CA1081693A - Kanamycin c derivatives - Google Patents

Abstract

NEW KANAMYCIN C DERIVATIVES

ABSTRACT OF THE DISCLOSURE

1-N-(L-4-amino-2-hydroxybutyryl) derivatives of kanamycin C, 3'-deoxykanamycin C and 3',4'-dideoxykanamycin C have been pre-pared which possess high antibacterial activity against a wide variety of drug-resistant bacteria. These new derivatives are prepared by reacting L-4-amino-2-hydroxybutyric acid or a functional equivalent thereof with the l-amino group of kanamycin C, 3'-deoxykanamycin C or 3',4'-dideoxykanamycin C.

Description

108169~

BACKGROUND OF THE INVENTION
Field of the invention This invention relates to new kanamycin C
derivatives active against a wide variety of kanamycin-resistant bacteria, and a process for the preparat.ion of these new kanamycin C derivativesO
More particularly, this invention relates to new and useful l-N-(L-4-amino-2-hydroxybutyryl) derivatives of kanamycin C, 3'-deoxykanamycin C
and 3',4'-dideoxykanamcyin C, and the preparation thereof.
Description of the prior art Butirosin B9 that is, 1-N--(L-4-amino-2-hydroxybutyryl)-ribostamycin may be produced by a fermentative method ("Tetrahedron Letters" 28, 2617-2620 (1971)), and 1-N-(L-4-amino-2-hydroxy-butyryl) derivatives of kanamycin A and kanamycin B
are synthesiæed (UOSo Patent No. 3,781,268 (1973)).
l-N-(L-4-amino-2-hydroxybutyryl) derivatives of other some aminoglycosidic antibiotics are also synthesized (UoK~ Patent NoO 1,426,908 (published in March 1976))o Furthermore~ deoxy derivati~es of kanamycins are also synthetized on the basis of the previous findings ~Jhich were obtained by H. Umezawa et al with respect to the mechanism of resistance of bacteria to aminoglycosidic antibiotics owing to various inactivating enzymes produced by the resistant bacteriaO For instance, 3',4'-dideoxykanamycin B and 3'-deoxykanamycin B
are synthesized which are active against the

- 2 -10~ 3 resistant bacteria producing aminoglycoside 3'-phosphotransferase (U. 5. Patent Nos. 3,753,873 and 3,929,762; and H. Umezawa's "Advances in Carbohydrate Chemistry and Biochemistry" 30, 183 (1974) and "Drug Action and Drug Resistance in Bateria" 2, 211 (1975)). 3',4'-Dideoxykanamycin B has been widely used in thera-peutic treatment of infections caused by a variety of the resis-tant bacteria, including Pseudomonas aeruginosa. However, it has been found that these deoxy derivatives of kanamycin B do not in-hibit the growth of such resistant bacteria which are capable or producing the aminoglycoside 6'-acetyltransferase and 2"-nucleo-tidyltransferase. As a result of a further research, we have succeeded in synthetically converting the 6'-amino group of kana-mycin B or its doexy derivatives into hydroxyl group and thereby producing kanamycin C and its deoxy derivatives which inherently cannot be inactivated by the 6'-acetyltansferase (see co-pending Canadian Patent Application S.N. 280,338 filed June 10, 1977).
However, kanamycin C, 3'-deoxykanamycin C and 3',4'-dideoxykana-mycin C so obtained are not able to inhibit the growth of such resistant bacteria producing the 2"-nucleotidyltransferase.
SUMMARY OF THE INVENTION
In these circumstances, we have made further research, and as a result we have succeeded in synthesizing new compounds, t l-N-(L-4-amino-2-hydroxybutyryl) derivatives of kanamycin C,3'-.' :, .'~

~ 30 . .

,:

3 --~081~;93 deoxykanamycin C ~nd 3',4'-dideoxykanamycin C which are active against a wide variety of aminoglycoside-resistant bacteria, including the 2"-nucleotidyl-transferase-producing bacteria.
Thus, an object of this invention is to provide such new kanamycin C derivatives which inherently cannot be inactivated by the 3'-phospho-transferase extensively distributed in a variety of the aminoglycoside-resistant bacteria and also cannot be inactivated by the 6'-acetyltransferase and the 2"-nucleotidyltransferase, which is accordingly active against not only the aminoglycoside-resistant bacteria producing the 3'-phosphotransferase, but also the aminoglycoside-resistant bacteria producing the 6'-acetyltransferase and the aminoglycoside-resistant bacteria producing the 2"-nucleotidyl-transferase and which exhibits a very much low toxicity. Another obJect of this lnvention is to provide a new process for the preparation of such new kanamycin C derivatives which can be operated in a facile way and with a reasonable efficiencyO Another objects will be seen from the following descriptionsO
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of this invention, there is provided the novel l-N-(L-4-amino-2-hydroxybutyryl) derivativeSof kanamycin C, 3'-deoxykanamycih C or 3',4'-dideoxy-kanamycin C represented by the general formula:

- 4 -.

10~1~93 6~
4 l 2' Rl 4 HO - ~ O \ H2N 7 \ R2 H2N ~ 6 OH 14 ~ CH20H

~ ~ ~\5--'-~

HN ~ 3~ MH2 (I) CO
(L) CHOH

wherein Rl and R2 are each hydroxyl, or Rl is hydrogen and R2 is hydroxyl or Rl and R2 are each hydrogen or a nontoxic, pharmaceutically acceptable acid-addition 6alt thereof.

The new compounds according to this invention include l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C ~the compound of the formula (I) where both R
and R2 are hydroxyl group); 1-N-(L-4-amino-2-hydroxybutyry~)-3' deoxykanamycin C ~the compound of the formula (I) where Rl is hydrogen atom and R2 is . hydroxyl); and l-N-(L-4-amino-2-hydroxybutyryl~-3',4'-dideoxykanamycin C ~the compound o~ the formula (I) where both Rl and R2 are hydrogen atom) as : well as pharmaceutically acceptable nontoxic acid-addition ~ salts thereofO The chemical, physical and biological properties of these new compounds are as follows:
l-N-(L-4-Amino-2-hydroxybutyryl)-kanamycin C
is a substance in the form of a colorless powder having no definite melting point but decomposing ~81t;93 at 167-180C. It shows a specific optical rotation ~~)D +91 (c 1, water). Its elemental analysis is coincident with the theoretical values of C22H43N5O13.H2O (C 43.77%, H 7.51%, N 11.60%).
This substance gives a single spot positive to ninhydrin at Rf.
0.18 in thin layer chromatography on silica gel (available under a trade marlc "ART 5721", a product of Merck Co., Germany) deve-loped with butanol-ethanol-chloroform-17% aqueous ammonia (4:5:2:8 by volume) and at Rf 0.19 in the same thin layer chromatography developed with chloroform-methanol -28% aqueous ammonia (1:4:2 by 10 volume) as the development solvent, respectively.
l-N-(L-4-Amino-2-hydroxybutyryl)-3'-deoxykanamycin C
is a substance in the form of a colorless powder having no definite melting point but decomposing at 151-160 C. It shows a specific optical rotation (~)D +83 (c 1, water). Its elemental analysis is coincident with the theoretical values of C22H43N5O12.H2O
(C 44.96%, H 7.7296, N 11.92%). This substance gives a single spot positive to ninhydrin at Rf 0.22 in the above-mentioned silica gel thin layer chromatography developed with the first-mentioned development solvent and at Rf 0.24 in the same thin layer chroma-20 tography developed with the second-mentioned development solvent, respectively.
L-N-(L-4-Amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C is a substance also in the form of a colorless pcwder having no definite melting , , .

