CA1083176A - .alpha.-SUBSTITUTED NEGAMYCIN DERIVATIVES AND SYNTHESES - Google Patents

Abstract

Division II
.delta.-SUBSTITUTED NEGAMYCIN DERIVATIVES
AND SYNTHESES

ABSTRACT
Compounds having the structure were found to be antibacterial agents. They were synthesized by a process which comprises a) catalytically hydrogenating a compound of the formula

Description

.
3:~1L7~

ACKGROUND OF_THE INVE_TION

1. Field of the Invention . . .
The no~el synthetic organic compounds of the present invention are antibacterial agents and are used as are other members o~ that class.

2. Description of the Prior Art Negamycin is an antibiotic di~covered by the present inventors (Japan 34827/1969, Japan 28835/1971) and is represented by the following formula (III):

.
(R) (R) O
H2NCH2fHcH2clHcH2cONH7c~2co (III) ~ -`

having a unique structure consisting of a hydrazide bond between (R,R)-~-hydroxy-~-lysine (IV) and l-methylhydra-zinoacetic acid (V) -- H2NCH2CECH2lECH2COOH ~IV) OH NH~

H2N INCH2COOH (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. Fletcher3 O. C. Dermer and R. B. Fox, p. 103, American Chemical Society, Washington, D.C., 1974).
See also U.S. Patents 3,679,742 and 3,743,580 and ~.
Hamada et al., A New Antibiotic, Negamycin, J. Antibiotics, 23(3), 170-71 (1970)-33:~l7~ -SUMMARY OF THE INVENTION

The present invention provides new derivatives o~ negamycin, that is, semisynthetic antlbiotics ha~ing the ability to inhibit Gram-positive and Gram-negative bacteria and being of quite stable character even in acidic aqueous solutions, which are represented by the following formula:

H2NCH2CHCH2CaCH2CONHNCH2COO~
D NH2 C~ .
and the acid and base salts thereof, wherein D is selected ~. .
, from the group conslsting of H (deoxynegamycin) and CH30-(O-methylnegamycin).
The subject of this divisional application is a process . . , ~
for the production of deoxynegamycin having the formula ~ :

. ` ~R) . :~.

2 2 2 2f 2 1 2 comprising the consecutive steps of (a) catalytically hydrogenating a compound of the : the formula A~ (R) B~ NCH21CHCH21CHCH2CONHICH2COE

; X N_,_A 3 ~ B
wherein X is bromo or iodo, A is hydrogen and B is a monovalent amino-protective group or A together with B are a divalent amino-protective group, and E is an easily removable ester forming group, and (b) removing any remaining ester forming and amino-protective groups.

3~76 One preferred aspect of such a process wherein the steps (a) and (b) are carried out simultaneously by introducing hydrogen gas in the presence of palladium-barium carbonate as the cataylst.
Preferably, X is iodo.
In a preferred embodiment of such a process E is an alkoxy group consisting of 1-4 carbon atoms or a benzyloxy group.
s In one embodiment the invention provides such a process further comprising a step of forming a pharmaceutically acceptable nontoxic salt or acid addition salt of deoxynegamycin.
Salts of deoxynegamycin and O-methylnegamycin include carboxylic acid salts including nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts~ e.g. salts of such nontoxic amines as trialkylamines, including tri-- ethylamine, procaine3 dibenzylamine, N-benzyl-~-phenethyl-amine, l-ephenamine, N~N'-dibenzylethylenediamine, dehydro-abietylamine~ N,N'-bis-dehydroabietylethylenediamine, N-(lower)alkylpiperidine, e.g. N-ethylpiperidi~e and 'L other amines which ha~e been used to form salts with benzylpenicillinj and the nontoxic, acid addition salts thereof, (i.e. the amine salts) including the mineral acid addition salts such as the hydrochloride, hydrobro~
mide, hydroiodide, sulfate, sulfamate and phosphate and the organic acid addition salts such as the maleate~
acetate, citrate, oxalate, succinate, benzoate, tartrate, fumarate, malate, mandelate~ ascorbate and the like.
Also included are the salts of deoxynegamycin and .
-~01~33~76 0-methylnegamycin with cinnamic acid, picric acid, p-hydroxyazobenze~e-p'-sul~onic acid3 phytic acid, livopimaric-6,6a-cis-endosuccinic aci.d, sulfamic acid and glycolic acld. For therapeutic purposes use is made of salts of 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 solutions and for disinfectant purposes where toxicity ls 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 ~ay. They are administered in dosage units containing, for example, 125 or 250 or 500 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. The dosage units 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 infections such as infections of 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 o~ 1% to 10%.

