79% av det subkutant injiserte 8^2A gjenvunnet i lopet av h timer. it is generally more active than cephalothin and about the same as cephaloridine, ampicillin or chloromycetin in protecting against infection by gram-negative organisms. It is remarkably non-toxic and is quickly excreted in the urine with approx. 79% of the subcutaneously injected 8^2A recovered within h hours.
I in vivo-undersokelser beskytter antibiotikum 842A mot in-feksjon av 3 arter av Proteus, 2 av Salmonella, 1 stamme av Escherichia coli, 2 av Klebsiella og også mot Paracolobactrum arizoniae, Aerobacter aerogenes, Pasteurella multocida og Diplococcus pneumoniae E<>>+00. In in vivo studies, antibiotic 842A protects against infection by 3 species of Proteus, 2 of Salmonella, 1 strain of Escherichia coli, 2 of Klebsiella and also against Paracolobactrum arizoniae, Aerobacter aerogenes, Pasteurella multocida and Diplococcus pneumoniae E<>>+ 00.
Resultatene av disse prover er beskrevet i Tabell Vllb. The results of these tests are described in Table Vllb.
8Lf2A- fermentering: Antibiotikum 8k2A dannes under den aerobe fermentering av passende vandige næringsmedier under regulerte betingelser ved inokulering med organismen Streptomyces lactamdurans. Generelt kan mange medier som er en kilde til carbon og nitrogen, anvendes ved fremstilling av 8U2A. 8Lf2A fermentation: Antibiotic 8k2A is formed during the aerobic fermentation of suitable aqueous nutrient media under controlled conditions by inoculation with the organism Streptomyces lactamdurans. In general, many media which are a source of carbon and nitrogen can be used in the production of 8U2A.
Den noyaktige mengde av carbohydrat- og nitrogenkildene vil avhenge av de andre bestanddeler som omfattes av fermenteringsmediet, men i alminnelighet er mengden av carbohydrat ca. 1-6 vekt% av mediet, og mengden av tilgjengelig nitrogen, enten alene eller i kombinasjon, er vanligvis fra ca. 0,2% til ca. 6 vekt% av mediet. Mediene beskrevet nedenfor er eksempler på dem som er egnet for. fremstilling av Antibiotikum 8<*>+2A. Disse medier er bare typiske eksempler på medier som kan anvendes. The exact amount of the carbohydrate and nitrogen sources will depend on the other components included in the fermentation medium, but in general the amount of carbohydrate is approx. 1-6% by weight of the medium, and the amount of available nitrogen, either alone or in combination, is usually from approx. 0.2% to approx. 6% by weight of the medium. The media described below are examples of those that are suitable for. production of Antibiotic 8<*>+2A. These media are only typical examples of media that can be used.
Fermenteringen utfores ved temperaturer fra ca. 20°C til 37°C, men for optimalt resultat foretrekkes det å utfore fermenteringen ved en temperatur fra ca. 2h til 32°C. pH-verdier av nærings-mediene egnet for dyrkning av Streptomyces lactamdurans (MB-2908) under dannelse av Antibiotikum 8k2A bor være i omr-V fra ca. 6,0 til ca. 8,0. The fermentation is carried out at temperatures from approx. 20°C to 37°C, but for optimal results it is preferred to carry out the fermentation at a temperature from approx. 2h at 32°C. pH values of the nutrient media suitable for the cultivation of Streptomyces lactamdurans (MB-2908) during the formation of Antibiotic 8k2A should be in the range V from approx. 6.0 to approx. 8.0.
Fermentering av Antibiotikum 8k2A i liten skala utfores be-kvemt ved å inokulere et passende næringsmedium med den antibiotikum-produserende stamme og tillate fermenteringen å forlope ved en konstant temperatur på ca. 28°C på en ryster i flere dager. Ved utlopet av inkuberingsperioden fjernes myceliet og den overstående væske analyseres. Fermentation of Antibiotic 8k2A on a small scale is conveniently carried out by inoculating a suitable nutrient medium with the antibiotic-producing strain and allowing the fermentation to proceed at a constant temperature of approx. 28°C on a shaker for several days. At the end of the incubation period, the mycelium is removed and the supernatant liquid is analysed.
I praksis utfores denne fermentering i en sterilisert kolbe over en et-, to-, tre- eller fire-trinns kimutvikling. Næringsmediet for kimtrinnene kan være en hvilken som helst egnet kombina-. sjon av carbon- og nitrogenkilder som f.eks. et hvilket som helst av mediene IX-XI ovenfor. Kimkolben rystes i et kammer med konstant temperatur på ca. 28°C i fra 1 til ca. 3 dager og den erholdte vekst anvendes for å inokulere enten et annet-trinns kim eller produksjonsmediet. Mellomliggende trinn av kimkolber, når de brukes, utvikles på i det vesentlige samme måte, dvs. innholdene av kolbene anvendes for å inokulere produksjonsmediet, de inokulerte kolber rystes ved konstant temperatur i flere dager og ved utlopet av inkubasjonstiden sentrifugeres innholdet av kolbene for å fjerne myceliet. Den overstående væske eller buljong konsentreres så og renses for å få Antibiotikum 81+2A. In practice, this fermentation is carried out in a sterilized flask over a one-, two-, three- or four-stage germ development. The nutrient medium for the germ stages can be any suitable combination. tion of carbon and nitrogen sources such as any of the media IX-XI above. The germ flask is shaken in a chamber with a constant temperature of approx. 28°C in from 1 to approx. 3 days and the growth obtained is used to inoculate either a second-stage germ or the production medium. Intermediate stages of seed flasks, when used, are developed in essentially the same way, i.e. the contents of the flasks are used to inoculate the production medium, the inoculated flasks are shaken at a constant temperature for several days and at the end of the incubation period the contents of the flasks are centrifuged to remove the mycelium. The remaining liquid or broth is then concentrated and purified to obtain Antibiotic 81+2A.
Ved arbeide i storre målestokk foretrekkes det å utfore fermenteringen i passende tanker forsynt med en rorer og anordning for luftning av fermenteringsmediet. I henhold til denne metode fremstilles næringsmediet i tanken og steriliseres ved oppvarmning ved temperaturer opp til ca. 120°C. Efter avkjoling inokuleres det steriliserte medium med den produserende kultur og fermenteringen tillates å forlope i flere dager, som f.eks. fra 2 til k dager, mens næringsmediet omrorBS og/eller luftes og temperaturen holdes ved ca. 28°C. Ved forandringer i inokulum-utviklingen og forandringer i produksjonsmedium er det også mulig å oppnå en flerdoblet forbed-ring i produksjon og okning i kraften av dette antibiotikum. When working on a larger scale, it is preferred to carry out the fermentation in suitable tanks equipped with a stirrer and device for aerating the fermentation medium. According to this method, the nutrient medium is prepared in the tank and sterilized by heating at temperatures up to approx. 120°C. After cooling, the sterilized medium is inoculated with the producing culture and the fermentation is allowed to proceed for several days, which e.g. from 2 to k days, while the nutrient medium is stirred and/or aerated and the temperature is kept at approx. 28°C. With changes in the inoculum development and changes in the production medium, it is also possible to achieve a multifold improvement in production and increase in the power of this antibiotic.
8i+ 2A- bestemmel. sesmetode under anvendelse av Vibrio percolans: Bestemmelser ble utfort ved skive-plate-metoden under anvendelse av 12,7 mm filtrerpapirskiver. Bestemmelsesplatene ble fremstilt under anvendelse av "Difco" næringsagar pluss 2,0 g/l "Difco" gjærekstrakt med 10 ml pr. plate. En overnatten-vekst av bestemmelsesorganismen, Vibrio percolans (MB-1272) i næringsbuljong og 0, 2% gjærekstrakt ble fortynnet i steril saltopplosning til en suspensjon 8i+ 2A- determinant. method using Vibrio percolans: Determinations were carried out by the disc-plate method using 12.7 mm filter paper discs. The determination plates were prepared using "Difco" nutrient agar plus 2.0 g/l "Difco" yeast extract at 10 ml per disc. An overnight growth of the target organism, Vibrio percolans (MB-1272) in nutrient broth and 0.2% yeast extract was diluted in sterile saline to a suspension
med hO% transmittans ved en bølgelengde på 660 nm. Denne suspensjon ble i en mengde på 20 ml/l tilsatt til mediet for helning av platene. with h0% transmittance at a wavelength of 660 nm. This suspension was added in an amount of 20 ml/l to the medium for tilting the plates.
