CA1052780A - 6'-n-methylkanamycin a and b derivatives - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

6'-N-METHYLKANAMYCIN A & B DERIVATIVES
This invention relates to semi-synthetic derivatives of kanamycin A and B, said compounds being known as l-N-[L-(-)-.beta.-amino-.alpha.-hydroxy-propionyl]-6'-N-methylkanamycin A or B, l-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]-6'-N-methyl-kanamycin A or B and l-N- [L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl]-6'-N-methylkanamycin A or B
and having the generic formula (IV) in which R3 is -OH or -NH2 and R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl, L-(-)-.beta.-amino-.alpha.-hydroxypropionyl or L-(-)-.delta.-amino-.alpha.-hydroxy-valeryl; or a pharmaceutically acceptable nontoxic salt thereof. Some R-factor mediated - a -kanamycin-resistant organisms, for example E. Coli K-12 R-5 and Ps. aeruginosa GN 315, inactivate the kanamycins by 6'-N-acetylation. The specification dicloses derivatives of kanamycin A and B which are resistant to this type of enzymatic inactivation by these kanamycin A and B resistant organisms, but still retain the majority of their biological activity and spectra.

- b -

Description

~5~,~7~(~
..
lET:IVLKANA~IYCIN A & _B DFRIVAT. IVES
This invention relat~s to semi-synthetic deri~atives of kanamycin A and B, said compounds being known as l-N-~L-(-)-~-amino-a-hydroxy-propionyl]-6'-N-methylka~amycin A or B, l-N-1L- (-) -r-amino-a-hydroxybut~ryl~-6'~N-methyl-kanamycin A or B and 1-N~.~L- (~ -amino-~-hydroxy~aleryl]-6'-N-methylkanamycin A or B
and having the generic formula ~:
4, 6' N~;E

H / H-R .

CH20H / , ~0~ , / ,~:

2~ /
(XV) .

in which R3 is -OH or -NH2 and R is L~
amino--hydroxybutyryl, L-(~ -amino-a- l hydroxypropionyl or L-(-)-~-amino--hydroxy- i valeryl; or a pharmaceutically aoceptable nontoxi~ salt thereof.
It is known that some R-factor mediated - . kanamycin-resistant organisms, for example - I--.' ~ '.~

:

1~2~S~

E. coli K-12 R-5 and Ps. aeruginosa GN 315, inactivate the kanamycins by 6'-N~acetylation.
It was, therefore, an object of the present invention to prepare derivatives of kanamycin A
and B which ~ould be resistant to this type of enzymatic inactivation by these kanamycin A and B resistant organisms, but ~ould still retain the majority of their biological activity and spectraO , .' ~ . :
The object of the present invention has been achieved by the synthesis of the compounds herein designated by the compound IV.
A preferxed embodiment of the present invention is the compound having the for~ula HO ~ CH3 3' HO ,~ \
~3 ~ ~

CH~OH ~ \
~O ~ ¦ H~ / ~ ~H-R

~2 H ~
i' ' :.

:
in which R is L~ y-amino-a-hydroxybutyryl, L- -amino-a-hydroxypropionyl or L~ amino-a-hydroxyvaleryl and R is OH or NH2; or a nontoxic . :

, . .
~2-~,.. . ..... ... . ... . . . .

:

~5;~78(~ :
..

. pharmaceutically acceptable ac:id addition salt thereof.
The most preferred embodLments are the .. . . .
compounds of formula IV in which 1) R is L~ y-amino-~-hydroxybutyryl and R is OH ~XVa);
2) R is L-( )-y-amino-~hydroxybutyryl and ~3 iS NH2(Ivb);
33 R is L~ amino-a-hydroxypropionyl and R is O~ (IVc);
4) R is L~ -amino-~-hydroxypropionyl and R is NH2 ¦~Vd)~ :
5) R is L~ amino-a~hydroxyvaleryl and R3 is OH (IVe); and 6) R is L-(-)-~-ami~o-a-hydroxyvaleryl and R is~NH2 ~IVf); or a non-toxic pharmaceutically acceptable acid addition salt thereof.
For the purpose of this disclosurer the term ~nontoxic pharmaceutically acceptable acid addition salt" shall mean a mono-, di-, tri- or tetrasalt formed by the interaction of 1 molecule of compound IV with 1-4 moles of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, ~ ' ' .

: -3-~, ,, ,' .
.1 ' i5Z7~) , . . .
. maleic, phosphoric, nitric, bydrobromic, ascorb;c, ~ malic and citric ac.id, an~ those o~her acids commonl~
used to make salts o~ amine containing pharmaceu-I . ticals s Other most preferred embodiments are the sulfate, hydrochloride, acet~te, maleate, citrate, . .
ascorbate, nitrate or phosphate salts of compound IV . ,", Another most preferred embod~ment is the mono-sulfate salt of compound IV.
Still another preerred embodiment is the -disulfate salt of compound IV.
:. ~... .
The objectives of the present invention hàve been achieved, by the provision accoraing to the present invention of the process for the preparation of the compound having tbe formula BO. ~ HCH3 HO ~ ~ i }~o C~20H )7~\ ' : ~ ~ H~ NH-R ¦
NH2~7--\b~// ' !
'' ' ~ ' -: `

. j ~

5~7~) : ~ which R ii5 L~ y-amino-~ hydroxybutyryl, amino-~-hydroxypropionyl or L~
~mino-~-hydroxyvale~yl, an~. R3 1~ -OH or -NH2' ~hich comprise~ the con3ecutive i~te!pQ o~
A) acylating ~.h~ compoolnd ha~ing the . formu~a : . .
~O ~ ~H3 ~10 ~ ,, -\ .
, HO ~ 2 ~2 ~

~, ln which R3 ~ ~O~ or -NH2 with an acylating agent' ha~,rlng the formula ~
.' 1~ f~ , , .
W-NH-~C~2)n C~-C-~ (VII) ~n whlch W ~8 ~ radical sele~ted from the sroup ~ompr~sing ; ~ }~2-- ~ CH3-~ ~ 2 ~2 .j .