The present invention provides the synthesis of ~-substituted negamycins which are much more stable in aqueous solution than negamycin itsel~ and have antibac-33~7~

terial activities Thus~ a constituent of negamycin,(R,R)-~-hydroxy-~-lysine (IV), 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 derivatives are protected, the acid coupled with 1-methylhydrazinoacetic acid and the protective groups for the amino groups are then removed to yield the desired ~-substituted 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 o~ the present invention -~
N,N'-di-protected-D-~-lysine (D = H in formula I) and N,N'-di-protected-(R3R)-~-methoxy-~-lysine (D = -OCH3 in formula I), which are represented by the following formula:

~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 deoxynegamycin (D = H in ~ormula II) or O-methylnegamycin (D = -OCH3 in formula II). ;

~0~331'76 (R) (R) H2NCrI2CHCH2lHCH2CONHNCH2COOH (II) ~ ~H2 C ~
Starting materials (I'), D-~-lysine (D = E) and (R,R)-~-methoxy-~-lysine (D = -OCH3), H2NCH2CHCH2CHCH2COOH (I') should be protected with known protective groups on their 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 ~-lyslne deriva-tive (I) and l-methylhydrazinoacetic acid by this procedure.
Suitable monovalent protective group~ for amino groups in the ~-lysine derivative are alkyloxycarbonyl groups, e.g. tert-butyloxycarbonyl group; cycloalkyloxy-carbonyl groupsg e.g. cyclohexyloxycarbonyl group; aryl-alkyloxycarbonyl groups, e.g. benzyloxycarbonyl and ~-~
p-methoxylbenzyloxycarbonyl groups; and ac~l 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 preparation of deoxynegamycin the ~
lysine used as the starting material is made9 for example by the treatment of (R,R)-~-hydroxy-~-lysine with red phosphorus and hydroiodic acid in a sealed tube at 150 C.

. .. .

~133~76 for 2 hours. The (R,R)-~-hydroxy-~-lysine ls obtained by hydrolysis of negamycin or by synthesis from D-galacturonic acld (J. Am. Chem. Soc., 94~ 4353, 1972). Amino-protective groups are introduced lnto 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-methylnegamyci~ 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)-~-methox~~~-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 temperature and the reaction mixture is concentrated to obtain the ~ethyl 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 dichloro~ethane followed by hydrolysis with sodium hydroxide in ethanol to yield di-N-benzyloxycar-bonyl-(R,R)-~-methoxy-~-lysine.
In the coupling process the condensation forming 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.

For instance, the di-N-benzyloxycarbonyl deriva-tive of the ~-substituted (R,R)-~-hydroxy-~-lysine is converted to an active ester by treatmen~ with N-hydroxy-succinimide and dicyclohexylcarbodiimlde in a nonagueous solvent. The active ester is condensed with 1-1.5 moles of l-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 bromlde 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 Schiff base the coupling products 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 -8-1~83~

formula:

A~ (R) (R) CH2CHCH2fHCH2CONHNCH2COOH (VI) B

wherein A is H and B is a monovalent amlno-protective group or A together with B is one divalent amino-protective group. As the amino-protective groups ~or negamycin,.all known amino-protectivè 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 ~
formula VI) in high yield. The 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 dichloromethane to obtain an ~ ?~`. .
amino-protected derivative of O-methylnegamycin methyl ester as shown by the following formula:

NCH21HCH21HCH2CONHICH2COO 3 (VII) ~ ~B CH~
wherein A and B mean the same groups described above. The _g_ .

.

-amino-protective group and methyl ester group are removed by hydrolysis followed by column chromatography with cation exchangers having carboxylic acid as the active group, and i.~ 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-methylnegamycin which comprises converslon of amino-protected derivatives of negamycin (VI) A~ (R) (R) f ~ A ¦ (VI) OH ~ B 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).