Bestemmelsesplatene ble holdt ved <!>+°C inntil anvendelse (maksimum 5 dager). Efter påføring av de antibiotikummettede be-stemmelsesskiver ble platene inkubert ved 28°C i fra 8 til 2h timer. Inhiberingssoner ble avlest som mm diameter. De ble anvendt for å bestemme relative styrker, eller ved sammenligning med en renset ■ referansestandard, styrken i <u,g/ml. Når en slik. bestemmelse utfores på kvantitativt vis, kan fra 1 til 2/£g/ml antibiotikum påvises. The determination plates were kept at <!>+°C until use (maximum 5 days). After application of the antibiotic-saturated determination plates, the plates were incubated at 28°C for from 8 to 2 hours. Inhibition zones were read as mm diameter. They were used to determine relative strengths, or by comparison with a purified ■ reference standard, the strength in <u.g/ml. When such a determination is carried out quantitatively, from 1 to 2/£g/ml of antibiotic can be detected.
Bakteriell inaktivering hos 842A: En in vitro-undersokelse var beregnet på å bestemme resistensen av Antibiotikum 8k2A overfor bakteriell inaktivering sammenlignet med cefalosporin C, cefaloridin og cefalothin. Disse undersøkelser viste at Antibiotikum 842A var mere stabil enn de sistnevnte overfor visse mikroorganismer. Bacterial inactivation in 842A: An in vitro study was designed to determine the resistance of Antibiotic 8k2A to bacterial inactivation compared to cephalosporin C, cephaloridine and cephalothin. These investigations showed that Antibiotic 842A was more stable than the latter against certain microorganisms.
De omdannende bakterier anvendt ved denne undersøkelse var The transforming bacteria used in this investigation were
to organismer kjent for fullstendig å inaktivere cefalosporin C, nemlig Alcaligenes faecalis (MB-9) og Alcaligenes viscosus (MB-12). two organisms known to completely inactivate cephalosporin C, namely Alcaligenes faecalis (MB-9) and Alcaligenes viscosus (MB-12).
(a) Fremstilling av bakterieceller: Alcaligenes viscosus (MB-12) og A. faecalis (MB-9)-celler ble fremstilt som folger: Innholdet av et L-ror ble blandet med noen få ml næringsbuljong inneholdende 0,2% gjærekstrakt. En lokkefull av denne suspensjon ble spredd over overflaten av en næringsagar-skråkultur og inkubert i 18 timer ved 37°C. Alle skråkulturer ble oppbevart ved 15°C og anvendt i lopet av en uke efter inkubering. En lokkefull av over-flateveksten fra hver skråkultur ble aseptisk overfort i 50 ml næringsbuljong inneholdende 0,2% gjærekstrakt og rysteinkubert i 18 timer ved 28°C. Kulturen ble så sentrifugert ved 4000 opm. i (a) Preparation of bacterial cells: Alcaligenes viscosus (MB-12) and A. faecalis (MB-9) cells were prepared as follows: The contents of an L tube were mixed with a few ml of nutrient broth containing 0.2% yeast extract. A ladleful of this suspension was spread over the surface of a nutrient agar slant culture and incubated for 18 hours at 37°C. All slant cultures were stored at 15°C and used within one week of incubation. A bowlful of the surface growth from each slant culture was aseptically transferred to 50 ml of nutrient broth containing 0.2% yeast extract and incubated on a shaker for 18 hours at 28°C. The culture was then centrifuged at 4000 rpm. in
10 minutter. Den overstående væske ble dekantert og den gjenværende pellet av celler ble vasket to ganger med steril 0,1 N fosfatpuffer, pH 7,5 (6,8 g kaliumfosfat, dvs. KH2P01+ og 7,1 g natrium-hydrogenfosfat pr. liter destillert vann). De vaskede celler ble så resuspendert; i 1/10 av det originale volum av en h mg/ml oppløs-ning av antibiotikum i 0,1 M fosfatpuffer. Forsoksblandingen ble så inkubert uten rystning i et vannbad innstilt på 37°C i h timer. Forsoksblandingene ble så sentrifugert ved 2000 opm. i 10 minutter og dette ga en klar overstående væske som ble dekantert i sterile ror og straks frosset i torris inntil ferdige for biobestemmelse, vanligvis i lopet av 3 timer. Kontrollprover ble inkubert på noy- 10 minutes. The supernatant was decanted and the remaining pellet of cells was washed twice with sterile 0.1 N phosphate buffer, pH 7.5 (6.8 g of potassium phosphate, i.e. KH 2 PO 1 + and 7.1 g of sodium hydrogen phosphate per liter of distilled water ). The washed cells were then resuspended; in 1/10 of the original volume of a h mg/ml solution of antibiotic in 0.1 M phosphate buffer. The trial mixture was then incubated without shaking in a water bath set at 37°C for h hours. The test mixtures were then centrifuged at 2000 rpm. for 10 minutes and this gave a clear supernatant which was decanted into sterile tubes and immediately frozen in dry ice until ready for biodetermination, usually within 3 hours. Control samples were incubated on noy-
aktig samme måte unntatt fraværet av celler. in much the same way except for the absence of cells.
(b) Grad av Antibiotikum 842A- inaktivering: De overstående væsker ble provet på antibakteriell aktivitet på folgende måte: 7 mm papirskiver ble fuktet med de overstående væsker og anbragt på overflaten av næringsagar-gjærekstrakt-(0,2%) plater som på forhånd var podet med angjeldende forsoksorganisme. B. subtilis (MB-964) bestemmelsesplater ble podet på folgende måte: 5 ml av en suspensjon av vaskede sporer i 0,9% saltlake ble tilsatt til hver 150 ml av en 2% næringsagar-gjærekstrakt (45°C) hvorav 5 ml så ble overfort i 15 x 100 mm petriskåler. Alle bestemmelsesskålene ble lagret ved 5°C og anvendt i lopet av 3 dager. Bestemmelsesskålene ble inkubert over natten ved 25°C for måling av inhiberingssonene rundt proveskivene. (b) Degree of Antibiotic 842A inactivation: The supernatants were tested for antibacterial activity as follows: 7 mm paper discs were moistened with the supernatants and placed on the surface of nutrient agar-yeast extract (0.2%) plates as previously was inoculated with the relevant test organism. B. subtilis (MB-964) determination plates were inoculated as follows: 5 ml of a suspension of washed spores in 0.9% saline was added to each 150 ml of a 2% nutrient agar-yeast extract (45°C) of which 5 ml then transferred to 15 x 100 mm Petri dishes. All assay dishes were stored at 5°C and used within 3 days. The assay dishes were incubated overnight at 25°C to measure the zones of inhibition around the test disks.
Cellefrie kontrollprover av hvert antibiotikum ble bestemt ved 1:1, 1:2, 1:4, 1:8, 1:16 og 1:32 fortynninger for å få en standard referansekurve. Opplesninger av forsoksantibiotika ble bestemt ved full styrke efter inkubering i nærvær av de vaskede bakterieceller. Alle prover ble utfort in triplo. Cell-free control samples of each antibiotic were determined at 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 dilutions to obtain a standard reference curve. Readings of trial antibiotics were determined at full strength after incubation in the presence of the washed bacterial cells. All samples were performed in triplicate.
(c) Resultater: Prosent av inaktivering ble beregnet ved å ta gjennomsnittet for de tre inhiberingssoner erholdt for hvert forsok og bestemme mengden av antibiotikum som er igjen i forsoks-opploshinggn .som vist ved standardkurven. Denne verdi trekkes så fra utgan&skonsentrasjonen (4 mg/ml) og resten divideres med ut-gangskonsentrasjonen og multipliseres med 100 for å få prosent inaktivering. Tabell VIII viser inaktiveringen erholdt for cefalosporin C og Antibiotikum 842A under de ovenfor angitte betingelser." (c) Results: Percent inactivation was calculated by averaging the three zones of inhibition obtained for each trial and determining the amount of antibiotic remaining in the trial solution as shown by the standard curve. This value is then subtracted from the initial concentration (4 mg/ml) and the remainder is divided by the initial concentration and multiplied by 100 to obtain percent inactivation. Table VIII shows the inactivation obtained for cephalosporin C and Antibiotic 842A under the above conditions."