~ - 6 -~ "~, . ",~ .

~ 081~3 point but decomposing at 142-158Co It shows a specific optical rotation ~)28 ~76 (c 1, water)D
Its elemental analysis is coincident with the theoretical values of C22H43N5011 H2 H 7D940~ N 12~25%)o It gives a single spot positive to ninhydrin at Rf 0O31 in the above-mentioned silica gelthinlayer chromatogr~phy developed with the first-mentioned development solvent and at Rf O.30 in the same thin layer chromatography developed with the second-mentioned development solvent~
The molecular structure of these new kanamycin C
derivatives has been identified by acid hydrolysis .and by lH and 13C nuclear magnetic resonance absorption spectraO
The minimum inhibitory concentrations (mcg/ml.) o~ l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C (abbreviated as AHB-KC), l-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C
(abbreviated as AHB-DKC) and l-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykana~ycin C (abbre-viated as AHB-DDKC) against various microorganisms were determined according to serial dilution method on nutrient agar medium at 37C, the estimation being effected after 18 hours incubationO
For comparison, the minimum inhibitory concentrations o~ l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin A
(amikacin) was also determined in the same manner as described aboveD
The antibacterial spectra of these substances are shown in Table 1 belowO

10~ ;93 .
:~ ~
. 1~ N ~ C-- 1~ ~ ~ ~ U~
O O O O O O O O ~1 0 0 0 ~j ~
_ I U~ ~J 11~ U~ U~ U~ U~
~ r-l ~1 ~1 ~1 ~I N N~ N

~q C) ~1 ~ U~ U~ U~ U~ U~
t~ ~ N U~ N N N N U~ N u~ u~ N r-l O $ ~0 N ~ L) ~D N ~D ~ N ~t) 1 ~ U~ ~ U~ Lr~ U~ U~
$ f q N r1 N U~ N N U~ 1-'\ U~ N I
,Q ~3 ~ ~U~ N ~O N

,~
fi ~ ~ u~
~ ~ o o o h h .. o ..
~ ~ ~ o _ = = - = = = = _ ~ t>
E~ o r~
O h ~t t) a) ~:
o ~ C~
.. ~
h . P1 0 td ':, ~

~O~i~93 O ~D~~D ~~ ~ u~
o o ~~ o ~ ~
o ,,o ~ ~~ ~ o ~, ,, --' L~
U~ ~ ~.
~oU~ooooo ,, ~ ~ ,1 ~ ,, ~o ~o OOOOO

, ~ ~ U~ Ln ~U~ooooo :
o~
o r.
~, ~ ~ ~ ~ 3 a' ~o ta o o ~P
o ~, ,.
~, ~ , P.
o~
.,1 ~ ~
h = ,1 = o ~ ~,q ,~

.0 P~

_ g _ lV81693 From the above Table, it is seen that the new compounds of this invention inhibit the growth of many kinds of bacterial strainsO The new compounds of this invention further exhibit a extremely low acute toxicity to animals and men.
It has been estimated that AHB-KC and AHB-DKC
have an LD50 value cf more than 400 mg/kg upon intravenous injection in miceO Accordingly, the new compounds of the invention are promising as chemotherapeutic agents for therapeutic treatment of infections caused by gram-negative and gram-positive bacteriaO
l-N-(L-4-Amino-2-hydroxybutyryl)-kanamycin A
(amikacin) and 1-N-(L-4-amino-2-hydroxybutyryl)-kanamycin B are synthetized by Kawaguchi et al (U.SO Patent NoO 3,781,268), and these known compounds are acetylated by a 6'-acetyltransferase which is obtained from aminoglycoside-resistant strains, such as Pseudomonas aeruginosa GN315 (see, M~ Yagisawa et al9 J. Antibiot., 28, 486 (1975)) and in this respect they are in contrast with the new compounds o~ this invention which are not inactivated by the 6'-acetyltransferaseO
The new compounds of the formula (I) according to this invention may readily be converted into the form of a pharmaceutically acceptable, nontoxic acid-addition salt such as the hydrochloride, sulfate, phosphate, nitrate 9 acetate, maleate, fumarate, succinate 9 tartarate, oxalate, citrate, ascorbate, methanesulfonate, ethanesulfonate and the like by 1081~93 reacting the free base form of l-N-(L-4-amino-2-hydroxybutyryl) derivative of kanamycin C, 3'-deoxykanamycin C or 3'94'-dideoxykanamycin C with the appropriate acid in ~queous mediumO The new kanamycin C compounds of this invention and their pharmaceutically acceptable acid-addition salt may be administered orally, intraperitoneally, intravenously, subcutaneously or intramuscularly using any pharmaceutical form known to the art for such administration and in a similar manner to the known kanamycins. For instance, the new compounds of this invention may be administered orally using any pharmaceutical form known to the art for oral administration. Examples of the pharmaceutical forms for oral administration are powders, capsules 9 tablets 9 syrup and the like.
A suitable dose of the new compounds of this invention for effective treatment of bacterial infections is in a range of 005 to 4 gO per person a day when it given orallyO It is preferred that said dose should be orally administered in three to four aliquots per day. The new compounds of this invention may also be administered by intramuscular injection at a dosage of 200 to 2000 mg per person two to four times per dayO
Moreover, the new`compounds of this invention may be formulated into an ointment for external application which contains the active compound at a concentration of 0.5-5% by weight in mixture with a known ointment base such as polyethylene . .

