CA1083175A - .alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESES - Google Patents
.alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESESInfo
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- CA1083175A CA1083175A CA332,525A CA332525A CA1083175A CA 1083175 A CA1083175 A CA 1083175A CA 332525 A CA332525 A CA 332525A CA 1083175 A CA1083175 A CA 1083175A
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- Prior art keywords
- amino
- group
- negamycin
- lysine
- methylnegamycin
- Prior art date
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
.delta.-SUBSTITUTED NEGAMYCIN DERIVATIVES
AND SYNTHESES
ABSTRACT
Compounds having the structure were found to be antibacterial agents and were synthesized by a process which comprises a) reacting diazomethane in the presence of boran trifluoride etherate with a conventional, easily removable ester of amino-protected negamycin to form the intermediate having the formula
AND SYNTHESES
ABSTRACT
Compounds having the structure were found to be antibacterial agents and were synthesized by a process which comprises a) reacting diazomethane in the presence of boran trifluoride etherate with a conventional, easily removable ester of amino-protected negamycin to form the intermediate having the formula
Description
3L~383~75 BACKGROUND OF THE INVENTION
_ _ 1. Field of the Invention The novel synthetic organic compounds of the present invention are antibacterial agents and are used as are ot~ler members of that class.
_ _ 1. Field of the Invention The novel synthetic organic compounds of the present invention are antibacterial agents and are used as are ot~ler members of that class.
2. Description of the Prior Art Negamycin is an antibiotic discovered by the present inventors (Japan 34827/1969, Japan 28835/1971) and ls represented by the following ~ormula (III):
.
(R) (R) (III) OH NH2 C~
having a unique structure consisting of a hydrazide bond between (R,R)-~-hydroxy-~-lysine (IV) and l-methylhydra-zinoacetic acid (V) (R) (R) H2NCH2fHCH2CIHCH2CE (IV) ~ ;
H2N7CH2COOH (V) (J. Am. Chem. Soc. 93, 6305, 1971). In the above formulae, a symbol (R) is indicated and used for stereo-chemistry (Ref. "Nomenclature of Organic Compounds" ed.
by J. H. Fletcher, O. C. Dermer and R. B. Fox, p. 103, American Chemical Society, Washington, D.C., 1974).
See also U.S. Patents 3,619,742 and 3,743,580 and M.
Hamada et al , A New Antibiotic, Negamycin, J. Antibiotics~
23(3), 170-71 (1970).
~83~ 5 SUMMARY OF TXE_INVENTION
The present invention provides new derivatives of negam~cin, that is, semisynthetic antlbiotics having the ability to inhibit Gram-positive and Gram-negative bacteria and belng o~ qulte stable character even in acidic aqueoUs solution~, which are represented by the ~ollowing ~ormula:
(R) (R) ~ :
H2NcH2fHcH2cHc~2coNHNc~2cooH (II) D NH2 C~
and the acid and base salts thereo~, wherein D is selected ~rom the group consisting o~ H (deoxynegamycin) and C~ O-(O-methylnegamycin).
The subject of this divisional application is a process for the production of O-methylnegamycin having the formula (R) (R) H2NcH2cHcH2fHcH2coNHNl cH2cooH ~`' . , OCH3 NH2 CH3 ~ .
which comprises the consecutive steps of a) reacting diazomethane in the presence of boron trifluoride etherate with a conventional, easily ;~ - .
removable ester of amino-protected negamycin to form the ;~
intermediate having the formula A ~ (R) (R) :~ -~NCH2IHCH2fHCH2COnHNCH2COOCH3 ' ,~
OCH3 N' CH : .
\ B 3 - : .~
wherein A is hydrogen and B is a monovalent amino-protective ~ ; ;
group or A together with B are a divalent amino-protective group and .
.... .
.. . . .
, ~831~
b) removing from said intermediate the amino-prOt~ctive groups A and s and the methyl ester group to form O-methylnegamycin.
Preferably in ~his process A i~ hydrogen and B is a benzyloxycarbonyl group.
Also preferably in such a process the amino-protective groups A and B and the methyl ester group are removed by hydrolysis followed by column chromatography with cation exchangers having carboxylic ~cid as the active group.
In one embodim~nt the invention provides such a process :
further comprising the step of forming a pharmaceutically acceptable nontoxi~ salt or acid addition salt of O-methylnegamycin. :-Salts o~ deoxynegamycln and O-methylnegamycin lnclude carboxylic acld salt~ lnclud1ng nontoxic metallic salts such as sodl~m, potassium, calcium and aluminum, the . ~ :
ammonium salt and substituted ammonium salts, e.g. salts o~ such nontoxic amines as trialkylamines, including tri~
ethylamine~ proc~ine, dibenzylamine, N-benzyl-~-phenethyl- `.
amine, l-ephenamine, N,N'-dibenzylethylenediamine, dehydro-ab~etylamine, N,N'-bis-dehydroabietylethylenedi~mine, ;~
N-(lower)alkylpiperldine, e.g. N-ethylplperidine and .
other amines whlch have been used to form salts with ~
benzylpenlcillin; and the nontoxic, acid additlon ~alts .
thereof, (l.e. the amine salt~) including the mineral acid addition 8alts such as the hydrochloride, hydrobro-mide, hydrolodide, sulfate, ~ul~amate and phosphate and ~
the organlc acld addition salts such as the maleate, : ~ :
acetate, citrate, oxalate, succinate, ben~oate, tartrate~
fumarate, malate, m~ndelate, ascorbate and the like.
Also ~ncluded are t~ 8al~8 Or deo~ynegamgcln and ~ ~
.~ ~ ...
.
(R) (R) (III) OH NH2 C~
having a unique structure consisting of a hydrazide bond between (R,R)-~-hydroxy-~-lysine (IV) and l-methylhydra-zinoacetic acid (V) (R) (R) H2NCH2fHCH2CIHCH2CE (IV) ~ ;
H2N7CH2COOH (V) (J. Am. Chem. Soc. 93, 6305, 1971). In the above formulae, a symbol (R) is indicated and used for stereo-chemistry (Ref. "Nomenclature of Organic Compounds" ed.
by J. H. Fletcher, O. C. Dermer and R. B. Fox, p. 103, American Chemical Society, Washington, D.C., 1974).
See also U.S. Patents 3,619,742 and 3,743,580 and M.
Hamada et al , A New Antibiotic, Negamycin, J. Antibiotics~
23(3), 170-71 (1970).
~83~ 5 SUMMARY OF TXE_INVENTION
The present invention provides new derivatives of negam~cin, that is, semisynthetic antlbiotics having the ability to inhibit Gram-positive and Gram-negative bacteria and belng o~ qulte stable character even in acidic aqueoUs solution~, which are represented by the ~ollowing ~ormula:
(R) (R) ~ :
H2NcH2fHcH2cHc~2coNHNc~2cooH (II) D NH2 C~
and the acid and base salts thereo~, wherein D is selected ~rom the group consisting o~ H (deoxynegamycin) and C~ O-(O-methylnegamycin).
The subject of this divisional application is a process for the production of O-methylnegamycin having the formula (R) (R) H2NcH2cHcH2fHcH2coNHNl cH2cooH ~`' . , OCH3 NH2 CH3 ~ .
which comprises the consecutive steps of a) reacting diazomethane in the presence of boron trifluoride etherate with a conventional, easily ;~ - .
removable ester of amino-protected negamycin to form the ;~
intermediate having the formula A ~ (R) (R) :~ -~NCH2IHCH2fHCH2COnHNCH2COOCH3 ' ,~
OCH3 N' CH : .
\ B 3 - : .~
wherein A is hydrogen and B is a monovalent amino-protective ~ ; ;
group or A together with B are a divalent amino-protective group and .
.... .
.. . . .
, ~831~
b) removing from said intermediate the amino-prOt~ctive groups A and s and the methyl ester group to form O-methylnegamycin.
Preferably in ~his process A i~ hydrogen and B is a benzyloxycarbonyl group.
Also preferably in such a process the amino-protective groups A and B and the methyl ester group are removed by hydrolysis followed by column chromatography with cation exchangers having carboxylic ~cid as the active group.
In one embodim~nt the invention provides such a process :
further comprising the step of forming a pharmaceutically acceptable nontoxi~ salt or acid addition salt of O-methylnegamycin. :-Salts o~ deoxynegamycln and O-methylnegamycin lnclude carboxylic acld salt~ lnclud1ng nontoxic metallic salts such as sodl~m, potassium, calcium and aluminum, the . ~ :
ammonium salt and substituted ammonium salts, e.g. salts o~ such nontoxic amines as trialkylamines, including tri~
ethylamine~ proc~ine, dibenzylamine, N-benzyl-~-phenethyl- `.
amine, l-ephenamine, N,N'-dibenzylethylenediamine, dehydro-ab~etylamine, N,N'-bis-dehydroabietylethylenedi~mine, ;~
N-(lower)alkylpiperldine, e.g. N-ethylplperidine and .
other amines whlch have been used to form salts with ~
benzylpenlcillin; and the nontoxic, acid additlon ~alts .
thereof, (l.e. the amine salt~) including the mineral acid addition 8alts such as the hydrochloride, hydrobro-mide, hydrolodide, sulfate, ~ul~amate and phosphate and ~
the organlc acld addition salts such as the maleate, : ~ :
acetate, citrate, oxalate, succinate, ben~oate, tartrate~
fumarate, malate, m~ndelate, ascorbate and the like.