Evnen til Antibiotikum 842A og cefalosporin C til å motstå den omdannende virkning av fire andre kulturer ble også bestemt. Disse kulturer er Escherichia coli 236, Proteus morganii 251, Proteus morganii 356 og Proteus mirabilis 2kl. Hver av disse er gram-negativ og resistent overfor Cefalosporin C. Ved utforelse av bestemmelsen ble individuelle blandinger av organismene og et av de antibiotiske stoffer provetatt efter 4 timers inkubering og analysert på gjenværende antibiotisk aktivitet. Metoden er den samme bestemmelsesmetode som beskrevet ovenfor mot Alcaligenes faecalis MB-9 og A. viscosus MB-12. De folgende tabeller viser prosent inaktivering av cefalosporin C og 842A på B. subtilis (MB-964) \ied denne metode: The ability of Antibiotic 842A and Cephalosporin C to resist the transforming action of four other cultures was also determined. These cultures are Escherichia coli 236, Proteus morganii 251, Proteus morganii 356 and Proteus mirabilis 2kl. Each of these is gram-negative and resistant to Cephalosporin C. When carrying out the determination, individual mixtures of the organisms and one of the antibiotic substances were sampled after 4 hours of incubation and analyzed for residual antibiotic activity. The method is the same determination method as described above against Alcaligenes faecalis MB-9 and A. viscosus MB-12. The following tables show the percentage inactivation of cephalosporin C and 842A on B. subtilis (MB-964) by this method:
De foregående data viser at Antibiotikum 842A øyensynlig er mere resistent enn Cefalosporin C overfor inaktivering av A. faecalis, A. viscosus, Escherichia coli 236, Proteus morganii 251, Proteus morganii 236 og Proteus mirabilis 241. The preceding data show that Antibiotic 842A is apparently more resistant than Cephalosporin C to the inactivation of A. faecalis, A. viscosus, Escherichia coli 236, Proteus morganii 251, Proteus morganii 236 and Proteus mirabilis 241.
Antibiotikum:842A erholdes ved foreliggende fermenteringsfremgangsmåte som en amfotær forbindelse med et tilsynelatende isoelektrisk punkt på*ca. pH 3,5, den er ustabil over pH 9,0, men er relativt stabil ved pH 1,5* Antibiotic: 842A is obtained by the present fermentation method as an amphoteric compound with an apparent isoelectric point of *approx. pH 3.5, it is unstable above pH 9.0, but is relatively stable at pH 1.5*
Da Antibiotikum 842A og dets salter effektivt inhiberer veksten av forskjellige arter av Salmonella, kan det anvendes som desinfeksjonsmiddel ved forskjellige husholdnings-og industrielle anvendelser. Eksempelvis oppviser 842A As Antibiotic 842A and its salts effectively inhibit the growth of various species of Salmonella, it can be used as a disinfectant in various household and industrial applications. For example, 842A exhibits
aktivitet mot Salmonella schottmuelleri 3010, S. gallinarum og S. typhosa og denne egenskap indikerer dens nytte som sanitært activity against Salmonella schottmuelleri 3010, S. gallinarum and S. typhosa and this property indicates its utility as a sanitary
middel i husholdningen og ved industrielle anvendelser. agent in the household and in industrial applications.
Rensning; Antibiotikum 842A kan renses ved adsorpsjon på en ionebytteharpiks som f.eks. på harpikser bestående av kvartære ammonium- eller sulfonsyrebyttemedier. Det adsorberte antibiotikum elueres fra harpiksadsorbatet med vandige oppløsninger eller med en vandig alkoholisk oppløsning av et passende salt som ammoniumklorid, nat riumklorid og lignende. Passende ionebytte-harpikser som kan anvendes, .innbefatter f.eks. polystyrenkjerne-sulfonsyreharpiksene (45% eller 53% vann) eller polystyrentrimethyl-benzylammoniumharpikser (43% vann) som er kjent som "Dowex 50" henholdsvis "Dowex 1". Om ønskes, kan eluatet erholdt ved den foregående metode renses ytterligere ved en annen eller tredje adsorpsjon og elueringstrinn. Alle de således erholdte eluater konsentreres så for å få det rensede 842A-produkt. Purification; Antibiotic 842A can be purified by adsorption on an ion-exchange resin such as e.g. on resins consisting of quaternary ammonium or sulfonic acid exchange media. The adsorbed antibiotic is eluted from the resin adsorbate with aqueous solutions or with an aqueous alcoholic solution of a suitable salt such as ammonium chloride, sodium chloride and the like. Suitable ion exchange resins which can be used include e.g. the polystyrene core sulfonic acid resins (45% or 53% water) or the polystyrene trimethyl-benzylammonium resins (43% water) which are known as "Dowex 50" or "Dowex 1" respectively. If desired, the eluate obtained by the preceding method can be further purified by a second or third adsorption and elution step. All the eluates thus obtained are then concentrated to obtain the purified 842A product.
Preparater Preparations
Antibiotikum 842A kan anvendes alene eller i kombinasjon som den aktive bestanddel av en hvilken som helst av en rekke farma-søytiske preparater. Disse antibiotika og deres tilsvarende salter kan anvendes i kapselform eller som tabletter, pulvere elle:c flytende oppløsninger eller som suspensjoner eller eliksirer. De kan .administreres oralt, intravenøst eller intramuskulært. Passende bærere som kan anvendes i preparatene, innbefatter f.eks. mannitol, sucrose, glucose eller sterile væsker som vann, saltlake, Antibiotic 842A can be used alone or in combination as the active ingredient of any of a number of pharmaceutical preparations. These antibiotics and their corresponding salts can be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. They can be administered orally, intravenously or intramuscularly. Suitable carriers that can be used in the preparations include e.g. mannitol, sucrose, glucose or sterile liquids such as water, saline,
glycoler og oljer av petroleum-, animalsk, vegetabilsk eller syntetisk opprinnelse som f.eks. jordnøttolje, mineralolje eller sesamolje. Foruten bæreren kan preparatene også innbefatte andre bestanddeler som stabilisatorer, bindemidler, antioxydanter, kon-serveringsmidler, smøremidler, suspenderingsmidler, viskositets-økende midler, smaksstoffer og lignende. Dessuten kan der i preparatet også innbefattes andre aktive bestanddeler som gir et bredere spektrum av antibiotisk aktivitet. glycols and oils of petroleum, animal, vegetable or synthetic origin such as e.g. peanut oil, mineral oil or sesame oil. In addition to the carrier, the preparations can also include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity-increasing agents, flavorings and the like. In addition, the preparation can also include other active ingredients that provide a wider spectrum of antibiotic activity.
Dosen som skal administreres, avhenger i stor utstrekning av den lidelse som behandles og pasientens vekt. Den parenterale vei foretrekkes for generelle infeksjoner og den orale vei for infeksjoner i fordøyelseskanalen. I alminnelighet består en dagsdose av fra ca. 15 til ca. 175 mg aktiv bestanddel pr. kg legemsvekt hos pasienten ved en eller flere administrasjoner pr. dag. Den foretrukne dagsdose for Antibiotikum 842A er i området fra ca. 40 til 80 mg aktiv bestanddel pr. kg legemsvekt. The dose to be administered depends to a large extent on the disorder being treated and the patient's weight. The parenteral route is preferred for general infections and the oral route for infections of the digestive tract. In general, a daily dose consists of from approx. 15 to approx. 175 mg of active ingredient per kg body weight in the patient with one or more administrations per day. The preferred daily dose for Antibiotic 842A is in the range from approx. 40 to 80 mg of active ingredient per kg body weight.
Preparatene kan administreres i flere enhetsdoseformer som f.eks. i fast eller flytende oralt inntagbar doseform. Preparatene, vil pr. enhetsdose, enten i flytende eller fast form, i alminnelighet inneholde fra ca. 15 mg til ca. 700 mg av den aktive bestanddel, men i alminnelighet foretrekkes det å anvende en dose i området fra ca. 80 mg til 320 mg. Ved parenteral administrasjon er enhetsdosen vanligvis den rene forbindelse i en steril vann-oppløsning eller i form av et oppløselig pulver beregnet på opp-løsning . The preparations can be administered in several unit dose forms, such as e.g. in solid or liquid orally ingestible dosage form. The preparations, will per unit dose, either in liquid or solid form, generally contain from approx. 15 mg to approx. 700 mg of the active ingredient, but in general it is preferred to use a dose in the range from approx. 80 mg to 320 mg. For parenteral administration, the unit dose is usually the pure compound in a sterile water solution or in the form of a soluble powder intended for dissolution.