- NCH2CHCH21HCH2CONHNC~2COOC~3 (VII) OCH N CH~
3 ~ B
In this procedure the reaction with diazomethane is carried but in an inert organic solvent~for instanceg halogenated hydrocarbons such as dichloromethane. ~he removal of the amino-protective groups and methyl ester group is carried out by ordinary procedures, for instanc~
by hydrolysis in alkaline conditîon.
Furthermore, in the synthesis of deoxynegamycin from the amino-protected derivative of negamycin (VI), the only free carboxyl group of amino-protected negamycin is primarily protected with a common ester form, for instance3 -1~31~76 methyl , ethyl or benzyl ester to obtain an ester of amino-protected negamycin. And then the hydroxyl group at the ~-position of the ester is sulfonylated by treat-ment with alkylsulfonylating agent such as mesyl chloride, ary].sulfonating agent such as p-toluenesulfonyl chloride, or arylmethylsulfonylating agent such as benzylsulfonyl chloride to obtain the sul~onic acid ester derlvative as shown by VIII.-A~ (R) (R) ~,NCH2fHCH2CHCH2CONHNCH2COE (VIII) S03G ~ C~ ;

wherein A and B are the same as described in the above; i E is an alkyloxy group especially consisting of 1-4 carbon atoms or a benzylox~ 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 phenyl or benzyl group. :
The product (VIII) is treated with an iodide or bromide of alkali 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 of 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 amino-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~ ~ 3~

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-ne~amycin 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) NCH2fHCH2fHCH2CONH I CH2COE ( IX ) .
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 formlng 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 ~ollows An amino-protected derivative of negamycin (VI) prepared as above is treated with an agent for esteriflcation 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 3'~4' . ~ . .

~al83~6 dideoxykanamycin B as described in Japan Patent 7595/1975. ;
The reaction to convert the sulfonyl group (-SO~G) to halogen group (X) by the effect of an iodide or bromide of an alkali metal and the procedure to remove the ~-halogen group by catalytic hydrogenation is also carried out in known manner.
The properties of deoxynegamycin and O-methyl-negamycin which are the final products obtained in this invention are as follows:
Deoxynegamycin is a white powder showing d.po 120-125 C. [a]23 = _5o (c 1.5, H20) and elemental analysis to coincide with the theoretical value for CgH20N40~-H20 (C 43.18~, H 8.86%, N 22.39~). On silica gel thin layer chromatography (Art. 5721, Merck Co., Germany3 developed with n-butanol-ethanol-chloroform-17% ? -aqueous ammonia (4:5:2:5 by volume) it gives a single spot (ninhydrin) at Rf 0.~4.
O-Methylnegamycin is a white powder showing d.p. 137-140 C. ~a]22 = _3o (c 1.5, H20), and elemental ~nalysis to coincide with the theoretical value for CloH22N404 H20 (C 42.84~ H 8.63~, N 19.99~). 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-negatlve 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 solutionO Those two compounds `
are low in toxicity (LD50 of both in mice, i.v., ~200 mg./kg.) and are expected to be used in the chemotherapy ,' . . .
,' .,:

1~83~
of infections caused by various Gram-positi~e and Gram-negative bacterla.

TABLE: Antimicrobial Spectra of O-Methylnegamycin and Deoxynegamycin _ _ _ __ Minimum Inhibitory~~ ~
Concentrations*(Mc~./ml.) O-Methyl- Deoxy-Test Organisms _ Staphylococcus aureus FDA209P 12.5 25 Staphylococ us aureus Smith 6~ 25 6.25 .
Sarcina lutea PCI1001 25 ~100 - .
Micrococcus flavus FDA16 50 50 :
...,.
Bacillus subtlllR NRRL B-558 50 l cobacterium smegmatis ATCC607 25 50 Escherichia coli NIHJ 6.25 6.25 Escherichia coli K-12 3.13 3.13 Escherichia coli K-12 ML1629 1.56 3.1~
Salmonella ty~hi T-63 1. 56 o .78 Proteus vul~ is OX-l9 1. 56 3.13 Proteus rettgeri GN311 6. 25 6.25 .. ~
Proteus ~ E~ GN466 3.13 3.13 Serratia marcescens 25 25 ......
-Klebsiella pneumoniae PCI602 6. 25 6.25 Pseudomonas fluorescens 1. 56 3.13 Pseudomonas aeruginosa A3 12. 5 25 :
Pseudomonas aeru inosa No. 12 25 5o .
*Minimum inhibitory concentrations were determined on a o ~ 5~ peptone agar b~ incubation at 37 C . ~or 17 hours.
: ' .

