DK156070B - METHOD OF ANALOGY FOR THE PREPARATION OF ANTIBIOTIC ACTIVE DERIVATIVES OF AMINOCYCLITOLS - Google Patents
METHOD OF ANALOGY FOR THE PREPARATION OF ANTIBIOTIC ACTIVE DERIVATIVES OF AMINOCYCLITOLS Download PDFInfo
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- DK156070B DK156070B DK018675AA DK18675A DK156070B DK 156070 B DK156070 B DK 156070B DK 018675A A DK018675A A DK 018675AA DK 18675 A DK18675 A DK 18675A DK 156070 B DK156070 B DK 156070B
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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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Description
1 DK 156070 B1 DK 156070 B
Opfindelsen angår en analogifremgangsmåde til fremstilling af antibakterielt aktive 1-N-substituerede derivater af 4,6-di(ami-noglycosyl)-1,3-diaminocyclitoler, hvori 1-N-substituenten er en aminohydroxyacylgruppe, og ikke-toksiske syreadditionssalte af sådanne forbindelser.The invention relates to an analogous process for the preparation of antibacterially active 1-N-substituted derivatives of 4,6-di (aminoglycosyl) -1,3-diaminocyclitols wherein the 1-N substituent is an aminohydroxyacyl group and non-toxic acid addition salts of such compounds.
US-patentskrift nr. 3.780.018 omhandler en fremgangsmåde, ved hvilken 1-[L-(-)-γ-amino-a-hydroxybutyryl3gentamicin og 2[L-(-)-γ-amino-a-rhydroxybutyryl]gentamicin fremstilles ved omsætning mellem gentamicin og en blokeret aktiv ester af L—(—)— γ-amino-a-hydroxysmørsyre efterfulgt af fjernelse af den blokerede gruppe ved i teknikken i og for sig kendte metoder og separering af reaktionsblandingen ad chromatografisk vej. US-patentskrift nr.U.S. Patent No. 3,780,018 discloses a process in which 1- [L - (-) - γ-amino-α-hydroxybutyryl-3gentamicin and 2 [L - (-) - γ-amino-α-rhydroxybutyryl] gentamicin are prepared by reaction of gentamicin with a blocked active ester of L - (-) - γ-amino-α-hydroxybutyric acid followed by removal of the blocked group by methods known in the art and separation of the reaction mixture by chromatographic route. U.S. Pat.
2 DK 156070B2 DK 156070B
3.796.698 omhandler en fremgangsmåde til fremstilling af 1 — [L—(—) — γ-amino-a-hydroxybutyryl]gentamicin C2, og US-patentskrift nr.No. 3,796,698 discloses a process for the preparation of 1 - [L - (-) - γ-amino-α-hydroxybutyryl] gentamicin C2, and U.S. Pat.
3.796.699 omhandler en fremgangsmåde til fremstilling af 1 — [L—(—) — γ-amino-a-hydroxybutyryl]-gentamic in .3,796,699 discloses a process for the preparation of 1- [L - (-) - γ-amino-α-hydroxybutyryl] -gentamic in.
De hidtil ukendte antibakterielle midler fremstillet ifølge opfindelsen har overraskende uventede egenskaber. Generelt er disse omhandlede forbindelser aktive over for bakteriestammer og/eller protozostammer. Endvidere er de aktive over for mange bakterier, som er blevet i det væsentlige ufølsomme over for . de ikke-afledte antibiotika.The novel antibacterial agents prepared according to the invention have surprisingly unexpected properties. Generally, these compounds are active against bacterial strains and / or protozoan strains. Furthermore, they are active against many bacteria which have become essentially insensitive to. the non-derived antibiotics.
Opfindelsen angår således en analogifremgangsmåde til fremstilling af antibiotisk aktive 1-N-substituerede derivater af 4,6-di-(aminoglycosyl)-1,3-diaminocyclitolerne, gentamicin B, gentamicin , gentamicin , gentamicin C·^, sisomicin, verdamicin, antibiotikum G-418, antibiotikum 66-40B, antibiotikum 66-40D, antibiotikum JI-20A, antibiotikum JI-20B og antibiotikum G-52, hvori 1-N-substituenten betegnes X og er enten i3-3-amino- 2- hydroxypropionyl eller S-4-amino-2-hydroxybutyryl, med det forbehold, at når der er tale om gentamicin , gentamicin C. og gentamicin C0, er X S-3-amino-2-hydroxypropionyl, samt de farmaceutiske acceptable syreadditionssalte af disse forbindelser. .The invention thus relates to an analogous process for the preparation of antibiotically active 1-N-substituted derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols, gentamicin B, gentamicin, gentamicin, gentamicin C, sisomicin, verdamicin, antibiotic G-418, antibiotic 66-40B, antibiotic 66-40D, antibiotic JI-20A, antibiotic JI-20B, and antibiotic G-52, wherein the 1-N substituent is X and is either 1-3-amino-2-hydroxypropionyl or S-4-amino-2-hydroxybutyryl, with the proviso that in the case of gentamicin, gentamicin C. and gentamicin CO, X is S-3-amino-2-hydroxypropionyl, as well as the pharmaceutically acceptable acid addition salts of these compounds. .
En foretrukken gruppe af forbindelser består af 1-N-substituerede derivater af 4,6-di(aminoglycosyl)-1,3-diamino-cyclitolerne, gentamicin B, gentamicin B^, sisomicin og verdamicin, hvori 1-N-substituenten er en del af gruppen bestående af S- 3- amino-2-hydroxypropionyl og S-4-amino-2-hydroxybutyryl, samt de farmaceutisk acceptable syreadditionssalte deraf.A preferred group of compounds consists of 1-N-substituted derivatives of the 4,6-di (aminoglycosyl) -1,3-diamino-cyclitols, gentamicin B, gentamicin B 2, sisomicin and verdamicin wherein the 1-N substituent is a part of the group consisting of S-3-amino-2-hydroxypropionyl and S-4-amino-2-hydroxybutyryl, as well as the pharmaceutically acceptable acid addition salts thereof.
Fra denne gruppe forbindelser foretrækkes især følgende specielt navngivne forbindelser: 1-N-(S-4-amino-2-hydroxybutyryl)gentamicin B, 1-N-(S-3-amino-2-hydroxypropionyl)gentamicin B, 1-N-(S-4-amino-2-hydroxybutyryl)gentamicin B^, 1-N-(S-3-amino-2-hydroxypropionyl)sisomicin, 1-N-(S-3-amino-2-hydroxypropionyl)verdamicin og de farmaceutisk acceptable syreadditionssalte deraf.Particularly preferred from this group of compounds are the following specially named compounds: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin B, 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin B, 1-N - (S-4-amino-2-hydroxybutyryl) gentamicin B1, 1- N- (S-3-amino-2-hydroxypropionyl) sisomicin, 1-N- (S-3-amino-2-hydroxypropionyl) verdamicin and the pharmaceutically acceptable acid addition salts thereof.
Forbindelserne 1-N-(S-4-amino-2-hydroxybutyryl)gentamicin B, 1-N—(S-3-amino-2-hydroxypropionyl)gentamicin B, 1-N-(S-3-amino-2-hydroxypropionyl)sisomicin, 1-N-(S-3-amino-2-hydroxypropionyl)verdamicin og de farmaceutisk acceptable syreadditionssalte deraf repræ-centerer en specielt foretrukken gruppe forbindelser, og forbindelserne 1-N-(S-4-amino-2-hydroxybutyryl)gentamicin B, 1-N-(S-3-amino-The compounds 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin B, 1- N- (S-3-amino-2-hydroxypropionyl) gentamicin B, 1-N- (S-3-amino-2- hydroxypropionyl) sisomicin, 1-N- (S-3-amino-2-hydroxypropionyl) verdamicin and the pharmaceutically acceptable acid addition salts thereof represent a particularly preferred group of compounds, and the compounds 1-N- (S-4-amino-2 hydroxybutyryl) gentamicin B, 1- N- (S-3-amino-
3 DK 156070B3 DK 156070B
2-hydroxypropionyl)gentamicin B og de farmaceutisk acceptable syre-additionssalte deraf er de mest foretrukne.2-hydroxypropionyl) gentamicin B and the pharmaceutically acceptable acid addition salts thereof are the most preferred.
Forbindelserne fremstillet ifølge opfindelsen har de i det følgende angivn strukturformler I, II og III, hvori X betegner 1-N-substituenten som angivet i det foregående.The compounds of the invention have the following structural formulas I, II and III, wherein X represents the 1-N substituent as set forth above.
nh2NH2
. jA \NHX. jA \ NHX
y-0—K^QH y iy-0 — K ^ QH y i
XX IXX I
hvori Y er en aminoglycosylgruppe valgt blandt følgende: CH3 ch2nh2 nh2-I— /' V (i 1N-X- K OH A_(i 1-N“X" nn\ l/L gentamicin B) hINL—J/" gentamicin B^)wherein Y is an aminoglycosyl group selected from the following: CH3 ch2nh2 nh2-I- / 'V (in 1N-X-K OH A_ (in 1-N "X" nn \ l / L gentamicin B) hINL-J / "gentamicin B ')
OH OHOH OH
ch3 ch3nh-- ch2nh2 (i 1-N-X- IX) (/-(i 1-N-X- I-f gentamicin C-,) ] [ gentamicin C, ) nh2 nh2 ch3 --NH2ch3 ch3nh-- ch2nh2 (i 1-N-X-IX) (- - (i 1-N-X-I-f gentamicin C-)] [gentamicin C,) nh2 nh2 ch3 --NH2
Ni i/1- ϋ i-η-*- , ] f verdamicin) NH2 4Ni i / 1- ϋ i-η - * -,] f verdamicin) NH2 4
DK 156070 BDK 156070 B
CH2NH2 CH2NH2 XI ,/L (i 1-N-X- ho\?H (± 1 i sisomicin) ' | antibiotikum JI-20A) nh2 nh2 CH3 --nh2 ch2nhch3 wn\?H y/ — (i 1-N-X- H0 ]-f antibiotikum N-< — NH2 JI-20B) > JjH antibiotikum ^ 2 G-52) og ch3 HO--CH2NH2 CH2NH2 XI, / L (i 1-NX- ho \? H (± 1 in sisomicin) | antibiotic JI-20A) nh2 nh2 CH3 - nh2 ch2nhch3 wn \? H y / - (i 1-NX-H0 ] -f antibiotic N- <- NH2 JI-20B)> JjH antibiotic ^ 2 G-52) and ch3 HO--
I^NII ^ NI
(i-N-X-antibiotikum G-418) nh2 1-N-X-antibiotikum 66-40D med følgende formel II CH0NH0 NH2 NH2 h°kS>lo(i-N-X antibiotic G-418) nh2 1-N-X antibiotic 66-40D of the following formula II CH0NH0 NH2 NH2 h ° kS> lo
OHOH
og i-N-X-gentamicin A og 1-N—X—antibiotikum 66—40B med følgende formel IIIand i-N-X-gentamicin A and 1-N-X-antibiotic 66-40B of the following formula III
55
DK 156070 BDK 156070 B
NH2NH2
λ-k HHXλ-k HHX
*.-o—Κ^ΐ_ρ i/^Vj hvori Y' betegner* .- o — Κ ^ ΐ_ρ i / ^ Vj wherein Y 'represents
AA
— i 1-N-X-antibiotikum 66--40B.- in 1-N-X antibiotic 66--40B.