Syntetiske metoder Synthetic methods
Fremgangsmåteforbindelsen kan også anvendes som utgangs - materialer ved fremstilling av derivater hvori 3-carbamoyloxy-gruppen av 842A ér erstattet med en rekke forskjellige substi-tuenter. I alminnelighet utføres denne utbytning eller substi-tusjon ved ganske enkelt å behandle 842A med et reagens som er istand til å overføre 3-carbamoyloxymethylgruppen til den ønskede 3-substituent. I praksis ér det imidlertid før erstatningen av 3-carbamoyloxyradikalet ofte nød-vendig å beskytte de frie amino- og carboxygrupper ved å behandle 842A med et nitrogenblokkerende middel som et N-laverealkoxy-carbonylfthalimid, og et forestringsmiddel for å få den tilsvarende 76(D-5-fthaloylamino-5-carboxyvaleramido)-3-(carbamoyloxy-methyl)-7-methoxy-3-cefem-4-carboxylsyre-diester. The process compound can also be used as starting materials in the production of derivatives in which the 3-carbamoyloxy group of 842A is replaced with a number of different substituents. Generally, this exchange or substitution is accomplished by simply treating 842A with a reagent capable of transferring the 3-carbamoyloxymethyl group to the desired 3-substituent. In practice, however, before the replacement of the 3-carbamoyloxy radical, it is often necessary to protect the free amino and carboxy groups by treating 842A with a nitrogen-blocking agent such as an N-lower alkoxycarbonylphthalimide, and an esterification agent to obtain the corresponding 76(D -5-phthaloylamino-5-carboxyvaleramido)-3-(carbamoyloxy-methyl)-7-methoxy-3-cephem-4-carboxylic acid diester.
Fremgangsmåteforbindelsen med formel (I) danner en lang rekke farmakologisk godtagbare salter med uorganiske og organiske båser. Disse innbefatter f.eks. metallsalter som dem som er avledet av alkalimetall- og jordalkalimetallhydroxyder, -carbonater og -bicar-bonater, og salter avledet av primære, sekundære og tertiære aminer som monoalkylaminer, dialkylaminer, trialkylaminer, laverealkanolaminer, di-laverealkanolaminer, laverealkylendiaminer, N,N-diaryl-laverealkylendiaminer, aralkylaminer, amino-substituerte laverealkanoler, N,N-di-laverealkylamino-substituerte laverealkanoler, amino-, polyamino- og guanidino-substituerte laverealkansyrer og nitrogenholdige heterocycliske aminer. Representative eksempler på disse salter innbefatter salter avledet av natriumhydroxyd, natrium-carboriat, natriumbicarbonat, kaliumcarbonat, kaliumhydroxyd og calciumcarbonat og salter avledet av slike aminer som tri- ' methylamin, triethylamin, piperidin, morfolin, kinin, lysin, prot-amin, arginin, prokain, ethanolamin, morfin, benzylamin, ethylen-diamin, N,N'-dibenzylethylendiamin, diethanolamin, piperazin, di-methylaminoethanol, 2-amino-2-methyl-l-propanol, theofyllin og N-methylglucamin . The process compound of formula (I) forms a wide range of pharmacologically acceptable salts with inorganic and organic bases. These include e.g. metal salts such as those derived from alkali metal and alkaline earth metal hydroxides, carbonates and bicarbonates, and salts derived from primary, secondary and tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylenediamines, N,N-diaryl -lower alkylenediamines, aralkylamines, amino-substituted lower alkanols, N,N-di-lower alkylamino-substituted lower alkanols, amino-, polyamino- and guanidino-substituted lower alkanoic acids and nitrogen-containing heterocyclic amines. Representative examples of these salts include salts derived from sodium hydroxide, sodium carboriate, sodium bicarbonate, potassium carbonate, potassium hydroxide and calcium carbonate and salts derived from such amines as tri-methylamine, triethylamine, piperidine, morpholine, quinine, lysine, prot-amine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol, theophylline and N-methylglucamine.
De forannevnte salter kan være monosalter som mononatriumsaltet erholdt f.eks. ved å behandle en ekvivalent natriumhydroxyd med en ekvivalent produkt (I), eller blandede disalter erholdt ved å behandle en ekvivalent av monosaltet med en ekvivalent av en for-skjellig base. Alternativt kan disse disalter erholdes ved å behandle en ekvivalent av en base med et toverdig kation som calciumhydroxyd, med en ekvivalent av produkt (I). Dessuten omfatter oppfinnelsen fremstillingen av blandede salter og estere som dem som fåes ved å behandle produktet (I) med en ekvivalent natriumhydroxyd og derpå The aforementioned salts can be monosalts such as the monosodium salt obtained e.g. by treating one equivalent of sodium hydroxide with one equivalent of product (I), or mixed disalts obtained by treating one equivalent of the monosalt with one equivalent of a different base. Alternatively, these disalts can be obtained by treating one equivalent of a base with a divalent cation such as calcium hydroxide, with one equivalent of product (I). Furthermore, the invention encompasses the preparation of mixed salts and esters such as those obtained by treating the product (I) with an equivalent of sodium hydroxide and then
med en ekvivalent melkesyre. with an equivalent of lactic acid.
Saltene som fremstilles ifolge oppfinnelsen, er farmakologisk godtagbare ikke-giftige derivater som kan anvendes som den aktive bestanddel i passende enhetsdose-farmasoytiske former. Dessuten kan de kombineres med andre droger for å få preparater med et bredt aktivitets spektrum. Dessuten har disse salter og også de tilsvarende ester- og amidderivater anvendelse som utgangsmaterialer ved fremstilling av carboxylsyreproduktet av formel I. Disse salter kan også anvendes ved fremstilling av andre farmasoytisk godtagbare salter. The salts produced according to the invention are pharmacologically acceptable non-toxic derivatives which can be used as the active ingredient in suitable unit dose pharmaceutical forms. Moreover, they can be combined with other drugs to obtain preparations with a broad activity spectrum. Moreover, these salts and also the corresponding ester and amide derivatives are used as starting materials in the preparation of the carboxylic acid product of formula I. These salts can also be used in the preparation of other pharmaceutically acceptable salts.
Foruten til salter kan fremgangsmåteproduktet (I) også over-fores til de tilsvarende mono- og diestere og mono- og diamider, In addition to salts, the process product (I) can also be converted to the corresponding mono- and diesters and mono- and diamides,
som f.eks. pivaloyloxymethyl- eller dibenzhydrylesterne, eller alkyl-, cycloalkyl-, aryl- eller aralkylestere som f.eks. methyl-, ethyl-, cyclohexyl-, fenyl- og benzylestere, eller amider, diamider, N-laverealkylamider, N,N-di-laverealkylamider, W-aralkylamider, N,N-diaralkylamider eller heterocycliske amider som N-methyl og N-ethyl-amid, N,N-dimethylamid, N,N-diethylamid, N-benzylåmid, N,N-dibenzyl-amid, piperidid, pyrrolidid eller morfolid. like for example. the pivaloyloxymethyl or dibenzhydryl esters, or alkyl, cycloalkyl, aryl or aralkyl esters such as e.g. methyl, ethyl, cyclohexyl, phenyl and benzyl esters, or amides, diamides, N-lower alkyl amides, N,N-di-lower alkyl amides, W-aralkyl amides, N,N-diaralkyl amides or heterocyclic amides such as N-methyl and N- ethyl amide, N,N-dimethylamide, N,N-diethylamide, N-benzylamide, N,N-dibenzylamide, piperidide, pyrrolidide or morpholide.
Fremgangsmåtene ved fremstilling av de ovennevnte estere og amidderivater innbefatter omsetning av carboxylsyreproduktet (I) eller det tilsvarende syrehalogenid med methanol, ethanol, cyclo-hexanol, fenol, benzylalkohol eller dibenzhydrol. På lignende måte kan amidderivatene erholdes ved å behandle det tilsvarende syrehalogenid med ammoniakk eller med det passende alkylamin, dialkyl-amin, aralkylamin eller heterocyclisk amin. ■ Disse og andre konven-sjonelle metoder for fremstilling av esterne og amidene er alminne-lig kjent. The methods for producing the above-mentioned esters and amide derivatives include reaction of the carboxylic acid product (I) or the corresponding acid halide with methanol, ethanol, cyclohexanol, phenol, benzyl alcohol or dibenzhydrol. In a similar manner, the amide derivatives can be obtained by treating the corresponding acid halide with ammonia or with the appropriate alkylamine, dialkylamine, aralkylamine or heterocyclic amine. ■ These and other conventional methods for producing the esters and amides are generally known.
De folgende eksempler belyser oppfinnelsen ytterligere. The following examples further illustrate the invention.