,, , . . ! '.

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~C~ C1~2-C}12-C--: :
' ' '..':~':
but pref erably ~12--C-! . ;. .
s a radical selected from ~he group consisting ::

_0--~ -o~N02 -o~J2 . . ,, , ::
:, :
-0---~ 9 -~-O-C-~ and ~ . ~

, ..:;,, . .
:', ~ '':

~X ~~~ but pref erably '~ O .;
, ,~: , , .
~.f ,.
,, ~ , , ' ' .

'-1~ . ', : l , . .
`~''1 , . ' ' ! -6- :

. : . .

~ N-O- or ~ N-O-~ O

in which n is an integer of 1 to 3; in a ratio of about 0.5 to about 1.4 mole of the compound VII per mole of compound II, and preferably in a ratio of about 0.8 to about 1.1, in a solvent selected from the group comprising a mixture of water and ethyleneglycol dimethyl ether, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran and propyleneglycol dimethyl ether, but preferably aqueous tetrahydrofuran, to produce the compound having the formula .
HO ~ H-CH3 ,. \
HO ~ \

HO ~ ~ NH-C=O

NH2~ / ( IH2) III NH
, . l .
' W ,, ~ in which n, R3 and W are as above; and ... ::.
':' ', :' ,'"~

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~ -7~

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sz~
B~ Removing the blocking group W from :~
compound III by methods commonly known in the art to produce a compound of formula IV.
This may preferably be done when W is a radical of the formula ~
. O .

:. ~ 2 .. ... . : .
by hydrogenating compound III with hydrogen in the 0 presence of a metal catalyst, preferably selected from ~ ~ :
: the group comprising palladium,platinum, Raney nickel, .- rhodium, ruthenium and nickel, but preferably palladium ..
` and most preferably palladium on charcoal, in a water- -~
water miscible solvent system, preferably selected from .~ . .. .
' the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether, propyleneglycol dimethyl ether, or the like, but preferably 1:1 water-dioxane, ~` and preferably in the presence of a catalytic amount jj~ of glacial acetic acid to produce the compound of `0 ~ormula IV. ~.
. ~ It should be apparent to ~hose knowledgeable ~' in the art that other agents can be used in the process :
-i above to acylate the amine functions of the inter~
~ mediate compounds of the instant invention. This ` `.~
,j ..
~ disclosure is meant to include all such ':1 , , ,1 " : ' ~'O , ~
,,. . :
'"'' .' , ' ` ;:: ' . ~ ' :

,t.,_ ': ' ~ ' ' ' , , ' ' ' , , ' . , ' , . . , '.'.. .. ' ' . ' .', ' ' , , ', ' ' ,"' ' "' , ', .. ,','`,,' '. ' ~ ' ; ' , . :': ' ' , . ' ' : . , . . ' . ' . ' :~ .' . '. ' , .

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acylating agents that produce labile amine blocking groups, said labile blocking groups commonly employed in the synthes is of peptides .
The labile blocking groups must be readily removable by methods commonly known in the art.
Examples of said labile blocking groups and their removal can be found in the review of A. Kapoor, Jr PharmO Sciences, 59, ppn 1-27 (1970)~

Functional equivalents as acylatin~ agent for primary amine groups would include corresponding carboxylic chlorides, bromides, acid anhydrides, including mixed anhydrides and particulaxly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of caxbonic acid, of alkyl and aryl sulfoni~ acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active . , , e~er of thio-ester ~e.g., with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioaoetic acid) may be used or the free acid itself may be :l coupled with the kanamycin derivative (II~ a~ter first reacting said free acid with N,N'-dimethylchloro- :

forminium chloride ~cf. Great Britain 1,008,170 and Novak and Weight, Experientia XXIj6, 360 (1965)~ or by the use j of enzymes or of an N,N'-carbo~yldiimidazole or an N,N'-carbonylditriazole lcf. Sheehan and Hess, J. Amer. Chem.
8OC.~ 77, 1067 (1955)~ or o~ alkynylamine reagent [c~.
R. Buijile and ~. G. VieheO Angew, Chem,, In~ernational Edition 3, 582 (lg64), or of a ketenimine r~agent lc~. C. L. Stevenes _9_ - .... . .. . - . .. . . .. ~ ........ . .

7~0 i and M. E. Monk, J. Am~r. Chem. Soc., 80, 4065 ~1958)~ or of aR isoxazolium salt reagent [cf~ R. B.
Woodward, R. A~ Olof son and H. Mayer, J. Amer.
Chem. Soc., 83, 1010 ~1961~]. Another equivalent o~ the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic ~ive memb2red ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole~ the triazoJes, benæimidazole, benzotriazole and their substituted dexivatives. As an example of the general method for the preparation of an , . .
azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proporticns at room temperature in tetrahydrofuran, chlorofo~m;
dimethylfoxmamide or a similar inert solvent to :.ï . . . .
' form the carboxylic acid imidazolide in practically `~ quantitative yield with liberation of carbon dioxide ~nd one ~ole o~ imidazole. Dicarboxylic acids yield , diimidazolides. The by~product imidazole, precipitates and may be s~parated and the imida~olide ~i isolated, but this is not essential. These reactions ! are well-known in the art ~cf. U~ S~ Patent No~.
~, 3,079~314, 3ill7~126 and 3,129~224 and British :.,~ . .
~ Patent Nos. 932,644, 957,570 and 959,054).