10131f~93 glycol. Furthermore 9 the new compounds of this invention are useful for sterilization of surgical instruments~
According to a second aspect of this invention9 therefore, there is provided an antibacterial composition comprising as the active ingredient l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C, l-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C or l-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C or an acid-addition salt thereof in an antibacterially effective amount to inhibit the growth of bacteria, in combination with a carrier or vehicle for the active ingredient compound.
According to a further aspect of this invention, there is provided a process for the preparation of the l-N-(L-4-amino-2-hydroxybutyryl) derivative of kanamycin C, 3'-deoxykanamycin C
or 3',4'-dideoxykanamycin C represented by the general formula (I) shown hereinbefore, which comprises the steps ofo-acylating the l-amino group of kanamycin C, 3'-deoxykanamycin C or 3',4'-dideoxykanamycin C represented by the general formula (II):

1081f~93 6~
4" CH20H O 2' Rl 4~
~z ~ ~ H2N~ ~R2 H2N ~ ~ 6H20H

.. 0~0 H2N ~ / ~ MH2 (II) wherein Rl and R2 are each hydroxyl, or Rl is hydrogen and R2 is hydroxyl or Rl and R2 are each hydrogen,or a nontoxic, pharmaceutically acceptable acid-addition salt thereof, by reaction with an amino-protected derivative of L-4-amino-2-hydroxybutyric acid or a functional equivalent thereof, to produce the l-N-acylation product of the starting compound (II), .:
and removing the amino-protecting group from the resulting 1-N-acylation product to give the compound of the formula (I). :
The present process may include a further step of reacting the compound of the formula (I) so obtained, with a pharmaceutically acceptable acid to produce the corresponding pharmaceuticaIly acceptable acid-addition salt of said compound .~ of the formula (I) 9 if desired.
In one embodiment of the process of this invention, the process may comprise the following steps of:-reacting the starting compound of the : formula (II) of which amino groupsare not protected, with an amino-protected derivat.i~e of L-4-amino-2-. -hydroxybutyric acid represented by the formula (III):

(L) / R3 Hooc-cH(oH)-cH2cH2- N~\ (III) ~R4 wherein R3 is a hydrogen atom and R4 is a known mono-valent amino-protecting group such as alkyloxy-carbonyl, cycloalkyloxycarbonyl and aralkyloxy-carbonyl, or R3 and R4 together form a divalent amino-protecting group such as phthaloyl group, or a Schiff base group (N=CHR5 in which R5 is a hydrogen atom, an alkyl group o~ 1-4 carbon atoms or an aryl group such as phenyl), in a manner known for the acylation of amino group, to produce the mixed acylation products comprising a l-N-(L-4-protected-amino-2-hydroxybutyryl)derivative of thestartln~ compound of the ~ormula tII).
treating the mixed acylation products in a known manner to remove the amino-protecting group therefrom, and then isolating the desired l-N-(L-4-amino-2-hydroxybutyryl) derivative of the antibiotic according to the ~ormula (I) by chromatographic separation of the acylation products from which the amino-protecting group has been removed, to recover the desired compound of the ~ormula (I).
In carrying out the process of this invention, the starting kanamycin C, 3'-deoxykanamycin C or 3',4'-dideoxykanamycin C o~ the formula (II) of which amino groups are not prctected, may be used either in the form o~ the free base or in the form ~081~93 of its acid-addition salt with an appropriate acid such as hydrochloric acid or sul~uric acidD In the acylation step of the present process, the reactionmay be conducted generally as ~escribed in the specification of U.K. Patent No~ 1,426,908 or U.S. Patent NoO 4,001,208. Preferably, the starting compound of the formula (II) is dissolved in water and theresulting aqueous solution is adjusted to a pH of' 6-8 and more conveniently at a pH o~ 6.5-7.0 by addition of an ordinary acid such as hydrochloric acid and sulfuric acid or an ordinary base such as aqueous sodium hydroxide and aqueous potassium hydroxideO To this solution of the above-mentioned partially protonated form of the startin~ compound is added a solution of an amino-protected derivative of L-4-amino-2-hydroxy-butyric acid according to the formula (III) or a reactive derivative thereof which acts as a functional equivalent of the aforesaid amino-protected butyric acid derivative (III). In this way, the l-amino group of the starting kanamycin C compound (II) is acylated with the acid (III).
The amino-protecting group available (for R3 and/or R4) in the amino-protected L-4-amino-2-hydroxybutyric acld derivative (III) employed in the acylation step of the present process may be a known amino-protecting group which is usually used in the conventional synthesis of peptides.
However, the a~ino-protecting group employed must be such one of the nature which is removable .