Eksempel Example
Antibiotikum 8U - 2A Antibiotic 8U - 2A
Trinn A: Rystekolbeproduksjon Step A: Shaker flask production
Et lyofilisert ror med Streptomyces lactamdurans (MA-2908) ble åpnet aseptisk. Roret ble så anvendt til å inokulere en 250 ml skovlet Erlenmeyerkolbe inneholdende 50 ml næringsmedium I ved å bryte roret i steril gas og overfore pelleten aseptisk i kolben. Medium I har folgende sammensetning: A lyophilized tube of Streptomyces lactamdurans (MA-2908) was opened aseptically. The stirrup was then used to inoculate a 250 ml scooped Erlenmeyer flask containing 50 ml nutrient medium I by breaking the stirrup in sterile gas and transferring the pellet aseptically into the flask. Medium I has the following composition:
Medium I: Medium I:
Denne kimkolbe ble rystet ved 28°C på en 220 opm roterende ryster med et 50 mm slag i 3 dager. 5 ml porsjoner (10% inbkulum) av denne vekst ble så overfort under anvendelse av sterile pipetter til fire annet-trinns kimkolber av samme storrelse inneholdende samme medium som angitt ovenfor, og disse kolber ble så rystet som ovenfor angitt. Innholdet av de annet-trinns kimkolber ble så slått sammen aseptisk i en kolbe og anvendt til å inokulere 11 to-liters skovlede Erlenmeyerkolber, hver inneholdende 350 ml av Medium II med 2-3% inokulum under anvendelse av sterile pipetter. Medium II har folgende sammensetning: This seed flask was shaken at 28°C on a 220 rpm rotary shaker with a 50 mm stroke for 3 days. 5 ml portions (10% inoculum) of this growth were then transferred using sterile pipettes to four second-stage seed flasks of the same size containing the same medium as above, and these flasks were shaken as above. The contents of the second-stage seed flasks were then pooled aseptically in a flask and used to inoculate 11 two-liter scooped Erlenmeyer flasks, each containing 350 ml of Medium II with 2-3% inoculum using sterile pipettes. Medium II has the following composition:
Produksjonskolbene ble så rystet ved 28°C på en 14-5 opm ryster med et 50 mm slag i fire dager. Ved utlopet av inkubasjonstiden ble innholdet av ti slike kolber forenet og en prove ble sentrifugert for å fjerne myceliet. The production flasks were then shaken at 28°C on a 14-5 rpm shaker with a 50 mm stroke for four days. At the end of the incubation period, the contents of ten such flasks were combined and a sample was centrifuged to remove the mycelium.
Nærværet av Antibiotikum 842A, dvs. produktet 76-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy~3-cefem-4-carboxylsyre (I), i fermenteringsvæsken ble bestemt ved agardiffusjonsbestemmelser utfort med 12,7 mm filtrerpapirskiver gjennom-trukket med fermenteringsvæsken og anbragt på overflaten av bestemmelsesplater inneholdende 10 ml næringsagar ("Difco") pluss 0,2% gjærekstrakt ("Difco") medium podet med det bakterielle inokulum. Inhiberingssonene ble målt i mm efter inkubering over natten.ved 28°C. Analysen av fermenteringsvæske hostet efter fermentering i 4 dager viste en inhiberingssone på 31,5 mm diameter på plater podet med Vibrio percolans (MB-1272). The presence of Antibiotic 842A, i.e. the product 76-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy~3-cephem-4-carboxylic acid (I), in the fermentation broth was determined by agar diffusion assays carried out with 12.7 mm filter paper discs permeated with the fermentation broth and placed on the surface of determination plates containing 10 ml nutrient agar ("Difco") plus 0.2% yeast extract ("Difco") medium inoculated with the bacterial inoculum. The inhibition zones were measured in mm after overnight incubation at 28°C. The analysis of fermentation fluid coughed up after fermentation for 4 days showed an inhibition zone of 31.5 mm diameter on plates inoculated with Vibrio percolans (MB-1272).
Trinn B: Adsorpsjon på en ionebytteharpiks Step B: Adsorption on an ion exchange resin
Den filtrerte fermenteringsvæske ble innstilt på pH 7,0 med fortynnet saltsyre og 2900 ml ble adsorbert på 100 ml av en sterkt basisk anionbytteharpiks med en styren-divinylbenzen-matrix ("Dowex 1 x 2" i kloridformen) ved 10 ml/min. Den forbrukte væske ble oppsamlet i 500 ml fraksjoner. Harpikskolonnen ble vasket med vann og eluert med 3% ammoniumklorid i 90% methanol. Eluatet ble oppsamlet i 100 ml fraksjoner. The filtered fermentation broth was adjusted to pH 7.0 with dilute hydrochloric acid and 2900 ml was adsorbed onto 100 ml of a strongly basic anion exchange resin with a styrene-divinylbenzene matrix ("Dowex 1 x 2" in the chloride form) at 10 ml/min. The consumed liquid was collected in 500 ml fractions. The resin column was washed with water and eluted with 3% ammonium chloride in 90% methanol. The eluate was collected in 100 ml fractions.
Skive-platebestemmelser mot Vibrio percolans (MB-1272) ble utfort på alle fraksjoner og sonediametrene er angitt nedenfor. Disk-plate determinations against Vibrio percolans (MB-1272) were carried out on all fractions and the zone diameters are indicated below.
Disse bestemmelser viser at ca. 60% av aktiviteten er i det forbrukte og ca. 18% er i eluatene. Videre viser de at harpiks-kapasiteten er bare 2 fraksjoner eller ti kolonnevolum av fermenteringsvæsken. Eluatfraksjonene 1-4 ble forenet og konsentrert for å fjerne methanol. Forbrukte fraksjoner 1-6 ble forenet hvorved man fikk 1960 ml opplosning. 1860 ml av opplbsningen ble innstilt fra pH 7,2 til 8,0 med fortynnet natriumhydroxyd og adsorbert på 100 ml av en sterkt basisk anionbytteharpiks med en styren-divinylbenzenmatrix ("Dowex 1x2" i kloridformen) ved 14 ml/min. Den forbrukte væske ble oppsamlet i 4 like store fraksjoner og analyser viser at 5% av aktiviteten var tilstede. Kolonnen ble vasket med vann og eluert med 5% vandig natriumklorid. Eluatet ble oppsamlet i 50 ml fraksjoner og analysert. Analysene viste at 90% av aktiviteten var tilstede i fraksjonene 3 - 16 og disse ble derfor forenet. These provisions show that approx. 60% of the activity is in what is consumed and approx. 18% is in the eluates. Furthermore, they show that the resin capacity is only 2 fractions or ten column volumes of the fermentation liquid. Eluate fractions 1-4 were combined and concentrated to remove methanol. Spent fractions 1-6 were combined, whereby 1960 ml of solution was obtained. 1860 ml of the solution was adjusted from pH 7.2 to 8.0 with dilute sodium hydroxide and adsorbed onto 100 ml of a strongly basic anion exchange resin with a styrene-divinylbenzene matrix ("Dowex 1x2" in the chloride form) at 14 ml/min. The consumed liquid was collected in 4 equal fractions and analyzes show that 5% of the activity was present. The column was washed with water and eluted with 5% aqueous sodium chloride. The eluate was collected in 50 ml fractions and analyzed. The analyzes showed that 90% of the activity was present in fractions 3 - 16 and these were therefore combined.
Trinn C: Adsorpsjon på en kationbytteharpiks Step C: Adsorption on a cation exchange resin
En 50 ml porsjon av konsentratet fremstilt i trinn B ble fortynnet til 500 ml, pH ble innstilt fra 8,8 - 2,0 med fortynnet saltsyre og adsorbert på 25 ml av en sterkt sur kationbytteharpiks av sulfonattypen med en styren-divinylbenzenmatrix ("Dowex 50 x 2" i hydrogenformen) ved 2,5 ml/min. Kolonnen ble vasket med 25 ml vann, derpå eluert med 2% pyridin inntil pH av kolonneavlopet steg til pH 7 (54 ml). Analyser av den forbrukte fraksjon og eluatet viste 9% av aktiviteten i det forbrukte og 90% i eluatet. Eluatet ble identifisert som pyridiniumsaltet av Antibiotikum 842A. A 50 ml portion of the concentrate prepared in step B was diluted to 500 ml, the pH was adjusted from 8.8 - 2.0 with dilute hydrochloric acid and adsorbed on 25 ml of a strongly acidic sulfonate type cation exchange resin with a styrene-divinylbenzene matrix ("Dowex 50 x 2" in the hydrogen form) at 2.5 ml/min. The column was washed with 25 ml of water, then eluted with 2% pyridine until the pH of the column effluent rose to pH 7 (54 ml). Analyzes of the consumed fraction and the eluate showed 9% of the activity in the consumed and 90% in the eluate. The eluate was identified as the pyridinium salt of Antibiotic 842A.