Also ~ncluded are t~ 8al~8 Or deo~ynegamgcln and ~ ~
.~ ~ ...
-3~
.-.: . . . ,, :.
, .. . . . . .
33:~75 0-methylnegamycin with cinnamic acid, picric acid, p hydroxyazobenzene-p'-sulfonic acid, phytic acid~
livopimaric-6,6a-cis-endosuccinic acid, sulfamic acid and glycolic acid. For therapeutic purposes use is made o~ salts o~ nontoxic acids but salts of toxic acids, e.g. p-hydroxyazobenzene-p'-sulfonic acid, are useful in isolation procedures, e.g. as precipitants from aqueous solut~ons and for disinfectant purposes where toxicity is not important.
In the treatment o~ bacterial infections in animals, including man, the compounds of this invention are administered parenterally in accordance with con-ventional procedures for antibiotic administration in ~:
an amount of from about 5 to 200 mg./kg./day and pre-ferably about 5 to 20 mg./kg./day in divided dosage, e.g. three to four times a da~. They are administered in dosage units containing, for example, 125 or 250 or 500 mg. of acti-~e ingredient with suitable physiologically acceptable carriers or excipients. The dosage unit-s are in the form of liquid preparations such as solutions or suspensions.
The compounds of the present invention are also used topically in the treatment of superficial lnfections such as infections o~ skin or mucous membrane. For this purpose use can be made of conventional ointments, (e.g.
1~ or 5~) or solutions and suspensions in aqueous media at concentrations of 1~ to 10~.
The present invention provides the synthesis of ~-substituted negamycins which are much more stable in aqueous solution than negamycin itself and have antibac-.4.
, 33~75 terial activities. Thus, a constituent of negamycin,(R,R)-~-hydroxy-~-lysine (rV), is converted to D-~-lysine by removing the hydroxyl group at ~-position or to (R,R~-~-methoxy-~-lysine by replacing the hydroxyl group with a methoxyl group at ~ position, and two amino groups in those derlvatives are protected, the acid coupled with -l-methylhydrazinoacetic acid and the protective groups ~or the amino groups are then removed to yield the desired ~-subqtituted negamycin derivatives, deoxynegamycin or O-methylnegamycin as shown as formula (II) which are found to be quite stable in aqueous solutlon and active against Gram-positive and Gram-negative bacteria. ;
In one aspect of the present invention N~N'-di-protected-D-~-lysine (D - H in formula I) and N,N'-di-protected-(R,R)-~-methoxy-~-lysine (D ~ -OCH~ in ~:
formula I), which are represented by the following formula:
D M ~
. B
wherein A is hydrogen and B is a monovalent protective group for an amino group or A and B are one divalent protective group for an amino group and D is hydrogen or methoxyl, are separately coupled with 1-methylhydrazino- ;
acetic acid and the protective groups removed to prepare deoxynega~ycin (D = H in formula II) or O-methylnegamycin (D = -OCH3 ~n formula II).
1~l33~5 ... ..
(R) (R) H2NcH2cHcH2lHcH2coNHNcH2cooH (II) Starting materials (I'), D-~-lysine (D = H) and (R,R)-~-methoxy-~-lysine (D - -OCH~
(R) (R) ~2NC~2lHCH2lHCH2COOH (I') ~ -should be protected with known protective groups on the1r two amino groups. Although use can be made of the usual amino-protective groups used for peptide synthesis as the known protective groups, the protective groups used should ~;~
be easily removed without any cleavage of hydrazide bond in a compound which is synthesized from a ~-lysine deriva-tive (I) and 1-methylhydrazinoacetic acid by this procedure.
Suitable monovalent protective groups for amino groups in the ~-lysine derivative are alkyloxycarbonyl groups, e.g. tert-butyloxycarbonyl group; cycloalkyloxy~
carbonyl groups, e.g. cyclohexyloxycarbonyl group; aryl~
alkyloxycarbonyl groups, e.g. benzyloxycarbonyl and p-methoxylbenzyloxycarbonyl groups; and acyl groups especially lower alkanoyl groups, e.g. trifluoroacetyl and o-nitrophenoxyacetyl groups. A divalent protective ~ `;
group is a Schiff base such as salicylidene group. Those protective groups are introduced by known procedure, e.g.
using acid halide, acid azide, active ester, etc. ~ ;
In the prepara-tion of deoxynegamycin the D~
lysine used as the starting material is made, for example by the treatment of (R,R)-~-hydroxy-~-lysine with red phosphorus and hydroiodic acid in a sealed tube at 150 C.
., . , .. .. ,. , .. .. . . : . -~11 83~S
for 2 hours. The (R,R)-~-hydroxy-~-lysine is obtained by hydrolysis of negamycin or by synthesis from D-galacturonic acid (J. Am. Chem. Soc., 94, 4353, 1972). Amino-protective g~oups are introduced into D-~-lysine as described above. -For instance, by the treatment of D-~-lysine with benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in the presence of triethylamine in water-dioxane solution, di-N-benzyloxy-carbonyl-D-~-lysine is obtained at excellent yield.
In the preparation of O-methylnegamycin the (R,R)-~-methoxy-~-lysine used as the starting material is also synthesized from (R,R)-~-hydroxy-~-lysine and con-verted directly to the di-N-benzyloxycarbonyl derivative of (R,R)-~-methoxy-~-lysine. For instance, the aqueous solution of (R,R)-~-hydroxy-~-lysine is treated with benzyl-oxycarbonyl chloride in the presence of sodium bicarbonate yielding a lactone of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine as crystals. The lactone is dissolved in 0.5%-hydrogen chloride in methanol, allowed to stand at room te~perature and the reaction mixture is concentrated to obtain the methyl ester of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine as crystals The ester is reacted with diazomethane in the presence of boron trifluoride etherate in dichloromethane followed by hydrolysis with sodium hydroxide in ethanol to yield di-N-benzyloxycar-bonyl-(R,R)-~-methoxy-~-lysine.
In the coupling process the condensation ~orming a hydrazide bond with the N-protected ~-lysine derivative (I) and l-methylhydrazinoacetic acid is carried out by known methods for amide synthesis such as methods using dicyclohexylcarbodiimide, mixed anhydride, azide, acid halide, active ester, etc.
1(~83~5 For instance, the di-N benzyloxycarbonyl deriva-tlve o~ the ~-substituted (R,R)-~-hydroxy-~-lyslne i8 converted to an active ester by treatment with N-hydroxy-succinlmlde and dicyclohexylcarbodiimide in a nonaqueous solvent. The active ester is condensed with 1-1.5 moles of 1 methylhydrazinoacetic acid in the presence of 1-2.5 moles of sodium bicarbonate in an aqueous solvent. The coupling product thus obtained is treated with 25~ hydro~
gen bromide in acetic acid or hydrogenated with platinum, palladium, etc. as the catalyst to remove the N-benzyloxy-carbonyl groups and to synthesize the desired products (II), that is ~-substituted negamycins.
The removal of the protective groups from the coupling product is carried out by a usual method as described above. When the protective groups are alkyloxy-carbonyl group or Schif~ base the coupling productæ are hydrolyzed with a weak acid to remove the amino-protective groups.
An arylalkyloxycarbonyl group as the protective group is removed by the treatment with 25~ hydrogen bro~
mide in acetic acid, and o-nitrophenoxyacetyl group is easily removed by catalytic hydrogenation with platinum, palladium, etc.
The products are purified with good recovery by ion exchange chromatography using carboxylic cation exchange resins such as "Amberlite CG-50" (Rohm and Haas Co. Ltd., U.S.A.) or "CM-Sephadex C 25" (Pharmacia, Sweden).
0-Methylnegamycin and deoxynegamycin are also able to be derived directly from an amino-protected derivative of negamycin itself as shown in the following *Trade Marks _8 ~(~83~L~75 ':
formula:
A~ (R) (R) CH2fHCH2CHCH2CONHTCH2COOH (VI) B
wherein A is H and B is a monovalent amino-protective group or A together with B is one dlvalent amino-protective group. As the amino-protective groups for negamycin, all known amino-protective groups used for D-~-lysine or (R,R)-~-methoxy-~-lysine above are available in the same procedure For instance, negamycin is treated with benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in the presence of triethylamine in a mixture of water-dioxane to obtain di-N-benzyloxycarbonylnegamycin (A = H, B - COOCH2C6H5 in ~ormula VI) in high ~ield. m e benzyloxycarbonyl groups in this derivative are removed by catalytic hydrogenation simultaneously removing halogen atom to prepare deoxynega-mycin as described later, and therefore this group is a useful amino-protective group.
O-Methylnegamycin is synthesized from an amino~
protected derivative of negamycin (VI) by the same reaction `
conditions used for O-methylation of amino-protected ~ -(R,R)-~-oxy-~-lysine, that is, treatment with diazomethane in the presence of boron trifluoride etherate in an inert organic solvent such as dlchloromethane to obtain an amino-protected derivative of O-methylnegamycin methyl ester as shown by the following formula:
A (R) NCH CHCH CHCH CONHNCH COOCH (VII ) B ~ 21 21 2 1 2 ~B CH~;
wherein A and B mean the same groups described above. The 1'7S
amino-protective group and methyl ester group are removed by hydrolysis followed by col D chromatography wlth cation exchangers having carboxylic acid as the active group, and if necessary are purified by a column chromatography on silica gel obtaining O-methylnegamycin as the final product.