NH2NH2
De ikke-afledte (forælder) antibiotika (defineret ved de foregående strukturformler I, II og III, hvori X betegner hydrogen), er alle kendte indenfor teknikken.The non-derived (parent) antibiotics (defined by the preceding structural formulas I, II and III, wherein X represents hydrogen), are all known in the art.
Antibiotikum 66-40B og antibiotikum 66-40D fremstilles sammen med sisomicin, som hovedproduktet ved fermenteringen af Micromo-nospora inyoensis (beskrevet i britisk patentskrift nr.Antibiotic 66-40B and antibiotic 66-40D are produced together with sisomicin, as the main product of the fermentation of Micromonospora inyoensis (described in British Pat.
1.274.518). Antibiotikum 66-40B og antibiotikum 66-40D kan skilles fra fermenteringsmediet ved anvendelse af specielle chromatografiske separationsteknikker som beskrevet i belgisk patentskrift nr. 811.370.1,274,518). Antibiotic 66-40B and antibiotic 66-40D can be separated from the fermentation medium using special chromatographic separation techniques as described in Belgian Patent Specification No. 811,370.
Analogifremgangsmåden ifølge opfindelsen er ejendommelig ved, at man benytter én af de efterfølgende fremgangsmåde-alternativer A) eller B): A) en af de ovenfor angivne 4,6-di-(aminoglycosyl)-1,3-diamino-cyclitoler, som kan have aminobeskyttende grupper i en vilkårlig stilling forskellig fra stilling 1, behandles med en syre med formlen HO-X' hvori X' er en gruppe som defineret for X, hvori amino-gruppen og/eller hydroxylgruppen kan være beskyttet, i nær- Λ 1 Π Λ « ·Ρ /—V 4- •«»ΚλαΙ ί ·Ι »W 1 Λ 1 1 Λν Λ·!· 1 Ttt ^ /-» V* 4 ΤΤΑ 4- 6The analogous process of the invention is characterized by using one of the following process alternatives A) or B): A) one of the 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitols mentioned above, which can be having amino protecting groups at any position other than position 1, treated with an acid of the formula HO-X 'wherein X' is a group as defined for X wherein the amino group and / or hydroxyl group may be protected, in the vicinity of Λ 1 Π Λ «· Ρ / —V 4- •« »ΚλαΙ ί · Ι» W 1 Λ 1 1 Λν Λ ·! · 1 Ttt ^ / - »V * 4 ΤΤΑ 4- 6
DK 156070 BDK 156070 B
af ovennævnte syre, eller B) en af de ovenfor angivne 4,6-di-(aminoglycosyl)-1,3- diaminocyclitoler, som er delvis neutraliseret ved dannelse af et syreadditionssalt, behandles med en syre med formlen HO-X' hvori X' er en gruppe som defineret for X, hvori amino-gruppen og/eller hydroxygruppen kan være beskyttet i nærværelse af et carbodiimid, eller med et reaktivt derivat af ovennævnte syre, hvilken fremgangsmåde A) eller B) efterfølges af fjernelse af alle tilstedeværende beskyttende grupper i molekylet, og isolering af derivatet som sådant, eller som et farmaceutisk acceptabelt syreadditionssalt.of the above acid, or B) one of the above 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols, which is partially neutralized by formation of an acid addition salt, is treated with an acid of the formula HO-X 'wherein X 'is a group as defined for X wherein the amino group and / or hydroxy group may be protected in the presence of a carbodiimide, or with a reactive derivative of the above acid, which method A) or B) is followed by removal of all protecting groups present. in the molecule, and isolation of the derivative as such, or as a pharmaceutically acceptable acid addition salt.
I fremgangsmådealternativ A) foretrækkes det almindeligvis at anvende de udgangsforbindelser, der har en 6'-CI^-NI^-gruppe, i form af deres 6'-N-beskyttede derivater. Eksempler på foretrukne beskyttelsesgrupper er trifluoracetyl og t-butoxycarbonyl. Gentamicin kan med fordel anvendes som det 2', 3-di-N-beskyt-tede derivat. Gentamicin B^, verdamicin, antibiotikum G-418, antibiotikum JI-20B og antibiotikum G-52 kan anvendes på deres beskyttede form, men kan også med fordel anvendes på fri form.In process alternative A), it is generally preferred to use the starting compounds having a 6'-C1 -NI2 group in the form of their 6'-N protected derivatives. Examples of preferred protecting groups are trifluoroacetyl and t-butoxycarbonyl. Gentamicin can advantageously be used as the 2 ', 3-di-N protected derivative. Gentamicin B 1, verdamicin, antibiotic G-418, antibiotic JI-20B and antibiotic G-52 can be used in their protected form, but can also be used in free form.
Ved fremgangsmådealternativ B) opnår man specifik acylering ved aminogruppen i stilling 1 frem for tilfældig acylering ved anvendelse af 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol, som er delvis neutraliseret ved dannelse af et syreadditionssalt.In Method Alternative B), specific acylation is achieved at the amino group at position 1 rather than random acylation using 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, which is partially neutralized by the formation of an acid addition salt.
Protonisering af en aminogruppe giver den samme virkning*som tilstedeværelsen af en "sædvanlig kemisk" blokeringsgruppe. Det har overraskende vist sig, at aminogruppen i stilling 1 i en 4,6— di-(aminoglycosyl) -1,3-diaminocyclitol er den sidste, der protoniseres ved tilsætning af syre, eller, hvis et persyreadditionssalt behandles med base, den første, som deprotoniseres. "Blokeringsgrupper" (i form af protoner) kan således let indføres i molekylet ved til sætning af en ønsket mængde syre til aminoglycosidet eller ved tilsætning af en ønsket basemængde til et persyreadditionssalt deraf. Dette fremgangsmådealternativ· er fordelagtig på grund af disse opdagelser, og den udgør således en hensigtsmæssig måde til fremstilling af 1-N-acyIderivater af 4,6-di-(aminoglycosyl)-1,3-diaminocycli-tolerne ved anvendelse af delvis neutraliserede og derfor delvis blokerede udgangsforbindelser.Protonation of an amino group gives the same effect * as the presence of a "usual chemical" blocking group. Surprisingly, it has been found that the amino group at position 1 of a 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol is the last to be protonated by the addition of acid, or, if a peracid addition salt is treated with base, the first , which is deprotonized. Thus, "blocking groups" (in the form of protons) can be readily introduced into the molecule by adding a desired amount of acid to the aminoglycoside or by adding a desired base amount to a peracid addition salt thereof. This process alternative is advantageous because of these discoveries, and thus provides a convenient way to prepare 1-N-acyl derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclic tolols using partially neutralized and therefore partially blocked output connections.
7 DK 156070B7 DK 156070B
Som anvendt heri betegner udtrykket "delvis neutraliseret ved dannelse af et syreadditionssalt", at hvert mol 4,6-di-(aminoglyco-syl)-l,3-diaminocyclitol derved har forbundet sig med mindre end det støkiometriske antal mol syre, som kræves til dannelse af persyreaddi· tionssaltet. Endvidere betyder dette udtryk, at hvert mol 4,6-di-(a-minoglycosyl)-1,3-diaminocyclitol har mindst ét mol syre knyttet til sig.As used herein, the term "partially neutralized by the formation of an acid addition salt" means that each mole of 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol is thereby associated with less than the stoichiometric number of moles of acid required. to form the peracid addition salt. Furthermore, this term means that each mole of 4,6-di- (α-minoglycosyl) -1,3-diaminocyclitol has at least one mole of acid attached to it.
F.eks. ville 1 ækvivalent gentamicin med 5 aminogrupper kræve 5 ækvivalenter syre til dannelse af persyreadditionssaltet. Fremgangsmåden ifølge opfindelsen udføres på et syreadditionssalt af gentamicin med mindre end 5 ækvivalenter og mindst 1 ækvivalent syre (f. eks. 4,5, 4,0, 3,5, 3,0, 2,5, 2,0, 1,5 eller 1,0 ækvivalenter syre).Eg. would 1 equivalent gentamicin with 5 amino groups would require 5 equivalents of acid to form the peracid addition salt. The process of the invention is carried out on an acid addition salt of gentamicin having less than 5 equivalents and at least 1 equivalent acid (e.g., 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1, 5 or 1.0 equivalents of acid).
Ifølge en foretrukken udførelsesform for analogifremgangsmåden ifølge opfindelsen neutraliseres udgangsmaterialet med (n-1) ækvivalenter syre, hvor n er antallet af aminogrupper i molekylet. Der neutraliseres således (n-1) aminogrupper ved dannelse åf et syreadditionssalt. Det må imidlertid forstås, at fremgangsmåden også med fordel kan udføres på delvis neutraliserede udgangsmaterialer, hvori mere eller mindre end (n-1) ækvivalenter syre bevirker dannelse af syreadditionssaltet inden for de i det foregående angivne grænser. Udtrykt i pH-områder kan fremgangsmåden udføres i et område på 5,0-9,0, fortrinsvis 5,0-8,0. Det mest foretrukne område for reaktionsmediet er en pH 6,5-7,5, specielt 6,8-7,2.According to a preferred embodiment of the analogous process of the invention, the starting material is neutralized with (n-1) equivalents of acid, where n is the number of amino groups in the molecule. Thus, (n-1) amino groups are neutralized by formation of an acid addition salt. However, it should be understood that the process can also be advantageously carried out on partially neutralized starting materials in which more or less than (n-1) equivalents of acid cause formation of the acid addition salt within the foregoing limits. Expressed in pH ranges, the process can be carried out in a range of 5.0-9.0, preferably 5.0-8.0. The most preferred range for the reaction medium is pH 6.5-7.5, especially 6.8-7.2.
Udtrykket "syreadditionssalt" omfatter sådanne salte, som kan dannes mellem den basiske 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol og en syre uden hensyn til, hvorvidt syren kan betegnes som uorganisk eller organisk. Eksempler på syrer, der omfattes af udtrykket, er svovlsyre, saltsyre, phosphorsyre, salpetersyre, eddikesyre, propionsyre, ravsyre, oxalsyre, cyclopropylcarboxylsyre, trimethyleddikesyre, ma-leinsyre, benzoesyre, phenyleddikesyre, trifluoreddikesyre eller lignende. Hvis det som udgangsmateriale er ønsket at anvende et syreadditionssalt, hvori (n-1) aminogrupper er protoniserede, fremstilles den- .ne forbindelse in situ ' ved, at man omsætter et persyre additionssalt med 1 ækvivalent stærk base, f.eks. triethylamin.The term "acid addition salt" includes such salts which may be formed between the basic 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol and an acid regardless of whether the acid may be referred to as inorganic or organic. Examples of acids encompassed by the term are sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, oxalic acid, cyclopropylcarboxylic acid, trimethylacetic acid, maleic acid, benzoic acid, phenylacetic acid, trifluoroacetic acid or the like. If it is desired to use as an starting material an acid addition salt in which (n-1) amino groups are protonated, this compound is prepared in situ by reacting a peracid addition salt with 1 equivalent strong base, e.g. triethylamine.
Det er også muligt at anvende delvis neutraliserede udgangsforbindelser, som har kemiske blokeringsgrupper. Dette er imidlertid ikke nødvendigt.It is also possible to use partially neutralized starting compounds having chemical blocking groups. However, this is not necessary.