'<8>42A-produktet er amfotært med et tilsynelatende isoelektrisk punkt ved ca. pH 3,5- Produktet er ustabilt over pH 7, men stabilt ved pH 1,5. Det således erholdte eluat ble innstilt på pH 8,0 med-' fortynnet natriumhydroxyd og inndampet under vakuum for å fjerne pyridin. Det således erholdte produkt ble identifisert som mono-,natriumsaltet av Antibiotikum 842A. Molekylvekten er <1>+68 basert på den empiriske formel. The '<8>42A product is amphoteric with an apparent isoelectric point at ca. pH 3.5- The product is unstable above pH 7, but stable at pH 1.5. The eluate thus obtained was adjusted to pH 8.0 with dilute sodium hydroxide and evaporated under vacuum to remove pyridine. The product thus obtained was identified as the mono-sodium salt of Antibiotic 842A. The molecular weight is <1>+68 based on the empirical formula.
Analyse for C-^H^N^SO^Na: Analysis for C-^H^N^SO^Na:
Beregn. C 4l,0%; H 4,5%; N 12,0%; S 6,8; Calculate. C 4l.0%; H 4.5%; N 12.0%; S 6.8;
0 30,8%; Na 4,9%. 0 30.8%; Na 4.9%.
Funnet: C 39,31%; H ^,76%; N 11,16%; S 6,46%; Found: C 39.31%; H ^.76%; N 11.16%; S 6.46%;
0 3^,12%; Na 4,19%. 0 3^.12%; Na 4.19%.
I in vitro undersøkelser inhiberer dette produkt, dvs. Antibiotikum 842A, veksten av folgende gram-negative bakterier: Escherichia coli, Proteus vulgaris., Alcaligenes faecalis, Brucella bronchiseptica, Salmonella gallinarum, Vibrio percolans og Xanth-omonas vesicatoria. Dessuten inhiberer produktet veksten av folgende gram-positive bakterier: In in vitro studies, this product, i.e. Antibiotic 842A, inhibits the growth of the following gram-negative bacteria: Escherichia coli, Proteus vulgaris., Alcaligenes faecalis, Brucella bronchiseptica, Salmonella gallinarum, Vibrio percolans and Xanthomonas vesicatoria. In addition, the product inhibits the growth of the following gram-positive bacteria:
Staphylococcus aureus, Sarcina lutea og Bacillus subtilis. Staphylococcus aureus, Sarcina lutea and Bacillus subtilis.
I in vivo undersøkelser i mus inhiberer Antibiotikum 842A folgende aktiviteter. Administrasjonen var ved subkutan injeksjon. Efter avslutning av forsøksperioden, vanligvis 7 dager efter administrasjon, ble mengden av produkt som kreves for å beskytte 50% av musene (ED^q) fra denne ellers dødelige injeksjon beregnet: In in vivo studies in mice, Antibiotic 842A inhibits the following activities. Administration was by subcutaneous injection. After the end of the experimental period, usually 7 days after administration, the amount of product required to protect 50% of the mice (ED^q) from this otherwise lethal injection was calculated:
Foruten de ovennevnte in vivo forsok med produktet ble der anvendt et klinisk isolat av Proteus morganii 356 som er resistent overfor cefalosporiner og istand til å nedbryte cefalosporin C,-anvendt i en musebeskyttelsesprove utfort på samme måte som angitt ovenfor. ED^q for disse forsok er som folger: In addition to the above-mentioned in vivo trials with the product, a clinical isolate of Proteus morganii 356, which is resistant to cephalosporins and capable of degrading cephalosporin C, was used in a mouse protection test carried out in the same manner as stated above. ED^q for these trials is as follows:
Eksempel 2 Example 2
Antibiotikum 8V2A Antibiotic 8V2A
Rystekolbeproduksjon Shaker flask production
Inokulumet ble fremstilt som beskrevet i Eksempel 8. Innholdet av to annet-trinns kimkolber ble forenet og fermenteringsvæsken anvendt til å inokulere (ved 1 ml/kolbe) 62"250 ml Erlenmeyerkolber som hver inneholdt 50 ml Medium "IH. Medium IH har folgende sammensetning : The inoculum was prepared as described in Example 8. The contents of two second-stage seed flasks were combined and the fermentation liquid used to inoculate (at 1 ml/flask) 62" 250 ml Erlenmeyer flasks each containing 50 ml of Medium "IH. Medium IH has the following composition:
Medium III; Medium III;
Kolbene ble rystet ved 28°C på en 220 opm ryster med 50 mm The flasks were shaken at 28°C on a 220 rpm shaker with 50 mm
slag i 5 dager. Ved utlopet av inkubasjonstiden ble innholdet av 60 slike kolber forenet og en prove ble sentrifugert for å fjerne myceliet. stroke for 5 days. At the end of the incubation period, the contents of 60 such flasks were combined and a sample was centrifuged to remove the mycelium.
Nærværet av Antibiotikum 8h2A ble bestemt ved fremgangsmåten beskrevet i Eksempel 1 via agardiffusjonsbestemmelser utfort på en 12,7 mm filtrerpapirbestemmelsesskive. Efter inkiibering i h dager ga en analyse av fermenteringsvæsken en inhiberingssone på 33 mm mot Vibrio percolans (MB-1272). The presence of Antibiotic 8h2A was determined by the method described in Example 1 via agar diffusion determinations carried out on a 12.7 mm filter paper determination disc. After inhibition for h days, an analysis of the fermentation liquid gave an inhibition zone of 33 mm against Vibrio percolans (MB-1272).
Eksempel 3 Example 3
Antibiotikum 842A Antibiotic 842A
Fermentering: Fermentation:
Trinn 1: Et lyofilisert ror med Streptomyces lactamdurans • (MA-2908) ble anvendt til å inokulere 50 ml sterilt Medium I i en skovlet 200 ml Erlenmeyerkolbe. Step 1: A lyophilized tube of Streptomyces lactamdurans • (MA-2908) was used to inoculate 50 ml of sterile Medium I in a shoveled 200 ml Erlenmeyer flask.
Medium I: Medium I:
Den inokulerte kolbe ble anbragt på en 220 opm roterende ryster med 50 mm slag og inkubert i 72 timer ved 28°C. The inoculated flask was placed on a 220 rpm rotary shaker with a 50 mm stroke and incubated for 72 hours at 28°C.
Trinn 2: Et inokulum på 10,0 ml av den dannede vegetative vekst ble så anvendt til å inokulere en 2 liters skovlet Erlenmeyerkolbe inneholdende 500 ml av det. steriliserte Medium I beskrevet 'ovenfor. Den inokulerte kolbe ble anbragt på en 220 opm roterende ryster og inkubert i 48 timer ved 28°C. Step 2: An inoculum of 10.0 ml of the vegetative growth formed was then used to inoculate a 2 liter scooped Erlenmeyer flask containing 500 ml thereof. sterilized Medium I described 'above. The inoculated flask was placed on a 220 rpm rotary shaker and incubated for 48 hours at 28°C.
Trinn 3: Innholdet av inokulumkolben ble så anvendt til å inokulere et 190 1 rustfritt fermenteringsapparat inneholdende 160 1 av det samme medium I beskrevet ovenfor. Det inokulerte medium ble inkubert ved 28°C i 48 timer under omroring mens der Step 3: The contents of the inoculum flask were then used to inoculate a 190 l stainless fermenter containing 160 l of the same medium I described above. The inoculated medium was incubated at 28°C for 48 hours with agitation while there
ble opprettholdt en luftstrom på 85 l/min. gjennom fermenteringsvæsken. I fermenteringstiden Me små mengder "Polyglycol 2000" tilsatt for å bekjempe skumning. an air flow of 85 l/min was maintained. through the fermentation liquid. During the fermentation period Me small amounts of "Polyglycol 2000" added to combat foaming.