The present invention also provides -the process for the preparation of O-methy~negamycin which comprises conversion of amino-protected derivatives of negamycin (VI) ~NCH2CHCH2CHCH2CONHNCH2COOH (~
OH N~ C~
to amino-protected derivative of O-methylnegamycin methyl ester (VII) by reaction with diazomethane in the presence of boron trifluoride etherate followed by removal of the amino-protected groups and methyl ester group from the product (VII).
A~
NCH2CHCH2CHCH2CONHNCH2C~OCH~
OCH3 N~ CH3 ~.
In this procedure the reaction with diazomethane is carried out in an inert organic solvent7for instance, ; ;
halogenated hydrocarbons such as dichloromethane. The removal of the amino-protective groups and methyl ester group is carried out by ordinary procedures, for instance~
by hydrolysis in alkaline condition.
Furthermore, in the synthesis of deoxynegamycln from the amino-protected derivative of negamycin (VI), the only free carboxyl group of amino-protected negamycin i~ `
primarily protected with a common ester form, for instance~
33~L7~i ~
methyl , ethyl or benzyl ester to obtain an ester of amino-protected negamycin. And then the hydroxyl group at the ~-position of the ester i8 sulfonylated by treat-ment with alkylsulfonylating agent such as mesyl chloride, arylsulfonating agent such as p-toluenesulfonyl chloride, or arylmethylsulfonylating agent such as benzylsulfonyl chloride to obtain the sulfonic acid ester derivative as shown by ~III.
A~ (R) (R) ~NcH2cHcH2THcH2coNHlcH2coE (VIII) S03G ~ CH3 wherein A and B are the same as described in the above;
E is an alkyloxy group especially consisting o~ 1-4 carbon atoms or a benzyloxy group which are ester forming radi-cals removable easily by hydrolysis to hydroxyl group; G
is an alkyl group and especially a lower alkyl group, phenyl group or alkyl substituted phengl or benzyl group.
The product (VIII) is treated with an iodide or bromide of alkaii metal such as sodium iodide or sodium bromide and the sulfonyl ester at the ~-position is replaced with iodine or bromine to obtain the ~-iodo- or ~-bromo-deriva-tive which is a mixture o~ R and S at the ~-position. The iodo- or bromo-derivative in water or methanol or mixed solvent of water and methanol is hydrogenated with pal-ladium or platinum as the catalyst to produce deoxynega-mycin derivative by dehalogenation. When the amin ~
protected group is the benzyloxycarboxyl group as described above the removal of the amino-protected group takes place simultaneously in the above-mentioned catalytic hydrogena-tion. The remaining amino-protected group is removed by the procedure described above. The ester group of the 1~83~7S
product is removed by hydrolysis under weakly alkaline conditions and the reaction mixture is purified by column chromatography on cation exchange resin having carboxylic acid as the active group to obtain deoxy-negamycin as the final product.
There is further provided by the present inven-tion the process for the preparation of deoxynegamycin whereby an amino-protected derivative of ~-halogenated negamycin ester (IX) is catalytically hydrogenated and the remaining amino-protective group in the compound is removed by a conventional method and the ester ~orming group (E) is converted to hydroxyl by hydrolysis.
A~ (R) NcH27HcH2THcH2coNH I CH2COE
X N CH
~ B 3 (wherein A is hydrogen and B is a monovalent amino-protective group or A together with B is one divalent amino-protective group, E is an ester forming group which is converted to hydroxyl by hydrolysis, and X is iodine or bromine atom.) ~-The this procedure an amino-protected deriva~
tive of ~-halogenated negamycin ester (IX) is prepared as follows. An amino-protected derivative of negamycin (VI) prepared as above is treated with an agent for -esterification such as diazomethane or diazoethane to convert it to negamycin ester derivative (carboxylate).
Next the ~-hydroxyl group of the derivative is sulfonylated (-S03G) and then halogenated. For instance, the sulfonyla-tion is carried out according to a similar procedure used to prepare an intermediate in the production of ~',4'--- 1~ _ ~8317~
dideoxykanamycin B as described in Japan Patent 7595/1975.
The reaction to convert the sulfonyl group (-S03G) to halogen group (X) by the effect of an iodide or bromide of an alkall metal and the procedure to remove the ~-halogen group by catalytic hydrogenation is also carried out in known manner.
The properties o~ deoxynegamycin and O-methyl-negamycin which are the final products obtained in this invention are as follows:
Deoxynegamycin is a white powder showing d.p.
120-125 C. [a]23 = 5o (c 1.5, H20) and elemental analysis to coincide with the theoretical value for CgH20N~03-H20 (C 43.18~, H 8.86%, N 22.39%). On silica gel thin lajer chromatography (Art. 5721, Merck Co., Germany) de~eloped with n-butanol-ethanol-chloroform-17%
aqueous amm~nia (4:5:2:5 by volume) it gives a single spot (ninhy_rin) at Rf 0.~4.
O-Methylnegamycin is a white powder showing d.p. 137-140 C. ~a]22 = -3 (c 1.5, H~O), and elemental ~nalysis to coincide with the theoretical value for CloH22N4C4 H20 (C 42.84~, H 8.63~, N 1~.99~). On the tlc described above it gives a single spot at Rf 0.33. As shown in the table, those two novel derivatives of nega mycin inhibited the growth of Gram-positive and Gram-negative bacteria. Those two compounds were completely stable in both aqueous or 0.02N HCl aqueous solution at 37 C. for one month while negamycin as the control was reduced in its activity to 63~ in aqueous solution and to 50~ in 0.02N HCl aqueous solution. Those two compounds are low in to~icity (LD50 of both in mice, i.v., >200 mg./kg.) and are expected to be used in the chemotherapy ~aQ831~S
of infections caused by various Gram-positive and Gram-negative bacteria.
TABLE: Antimicrobial Spectra of O-Methylnegamycin and Deoxynegamycin Min~mum Inhibi~ory Concentrations*(Mcg./ml.) O-Methyl-Deoxy-Test Organisms _ ~Y~ æ~ YD5 StaPhylococcus aureus FDA209P 12.5 25 Staphylococcus aureus Smith 6. 256.25 Sarcina lutea PCI1001 25 ~100 ;
Micrococcus ~lavus FDA16 50 50 Bacillus subtilis NRRL B-5s8 50 100 Mycobacterium smegmatis ATCC607 25 50 Escherichia coli NIHJ 6 256. 25 . , ~
Escherichia coli K-12 3-13 3.13 Escherichia coli K-12 ML1629 1 56 3013 .
Salmonella t~phi T-63 1 - 56 o . 78 Proteus ulgaris OX-l9 1- 56 3.13 Proteus rett~eri GN311 6.25 6.25 Proteus rett~eri GN466 3.13 3.13 Serratia marcescens 25 25 Klebsiella pneumoniae PCI602 6.25 6.25 Pseudomonas :~luore~cens 1- 56 3-13 Pseudomonas aeruginosa A3 12.5 25 ~i Pseudomonas aeruginosa No. 12 25 50 . .
.. _ ._ *Minimum inhibitory concentrations were determined on a 0.5% peptone agar by incubatlon at 37 C. for 17 hours.
- 1~
10831~5 DESCRIPTION _F THE PREFE _ED EMBODIMENTS
(a) Synthesis of Di-N-benzyloxycarbon~vl-D-~-lysine-.
To ~ solution containing 100 mg. (o.6g mmolej D-~-lysine ([~]25 = -22.5 (c o.8, lN HC1) in 1 ml. of water, 104 mg. (1.0 mmole) of triethylamine at first and then a solution containing 410 mg. (1.5 mmoles) benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate (Kokusan Chemical Works~ Tokyo) in 1 ml. o~ dioxane were added and stirred at 30 C. for 17 hours. To the reaction mixture 3 ml. of water was added and the mix-ture was washed with 6 ml. of ether. The aqueous layer was adjusted to pH 2 with 6N HCl, saturated with sodium chloride and extracted with 13 ml. of ethyl acetate. The ethyl acetate layer was washed with 4 ml. o~ 5% HCl in water saturated with sodlum chloride and with 4 ml. of water saturated with sodium chloride, dried with anhydrous -sodium sulfate and concentrated to dryness under reduced pressure to obtain 219 mg. of di-N-benzyloxycarbonyl-D-~-lysine as colorless crystals, d.p. 149-151 C`., [a]D6 =
l6 (c 1.3, MeOH), 73~ yield.
(bj Synthesis of Deoxynegamycin~
To a solution containing 79 mg. of di-N-benzyl-oxycarbonyl-D-~-lysine obtained in Example 1 (a) dissolved in 3 ml. of 1,2-dimethoxyethane, 22 mg. (0.19 mmole) of N-hydroxysuccinimide and 39 mg. (0.19 mmole) of dicyclo-hexylcarbodiimide were added under ice-cooling. After the reaction mixture was allowed to stand at 5 C. for 17 hours crystals of dicyclohexylurea deposited and were removed by filtration and the filtrate was concentrated 3L~83:~L75 to dryness to obtaln 97 mg. of N-hydroxysuccinimide ester of di-N-benzyloxycarbonyl-D-~-lysine as colorless crystals.
