DK143111B - PROCEDURE FOR PREPARING AMINOCYCLOTOL COMPOUNDS OR ACID ADDITION SALTS - Google Patents
PROCEDURE FOR PREPARING AMINOCYCLOTOL COMPOUNDS OR ACID ADDITION SALTS Download PDFInfo
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- DK143111B DK143111B DK63176AA DK63176A DK143111B DK 143111 B DK143111 B DK 143111B DK 63176A A DK63176A A DK 63176AA DK 63176 A DK63176 A DK 63176A DK 143111 B DK143111 B DK 143111B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/22—Cyclohexane rings, substituted by nitrogen atoms
- C07H15/222—Cyclohexane rings substituted by at least two nitrogen atoms
- C07H15/226—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings
- C07H15/234—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings attached to non-adjacent ring carbon atoms of the cyclohexane rings, e.g. kanamycins, tobramycin, nebramycin, gentamicin A2
- C07H15/236—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings attached to non-adjacent ring carbon atoms of the cyclohexane rings, e.g. kanamycins, tobramycin, nebramycin, gentamicin A2 a saccharide radical being substituted by an alkylamino radical in position 3 and by two substituents different from hydrogen in position 4, e.g. gentamicin complex, sisomicin, verdamycin
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/56—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds from heterocyclic compounds
- C07C45/57—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds from heterocyclic compounds with oxygen as the only heteroatom
- C07C45/58—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds from heterocyclic compounds with oxygen as the only heteroatom in three-membered rings
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/385—Saturated compounds containing a keto group being part of a ring
- C07C49/487—Saturated compounds containing a keto group being part of a ring containing hydroxy groups
- C07C49/497—Saturated compounds containing a keto group being part of a ring containing hydroxy groups a keto group being part of a six-membered ring
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
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- C07—ORGANIC CHEMISTRY
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- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
- C07D303/02—Compounds containing oxirane rings
- C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
- C07D303/14—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by free hydroxyl radicals
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Description
(11) FREMLÆGGELSESSKRIFT 1431 1 1 DANMARK ln,-c'·' ® J* Hp t(21) Ansøgning nr. 65 1 /76 (22) Indleveret den 17· feb. 1976 (24) Ubedag 1 7. feb. 1976 (44) Ansøgningen fremlagt og fremlæggelsesskriftet offentliggjort den JO . Π13.Γ · 1 9@1(11) PRESENTATION 1431 1 1 DENMARK ln, -c '·' ® J * Hp t (21) Application No. 65 1/76 (22) Filed on 17 · Feb. 1976 (24) Unwind 1 Feb 7 1976 (44) The application presented and the petition published on JO. Π13.Γ · 1 9 @ 1
DIREKTORATET FORDIRECTORATE OF
PATENT- OG VAREMÆRKEVÆSENET <30> Wontet begæret fra denPATENT AND TRADE MARKET <30> Wont be requested from it
18. feb. 1975* 550275* USFeb 18 1975 * 550275 * US
22. sep. 1975* 615595* USSep 22 1975 * 615595 * US
<71> STERLING DRUG INC., 9O Park Avenue, New York, N.Y., US.<71> STERLING DRUG INC., 9O Park Avenue, New York, N.Y., US.
(72) Opfinder: Sol Jacob Dautn, 468 West Lawrence Street, Albany, New York, US: Robert La Grone TTlarke, 5 Sunset Drive, Delmar, New York, US.(72) Inventor: Sol Jacob Dautn, 468 West Lawrence Street, Albany, New York, US: Robert La Grone TTlarke, 5 Sunset Drive, Delmar, New York, US.
(74) Fuldmægtig under sagens behandling:(74) Plenipotentiary in the proceedings:
Ingeniørfirmaet Hofman-Bang & Bout ard._ (54) Fremgangsmåde til fremstilling af aminocyclltol-forbindelser eller syreadditionssalte deraf.The Hofman-Bang & Bout engineering firm (54) Process for the preparation of aminocyclytol compounds or acid addition salts thereof.
Opfindelsen angår en fremgangsmåde til fremstilling af hidtil u-kendte aminocyclitol-forbindelser med den i krav 1's indledning angivne almene formel I eller syreadditionssalte deraf, hvilken fremgangsmåde er ejendommelig ved det i krav l’s kendetegnende del anførte.The invention relates to a process for the preparation of novel aminocyclitol compounds having the general formula I or acid addition salts thereof according to the preamble of claim 1, which is characterized by the characterizing part of claim 1.
Den anvendte dyrkningsmetode svarer til den metode, som er beskrevet i US-patentskrift nr. 5 669 858. Som mikroorganisme anvendes en mutant af Micromonospora purpurea, fortrinsvis organismen betegnet Micromonospora purpurea ATTCC 51 119.The culture method used is similar to that described in U.S. Patent No. 5,669,858. As a microorganism, a mutant of Micromonospora purpurea, preferably the organism designated Micromonospora purpurea ATTCC 51 119, is used.
15 88
De forbindelser med formlen I, hvori en af R , R og R er en 2 U3111 |n/-amino-cc-hydroxy-lavere-alkanoylgruppe, fremstilles ved den metode, som er beskrevet i US-patentskrift nr. 3 780 018, og som består i at omsætte en ved dyrkningen opnået forbindelse med form-len I, hvori R , Ir og R alle er hydrogen, med en N-hydroxysucci-nimidester med formlen _ o 5-, f/ \ I /The compounds of formula I wherein one of R, R and R is a 2-amino-cc-hydroxy-lower alkanoyl group are prepared by the method described in U.S. Patent No. 3,780,018, and consisting of reacting a compound of formula I, wherein R, Ir and R are all hydrogen, with an N-hydroxysuccinimide ester of the formula
// CH20-C-NHCH2(CH2)nCH0HC0-0-N// CH20-C-NHCH2 (CH2) nCHOHCO0-N
\=/ V-1 -iv\ = / V-1 -iv
OISLAND
hvori n betyder 0 eller 1. Den resulterende blanding af forbindel-wherein n is 0 or 1. The resulting mixture of
" 4 «2 Q„4« 2 Q
seme med formlen I, hvori en af R , R og R er q,—(N—benzyloxy— carbonyl)amino-a-hydroxy-lavere-alkanoylgruppen: /~~\ *jj / v CH20-C-NHCH2(CH2)nCHOHCO- og de to andre er hydrogen, underkastes derpå hydrogenolyse af ben-zyloxycarbonylgruppen med hydrogen over en katalysator.a scheme of formula I wherein one of R, R and R is q, - (N-Benzyloxy-carbonyl) amino-α-hydroxy-lower-alkanoyl group: - (CH 2) -CH 2 -C-NHCH 2 (CH 2) nCHOHCO- and the other two are hydrogen, are then subjected to hydrogenolysis of the benzyloxycarbonyl group with hydrogen over a catalyst.
Der opnås som angivet ovenfor en blanding af de tre mulige isomere 1 g monoamider, hvori henholdsvis R -, R - og R -aminhydrogenatomet er erstattet med W-(N-benzyloxycarbonyl)-amino-a-hydroxy-lavere-alka-noylgruppen. Når der ønskes individuel karakterisering og undersøgelse af disse produkter, må de selvfølgelig adskilles fra hverandre. Acyleringsreaktionen udføres ved omsætning af molært ækvivalente mængder af forbindelsen med formlen I og N-hydroxysuccin-imidesteren, fortrinsvis ved en temperatur fra omkring -10 til omkring 10°C og i en vandig opløsning af et inert organisk opløsningsmiddel, f.eks. tetrahydrofuran, dioxan, ethylenglycol-dimethyl-ether, dimethylacetamid, dimethylformamid, propylenglycol-dimethyl-ether og lignende.As indicated above, a mixture of the three possible isomeric 1 g monoamides is obtained in which the R, R, and R amine hydrogen atoms respectively are replaced by the W- (N-benzyloxycarbonyl) amino-α-hydroxy-lower alkanoyl group. Of course, when individual characterization and examination of these products is desired, they must be separated from one another. The acylation reaction is carried out by reacting molar equivalent amounts of the compound of formula I and the N-hydroxysuccin imide ester, preferably at a temperature of from about -10 to about 10 ° C and in an aqueous solution of an inert organic solvent, e.g. tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dimethylacetamide, dimethylformamide, propylene glycol dimethyl ether and the like.
3 1631113 163111
Hydrogenolysen af benzyloxyearbonylgruppen udføres over en palladi-um-på-kul-katalysator i et inert, med vand "blandbart organisk opløsningsmiddel, f.eks. methanol, ethanol, dioxan, tetrahydrofuran, ethylenglycol-dimethylether, propylenglycol-dimethylether og lignende .The hydrogenolysis of the benzyloxyearbonyl group is carried out over a palladium-on-carbon catalyst in an inert, water-miscible organic solvent, for example, methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, propylene glycol dimethyl ether and the like.
De aminocyclitoler, som anvendes ved udøvelse -af opfindelsen, er kendte forbindelser. Disse er f.eks.: streptamin-sulfat [Peck et al., J. Am. Chem. Soc. 68, 776 (1946)]; 2-epistreptamin-dihydrochlorid [Suami et al., J, Org. Chem. 33, 2831 (1968)]; 2,5-dideoxystreptamin-dihydrochlorid, smp. > 300°C; og 6,7-diazabicyclo[3,2,1]octan-2,4-diol (exo, exo), smp. 185 - 193°C [De sidstnævnte to forbindelser er angivet af Testa et al., J. Antibiotics 27, 917-921 (1974)].The aminocyclitols used in the practice of the invention are known compounds. These are, for example: streptamine sulfate [Peck et al., J. Am. Chem. Soc. 68, 776 (1946)]; 2-Epistreptamine dihydrochloride [Suami et al., J, Org. Chem. 33, 2831 (1968)]; 2,5-dideoxystreptamine dihydrochloride, m.p. > 300 ° C; and 6,7-diazabicyclo [3,2,1] octane-2,4-diol (exo, exo), m.p. 185 - 193 ° C [The latter two compounds are reported by Testa et al., J. Antibiotics 27, 917-921 (1974)].