1 . .
, ~, .
.

278(:1 The most preferred process for the preparation of compounds having formula IV comprises the consecutive steps of A) acylating the compound having formula II
with an acylating agent having the formula ..

2)n H C o--1 or ~3CH2-o-C-NH- ~CH2 ) -hC--C-- ~

in which n is an integer of 1 to 3 in an aqueous tetrahydrofuran or aqueou~ dimethylformamide-~oetone solvent system, at about room ~emperature to produce the compound having the formula ~' '.' '^'., ~C~5~78(~

~10 ~ CH3 I .
HO ~ _ HO CH2H HO ~ \
(C~2 ) n III l~ `

o I '~ .
CH2 ¦

in which n is an integer of 1 to 3; and B) hydrogenating compound III in aqueous tetrahydro~uran in the presence o~ palladium on charcoal at about a~mospheric pressure to produce compound IVo Al~ernatively, in step A, the acylating agent VII may be generated in situ by mixing the compound :
having the formula .

-O-C-NH-(CH2)n-C - C -OH
:
.:
with equimolar quantities of N-hydroxy-5-norbornene-2~3-dicarboximide and dicyclohexylcarbodiimide in a .
small q~lantity of anhydrous tetrahydrofuran. The . .

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. .
resultant mixture is filtered and the filtrate added to an aqueous tetrahydrofuran mixture of the 6'-N-kan~mycin A or B to produce the appropriate compound III.
The compounds IV are valuable an anti bacterial agent~, nutritional supplements in animal feeds, therapeutic agents in poultry and animals~ ineluding man, and are especially valuable in the trea~ment of infectious diseases caused by Gram-positive and Gram-negative bacteria.
The compounds IV when a~ministered orally are useful as an adjunctive rea~ment fox pre operative sterilizatio~ o~ the bowel~ Both aerobic and anaerobi~ flora which are suseptible to these dxugs are reduced in the large i~testineO
~hen accompanied by adequate mechanical cleansing, they are useful in preparing ~or colonic suxgery.
.The compounds IV are e~fective in the treat-ment of systemic bacterial infections when administered parenterally in the dosage range of ,bout 250 mg. to about 3000 mgO per day in divided doses three to four times a day. Generally the c~mpounds are effective when ad~inistered at a dosage of about 5.0 to 7.5 mg./kg. of body weight e~ery 1~ hoursO ;
I~he compounds comprising the genus o~ compound TY, ,specific~ally BB-K28, 142, 14,3, 162 and 163, all '',, . . .
. . .

~5~7~0 possess substantially improved activities agains~ a wider spectrum o~ microorganisms as compared to the parent ~ompounds fr~m which they are derived, i.e., kanamycin A and B.
Illustrated below is Table I sho~ing the minimal inhibitory concentrations (MIC's) of kanamycin A and B and BB-K28~ 142, 148, 162 and 163 against a variety of Gram positive and Gram~
nega~ive bacteria as obtained by the Steers agar-dilution method on Mueller-Hinton Agar ,~
Medium.

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~. . I m "' O O O ~ ) ,~ ~ Lt~ ~1 U~ O

a~ D ~ N ~1 ~D ~D ~ t~ O O O t~ O N ~ ~D ~ O ~) ~ Lt) O
. ' ' ~ .

0 0 0 0 0 t~l o o ~ O O ~ ~ O

1 N ~ o ~ A
~ ¦ O O ~1 0 0 0 0 C~ 0 O ~ O ~ ~ O O ~ U~ N O O
. ,. , I .

~I ~ ~ o ~o o _I o ~ o ~ o ,~ N N C~ N N N O o o o ~ ~ N N

W ~ L; " N

V ~ K P~ 1~ ~ ~ ~ Q' , . `;'.

u ,9 0 o o o o D u (~ ~ X ~ ' X

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L o o c:~ o o o o o o o o o ~
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a~ u~ ~
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.

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K I N cc~
N O O O OO _I ~ O O O O O O
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r~ h h q) C ~ ~ ~ h ~1 r1 ~ h ~ F~ `- ~
E~ ~ ~ h . C"~, :

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O P. a~
. ~' .. -1~-, " , ........... . ..

- ~5'~8() From our vast experience with kanamycin A
and B and its chemical reactivity, it was expected that o~ the 4 or 5 primary amine functions ~ound in kanamycin A and B, respectively, that the ~'-amino func~ion is the most reactive for steric reasons. It was, therefore, postulaked that when the 6'~amino function was converted to a 6'-N-methyl secondary amine, its reactivity wi~h an acylating agent wou~d be substantially decreased and the 1-N primary amine function would ~e the most reactive of the remaining primary amine for steric r~asons. Therefore, S'-N-methylkanamycin A or B was directiy acylated with an appropriate side chain acylating agent without ~irst blocking the other amine ~unc~ions of the molecule. Control of the molar quantities of acylating agent used determines the degrce of acylation occurr-ng at other positions.