De ved fremgangsmåderne ifølge opfindelsen anvendte hydroxyamino-acyleringsmidler anvendes fortrinsvis i form af reaktive derivater,The hydroxyamino acylating agents used in the methods of the invention are preferably used in the form of reactive derivatives,
8 DK 156070 B8 DK 156070 B
f.eks. i form af aktive estere, anhydrider, azider eller imidazolde-rivater. Det foretrækkes endvidere, at aminogruppen i hydroxyamino-acyleringsmidlet er blokeret. Når det ønskede hydroxyaminoacylpro-dukt er iso-serinylderivatet, vil det foretrukne acyleringsmiddel antage følgende formeg. in the form of active esters, anhydrides, azides or imidazole derivatives. It is further preferred that the amino group of the hydroxyamino acylating agent is blocked. When the desired hydroxyaminoacyl product is the iso-serinyl derivative, the preferred acylating agent will take the following form
Lg-C-CH-CH- I i 2 OH NH-Bg hvori carboxylgruppen fortrinsvis er aktiveret i fom af en aktiv ester (forladende gruppe Lg), og aminogruppen er fortrinsvis blokeret (blokeringsgruppen Bg) af en benzyloxycarbonylgruppe, som let fjernes ved hydrogenolyse, eller en t-butoxycarbonylgruppe eller en trifluoracetylgruppe, idet førstnævnte hensigtsmæssigt fjernes med syre og sidstnævnte med base.Lg-C-CH-CH-I in 2 OH NH-Bg wherein the carboxyl group is preferably activated in the form of an active ester (leaving group Lg) and the amino group is preferably blocked (blocking group Bg) by a benzyloxycarbonyl group which is readily removed by hydrogenolysis , or a t-butoxycarbonyl group or a trifluoroacetyl group, the former being conveniently removed with acid and the latter with base.
Hvis der ønskes farmaceutisk acceptable syreadditionssalte af forbindelserne fremstillet ifølge opfindelsen, kan de fremstilles ved at indstille en vandig opløsning af midlet på pH 4,0 efterfulgt af lyofilisering.If pharmaceutically acceptable acid addition salts of the compounds of the invention are desired, they may be prepared by adjusting an aqueous solution of the pH 4.0 agent followed by lyophilization.
De derved dannede salte er funktionelle ækvivalenter til den frie nitrogenbase, Eksempler på syrer, der kan anvendes til saltdannelse, er svovlsyre, saltsyre, phosphorsyre, salpetersyre, eddikesyre, pro-pionsyre, ravsyre, oxalsyre og maleinsyre.The salts thus formed are functional equivalents to the free nitrogen base. Examples of acids which can be used for salt formation are sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, oxalic acid and maleic acid.
Som anvendt heri betegner udtrykkene "blokeringsgruppe" eller "beskyttelsesgruppe" grupper, som gør de blokerede eller beskyttede aminogrupper inerte over for efterfølgende ønsket . kemisk behandling, men som let kan fjernes ved afslutningen af syntesesekvensen uden spaltning af den ønskede N-aminohydroxyacylgruppe.As used herein, the terms "blocking group" or "protecting group" refers to groups which make the blocked or protected amino groups inert to the subsequent desired. chemical treatment but which can be easily removed at the end of the synthesis sequence without cleavage of the desired N-aminohydroxyacyl group.
Aminobeskyttelsesgrupper er almindeligt kendt i teknikken. Til den foreliggende frangangsmåde er trifluoracetyl-, 2,2,2-trichlorethoxycarbonyl-,t-butoxycarbonyl-, benzyloxycarbonyl- og 4-methoxybenzyloxycarbonyl-grupper imidlertid foretrukne grupper. Specielt foretrukne af de nævnte er trifluoracetyl-, t-butoxycarbonyl- og benzyloxycarbonylgrup-per.Amino protecting groups are well known in the art. However, for the present method trifluoroacetyl, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl and 4-methoxybenzyloxycarbonyl groups are preferred groups. Particularly preferred of the above are trifluoroacetyl, t-butoxycarbonyl and benzyloxycarbonyl groups.
I blokeringsprocessen anvendes blokeringsgruppen sædvanligvis i form af et syreimidazolderivat, et syreazid eller som aktive 1 estere, såsom ethylthioltrifluoracetat, t-butoxycarbonylazid, N-ben-zyloxycarbonyloxysuccinimid eller p-nitrophenyltrichlorethylcarbonat. ;In the blocking process, the blocking group is usually used in the form of an acid imidazole derivative, an acid azide or as active 1 esters such as ethyl thiol trifluoroacetate, t-butoxycarbonylazide, N-benzyloxycarbonyloxysuccinimide or p-nitrophenyltrichloroethyl carbon. ;
VV
9 DK 156070 B9 DK 156070 B
De efterfølgende eksempler forklarer opfindelsen nærmere.The following examples further explain the invention.
Eksempel 1 1-N- (S—3 -Amino-2-hydroxy.propionyl) sisomicin.Example 1 1-N- (S-3-Amino-2-hydroxy-propionyl) sisomicin.
A. S-3 -Trifluoracetamido-2-hydroxy,propionsyre.A. S-3-Trifluoroacetamido-2-hydroxy, propionic acid.
5 g S-3-benzyloxycarbonylamino-2-hydroxypropibnsyre opløses i en blanding af 80 ml dioxan og 20 ml vand. Der tilsættes 200 ml 30 %'s palladium-på-kul og hydrogeneres i 3 timer ved omgivelsernes temperatur og 3,4 atmosfærer. Katalysatoren fjernes ved filtrering, og filtratet koncentreres til en rest i vakuum. Resten tørres i højvakuum i 72 timer. Den tørrede rest opløses i 30 ml kold trifluored-dikesyreanhydrid under omrøring. Opløsningen omrøres i 3 timer ved omgivelsernes temperatur, hvorpå den i vakuum koncentreres til en rest. Resten tritureres med benzen, hvorved man opnår et gråt fast stof, som isoleres ved filtrering, vaskes med benzen og tørres til opnåelse af produktet ifølge dette trin.Dissolve 5 g of S-3-benzyloxycarbonylamino-2-hydroxypropyric acid in a mixture of 80 ml of dioxane and 20 ml of water. 200 ml of 30% palladium on charcoal is added and hydrogenated for 3 hours at ambient temperature and 3.4 atmospheres. The catalyst is removed by filtration and the filtrate is concentrated to a residue in vacuo. The residue is dried in high vacuum for 72 hours. The dried residue is dissolved in 30 ml of cold trifluoroacetic anhydride with stirring. The solution is stirred for 3 hours at ambient temperature and then concentrated in vacuo to a residue. The residue is triturated with benzene to give a gray solid which is isolated by filtration, washed with benzene and dried to obtain the product of this step.
B. N-(S-3-Trifluoracetamido-2-hydroxypropionyloxy)succinimid.B. N- (S-3-Trifluoroacetamido-2-hydroxypropionyloxy) succinimide.
20 mmol af produktet ifølge trin A opløses i 50 ml ethylacetat, der tilsættes 2,31 g N-hydroxysuccinimid under omrøring, og den resulterende opløsning afkøles i et isbad.Der tilsættes 4,3 g dicyclo-hexylcarbodiimid til opløsnirgen pg reaktionsblandingen omrøres i 3 timer ved stuetemperatur. Der filtreres til fjernelse af bundfaldet, og filtratet koncentreres til tørhed, hvilket giver titelforbindelsen, som tørres i højvakuum og anvendes i trin D.Dissolve 20 mmol of the product of step A in 50 ml of ethyl acetate, add 2.31 g of N-hydroxysuccinimide with stirring and cool the resulting solution in an ice bath. Add 4.3 g of dicyclohexylcarbodiimide to the solution gene and stir the reaction mixture for 3 hours. hours at room temperature. Filtrate to remove the precipitate and concentrate the filtrate to dryness to give the title compound which is dried in high vacuum and used in step D.
C. 61-N-Trifluoracetylsisomicin.C. 61-N-Trifluoroacetylsisomicin.
20 g sisomicin opløses i 1,2 liter vandfri methanol, og dråbevis tilsættes en opløsning af 6 ml ethylthioltrifluoracetat i 60 ml methanol i løbet af et tidsrum på 3 timer under omrøring. Reaktionen får lov at skride frem i 18 timer ved stuetemperatur, hvorefter opløsningsmidlet fjernes i vakuum, hvilket giver en rest på 23,8 g af produktet med en tilnærmelsesvis renhed på 95 % og følgende fysiskkemiske egenskaber:Dissolve 20 g of sisomicin in 1.2 liters of anhydrous methanol and dropwise add a solution of 6 ml of ethyl thiol trifluoroacetate in 60 ml of methanol over a period of 3 hours with stirring. The reaction is allowed to proceed for 18 hours at room temperature, after which the solvent is removed in vacuo to give a residue of 23.8 g of the product with an approximate purity of 95% and the following physicochemical properties:
Massespektraldata: m/e 543 M+, andre afgørende spidser ved m/e 413, 395, 385, 362, 223 og 126.Mass spectral data: m / e 543 M +, other crucial peaks at m / e 413, 395, 385, 362, 223 and 126.
NMR (60 MHz, D20) ^5,37 dublet, J = 2 Hz, H-l'f 5,12 dublet, J = 4 Hz, H-l"; 4,96 bred singlet, H-4'; 2,57, singlet, N-CH-.NMR (60 MHz, D 2 O) δ 5.37 doublet, J = 2 Hz, H-l'f 5.12 doublet, J = 4 Hz, H1 "; 4.96 wide singlet, H-4 '; 2.57 , singlet, N-CH-.
1,26 singlet, C-CH^.1.26 singlet, C-CH
1010
DK 156070 BDK 156070 B
D. 1-N- (S- 3-Amino-2-hydroxypropionyl) sisomicin.D. 1-N- (S-3-Amino-2-hydroxypropionyl) sisomicin.
814 mg (1,5 mmol) 6'-N-trifluoracetylsisomicin opløses i 12 ml 50%1 s vandig methanol, og der tilsættes dråbevis under omrøring en opløsning af 1,5 mmol af produktet fra trin B opløst i 3 ml dime-thylformamid. Den resulterende opløsning omrøres ved stuetemperatur i 18 timer og koncentreres i vakuum til en rest. Resten opløses i 10 ml koncentreret ammoniumhydroxid og får lov at henstå i 2 timer (til fjernelse af trifluoracetylgrupperne). Opløsningsmidlet afdampes til opnåelse af titelforbindelsen gom en rest. Resten chromatograferes på en silicagelsøjle under anvendelse af den nedre fase af et chloro-form:methanol:ammoniumhydroxid-opløsningsmiddelsystem (2:1:1) som elu-eringsmiddel. Fraktioner indeholdende titelforbindelsen forenes og koncentreres til en rest, som opløses i vand og lyofiliseres, hvilket giver et amorft hvidt fast stof.Dissolve 814 mg (1.5 mmol) of 6'-N-trifluoroacetyl isomicin in 12 ml of 50% 1 sec aqueous methanol and add dropwise with stirring a solution of 1.5 mmol of the product of step B dissolved in 3 ml of dimethylformamide. . The resulting solution is stirred at room temperature for 18 hours and concentrated in vacuo to a residue. The residue is dissolved in 10 ml of concentrated ammonium hydroxide and allowed to stand for 2 hours (to remove the trifluoroacetyl groups). The solvent is evaporated to give the title compound from a residue. The residue is chromatographed on a silica gel column using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (2: 1: 1) as eluent. Fractions containing the title compound are combined and concentrated to a residue which is dissolved in water and lyophilized to give an amorphous white solid.