~081~;93 readily by such a procedure and such reaction conditions which well not break or damage sub--stantially the amido linkage present be~ween the L-4-amino-2-hydroxybutyryl substituent and the l-amino group of the am,inoglycosidic moiety of the resulting acylation product when the removal of the amino-protecting gro~p from the l-N-amino-alkanoyl substituent of the acylation product is operatedO Suitable examples of the mono-valent amino-protecting group which are available (as the group R4) for the above purpose include an alkoxycarbonyl group of 2-6 carbon atoms such as tert-butoxycarbonyl ard tert-amyloxycarbonyl;
,a cycloalkyloxycarbonyl grou~ of 3-7 carbon atoms such as cyclohexyloxycarbonylg an aralkyloxy-carbonyl group sv.ch as benzyloxycarbonyl and p-methoxybenzyloxycarbonyl; and a substituted alkanoyl gro~p of ~-5 carbon atoms such as tri-fluoroacet~l and o-nitrophenoxyacetyl~ Preferred examples of the diva].ent amino-protecting group available (as the groups R3 and R4 taken together) for the above-mentioned prupose include phthaloyl group and a group of ~chiff base type such as salicylidene~ The-ntroductiQn of the amino-'~ protecting group into L--4-arnino-~-hydroxybutyric acid may be achieved by reacting the,latter with an appropriate reagent for the introduction of the amino-p.ro-tecting group which is in the form of an acid halide~ acid azide 9 active ester or acid anhydrideg in the same manner as described , - 16 -1081f~93 e gO in the specifications of UOSo Patent NosO
~,929,762 and 3,939,143 as well as the aforesaid UoK~ Patent NoO 1,42699080 The acylation of the l-amino gro-up of the starting kanamycinC compound of the formula (II) with the amino-protected L-4-amino-2-hydroxy-butyric acid (III) may be conducted according to the conventional methods for the synthesis of amides using the acylating reagent (III) in the form of active ester such as N-hydroxysuccinimide ester, mixed acid anhydride or acid azideO As the starting kanamycin C compound (II) is insoluble or sparingly soluble in any organic solvent but soluble in water, it is pre~erred that the acylation . reaction should be carried out in an aqueous reaction medium using the acylating agent (III) in the form of its active esterO For instance, a solution containing a 1-2 molar proportion of the N-hydroxysuccinimide ester of L-4-tert-butoxy-carbonylamino-~-hydroxybutyric acid in a water-miscible organic solvent such as 1,2-dimethoxyethane and dimethylformamide may be admixed with an aqueous solution of 1 molar proportion of the starting kanamycin C compound (II) at a pH of 6-8 at ambient temperature under stirring to effect the desired acylationO The reaction temperature may be elevated, if desiredO The reaction time may be for several hours and preferably for 5-6 hoursO
The acylation product so obtained is, in fact, in the form of the mixed acylation products ;:
~ - 17 -1081~i93 comprising the desired 1-N-(L-4-protected amino-2-hydroxybutyryl) derivative as well as the undesired, otherwise mono-N-acylated, di-N-acylated and poly-N-acylated products in which one or more of the l-amino, 3-amino, 2'-amino and possibly 3"-amino groups of the kanamycin C moiety has or have been bonded with the L-4!protected amino-2-hydroxybutyryl substituent. The acylation product (the mixed acylation products) as such may be directly treated in the second step of the present process for the removal of the amino-protecting group, which is carried out in a manner known per se in the peptide synthesisO All of .the amino-protecting groups of .the above-mentioned nature may be removed readily by weak acid hydro-lysis using an aqueous solution of trifluoroacetic acid or acetic acid or dilute hydrochloric acid.
When the amino-protecting group is an aralkyloxy-carbonyl group9 the latter may be removed also by an ordinary hydrogenolysisO
The deprotected acylation product obtained from the second step of the present process is actually also in the from ofthe mixed products containing the desired l-N-(L-4-amino-2-hydroxy-butyryl)-kanamycin C 9 -3'-deoxykanamycin C or -3~,4'-dideoxykanamycin C of the formula (I) andthe undesired, otherwise acylated derivatives of the starting kanamycin C compound, together with the unreacted starting kanamycin C compound (II). In order to isolate the desired l-N-10~16i93 acylation product (I) from the above mixed products, the latter may be sub~ected to a chromatographic separation method, such as ion-exchange chromato-graphy uæing a cation-exchanger containing carbo-xylic functions, for example, Amberlite CG-50 (a product of Rohm & Haas Co., U.S.A.), CM-sephadex C-25 (a product of Pharmacia Co., Sweden) and carboxymethylcellulose; ion-exclusion chromato-graphy using strong anion-exchange resin, for example, Dowex l-X2 (a product of Dowex Co., U.S.A.); and column chromatography using silica gel.
In th~s way, the isolation and recovery of the desired product (I) may be achieved with high efficiencyO Particularly, it is recommendable that the deprotected mixed acylation product is chromatographed on Amberlite CG-50 (a weak cation-exchange resin containing carboxylic functions) using diluted aqueous ammonia as the development solvent, because this chromatographic procedure enables the desired product (I) as well as the unreacted starting kanamycin C compound (II) to be recovered efficiently and in pure state.
According to these chromatographic methods, the desired kanamycin C derivative of the formula (I) is usually recovered ir, the form of its free base, hydrate or carbonateO
*
"Sephadex LH-20" is a lyophilic insoluble molecular-sieve chromatographic medium made by cross-linking dextran and marketed by Pharmacia, Uppsala, Sweden. Sephedex LH-20 can be replaced by other æimilar gel-filtration agents, e.g. Sephadex G25 to *Trade Marks 1 ~ 8 1 ~9 ~

G200, Sepharose 4B and 6B (Pharmacia Fine Chemicals AB, ~ppsala, Sweden) and Bio-Gel Al.5 m (Bio Rad Co.). Pre-ferred gel-filtration agents include the carboxy-methyl substituted cross-linked dextran gels described in columns 3 and 4 of U.S. Patent No. 3,819,836.
Dowex l-X2(OH ) is the basic or hydroxlde form of cholestyramine resin ~hich in its chloride form is a synthetic, strongly basic anion exchange resin containing quaternary ammonium functional groups which are attached to a styrene-divinylbenzene copolymer. Main constituent:
Polystyr~ne trimethylbenzylammonium as Cl anion, also contains divinylbenzene (about 2%) and water ;. . _ _ . ' . :
... -CH C~2 CH CH2 ^-CN CN2N+(CN~)~Cl ~ :

,-typi~ied ~tructure o~ main polymeric group~

(about 43%). Cros~ l~nkage ~ 10.. Particle size: 50-lOO mesh. Percent volume increa3e, new to exhausted (Cl to OH ) - 20%. Stable at temperatures up to 150. Capacity: 3.5 meq/g : dry, 1.33 meq/ml wet.
*Trade Marks t~`7 -20-I
,. .

~(:)8~3 "Amberlite" is a registered trademark o~ the Rohm and Haas Company, Philadelphia, Pennsylvania.
Amberlite IRC-50 and CG-50 are tradenames for weakly acidic cation exchange resins of a carboxylic-polymethacrylic type.
The invention is now illustrated with ', reference to the following ExamplesO
E~ample 1 Synthesis of l-N-(L-4-amino-2-hydroxy-butyryl)-kanamycin C
A solution of 500 mg (1002 millimole) of kanamycin C free base (hemihydrate) in 10 ml.
of water was adjusted to pH 6.35 by addition of ,3.2 mlO of lN hydrochloric acid. To this solution was dropwise added over 5 minutes a solution of 500 mg (1058 millimole) o~ N-hydroxy-succinimide ester of L-4-tert-butoxycarbonylamino-2-hydroxybutyrlc acid ln 5 ml of dimethylformamide at ambient temperature under stirringO The admixture so obtained was stirred further for 6 hours to effect the acylation. The reaction solution comprising l-N-(L-4-tert-butoxycarbonylamino-2-hydroxybutyryl)-kana.~ycin C so formed was con-centrated to dryness under reduced pressure, and the solid residue ras admixed with 16 ml of an aqueous solution of 90% trifluoroacetic acidO
The mixture was ~gitated for 1 hour at ambient temperatureto effect the removal of the amino-protecting tert-butoxycarbonyl groupO The reaction solution was concentrated to dryness ' ;, -21-. .