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r ~r~ Ls Preparat.ion o~ L~ y-benzyloxy ,~ c~rbonylamino-~-hydroxybutyric acid (VIl.
1~ L-(-J-y-amino-a-hydroxybutyric acid (7.4 g., 0.062 mole was added to a solution .. . .
of 5.2 g. ~0.13 mole) of sodium hydroxide in 50 mlr of water. To the stirred solution was added dropwise at 0-5~ C. over a period of 0.5 hour, 11.7 g. (0~068 mble) of carboben-zoxy chloride and tAe mixture w~s continued to stir for one hour at the same tempexature.
The reaction mixture was washed with 50 ml.
of ether, adjusted to pH 2 with dilute hydro- :
chloric acid and extracted with our ~0-ml.
poxtions of ether. The ethereal extracts were ~ombined, washed with a small amount of saturated ~odium chloride solution, dried with anhydrous sodium sulfate and filteredO The filtrate was e~aporated in vacuo and the resulting residue was crystallized from benzene to give 11.6 g.
(i4~) fo colorless plates; melting point 78.5-79.5 C., la]D - 4.5 ~c. = 2, CH30H)~ Infrared SIR) l~Br]: IR ~Br) yc=o 1740, 1690 cm. 1 Nuclear magnetic resonance (NMR) (acetone-d (in p.p.m. fxom T~S) 200 ~2~, m.), 3O29 (2H, d-d, J=6.7 and 12 Hz.), 4~16 (lH, d-d, ..
J=4.5 and B Hz.), 4.99 ~2~, s.~, 602 (2H, ~road), 7.21 ~SH, s.).
, ~1~)5Z7~ [I

AnalYsis - Calcd~ for C12H15NOS (percent):
C~ 56.91; H, 5.97; N, 5.53. ~ound (percent~:
C, 56.66; H, 5.97; N, 5.47.
2) N--hydrox~succinimide ester o~ L~ y-benzyloxycarbonylamino-~-hydroxybutyric acid ~VII). - A solution o~ 10.6 g. ~0.042 mole~
o~ VI and 4.8 g~ (0.042 mole) of N-hydroxy- i succinimidel in 200 ml. of ethyl acetate was cooled to 0 C. and then 8.6 g. ~0.042 mole) of dicycloh~xylcarbodiimide was added. The mixture was ~ept overnight in a refrigerator. The ~cyclohexylurea which separated was filtered off and the filtrate was concentrated to about 50 ml.
under xeduced pressure to give colorless crystals of VII which were collected.by ~i~tration; 604 g.
M.P. 121-122.5~C. The filtrate was evaporated to dryness in vacuo and the crystalline residue '.:
was~washed with 20 ml. of a benzene-n-hexane mix- .:
tuxe t~ give an additional amount of VII. The ^.
total yield was 13.4 g. (92~ a1D 1.5 (C.= .
2, CHC13?. ~R tRBr) yc=o 1810, 1755, 1740, 1680 cm lo ~:
NMR (acetone-d6) ~(in p.p.m. from TMS) 2.0 t2H, m.), 2.83 (4H, s.), 3.37 (2H, d-d~ J=6.5 and 12.5 Hz.), 4.56 (lH" m.3~ 4.99 (2H, s.), 6.3 (2H~ broad), 7.23 t4}~, s.).

- 1 9- : :

~5~7~0 ~:
A~lysls. - Calc'd. for C16Hl~N2O7 (percent):
C, 54.85 Il, 5.18; N, 8~00. Found ~percent): C, 54.79, 54.70; H, 5.21, S~20; N, 8.14, 8.120

3) PreParation of N-(benzy~oxycarbon~loxy)-succinimide. - N-Hydroxy~uccinimide2 ~23 g. r 0.2 mole) was dissolved in a solution of g g. (0.22 mole) of sodium hydroxide in 200 ml. of water.
To the stirred solution was added dropwise 34 g.
0~2 mole) of carbobenzoxy chloride wi~h water-cooling and then the mixture was stirred at room temperature overn~ght to separate the carboben-zoxy derivative which was collected by filtration, washed with water and air-dried. Yield 41~1 g.
ii , .
(82~). Recrysta~lization from benzene-n-hexane ~10:1) gave colorless prisms melting at 78-79 C.

4) ~ Preparation of S'-carbobenzoxykanamycin A. -A solution of 42.5 g. ~90 mmoles) of kanamycin A free base in 450 ml. of water and 500 mlO of dimethylfoxmamide ~DMF) was cooled ' below 0 C. and stirred ~igorously. To the solution was added dropwise over a period of a~out two hours a solution of Z2.4 g. t90 mmoles) ~f N-~benzyloxycarbonyloxy)succillimide in 500 ml.
of D~Fo The mixture was stirred at -10 to 0 C.
o~ernight and then at room temperature for one day.
'' ~ ' .

~ 2G W. Anderson et al~, ~. Am. Chem. Soc., 86, , ~S~780 The reaction mixture was evaporated under reduced pressure below about 50 C. The oily residue wa~ dissolved in a mixture of 500 ml. water and 500 ml~ butanol, the mixture being filtered to remove insoluble material and separated into two layers. The butanol and aqueous layers were treated with butanol-saturated water (500 ml. x 2) and water-saturated butan~l (500 ml. x 2), xespectively, using a technique similar to counter current distribution~ Tha three aqueous layers were combined and evaporated to dryness under reduced pressure to give an oily residue, a part of whIch crystallized on standlng at room tempera-t~sre. To the residue including the crystals was added about 100 ml. of metharlo~, which dissolved the oil and separated it from the crystalsO A~ter adding about 300 ml. of ethanol, the mixture was kept at room t~mperature overnight to gi~ a crystalline mass which was collected by filtration. i~
It weighed 44 g. The product contained a small ~mount of kanamycin ~ as indicated by thin iayer :~
chromatography using n-propanol-pyridine-acetic ~ :
acid-water (15:10:3:12) as the solvent system and ~inhydrin as the spray reagPnt.
The crude product was dissolved in 300 ml. o water and chromatographed on a column ~30 mmO
diameLer) o~ CG-50 ion-exchange resin (NH~ t~pe, '' -21- .