-: ~: . . . . . .

1~83~ 6 :
DESCRIPTION OF THE PREFERRED E~ODIMENTS

(a) Synthesis of Di-N-benzyloxycarbonyl-D~ sine:
To a solution containing 100 mg. (o.69 mmole) D-~-lysine (~a]D5 = -22.5 (c o.8, lN HCl) 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-436-dimethylpyrimidin-2-ylthiolcarbonate (Kokusan Chemical Works, Tokyo) in 1 -ml. of 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 ad~usted to pH 2 with 6N HCl, saturated with sodium chloride and extracted with 13 ml. o~ ethyl acetate. The ethyl acetate layer was washed with 4 ml. of 5% HCl in water saturated with sodium chloride and wlth 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]26 =
-~6 (c 1.~, MeOH), 73~ yield. ~
.. :
(b) 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. A~ter the reaction mixture was allowed to stand at 5 C. for 17 ~-hours crystals of dicyclohexylurea deposited and were removed by ~iltration and the filtrate was concentrated .. , ~ ......... . . . . ~ . .. . . .

~831~7S

to dryness to obtain 97 mg. of N-hydroxysuccinimide ester of di~N-benzyloxycarbonyl-D-~-lysine as colorless crystals.
A solution containing the cr~stals obtained in the above dissolved in 2 ml. of 1~2-dimethoxyethane was graduall~ added to a solution containing 19.8 mg. (0.19 mmole) of l-methylhydrazinoacetic acid and ~2 mg. (o.38 mmole) o~ sodium bicarbonate in 0.5 ml. o~ water under stirring at room temperature. After 18 hours 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 O.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-carbonO After the mixture was stirred for 3 hours under an atmosphere of hydrogen the catalyst was removed by ~iltration and the filtrate was concentrated ,~ ' to dryness. The residue was dissolved in 2 ml. of water ad~usted to pH 8.4 with 5N ammonia-water and passed through and adsorbed on a column containing 8.5 ml. Amberlite CG-50 (NH4 ) resin. The column was washed with 20 ml. water and eluted with 90 ml. o~ 1~ ammonia-water. The eluate was collected as 1 Inl~ fractions.
Fractions No~ 57-62 having antibacterial activity to E.
coli K-12 were collected and concentrated to dr~ness to obtain 20.5 mg. o~ deoxynegamycin as a white powder.
Yield was 47~.

~ ~ _ .

~0831~76 (a) Synthesis o~ Di-N-benzyloxycarbonyl-(R,R)-~-h~droxy-~-lysine Methyl 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. of 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 44.3g. of di-N-benzyloxycarbonyl-(R,R)-~-hydroxy-~-lysine lactone as colorless crystals.
m.p. 128-130 C., ~a]22 = -13 (c 3.4, chloroform), 70%
yield. The crystals (34.2 g.) were dissolved in 360 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 recrystalllzed 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, chlorofor~, 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~

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 sulfate and concentrated to dryness under reduced pres-sure yielding 295 mg. of a pale yellow oily material.
The oily 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. fractions. 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. ~2~ 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 for

4 hours the mixture was acidified by the addition o~
0.28 ml. of lN HCl and extracted with 3 ml. of ethyl acetate. The ethyl acetate layer was washed with 1 ml. of water, dehydrated with anhydrous sodium sulfate and concentrated to dryness yielding 80 mg. of di-N-benzyloxycarbonyl-(R,R)-~-methoxy-~-lysine as a color-less oil. 98% yield.

(c) Synthesis of O-Met~ gamycin:
To a solution containing 80 mg. (0.18 mmole) of di-N-benzyloxycarbonyl-(R,R)-~-methoxy-~lgsine which was obtained in Example 2 (b), in 1.6 ml. of 1,2-dimethoxyethane, 21 mg. (0.18 mmole) of N-hydroxy-succinimide were added under ice cooling. After the reaction mixture was allowed to stand at 5 C. for 17 ~ 8~ /6 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-less 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) of l-methylhydrazinoacetic acid and 30 mg. (o.36 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. m e precipitate was dissolved ir 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 ~ 33~76 activity were combined and concentrated under reduced pressure to dryness to provide 20.8 mg. of O-methyl-negamycin as a white powder~ 44~ yield.