26 o 1-N- (S-*3-amino-2-hydroxypropionyl) sisomicin, [a]D +139 (c = 0,3 i vand). Analyse: beregnet for C22H42°9N6r ^2 H2C03: C = 47/74f H - 7,66, N = 14,85? fundet: C = 47,39, H = 7,14, N = 15,16.pmr 100 MHz D20: il,22 (s), C-Me? £2,60 (s) N-Me 4,22 (q), J = 4 Hz, 7,5 Hz? £5,09 (d), H-l", J1If 2n = 4'° Hz* ^5'34 (d)' Π"1’' Jl\2' = 2 Hz26 o 1-N- (S- * 3-amino-2-hydroxypropionyl) sisomicin, [α] D + 139 (c = 0.3 in water). Analysis: Calculated for C 22 H 42 ° 9 N 6 R 2 H 2 CO 3: C = 47 / 74f H - 7.66, N = 14.85? found: C = 47.39, H = 7.14, N = 15.16.pmr 100 MHz D20: il, 22 (s), C-Me? £ 2.60 (s) N-Me 4.22 (q), J = 4 Hz, 7.5 Hz? £ 5.09 (d), H-1 ", J1If 2n = 4 '° Hz * ^ 5'34 (d)' Π" 1 '' Jl \ 2 '= 2 Hz
Fremstillet på tilsvarende måde: 1-N-(S-3-amino-2-hydroxypropionyl)gentamicin Cla, pmr 100 MHz, D20: Si,22, C-Me? 2,59, N-Me? [a]26+115,4° (c = 0,3 i vand)? 5,09, H-l", J-Lii/2*" = 4 Hz? 5,17, H-l", J^, 2, = 3,5 Hz. Analyse: beregnet for C22H44°9N6' H20/ C02: C = 45'01' H = 7/39/ N = 13,70? fundet: C = 44,98, H = 7,81, N = 13,68%, 1-N-(S-3-amino-2-hydroxypropionyl)gentamicin B, pmr 100 MHz, D20 + DC1: £1,18 C-Me (s)? 2,77 N-Me (s)? £4,35 (q) , J = 4,5, 8,0,* H-l', £5,01 (d), Jx,^21 = 4,0 Hz? H-l", 5,43 (d), Jin 2„ = 3,5 Hz,Prepared similarly: 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin Cla, pmr 100 MHz, D 2 O: Si, 22, C-Me? 2.59, N-Me? [α] 26 + 115.4 ° (c = 0.3 in water)? 5.09, H-l ", J-Lii / 2 *" = 4 Hz? 5.17, H1 ", J2, 2, = 3.5 Hz. Analysis: calculated for C 22 H 44 ° 9 N 6 'H 2 O / CO 2: C = 45'01' H = 7/39 / N = 13.70? Found: C = 44.98, H = 7.81, N = 13.68%, 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin B, pmr 100 MHz, D20 + DC1: £ 1.18 C -Me (s)? 2.77 N-Me (s)? 4.35 (q), J = 4.5, 8.0, * H-1 ', £ 5.01 (d), Jx, Δ 21 = 4.0 Hz? HI ", 5.43 (d), Jin 2" = 3.5 Hz,
Eksempel 2 1-N-(S-4-Amino-2-hydroxybutyryl)gentamicin B.Example 2 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin B.
A. 6'-N-t-Butoxycarbonylgentamicin B.A. 6'-N-t-Butoxycarbonylgentamicin B.
1 g gentamicin B opløses i 30 ml 50%'s vandig methanol, og der afkøles til 5°C. Der tilsættes 0,297g t-butoxycarbonylazid dråbevis under omrøring efterfulgt af 0,186 ml triethylamin, og den resulterende opløsning omrøres i 18 timer. Reaktionsblandingen inddampes i vakuum til en rest, og resten chromatograferes på 100 g silicågel under anvendelse af den nedre fase af et chloroform:methanolkoncentreret ammoniumhydroxid-opløsningsmiddelsystem (2:1:1) som eluerings-Dissolve 1 g of gentamicin B in 30 ml of 50% aqueous methanol and cool to 5 ° C. 0.297g of t-butoxycarbonylazide is added dropwise with stirring followed by 0.186ml of triethylamine and the resulting solution is stirred for 18 hours. The reaction mixture is evaporated in vacuo to a residue and the residue is chromatographed on 100 g of silica gel using the lower phase of a chloroform: methanol concentrated ammonium hydroxide solvent system (2: 1: 1) as eluent.
n DK 156070Bn DK 156070B
middel. Fraktioner på 2 ml opsamles, og søjleefluentet undersøges ved tyndtlagschromatografi. Fraktioner indeholdende samme materiale samles (fraktionerne 180-230) og inddampes til opnåelse af 0,830 g 6'-N-t-butoxycarbonylgentamicin B med følgende fysiske konstanter: pmr (60 MHz, D20) : <Jl,21 (3H, s, C-CH3) , 1,42 (9H, s, C(CH3)3), 2,53 (3H, s, N-CH3), 5,2 (IH, d, J = 4,5 HzT H-l"), 5,23 (IH, d7"j = 3,0agent. 2 ml fractions are collected and the column effluent is assayed by thin layer chromatography. Fractions containing the same material are collected (fractions 180-230) and evaporated to give 0.830 g of 6'-Nt-butoxycarbonylgentamicin B with the following physical constants: pmr (60 MHz, D20): <J1, 21 (3H, s, C-CH3 ), 1.42 (9H, s, C (CH 3) 3), 2.53 (3H, s, N-CH 3), 5.2 (1H, d, J = 4.5 HzT H 23 (1H, d 7 "j = 3.0)
Hz, H-l') PPM, analyse beregnet for C24H46N4°i2,C02,H20: C = 46'57' H = 7,51, N = 8,69%, fundet: C = 46,80, H = 7,82, N = 8,54%, [a]^+124° (c = 1 i methanol).Hz, H-1 ') PPM, analysis calculated for C24H46N4 ° i2, CO2, H2O: C = 46'57' H = 7.51, N = 8.69%, found: C = 46.80, H = 7 , 82, N = 8.54%, [α] 20 + 124 ° (c = 1 in methanol).
B. 1-N-(S-4-Amino-2-hydroxybutyryl)-61-N-t-butoxycarbonyl-gentamicin B.B. 1-N- (S-4-Amino-2-hydroxybutyryl) -61-N-t-butoxycarbonyl-gentamicin B.
0,582 g 6'-N-t-butoxycarbonylgentamicin B opløses i 25 ml metha-nol:vand (1:3). Der tilsættes en opløsning af 0,334 g N-(S-4-carbo-benzyloxyamino-2-hydroxybutyryloxy)succinimid i 5 ml dimethylforma-mid dråbevis under omrøring. Reaktionsblandingen omrøres ved 5°C i 5 timer, hvorpå der inddampes til tørhed i vakuum. Resten chromato-graferes på 60 g silicagel under anvendelse af den nedre fase af et chloroform:methanol:ammoniumhydroxid-opløsningsmiddelsystem (2:1:1) som elueringsmiddel. Der opsamles fraktioner på 3 ml, hvis indhold bestemmes ved tyndtiagschromatografi. Fraktionerne 160-235 forenes, og der inddampes til en rest på 0,280 g, der vandrer som en enkelt plet ved tyndtlagschromatografi. Resten opløses i 8 ml methanol og 10 ml vand og der hydrogeneres ved 3,7 atmosfærer over 60 mg 5%'s palla-dium-på-kul-katalysator. Katalysatoren fjernes ved filtrering, filtratet inddampes til en rest, der chromatograferes på 20 g silicagel under anvendelse af den nedre fase af et chloroform:methanol:ammoni-umhydroxid-opløsningsmiddelsystem (1:1:1) som elueringsmiddel. Fraktionerne 79-127 (hver på 2 ml) bestående af 1-N-(S-4-amino-2-hy-droxybutyryl)-61-N-t-butoxycarbonyl-gentamicin B forenes. Udbytte 86 mg. pmr: $1,21 (3H, s, C-CH3), 1,43 (9H, s, C-(CH3)3), 2,51 (3H, s, N-CH3), 5,08 (IH, d, J = THz, H-l"), 5,25 (lH,~d7 J = 3,5 Hz, H-l')"PPM.Dissolve 0.582 g of 6'-N-t-butoxycarbonylgentamicin B in 25 ml of methanol: water (1: 3). A solution of 0.334 g of N- (S-4-carbo-benzyloxyamino-2-hydroxybutyryloxy) succinimide in 5 ml of dimethylformamide is added dropwise with stirring. The reaction mixture is stirred at 5 ° C for 5 hours, then evaporated to dryness in vacuo. The residue is chromatographed on 60 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (2: 1: 1) as eluent. 3 ml fractions are collected, the contents of which are determined by thin-layer chromatography. Fractions 160-235 are combined and evaporated to a residue of 0.280 g, which migrates as a single spot by thin layer chromatography. The residue is dissolved in 8 ml of methanol and 10 ml of water and hydrogenated at 3.7 atmospheres over 60 mg of 5% palladium-on-carbon catalyst. The catalyst is removed by filtration, the filtrate is evaporated to a residue which is chromatographed on 20 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (1: 1: 1) as eluent. Fractions 79-127 (each of 2 ml) consisting of 1-N- (S-4-amino-2-hydroxybutyryl) -61-N-t-butoxycarbonyl-gentamicin B are combined. Yield 86 mg. pmr: $ 1.21 (3H, s, C-CH3), 1.43 (9H, s, C- (CH3) 3), 2.51 (3H, s, N-CH3), 5.08 (1H, d, J = THz, H1 "), 5.25 (1H, ~ d7 J = 3.5 Hz, H-1 ')" PPM.
C. 1-N-(S-4-Amino-2-hydroxybutyryl)gentamicin B.C. 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin B.
Produktet fra trin B opløses i 0,3 ml trifluoreddikesyre, og efter 5 minutters forløb tilsættes 30 ml ethylether. Et bundfald af forbindelsen ifølge dette eksempel dannes i form af sit trifluoracetatsalt og isoleres ved filtrering. Bundfaldet opløses i vand, og opløsningen passeres gennem en basisk ionbytterharpiks-kolonne på hydroxyl-ionform til overføring af syreadditionssaltet i den frie base. Kolon-The product of step B is dissolved in 0.3 ml of trifluoroacetic acid and after 5 minutes 30 ml of ethyl ether is added. A precipitate of the compound of this example is formed in the form of its trifluoroacetate salt and is isolated by filtration. The precipitate is dissolved in water and the solution is passed through a basic hydroxyl ion exchange resin column to transfer the acid addition salt into the free base. colon
12 DK 156070B12 DK 156070B
neeffluentet lyofiliseres, hvilket giver forbindelsen ifølge dette eksempel i form af et hvidt,amorft fast stof. Udbytte 72 mg. pmr D20 100 MHz: £l,14 C-methyl (s), 2,45 (s) N-methyl, 5,04 (d), H-l", = 4/0 Hz; 5,30 (d) , H-l', 2, = 3,5 Hz.the neofluent is lyophilized to give the compound of this example in the form of a white, amorphous solid. Yield 72 mg. pmr D20 100 MHz: δ.14 C-methyl (s), 2.45 (s) N-methyl, 5.04 (d), H1 "= 4/0 Hz; 5.30 (d), H -l ', 2, = 3.5 Hz.