N-hydroxysuccinimidesterne med formlen IV er en almen kendt klasse af forbindelser.The N-hydroxysuccinimide esters of formula IV are a generally known class of compounds.
Forbindelserne med formlen I er blevet prøvet ved en standard successiv fortyndingsprøvning og har vist sig at have antibakteriel aktivitet, især over for gentamicin-resistente organismer. Forbindelserne er således nyttige som antibakterielle midler.The compounds of formula I have been tested by a standard successive dilution assay and have been shown to have antibacterial activity, especially against gentamicin-resistant organisms. Thus, the compounds are useful as antibacterial agents.
Forbindelserne med formlen I er hovedsageligt beregnet til oral, topisk eller parenteral indgivning og kan præpareres til brug med en pharmaceutisk bærer ved suspension, enten i form af deres frie baser eller som pharmaceutisk acceptable, ikke-toxiske syreadditionssalte, i en inert bærer, såsom polyethylenglycol, eller ved tablettering eller indkapsling til oral indgivning enten alene eller med egnede tilsætningsstoffer, eller de kan alternativt sammensættes med konventionelle cremer eller vaseliner til topisk påføring .The compounds of formula I are mainly intended for oral, topical or parenteral administration and can be prepared for use with a pharmaceutical carrier by suspension, either in the form of their free bases or as pharmaceutically acceptable, non-toxic acid addition salts, in an inert carrier such as polyethylene glycol. , or by tableting or encapsulation for oral administration either alone or with suitable additives, or alternatively they may be formulated with conventional creams or vaselines for topical application.
4 1431114 143111
Molekylstrukturerne af forbindelserne med formlen I blev opstillet på basis af deres chromatografiske egenskaber bestemt ved tyndt-lagschromatografisk analyse, deres magnetiske kerner e s onans-(NMR)-og massespektrum, nedbrydning til kendte forbindelser samt overensstemmelse mellem beregnede og fundne værdier for elementanalyser af forbindelserne.The molecular structures of the compounds of formula I were established on the basis of their chromatographic properties determined by thin-layer chromatographic analysis, their magnetic nucleation (NMR) and mass spectrum, decomposition to known compounds, and agreement between calculated and found values for elemental analyzes of the compounds.
De følgende udførelseseksempler tjener til nærmere at belyse fremgangsmåden ifølge opfindelsen og fremstillingen af ndgangafn-pbl udel-seme for denne.The following exemplary embodiments serve to elucidate in detail the process of the invention and the preparation of the ndgangafn-pbl except for it.
MUT A TTOTSrSl'RSMGANGSMÅDERMUT A TTOTSrSl'MEcEmEs
Yed de følgende procedurer blev der anvendt forskellige medier sammensat som følger:The following procedures used different media composed as follows:
Medium 1: N-Z Arnin g/3_Medium 1: N-Z Arnin g / 3_
Glucose 10Glucose 10
Opløselig stivelse 20 Gærekstrakt 5 N-Z-Amin-Iype A (Difco) 5Soluble Starch 20 Yeast Extract 5 N-Z-Amine Type A (Difco) 5
CaCO^ 1CaCO1
Agar 15Agar 15
Medium 2: Spiremedium (i destilleret vand) Kødekstrakt 0,3 <foMedium 2: Sprouting medium (in distilled water) Meat extract 0.3 <fo
Trypton 0,5 1°Tryptone 0.5 1 °
Dextrose 0,1 $Dextrose 0.1 $
Opløselig stivelse 2,4 i» Gær ekstrakt 0,5 iaSoluble starch 2.4 in »Yeast extract 0.5 ia
CaC03 0,4 1°CaCO 3 0.4 1 °
Medium 3: Sojabønne-Glucose EliMedium 3: Soybean Glucose Eli
Sojabønnemel 30Soybean meal 30
Dextrose (cerelose) 40Dextrose (Cerelose) 40
Ca003 1 5 143111Ca003 1 5 143111
Medium 4: TGE g/1Medium 4: TGE g / l
Trypticase-glucose-ekstrakt 5,0Trypticase glucose extract 5.0
Trypticase-pepton 3,0Trypticase peptone 3.0
Glucose 1,0Glucose 1.0
Agar 15,0Agar 15.0
Organismen Micromonospora purpurea "blev fremskaffet fra the U.S.The organism Micromonospora purpurea "was obtained from the U.S.
Dept, of Agriculture som NERL 2953 og holdt på N-Z-amin-skråsubstrater (Medium 1). Submerse gæringer blev gennemført i kolber indeholdende spiremedium (Medium 2) i 4 dage ved 37°C på en roterende ryster. Era dette første stadium blev der overført et 10 pct. podemateriale til spiremediet (Medium 2), og gæringen fortsattes som ovenfor ved 28°C i 7 dage.Dept, of Agriculture as NERL 2953 and held on N-Z-amine oblique substrates (Medium 1). Submerged ferments were carried out in flasks containing germ medium (Medium 2) for 4 days at 37 ° C on a rotary shaker. In this first stage, a 10 per cent transfer was made. graft material to the germinating medium (Medium 2) and fermentation was continued as above at 28 ° C for 7 days.
Med henblik på at fastslå organismens evne til at biosyntetisere gentamicin i fravær af tilsat deoxystreptamin udførtes en tredje stadium-gæring under anvendelse af et 5 pct. podemateriale i en 10 liter fermentor i et sojabønne-glucose-medium (Medium 3) ved 28°C}/ idet der omrørtes ved 200 omdr./min. og luftedes ved 2 liter/min. med filtreret luft. Efter 6 dage blev tankindholdene gjort sure til pH 2,0 med 6N svovlsyre, filtreret, og en 500 ml portion neutraliseret med ammoniumhydromid og sendt igennem en "IRC-50"-ion-bytterharpiks (Na+-form). Søjlen blev derpå skyllet med vand og elueret med 2N svovlsyre. Ifølge den procedure, som er beskrevet i US-patentskrift nr. 3 091 572, blev der isoleret en 300 mg prøve af råt gentamicin, som fandtes at være biologisk aktivt og indeholdt tre komponenter, der svarede til gentamicin-C1, -Cg og -C^a ved tyndtlagskromatografisk undersøgelse.In order to establish the organism's ability to biosynthesize gentamicin in the absence of added deoxystreptamine, a third-stage fermentation was performed using a 5 per cent. graft in a 10 liter fermentor in a soybean glucose medium (Medium 3) at 28 ° C, stirring at 200 rpm. and aerated at 2 liters / min. with filtered air. After 6 days, the tank contents were acidified to pH 2.0 with 6N sulfuric acid, filtered and a 500 ml aliquot neutralized with ammonium hydromide and passed through an "IRC-50" ion exchange resin (Na + form). The column was then rinsed with water and eluted with 2N sulfuric acid. According to the procedure described in U.S. Patent No. 3,091,572, a 300 mg sample of crude gentamicin was found which was found to be biologically active and contained three components corresponding to gentamicin-C1, -Cg and - C ^ a by thin layer chromatographic examination.
Med henblik på mutering af organismen blev væskekulturer dyrket i Medium 2 (37°C i 3 dage), og de resulterende celler indhøstet ved centrifugering, vasket og gensuspenderet i pufret saltvand. Denne suspension blev behandlet med det mutagene middel, N-methyl-N’-nitro-N-nitrosoguanidin. Prøver af den mutagenerede kultur blev sat på plader i Medium 4 ved 37°C, indtil kolonier var tydelige (sædvanligvis omkring en uge). Kolonier blev udsået på tilsvarende plader (Medium 4), hvoraf et sæt blev overhældt med en sporesuspension af B. subtilis. Efter inkubering ved 37°C i 18-20 timer blev de 6 143111 "såninger”, som ikke viste nogen inhiberingszone på B. subtilis-pladen, overført fra hovedpladen (ingen B. subtilis) til Medium 1-skråsubstrater og inkuberet, indtil fuld vækst var tydelig.For mutation of the organism, liquid cultures were grown in Medium 2 (37 ° C for 3 days) and the resultant cells harvested by centrifugation, washed and resuspended in buffered saline. This suspension was treated with the mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine. Samples of the mutagenic culture were placed on plates in Medium 4 at 37 ° C until colonies were evident (usually about a week). Colonies were seeded on similar plates (Medium 4), one set of which was poured with a spore suspension of B. subtilis. After incubation at 37 ° C for 18-20 hours, the 6 143111 "seeds" showing no inhibition zone on the B. subtilis plate were transferred from the main plate (no B. subtilis) to Medium 1 oblique substrates and incubated until full. growth was evident.