____ _s_ -1~5~8~

500 ml.). The column was irriga*ed with 0.1 N
ammonium hydroxide solution and the eluate was collected in 10-ml. ~raction. The desired product was contained in tube numbers lV 100, while kanamyci.n A recovered from slower-moving fractions and the pos~tion isomer(s~ of the product seemed to be contained in the faster-moving ~rac~ionsO The ~ractions 10~110 were combined and evaporated to dxyness under reduced pressure ~o give 24.6 g. ~45%) of a colorless product 6-~arbo~enzoxykanamycin A SII) t6'-Cbz--kanamycin ~], which began to melt and colox at 204 C. and decomposed~at 212 CO with gas evolution.

1~1D ~ 106 (C.=2, H2O) ~ -- .

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~Z780 The final product was found to be accompanied ~y two minor components by TI,C with one o thP
solv~nt systems tested. However, the final product was used without fur~her purification ~or the pxeparation of co~pound II.
S) ~ aration o~ L~ y-amino-~-h~droxy . .
.' .~ ~ ''.
thereof. .
~mbutyrosin A t5.0 gm.) lU. S~ Patent No~
3,541~078, issued No-~ember 17, 1~70i was refluxed .
with 160 ml. of 0~5 N sodium hydroxide fo~ one hour. The hydrolysate was ~eu~ralized with 6 N
HCl and chromatographed on a column of CG-50 ~NH4~type)0 The desired L~ y-amino ~-h~droxy-butyric a~id was isolated by developing the col~mn with~water and removing the water by freeze .
I ~ drylng. The L~ y-amino-a-hydroxybutyric acid is ~haracterized as a crystalline ma~erial having a M.P. o~ 212.5-214.5 C. lcolumn 2, lines 31-38, U. SO Patent No. 3,541,0~8].
: 6) Prepar carbob ~ in ~.
. To a chilled solution of 801 g~ (0.0I68 mole~ of kanamycin B in 120 ml. of water and 80 : ml. of 1,2-dimethoxyethane was added dropwise with stirring a solution of 4.2 g~ ~0.0168 mole) ,, ~``, .

~ .~

l~S'~
of N-(benzyloxycarbonyloxy)succinimide in 40 ml. of 1,2-dimethoxyethane. The reaction mixture was stirred overnight and evaporated under reduced pressure. ~he residue was dis-solved in 100 ml. of water and shaken twice with 50 ml. of water-saturated n-butanol. The aqueous layer was separated and adsorbed on a column of 100 ml. of CG-50 (NH4+type).
The column was washed with 200 ml. of water, eluted with 0.0S
~ N N~40H. The eluate was collected in 10-ml. fraction. Frac-- tions 121 to 180 were collected, evaporated and freeze-dried ; 0 to give 1.58 g (15%) of th~ desired product. Fractions 1 to 120 were evaporated and re-chromatographed on CG-50 (NH4+) ~` to give 1.21 g. (12%) of the product. M.P. 151-152 C. (dec.).
~- [a]D24 + 104 (c. = 2.5, H20). y~=o 1710 cm. 1.
AnalySiS. - Calc'd. for C26H43N512 (percent) ~L5 C, 50.56; H, 7.02; N, 11.34. Found (percent)~
`~ C, 50.71; H, 7.38; N, 11.48.
TLC (silica gel F254), Rf 0.03 in n-PrOH-pyridine-AcOH-H2O ~15:10:3:12); Rf 0.16 in acetone-AcOH-H~O (20:6:74).
7) ~ ratlon of L-(-)-y- ~ oxybutyric-acid from Z0 DL-a-hydroxy-y~phthalimidobutyric acid. ~ ;
A) Dehydroabietylammonium L-a-hydroxy-y-phthalimi-dobutyrate: To a solution of 25 g. (0.1 mole) of a-hydroxy- ;~
y-phthalimidobutyric acid3 in ~00 ml. of ethanol was added a solution . .

-25~ ~
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.. .
.
vf 29 g. (0.1 mole) of dehydroalbietylamine in 130 ml. of ethanol., The soluti.on was shaken vigorou~ly for a miriute and stood at room temperature for f ive hours during which time ~in~ needl~s.crystallized out. The crystals wexe ccllected by filtration, washed with 50 ml.
. o~ ethanol and air-dried to obtain 3001 g.
~56%) of a diastereomer of the dehydroabiethy-amine salt. M~.P. 93-94 '~ C. tal 24 ~ 15~
lc. = 2~5, MeOH3 . Recrystalliza~ion ~rom 300 ml.
of ethanol gave 23 . 2 g. ~4396) o~ the pure product, . M.P. 94-95 C. lalD24 + 1008 (c. = 2.5, MeOH) O
~uxthex recrystallization did not change the ~, ~elting point and the speci~ic rotation.
AnalYs1s - Calc~d. for C3;~H42N~05,.}~20 5percent): C, 69.54; H~ 8.02; N, 5,07. Foun~ ..
- ~(perce~tj: C~ 69.58;~ H~ 8.08~ N, 5~07O
~a . B3 ~ ~ _ . To a ~olution o~ 1.5 gO (0.014 mole) o~
80~ ~um carbona~e in 40 ml. o~ wat~r were added 5.3 gv (0.01 mole) o dehydxoabiethyammonium L-hydroxy-yophthal~midobutyrate and 60 ml. of etherO The mix~- re was shaken vigorously .~ , .
.i until a~l o~ the solid. had ~issolvedO The e he~
layer was separatad. The aqueous solution was ~.i washed twice with 20-1rllO portions o~ ether and .',1 , ;
~ .
~; --2 6--; ' . . .