Eksempel 3 1-N-(S-3-Amino-2-hydroxypropionyl)gentamicin .Example 3 1-N- (S-3-Amino-2-hydroxypropionyl) gentamicin.
A. 1-N-(S-3-Carbobenzyloxyamino-2-hydroxypropionyl)-21-3-di-N-tri-fluoracetyIgentamicin C^.A. 1-N- (S-3-Carbobenzyloxyamino-2-hydroxypropionyl) -21-3-di-N-trifluoroacetylgentamicin C
0,84 g 2',3-di-N-trifluoracetylgentamicin opløses i 40 ml tetra- hydrofuran, og der tilsættes en opløsning- af 1,19 g N- (S-3-carbobenzyloxyami-no-2-hydroxypropionyloxy)succinimid i 24 ml tetrahydrofuran dråbevis under omrøring. Reaktionsblandingen omrøres i 24 timer, hvorpå den inddampes til en rest. Resten chromatograferes på silicagel under anvendelse af den nedre fase af et chloroform:methanolikoncentreret ammoniumhydroxidivand-opløsningsmiddelsystem (2:1:0,2:0,8) v/v som elueringsmiddel. Chromatogrammet undersøges ved tyndtlagschromatogra-fi, og fraktioner indeholdende samme produkt forenes til opnåelse af hovedproduktet af reaktionen med følgende konstanter: Smp.: 125-131°C, [a]^6= +93° (CH^OH), analyse beregnet for dihydratet: C = 47,47, H = 6,20, N = 9,23%, fundet: C = 47,85, H = 6,67, N = 9,08%.Dissolve 0.84 g of 2 ', 3-di-N-trifluoroacetylgentamicin in 40 ml of tetrahydrofuran and add a solution of 1.19 g of N- (S-3-carbobenzyloxyamino-2-hydroxypropionyloxy) succinimide in 24 ml of tetrahydrofuran dropwise with stirring. The reaction mixture is stirred for 24 hours and then evaporated to a residue. The residue is chromatographed on silica gel using the lower phase of a chloroform: methanolic concentrated ammonium hydroxydivand solvent system (2: 1: 0.2: 0.8) v / v as eluent. The chromatogram is examined by thin layer chromatography, and fractions containing the same product are combined to give the main product of the reaction with the following constants: mp: 125-131 ° C, [.alpha.] @ 6 = + 93 ° (CH2 OH), analysis calculated for dihydrate: C = 47.47, H = 6.20, N = 9.23%, found: C = 47.85, H = 6.67, N = 9.08%.
B. 1-N-(S-3-Carbobenzyloxyamino-2-hydroxypropionyl) gentamicin C-^.B. 1-N- (S-3-Carbobenzyloxyamino-2-hydroxypropionyl) gentamicin C
0,55 g af produktet fra trin A opløses i 55 ml methanol, og der tilsættes 25 ml koncentreret ammoniumhydroxid under omrøring. Der omrøres i 3 dage, hvorefter tyndtlagschromatografi på silicagelplader under anvendelse af den nedre fase af et chloroform:methanolikoncentreret ammoniumhydroxid-opløsningsmiddelsystem (1:1:1) som mobil fase indikerer i det væsentlige fuldstændig fjernelse af trifluoracetyl-blokeringsgrupperne. Opløsningen inddampes til tørhed i vakuum. Udbytte 0,2 g, smp.: 109-112°C, [a]^6= +73° (H20).Dissolve 0.55 g of the product of step A in 55 ml of methanol and add 25 ml of concentrated ammonium hydroxide with stirring. Stir for 3 days, after which thin layer chromatography on silica gel plates using the lower phase of a chloroform: methanolic concentrated ammonium hydroxide solvent system (1: 1: 1) as mobile phase indicates substantially complete removal of the trifluoroacetyl blocking groups. The solution is evaporated to dryness in vacuo. Yield 0.2 g, mp: 109-112 ° C, [α] D = 6 = + 73 ° (H 2 O).
C. 1-N-(S-3-Amino-2-hydroxypropionyl)gentamicin C^.C. 1-N- (S-3-Amino-2-hydroxypropionyl) gentamicin C
0,153 g af resten fra trin B opløses i 8 ml eddikesyre, og der tilsættes 0,05 g 10%'s palladium-på-kul-katalysator. Opløsningen hydrogeneres ved 4,0 atmosfærer og 25°C, indtil tyndtlagschromatografi (under anvendelse af det i trin B anvendte opløsningsmiddelsystem) indikerer fuldstændig omsætning til titelforbindelsen (dvs. 16 timer til 3 dage). Katalysatoren fjernes ved filtrering, og opløsningenDissolve 0.153 g of the residue from step B in 8 ml of acetic acid and add 0.05 g of 10% palladium-on-carbon catalyst. The solution is hydrogenated at 4.0 atmospheres and 25 ° C until thin layer chromatography (using the solvent system used in step B) indicates complete reaction to the title compound (i.e., 16 hours to 3 days). The catalyst is removed by filtration and the solution
DK 156070 BDK 156070 B
13 inddampes til en rest i vakuum. Resten chromatograferes på 7 g silicagel under anvendelse af den nedre fase af en chloroform:methanol:ammoni-umhydroxid-blanding (2:1:1) som elueringsmiddel. Søjlen undersøges under anvendelse af tyndtlagschromatografi, lignende materialer kombineres , hvorved produktet ifølge dette eksempel opnås. Udbytte 112 mg, smp.: l09-119°Cf [a]^= +98° (E^O), analyse beregnet for monohydra-tet: C = 49,47, H = 8,65, N = 14,42%, fundet: C = 49,24, H = 8,53, N = 14,10%.13 is evaporated to a residue in vacuo. The residue is chromatographed on 7 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide mixture (2: 1: 1) as eluent. The column is examined using thin layer chromatography, combining similar materials to give the product of this example. Yield 112 mg, mp: 109-119 ° C [α] D = + 98 ° (E₂O), analysis for the monohydrate: C = 49.47, H = 8.65, N = 14.42 %, found: C = 49.24, H = 8.53, N = 14.10%.
Eksempel 4 1-N-(S-4-Amino-2-hydroxybutyry1)verdamicin.Example 4 1- N- (S-4-Amino-2-hydroxybutyryl) verdamicin.
A. 1-N-(S-4-Phtha1imido-2-hydroxybutyry1)verdamicin.A. 1-N- (S-4-Phthalimido-2-hydroxybutyryl) verdamicin.
4,61 g verdamicin opløses i 50 ml af en methanol:vand-opløsning (1:3), og der afkøles til 0-5°C. Der tilsættes en opløsning af 3,81 g N-(S-4-phthalimido-2-hydroxybutyryloxy)succinimid i 20 ml dimethyl-formamid dråbevis under omrøring til den antibiotiske opløsning. Der omrøres i yderligere 16 timer, hvorpå der koncentreres til en rest i vakuum. Resten chromatograferes på en søjle af 250 g silicagel med den nedre fase af en chloroform:methanol:koncentreret ammoniumhydro-xid-blanding (2:1:1) som elueringsmiddel. Eluatet undersøges ved tyndtlagschromatografi,og fraktioner indeholdende lignende materialer forenes. De forenede fraktioner, der indeholder hovedproduktet, inddampes til opnåelse af 1-N-(S-4-phthalimido-2-hydroxybutyryl)verda“ micin og en isomer deraf.Dissolve 4.61 g of verdamicin in 50 ml of a methanol: water solution (1: 3) and cool to 0-5 ° C. A solution of 3.81 g of N- (S-4-phthalimido-2-hydroxybutyryloxy) succinimide in 20 ml of dimethylformamide is added dropwise with stirring to the antibiotic solution. Stir for an additional 16 hours, then concentrate to a residue in vacuo. The residue is chromatographed on a column of 250 g of silica gel with the lower phase of a chloroform: methanol: concentrated ammonium hydroxide mixture (2: 1: 1) as eluent. The eluate is assayed by thin layer chromatography and fractions containing similar materials are combined. The combined fractions containing the main product are evaporated to give 1-N- (S-4-phthalimido-2-hydroxybutyryl) digestion and an isomer thereof.
B. 1-N-(S-4-Amino-2-hydroxybutyry1)verdamicin.B. 1-N- (S-4-Amino-2-hydroxybutyryl) verdamicin.
4,0 g af produktet fra det foregående trin opløses i 35 ml ethanol, og der tilsættes 1,0 g hydrazinhydrat. Opløsningen, koges under tilbagesvaling i 3 timer, hvorpå den inddampes til tørhed i vakuum. Resten chromatograferes over 160 g silicagel, idet man eluerer med den nedre fase af en chloroform:methanol:koncentreret ammoniumhydro-xid-blanding (1:1:1) (v/v). De fraktioner, der indeholder hovedkom ponenten for reaktionen som vist ved tyndtlagschromatografi, kombineres og inddampes til opnåelse af forbindelsen ifølge dette eksempel som et hvidt amorft fast stof.Dissolve 4.0 g of the product of the previous step in 35 ml of ethanol and add 1.0 g of hydrazine hydrate. The solution is refluxed for 3 hours and then evaporated to dryness in vacuo. The residue is chromatographed over 160 g of silica gel eluting with the lower phase of a chloroform: methanol: concentrated ammonium hydroxide mixture (1: 1: 1) (v / v). The fractions containing the main component of the reaction as shown by thin layer chromatography are combined and evaporated to give the compound of this example as a white amorphous solid.
CMR (25.2 MH, D20) 147,2, 101,5, 100,5, 99,0, 86,2, 83,9, 75,2, 70,9, 68,9, 68,3, 64,6, 51,2, 49,6, 46,4, 43,0, 35,6,32,8, 23,4, 21,7, 16,8.CMR (25.2 MH, D 2 O) 147.2, 101.5, 100.5, 99.0, 86.2, 83.9, 75.2, 70.9, 68.9, 68.3, 64.6 , 51.2, 49.6, 46.4, 43.0, 35.6, 32.8, 23.4, 21.7, 16.8.