Bisse potentielt ikke-producerende mutantstammer Blev derpå udsat for deoxystreptamin i et forsøg på at stimulere antiBiotisk Biosyntese som følger. Lagerkulturer af de potentielle mutantstammer Blev udstrøget som Bånd på overfladen af Medium 4-plader og inku-Beret ved 37°C, indtil vækst var tydelig (omkring 3-4 dage). Eil-terpapirskiver Blev derpå dyppet i en opløsning af deoxystreptamin (500 pg/ml) og anBragt ovenpå kulturstregen. Efter inkuBation i 24 timer Blev pladens overflade podet med B. suBtilis ved overlægningsteknikken, og inkubationen fortsattes i yderligere 18-20 timer. Isolater, som viste inBiBeringszoner omkring skiven, Blev Betegnet som deoxystreptamin-mutanter. En sådan mutant, Betegnet mutant YIB og deponeret i the American Type Culture Collection, 12301 Parklawn Brive, Rockville, Md. 20852 som Micromonospora purpurea ATCC 31 119, anvendtes til fremstilling af antiBiotica af gentamicin-type som Beskrevet nedenfor.Pieces of Potentially Non-Producing Mutant Strains Then were exposed to deoxystreptamine in an attempt to stimulate antiBiotic Biosynthesis as follows. Stock cultures of the potential mutant strains were plated as Bands on the surface of Medium 4 plates and incubated at 37 ° C until growth was evident (about 3-4 days). Eilter paper slices were then dipped in a solution of deoxystreptamine (500 µg / ml) and placed on top of the culture line. After incubation for 24 hours, the plate surface was seeded with B. suBtilis by the overlay technique and the incubation was continued for an additional 18-20 hours. Isolates showing inbibition zones around the disc were designated as deoxystreptamine mutants. One such mutant, designated YIB Mutant and deposited in the American Type Culture Collection, 12301 Parklawn Brive, Rockville, Md. 20852 as Micromonospora purpurea ATCC 31 119 was used to prepare gentamicin-type antibiotics as described below.
BIOSYNTESER MED M. PURPUREA ATCC 31 119 Eksempel 1BIOSYNTHESES WITH M. PURPUREA ATCC 31 119 Example 1
Mutantorganismen Blev holdt på N-Z-amin-agar-skråsuBstrater (Medium 1), hvorfra der foretoges overførsler til kolher indeholdende 500 ml spiremedium (Medium 2). Kolberne Blev inkuberet ved 28°C i 4 dage på en roterende ryster (5 cm slag) ved 225 omdr./min.The mutant organism was kept on N-Z-amine agar slant substrates (Medium 1) from which transfers were made to flasks containing 500 ml of germinating medium (Medium 2). The flasks were incubated at 28 ° C for 4 days on a rotary shaker (5 cm stroke) at 225 rpm.
10 volumenprocent podemateriale fra spirestadiet Blev aseptisk overført til 14 liters fermentorer indeholdende 9 liter sterilt spiremedium (Medium 2). Bisse Blev omrørt med 450 omdr./min. ved 28-29°C og luftet med filtreret luft ved 5 liter/minut. Ved podningstidspunktet tilsattes 200 mg/liter streptaminsulfat som en suspension i sterilt destilleret vand. Gæringen fortsattes i 8 dage.10% by volume seed graft from the germination stage Was aseptically transferred to 14 liter fermentors containing 9 liters of sterile germ medium (Medium 2). Bisse Stirred at 450 rpm. at 28-29 ° C and aerated with filtered air at 5 liters / minute. At the time of inoculation, 200 mg / liter of streptamine sulfate was added as a suspension in sterile distilled water. The fermentation was continued for 8 days.
10 liter 24 timer gammelt podemateriale, fremstillet som ovenfor, Blev aseptisk overført til 130 liters fermentorer indeholdende 7 163111 70 liter sterilt spiremedium (Medium 2), og der tilsattes 0,31 g/li-ter streptaminsulfat suspenderet i sterilt destilleret vand. Aero-gæring gennemførtes ved 29°C i 7 dage.10 liter 24 hour old seed material prepared as above was aseptically transferred to 130 liter fermenters containing 70 liters of sterile germ medium (Medium 2) and 0.31 g / liter streptamine sulfate suspended in sterile distilled water was added. Aero fermentation was performed at 29 ° C for 7 days.
Gæringerne "blev afsluttet ved tilsætning af 10N svovlsyre til pHThe ferments were terminated by adding 10N sulfuric acid to pH
2,0 og filtrering under anvendelse af et filterhjælpemiddel til fjernelse af mycelier. Den filtrerede væske "blev indstillet til pH 7,0, og der tilsattes 1,56 g oxalsyre pr. gram tilstedeværende calciumcarbonat i mediet for at fjerne calcium. Blandingen fik lov at stå natten over, og den klarede væske blev fradekanteret og sendt igennem "Bio-Rex 70" (svag kationbytterharpiks) i Na+-formen under anvendelse af omkring 14 g harpiks pr. liter væske. Søjlen blev derpå vasket med destilleret vand og elueret med 2N svovlsyre. Alle fraktioner, som udviste antibiotisk aktivitet, blev kombineret, neutraliseret og koncentreret under vakuum ved under 50°C til det punkt, hvor saltkrystallisation blev tydelig (omkring en tredjedel volumen). pH-værdien blev derpå indstillet til 10,5, og der tilsattes 4 volumener acetone for at udfælde uorganisk materiale, som blev fjernet ved filtrering. Filtratet blev indstillet til pH 5,0 med 6N svovlsyre, koncentreret under vakuum til omkring 1/20 af det oprindelige volumen og afkølet.2.0 and filtration using a filter aid for removing mycelia. The filtered liquid "was adjusted to pH 7.0 and 1.56 g of oxalic acid per gram of calcium carbonate present in the medium was removed to remove calcium. The mixture was allowed to stand overnight and the clarified liquid was decanted off and passed through" Bio-Rex 70 "(weak cation exchange resin) in the Na + form using about 14 g of resin per liter of liquid. The column was then washed with distilled water and eluted with 2N sulfuric acid. All fractions showing antibiotic activity were combined, neutralized and concentrated in vacuo at below 50 ° C to the point where salt crystallization became apparent (about one-third volume), the pH was then adjusted to 10.5 and 4 volumes of acetone added to precipitate inorganic material which was removed by filtration The filtrate was adjusted to pH 5.0 with 6N sulfuric acid, concentrated in vacuo to about 1/20 of the original volume and cooled.
Der udfældedes et hvidt krystallinsk materiale, som blev opsamlet ved filtrering og smeltede ved over 300°C. Det fandtes ud fra dets tyndtlagskromatografiske egenskaber og dets infrarøde spektrum at være identisk med streptaminsulfat. Fra to 10 liters fermenteringer, forarbejdet som ovenfor, blev der opnået i alt 0,7 g streptaminsulfat, og fra to 80 liters fermenteringer blev der opnået i alt 21 g streptaminsulfat i dette trin.A white crystalline material precipitated, which was collected by filtration and melted at over 300 ° C. Based on its thin-layer chromatographic properties and its infrared spectrum, it was found to be identical to streptamine sulfate. From two 10-liter fermentations, processed as above, a total of 0.7 g of streptamine sulfate was obtained, and from two 80 liter of fermentations a total of 21 g of streptamine sulfate was obtained in this step.
Filtratet blev yderligere koncentreret, og der tilsattes 10 volumener methanol, hvilket gav det første rå faste antibioticum. Fra to 10 liters fermenteringer blev der opnået 7 g, og fra to 80 liters fermenteringer blev der opnået 24 g.The filtrate was further concentrated and 10 volumes of methanol were added to give the first crude solid antibiotic. From two 10 liter fermentations 7 g was obtained and from two 80 liter fermentations 24 g was obtained.
Med henblik på at identificere antibiotiske komponenter under rensningsprocedurerne bestemtes kromatografiske mobilitetsværdier opnået ved papirkromatografi og tyndtlagskromatografi for gentamicin-C^, -Cg og -C-]a og for hver af de tilsvarende amino -cyclitol-analoge, fremstillet som angivet ovenfor, hvor den kro 8 143111 matografiske mobilitet (i det følgende betegnet C.M.) er udtrykt som: q jj _ komponentens afstand fra oprindelsesstedet gentamicin-O^1 s afstand fra oprindelsesstedetIn order to identify antibiotic components during the purification procedures, chromatographic mobility values obtained by paper chromatography and thin layer chromatography for gentamicin-C ^, -Cg and -C-] α and for each of the corresponding amino-cyclitol analogs prepared as above were determined. Inn 8 143111 matographic mobility (hereinafter referred to as CM) is expressed as: q jj - the distance of the component from the origin of the gentamicin-O
De anvendte kromatografiske systemer var som følger: S^s t em_ 1_:The chromatographic systems used were as follows: S ^ s t em_ 1_:
Whatman nr. 1 papir mættet med 0,95M sulfat-hydrogensulfat og udviklet på nedadgående måde i 80 pct. vandigt ethanol + 1,5 i° NaCl og efterfølgende bioautografi under anvendelse af B. subtilis som prøve or gani sme.Whatman No. 1 paper saturated with 0.95M sulfate hydrogen sulfate and developed downwards in 80 per cent. aqueous ethanol + 1.5 in ° NaCl and subsequent bioautography using B. subtilis as a sample or genome sme.
System 2:System 2:
Silicagel I 254-plade udviklet i nedre fase af 1:1:1 chloroform/me-thanol/koncentreret ammoniumhydroxid (28 $). Komponenterne blev lokaliseret med en nihhydrin-sprøjtning efter opvarmning.Silica gel I 254 plate developed in the lower phase of 1: 1: 1 chloroform / methanol / concentrated ammonium hydroxide ($ 28). The components were localized with a nihhydrin spray after heating.