EXAMPLE_3 (a) S~nthesis of Di-N-benzyloxycarbonylnegamycin-To a solution containing 4 g. (16.1 mmoles) ofnegamycin ln 20 ml. of water and 2.44 g. (24.2 mmoles) of triethylamine~ 9.72 g. (~5.5 mmoles) o~ benzyl S-4,6-dimethylpyrimidin-2-ylthiolcarbonate in 20 ml. of dioxane was added and the reaction mixture was stirred at room temperature for 17 hours. To the reaction mixture ~0 ml. of water was added and the mixture was washed with two por-tions of 60 ml. of ethyl acetate. The aqueous layer was adjusted to pH ? 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]26 = ~4.2 (c 6.3, CH~OH), 92~ yield.

(b) Synthesis of O-Methylnegamycin:
To a solution containing 449 mg. (0.87 mmole) of di-N-benzyloxycarbonylnegamycin which was obtained in Example 3(a), in 4.5 ml. of dichloromethane, 62 mg, (0.44 mmole) of boron trifluoride etherate was added with ice-cooling and stirring followed hy the addition of 1.5~ diazomethane-ether solution until the yellow color ,.. , .. : . I . . .

~ 33~
of the reaction mlxture 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 mI. of ethyl ether and dried to obtain a yellow powder. The powder was dissolved with 20 ml. of water, adjusted 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. 32-34 giving 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.

~83 E~AMPIE 4 _ (a) Synthesis of Di-N-benzyloxycarbonylnegamycin Methyl Ester:
To a solution containing 6 g. (11.6 mmoles) of dl-N-benzyloxycarbonylnegamycin which was obtained in Ex~mple 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 di-N-benzyloxycarbonylnegamycin methyl ester which ~ -was obtained in Example 4 (a), in 150 ml. of pyridine a solution containg 6.43 g (56.4 mmoles) of methanesul-fonyl 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 wlth 150 ml. of ice water and extracted with 240 ml. o~
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 yielding 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) ~

~ 33~6 of the pale yellow powder so obtained in 225 ml. of acetone, 7.2 g. ~48 mmoles) of sodium lodide, which was thoroughly dried at 50-60 C. overnight, was added.
The mixture was refluxed at 65 C. for 15 hours, diluted with 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 benæene-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 1.3 g. (2 mmoles) of ~-iodo-derivati~e (a mixture of 2 kinds of stereoisomer). 22%
yield.
To a solution containing 937 mg. (1.46 mmoles) of the ~-iodo-derivative thus obtained in 20 ml. of methanol and 8 ml. of water, 10 g. of 5~ palladium-barium carbonate was added at room temperature under -stirring and introduction of hydrogen gas to carry out simultaneously 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 pH 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.

' , . . . .
. . .

1q)831~

fractions. Fractions No. 20-50 having antibacterial activity versus E. coli K-12 and positive ninhydrin reaction were combined and concentrated to dryness under reduced pressure to yield 95 mg. of deoxynega-mycin as a whita powder. 28~ yield.

.:

, .

Claims (6)

Division II

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the production of deoxynegamycin having the formula comprising the consecutive steps of (a) catalytically hydrogenating a compound of the formula wherein X is bromo or iodo A is hydrogen and B is a monovalent amino-protective group or A together with B are a divalent amino-protective group, and E is an easily removable ester forming group and then (b) removing any remaining ester forming and amino-protective groups.

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 X is iodo.

4. The process according to Claim 1, 2 or 3 wherein the steps (a) and (b) are carried out simultaneously by introducing Division II
hydrogen gas in the presence of palladium-barium carbonate as the catalyst.

5. The process according to Claim 1 wherein E is an alkoxy group consisting of 1-4 carbon atoms or a benzyloxy group

6. The process according to Claim 1, 2 or 3, further comprising a step of forming a pharmaceutically acceptable nontoxic salt or acid addition salt of deoxynegamycin.