~5Z~8~

evaporated to 15 ml. under reduced pressure.
;. . To the concentrate was added 10 ml. of concen-. trated hyarochloric acid and the mixture was : refluxed for ten hour~. After cooling, : s~parated phthalic acid was removed by filtration.
:
` The filtra~e was e~aporated under xeduced pres~ureO
The residue was dissolved in 10 ml. of water and the solution was evaporated to dryness. ~his ~: operation was repeated twice to remove excess hydrochloric acid. The residual ... ... _ .... .
syrup was d~ssolved in 10 ml. of water and fiitered ; to remove a small amount of insoluble phthalic acidO

The filtrate was adsorbed on a column of ~R-120 . (H~ 1 cm. x 35 cm)~ the column was washed with 'J~ , 300 ml. of water and eluted with 1 N ammonium :.
hydrox~de solution. The ninhydrin positive fsactiona 10 to 16 wexe combined and evaporated under reduced pressure to give a syrup which ~rystallized gradually. The crystals w~re triturated with ethanol, f iltered and dried in a va~uum desiccator ~o give 0.78 g. (66%) of L~ y-amino-a-hydroxybutyric acid. M.P. 206 207`' C.

¦a]24. -29 (c. = 2.5, ~2)- The IR spectrl1m was identical ~l1ith the authentic sample which was obtainëd from ambutyxosin.

! , .

.

; I

:

~ 5~78(~
droXypropionic-Acid. XX~
L-~-Amino--hydroxypropionic acid* ~8.2 gO, 0,t078 mole) was dissolved in a solution of 6.56 g~
~0 . 0164 mol~) of sodium hydroxide and in 60 ml . of water. To the stirred solution was aslded dropwise 14.7 g. (00086 mole) qf carbobenzoxy chloride below 5 C. The mixture was stirred for an hour at room t~nperature, washed with 6 0 ml . of ~ther and adjusted to pH 2 with dilute HCl~ The precipitc.te was c~ollected by filt:rationr washed ~ .
with water and air-dried to give 9.65 ~. (52%) :
of XX. The filtrate was extxacted with five 100-ml. portions o~ ether. The ethereal solution was washed with water, dried over sodium sul~ate and evaporated to dryness in vacuo to give additional 2.0 g. (11%~ of XX. A total of 11.65 g. of VI was , . .
crystallized ~rom 500 mlO of ber~zene-ethyl acetate (4sl~ to give ~.36 g. (50%~ of pure XX, m. p. 128.5-129.5 C. ~In~rared ~IR) (~Br): y~,O 1745, 1690 cm 1.
]D5 ~ 209 ~C 5.0, MeOH). Nuclear Magnetic Resonance ~pectra tNMR (DMSO-d6)~ in ppm) 3~05-3~5 ~2H~ ~ CH2N) t 4.05 (lH, d-d~ -O-CH ~O~
5.03 ~2H, s, CH2Ar) 7.18 (lH~ broad, NH) " .36 (5~ ring H~o . ~ .

., . ~ .
., ,, . ~ ' ' ' . .

:

h .

s~æ7~ `

. Analys-is calc'd. for CllH13NO5: C~ 55-23;
B, 5.48; N~ 5~86.
Found: C~ 55~34; H, S.49, N, 5.87.
''`
:, . .
. *Ro ~reudenberg, Ber., 47, 2027 ~1914~.
; : ,' . ~
9~ dro~succinimide Ester of L-~-benzyloxy-: To a chilled and stirxed solu~ion of 478 ~ng.
(? m. moles) of xx and 230 mg. ~2 m.moles) of N-hydroxysuccinimide in 10 ml. of tetr~hydro~uran (THF) was added ~12 mg. (2 m.moles) of dicyclo-hexylcarbodimide. The mixture was stirred ~or an .
hour at 0-5 C., for two hours at roo~ temp~rature and then filtered to remove the N,N'-dicyclohexylurea.
The ~iltrate containing ~he title produc~ was used for the next reaction without isolation.
10) Preparation of L ~-Benz~loxycarbonylamino-a~

To a stirred solution o~ 400 mg (3.0 m moles) of L-~-amino-~ hydroxy~aleric acid* and 250 mg ~6.5 m moles) of sodium hydroxide in 25 ml of water was add~d dropwise 580 mg (3.3 m moles) vf o~rbo-benzQxy chloride over a period of 30 minutes at 0-5 C. The mixture was stirr.ed for an hour at 5-15C, washed with 25 ml of èther, adjusted to p~ 2 ' : .
' .