1C181~;93 under reduced pressure and the residue was washed twice with 20 ~lof ethylether to give 1u55 g of a colorless powder comprising a crude product of l-N-(L-4-amino-2-hy~roxybutyryl)-kanamycin C0 T~.is cru~e product was taken up in 15 ml Of water and the resulting solution was adjusted to pH 702 by addition of lN aqueous ammonia and then rassed through a column (inner diameter 20 mm) of 190 ml of Amberlite CG-50 (NH4-form) for the adsorption of the desired productO After the column was washed with water (660 ml), it was eluted with 1940 ml of O.lN aqueous ammonia and then with 1540 ml of 0.4N aqueous ammonia. The eluate was collected in 20 ml-fractionsO The fractions Nos. 75-106 of the eluate obtained using OolN
aqueous ammonia were combined together and con-centrated to dryness under reduced pressure, recovering 199 mg of unreacted kanamycin C
(recovery yield,40%). The fractions Nos. 152-175 of the eluate obtained using 004N aqueous ammonia were combined together and concentrated to dryrless under reduced pressure to give 214 mg of a colorless powder containing the desired product~ This powder was suspended in 2 ml of a mixed solvent of chloroform-methanol-17% aqueous ammonia (1:4:2 by volume), and the resulting suspension was passed through a column (inner diameter 14 mm) of 50 g of silica gelO The silica gel column was then developed with the above-mentioned mixed solvent, and the eluate was collected in 10 ml-frac-tions. The fractions Nos. 86-114 were combined together , 108~6i93 and concentrated to dryness under reduced pressure,and the residue was taken up into water~ The resulting aqueous solution was chromatographed in a column of 5 ml of Amberlite CG-50 (NH4-form) using 0.5N aqueous ammonia as eluent. The eluate was concentrated to dryness under reduced pressure, affording 49 mg of pure 1-N-(L-4-amino-2-hydroxy-butyryl)-kanamycin C in the form of a colorless powderO Yield 800%o This powder had no definite melting point and deoomposed at 167-180C.

~a)D ~91% (c 1, water).
Example 2 Synthesis of l-N-(L-4-amino-2-hydroxy-butyryl)-3'-deoxykanamycin C
A solution of 440 mg (0.~2 millimole) o~
3'-deoxykanamycin C free base (hemihydrate) in 808 ml of water was adjusted to pH 6060 by addition of 2.75 ml of lN hydrochloric acid. To this resulting solution was dropwise added over ,10 minutes a solution of 449 mg (1.42 millimole) of N-hydroxysuccinimide ester of L-4-tert-butoxy-carbonylamino-2-hydroxybutyric acid in 407 ml of dimethylformamide at ambient temperature under stirringO The admixture so obtained was stirred further for 6 hours at ambient temperature to effect the acylation~ The reaction solution containing 1-N-~L-4-tert-butoxycarbonylamino-2-hydroxybutyryl)-3'-deoxykanamycin C so formed was concentrated to dryness under reduced pressure.
The solid residue was admixed wi~h 11 ml of an aqueous solution of 90% trifluoroacetic acid, and 1081~;93 the mixture was agitated at ambient temperature for 1 hour to effect the removal of the amino-protecting tert-butoxycarbonyl group. The reaction solution was concentrated to dryness under reduced pressure and the residue was washed twice with 20 ml o~ ethylether to give 2.12 g of a crude product of l-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C as colorless powderO
This crude product was taken up into 15 ml of water, and the resultant aqueous solution was adjusted to pH 7.8 by addition of lN aqueous ammonia and then passed through a column (inner diameter 20 mm) of 220 ml of Amberlite CG-50 (NH4-form) to e~fect the adsorption of the desired product.
After the column was washed with 1040 ml of water, it was eluted with 1340 ml of 0.2N aqueous ammonia and then with 1320 ml of 0c5N a~ueous ammoniaO
The eluate was collected in 20 ml-fractionsO
The fractions Nos. 71-~0 of the eluate obtained using 0.2N a~ueous ammonia were combined together and concentrated to dryness under reduced pressure, recovering 149 mg of unreacted 3' deoxykanamy~in C
(recovery yield 34%)0 The fractions Nos. 134-145 of the eluate obtained with 005N aqueous ammonia were combined together and concentrated to dryness under reduced pressure to give 155 mg of a colorless powder containing the desired productO This powder was suspended in 2 ml of a mixed solvent of chloro-form-ethanol-17% aqueous ammonia (1:4:2 by volume), and the suspension so obtained was passed through a column (inner diameter 10 mm) of 25 g of silica gel, ' .
~ ~, 1081~;93 which was then developed with the above-mentioned mixed solvent. The effluent was collected in 3O2 ml-fractionsO The combined fractions Nos. 99-140 were concentrated to dryness under reduced pressure, and the solid residue was taken up into water and chromatographed in a column of 5 ml of Amberlite CG-50 (NH4-form) using 0O5N aqueous ammonia as the eluent The eluate was concentrated to dryness under reduced pressure. Pure l-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C was obtained as a colorless powder. Yield 46 mg (80 5%). This powder had no definite melting point but decomposed at 151-160C. (a)D7 +83 (c 1, water)0 Example 3 Synthesis of l-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C
A solution of 340 mg (0.74 millimole) of 3',4'-dideoxykanamycin C free base (hemihydrate) in 7.5 ml of water was adjusted to pH 6.70 by addition of 2.45 ml of lN hydrochloric acid.
To this solution was dropwise added over 5 minutes a solution of 373 mg (1.18 millimole) of N-hydroxy-succinimide ester of L-4-tert-butoxycarbonylamino-2-hydroxybutyric acid in 3.75 ml of dimethylformamide at ambient temperature under stirring. The admixture so obtained wa~ stirred further for 6 hours to effect the acylation. The reaction solution containing l-N-(L-4-tert-butoxycarbonylamino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C so formed was concentrated to dryness under reduced pressure, and the solid residue was admixed with 8.5 ml of an aqueous solution of 90% trifluoroacetic acidO

~081~;93 The mixture was stirred for 1 hour at ambient temperature to effect the removal of the amino-protecting tert-butoxycarbonyl groupO The reaction mixture was then concentrated to dryness under reduced pressure and the residue was wa~hed twice with 20 ml of ethylether to give 1.46 g of a crude product of l-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C as colorless powder.
This crude product was taken up into 14 ml of water and the resulting aqueous solution adjusted to pH 7.8 by addition of lN aqueous ammoniaO
The solution was then passed through a column (inner diameter 20 mm) of 220 ml of Amberlite CG-50 (NH4-form) for adsorption of the active compounds. After the column was washed with water (1040 ml), ~t was eluted with 1560 ml of 0.2N
aqueous ammonia and then with 1460 ml of 0.5N
aqueous ammonia so that the eluate was collected in 20 ml-fractions. The fractions Nos. 74-93 of the eluate obtained with 0.2N aqueous ammonia were combined together and concentrated to dryness under reduced pressure to recover 153 mg of unreacted 3',4'-dideoxykanamycin C (recovery yield 45%?o The fractions Nos. 152-168 of the eluate obtained with 0.5N aqueous ammonia were combined together and concentrated to dryness under reduced pressure to give 116 mg of a colorless powder containing the desired product.
This powder was suspended in 2 ml of a mixed solvent of chloroform-ethanol-17% aqueous ammonia (1:4:2 by volume), and the resulting suspension was placed in a column (inner diameter 10 mm) of . .