A solution containing the crystals obtained in the above dissolved in 2 ml. of 1~2-dimethoxyethane was gradually added to a solution containing 19.8 mg. (0.19 mmole) of l-~methylhydrazinoacetic acid and 32 mg. (o.~8 mmole) of sodium bicarbonate in 0.5 ml. of water under stirring at room temperature. After 18 hour~ stirring the reaction mixture was concentrated to dryness under reduced pressure and the residue was dissolved in 0.5 ml. of water. To this solution cooled in ice was added 0.42 ml. of lN HCl forming a white precipitate. The precipitate was collected by filtration, washed with water and dried to obtain 108 mg~ of di-N~benzyloxycar-bonyldeoxynegamycin as a white powder.
The white powder was dissolved in a mixture containing 1 ml. of acetic acid, 0.8 ml. methanol and 0.2 ml. water. To the solution was added 50 mg. of 5 palladium-carbon. After the mixture was stirred for 3 hours under an atmosphere of hydrogen the catalyst was removed by filtration and the filtrate was concentrated to dryness. The residue was dissolved in 2 ml. of water adjusted to pH 8.4 with 5N ammonia-water and passed through and adsorbed on a column containing 8.5 ml. Am~erlite CG-50 (NH4 ) resin. The column was washed ?
with 20 ml. water and eluted with 90 ml. of 1~ ammonia--water. The eluate was collected as 1 ml. fractions.
Fractions No. 57-62 having antibacterial activity to E.
coli K-12 were collected and concentrated to dryness to obtain 20.5 mg. of deoxynegamycin as a white powder.
Yield was 47~. _ 16 ~
.
1~83~
(a) Synthesis of Di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine Meth~l Ester:
To a solution containing 26 g. of (R,R)-~-hydroxy-~-lysine and 63 g. of sodium bicarbonate in 450 ml. of water was added dropwise 77 g. o~ benzyloxycarbonyl chloride at room temperature. After stirring for another 2 hours the mixture was allowed to stand at 5 C. for 1 hours. Crystals deposited and were collected by filtra-tion, washed with 200 ml. of water and with 350 ml. of ether and dried to obtain 44.~g. of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine lactone as colorless crystals.
m.p. 128-130 C., [a]D2 = -13 (c 3.4, chloroform), 70%
yield. m e crystals (~4.2 g.) were dissolved in ~60 ml.
of 0.5% HCl-CH30H and allowed to stand at room tempera-ture for 16 hours. The reaction mixture was concentrated and the crystals which deposited were recrystallized from a mixture of methanol-ether to obtain 28.3 g. of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine methyl ester as colorless crystals. m.p. 111 112 C ~ [a]D8 = +11 (c 1.2, chlorofo ~, 77% yield.
(b) Synthesis of Di-N-benzyloxycarbonyl-(R~R)-~-methoxy-~-lysine:
The 266 mg. (o.6 mmole) of di-N-benzyloxycar-bonyl-(R,R)-~-hydroxy-~-lysine methyl ester obtained in Example 2 (a) was dissolved in 4 ml. of dichloromethane To that solution under stirring at ice-cooling was added 17 mg. (0.12 mmole) of boron trifluoride etherate and to the mixture 1.5% diazomethane-ether was added until the yellow color remained in the reaction mixture (4 hours).
~83~L~S
After stirring at room temperature for 16 hours the reaction mixture was washed with 10 ml. of 10~ sodium bicarbonate-water and 20 ml. of water successively and the solvent layer was dehydrated with anhydrous sodium sul~ate and concentrated to dryness under reduced pres-sure yielding 295 mg. of a pale yellow oily material.
The olly material was chromatographed on a column con taining 30 g. of silica gel developed with benzene-methyl ethyl ketone (10:1 by volume) and collected in 9 ml. ~ractions. FractionSNo. 31-59 were collected and concentrated to dryness yielding 87 mg. of di-N-benzyl-oxycarbonyl-(R~R)~~-methoxy-~-lysine methyl ester as a colorless oil. 32% yield.
To a solution containing 85 mg. (0.19 mmole) of the oil in 0.5 ml. of ethanol~O.22 ml. of lN NaOH was added. After allowing to stand at room temperature ~or
.-.: . . . ,, :.
, .. . . . . .
33:~75 0-methylnegamycin with cinnamic acid, picric acid, p hydroxyazobenzene-p'-sulfonic acid, phytic acid~
livopimaric-6,6a-cis-endosuccinic acid, sulfamic acid and glycolic acid. For therapeutic purposes use is made o~ salts o~ nontoxic acids but salts of toxic acids, e.g. p-hydroxyazobenzene-p'-sulfonic acid, are useful in isolation procedures, e.g. as precipitants from aqueous solut~ons and for disinfectant purposes where toxicity is not important.
In the treatment o~ bacterial infections in animals, including man, the compounds of this invention are administered parenterally in accordance with con-ventional procedures for antibiotic administration in ~:
an amount of from about 5 to 200 mg./kg./day and pre-ferably about 5 to 20 mg./kg./day in divided dosage, e.g. three to four times a da~. They are administered in dosage units containing, for example, 125 or 250 or 500 mg. of acti-~e ingredient with suitable physiologically acceptable carriers or excipients. The dosage unit-s are in the form of liquid preparations such as solutions or suspensions.
The compounds of the present invention are also used topically in the treatment of superficial lnfections such as infections o~ skin or mucous membrane. For this purpose use can be made of conventional ointments, (e.g.
1~ or 5~) or solutions and suspensions in aqueous media at concentrations of 1~ to 10~.
The present invention provides the synthesis of ~-substituted negamycins which are much more stable in aqueous solution than negamycin itself and have antibac-.4.
, 33~75 terial activities. Thus, a constituent of negamycin,(R,R)-~-hydroxy-~-lysine (rV), is converted to D-~-lysine by removing the hydroxyl group at ~-position or to (R,R~-~-methoxy-~-lysine by replacing the hydroxyl group with a methoxyl group at ~ position, and two amino groups in those derlvatives are protected, the acid coupled with -l-methylhydrazinoacetic acid and the protective groups ~or the amino groups are then removed to yield the desired ~-subqtituted negamycin derivatives, deoxynegamycin or O-methylnegamycin as shown as formula (II) which are found to be quite stable in aqueous solutlon and active against Gram-positive and Gram-negative bacteria. ;
In one aspect of the present invention N~N'-di-protected-D-~-lysine (D - H in formula I) and N,N'-di-protected-(R,R)-~-methoxy-~-lysine (D ~ -OCH~ in ~:
formula I), which are represented by the following formula:
D M ~
. B
wherein A is hydrogen and B is a monovalent protective group for an amino group or A and B are one divalent protective group for an amino group and D is hydrogen or methoxyl, are separately coupled with 1-methylhydrazino- ;
acetic acid and the protective groups removed to prepare deoxynega~ycin (D = H in formula II) or O-methylnegamycin (D = -OCH3 ~n formula II).
1~l33~5 ... ..
(R) (R) H2NcH2cHcH2lHcH2coNHNcH2cooH (II) Starting materials (I'), D-~-lysine (D = H) and (R,R)-~-methoxy-~-lysine (D - -OCH~
(R) (R) ~2NC~2lHCH2lHCH2COOH (I') ~ -should be protected with known protective groups on the1r two amino groups. Although use can be made of the usual amino-protective groups used for peptide synthesis as the known protective groups, the protective groups used should ~;~
be easily removed without any cleavage of hydrazide bond in a compound which is synthesized from a ~-lysine deriva-tive (I) and 1-methylhydrazinoacetic acid by this procedure.
Suitable monovalent protective groups for amino groups in the ~-lysine derivative are alkyloxycarbonyl groups, e.g. tert-butyloxycarbonyl group; cycloalkyloxy~
carbonyl groups, e.g. cyclohexyloxycarbonyl group; aryl~
alkyloxycarbonyl groups, e.g. benzyloxycarbonyl and p-methoxylbenzyloxycarbonyl groups; and acyl groups especially lower alkanoyl groups, e.g. trifluoroacetyl and o-nitrophenoxyacetyl groups. A divalent protective ~ `;
group is a Schiff base such as salicylidene group. Those protective groups are introduced by known procedure, e.g.
using acid halide, acid azide, active ester, etc. ~ ;
In the prepara-tion of deoxynegamycin the D~
lysine used as the starting material is made, for example by the treatment of (R,R)-~-hydroxy-~-lysine with red phosphorus and hydroiodic acid in a sealed tube at 150 C.
., . , .. .. ,. , .. .. . . : . -~11 83~S
for 2 hours. The (R,R)-~-hydroxy-~-lysine is obtained by hydrolysis of negamycin or by synthesis from D-galacturonic acid (J. Am. Chem. Soc., 94, 4353, 1972). Amino-protective g~oups are introduced into D-~-lysine as described above. -For instance, by the treatment of D-~-lysine with benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in the presence of triethylamine in water-dioxane solution, di-N-benzyloxy-carbonyl-D-~-lysine is obtained at excellent yield.