Trinn 4: Et inokulum på 43 1 av den erholdte vekst ble så anvendt til å inokulere et 760 1 rustfri stål-fermenteringsapparat inneholdende 467 1 av et sterilt medium IV med folgende sammensetning: ; Step 4: An inoculum of 43 1 of the growth obtained was then used to inoculate a 760 1 stainless steel fermenter containing 467 1 of a sterile medium IV with the following composition: ;
Fermenteringen ble utfort ved en temperatur på 28°C under omroring mens der ble opprettholdt en luftstrom på'280 l/min i 72 timer. Under fermenteringen ble der tilsatt små mengder av et antiskumningsmiddel, "Polyglycol 2000", for å forhindre forsterk skumning. Satsen ble hostet og aktiviteten ble bestemt ved skive-plate-metoden. Fermenteringsvæsken ble så filtrert gjennom diatomé-jord ved en pH på 7,8 og det således erholdte produkt ble identifisert som 842A . Skive-plate- The fermentation was carried out at a temperature of 28°C with stirring while an air flow of 280 l/min was maintained for 72 hours. During the fermentation, small amounts of an antifoam agent, "Polyglycol 2000", were added to prevent increased foaming. The batch was coughed up and the activity was determined by the disc-plate method. The fermentation liquid was then filtered through diatomaceous earth at a pH of 7.8 and the product thus obtained was identified as 842A. disc-plate-
analyser av en 1:10 fortynning ga en inhiberingssone på 21,5 mm mot Vibrio percolans (MB-1272). assays of a 1:10 dilution gave a zone of inhibition of 21.5 mm against Vibrio percolans (MB-1272).
Eksempel Example
Rensning; Mononatriumsalt av 7p-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cefem- 4- carboxylsyre Purification; Monosodium salt of 7p-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cephem- 4- carboxylic acid
Adsorpsjon på carbon: Fire fermenteringssatser hostet i henhold til Eksempel 1 ble hver adsorbert på 100 ml av en sterkt basisk anionbytteharpiks med en styren-divinylbenzenmatrix ("Dowex 1x2" i kloridformen) og fortynnet med 1% vandig natriumklorid. Eluatet ble oppsamlet i 50 ml fraksjoner og analysert. Eluatfraksjonene fra alle fire satser ble innstilt på pH 5 med fortynnet saltsyre og forenet hvorved man fikk 4300 ml opplosning. 4200 ml av denne opplosning ble omrort med 42 g carbon ("Darco G-60") i en halv time. Carbonet ble oppsamlet ved filtrering og vasket med vann. Filtratet og vaskevæsken var uten aktivitet. Carbonkaken ble eluert to ganger med en liters porsjoner 60%-ig vandig aceton ved å rore blandingen i 1/2 time og filtrere hver gang. Eluatene ble inndampet under vakuum til henholdsvis 108 ml og 100 ml. Analyser viste at det forste eluat inneholdt 76% av aktiviteten, Adsorption on carbon: Four fermentation batches prepared according to Example 1 were each adsorbed on 100 ml of a strongly basic anion exchange resin with a styrene-divinylbenzene matrix ("Dowex 1x2" in the chloride form) and diluted with 1% aqueous sodium chloride. The eluate was collected in 50 ml fractions and analyzed. The eluate fractions from all four batches were adjusted to pH 5 with dilute hydrochloric acid and combined, whereby 4300 ml of solution was obtained. 4200 ml of this solution was stirred with 42 g of carbon ("Darco G-60") for half an hour. The carbon was collected by filtration and washed with water. The filtrate and washing liquid were without activity. The carbon cake was eluted twice with one liter portions of 60% aqueous acetone by stirring the mixture for 1/2 hour and filtering each time. The eluates were evaporated under vacuum to 108 ml and 100 ml, respectively. Analyzes showed that the first eluate contained 76% of the activity,
atten ganger så sterkt som utgangsmaterialet, og at det annet inneholdt ' 17% av aktiviteten, fjorten ganger styrken av utgangsmaterialet. De to konsentrater ble forenet og konsentrert ytterligere til 61 ml og innstilt fra pH 4 til pH 5 med fortynnet natriumhydroxyd. Dette konsentrat inneholdt 40 mg/ml torrstoff og ga en 25 mm sone mot MB-1272 ved en fortynning på 1:100 (400 mcg/ml). Produktet ble identifisert som mononatriumsaltet av 7B-(D-5-amino- eighteen times as strong as the starting material, and that the other contained ' 17% of the activity, fourteen times the strength of the starting material. The two concentrates were combined and further concentrated to 61 ml and adjusted from pH 4 to pH 5 with dilute sodium hydroxide. This concentrate contained 40 mg/ml dry matter and gave a 25 mm zone against MB-1272 at a dilution of 1:100 (400 mcg/ml). The product was identified as the monosodium salt of 7B-(D-5-amino-
5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cef em- k-carboxylsyre (I). 5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cef em-k-carboxylic acid (I).
Eksempel 5 Example 5
Rensning; Mononatriumsalt av 7p-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cefem- 4- carboxylsyre Purification; Monosodium salt of 7p-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cephem- 4- carboxylic acid
Adsorpsjon på gel: En 22 ml porsjon av eluatet erholdt i henhold til Eksempel 1, trinn B, ble innstilt på pH 7,0 med fortynnet natriumhydroxyd og kromatografert på en kolonne inneholdende 388 ml "BioGel P-2". Kolonnen ble utviklet med vann, avlopet ble kontrollert med et differensialrefraktometer og 5 ml fraksjoner Adsorption on gel: A 22 ml portion of the eluate obtained according to Example 1, step B, was adjusted to pH 7.0 with dilute sodium hydroxide and chromatographed on a column containing 388 ml of "BioGel P-2". The column was developed with water, the effluent was checked with a differential refractometer and 5 ml fractions
•oppsamlet automatisk og bioanalysert. Bioaktiviteten viste seg •collected automatically and bioanalyzed. The bioactivity was shown
i fraksjonene K7 - 63, mens natriumklorid viste.seg i fraksjonene 62 - 72. Fraksjonene 50 - 60 ble forenet, analysert og inndampet til torrhet hvilket ga 10,8 mg residuum som ble identifisert som mononatriumsaltet av 7p-(D-5-amino-5-carboxyvaleramido)-3-(carb-amoyloxymethyl) -7-methoxy-3-cefem-4-carboxylsyre (I). Ved bestemmelse med Vibrio percolans ga dette produkt en 25 mm sone ved 8 mcg/ml. in fractions K7 - 63, while sodium chloride appeared in fractions 62 - 72. Fractions 50 - 60 were combined, analyzed and evaporated to dryness yielding 10.8 mg of residue which was identified as the monosodium salt of 7β-(D-5-amino -5-carboxyvaleramido)-3-(carb-amoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid (I). When determined with Vibrio percolans, this product gave a 25 mm zone at 8 mcg/ml.
Eksempel ' 6 Example ' 6
7p-(D-5-amino-5-carboxyvaleramido)-3~(carbamoyloxymethyl)-7- methoxy- 3- cefem- 4- carboxylsyre 7p-(D-5-amino-5-carboxyvaleramido)-3~(carbamoyloxymethyl)-7- methoxy- 3- cephem- 4- carboxylic acid
Modifisert fermenteringsfremgangsmåte: Modified fermentation procedure:
Trinn A: Skråkulturer Step A: Slant cultures
Et lyofilisert ror med Streptomyces lactamdurans (MA-2908) ble åpnet aseptisk og organismen overfort til et medium av folgende sammensetning: A lyophilized tube with Streptomyces lactamdurans (MA-2908) was opened aseptically and the organism transferred to a medium of the following composition:
Medium V: Medium V:
1% "Blackstrap Molasses" 1% "Blackstrap Molasses"
1% "National Brewer's" gjær 1% "National Brewer's" yeast
Vann til volum Water to volume
Skråkulturene ble inkubert i 7 dager ved 28°C. Når lagret koldt var skråkulturene stabile i mere enn 13 uker. The slant cultures were incubated for 7 days at 28°C. When stored cold, the slant cultures were stable for more than 13 weeks.
Trinn B: Kimtrinn: Totrinnssystern Stage B: Germ stage: Two-stage sister
Forste kim: Det fbrste kim ble inokulert direkte, fra skrå-kulturen fra, Trinn A til h0 ml 1% "Primary Dried Yeast N.F." pH 7,0 i en 250 ml skovlet Erlenmeyerkolbe. Kolbene ble så rystet på en 220 opm roterende ryster med 50 mm slag ved 28°C i et tidsrom fra First germ: The first germ was inoculated directly, from the slant culture from, Stage A to h0 ml 1% "Primary Dried Yeast N.F." pH 7.0 in a 250 ml scooped Erlenmeyer flask. The flasks were then shaken on a 220 rpm rotary shaker with a 50 mm stroke at 28°C for a period of
to til tre dager. two to three days.
Annet kim: Et'2,5% inokulum fra det forste kimtrinn ble tilsatt til en kolbe inneholdende et 2% Fleischmann S-150" gjærauto-lysat, pH 7,0. Veksten i dette trinn er karakteristisk lys og in-kubasjonen, utfort som i forste trinn, ble ikke strukket utover 48 timer. Second germ: A 2.5% inoculum from the first germ stage was added to a flask containing a 2% Fleischmann S-150" yeast autolysate, pH 7.0. The growth in this stage is characteristically light and the incubation, continued as in the first stage, was not extended beyond 48 hours.