På lignende måde fremstilles: tSimilarly prepared: t
14 DK 156070 B14 DK 156070 B
1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin B1 PMR (60 MH , D20) 6 1,28, s, C~.Jie; 1,26, d, CH-CH,; 1,7-2,3,m, 2xH-3"' og H-2"eq; 2,74, s, N-Me; 5,18, d, J=4HZ, H-1", 5,53, d, J=3H , H-1' ppm. 1-N-(S-4-amino-2-hydroxybutyryl)-antibiotikum JI-20B m.p. 128-138°C;[a]2® + 126,4 (C , 0,.28 i vann) . PMR (100 MH , D20) 61,12, s, C-Me; 1,21, d, CH-Me; 2,48, s, N-Me; 5,08, d, J=3H , H-1"? 5r25, d, J=4Hz, H-1' ppm og 1-N-(S-3-amino-2-hydroxy-propionyl)-gentamicin PMR (D20): 6 1,08 (3H, d, J= 7,0 Hz, CHCH^), l, 20 (3H, s, C-CH3), 1,38 (1H, q,J= 13,0 Hz, H-2ax,),1,93 (1H, m, H„ ), 2,48 (1H, d, J=10,5 Hz, H-3"), 2,5 (3H, s, N-CH-,) , 3,93- 2eq 4,28 (m, 2H, H-5" og H-2",)f 5,05 (1H, d, J= 4.0 Hz, H-1"),5,25 -(1H, d, J= 3,0 Hz, H-1').1-N- (S-4-amino-2-hydroxybutyryl) -gentamicin B1 PMR (60 MH, D 2 O) δ 1.28, s, C ~JJ; 1.26, d, CH-CH 2; 1.7-2.3, m, 2xH-3 "and H-2" eq; 2.74, s, N-Me; 5.18, d, J = 4HZ, H-1 ", 5.53, d, J = 3H, H-1 'ppm. 1-N- (S-4-amino-2-hydroxybutyryl) antibiotic JI- 20B mp 128-138 ° C; [a] 2 + + 126.4 (C, 0.28 in water) PMR (100 MH, D 2 O) 61.12, s, C-Me; 1.21, d , CH-Me; 2.48, s, N-Me; 5.08, d, J = 3H, H-1 "? 5r25, d, J = 4Hz, H-1 'ppm and 1-N- (S-3-amino-2-hydroxy-propionyl) -gentamicin PMR (D20): 6 1.08 (3H, d, J = 7 ), 1.28 (3H, s, C-CH 3), 1.38 (1H, q, J = 13.0 Hz, H-2ax,), 1.93 (1H, m, H 2), 2.48 (1H, d, J = 10.5 Hz, H-3 "), 2.5 (3H, s, N-CH-,), 3.93-2eq 4.28 (m , 2H, H-5 "and H-2",) f 5.05 (1H, d, J = 4.0 Hz, H-1 "), 5.25 - (1H, d, J = 3.0 Hz, H-1 ').
CMR (D20),6 (rel TMS, PPM): 179,1, 102,4, 99,1, 89,6, 80,3, 76,1, 75.1, 74,7, 73,5, 73,1, 72f3, 69,3, 68,6, 64,1, 50,2, 49,4, 46,7, 46.1, 38,0, 22,7, 16,9.CMR (D20), δ (rel TMS, PPM): 179.1, 102.4, 99.1, 89.6, 80.3, 76.1, 75.1, 74.7, 73.5, 73.1 , 72f3, 69.3, 68.6, 64.1, 50.2, 49.4, 46.7, 46.1, 38.0, 22.7, 16.9.
Eksempel 5 1. N-(S-3-t-Butoxycarbonylamino-2-hydroxypropionyloxy)succinimid.Example 5 1. N- (S-3-t-Butoxycarbonylamino-2-hydroxypropionyloxy) succinimide.
A. S-3-t-Butoxycarbonylamino-2-hydroxypropionsyre.A. S-3-t-Butoxycarbonylamino-2-hydroxypropionic acid.
8,0 g S-3-carbobenzyloxyamino-2-hydroxypropionsyre opløses i 110 ml dioxan:vand (4:1). Der tilsættes 200 mg palladium-på-kul-katalysa-tor og hydrogeneres ved 3,4 atmosfærer i 3 timer. Katalysatoren fra-filtreres, og filtratet inddampes til en rest omfattende S-3-amino-2-hydroxypropionsyre. Syren opløses i methanol (40 ml) og triethyl-amin (8,5 ml), opløsningen afkøles i et isbad og t-butoxycarbonylazid (4,76 g) tilsættes Blandingen henstår natten over under gradvis opvarmning til stuetemperatur. Det meste af methanolen afdampes, der fortyndes med vand og syrnes med fortyndet saltsyre til pH 3. Den vandige blanding ekstraheres flere gange med ethylacetat, ethylacetatekstrak-terne kombineres og tørres og inddampes derefter til en rest i vakuum. Resten omkrystalliseres af ethylacetat:hexan, hvilken giver titelforbindelsen fra dette eksempel. Udbytte 3,5 g, smp. 92-94°C, [a]26 +14,9°, (c = 0,68, ^0) , analyse beregnet for CgH^NO^: C = 46,82, H * 7,37, N = 6,83%, fundet: C = 46,81, H = 7,61, N = 6,63%.Dissolve 8.0 g of S-3-carbobenzyloxyamino-2-hydroxypropionic acid in 110 ml of dioxane: water (4: 1). 200 mg of palladium-on-carbon catalyst is added and hydrogenated at 3.4 atmospheres for 3 hours. The catalyst is filtered off and the filtrate is evaporated to a residue comprising S-3-amino-2-hydroxypropionic acid. The acid is dissolved in methanol (40 ml) and triethylamine (8.5 ml), the solution is cooled in an ice bath and t-butoxycarbonylazide (4.76 g) is added. The mixture is allowed to stand overnight at room temperature. Most of the methanol is evaporated, diluted with water and acidified with dilute hydrochloric acid to pH 3. The aqueous mixture is extracted several times with ethyl acetate, the ethyl acetate extracts are combined and dried and then evaporated to a residue in vacuo. The residue is recrystallized from ethyl acetate: hexane to give the title compound of this example. Yield 3.5 g, m.p. 92-94 ° C, [α] 26 + 14.9 °, (c = 0.68, 0), analysis calculated for C CH ^NO ^: C = 46.82, H * 7.37, N = 6 , 83%, found: C = 46.81, H = 7.61, N = 6.63%.
B. N-(S-3-t-Butoxycarbonylamino-2-hydroxypropionyloxy)succinimid.B. N- (S-3-t-Butoxycarbonylamino-2-hydroxypropionyloxy) succinimide.
2,3 g S-3-t-butoxycarbonylamino-2-hydroxypropionsyre opløses i en blanding af tetrahydrofuran (25 ml) og ethylacetat (60 ml). Under omrøring tilsættes 1,42 g N-hydroxysuccinimid, efterfulgt af en opløsning af dicyclohexylcarbodiimid (2,51 g) i ethylacetat (10 ml).2.3 g of S-3-t-butoxycarbonylamino-2-hydroxypropionic acid are dissolved in a mixture of tetrahydrofuran (25 ml) and ethyl acetate (60 ml). With stirring, 1.42 g of N-hydroxysuccinimide is added, followed by a solution of dicyclohexylcarbodiimide (2.51 g) in ethyl acetate (10 ml).
Blandingen omrøres i 16 timer, hvorpå det udfældede faste stof fra-The mixture is stirred for 16 hours, after which the precipitated solid is removed.
,5 DK 156070 B, 5 DK 156070 B
filtreres. Filtratet inddampes til opnåelse af produktet ifølge dette eksempel. Udbytte 3,56 g.filtered. The filtrate is evaporated to give the product of this example. Yield 3.56 g.
2. 2' ,3-di-N-Trifluoracetylgentamicin C^.2. 2 ', 3-di-N-Trifluoroacetylgentamicin C
A. 2'-N-Trifluoracetylgentamicin C·^.A. 2'-N-Trifluoroacetylgentamicin C · ^.
1.7 g gentamicin opløses i 20 ml methanol, blandingen afkøles til 4°C, og der tilsættes 0,46 ml (0,563 g) ethylthioiltrif luoracetat under omrøring. Reaktionen får lov at fortsætte i 2 timer, og opløsningen koncentreres til en rest i vakuum. Produktet chromatograferes på 80 g silicagel G under anvendelse af den nedre fase af en chlo-roformimethanol:vand:ammoniumhydroxid-blanding i volumenforholdet 10:5:4:1 som elueringsmiddel. De fraktioner, der indeholder hovedkomponenten, kombineres og koncentreres til opnåelse af 1,4 g af titelforbindelsen, smp. 108-111°C, [a]^= +128° (c = 0,3%, E^O). Analyse for C23H42N508F3,H20 kræver C = 46,69, H = 7,50, N = 11,84, F = 9,63%. Fundet: C = 46,66, H = 7,65, N = 11,60, F = 9,24%.Dissolve 1.7 g of gentamicin in 20 ml of methanol, cool the mixture to 4 ° C and add 0.46 ml (0.563 g) of ethylthioiltrifluoroacetate with stirring. The reaction is allowed to continue for 2 hours and the solution is concentrated to a residue in vacuo. The product is chromatographed on 80 g of silica gel G using the lower phase of a chloroformimethanol: water: ammonium hydroxide mixture in volume ratio of 10: 5: 4: 1 as eluent. The fractions containing the main component are combined and concentrated to give 1.4 g of the title compound, m.p. 108-111 ° C, [α] D = + 128 ° (c = 0.3%, E + O). Analysis for C 23 H 42 N 5 O 8 F 3, H 2 O requires C = 46.69, H = 7.50, N = 11.84, F = 9.63%. Found: C = 46.66, H = 7.65, N = 11.60, F = 9.24%.
B. 2f,3-di-N-Trifluoracetylgentamicin C^.B. 2f, 3-di-N-Trifluoroacetylgentamicin C
0,66 g af produktet fra trin A opløses i 10 ml methanol, blandingen afkøles til 4°C, og der tilsættes 0,148 ml (0,182 g) ethylthiol-trifluoracetat opløst i 3 ml methanol. Reaktionsblandingen omrøres i ca. 16 timer, og der koncentreres i vakuum til en rest. Produktet chroma tograferes på 30 g silicagel som beskrevet under trin A. Søjlen undersøges ved tyndtlagschromatografi, de egnede fraktioner kombineres, og der koncentreres til opnåelse af 0,32 g af titelforbindelsen, smp. 121-129°C, [α]ρβ= 121° (c = 0,3%, H20). Analyse for Ο^Η.-,Ν,-ΟοΕ,- kræver C = 44,84, H = 6,17, N = 10,46%. Fundet: C = 44,94, Δο 4i o y b H = 6,35, N = 10,17%.Dissolve 0.66 g of the product of step A in 10 ml of methanol, cool the mixture to 4 ° C and add 0.148 ml (0.182 g) of ethylthiol trifluoroacetate dissolved in 3 ml of methanol. The reaction mixture is stirred for approx. 16 hours and concentrated in vacuo to a residue. Chromatograph the product on 30 g of silica gel as described in step A. The column is assayed by thin layer chromatography, the appropriate fractions are combined and concentrated to give 0.32 g of the title compound, m.p. 121-129 ° C, [α] ρβ = 121 ° (c = 0.3%, H 2 O). Analysis for Ο ^ Η .-, Ν, -ΟοΕ, - requires C = 44.84, H = 6.17, N = 10.46%. Found: C = 44.94, Δο 4i o y b H = 6.35, N = 10.17%.
Eksempel 6 l-N-(S-4- ftmino-2-hydroxybutyry1)gentamicin C^a· A. 1-N-(S-4-Benzyloxycarbonylamino-2-hydroxybutyryl)gentamicin Cla.Example 6 1- N- (S-4-amino-2-hydroxybutyryl) gentamicin C ^ a C A. 1-N- (S-4-Benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin Cla.