C.M.-værdierne for de overvejende antibiotiske komponenter ifølge eksemplet i sammenligning med et refer ene e-gentamic in-komplex, alt i forhold til gentamicin-C-^, er vist i tabel I, hvor forbindelserne betegnet komponent 1, komponent 2 og komponent 3 skal forstås at være henholdsvis: 0-3-deoxy-4-C-methyl-5-methylamino-p-I-arabinopyranosyl-(1*6)- 0-[2-amino-6-methylamino-6-C-me thy1-2,5,4,6-tetradeoxy-a-D-ery-thro-glucopyranosyl-( 1*4) ] -D-streptamin; 0-5-deoxy-4-C-methyl-3-methylamino-P-l-arabinopyranosyl-( 1*6) -0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythroglucopyrano-syl-( 1*4) j -D-streptamin; og 0-3-deoxy-4-C-methyl~3-methylamino-P-L-arabinopyranosyl-( 1*6) - 0-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-( 1*4)]- D-streptamin.The CM values of the predominant antibiotic components of the example in comparison with a reference e-gentamic in-complex, all relative to gentamicin-C-1, are shown in Table I, where the compounds are designated component 1, component 2 and component 3 is meant to be respectively: 0-3-deoxy-4-C-methyl-5-methylamino-pI-arabinopyranosyl- (1 * 6) - O- [2-amino-6-methylamino-6-C-thyme- 2,5,4,6-tetradeoxy-α-D-γ-thro-glucopyranosyl- (1 * 4)] -D-streptamine; 0-5-Deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1 * 6) -0- [2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy -aD-erythroglucopyrano-syl- (1 * 4) j -D-streptamine; and 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1 * 6) -O- [2,6-diamino-2,3,4,6-tetradeoxy-α-D glucopyranosyl- (1 * 4)] - D-streptamine.
9 1431119 143111
TABEL ITABLE I
C.M. System 1 C.M. System 2 G-entamlcin-C^ 1 1 G-entamlcin-02 0,89 0,83C.M. System 1 C.M. System 2 G-entamyclin-C1-1 G-entamycin-02 0.89 0.83
Gentamicin-C., 0,50 0,67Gentamicin C, 0.50 0.67
Komponent 1 (overvejende) 0,96 0,92Component 1 (predominantly) 0.96 0.92
Komponent 2 (mindre) 0,76 0,75Component 2 (minor) 0.76 0.75
Komponent 3 (mindre) 0,50 0,61Component 3 (minor) 0.50 0.61
Era 10 liters fermentoreme "blev det rå faste stof (7 g), som viste antibakteriel aktivitet, suspenderet i 200 ml methanol og 10 ml koncentreret ammoniumhydroxid (28 $), og "blandingen blev omrørt i 30 minutter og filtreret. Dette blev gentaget to gange til, og filtraterne blev kombineret og koncentreret under vakuum til opnåelse af en bleggul olie, som vejede 0,9 g. De "forbrugte salte" var i hovedsagen fri for antibiotisk aktivitet.In the 10 liter fermentor ", the crude solid (7 g) showing antibacterial activity was suspended in 200 ml of methanol and 10 ml of concentrated ammonium hydroxide (28 $), and" the mixture was stirred for 30 minutes and filtered. This was repeated two more times, and the filtrates were combined and concentrated in vacuo to give a pale yellow oil weighing 0.9 g. The "spent salts" were essentially free of antibiotic activity.
Den olieagtige base blev blandet med 4 g silicagel (Davison grade 923, partikelstørrelse 0,074 - 0,149 mm) og sat på en silicagelsøjle, som målte 1,8 x 28 cm. Søjlen var fremstillet som en opslæmning under anvendelse af den nedre fase af isopropylalkohol/chloroform/17 pct. vandigt ammoniumhydroxid (1:2:1). Søjlen blev udviklet med dette opløsningsmiddel, og der blev opsamlet 50 ml fraktioner.The oily base was mixed with 4 g of silica gel (Davison grade 923, particle size 0.074 - 0.149 mm) and placed on a silica gel column measuring 1.8 x 28 cm. The column was prepared as a slurry using the lower phase of isopropyl alcohol / chloroform / 17 per cent. aqueous ammonium hydroxide (1: 2: 1). The column was developed with this solvent and 50 ml fractions were collected.
Fraktionerne 8 og 9 indeholdt en enkelt ninhydrin-positiv komponent, som gav 20 mg som en bleggul olie efter fjernelse af opløsningsmidlet. Massespektret for dette materiale, betegnet komponent 1, viste en molekylær ion og hovedfragmenter, som hver var 16 masseenheder (dvs. et oxygenatom) større end det, der opnås fra gentamicin-C1 som følger: 10 U3111Fractions 8 and 9 contained a single ninhydrin positive component which gave 20 mg as a pale yellow oil after removal of the solvent. The mass spectrum of this material, designated Component 1, showed a molecular ion and major fragments, each of which were 16 mass units (i.e., an oxygen atom) larger than that obtained from gentamicin-C1 as follows:
Reference gentamicin C^: M+ 4-77, 420, 60, 350, 347, 322, 319, 304Reference gentamicin C ^: M + 4-77, 420, 60, 350, 347, 322, 319, 304
Komponent 1: M 493, 436, 376, 366, 363, 338, 335, 320.Component 1: M 493, 436, 376, 366, 363, 338, 335, 320.
Dette materiale blev omdannet til dets sulfatsalt ved opløsning i ethanol ved tilsætning af nogle få dråber ethanol indeholdende svovlsyre. Det resulterende hvide faste stof blev opsamlet og tørret, hvorved der blev opnået 22 mg af komponent 1, 0-3-deoxy-4-C-me thy1-3-methylamino-β-1-arabinopyranosyl-( 1-*6)-0-[2-amino-6-methyl-amino—6—0—methyl—2,3 , 4 , 6—tetradeoxy—u—D—erythro—glueopyranysol— (1“*4)]-D-streptamin som di-base*heptasulfat*decahydratet, smp.This material was converted to its sulfate salt by dissolving in ethanol by adding a few drops of ethanol containing sulfuric acid. The resulting white solid was collected and dried to give 22 mg of component 1, 0-3-deoxy-4-C-thyme-3-methylamino-β-1-arabinopyranosyl- (1- * 6) - O- [2-amino-6-methylamino-6-O-methyl-2,3, 4, 6-tetradeoxy-u-D-erythro-glueopyranysole - (1 "* 4)] - D-streptamine as di -base * heptasulfate * decahydrate, m.p.
>300°C.> 300 ° C.
Analyse beregnet for 0: 0 27,22 - H 6,53 - N 7,55 - S 12,09Analysis calculated for 0: 0 27.22 - H 6.53 - N 7.55 - S 12.09
Rundet: C 27,15 - H 6,67 - N 7,79 - S 12,76.Found: C 27.15 - H 6.67 - N 7.79 - S 12.76.
Erakti oneme 10-13 gav en mere polær ninhydrin-positiv komponent, betegnet komponent 2, 0-3-deoxy-4-0-methyl-3-methylamino-P-L-arabino-pyranosyl-( 1-*>6) -0-[ 2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(l-M)]D-streptamin, som udviste antibiotisk aktivitet.The actions 10-13 yielded a more polar ninhydrin positive component, designated component 2, 0-3-deoxy-4-O-methyl-3-methylamino-PL-arabino-pyranosyl- (1-6> -0) [2,6-Diamino-6-C-methyl-2,3,4,6-tetradeoxy-αD-erythro-glucopyranosyl (1M)] D-streptamine, which showed antibiotic activity.
Fraktionerne 15-26 gav en tredje mere polær komponent, som udviste antibiotisk aktivitet. Massespektret for denne komponent viste karakteristiske sukkermaksima svarende til gentamicin-Cήo ved 129 (purpurosamin) og 160p(garosamin), og den betegnes komponent 3, 0-3-deoxy-4-C-methy 1-3-me thy lamino-6-L-arabinopyranosyl-( 1-*6)-0-[ 2,6-diamino-2,3,4,6-tetradeoxy-oc-D-erythro-glucopyranosyl-( 1-*4) 1 -D-streptamin.Fractions 15-26 provided a third more polar component which exhibited antibiotic activity. The mass spectrum of this component showed characteristic sugar maxima similar to gentamicin-CήO at 129 (purpurosamine) and 160β (garosamine), and it is designated component 3, 0-3-deoxy-4-C-methyl 1-3-methyl thyro-6- L-arabinopyranosyl- (1- * 6) -O- [2,6-diamino-2,3,4,6-tetradeoxy-oc-D-erythro-glucopyranosyl- (1- * 4) 1- D-streptamine.
Alternativt blev den nye komponent 1 isoleret som følger: En blan ding af rå antibiotieumbase, fremstillet som ovenfor (0,9 g), blev opløst i 7 ml vand, og pH-værdien blev indstillet til 4,5 med 1N svovlsyre. Opløsningen blev sendt igennem en stærk anionbyt-terkolonne ('IRA 401") i 0H“~formen (lagets mål = 0,7 x 10 cm). Søjlen blev elueret med vand, og eluatet inddampet i vacuum ved 35°C.Alternatively, the new component 1 was isolated as follows: A mixture of crude antibiotic base, prepared as above (0.9 g), was dissolved in 7 ml of water and the pH was adjusted to 4.5 with 1N sulfuric acid. The solution was passed through a strong anion exchange column ('IRA 401') in the OH form (layer size = 0.7 x 10 cm). The column was eluted with water and the eluate evaporated in vacuo at 35 ° C.
Den resulterende remanens blev udrevet med 50 ml af den nedre fase 11 143111 af et opløsningsmiddel sammensat af 17 pct. vandigt ammoniumhydroxid/ isopropylalkohol/chloroform (1:1:2). Opløsningen blev dekanteret fra og koncentreret under vakuum, hvorved der blev opnået en olie-remanens, som vejede 140 mg. Massespektret for dette materiale svarer til spektret fra fraktionerne 8 og 9 ovenfor, dvs.The resulting residue was triturated with 50 ml of the lower phase 11 of a solvent composed of 17 per cent. aqueous ammonium hydroxide / isopropyl alcohol / chloroform (1: 1: 2). The solution was decanted off and concentrated in vacuo to give an oil residue weighing 140 mg. The mass spectrum of this material corresponds to the spectrum of fractions 8 and 9 above, i.e.