` 29 : J

` : :

Z78~.
with hydrochloric acid and extracted with three 30~ml portions of ether. The combined ethereal solu~ion was shaken with 10 ml of a saturated sodium chloride solution~ dr:ied over anhydrous sodium s~ifate and evapora~ed in vacuo to give cr~stals which were recrystallized from .
~enzene to yield 631~ mg (78%) of XXII, mp 110-111~ C.; infrared spectr~n ~IR5K~r)]: 3460r 3350, 1725, 1685, 1535, 1280~ 730, 690cm 1. :

Nuclear magnetic resonance spectrum [NM~

~acetone-d6)]~ ppm) 1~70 ~4H, m) 4.14 ~2H, qf J=4.5EIzl, 4.19(lH, m?, 4.82(2H, s),

6.2(3H, broad), 7.25(5H, s1. [~125 ~ 1.6 (c 10, N~ON). ~.

Anal~ysis calc'dO for C~3H17N05: C, S8.42; H.6.41; N, 5.24.

. ~ound: C, 58~36; H~ 6.50; N, : So27~

,, *S. Ohshiro et al., Yakuga~u Zasshi, 87, 1184 ~ 6~).

,,~` ' , .

. -30-, .' .

. ~

~ ~5~7~

ni~ e:;ter of L-~-benzyl-~. To a stirred and chillecl solution o~ 535 mg `~ 12.0 m moles) of ~XII and 230 mg ~200 ~ moles~ of - N-hydroxysuccinimide in 55 ml of ethyl acetate : .
. was added 41~ mg ~2.0 m moles) of N, N'- dicyclo--- hexylcarbodiimide (DCC). The mixture was stirred for 3 hours at room temperature and filtered to .~ remove precipitated N, N'-dicyclohexyIurea. The -~ - filtrate was evaporated in ~acuo to yield 780 mg ;~ ~100~) of viscous syrup XXIII~ IR~Neat): .
C=O~ , 1785, 1725 cm-lo .~ , . .
i ., -', ' ' . .
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. .
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.. . .
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;~l " .

~5'~7~ ~
Description of the Preferred ~m odiments .
ethod A
1. To a suspension of 610 mq of I.i~lH4 in :.
10 ml of dry dioxane was added dropwise at 70 C
a ~uspension of 618 mg of 6'-N-carbobenzoxykanamycin A
~6'-N-Cbz-kanamycin A). in 30 ml of dry dioxane. The mixture was stirred at 70 C. for 20 hours an~ then ~ooled to -5 - 0 C. To the reaction mixture was ~ ~ -adde~ cautiously about 20 ml of cold water. After the addition was ~ompleted, the mixture was - : ~' neutralized with ~ ~ ~Cl and- then svaporated into ~.
. dryness under reduced pressure. The residue was ~.
washed with a large amount of EtOH to give 53~
. mg ~f the crude productt which was dissolved in a 9mall amount cf water and chromatographed on a ~olumn of ~G-50 ion-exchange resin (N~ type9 20 ml). The column was irrigated with water, 1 L
of 0.1 N NH~OH, ~00 ml of 0.2 N ~40~ and finally ~ ~;
500 ml of 0.5 N N~40H. Ten milliliter fra~tions ;~
were collected and subjected to ninhydrin spot test, ~isc assay ~B. oubtili~ ~I 21g~ and T~C . .
(thin,layer chromatography) silica gel plate;
~olvent system , S-llO ~CHC13-MeO~-28~ NH40H-~2 ~ 1 : 4 : 2 ~ he ~ractions which gave .
ni~hydrin-po~itive and ~io-active spot at R~

. *Trade Marks !~
,: ' .: ,"

~ -32- ~

. ,. . : , ,, , . , ,., ; ;

,'''~
, ` ' ' ' .,' ' ~

~5~7~
O.SO by TLC were com~lned and evaporated into dryness under re~uced pre~su~e to give 137 mg (27g) BB~K 25, m.p~ 183-187C (dec.).

NMR(D2O): ~ ~ppm), ~Sl ~3 H, s, 6'~N CH3)~

4~95 (1~, d~ 4 Hz, l~-H), 5.01 ~lH, d, 3 Hz, 1 3 -Hl .

for Cl~H38N~lllt~C3:
C, 42.85; ~ gt N, 9~9~
Found: C, 42.97; H, 7~27; N, 9.63.
2. To a sUspe~sion of 8.8 g of LiAlH4 in 100 ml o~ dry dioxane was added a .suspension of ~.0 g o~ 6'-N Cb~-kanamycin A in 2bo ml of dry dioxane and the reaction mixture was stirxed for 4 day~ .
at 80 C. ~he reaction mixture was cooled to ~.
10 C, treated cautiously ~Jith 200 ml of~water and iltered to remo~e in~oluble material~ The ~ltrate was neutralized with N HCl and evaporated in vacuo. The xesidue was washed several times with EtOH and dissolved in a small amount o~ water.
The aqueous solution wa~ chromatographed on a column of CG-50 (NH~ ~ , 200 ml), which was washed with 100 ml of water and eluted ~uccessively with 2.0 L of 0.1 N N~OH and 1.5 L o~ 0~2 N NH401~.
The e~uate was collected in 20-ml fraction.
~ractions 119 to 144 showed a bioactive and ninhydxin .. . . .
~,'` ' ' ~ -.
:, :

.. . .
, .' , ,.

. . .

.

~5;Z7~

positive spot at Rf. 0.45 in TLC (silica ~el ~ plate; CHC13-C~I-28% NH4OH-H2O = 1 : 4 : 2 : 1).
. They were combined~ concentrated under reduced pressure and finally lycphilized to give 1.139 g (16%~ of BB-X 25 which was identical with that prepared in Meth4d A~(l)o Example 2 .~ .