2.8 g (4 mmol) gentamicin C^sulfat opløses i 30 ml vand, og der tilsættes 15 ml methanol. Dernæst tilsættes 0,56 ml (4 mmol) triethyl-amin,og der amrøres i 10 minutter. Derefter tilsættes en opløsning indeholdende 4 mmol N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryloxy)-succinimid i 20 ml tør dimethylformamid dråbevis under omrøring til den antibiotiske opløsning. Blandingen omrøres natten over (16 timer)Dissolve 2.8 g (4 mmol) of gentamicin C sulfate in 30 ml of water and add 15 ml of methanol. Then 0.56 ml (4 mmol) of triethylamine is added and stirred for 10 minutes. Then, a solution containing 4 mmol of N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryloxy) -succinimide in 20 ml of dry dimethylformamide is added dropwise with stirring to the antibiotic solution. The mixture is stirred overnight (16 hours)
,6 DK 156070B, 6 DK 156070B
ved omgivelsernes temperatur. Tyndlagschromatografi af reaktionsblandingen via TLC på silicagel under anvendelse af den nedre fase af et chloroform:methanol:ammoniumhydroxid-opløsningsmiddelsystem (1:1:1) viser tilstedeværelsen af mindre mængder af flere komponenter og én hovedkomponent. Reaktionsblandingen koncentreres i vakuum til en rest, og resten tritureres med methanol, hvilket givet 3,2 g hvidt fast stof indeholdende de tidligere ved chromatografi observerede komponenter.at ambient temperature. Thin layer chromatography of the reaction mixture via TLC on silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (1: 1: 1) shows the presence of smaller amounts of several components and one major component. The reaction mixture is concentrated in vacuo to a residue and the residue is triturated with methanol to give 3.2 g of white solid containing the components previously observed by chromatography.
150 mg af produktet chromatograferes på 50 g silicagel under anvendelse af den nedre fase af et chloroform:methanol:ammoniumhydro-xid-opløsningsmiddelsystem (2:1:1). De fraktioner, der indeholder hovedkomponenten,forenes og lyofiliseres, hvilket giver l-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl)gentamicin C-^. Udbytte 70 mg. NMR (D20): £1,15 (3H, s, C-CH3)} 2,49 (3H, s, NCH3); 4,10 (IH, dd, J = 8,0, 4,0 Hz, sidekæde H-2); 7,36 (5H, m, phenyl)« B. 1-N-(S-4-Amino-2-hydroxybutyryl)gentamicin Cla·150 mg of the product is chromatographed on 50 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (2: 1: 1). The fractions containing the major component are combined and lyophilized to give 1-N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin C Yield 70 mg. NMR (D 2 O): δ 1.15 (3H, s, C-CH 3)} 2.49 (3H, s, NCH 3); 4.10 (1H, dd, J = 8.0, 4.0 Hz, side chain H-2); 7.36 (5H, m, phenyl) B. B. 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin Cla ·
Produktet fra trin A opløses i en blanding af 12 ml methanol og 3 ml vand, der tilsættes 20 mg 10%'s palladium-på-kul og hydrogeneres ved 4 atmosfærer og stuetemperatur. Efter 3 timers forløb er reaktionen i det væsentlige fuldstændig. Katalysatoren fjernes ved filtrering, og filtratet lyofiliseres til opnåelse af 46 mg 1-N-(S-4-amino-2-hy-droxybutyryl)gentamicin . NMR (D20): £l,17 (3H, s, C-CH3); 2,48 (3H, s, NCH3); 4,22 (IH, dd, J = 9,5 Hz, 4,0 Hz, sidekæde CHOH)j 5,04 (2H, m, H-Tr og H-l"). Massespektraldata: (M-H20) m/e 532.The product of Step A is dissolved in a mixture of 12 ml of methanol and 3 ml of water, added to 20 mg of 10% palladium-on-charcoal and hydrogenated at 4 atmospheres and room temperature. After 3 hours, the reaction is essentially complete. The catalyst is removed by filtration and the filtrate lyophilized to give 46 mg of 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin. NMR (D 2 O): δ 1.17 (3H, s, C-CH 3); 2.48 (3H, s, NCH 3); 4.22 (1H, dd, J = 9.5 Hz, 4.0 Hz, side chain CHOH) j 5.04 (2H, m, H-Tr and H1 "). Mass spectral data: (M-H 2 O) m / e 532nd
Eksempel η 1-N-(S-4-Amino-2-hydroxybutyryl)gentamicin B.Example η 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin B.
A. 1-N-(S-4-Benzyloxycarbonylamino-2-hydroxybutyryl)gentamicin B.A. 1-N- (S-4-Benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin B.
3,39 g gentamicin B-sulfat opløses i 48,4 ml vand og fortyndes med 23,7 ml methanol. Der tilsættes 0,7 ml triethylamin dråbevis under omrøring. 1,67 g N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl-oxy)succinimid opløses i dimethylformamid, og opløsningen sættes dråbevis under omrøring til den antibiotiske opløsning. Den resulterende opløsning omrøres ved stuetemperatur i 18 timer, hvorpå den koncentreres til en rest i vakuum. Resten opløses i vand og behandles med fortyndet bariumhydroxidopløsning under omrøring, indtil pH når ca.3.39 g of gentamicin B sulfate are dissolved in 48.4 ml of water and diluted with 23.7 ml of methanol. 0.7 ml of triethylamine is added dropwise with stirring. 1.67 g of N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryl-oxy) succinimide are dissolved in dimethylformamide and the solution is added dropwise with stirring to the antibiotic solution. The resulting solution is stirred at room temperature for 18 hours, then concentrated to a residue in vacuo. The residue is dissolved in water and treated with dilute barium hydroxide solution with stirring until the pH reaches approx.
8,0. Det udfældede bariumsulfat fjernes ved filtrering under anvendelse af et filterhjælpestof. Bundfaldet vaskes med vand, filtratet og vaskevandet forenes og koncentreres til tørhed i vakuum. Resten chroma-8.0. The precipitated barium sulfate is removed by filtration using a filter aid. The precipitate is washed with water, the filtrate and the wash water are combined and concentrated to dryness in vacuo. Remaining chromatic
17 DK 156070 B17 DK 156070 B
tograferes på en søjle indeholdende 600 g silicagel under anvendelse af den nedre fase af et chloroform:methanol:ammoniumhydroxid-opløs-ningsmiddelsystem (1:1:1) som elueringsmiddel. Det materiale, der elue-res umiddelbart før gentamicin B, forenes, og de forenede fraktioner koncentreres til tørhed, hvorved man opnår 1-N-(S-4-benzyloxycarbo-nylamino-2-hydroxybutyryl)gentamicin B som et amorft fast stof. Udbytte: 0,2 g. NMR (D20): £1,27 (3H, s, C-CH3)? 2,51 (3H, S, NCH3)? 5,08 (IH, d, J = 4 Hz)? 5,25 (IH, d, J = 3,5 Hz).Charge onto a column containing 600 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (1: 1: 1) as the eluant. The material eluted immediately before gentamicin B is combined and the combined fractions are concentrated to dryness to give 1-N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin B as an amorphous solid. Yield: 0.2 g. NMR (D 2 O): δ 1.27 (3H, s, C-CH 3)? 2.51 (3H, S, NCH 3)? 5.08 (1H, d, J = 4 Hz)? 5.25 (1H, d, J = 3.5 Hz).
B. 1-N-(S-4-Amino-2-hydroxybutyryl)gentamicin B.B. 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin B.
Produktet fra trin A opløses i en blanding bestående af 20 ml vand og 8 ml methanol. Produktet hydrogeneres i nærværelse af €0 mg 5%'s palladium-på-kul ved 3,4 atmosfærer og stuetemperatur i 3 timer. Katalysatoren fjernes ved filtrering ved hjælp af et filterhjælpestof. Filter-kagen vaskes med vand, og filtratet og vaskevandet kombineres. Det kombinerede filtrat og vaskeva^id koncentreres til tørhed i vakuum. Resten chromatograferes på en silicagelsøjle indeholdende 10 g silicagel under anvendelse af en chloroform:methanol:ammoniumhydroxid-op-løsning (1:2:1) som elueringsmiddel. De fraktioner, der indeholder de mest polære komponenter, forenes, koncentreres og lyofiliseres til opnåelse af 1-N-(S-4-amino-2-hydroxybutyryl)gentamicin B. Udbytte 12 mg.The product of step A is dissolved in a mixture of 20 ml of water and 8 ml of methanol. The product is hydrogenated in the presence of € 0 mg of 5% palladium-on-charcoal at 3.4 atmospheres and room temperature for 3 hours. The catalyst is removed by filtration using a filter aid. The filter cake is washed with water and the filtrate and wash water are combined. The combined filtrate and washings are concentrated to dryness in vacuo. The residue is chromatographed on a silica gel column containing 10 g of silica gel using a chloroform: methanol: ammonium hydroxide solution (1: 2: 1) as eluent. The fractions containing the most polar components are combined, concentrated and lyophilized to give 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin B. Yield 12 mg.
Pmr D20 100 MHz: >5 1,14 c-methyl (S), 2,45 (S), N-methyl, 5,04 (d), H-1", Jr.f2"=4'° HZ; 5,30 (d)' H~1'' Jl'» 2’=3'5 Hz*Pmr D 2 O 100 MHz:> δ 1.14 c-methyl (S), 2.45 (S), N-methyl, 5.04 (d), H-1 ", Jr.f2" = 4 '° HZ; 5.30 (d) 'H ~ 1' 'Jl' »2 '= 3'5 Hz *
De ved analogifremgangsmåden ifølge opfindelsen fremstillede forbindelser er bredspektrede antibakterielle midler, der udviser aktivitet over for mange organismer, som er resistente over for 1-N-usubstituerede forstadier dertil. Forbindelserne fremstillet ifølge opfindelsen kan således anvendes alene eller i kombination med andre antibiotiske midler til undgåelse af vækst eller reduktion af antallet af bakterier i forskellige miljøer. Forbindelserne fremstillet ifølge opfindelsen er mere aktive over for mange organismer, som inaktiverer de tilsvarende antibiotika ved acetylering af 3-aminogruppen og/eller ved adenylering af 2"-hydroxylgruppen.The compounds prepared by the analogous process of the invention are broad-spectrum antibacterial agents that exhibit activity against many organisms resistant to 1-N unsubstituted precursors thereof. Thus, the compounds of the invention can be used alone or in combination with other antibiotic agents to avoid growth or reduce the number of bacteria in different environments. The compounds of the invention are more active against many organisms which inactivate the corresponding antibiotics by acetylation of the 3-amino group and / or by adenylation of the 2 "hydroxyl group.
De ved analogifremgangsmåden ifølge opfindelsen fremstillede forbindelser har således mulighed for at kunne anvendes som antibakterielle midler og kan anvendes til samme formål som deres (forælder) ikke-afledte antibiotika, f.eks. kan de anvendes som bakte-riostatiske rensemidler til hospitalglasvarer, operationsinstrumen-Thus, the compounds prepared by the analogous process of the invention have the potential to be used as antibacterial agents and can be used for the same purpose as their (parent) non-derived antibiotics, e.g. they can be used as bacteriostatic cleaning agents for hospital glassware, surgical instruments,
,8 DK 156070B, 8 DK 156070B
ter, badekar eller til rengøring af områder, hvor der huses laboratoriedyr, eller lignende.utensils, bathtubs or for cleaning areas where laboratory animals are housed, or the like.
Minimum inhiberende koncentration (MIC) in vitro.Minimum inhibitory concentration (MIC) in vitro.