In the preparation of O-methylnegamycin the (R,R)-~-methoxy-~-lysine used as the starting material is also synthesized from (R,R)-~-hydroxy-~-lysine and con-verted directly to the di-N-benzyloxycarbonyl derivative of (R,R)-~-methoxy-~-lysine. For instance, the aqueous solution of (R,R)-~-hydroxy-~-lysine is treated with benzyl-oxycarbonyl chloride in the presence of sodium bicarbonate yielding a lactone of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine as crystals. The lactone is dissolved in 0.5%-hydrogen chloride in methanol, allowed to stand at room te~perature and the reaction mixture is concentrated to obtain the methyl ester of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine as crystals The ester is reacted with diazomethane in the presence of boron trifluoride etherate in dichloromethane followed by hydrolysis with sodium hydroxide in ethanol to yield di-N-benzyloxycar-bonyl-(R,R)-~-methoxy-~-lysine.
In the coupling process the condensation ~orming a hydrazide bond with the N-protected ~-lysine derivative (I) and l-methylhydrazinoacetic acid is carried out by known methods for amide synthesis such as methods using dicyclohexylcarbodiimide, mixed anhydride, azide, acid halide, active ester, etc.
1(~83~5 For instance, the di-N benzyloxycarbonyl deriva-tlve o~ the ~-substituted (R,R)-~-hydroxy-~-lyslne i8 converted to an active ester by treatment with N-hydroxy-succinlmlde and dicyclohexylcarbodiimide in a nonaqueous solvent. The active ester is condensed with 1-1.5 moles of 1 methylhydrazinoacetic acid in the presence of 1-2.5 moles of sodium bicarbonate in an aqueous solvent. The coupling product thus obtained is treated with 25~ hydro~
gen bromide in acetic acid or hydrogenated with platinum, palladium, etc. as the catalyst to remove the N-benzyloxy-carbonyl groups and to synthesize the desired products (II), that is ~-substituted negamycins.
The removal of the protective groups from the coupling product is carried out by a usual method as described above. When the protective groups are alkyloxy-carbonyl group or Schif~ base the coupling productæ are hydrolyzed with a weak acid to remove the amino-protective groups.
An arylalkyloxycarbonyl group as the protective group is removed by the treatment with 25~ hydrogen bro~
mide in acetic acid, and o-nitrophenoxyacetyl group is easily removed by catalytic hydrogenation with platinum, palladium, etc.
The products are purified with good recovery by ion exchange chromatography using carboxylic cation exchange resins such as "Amberlite CG-50" (Rohm and Haas Co. Ltd., U.S.A.) or "CM-Sephadex C 25" (Pharmacia, Sweden).
0-Methylnegamycin and deoxynegamycin are also able to be derived directly from an amino-protected derivative of negamycin itself as shown in the following *Trade Marks _8 ~(~83~L~75 ':
formula:
A~ (R) (R) CH2fHCH2CHCH2CONHTCH2COOH (VI) B
wherein A is H and B is a monovalent amino-protective group or A together with B is one dlvalent amino-protective group. As the amino-protective groups for negamycin, all known amino-protective groups used for D-~-lysine or (R,R)-~-methoxy-~-lysine above are available in the same procedure For instance, negamycin is treated with benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in the presence of triethylamine in a mixture of water-dioxane to obtain di-N-benzyloxycarbonylnegamycin (A = H, B - COOCH2C6H5 in ~ormula VI) in high ~ield. m e benzyloxycarbonyl groups in this derivative are removed by catalytic hydrogenation simultaneously removing halogen atom to prepare deoxynega-mycin as described later, and therefore this group is a useful amino-protective group.
O-Methylnegamycin is synthesized from an amino~
protected derivative of negamycin (VI) by the same reaction `
conditions used for O-methylation of amino-protected ~ -(R,R)-~-oxy-~-lysine, that is, treatment with diazomethane in the presence of boron trifluoride etherate in an inert organic solvent such as dlchloromethane to obtain an amino-protected derivative of O-methylnegamycin methyl ester as shown by the following formula:
A (R) NCH CHCH CHCH CONHNCH COOCH (VII ) B ~ 21 21 2 1 2 ~B CH~;
wherein A and B mean the same groups described above. The 1'7S
amino-protective group and methyl ester group are removed by hydrolysis followed by col D chromatography wlth cation exchangers having carboxylic acid as the active group, and if necessary are purified by a column chromatography on silica gel obtaining O-methylnegamycin as the final product.
The present invention also provides -the process for the preparation of O-methy~negamycin which comprises conversion of amino-protected derivatives of negamycin (VI) ~NCH2CHCH2CHCH2CONHNCH2COOH (~
OH N~ C~
to amino-protected derivative of O-methylnegamycin methyl ester (VII) by reaction with diazomethane in the presence of boron trifluoride etherate followed by removal of the amino-protected groups and methyl ester group from the product (VII).
A~
NCH2CHCH2CHCH2CONHNCH2C~OCH~
OCH3 N~ CH3 ~.
In this procedure the reaction with diazomethane is carried out in an inert organic solvent7for instance, ; ;
halogenated hydrocarbons such as dichloromethane. The removal of the amino-protective groups and methyl ester group is carried out by ordinary procedures, for instance~
by hydrolysis in alkaline condition.
Furthermore, in the synthesis of deoxynegamycln from the amino-protected derivative of negamycin (VI), the only free carboxyl group of amino-protected negamycin i~ `
primarily protected with a common ester form, for instance~
33~L7~i ~
methyl , ethyl or benzyl ester to obtain an ester of amino-protected negamycin. And then the hydroxyl group at the ~-position of the ester i8 sulfonylated by treat-ment with alkylsulfonylating agent such as mesyl chloride, arylsulfonating agent such as p-toluenesulfonyl chloride, or arylmethylsulfonylating agent such as benzylsulfonyl chloride to obtain the sulfonic acid ester derivative as shown by ~III.
A~ (R) (R) ~NcH2cHcH2THcH2coNHlcH2coE (VIII) S03G ~ CH3 wherein A and B are the same as described in the above;
E is an alkyloxy group especially consisting o~ 1-4 carbon atoms or a benzyloxy group which are ester forming radi-cals removable easily by hydrolysis to hydroxyl group; G
is an alkyl group and especially a lower alkyl group, phenyl group or alkyl substituted phengl or benzyl group.
The product (VIII) is treated with an iodide or bromide of alkaii metal such as sodium iodide or sodium bromide and the sulfonyl ester at the ~-position is replaced with iodine or bromine to obtain the ~-iodo- or ~-bromo-deriva-tive which is a mixture o~ R and S at the ~-position. The iodo- or bromo-derivative in water or methanol or mixed solvent of water and methanol is hydrogenated with pal-ladium or platinum as the catalyst to produce deoxynega-mycin derivative by dehalogenation. When the amin ~
protected group is the benzyloxycarboxyl group as described above the removal of the amino-protected group takes place simultaneously in the above-mentioned catalytic hydrogena-tion. The remaining amino-protected group is removed by the procedure described above. The ester group of the 1~83~7S
product is removed by hydrolysis under weakly alkaline conditions and the reaction mixture is purified by column chromatography on cation exchange resin having carboxylic acid as the active group to obtain deoxy-negamycin as the final product.
There is further provided by the present inven-tion the process for the preparation of deoxynegamycin whereby an amino-protected derivative of ~-halogenated negamycin ester (IX) is catalytically hydrogenated and the remaining amino-protective group in the compound is removed by a conventional method and the ester ~orming group (E) is converted to hydroxyl by hydrolysis.
A~ (R) NcH27HcH2THcH2coNH I CH2COE
X N CH
~ B 3 (wherein A is hydrogen and B is a monovalent amino-protective group or A together with B is one divalent amino-protective group, E is an ester forming group which is converted to hydroxyl by hydrolysis, and X is iodine or bromine atom.) ~-The this procedure an amino-protected deriva~
tive of ~-halogenated negamycin ester (IX) is prepared as follows. An amino-protected derivative of negamycin (VI) prepared as above is treated with an agent for -esterification such as diazomethane or diazoethane to convert it to negamycin ester derivative (carboxylate).
Next the ~-hydroxyl group of the derivative is sulfonylated (-S03G) and then halogenated. For instance, the sulfonyla-tion is carried out according to a similar procedure used to prepare an intermediate in the production of ~',4'--- 1~ _ ~8317~
dideoxykanamycin B as described in Japan Patent 7595/1975.
The reaction to convert the sulfonyl group (-S03G) to halogen group (X) by the effect of an iodide or bromide of an alkall metal and the procedure to remove the ~-halogen group by catalytic hydrogenation is also carried out in known manner.
The properties o~ deoxynegamycin and O-methyl-negamycin which are the final products obtained in this invention are as follows:
Deoxynegamycin is a white powder showing d.p.
120-125 C. [a]23 = 5o (c 1.5, H20) and elemental analysis to coincide with the theoretical value for CgH20N~03-H20 (C 43.18~, H 8.86%, N 22.39%). On silica gel thin lajer chromatography (Art. 5721, Merck Co., Germany) de~eloped with n-butanol-ethanol-chloroform-17%
aqueous amm~nia (4:5:2:5 by volume) it gives a single spot (ninhy_rin) at Rf 0.~4.