, 1081f~93 25 g of sllica gel, which was then eluted with the above-mentioned mixed solvent. The effluent was collected in 3.2 ml-fractions, and the fractions Nos. 71-96 were combined together and concentrated to dryness under reduced pressureO The residue was dissolved in water and the solution was chromato-graphed in a column of 5 ml of Amberlite CG-50 (NH4-form) ln the same manner as above using 0.5N
aqueous ammonia as the eluentO The eluate was concentrated to dryness under reduced pressure to afford 35 mg of 1-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C as colorless powder.
Yield 8.3%. This product showed no definite melting point but decomposed at 142-158C. ~)28 +76 (c 1, water)0 Example 4 The following tests were made to estimate how the pH of the reaction medium where the acylation reaction proceeded affected the yield of the , j .
desired acylation product 1-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C
Solutions each containing 50 mg (0.10 millimole) of kanamycin C free base (hemihydrate) in 0.5-1.0 ml of water were prepared and adjusted to different pH values by addition of varying volumes of lN hydrochloric acid. To each solution was added dropwise a solution of 49 mg (0.15 milli-mole) of N-hydroxysuccinimide ester of L-4-tert-butoxycarbonylamino-2-hydroxybutyric acid at ambient te~perature under stirring. The admixture so obtained was stirred for 6 hours to effect the acyl~tion. The reaction solution was concentr&ted ~ 9 3 to dryness under reduced pressure and the solid residue was taken up into 1.2-1.4 ml of aqueous 90% trifluoroacetic acidO The resultant solution was stirred for 1 hour at ambient temperature, and the reaction solution was concentrated to dryness under reduced pressure. The residue was washed with ethylether to give a crude productO
This crude product was chromatographed in the same manner as in Example 1 using a column (inner diameter 12 mm) of 20 ml of Amberlite CG-50 (NH4-form), so that the unreacted kanamycin C was recovered in the pure state and a crude powder containing l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C was obtained~ The contents of l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C in the crude powders so obtained were determined by measuring the antibacterial potency according to a standard cup-plate assay method using Bacillus subtilis PCI Zl9 as the test organismO In Table 2 below, there are shown the yield of unreacted kanamycin C recovered and the yield of the desired product formed, together with the pH value of the aqueous solution of the starting kanamycin C, the volume of water employed for the dissolution of the starting kanamycin C and the volume of the lN
hydrochloric acid added for the adjustment of the pH value~

.

.. , ~.
-:

1~38169~

Table 2 Volume Volume Yield of Yield of of of lN kanamycin C desired water HCl recoverd product ~H (ml) (ml) ) formed 6.30 005 0.32 43 18 6.75 1.0 0.30 38 23 7.40 0.5 0.21 27 19 8.35 1.0 0.10 22 12 3'-Deoxykanamycin C and 3',4'~dideoxykanamycin C used in Examples 2 and 3 are new compounds which may be prepared by the following procedure (see u.S. Patent 4,120,955 issued October 17, 1978):
3'-deoxykanamycin B or 3',4'-dideoxy-kanamycin B is reacted with tert-butyl chloroformate or tert-butyl S-4,6-dimethylpyrimid-2-ylthiocarbonate which is known as a reagent for introducing the amino-protecting tert-butoxycarbonyl group.
In this way, there is prepared 6'-N-tert-butoxy-carbonyl-3'-deoxykanamycin B or -3',4'-dideoxy-kanamycin B. The latter is then reacted with acetic --anhydride to protect the 1,3,2' and 3"-amino group of the deoxykanamycin B compound with the another kind of the amino-protecting group, the acetyl group, whereby 6'-N-tert-butoxycarbonyl-tetra-N-acetyl-3'-deoxykanamycin B or -3',4'-dideoxy-kanamycin B is prepared. When this product is treated with aqueous 90% trifluoroacetic acid, the preferential removal of the tert-butoxycarbonyl group takes place, giving 1,3,2',3"-tetra-N-acetyl-~ .

3'-deoxykanamycin B or -3',4'-dideoxykanamycin B.
1,3,2',3"-Tetra-N-acetyl-3'-deoxykanamycin B or -3',4'-dideoxykanamycin B is dissolved in an aqueous acetic acid, and the resulting solution is treated with an aqueous sodium nitrite, when the 6'-amino group is converted into the 6'-hydroxyl group, giving 19392',3"-tetra-N-acetyl-3'-deoxy-kanamycin C or -3'94'-dideoxykanamycin CO When the latter is treated forthe removal o~ the amino-protecting acetyl groups 9 ~or example, by alkaline hydrolysis with 2N aqueous sodium hydroxide, there is produced 3'-deoxykanamycin C or 3',4'-dideoxy-kanamycin C.
The preparation of 3'-deoxykanamycin C or 3',4'-dideoxykanamycin C may be achieved in a similar way to the above-mentioned procedure by protecting the 6'-amino group of 3'-deoxykanamy~cin B or 3',4'-dideoxy~anamycin B with benzyloxycarbonyl group. For this purpose, benzyloxycarbonyl chloride is employed as the reagent for introducing the amino-protecting benzyloxycarbonyl groupO In this case, the removal of the benzyloxycarbonyl group from 6'-N-benzyloxycarbonyl-tetra-N-acetyl-3'-deoxykanamycin B or -3' 7 4'-dideoxykanamycin B
which is formed as the intermediate product may be conducted by corventional hydrogenolysis over palladium catalyst or platinum catalyst.
The preparation o~ 3'-deoxykanamycin C
and 3',4'-dideoxykanamycin C is now illustrated below as Examples 5-6.

-~0-~0~31693 Example 5 Synthesis of 3'-deoxykanamycin ~
(a) A solution o~ 200 g (403 millimole) of 3'-deoxykanamycin B in 40 ml o~ water was admixed with a solution of 1.03 g (407 millimole) of t-butyl S-4,6-dimethylpyrimid-2-ylthiocarbonate in 40 ml of dioxane, and the admixtureso obtained was stirred for 24 hours at ambie.nt temperature. The reaction mixture was then concentrated to dryness under reduced pressure, and the solid residue was taken up into 32 ml of waterO The resultant aqueous solution was passed through a column of 160 ml of a cation-exchange resin, Amberlite CG-50 (ammonium form) for adsorptionof the formed 6'-N-t-butoxy-carbonyl-3'-deoxykanamycin Bo The resin column was washed with 800 ml of water and then eluted with 800 ml of OolN aqueous ammonia. The elu~te was collected in 15 ml-fractions,and the fraction Nos. 26 to 42 were combined together and concentrated to dryness under reduced pressure to give 1.06 g o~ a white colored powder comprising 6'-N-t-butoxy carbonyl-3'-deoxykanamycin BD Yield 44%. The resin column was further eluted with 0.5N aqueous ammonia to recover 452 mg of unreacted 3'-deoxy-kanamycin B.
(b) A solution of 211 mg (0.37 millimole~ of 6l-N-t-butoxycarbonyl-3'-deoxykanamycin B in 5 ml o~
methanol was admixed with 2~5 ml of acetic anhydride, and the admixture was agitated ~or 5 hours at ambient temperature for the acetylation of the remaining .