Benævnelser på forbindelser i tabel I: a) kendte forbindelserNames of compounds in Table I: (a) known compounds
Forbindelse 1: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin C1a (US-patent 3,796,699)Compound 1: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin C1a (U.S. Patent 3,796,699)
Forbindelse 2: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin C1 (US-patent 3,780,018) b) Omhandlede forbindelserCompound 2: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin C1 (U.S. Patent 3,780,018) b)
Forbindelse 3: 1-N-(S-3-amino-2-hydroxypropionyl) gentamicin C1Compound 3: 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin C1
Forbindelse 4: 1-N-(S-3-amino-2-hydroxypropionyl) gentamicin BCompound 4: 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin B
Forbindelse 5: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin B1Compound 5: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin B1
Forbindelse 6: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin BCompound 6: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin B
Forbindelse 7: 1-N-(S-4-amino-2-hydroxybutyryl) antibiotiskCompound 7: 1-N- (S-4-amino-2-hydroxybutyryl) antibiotic
JI-20BJI-20B
Forbindelse 8: 1-N-(S-3-amino-2-hydroxypropionyl) sisomicinCompound 8: 1-N- (S-3-amino-2-hydroxypropionyl) sisomicin
Forbindelse 9: 1-N-(S-3-amino-2-hydroxypropionyl) verdamicinCompound 9: 1- N- (S-3-amino-2-hydroxypropionyl) verdamicin
Anvendt testmetode til opnåelse af resultaterne er angivet i tabel I.Test method used to obtain the results is given in Table I.
200 ml Mueller-Hinton-medium indstillet til pH 7,2 steriliseres. 5 ml af det sterile medium overføres til hver af de 120 sterile, 16x150 run testrør med bomuldspropper. Rørene anbringes i 10 grupper med 12 4 rør i hver. Til hvert rør sættes 10 celler af en af de bakteriestammer som skal testes. Til hver gruppe sættes en vandig opløsning af det antibiotikum scm skal testes under dannelse af efterfølgende slutkoncentration pr. rør: 50 mcg/ml, 25 mcg/ml, 10 mcg/ml, 5 mcg/ml, 2 mcg/ml, 1 mcg/ml, 0,5 mcg/ml, 0,2 mcg/ml, 0,1 mcg/ml, 0,05 mcg/ml, 0,01 mcg/ml, 0,005 mcg/ml og 0,0 mcg/ml (kontrol). Rørene inkuberes i 24 timer ved 37°C. For hver gruppe af rør bestemmes visuelt den laveste koncentration af antibiotikum, som inhiberer bakterievækst og den højeste koncentration af antibiotikum, som tillader bakterievækst.200 ml of Mueller-Hinton medium adjusted to pH 7.2 is sterilized. Transfer 5 ml of the sterile medium to each of the 120 sterile, 16x150 run cotton plug test tubes. The tubes are placed in 10 groups with 12 4 tubes in each. To each tube are added 10 cells of one of the bacterial strains to be tested. To each group is added an aqueous solution of the antibiotic scm to be tested to produce subsequent final concentration per day. tubes: 50 mcg / ml, 25 mcg / ml, 10 mcg / ml, 5 mcg / ml, 2 mcg / ml, 1 mcg / ml, 0.5 mcg / ml, 0.2 mcg / ml, 0.1 mcg / ml, 0.05 mcg / ml, 0.01 mcg / ml, 0.005 mcg / ml and 0.0 mcg / ml (control). The tubes are incubated for 24 hours at 37 ° C. For each group of tubes, visually the lowest concentration of antibiotic which inhibits bacterial growth and the highest concentration of antibiotic permitting bacterial growth is determined.
Den minimale inhiberende koncentration til test af antibiotika overfor hver af de testede bakterier bestemmes ved at beregne middelværdien for de to fundne værdier.The minimum inhibitory concentration for antibiotic testing against each of the bacteria tested is determined by calculating the mean of the two values found.
19 DK 156070 B19 DK 156070 B
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21 DK 156070B21 DK 156070B
I almindelighed afhænger den indgivne dosis af forbindelserne fremstillet ifølge opfindelsen, af alderen og vægten af de dyrearter, der behandles, indgiftsmåden og af typen og styrken af den bakterieinfektion, der skal undgås eller reduceres. I almindelighed vil dosis af de derivater af 4,6-di-(aminoglycosyl)- 1,3-diaminocyclitolerne, der anvendes til bekæmpelse af en given bakterieinfektion, svare til dosiskravene for de tilsvarende 1-N-usubstituerede 4,6-di-(aminoglycosyl)-1,3-di-aminocyclitoler.In general, the dose administered of the compounds of the invention depends on the age and weight of the species being treated, the mode of administration, and the type and severity of the bacterial infection to be avoided or reduced. In general, the dose of the derivatives of the 4,6-di- (aminoglycosyl) - 1,3-diaminocyclitols used to control a given bacterial infection will correspond to the dose requirements for the corresponding 1-N unsubstituted 4,6-di- (aminoglycosyl) -1,3-di-aminocyclitols.
Forbindelserne fremstillet ifølge opfindelsen kan indgives oralt. De kan også påføres topisk i form af salver, såvel hydrofile som hydrofobe, i form af lotioner, der kan være vandige, ikke-vandige eller af emuisionstypen, eller i form af cremer. Farmaceutiske bærere, der er nyttige ved fremstillingen af sådanne formuleringer, omfatter f.eks. stoffer, såsom vand, olier, fedtstoffer, polyestere, polyoler og lignende.The compounds of the invention can be administered orally. They can also be applied topically in the form of ointments, both hydrophilic and hydrophobic, in the form of lotions which may be aqueous, non-aqueous or emuction type, or in the form of creams. Pharmaceutical carriers useful in the preparation of such formulations include, e.g. substances such as water, oils, fats, polyesters, polyols and the like.
Til oral indgift kan forbindelserne fremstillet ifølge opfindelsen formuleres i form af tabletter, kapsler, eliksirer eller lignende, eller de kan blandes med dyrefoder. Det er i disse doseringsformer, at de antibakterielle stoffer er mest effektive til behandling af bakterieinfektioner i mave-tarmkanalen, hvilken infektioner forårsager diarrhoea.For oral administration, the compounds of the invention may be formulated in the form of tablets, capsules, elixirs or the like, or may be mixed with animal feed. It is in these dosage forms that the antibacterial agents are most effective in treating bacterial infections of the gastrointestinal tract which infections cause diarrhea.
I almindelighed indeholder de topiske præparater fra ca. 0,1 til ca. 3,0 g af forbindelserne fremstillet ifølge opfindelsen pr. 100 g salve, creme eller lotion. De topiske præparater påføres sædvanligvis forsigtigt på læsioner ca. 2 til ca. 5 gange om dagen.Generally, the topical compositions contain from ca. 0.1 to approx. 3.0 g of the compounds of the invention per 100 g ointment, cream or lotion. The topical compositions are usually applied gently to lesions of approx. 2 to approx. 5 times a day.
De antibakterielle midler fremstillet ifølge opfindelsen kan anvendes på væskeform, såsom opløsninger, suspensioner og lignende, til brug i ører og øjne og kan også administreres parenteralt ved intramuskulær injektion. Den injicerbare opløsning eller suspension vil sædvanligvis blive indgivet i en mængde på fra ca. 1 mg til ca. 15 mg antibakterielt middel pr. kg legemsvægt pr. dag opdelt i ca. 2 til ca. 4 doser. Den præcise dosis afhænger af stadiet og styrken af infektionen, den inficerede organismes følsomhed over for det antibakterielle middel og de individuelle egenskaber hos de dyrearter, der behandles.The antibacterial agents of the invention can be used in liquid form, such as solutions, suspensions and the like, for use in the ears and eyes and can also be administered parenterally by intramuscular injection. The injectable solution or suspension will usually be administered in an amount of from ca. 1 mg to approx. 15 mg antibacterial agent per kg body weight per day divided into approx. 2 to approx. 4 doses. The exact dose depends on the stage and severity of the infection, the sensitivity of the infected organism to the antibacterial agent and the individual characteristics of the species of animal being treated.
Claims (2)
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
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US45257174A | 1974-03-19 | 1974-03-19 | |
US45258674 | 1974-03-19 | ||
US05/452,586 US4029882A (en) | 1974-03-19 | 1974-03-19 | Selective acylation of the C-1 amino group of aminoglycoside antibiotics |
US45257174 | 1974-03-19 | ||
CH1060174 | 1974-07-31 | ||
CH1060174 | 1974-07-31 |
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AT (1) | AT341088B (en) |
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DK (1) | DK156070C (en) |
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HU (1) | HU175673B (en) |
IT (1) | IT1049348B (en) |
LU (1) | LU71687A1 (en) |
NL (1) | NL183826C (en) |
NO (1) | NO140426C (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2390255B1 (en) * | 2006-04-03 | 2016-07-13 | Technion Research & Development Foundation Ltd. | Novel aminoglycosides and uses thereof in the treatment of genetic disorders |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3780018A (en) * | 1972-06-26 | 1973-12-18 | Bristol Myers Co | Derivatives of gentamicin c1 |
US3796698A (en) * | 1972-10-18 | 1974-03-12 | Bristol Myers Co | Antibiotic derivatives of gentamicin c2 |
US3796699A (en) * | 1972-11-06 | 1974-03-12 | Bristol Myers Co | Antibiotic derivatives of gentamicin c1a |
-
1975
- 1975-01-21 ES ES433990A patent/ES433990A1/en not_active Expired
- 1975-01-21 IT IT19435/75A patent/IT1049348B/en active
- 1975-01-21 FI FI750141A patent/FI60714C/en not_active IP Right Cessation
- 1975-01-21 AT AT42975A patent/AT341088B/en not_active IP Right Cessation
- 1975-01-21 LU LU71687*A patent/LU71687A1/xx unknown
- 1975-01-21 NO NO750168A patent/NO140426C/en unknown
- 1975-01-21 NL NLAANVRAGE7500690,A patent/NL183826C/en not_active IP Right Cessation
- 1975-01-22 JP JP50009654A patent/JPS5843398B2/en not_active Expired
- 1975-01-22 DK DK018675A patent/DK156070C/en not_active IP Right Cessation
- 1975-01-23 HU HU75SCHE507A patent/HU175673B/en unknown
- 1975-01-23 DD DD183784A patent/DD117871A5/xx unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3780018A (en) * | 1972-06-26 | 1973-12-18 | Bristol Myers Co | Derivatives of gentamicin c1 |
US3796698A (en) * | 1972-10-18 | 1974-03-12 | Bristol Myers Co | Antibiotic derivatives of gentamicin c2 |
US3796699A (en) * | 1972-11-06 | 1974-03-12 | Bristol Myers Co | Antibiotic derivatives of gentamicin c1a |
Also Published As
Publication number | Publication date |
---|---|
DD117871A5 (en) | 1976-02-05 |
NL183826B (en) | 1988-09-01 |
AT341088B (en) | 1978-01-25 |
JPS5843398B2 (en) | 1983-09-27 |
JPS50126640A (en) | 1975-10-04 |
FI60714B (en) | 1981-11-30 |
IT1049348B (en) | 1981-01-20 |
NL183826C (en) | 1989-02-01 |
NO750168L (en) | 1975-09-22 |
ATA42975A (en) | 1977-05-15 |
DK18675A (en) | 1975-09-20 |
LU71687A1 (en) | 1975-12-09 |
FI750141A (en) | 1975-09-20 |
FI60714C (en) | 1982-03-10 |
NL7500690A (en) | 1975-09-23 |
NO140426B (en) | 1979-05-21 |
NO140426C (en) | 1979-08-29 |
HU175673B (en) | 1980-09-28 |
ES433990A1 (en) | 1977-03-01 |
DK156070C (en) | 1989-11-06 |
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Legal Events
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PUP | Patent expired |