O-Methylnegamycin is a white powder showing d.p. 137-140 C. ~a]22 = -3 (c 1.5, H~O), and elemental ~nalysis to coincide with the theoretical value for CloH22N4C4 H20 (C 42.84~, H 8.63~, N 1~.99~). On the tlc described above it gives a single spot at Rf 0.33. As shown in the table, those two novel derivatives of nega mycin inhibited the growth of Gram-positive and Gram-negative bacteria. Those two compounds were completely stable in both aqueous or 0.02N HCl aqueous solution at 37 C. for one month while negamycin as the control was reduced in its activity to 63~ in aqueous solution and to 50~ in 0.02N HCl aqueous solution. Those two compounds are low in to~icity (LD50 of both in mice, i.v., >200 mg./kg.) and are expected to be used in the chemotherapy ~aQ831~S
of infections caused by various Gram-positive and Gram-negative bacteria.
TABLE: Antimicrobial Spectra of O-Methylnegamycin and Deoxynegamycin Min~mum Inhibi~ory Concentrations*(Mcg./ml.) O-Methyl-Deoxy-Test Organisms _ ~Y~ æ~ YD5 StaPhylococcus aureus FDA209P 12.5 25 Staphylococcus aureus Smith 6. 256.25 Sarcina lutea PCI1001 25 ~100 ;
Micrococcus ~lavus FDA16 50 50 Bacillus subtilis NRRL B-5s8 50 100 Mycobacterium smegmatis ATCC607 25 50 Escherichia coli NIHJ 6 256. 25 . , ~
Escherichia coli K-12 3-13 3.13 Escherichia coli K-12 ML1629 1 56 3013 .
Salmonella t~phi T-63 1 - 56 o . 78 Proteus ulgaris OX-l9 1- 56 3.13 Proteus rett~eri GN311 6.25 6.25 Proteus rett~eri GN466 3.13 3.13 Serratia marcescens 25 25 Klebsiella pneumoniae PCI602 6.25 6.25 Pseudomonas :~luore~cens 1- 56 3-13 Pseudomonas aeruginosa A3 12.5 25 ~i Pseudomonas aeruginosa No. 12 25 50 . .
.. _ ._ *Minimum inhibitory concentrations were determined on a 0.5% peptone agar by incubatlon at 37 C. for 17 hours.
- 1~
10831~5 DESCRIPTION _F THE PREFE _ED EMBODIMENTS
(a) Synthesis of Di-N-benzyloxycarbon~vl-D-~-lysine-.
To ~ solution containing 100 mg. (o.6g mmolej D-~-lysine ([~]25 = -22.5 (c o.8, lN HC1) in 1 ml. of water, 104 mg. (1.0 mmole) of triethylamine at first and then a solution containing 410 mg. (1.5 mmoles) benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate (Kokusan Chemical Works~ Tokyo) in 1 ml. o~ dioxane were added and stirred at 30 C. for 17 hours. To the reaction mixture 3 ml. of water was added and the mix-ture was washed with 6 ml. of ether. The aqueous layer was adjusted to pH 2 with 6N HCl, saturated with sodium chloride and extracted with 13 ml. of ethyl acetate. The ethyl acetate layer was washed with 4 ml. o~ 5% HCl in water saturated with sodlum chloride and with 4 ml. of water saturated with sodium chloride, dried with anhydrous -sodium sulfate and concentrated to dryness under reduced pressure to obtain 219 mg. of di-N-benzyloxycarbonyl-D-~-lysine as colorless crystals, d.p. 149-151 C`., [a]D6 =
l6 (c 1.3, MeOH), 73~ yield.
(bj Synthesis of Deoxynegamycin~
To a solution containing 79 mg. of di-N-benzyl-oxycarbonyl-D-~-lysine obtained in Example 1 (a) dissolved in 3 ml. of 1,2-dimethoxyethane, 22 mg. (0.19 mmole) of N-hydroxysuccinimide and 39 mg. (0.19 mmole) of dicyclo-hexylcarbodiimide were added under ice-cooling. After the reaction mixture was allowed to stand at 5 C. for 17 hours crystals of dicyclohexylurea deposited and were removed by filtration and the filtrate was concentrated 3L~83:~L75 to dryness to obtaln 97 mg. of N-hydroxysuccinimide ester of di-N-benzyloxycarbonyl-D-~-lysine as colorless crystals.
A solution containing the crystals obtained in the above dissolved in 2 ml. of 1~2-dimethoxyethane was gradually added to a solution containing 19.8 mg. (0.19 mmole) of l-~methylhydrazinoacetic acid and 32 mg. (o.~8 mmole) of sodium bicarbonate in 0.5 ml. of water under stirring at room temperature. After 18 hour~ stirring the reaction mixture was concentrated to dryness under reduced pressure and the residue was dissolved in 0.5 ml. of water. To this solution cooled in ice was added 0.42 ml. of lN HCl forming a white precipitate. The precipitate was collected by filtration, washed with water and dried to obtain 108 mg~ of di-N~benzyloxycar-bonyldeoxynegamycin as a white powder.
The white powder was dissolved in a mixture containing 1 ml. of acetic acid, 0.8 ml. methanol and 0.2 ml. water. To the solution was added 50 mg. of 5 palladium-carbon. After the mixture was stirred for 3 hours under an atmosphere of hydrogen the catalyst was removed by filtration and the filtrate was concentrated to dryness. The residue was dissolved in 2 ml. of water adjusted to pH 8.4 with 5N ammonia-water and passed through and adsorbed on a column containing 8.5 ml. Am~erlite CG-50 (NH4 ) resin. The column was washed ?
with 20 ml. water and eluted with 90 ml. of 1~ ammonia--water. The eluate was collected as 1 ml. fractions.
Fractions No. 57-62 having antibacterial activity to E.
coli K-12 were collected and concentrated to dryness to obtain 20.5 mg. of deoxynegamycin as a white powder.
Yield was 47~. _ 16 ~
.
1~83~
(a) Synthesis of Di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine Meth~l Ester:
To a solution containing 26 g. of (R,R)-~-hydroxy-~-lysine and 63 g. of sodium bicarbonate in 450 ml. of water was added dropwise 77 g. o~ benzyloxycarbonyl chloride at room temperature. After stirring for another 2 hours the mixture was allowed to stand at 5 C. for 1 hours. Crystals deposited and were collected by filtra-tion, washed with 200 ml. of water and with 350 ml. of ether and dried to obtain 44.~g. of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine lactone as colorless crystals.
m.p. 128-130 C., [a]D2 = -13 (c 3.4, chloroform), 70%
yield. m e crystals (~4.2 g.) were dissolved in ~60 ml.
of 0.5% HCl-CH30H and allowed to stand at room tempera-ture for 16 hours. The reaction mixture was concentrated and the crystals which deposited were recrystallized from a mixture of methanol-ether to obtain 28.3 g. of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine methyl ester as colorless crystals. m.p. 111 112 C ~ [a]D8 = +11 (c 1.2, chlorofo ~, 77% yield.
(b) Synthesis of Di-N-benzyloxycarbonyl-(R~R)-~-methoxy-~-lysine:
The 266 mg. (o.6 mmole) of di-N-benzyloxycar-bonyl-(R,R)-~-hydroxy-~-lysine methyl ester obtained in Example 2 (a) was dissolved in 4 ml. of dichloromethane To that solution under stirring at ice-cooling was added 17 mg. (0.12 mmole) of boron trifluoride etherate and to the mixture 1.5% diazomethane-ether was added until the yellow color remained in the reaction mixture (4 hours).
~83~L~S
After stirring at room temperature for 16 hours the reaction mixture was washed with 10 ml. of 10~ sodium bicarbonate-water and 20 ml. of water successively and the solvent layer was dehydrated with anhydrous sodium sul~ate and concentrated to dryness under reduced pres-sure yielding 295 mg. of a pale yellow oily material.
The olly material was chromatographed on a column con taining 30 g. of silica gel developed with benzene-methyl ethyl ketone (10:1 by volume) and collected in 9 ml. ~ractions. FractionSNo. 31-59 were collected and concentrated to dryness yielding 87 mg. of di-N-benzyl-oxycarbonyl-(R~R)~~-methoxy-~-lysine methyl ester as a colorless oil. 32% yield.
To a solution containing 85 mg. (0.19 mmole) of the oil in 0.5 ml. of ethanol~O.22 ml. of lN NaOH was added. After allowing to stand at room temperature ~or
4 hours the mixture was acidified by the addition of 0.28 ml. o~ lN HCl and extracted with ~ ml. of ethyl acetate. The ethyl acetate layer was washed with 1 ml. of water, dehydrated with anhydrous sodium sul~ate and concentrated to dryness yielding 80 mg. of di-N-benzyloxycarbonyl-(RgR)-~-methoxy-~-lysine as a color- ?
less oil. 98% yield.
(c) Synthesis of O-Methylnegamycin:
To a solution containing 80 mg. (0.18 mmole) o~ di-N-benzyloxycarbonyl-(R,R)-~-methoxy-~-lysine which was obtained in Example 2 (b), in 1.6 ml. of 1,2-dimethoxyethane, 21 mg. (0.18 mmole) o~ N-hydroxy-succinimide were added under ice cooling. A~ter the reaction mixture was allowed to stand at 5 C. for 17 ~33~S
hours dicyclohexylurea appeared and was removed by filtration and the filtrate was concentrated to dryness yielding 98 mg. of N-hydroxy succinimide ester of di-N-benzyloxycarbonyl-~R,R)-~-methoxy-~-lysine as a color-les~ oil.