~081693 amino groupsu The reaction solution was admixed with a volume of water and then concentrated to dryness under reduced pressure to give a powder comprising 6'~N-t-butoxycarbonyl-tetra-N-acetyl-3'-deoxykanamycin Bo Yield 296 mg.
(c) The product (235 mg) obtained in the preceding step (b) of this Example was dissolved in 2 ml of an aqueous solution of 90% trifluoroacetic acid and the resulting mixture was allowed to stand for 45 m$nutes at ambient temperature to effect the removal of the 6'-butoxycarbonyl groupO The reaction mixture was concentrated to dryness under reduced pressure, and the solid residue obtained was washed with about 2 ml of ethyl ether to give 227 mg of a white colored powder comprising the tetra-N-acetyl derivative, that is 9 1,3,2',3"-tetra-N-acetyl3'-deoxykanamycin Bo (d) The powdery product (193 mg) obtained in the preceding step (c) of this F,xample was dissolved in 3.2 ml of an aqueous solution of 33% acetic acid, and to the resulting solution was added a solution of 265 mg of sodium nitrite in 302 ml of water and then 1.6 ml of acetic acid under ice-cooling and stirringO The mixture so obtained was stirred for 1 hour under ice-cooling and then for 16 hours at ambient temperature to effect the reaction where the 6'-amino group was converted into the 6'-hydroxyl group. The reaction solution was concen-trated to dryness under reduced pressure to obtain 240 mg of a solid residueO This solid comprising -~2-iO~ i93 1,3,2',3"-tetra-N-acetyl-3'-deoxykanamycin C was taken up into 4 ml of 2Naqueous sodium hydro~ide, and the resulting mixture was heated for 7 hours under reflux to effect the removal of the acetyl groups.
The reaction solution so obtained was admixed with 200 ml of water and then passed through a column (inner diameter i~6 cm) of 50 ml of a catlon-exchange resin, Amberlite CG-50 (70%
ammonium form) for the absorption of the formed kanamycin C derivative. The resin column was washed with 250 ml of water and then eluted with 0 5N aqueous ammoniaO The eluate was collected in 10 ml-fractions, and the fraction Nos. 58 and 59 were combined together and concentrated to dryness under reduced pressure to give 89 mg of a crude powder of 3'-deoxykanamycin C. This crude powder was taken up into 2 ml of water, and the aqueous solution obtained was again chromatographed using a column (inner diameter 0075 cm) of 10 ml of Amberlite CG-50 (ammomium form) in such a manner that after washing with 30 ml of water, the .
resin column was eluted with 45 ml of OolN aqueous ammonia, and then with 45 ml of 0~2N aqueous ammoniaO
The eluate was collected in 1 ml-fraction9 and the fraction NosO 78 to 91 as combined together were concentrated to dryness under reduced pressureO
A colorless purified powder of 3'-deoxykanamycin C
(54 mg; 0 11 mlllimole) was obtained Yield 45~.

1(~8~693 Example 6 Synthesis of 3'94'-dideoxykanamycin C
(a) To a solution of 13053 g (30 millimole) of 3',4'-dideoxykanamycin B in 135 ml of water was dropwise added over 1 hour 5061 g (33 millimole) of benzyloxycarbonyl chloride under ice-cooling and stirringO After th~ dropwise addition was completed, the admixture so obtained was stirred for 1 hour at ambient temperature and the precipitate which ~ormed was removed by ~iltration. The filtrate was washed with 135 ml of ethyl ether, and the aqueous layer was neutralized by addition of aqueous ammonia ard then concentrated under reduced pressureO The concentated solution so obtained was passed through a column of 480 ml of a cation-exchange resin, Amberlite CG-50 (ammonium form) for adsorption of the formed 6'-N-benzyloxycarbonyl-3',4'-dideoxykanamycin Bo The resin column was washed with 1920 ml of water and then eluted with O.lN aqueous ammoniaO The first running (960 ml) of the eluate was discarded and the subsequent running (780 ml) was collected and concentrated to dryness under reduced pressure to give 5043 g of a white colored powder comprising 6'-N-benzyloxy-carbonyl-3',4'-dideoxykanamycin Bo Yield 31%o The column was further eluted with 0.5N aqueous ammonia to recover 2.7 g of unreacted 3',4'-dideoxykanamycin Bo (b) 6'-N-Benzyloxycarbonyl-3',4'-dideoxykanamycin B (1059 g; 2072 milimole) was admixed with 160 ml of acetic anhydride and 16 g of sodium acetate, ~01~1~93 and the admixture so obtained was heated for 2 hGurs under reflux (at 110 C) to effect the acetylation. The reaction mixture was concentrated to dryness under reduced pressure, and the solid resi-due was extracted with about 100 ml of acetone. The extract in acetone was concentrated to dryness under reduced pressure, leaving a solid (2.5 g). This solid was taken up into 10 ml of chloroform, and the resulting solution was passed through a column (inner dia-meter 2.6 cm) of 150 g of silica gel for adsorption of the formed acetylation product. The silica gel column was washed with 350 ml of~chloroform and then eluted successively with 900 ml of chloro-form-methanol (30:1 bY volume), with 900 ml of chloroform-methanol tl5:1 by volume) and with chloroform-methanol(10:1 by volume).
The eluate was collected in about 14 ml-fractions. The fraction Nos. 91 to 149 as combined together were concentrated to dryness under reduced pressure to give 1.80 g of a white colored powder comprising 6'-N-benzyloxycarbonyl-tetra-N-acetyl-tetra-O-acetyl-3', 4'-dideoxykanamycin B.
(c) The white colored powder (1.18 g) obtained in the above step (b) of this Example was dissolved in a mixture of 20 ml of methanol and 5 ml of water, and the resulting solution was subjected to catalytic reduction for 45 minutes under a stream of hydrogen over 1.61 g of 5~ palladium-on barium carbonate added to said solu-tion, so that the benzyloxycarbonyl group was removed. After removal of the catalyst , .