Trinn C: Produksjonsmedium Step C: Production medium
Produksjonsmediet inneholdt pr. liter destillert vann: 30 g "Distiller's Solubles", 7,5 g "Primary Dried Yeast N.F." og 0,25 volum% "Mobilpar-S" antiskumningsmiddel. Mediet ble innstilt på The production medium contained per liter of distilled water: 30 g "Distiller's Solubles", 7.5 g "Primary Dried Yeast N.F." and 0.25% by volume "Mobilpar-S" antifoam agent. The medium was set to
pH 7,0 med en liten mengde konsentrert natriumhydroxydopplosning, anbragt i Erlenmeyerkolber og behandlet i. autoklav i 15-20 minutter ved 121°C. Efter avkjoling ble mediet tilsatt et 2,5% inokulum av kimet erholdt i trinn B. Inkubasjonstiden kan variere fra ca. 50 timer til 100 timer, men en inkuberingstid på ca. 72 timer foretrekkes. Volumet av mediet i hver kolbe kan variere fra 30 til 50 ml, men 40 ml anvendes rutinemessig. Mengden av inokulum kan variere fra. 1% til 5%, men i praksis anvendes i alminnelighet 2,5%. pH 7.0 with a small amount of concentrated sodium hydroxide solution, placed in Erlenmeyer flasks and treated in an autoclave for 15-20 minutes at 121°C. After cooling, a 2.5% inoculum of the germ obtained in step B was added to the medium. The incubation time can vary from approx. 50 hours to 100 hours, but an incubation time of approx. 72 hours is preferred. The volume of medium in each flask can vary from 30 to 50 ml, but 40 ml is routinely used. The amount of inoculum can vary from 1% to 5%, but in practice 2.5% is generally used.
Trinn D: Analyse Step D: Analysis
Når fermenteringen er avsluttet, fjernes cellene ved sentri-fuger ing og fermenteringsvæsken fortynnes med fosfatpuffer, pH 7,0. Konsentrasjonen av 7p-(D-5-amino-5-carboxyvaleramido) *-3-( carbamoyl-oxymethyl) -7-methoxy-3-cefem-4-carboxylsyre i fermenteringsvæsken ble bestemt ved den standard biologiske skivebestemmelsesmetode. Den anvendte bestemmelsesorganisme var Vibrio percolans (ATCC 8461). Filtrerpapirskiver ble neddykket i de fortynnede fermenteringsvæsker og anbragt på overflaten av agarholdige petriskåler som var inokulert med bestemmelsesorganismen Vibrio percolans (ATCC 8461). På disse petriskåler ble også anbragt skiver som på forhånd-var dyppet i standard opplosninger inneholdende kjente konsentrasjoner av 842A. Skivene ble inkubert over natten ved 28°C og diameteren på inhiberingssonene ble notert. Konsentrasjonen av 842A og den ferm-enterte væske ble beregnet ved interpolering fra.standardkurven som angir forholdet mellom sonediametere og kjente konsentrasjoner av standard 842A opplosninger. Ved denne metode ble det beregnet at Streptomyces lactamdurans MB-2908 produserte 78,6 Mg/ml 842A When the fermentation is finished, the cells are removed by centrifugation and the fermentation liquid is diluted with phosphate buffer, pH 7.0. The concentration of 7β-(D-5-amino-5-carboxyvaleramido)*-3-(carbamoyl-oxymethyl)-7-methoxy-3-cephem-4-carboxylic acid in the fermentation broth was determined by the standard biological disc determination method. The test organism used was Vibrio percolans (ATCC 8461). Filter paper discs were immersed in the diluted fermentation fluids and placed on the surface of agar-containing Petri dishes inoculated with the target organism Vibrio percolans (ATCC 8461). Discs that had previously been dipped in standard solutions containing known concentrations of 842A were also placed on these Petri dishes. The slices were incubated overnight at 28°C and the diameter of the inhibition zones was noted. The concentration of 842A and the fermented liquid was calculated by interpolation from the standard curve indicating the relationship between zone diameters and known concentrations of standard 842A solutions. By this method it was calculated that Streptomyces lactamdurans MB-2908 produced 78.6 Mg/ml 842A
ved den modifiserte fermenteringsmetode. by the modified fermentation method.
Eksempel 7 Example 7
Rensning; mononatriumsalt av 7B-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cefem- 4- carboxylsyre Purification; monosodium salt of 7B-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cephem- 4- carboxylic acid
1,0 g mononatriumsalt av 7B-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cefem-4-carboxylsyre fremstilt ifolge eksempel 5 ble opplost i 20 ml 1%- lg vandig n-butanol og kromatografert på en kolonne inneholdende 2530 ml "Sephadex G-10", en modifisert dextrangel i perleform. Kolonnen ble utviklet med 1%- lg vandig n-butanol ved 10 ml/min og 10,5 ml fraksjoner ble oppsamlet automatisk. Avlopet'ble overvåket med et skrivende refrakto-meter og fraksjonene ble bioanalysert. Bioaktiviteten viste seg i fraksjonene 99 - 122, og disse ble slått sammen og inndarapet til torrhet hvorved man fikk 670 mg produkt inneholdende hovedsakelig mononatriumsaltet av 7B-(D-5-amino-5-carboxyvaleramido)~3-(carb-amoyloxymethyl) -7-methoxy-3-cefem-4-carboxylsyre. 1.0 g of monosodium salt of 7B-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid prepared according to example 5 was dissolved in 20 ml of 1%-lg aqueous n-butanol and chromatographed on a column containing 2530 ml of "Sephadex G-10", a modified dextran in bead form. The column was developed with 1% lg aqueous n-butanol at 10 ml/min and 10.5 ml fractions were collected automatically. The effluent was monitored with a recording refractometer and the fractions were bioanalysed. The bioactivity was evident in fractions 99 - 122, and these were combined and evaporated to dryness, whereby 670 mg of product containing mainly the monosodium salt of 7B-(D-5-amino-5-carboxyvaleramido)~3-(carb-amoyloxymethyl)- 7-Methoxy-3-cephem-4-carboxylic acid.
Kolonnen ble senere kalibrert ved kromatografi på en blanding av "Blue Dextran 2000" og natriumklorid under Identiske betingelser. "Blue Dextran 2000" ble påvist i fraksjonene 85 - 93 og natriumklorid ble påvist i fraksjonene lkO - 155, hvilket viser at det bioaktive produkt kan skilles fra forurensninger av denne natur. The column was later calibrated by chromatography on a mixture of "Blue Dextran 2000" and sodium chloride under identical conditions. "Blue Dextran 2000" was detected in fractions 85 - 93 and sodium chloride was detected in fractions 10 - 155, which shows that the bioactive product can be separated from contaminants of this nature.
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US9659470A | 1970-12-09 | 1970-12-09 | |
US05/115,779 US4302578A (en) | 1970-12-09 | 1971-02-16 | Cephalosporin antibiotics |
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CH (5) | CH589655A5 (en) |
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DE (3) | DE2166463A1 (en) |
DK (1) | DK131639C (en) |
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IT (1) | IT1045524B (en) |
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CA1040620A (en) * | 1972-11-14 | 1978-10-17 | Frank J. Urban | 6-alkoxy-6-acylamidopenicillins 7-alkoxy-7-acylamido acetoxycephalosporins and process |
US4065356A (en) | 1976-02-12 | 1977-12-27 | Merck & Co., Inc. | Production of antibiotic FR-02A (efrotomycin) by streptomyces lactamdurans |
US4327093A (en) | 1978-10-24 | 1982-04-27 | Fujisawa Pharmaceutical Co., Ltd. | 3,7-Disubstituted-2 or 3-cephem-4-carboxylic acid compounds |
JPS5931517B2 (en) * | 1978-12-12 | 1984-08-02 | 山之内製薬株式会社 | New 7↓-methoxycephalosporin derivative |
US4379920A (en) * | 1979-10-31 | 1983-04-12 | Glaxo Group Limited | Cephalosporins |
EP0137365A3 (en) * | 1983-09-06 | 1986-01-15 | Takeda Chemical Industries, Ltd. | Cephalosporins and their production |
EP0160745A3 (en) * | 1984-05-07 | 1986-11-05 | Takeda Chemical Industries, Ltd. | Cephem compounds and their production |
WO2004106347A1 (en) | 2003-05-28 | 2004-12-09 | Dsm Ip Assets B.V. | Cephem compound |
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