A solution of the oil in 1.5 ml. of 1~2-dimethoxyethane was gradually added at room temperature under stirring to a solution containing 18.7 mg. (0.18 mmole) o~ l-methylhydrazinoacetic acid and 30 mg. (o.~6 mmole) of sodium bicarbonate in 0.5 ml. of water. After stirring for 20 hours the reaction mixture was concen-trated to dryness, dissolved in 0.5 ml. of water and a white powder was precipitated by the addition of 0.4 ml.
lN HCl under ice-cooling. The precipitate was dissolved in 3 ml. of ethyl acetate and washed with 0.5 ml. of water. The ethyl acetate layer was dehydrated with anhy-drous sodium sulfate and concentrated to dryness yielding 94 mg. of di-N-benzyloxycarbonyl-0-methylnegamycin as a white powder.
To a solution containing the white powder ob-tained above in a mixture containing 1 ml. of acetic acid, o.8 ml. of methanol and 0.2 ml. of water, 24 mg.
of 5~ palladium-carbon was added and the mixture was stirred with hydrogen gas for 4 hours. After removal of catalyst by filtration the filtrate was concentrated to dryness. The residue was dissolved in 2 ml. of water,adjusted to pH 8.8 with 5N ammonia and charged into a column containing 10 ml. of Amberlite CG-50 (NH4 ). The column was washed with 20 ml. of water and eluted with 100 ml. of 0.1~ ammonia-water collecting 1 ml. fractions. Fractions No. 45-57 having antibacterial _ 19_ . .
31331~5 activity were combined and concentrated under reduced pressure to dryness to provide 20 8 mg. of 0-methyl-negamycin as a white powder. 44% yield.
(a) S~nthesis o~ Di-N-benzyloxycarbonylnegamycin:
To a solution containing 4 g (16.1 mmoles) of negamycin in 20 ml. of water and 2.44 g. (24.2 mmoles) of triethylamine, 9.72 g~ (35.5 mmoles) of benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in 20 ml. o~ dioxane was added and the reaction mixture was stirred at room temperature for 17 hours. To the reaction mixture 30 ml. of water was added and the mixture was washed with two por-tions of 60 ml. of ethyl acetate. The aqueous layer was ad~usted to pH 2 with 6N HCl and extracted with two por-tions of 60 ml. of ethyl acetate after saturation with sodium chloride. The ethyl acetate layer was washed with 80 ml. o~ water saturated with sodium chloride dehydrated with anhydrous sodium sulfate and concentrated to dryness under reduced pressure to yield 7.6 g of di N-benzyl-oxycarbonylnegamycin as a white powder. d.p. 110-113 C., [a]25 = +4.2 (c 6.3, CH30H), 92% yield.
(b) Synthesis of 0-Methylnegamycin .
To a solution containing 449 mg (0.87 mmole) of di-N-benzyloxycarbonylneg~mycin which was obtained in Example 3(a), in 405 ml. of dichloromethane, 62 mg.
(0.44 mmole) of boron trifluoride etherate was added with ice-cooling and stirring followed by the addition of 1.5~ diazomethane-ether solution until the yellow color _20_ ~83~75 of the reaction mixture remained (for two hours). The reaction mixture was concentrated to dryness yielding di~N-benzyloxycarbonyl-0-methylnegamycin methyl ester as a crude powder. The powder was dissolved in 5 ml. of 25~
hydrogen bromide-acetic acid and stirred at room tempera-ture for 20 minutes to remove the amino-protective groups.
To the solution 50 ml. of ethyl ether was added and a precipitate appeared which was collected by fil-tration, washed with 20 ml. o~ ethyl ether and dried to obtain a yellow powder. The powder was dissolved with 20 ml. o~ water, ad~usted to pH 8.8-9.o with 5N ammonia-water and charged into a column containing 40 ml. of Amberlite CG-50 (NH4 ). The column was washed with 80 ml. of water and eluted with 200 ml. of 0.9~ ammonia-water collecting 4 ml. fractions. Fractions No. ~2-34 glving positive ninhydrin were collected and concentrated to dryness under reduced pressure to yield 141 mg. of 0-methylnegamycin as a crude powder. The crude powder was charged into a column of silica gel (14 g.) and developed with n-butanol-ethanol-chloroform-17~ ammonia in water (4:5:2:3 by volume) collecting 4 ml. fractions.
Fractions No. 24-28 having positive ninhydrin reaction and antibacterial activity versus E. coli K-12 were combined and concentrated to dryness under reduced pres-sure to yield 30 mg. of 0-methylnegamycin as a white powder. 13~ yield.
_21 3~L75 E~AMPIE 4 (a) .Synthesis of Dl-N-benzyloxycarbonylnegamycin Methyl Ester: -To a solution containing 6 g. (11.6 mmoles) of di-N-benzyloxycarbonylnegamycln which was obtained in Example 3 (a), in 120 ml. of methanol, 1.5~ diazomethane-ethanol-ether solution was added until the yellow color of the mixture remained. After stirring for 45 minutes the reaction mixture was concentrated to dryness under `~
reduced pressure to yeild 5.98 g of di-N-benzyloxycar-bonylnegamycin methyl ester. d.p. 107-112 C., ~a]26 =
~4.0 (c 5.0, CH30H), 97~ yield. ?
(b) Synthesis of Deoxynegamycin:
To a solution containing 5.98 g. (11.3 mmoles) of dl-N-benzyloxycarbonylnegamycin methyl ester which was obtained in Example 4 (a), in 150 ml. of pyridine a solution containg 6.4~ g. (56.4 mmoles) of methanesul-~onyl chloride in 150 ml. of pyridine was added with ice-cooling for 15 minutes. After stirring at room tempera--ture for 5 hours the reaction mixture was diluted with 150 ml. of ice water and extracted with 240 ml. of chloroform. m e chloroform layer was washed successively with 60 ml. portions of 0.5M sodium bisulfite, water, lM sodium bicarbonate and water. After dehydration with anhydrous sodium sulfate the chloro~orm solution was ;
concentrated to dryness under reduced pressure ylelding 6 17 g. (10.1 mmoles) of di-N-benzyloxycarbonyl-0-methanesulfonylnegamycin methyl ester as a pale yellow powder.
To a solution containing 4.9 g. (8.1 mmoles) ' -22- ~
... . . . . . . . . .. . .
1~383~
of the pale yellow powder so obtained in 225 ml. of acetone, 7.2 g. (48 mmoles) of sodium iodide, which was thoroughly dried at 50-60 C. overnight, was added.
The mixture was refluxed at 65 C. for 15 hours, diluted wi.th 1,120 ml. of water and extracted with 1,350 ml. of ethyl acetate. The ethyl acetate layer was washed with 450 ml. of water, dehydrated with anhydrous sodium sulfate and concentrated to dryness under reduced pressure to yield 3.98 g. of a pale yellow powder.
The powder was subjected to a column chromatography on silica gel (400 g.) using benzene-methyl ethyl ketone (1:1 by volume) as the developing solvent. The eluate was collected in 18 ml. fractions and Fractions No. 77-170 were combined and concentrated to dryness under reduced pressure to yield l.~ g~ (2 mmoles) of ~-iodo-derivative (a mlxture of 2 kinds of stereoisomer). 22 yield.
To a solution containing 9~7 mg. (1.46 mmoles) of the ~-iodo-derivative thus obtained in 20 ml. of methanol and 8 ml. of water, lO g. of 5~ palladium- -barium carbonate was added at room temperature under stirring and introduction of hydrogen gas to carry out simu1taneously dehalogenation and removal of benzyloxy-carbonyl groups. The catalyst was removed by filtra-tion and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 16 ml. of water, adjusted to p~ 9 2-9.4 with 5N
ammonia-water and charged into a column containing 97 ml. of Amberlite CG-50 (NH4 ). After washing with -water the column was eluted with 650 ml. of 0.2 ammonia-water and the eluate was collected in 5 ml.
~3_ ~083~'75 fractions, Fractions No. 20-50 having antibacterial activity versus E. coli K-12 and positive ninh~drin reaction were combined and concentrated to dryness under reduced pressure to yield g5 mg. of deoxynega-mycin as a white powder, 28~ yield.
24 _
less oil. 98% yield.
(c) Synthesis of O-Methylnegamycin:
To a solution containing 80 mg. (0.18 mmole) o~ di-N-benzyloxycarbonyl-(R,R)-~-methoxy-~-lysine which was obtained in Example 2 (b), in 1.6 ml. of 1,2-dimethoxyethane, 21 mg. (0.18 mmole) o~ N-hydroxy-succinimide were added under ice cooling. A~ter the reaction mixture was allowed to stand at 5 C. for 17 ~33~S
hours dicyclohexylurea appeared and was removed by filtration and the filtrate was concentrated to dryness yielding 98 mg. of N-hydroxy succinimide ester of di-N-benzyloxycarbonyl-~R,R)-~-methoxy-~-lysine as a color-les~ oil.
A solution of the oil in 1.5 ml. of 1~2-dimethoxyethane was gradually added at room temperature under stirring to a solution containing 18.7 mg. (0.18 mmole) o~ l-methylhydrazinoacetic acid and 30 mg. (o.~6 mmole) of sodium bicarbonate in 0.5 ml. of water. After stirring for 20 hours the reaction mixture was concen-trated to dryness, dissolved in 0.5 ml. of water and a white powder was precipitated by the addition of 0.4 ml.
lN HCl under ice-cooling. The precipitate was dissolved in 3 ml. of ethyl acetate and washed with 0.5 ml. of water. The ethyl acetate layer was dehydrated with anhy-drous sodium sulfate and concentrated to dryness yielding 94 mg. of di-N-benzyloxycarbonyl-0-methylnegamycin as a white powder.