:

~ - 35 -lV~ 9~

by filtration, the reaction mixture was con-centrated to dryness under reduced pressure, .
affording 942 mg of a white colored powder of the 6'-amino derivative, that is, 1,3,2',3"-tetra-N-cetyl-5,2",4",6"-tetra-0-acetyl-3',4'-dideoxy-kanamycin Bo (d) The white colored powder (942 mg) obtained in the above step (c) of this Example was dissolved in 16 ml of a solution of 33% acetic acid in water, and to the resulting solution were added 16 ml of a solution of 1.24 g of sodium nitrite and then 8 ml of acetic acid under ice-cooling and stirring~ The admixture so obtained was stirred for 1 hour under ice-cooling and then for 3 hours at ambient temperature to effect the conversion of the 6'-amino group into 6'-hydroxyl groupO
The reaction mixture was concentrated to dryness . under reduced pressure, and the solid residue was dissolved in 3 ml of chloroform~ The solution in chloroform was passed through a column (inner diameter 2 cm) of 100 g of silica gel, which waq then washed with 210 ml of chloroform and thereafter eluted successively with 660 ml of chloroform-methanol (50:1 by volume)~ with 1750 ml o~ chloroform-methanol (30:1 by volume), with 900 ml of chloroform-methanol (10:1 by volume) and with 700 ml of chloroform-methanol (5:1 by volume). The eluate was collected in about 14 ml-fractionsO The fraction NosO 220 to 270 as combined together were concentrated to dryness under reduced pressure, 1()8i693 giving 587 mg of a white colored powder of tetra-N-acetyl-tetra-O-acetyl-3',4'-dideoxykanamycin CO
This ~rhite colored powder (234 mg) was taken up into 4 ml of 2N aqueous sodium hydroxide, and the resulting solution was heated for 7 hours under reflux to effect the removal of the acetyl groupsO The reaction solution was dissolved in 200 ml of water and then passed through a column (inner diameter 1.6 cm) of 50 ml of a cation-exchange resin, Amberlite CG-50 (70% ammonium form) for adsorption of the desired productO
After washing with 250 ml of water, the resin - column was eluted with 005N aqueous ammonia to yield 122 mg of a crude powder of 3',4'-dideoxy-kanamycin CO A solution of this crude powder in 2 ml of water was passed through a column (inner diameter 0.8 cm) of 14 ml of Amberlite CG-50 (NH4-form) for adsorption of the desired productO
After washing with 45 ml of water, the resin column was eluted with 40 ml of 0005N aqueous ammonia, then with 70 ml of OolN aqueous ammonia and finally with 70 ml of 002N aqueous ammonia~
The eluate was collected in 1 ml-fractions, and the fractions NosO 119 to 146 were combined together and concentrated to dryness under reduced pressure to give gO mg of à colorless purified powder of 3',4'-dideoxykanamycin CO Overall yield 23%o -~7-

Claims (17)

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

1. A process for the preparation of a compound of formula (I) (I) wherein R1 and R2 are each hydroxyl, or R1 is hydrogen and R2 is hydroxyl or R1 and R2 are each hydrogen, or a nontoxic, pharmaceutically acceptable acid-addition salt thereof, which comprises the steps of acylating the 1-amino group of kanamycin C, 3'-deoxykana-mycin C or 3',4'-dideoxykanamycin C represented by the formula:

(II) wherein R1 and R2 are as set out above, by reaction with an amino-protected derivative of L-4-amino-2-hydroxy-butyric acid or a functional equivalent thereof, to produce the 1-N-acylation product of the starting compound (II), and removing the amino-protecting group from the resulting 1-N-acylation product to give the compound of the formula (I), and, if desired, forming a non-toxic, pharmaceutically accept-able acid-addition salt of the product.

2. A process according to Claim 1 which comprises the steps of:-reacting the starting compound of the formula (II) of which amino groups are not protected, with an amino-protected derivative of L-4-amino-2-hydroxybutyric acid represented by the formula:

( III) wherein R3 is a hydrogen atom and R4 is a known mono-valent amino-protecting group, or R3 and R4 together form a divalent amino-protecting group selected from phthaloyl group and a Schiff base group (N=CHR5 in which R5 is a hydrogen atom, an alkyl group of 1-4 carbon atoms or an aryl group), in a manner known for the acylation of amino group, to produce the mixed acylation products comprising a 1-N-(L-4-protected amino-2-hydroxy-butyryl) derivative of the starting compound of the formula (II), treating the mixed acylation products in a known manner to remove the amino-protecting group therefrom, and then isolating the desired 1-N-(L-4-amino-2-hydroxybutyryl) derivative of the antibiotic according to the formula (I) by chromatographic separation of the acylation products from which the amino-protecting group has been removed, to recover the desired compound of the formula (I).

3. A process according to Claim 1 in which the amino-protecting group present in the amino-protected derivative of L-4-amino-2-hydroxybutyric acid employed is an alkoxy-carbonyl group of 2-6 carbon atoms, a cycloalkyloxycarbonyl group of 3-7 carbon atoms, an aralkyloxycarbonyl group or salicylidene group.

4. A process according to Claim 1 in which kanamycin C, 3'-deoxykanamycin C or 3',4'-dideoxykanamycin C is used without any protection of the amino groups as the starting compound in the acylation step.

5. A process according to Claim 1 in which kanamycin C, 3'-deoxykanamycin C or 3',4'-dideoxvkanamycin C is acylated with N-hydroxysuccinimide ester of L-4-protected amino-2-hydroxybutyric acid in an aqueous solution at pH 6-8.

6. A compound of the following formula (I) wherein R1 and R2 are each hydroxyl, or R1 is hydrogen and R2 is hydroxyl or R1 and R2 are each hydrogen, or a nontoxic, pharmaceutically acceptable acid-addition salt thereof, whenever prepared or produced by a process according to Claim 1 or by an obvious chemical equivalent thereof.

7. A process as in Claim 1, in which the compound of formula I is recovered.

8. A compound of the following formula I

(I) wherein R1 and R2 are each hydroxyl, or R1 is hydrogen and R2 is hydroxyl, or R1 and R2 are each hydrogen, whenever prepared or produced by a process according to Claim 2 or 7 or by an obvious chemical equivalent thereof.

9. A process as in Claim 4 wherein the starting material is kanamycin C.

10. A process as in Claim 5 wherein the starting material is kanamycin C.

11. 1-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C or a nontoxic, pharmaceutically acceptable acid-addition salt thereof, whenever prepared or produced by the process of Claim 9 or 10 or by an obvious chemical equivalent thereof.

12. A process as in Claim 4 in which the starting material is 3'-deoxykanamycin C.

13. A process as in Claim 5 in which the starting material is 3'-deoxykanamycin C.

14. 1-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C
or a nontoxic, pharmaceutically acceptable acid-addition salt thereof, whenever prepared or produced by the process of Claim 12 or 13, or by an obvious chemical equivalent thereof.

15. A process as in Claim 4 wherein the starting material is 3',4'-dideoxykanamycin C.

16. A process as in Claim 5 wherein the starting material is 3',4'-dideoxykanamycin C.

17. 1-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxy-kanamycin C or a nontoxic, pharmaceutically acceptable acid-addition salt thereof, whenever prepared or produced by the process of Claim 15 or 16, or by an obvious chemical equivalent thereof.