M+ 493, 436, 376, 366, 363, 338, 335, 320.M + 493, 436, 376, 366, 363, 338, 335, 320.
Det magnetiske kemeresonansspektrum for komponent 1 stemte også overens med den antagne struktur svarende til 0-3-deoxy-4-C-methyl- 3-me thy lamino - β-L-ar abino pyr ano sy 1- (1-*6)-0-[ 2-amino-6-methylamino- 6-C-methyl-2,3,4,5-tetradeoxy-a-D-erythro-glueopyranosyl-(1-*4)]-D-streptamin og er sammenfattet i tabel II.The magnetic chemistry resonance spectrum of component 1 also matched the assumed structure corresponding to 0-3-deoxy-4-C-methyl-3-methyl-lamino - β-L-ar abino pyr ano sy 1- (1- * 6) O- [2-Amino-6-methylamino-6-C-methyl-2,3,4,5-tetradeoxy-αD-erythro-glueopyranosyl- (1- * 4)] - D-streptamine and is summarized in Table II.
TABEL IITABLE II
_S_ Integration _Tilskrivelse_ 5,87; 5,60 2 anomere OCHO’-grupper_S_ Integration _ Attribution_ 5.87; 5.60 2 anomeric OCHO 'groups
5,22 12 udskifteligt H5.22 12 interchangeable H
3,09; 3,15 6 NCH3 x 23.09; 3.15 6 NCH3 x 2
2.9 - 4,8 13 -OHO x 6, -CHN x 5, -OHgO2.9 - 4.8 13 -OHO x 6, -CHN x 5, -OHgO
1.9 - 2,6 4 -CH2CH2-1.9 - 2.6 4 -CH2CH2-
1,72 6 -CHjO-, CHjCH1.72 6 -CH₂O-, CH₂CH
Alternativt blev en 4 g prøve af det rå antibioticumsalt opløst i 50 ml vand og sendt igennem en anionbytterharpiks "AG1X8" (harpikslag 1 x 27 cm). Antibioticumet blev elueret med vand, og alle aktive fraktioner kombineret og inddampet under vakuum. Yderligere af-saltning blev udført på remanensen ved ekstraktion med methanolisk natriumhydroxid. Den frigjorte base blev blandet med 7 g silicagel og sat på en 50 g silicagelsøjle. Denne søjle blev udviklet med chloroform/methanol/koncentreret (28 #) ammoniumhydroxid (3:2:1), og der blev opsamlet 25 ml fraktioner. Tidlige fraktioner gav den nye mindre polære komponent 1, men blandet med flere mindre polære urenheder som påvist ved tyndtlagskromatografi. Disse fraktioner blev kombineret, og koncentratet sat på en 50 g silicagel-søjle som ovenfor. Denne søjle blev udviklet med chloroform/methanol/kon-centreret (28 #>) ammoniumhydroxid (5:3:1), og der blev opsamlet 25 ml fraktioner. Fraktionerne 6-12 indeholdt den ønskede kompo 12 143111 nent fri for åbenbare urenheder. Efter fjernelse af opløsningsmid-let blev den resulterende olie omdannet til sulfatsaltet i ethano-lisk svovlsyre som beskrevet ovenfor, hvorved der blev opnået 0,124 g af komponent 1 som di-base*heptasulfat*decahydratet, smp. >300°C, som beskrevet ovenfor.Alternatively, a 4 g sample of the crude antibiotic salt was dissolved in 50 ml of water and passed through an anion exchange resin "AG1X8" (resin layer 1 x 27 cm). The antibiotic was eluted with water and all active fractions combined and evaporated under vacuum. Further desalting was performed on the residue by extraction with methanolic sodium hydroxide. The released base was mixed with 7 g of silica gel and placed on a 50 g of silica gel column. This column was developed with chloroform / methanol / concentrated (28 #) ammonium hydroxide (3: 2: 1) and 25 ml fractions were collected. Early fractions gave the new minor polar component 1, but mixed with several minor polar impurities as evidenced by thin layer chromatography. These fractions were combined and the concentrate was put on a 50 g silica gel column as above. This column was developed with chloroform / methanol / concentrated (28 #>) ammonium hydroxide (5: 3: 1) and 25 ml fractions were collected. Fractions 6-12 contained the desired compo 12 free of obvious impurities. After removal of the solvent, the resulting oil was converted to the sulfate salt in ethanolic sulfuric acid as described above to give 0.124 g of component 1 as the di-base * heptasulfate * decahydrate, m.p. > 300 ° C, as described above.
Ted dyrkning af en passende aminocyclitol med mutantstammen Micro-monospora purpurea AICC 31 119 i spiremedium (Medium 2) og isolering af produkterne som beskrevet ovenfor i eksempel 1 fremstilles på samme måde de følgende forbindelser med formlenl:Ted culture of a suitable aminocyclitol with the mutant strain Micromonospora purpurea AICC 31 119 in germination medium (Medium 2) and isolation of the products as described above in Example 1 are similarly prepared by the following compounds of formula I:
Eksempel 2 0-3-deoxy-4-C-methyl-3-methylamino-P-l-arabinopyranosyl-( 1-*6)-0-[ 2-amino-6-meth.ylamino-6-C-meth.yl-2,3,4» 6-tetrahydroxy-a-E-erythro-glucopyranosyl-( 1-»4)]epistreptami.n (C.M. i forhold til gentamicin-C^ s System 1 = 0,59, System 2 = 0,63); 0-3-deoxy-4-C-methyl-3-methylamino-P-l-arabinopyranosyl-( 1-*6) -0-[ 2,6-diamino-6-C-methyl-2,3,4» 6-tetradeoxy-oc-D-erythro-glueopyranosyl-(1-M-)]epistreptamin; og 0-3-deoxy-4-C-methyl-3-methylamino-P-Ii-arabinopyranosyl-( 1 ->6)- 0-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1-*4)]-epistreptamin opnået ved anvendelse af epistreptamin i stedet for streptamin ved gæringsproceduren.Example 20 0-3-Deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1- * 6) -0- [2-amino-6-methylamino-6-C-methyl 2,3,4 »6-tetrahydroxy-αE-erythro-glucopyranosyl (1-4)] epistreptamine (CM relative to gentamicin-C 2 s System 1 = 0.59, System 2 = 0.63) ; 0-3-Deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1- * 6) -0- [2,6-diamino-6-C-methyl-2,3,4 »6- tetradeoxy-.alpha.-D-erythro-glueopyranosyl- (M-1 -)] epistreptamin; and 0-3-deoxy-4-C-methyl-3-methylamino-β-1-arabinopyranosyl- (1-6) -O- [2,6-diamino-2,3,4,6-tetradeoxy-αD -erythro-glucopyranosyl- (1- * 4)] - epistreptamine obtained by using epistreptamine instead of streptamine in the fermentation procedure.
Eksempel 3 0-3-deoxy-4-0-methyl-5-methylamino-p-Ii-arabinopyranosyl-( 1-»6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(l-H) ]-2,5-dideoxystreptamin (C.M. i forhold til gen-tamicin-C^: System 1 = 0,95» System 2 = 0,98); 0-3-deoxy-4-C-methyl- 3-methylamino-p-l-arabinopyranosyl-(1-*6)-0-[2,6-diamino-6-C-methyl-2,3»4»6-tetradeoxy-oc-E-erythro-glucopyranosyl-( 1-*4)]-2,5-cLideoxy-streptamin (C.M. i forhold til gentamicin-C^: System 1 = 0,66,Example 30 0-3-Deoxy-4-O-methyl-5-methylamino-p-1-arabinopyranosyl- (1-6) -O- [2-amino-6-methylamino-6-C-methyl-2, 3,4,6-tetradeoxy-αD-erythro-glucopyranosyl- (1H)] -2,5-dideoxystreptamine (CM relative to gene tamicin-C ^: System 1 = 0.95 »System 2 = 0.98) ; 0-3-Deoxy-4-C-methyl-3-methylamino-p-arabinopyranosyl- (1- * 6) -0- [2,6-diamino-6-C-methyl-2,3 »4» 6- tetradeoxy-α-E-erythro-glucopyranosyl (1- * 4)] - 2,5-cLideoxy-streptamine (CM relative to gentamicin-C C: System 1 = 0.66,
System 2 = 0,73); og 0-3-deoxy-4“C-methyl-3-methylamino-p-L-arabino-pyranosyl-(l-»6)-0-/2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-gluco-pyranosyl-(1-^4)1-2,5-dideoxystreptamin opnået ved anvendelse af di-deoxystrepamin i stedet for streptamin ved gæringsproceduren.System 2 = 0.73); and 0-3-deoxy-4 "C-methyl-3-methylamino-pL-arabino-pyranosyl- (1-6) -O- / 2,6-diamino-2,3,4,6-tetradeoxy-αD -erythro-gluco-pyranosyl- (1-4) 1-2,5-dideoxystreptamine obtained by using di-deoxystrepamine instead of streptamine in the fermentation procedure.