To a solution containing the white powder ob-tained above in a mixture containing 1 ml. of acetic acid, o.8 ml. of methanol and 0.2 ml. of water, 24 mg.
of 5~ palladium-carbon was added and the mixture was stirred with hydrogen gas for 4 hours. After removal of catalyst by filtration the filtrate was concentrated to dryness. The residue was dissolved in 2 ml. of water,adjusted to pH 8.8 with 5N ammonia and charged into a column containing 10 ml. of Amberlite CG-50 (NH4 ). The column was washed with 20 ml. of water and eluted with 100 ml. of 0.1~ ammonia-water collecting 1 ml. fractions. Fractions No. 45-57 having antibacterial _ 19_ . .
31331~5 activity were combined and concentrated under reduced pressure to dryness to provide 20 8 mg. of 0-methyl-negamycin as a white powder. 44% yield.
(a) S~nthesis o~ Di-N-benzyloxycarbonylnegamycin:
To a solution containing 4 g (16.1 mmoles) of negamycin in 20 ml. of water and 2.44 g. (24.2 mmoles) of triethylamine, 9.72 g~ (35.5 mmoles) of benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in 20 ml. o~ dioxane was added and the reaction mixture was stirred at room temperature for 17 hours. To the reaction mixture 30 ml. of water was added and the mixture was washed with two por-tions of 60 ml. of ethyl acetate. The aqueous layer was ad~usted to pH 2 with 6N HCl and extracted with two por-tions of 60 ml. of ethyl acetate after saturation with sodium chloride. The ethyl acetate layer was washed with 80 ml. o~ water saturated with sodium chloride dehydrated with anhydrous sodium sulfate and concentrated to dryness under reduced pressure to yield 7.6 g of di N-benzyl-oxycarbonylnegamycin as a white powder. d.p. 110-113 C., [a]25 = +4.2 (c 6.3, CH30H), 92% yield.
(b) Synthesis of 0-Methylnegamycin .
To a solution containing 449 mg (0.87 mmole) of di-N-benzyloxycarbonylneg~mycin which was obtained in Example 3(a), in 405 ml. of dichloromethane, 62 mg.
(0.44 mmole) of boron trifluoride etherate was added with ice-cooling and stirring followed by the addition of 1.5~ diazomethane-ether solution until the yellow color _20_ ~83~75 of the reaction mixture remained (for two hours). The reaction mixture was concentrated to dryness yielding di~N-benzyloxycarbonyl-0-methylnegamycin methyl ester as a crude powder. The powder was dissolved in 5 ml. of 25~
hydrogen bromide-acetic acid and stirred at room tempera-ture for 20 minutes to remove the amino-protective groups.
To the solution 50 ml. of ethyl ether was added and a precipitate appeared which was collected by fil-tration, washed with 20 ml. o~ ethyl ether and dried to obtain a yellow powder. The powder was dissolved with 20 ml. o~ water, ad~usted to pH 8.8-9.o with 5N ammonia-water and charged into a column containing 40 ml. of Amberlite CG-50 (NH4 ). The column was washed with 80 ml. of water and eluted with 200 ml. of 0.9~ ammonia-water collecting 4 ml. fractions. Fractions No. ~2-34 glving positive ninhydrin were collected and concentrated to dryness under reduced pressure to yield 141 mg. of 0-methylnegamycin as a crude powder. The crude powder was charged into a column of silica gel (14 g.) and developed with n-butanol-ethanol-chloroform-17~ ammonia in water (4:5:2:3 by volume) collecting 4 ml. fractions.
Fractions No. 24-28 having positive ninhydrin reaction and antibacterial activity versus E. coli K-12 were combined and concentrated to dryness under reduced pres-sure to yield 30 mg. of 0-methylnegamycin as a white powder. 13~ yield.
_21 3~L75 E~AMPIE 4 (a) .Synthesis of Dl-N-benzyloxycarbonylnegamycin Methyl Ester: -To a solution containing 6 g. (11.6 mmoles) of di-N-benzyloxycarbonylnegamycln which was obtained in Example 3 (a), in 120 ml. of methanol, 1.5~ diazomethane-ethanol-ether solution was added until the yellow color of the mixture remained. After stirring for 45 minutes the reaction mixture was concentrated to dryness under `~
reduced pressure to yeild 5.98 g of di-N-benzyloxycar-bonylnegamycin methyl ester. d.p. 107-112 C., ~a]26 =
~4.0 (c 5.0, CH30H), 97~ yield. ?
(b) Synthesis of Deoxynegamycin:
To a solution containing 5.98 g. (11.3 mmoles) of dl-N-benzyloxycarbonylnegamycin methyl ester which was obtained in Example 4 (a), in 150 ml. of pyridine a solution containg 6.4~ g. (56.4 mmoles) of methanesul-~onyl chloride in 150 ml. of pyridine was added with ice-cooling for 15 minutes. After stirring at room tempera--ture for 5 hours the reaction mixture was diluted with 150 ml. of ice water and extracted with 240 ml. of chloroform. m e chloroform layer was washed successively with 60 ml. portions of 0.5M sodium bisulfite, water, lM sodium bicarbonate and water. After dehydration with anhydrous sodium sulfate the chloro~orm solution was ;
concentrated to dryness under reduced pressure ylelding 6 17 g. (10.1 mmoles) of di-N-benzyloxycarbonyl-0-methanesulfonylnegamycin methyl ester as a pale yellow powder.
To a solution containing 4.9 g. (8.1 mmoles) ' -22- ~
... . . . . . . . . .. . .
1~383~
of the pale yellow powder so obtained in 225 ml. of acetone, 7.2 g. (48 mmoles) of sodium iodide, which was thoroughly dried at 50-60 C. overnight, was added.
The mixture was refluxed at 65 C. for 15 hours, diluted wi.th 1,120 ml. of water and extracted with 1,350 ml. of ethyl acetate. The ethyl acetate layer was washed with 450 ml. of water, dehydrated with anhydrous sodium sulfate and concentrated to dryness under reduced pressure to yield 3.98 g. of a pale yellow powder.
The powder was subjected to a column chromatography on silica gel (400 g.) using benzene-methyl ethyl ketone (1:1 by volume) as the developing solvent. The eluate was collected in 18 ml. fractions and Fractions No. 77-170 were combined and concentrated to dryness under reduced pressure to yield l.~ g~ (2 mmoles) of ~-iodo-derivative (a mlxture of 2 kinds of stereoisomer). 22 yield.
To a solution containing 9~7 mg. (1.46 mmoles) of the ~-iodo-derivative thus obtained in 20 ml. of methanol and 8 ml. of water, lO g. of 5~ palladium- -barium carbonate was added at room temperature under stirring and introduction of hydrogen gas to carry out simu1taneously dehalogenation and removal of benzyloxy-carbonyl groups. The catalyst was removed by filtra-tion and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 16 ml. of water, adjusted to p~ 9 2-9.4 with 5N
ammonia-water and charged into a column containing 97 ml. of Amberlite CG-50 (NH4 ). After washing with -water the column was eluted with 650 ml. of 0.2 ammonia-water and the eluate was collected in 5 ml.
~3_ ~083~'75 fractions, Fractions No. 20-50 having antibacterial activity versus E. coli K-12 and positive ninh~drin reaction were combined and concentrated to dryness under reduced pressure to yield g5 mg. of deoxynega-mycin as a white powder, 28~ yield.
24 _
Claims (4)
1. A process for the production of O-methylnegamycin having the formula which comprises the consecutive steps of a) reacting diazomethane in the presence of boron trifluoride etherate with a conventional, easily removable ester of amino-protected negamycin to form the intermediate having the formula wherein A is hydrogen and B is a monovalent amino-protective group or A together with B are a divalent amino-protective group and b) removing from said intermediate the amino-protective groups A and B and the methyl ester group to form O-methylnegamycin.
2. The process according to Claim 1 wherein A is hydrogen and B is a benzyloxycarbonyl group.
3. The process according to claim 1 wherein the amino-protective groups A and B and the methyl ester group are removed by hydrolysis followed by column chromatography with cation exchangers having carboxylic acid as the active group.
4. A process according to claim 1 or 2 further comprising the step of forming a pharmaceutically acceptable nontoxic salt or acid addition salt of O-methylnegamycin.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA332,525A CA1083175A (en) | 1975-11-11 | 1979-07-25 | .alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESES |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP50134710A JPS5259112A (en) | 1975-11-11 | 1975-11-11 | Process for preparation of delta-substituted negamycine derivatives |
JP134710/1975 | 1975-11-11 | ||
CA264,405A CA1083174A (en) | 1975-11-11 | 1976-10-28 | .alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESES |
CA332,525A CA1083175A (en) | 1975-11-11 | 1979-07-25 | .alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESES |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1083175A true CA1083175A (en) | 1980-08-05 |
Family
ID=27164730
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA332,525A Expired CA1083175A (en) | 1975-11-11 | 1979-07-25 | .alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESES |
Country Status (1)
Country | Link |
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CA (1) | CA1083175A (en) |
-
1979
- 1979-07-25 CA CA332,525A patent/CA1083175A/en not_active Expired
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