13 14311113 143111
Det samme 0-3-deoxy-4-C-methyl-3-methylamino-P-L-arabinopyranosyl-( 1-j6 )-0-/S-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythroglucopyranosyl-(144)7-2,5-dideoxystreptamin og 0-3-deoxy- 4- C-methyl-3-methylamino-P-L-arabinopyranosyl-(1^6)-0-/5,6-diamino- 6-C-methyl-2,3»4,6-tetradeoxy-oc-D-erythro-glucopyranosyl-(144 )]- 2,5-dideoxystreptamin som beskrevet ovenfor og med de samme C.M. værdier som angivet ovenfor blev opnået ved anvendelse af 6,7* diazabicyclo[-3.2,l]octan-2,4-diol(exo,exo) 1 stedet for strep-tamin ved gæringsproceduren.The same 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-6) -O- / S-amino-6-methylamino-6-C-methyl-2,3,4 , 6-tetradeoxy-αD-erythroglucopyranosyl- (144) 7-2,5-dideoxystreptamine and 0-3-deoxy-4- C-methyl-3-methylamino-PL-arabinopyranosyl- (1 ^ 6) -0- / 5 , 6-Diamino-6-C-methyl-2,3 »4,6-tetradeoxy-oc-D-erythro-glucopyranosyl- (144)] - 2,5-dideoxystreptamine as described above and with the same CM values as indicated above were obtained using 6,7 * diazabicyclo [-3.2, 1] octane-2,4-diol (exo, exo) in place of streptamine in the fermentation procedure.
SYNTESE AE AMINO-HYDROXY-MVERE-ÅltKANPYL-DERIYATERSYNTHESY AE AMINO-HYDROXY-MVERE-ALTCANPYL DERIYATES
Eksempel 4Example 4
En opløsning af 270 mg (0,54 milllmol) 0-3-deoxy-4-C-methyl-3-methyl-amino-β-L-arablnopyrano syl-(1-+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-<x-D-erythro-glucopyranosyl-( 1-H-) ]-D-strep-tamin, fremstillet som beskrevet ovenfor i eksempel 1 (komponent 1), opløst i 5 ml 50 % vandigt tetrahydrofuran blev afkølet til 5°C i et isbad og behandlet med 208 mg (0,59 millimol) af N-hydroxysuccinimid-esteren af S-(-)-Y-(N-benzyloxycarbonyl)amino-a-hydroxysmørsyre (Koni-shi et al US patentskrift nr. 3 780 018), og blandingen blev omrørt ved 5°C i 20 timer. Blandingen blev derpå koncentreret til 10 ml i vakuum. Der tilsattes 25 ml n-butanol og 10 ml vand, og lagene blev adskilt.A solution of 270 mg (0.54 ml) of 0-3-deoxy-4-C-methyl-3-methyl-amino-β-L-arablnopyranoyl- (1- + 6) -O- [2-amino] 6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy- [xD-erythro-glucopyranosyl- (1-H-)] -D-strep -amine, prepared as described above in Example 1 (component 1), dissolved in 5 ml of 50% aqueous tetrahydrofuran, was cooled to 5 ° C in an ice bath and treated with 208 mg (0.59 millimole) of the N-hydroxysuccinimide ester of S - (-) - Y- (N-benzyloxycarbonyl ) amino-α-hydroxybutyric acid (Koni-shi et al. U.S. Patent No. 3,780,018) and the mixture was stirred at 5 ° C for 20 hours. The mixture was then concentrated to 10 ml in vacuo. 25 ml of n-butanol and 10 ml of water were added and the layers were separated.
Det vandige lag blev vasket igen med 10 ml n-butanol. De kombinerede organiske lag blev inddampet og efterlod en remanens på 513 mg råt produkt, som blev stillet til side.The aqueous layer was washed again with 10 ml of n-butanol. The combined organic layers were evaporated leaving a residue of 513 mg of crude product which was set aside.
Det vandige lag blev inddampet til tørhed, hvorved der blev opnået 304 mg remanens, som blev opløst i 25 ml 50 # vandigt tetrahydrofuran og behandlet med yderligere 208 mg af N-hydroxysuccinimidesteren af 5- (_)-Y-(N-benzyloxycarbonyl)-amino-cc-hydroxysmørsyre som før. Oparbejdning af reaktionsblandingen gav yderligere 435 mg råt produkt, som blev kombineret med de tidligere opnåede 513 mg og kro-matograferet på syv 1 mm tykke silicagelplader på 40 x 20 cm. Systemet blev udviklet med et opløsningssystem bestående af den nedre fase af en blanding af chloroform/methanol/koncentreret (28 $) ammoniumhydroxid (3:1*1). Syv passager i dette opløsningsmiddelsystem var nødvendige, og efter eluering af produktbåndet, som var 143111 14 ultravioletsynligt, blev der opnået 229,5 mg af en rå blanding af de tre monoacylerede produkter.The aqueous layer was evaporated to dryness to give 304 mg of residue which was dissolved in 25 ml of 50 # aqueous tetrahydrofuran and treated with an additional 208 mg of the N-hydroxysuccinimide ester of 5- (_) - Y- (N-benzyloxycarbonyl) -amino-cc-hydroxybutyric acid as before. Workup of the reaction mixture yielded an additional 435 mg of crude product which was combined with the previously obtained 513 mg and chromatographed on seven 40 mm x 20 cm silica gel plates. The system was developed with a solution system consisting of the lower phase of a mixture of chloroform / methanol / concentrated (28 $) ammonium hydroxide (3: 1 * 1). Seven passages in this solvent system were necessary and, after elution of the product band which was ultraviolet visible, 229.5 mg of a crude mixture of the three monoacylated products was obtained.
Blandingen af acylerede produkter blev sat på'tre 1 mm tykke silica-gelplader på 40 x 20 cm, og pladerne blev udviklet fem gange med den nedre fase af chloroform/isopropanol/koncentreret (28 f) ammoniumhydroxid (4:1:1), to gange med den nedre fase af chloroform/iso-propanol/koncentreret (28 fo) ammoniumhydroxid (3:1:1) og ni gange med den nedre fase af chloroform-methanol/koncentreret (28 fo) ammoniumhydroxid (4:1:1). Der blev opnået tre adskilte bånd, som var synlige under ultraviolet bestråling, og som blev udskåret separat og elueret fra silicagelen med den nedre fase af chloroform/methanol/ koncentreret (28 fo) ammoniumhydroxid (1:1:1), hvorved der blev opnået tre komponenter: A 90,9 mg; B 59,1 mg; og C 48,5 mg, som er 5- (-)-Y-(N-benzyloxycarbonyl)-amino-a-hydroxysmørsyreamid-deriva-teme i henholdsvis 2'-, 1- og 3-Stilling af 0-3-deoxy-4-C-methyl- 3-methylamino-P-Ii-arabinopyranosyl-( 1->6)-0-[ 2-amino-6-methylamino- 6- C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-( 1Ή) ] -D-streptamin. R^-vssrdierne for komponent A, B og C efter udvikling fem gange på silicagel med den nedre fase af et system bestående af chloroform/methanol/koncentreret (28 f) ammoniumhydroxid (4:1:1) var: A - 0,48 B - 0,62 C - 0,70The mixture of acylated products was placed on three 1 mm thick 40 x 20 cm silica gel plates and developed five times with the lower phase of chloroform / isopropanol / concentrated (28 f) ammonium hydroxide (4: 1: 1). twice with the lower phase of chloroform / iso-propanol / concentrated (28 fo) ammonium hydroxide (3: 1: 1) and nine times with the lower phase of chloroform-methanol / concentrated (28 fo) ammonium hydroxide (4: 1: 1 ). Three distinct bands visible under ultraviolet irradiation were obtained which were excised separately and eluted from the silica gel with the lower phase of chloroform / methanol / concentrated (28 fo) ammonium hydroxide (1: 1: 1) to give three components: A 90.9 mg; B, 59.1 mg; and C 48.5 mg which is 5- (-) - Y- (N-benzyloxycarbonyl) amino-α-hydroxybutyric acid amide derivatives in 2'-, 1- and 3-position of 0-3-deoxy, respectively -4-C-methyl-3-methylamino-β-1-arabinopyranosyl- (1-> 6) -0- [2-amino-6-methylamino-6- C-methyl-2,3,4,6-tetradeoxy -αD-erythro-glucopyranosyl- (1Ή)] -D-streptamine. The R ^ values of components A, B and C after development five times on silica gel with the lower phase of a system consisting of chloroform / methanol / concentrated (28 f) ammonium hydroxide (4: 1: 1) were: A - 0.48 B - 0.62 C - 0.70
Komponent B (1-amidet,56,1 mg) opløst i 25 ml 50 fo vandigt ethanol og tilsat 20 mg 10 fo palladium-på-kul blev rystet på en Parr-ryster, idet den blev holdt ved et hydrogentryk på 3,85 kp/cm2 i 5 1/2 time, hvorefter katalysatoren blev fjernet ved filtrering igennem filterhjælpemiddel. Afdampning af opløsningsmidlet gav 34,3 mg af et hvidt glasagtigt stof, som blev opløst i 2,5 ml vand og behandlet med 11,4 mg svovlsyre i 0,1 ml vand. Tilsætning af 10 ml ethanol udfældede 1-[S-(-)-Y-amino-o:-hydroxybutyryl]-0-3-deoxy-4-C-methyl-3 -me thylamin o - β -1-arabino pyr ano sy 1- (1->6) —0—[ 2-amino-6-methylamino- 6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-( 1Ή) ] - 15 143111 D-streptamin som pentasulfatsaltet (32 mg), smp. 230 - 235°C (dekomp,); tyndtlagskromatografi, R^ = 0,18 (silicagel; chloro-form/methanol/koncentreret (28 ¢) ammoniumhydroxid/vand (1:4:2:1); gentamicin-C^-standard =0,73.Component B (1-amide, 56.1 mg) dissolved in 25 ml of 50 µl aqueous ethanol and 20 mg of 10 µl palladium-on-charcoal was shaken on a Parr shaker, maintained at a hydrogen pressure of 3.85 kp / cm 2 for 5 1/2 hours, after which the catalyst was removed by filtration through filter aid. Evaporation of the solvent gave 34.3 mg of a white glassy substance which was dissolved in 2.5 ml of water and treated with 11.4 mg of sulfuric acid in 0.1 ml of water. Addition of 10 ml of ethanol precipitated 1- [S - (-) - γ-amino-o: -hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methyl-o-β-1-arabino pyrano silk 1- (1-> 6) -O- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-αD-erythro-glucopyranosyl- (1Ή)] - 143111 D-streptamine as the pentasulfate salt (32 mg), m.p. 230 DEG-235 DEG C. (decomp.); thin layer chromatography, R f = 0.18 (silica gel; chloroform / methanol / concentrated (28 ¢) ammonium hydroxide / water (1: 4: 2: 1); gentamicin C ^ standard = 0.73).
Analyse teregnet for C25H50°10N6*5H2S04: C 27,67 - H 5,57 - N 7,75Analysis calculated for C 25 H 50 ° 10 N 6 * 5H 2 SO 4: C 27.67 - H 5.57 - N 7.75
Rundet: C 27,50 - H 5,58 - N 7,42.Found: C 27.50 - H 5.58 - N 7.42.
Komponent A og C blev behandlet på samme måde. A gav 47 mg 2'-[S-(_)_Y_amlno-a-hydroxybutyryl]-0-3-deoxy-4-C-methyl-3-methylamlno-P-l-arabinopyranosyl-( 1-»6)-0-[ 2-amlno-6-me thylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-( 1-»4) ]-D-streptamin som bis-base*tetrasulfat ·heptahydratet, smp. 237 - 241°C (dekomp.); tyndtlagskromatografi, Rf = 0,33 (silicagel; chloroform/methanol/ koncentreret (28 ¢) ammoniumhydroxid/Tand (1:4:2:1); R^ gentand.cin--standard =0,75).Components A and C were treated in the same way. A gave 47 mg of 2 '- [S - (_) _ Y_amino-α-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1-6) -0- [ 2-Amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl (1-4)] -D-streptamine as the bis-base * tetrasulfate · heptahydrate , m.p. 237 - 241 ° C (decomp); thin layer chromatography, R f = 0.33 (silica gel; chloroform / methanol / concentrated (28 ¢) ammonium hydroxide / Tooth (1: 4: 2: 1); R
Analyse beregnet for (C2^H^0Ng0.j0)2*4H2S0^· H20: B 35,16 - H 7,20 - N 9,84Analysis calculated for (C₂ ^H ^ 0NgO.j0) 2 * 4H₂SO ^ · H₂O: B 35.16 - H 7.20 - N 9.84
Rundet: C 35,38 - Ξ 7,08 - N 9,49.Round: C 35.38 - Ξ 7.08 - N 9.49.
Komponent C gav 26 mg 3-[ S-(-) -γ-amino-oc-hydroxybutyryl] -0-3-deoxy- 4-C-methyl-3-methylamlno-p-L-arabinopyranosyl-( 1->6)-0-[ 2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosy l-( 1->4) ] -D-streptamin som bis-base .pentasulfat*trihydratet, smp.Component C gave 26 mg of 3- [S - (-) -γ-amino-oc-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (1-> 6) - O- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl 1- (1-> 4)] -D-streptamine as bis-base. The pentasulfate * trihydrate, m.p.
220 - 230°C (dekomp.); tyndtlagskromatografi, Rf = 0,30 [silicagel; chloroform/methanol/koncentreret (28 $) ammoniumhydroxid/vand (1:4:2:1); R^ gentamicin-C1-standard = 0,73].220 DEG-230 DEG C. (decomp.); thin layer chromatography, R f = 0.30 [silica gel; chloroform / methanol / concentrated ($ 28) ammonium hydroxide / water (1: 4: 2: 1); R gentamicin C1 standard = 0.73].
Analyse beregnet for (Cr^H^øNgO.^),,·5H2SO^· 350,0: C 34,64 - H 6,74 - N 9,67 Rundet: C 34,35 - H 6,38 - N 8,60.Analysis calculated for (Cr 2 H 2 ON 2 O 3) δ · 5H 2 SO 4 · 350.0: C 34.64 - H 6.74 - N 9.67 Round: C 34.35 - H 6.38 - N 8 , 60th
16 16311116 163111
ANTIBAKTERIELLE PRØVNINGSRESULTATERANTIBACTERIAL TEST RESULTS
De ifølge eksempel 1 fremstillede forbindelser 2-hydroxygentamicin-C^_, 2-hydroxygentamicin-Cg og 2-hydroxygentamicin-C-jyCg-komplex (i det følgende betegnet henholdsvis Oxy-C-^, Oxy-Cg og Oxy-C^/Cg) og de ifølge eksempel 3 fremstillede forbindelser 5-deoxygentamicin-og 5-deoxygentamicin-C1/C2/C2_a-komplex (i det følgende betegnet henholdsvis Deoxy-C^ og Deoxy-C-, /Cg/C-j ) blev prøvet i sammenligning med gentamicin-C^, gentamicin-Cg og gentamicin-C-^/C2/Cla-komplex (i det følgende betegnet henholdsvis G-C·^, G-Cg og G-C-^/Cg/C^a) over for et antal mikroorganismer efter den følgende procedure.The compounds of Example 1 prepared 2-hydroxygentamicin-C ^, 2-hydroxygentamicin-Cg, and 2-hydroxygentamicin-C-yCCg complex (hereinafter referred to as Oxy-C-, Oxy-Cg and Oxy-C ^, respectively). Cg) and the compounds of Example 3 prepared from Example 3 and 5-deoxygentamicin-C1 / C2 / C2_a complex (hereinafter referred to as Deoxy-C1 and Deoxy-C1 / Cg / C with gentamicin-C2, gentamicin-Cg and gentamicin-C - ^ / C2 / Cla complex (hereinafter referred to as GC · ^, G-Cg and GC - ^ / Cg / C following the following procedure.
lageropløsninger af hver forbindelse indeholdende 200 μg/ml base blev fremstillet i destilleret vand og filtersteriliseret. Kulturer af prøveorganismerne blev dyrket i 24 timer ved 37°C i 10 ml glas med tryptosephosphat eller Miller-Hinton-væske. Hver kultur blev indstillet med væske til optisk tæthed 0,1 på en SpectronicStock solutions of each compound containing 200 μg / ml base were prepared in distilled water and filter sterilized. Cultures of the sample organisms were grown for 24 hours at 37 ° C in 10 ml glass with tryptose phosphate or Miller-Hinton liquid. Each culture was adjusted with optical density fluid 0.1 on a Spectronic
OISLAND
20 (tilnærmelsesvis 10 celler/ml). De indstillede kulturer blev fortyndet 1:500 i væske til brug som podemateriale (endelig celle-20 (approximately 10 cells / ml). The set cultures were diluted 1: 500 in liquid for use as graft material (final cell culture).
CC
koncentration tilnærmelsesvis 2 x 103 celler/ml). Prøveforbindelserne blev prøvet for antibakteriel aktivitet ved en enkelt række fortyndingsmetoder. Dobbeltfortyndingsrækker blev fremstillet i væske fra lageropløsningerne, og 0,2 ml af hver antibioticumkoncen-tration blev anbragt i 17 13 x 100 mm glas. Alle glas blev podet med 0,2 ml af den passende fortyndede kultur (endelig cellekoncentration pr. glas = 105 celler/ml). Minimale inhiberende koncentrationer (laveste antibioticumkoncentration, som ikke viste nogen synlig vækst) blev aflæst efter 16 timers inkubation ved 37°C.concentration approximately 2 x 10 3 cells / ml). The test compounds were tested for antibacterial activity by a single series of dilution methods. Double dilution rows were prepared in liquid from the stock solutions and 0.2 ml of each antibiotic concentration was placed in 17 13 x 100 mm glass. All glasses were seeded with 0.2 ml of the appropriate diluted culture (final cell concentration per glass = 105 cells / ml). Minimum inhibitory concentrations (lowest antibiotic concentration showing no visible growth) were read after 16 hours of incubation at 37 ° C.
Resultaterne er anført i tabel III nedenfor. Forbindelserne blev betragtet som inaktive ved inhiberende koncentrationer på over 100 jag/ml.The results are listed in Table III below. The compounds were considered inactive at inhibitory concentrations above 100 µg / ml.
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Inden for antibioticum-området, hvor mikroorganismer udvikler resi·? stens over for antibioticum-terapi ved mutation, måles fremskridt ved nye antibioticas evne til at bekæmpe mikroorganismer, som af en eller anden grund er resistente over for'kendte antibiotica. Sammenligninger af prøvningsresultaterne i den ovenstående tabel III mellem henholdsvis 2-hydroxygentamicin-C-^ og gentamicin-C-^, 2-hydroxygentamic in-C^ og gentamicin-C^, 2-hydr oxygentamic in-C-^/C2-komplex og gentamiGin-C-^/C^/C^-komplex, 5-deoxygentamicin-C^ og gentamicin-C^ samt 5-deoxygentamicin-C-j /Op/C·^-komplex og gentami-cin-C^/C2/Cla~komplex viser, at de ifølge opfindelsen fremstillede forbindelser er aktive over for fra 4 til 8 mikroorganismer, som er resistente over for den tilsvarende gentamicin. Disse sammenligninger er sammenfattet i den nedenstående tabel III-A, hvor de i tabel III anførte organismer er nummereret i rækkefølge og identificeret ved de således tildelte numre, og hvor bedre aktivitet for en bestemt angivet 2-hydroxy- eller 5-de oxygentamic in eller komplex deraf i sammenligning med den tilsvarende gentamicin eller komplex deraf er angivet ved +.In the field of antibiotics where microorganisms develop resi? resistance to antibiotic therapy by mutation, progress is measured by the ability of new antibiotics to control microorganisms that are for some reason resistant to known antibiotics. Comparisons of the test results in the above Table III between 2-hydroxygentamicin-C- and gentamicin-C-, 2-hydroxygentamic in-C ^ and gentamicin-C ^, 2-hydr oxygentamic in-C - - / C₂ complex, respectively and gentamiGin-C - ^ / C ^ / C ^ complex, 5-deoxygentamicin-C ^ and gentamicin-C ^ as well as 5-deoxygentamicin-Cj / Op / C · ^ complex and gentamicin-C ^ / C2 / Clax complex shows that the compounds of the invention are active against from 4 to 8 microorganisms that are resistant to the corresponding gentamicin. These comparisons are summarized in Table III-A below, where the organisms listed in Table III are numbered in sequence and identified by the numbers thus assigned, and where better activity for a particular stated 2-hydroxy or 5-de oxygentamic in or complex thereof in comparison with the corresponding gentamicin or complex thereof is indicated by +.
19 14311119 143111
Organisme OxyC1 Oxy-C2 Oxy-c^/C., Deoxy-c^ Deoxy-c^/CVC, 2 3++4- 4 5 + + 4- 6 + 7 8 + + 4- 4- 9 + + + + 10 11 12 + + 4- 4- 13 + + 4. + 14 + + + 4.Organism OxyC1 Oxy-C2 Oxy-c ^ / C., Deoxy-c ^ Deoxy-c ^ / CVC, 2 3 ++ 4- 4 5 + + 4- 6 + 7 8 + + 4- 4- 9 ++ + 10 11 12 + + 4- 4- 13 + + 4. + 14 +++ + 4.
15 + 16 • 17 1815 + 16 • 17 18
Den samme komponent 1 som beskrevet ovenfor i eksempel 1, 0-3-de-ozy-4-C-methyl-3-methylamino-P-L-arabinopyranosyl-(1-*6)-0-[2-amino- 6-methylamino-6-C-methyl-2,3,4,6-t e tradeoxy-cc-D-ery thro-glucopyrano-syl-(1-*4)]-D-streptamin, blev prøvet i sammenligning med gentamicin-kompleks (C^ C2 og G^a) og gentamicin-C1; og 1-, 3- og 2'-[S-(-)-Y-amino-a-hydroxy-butyryl]amiderne af komponent 1, beskrevet ovenfor i eksempel 4 og betegnet henholdsvis komponent 1 (1-HABA), komponent 1 (3-HABA) og komponent 1 (2’-HABA), blev prøvet i sammenligning med de tilsvarende 1-, 3- og 2,-[S-(-)-Y-amino-a-hydroxybu-tyryl]amider af gentamicin-C1 (alle beskrevet af Konishi et al. i 20 143111 US patentskrift nr. 3 780 018, udstedt den 18. december 1973), betegnet henholdsvis (1-HABA), (3-HABA) og (2'-HABA).The same component 1 as described above in Example 1, 0-3-de-ozy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1- * 6) -0- [2-amino-6-methylamino -6-C-methyl-2,3,4,6-te tradeoxy-cc-D-ery thro-glucopyrano-sil- (1- * 4)] - D-streptamine, was tested in comparison with gentamicin complex ( C1-C2 and G3a) and gentamicin-C1; and the 1-, 3- and 2 '- [S - (-) - γ-amino-α-hydroxy-butyryl] amides of component 1, described above in Example 4 and designated component 1 (1-HABA), component 1, respectively (3-HABA) and component 1 (2'-HABA), were tested in comparison with the corresponding 1-, 3- and 2, - [S - (-) - γ-amino-α-hydroxybutyrryl] amides of gentamicin-C1 (all described by Konishi et al. in U.S. Patent No. 3,780,018, issued December 18, 1973), designated (1-HABA), (3-HABA) and (2'-HABA, respectively). .
Disse resultater er anført i tabel IY nedenfor, hvor prøveorganis-meme 1, 2, 3, 4, 5 og 6 identificerer henholdsvis B. subtilis ATCC 6633, S. aureus Smith, E. coli JR 76.2, Ent. cloacae A-20960, K. pneumoniae A-20636 og Ps. aeruginosa A-20987.These results are set forth in Table IY below, where test organisms 1, 2, 3, 4, 5 and 6 identify B. subtilis ATCC 6633, S. aureus Smith, E. coli JR 76.2, Ent. cloacae A-20960, K. pneumoniae A-20636 and Ps. aeruginosa A-20987.
TABEL· 17*TABLE · 17 *
PrøveorganismerTest organisms
Eorbindelse 1 23456Compound 1 23456
Gentamicin-C^, -Cg, -C^& 0,024 0,39 50 12,5 25 >100Gentamicin-C ^, -Cg, -C ^ & 0.024 0.39 50 12.5 25> 100
Gentami cin-C,] 0,049 0,78 50 12,5 25 >100Gentami cin-C, 0.049 0.78 50 12.5 25> 100
Komponent 1 0,098 1,56 6,25 0,78 3,15 25 C1 (1-HABA) 0,39 3,13 12,5 3,13 6,25 >100Component 1 0.098 1.56 6.25 0.78 3.15 C1 (1-HABA) 0.39 3.13 12.5 3.13 6.25> 100
Komponent 1 (1-HABA) 0,78 6,25 12,5 6,25 12,5 >100 01 (3-ΞΑΒΑ) 3,13 25 >100 100 >100 >100Component 1 (1-HABA) 0.78 6.25 12.5 6.25 12.5> 100 01 (3-ΞΑΒΑ) 3.13 25> 100 100> 100> 100
Komponent 1 (3-HABA) 6,25 50 100 50 50 >100 01 (21 -HABA) 6,25 50 100 25 50 >100Component 1 (3-HABA) 6.25 50 100 50 50> 100 01 (21-HABA) 6.25 50 100 25 50> 100
Komponent 1 (2'-HABA) 1,56 25 50 25 50 >100 * Dyrket i Mueller-Hinton-væske.Component 1 (2'-HABA) 1.56 25 50 25 50> 100 * Cultured in Mueller-Hinton liquid.
Claims (2)
Priority Applications (2)
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DK177179A DK143607C (en) | 1975-09-22 | 1979-04-30 | PROCEDURE FOR PREPARING AMINOCYCLITOL ANTIBIOTICA OR ACID ADDITION SALTS |
DK177279A DK143413C (en) | 1975-09-22 | 1979-04-30 | PROCEDURE FOR PREPARING AMINOCYCLITOL COMPOUNDS OR ACID ADDITION SALTS |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US05/550,273 US3972930A (en) | 1975-02-18 | 1975-02-18 | Aminocyclitol antibiotics |
US55027375 | 1975-02-18 | ||
US61559375 | 1975-09-22 | ||
US05/615,593 US3982996A (en) | 1975-09-22 | 1975-09-22 | Process for preparing aminocyclitol antibiotics |
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Publication Number | Publication Date |
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DK63176A DK63176A (en) | 1976-08-19 |
DK143111B true DK143111B (en) | 1981-03-30 |
DK143111C DK143111C (en) | 1981-11-16 |
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AR (3) | AR221027A1 (en) |
AU (1) | AU503105B2 (en) |
CA (1) | CA1061731A (en) |
CH (3) | CH617964A5 (en) |
DE (1) | DE2606517A1 (en) |
DK (1) | DK143111C (en) |
EG (1) | EG12994A (en) |
ES (1) | ES445274A1 (en) |
FI (1) | FI56026C (en) |
FR (1) | FR2301265A1 (en) |
GB (1) | GB1529376A (en) |
GR (1) | GR60050B (en) |
IE (1) | IE42807B1 (en) |
IL (1) | IL49053A (en) |
IN (2) | IN147046B (en) |
NL (1) | NL7601655A (en) |
NO (3) | NO147308C (en) |
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UA97245C2 (en) | 2006-06-02 | 2012-01-25 | Меиджи Сеика Фарма Ко., Лтд. | Aminoglycoside antibiotics |
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- 1976-02-17 CH CH193476A patent/CH617964A5/en not_active IP Right Cessation
- 1976-02-17 AU AU11177/76A patent/AU503105B2/en not_active Ceased
- 1976-02-17 SE SE7601810A patent/SE7601810L/en not_active Application Discontinuation
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- 1976-02-18 EG EG100/76A patent/EG12994A/en active
- 1976-02-18 JP JP51016876A patent/JPS51108041A/ja active Pending
- 1976-02-18 DE DE19762606517 patent/DE2606517A1/en not_active Ceased
- 1976-02-18 ES ES445274A patent/ES445274A1/en not_active Expired
- 1976-02-18 NL NL7601655A patent/NL7601655A/en not_active Application Discontinuation
- 1976-02-18 AR AR262294A patent/AR221027A1/en active
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1977
- 1977-02-10 AR AR266507A patent/AR227266A1/en active
- 1977-02-10 AR AR266506A patent/AR217417A1/en active
- 1977-09-20 IN IN1416/CAL/77A patent/IN147046B/en unknown
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1978
- 1978-11-01 CH CH1126778A patent/CH618215A5/en not_active IP Right Cessation
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1979
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1980
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