. To a stirred suspension of 618 mg ~1 m mole) of 6'-N-Cbz-kanamycin in 30 ml of dry pyridine were added 7 ml o~ trimethylchlorosilane and 14 ml of he~amethyldisilazane at 70C. The reaction !
.~ mixture was stirred o~ernight at the same tempera-ture and evaporated in vacuo. The residue was '~ treated with dry tetrahydrofuran (THF). The ,, ~
insoluble material was filtered a~d washed with dry TI~. The filtrate and the washings were combined and evaporated in vacuo to give 1.567 g o~ the trimethylsilylated product, which was d~olved in 30 ml of dry ~HF. The solution was added to a suspension of 758 mg of lithium aluminum h~rdride in 70 ml of dry THF. The mixture was re~luxed for 22 hrsO under stirring. After cooling to ca. O~C., the reaction ~ixture was reated ~autiously with 20 ml o~ ice water and filtered to remove~insoluble material. The ~iltrate was ~eutralized with 6 N HCl and evaporated to dryness ~n v~cuo. The residue was washed several times with :, .

;

. -34-. ~ .. . . . .

~C~5Z~O

.` i ethanol, dissolved in a small amount of water and .
chromatographed on a column of C~-50 (N~14 ~, 50 ml).
~, The column was washed with 50 ml of water and eluted successively with 1 L of 0.1 N NH40H and 600 ml of .' 0~2 N NH~OH. The eluate was collected in 20-ml fractions and monitored as described in Method ~
i Practions 47 - 72 which gave a ninhydrin positive and bioactive spot a~ Rf 0.50 b~ TLC were combined, , evaporated under reduced pressure and ~yophilized .~ to give 258 mg t52% from 6'-Cbzokanamycin A) of .. . . .. .
BB-K ~-5, m.p. 183-187C.

.' . ~ . ,.. , . ~ .
.j ~, ' ' .i ' ~ I .
To a solution of 750 mg of 6'-N-methylkanamycin A
~BB-K 25)~in 30 ml o~ 60% aq THF was added 525 mg of . N-hydroxysuccinimide ester of N-Cbz-L-r-amino-~-i~ ~ hydroxybutyric acid. The reaction mixture was hydrogenatated overni.ght at room temperature under atmospheric pressure in the presence o~ 500 mg of 10% palladium on charcoal. The react.ion mix ure was filtered and evaporatea under reduced pressure.
The residue was dlssolved in a small amount of water :
and adsorbed on a column of CG-S0 (NH4 ~, 70 ml).
The column was washed with water and irrigated ~
uccessively with 850 ml of 0.1 N ammonia, (tube ..
nos. 1-43 w~re colleoted in 20~ml fractions), .
;~ , ' . ':''. ' . , ' "' :
,, , ` ' .

,, ~ , .

`~ 1450 ml of 0.2 N ammonia (tube nos. 44-115 in 20-ml fraction) and finally 100 ml of O. 5 N anunonia ~tube nos . 11~-215 in ..10-ml .1 fraction).. Fractions 152 to 161 which showed -~ 5 a bioacti~re and ninhydrin positive spot at Rf. 0.17 in TLC (silica gel, CHC13-CH~OH-28%
.
NH.~,OH-H;~O = 1: 4: 2 :1 ) were combined, evaporated - under reduced pre~sure and lyophilized to give 149 mg ~169~) of BB-K 28, mp. 187-189 (dec) ,, Infrared lIR~KBr)]: I~c_o 1650 cm~
NM~ (D20) . 2. 70 pp~ 3~I, s, N-C~3) .
~ calc'd ~or ~ }I N O 2~ CO
`` 2~120: C, 39.52; H, 7903; N, 9~23.
~ound: C, 39.26, 39O00; H, 6.69, 6.543 N, 9.69, 9.20.
In order to remove a trace of BB-~ 11 type ~s~mer (3a-N acylated isomer) ~ BB-K 28 ~as sub-Jected to column chromatography w;th tetramine - t:opper ~TACu) type of Am}~erlite CG-50 ion e~change resin. BB-X 28 ~73 mg) was dissolved in a small ~noun~ ~ water and cllromatographed on a column o~ CG-50 ~TA::u type, 3 ml) . The column was w~shed with 20 ml of water and then eluted with 100 ml o~ 0.2 N N~3O11, and finally 100 ml of ~.0 N hE14OH., The eluatei was collected in 7-ml ~ractions and monito~ed with ninhydxin spot test, ~,~ , . .
*Trade Mark .. . .
~, .

~ -36-'_.'`~' ~' ,~ . , , , - ~, , ~52,~80 di~c assay (B. subtillis PCl 219 and Pseudomonas .. ae~inosa) and TLC on silica gel rS-llO, ninhydrin).
; ~ractions 21 - 24 showed a ninhydrin-positive and bio~active (against the P. ~ osa strain) spot at Rf 0.2, ~hey were combined and evaporated in vacuo to give 30 mg of blue powder. The blue-colored residue (30 mg~ wa~ dissolved in a small amount of water ~nd adsorbed on a column of CG 50 -~N~ ~, 3 ml), which was irrigated;with 20 ml of ` 0.2 ~ NH40H and 200 ml of 0.5 N NH~OH. The eluate was collected in 7-ml fractlons. Fractions 19-23 showing positive ninhydrin test were combinedy evaporated in vacuo and free2e-dried to give 20 mg of pure B~-K 28; m.p. lB7~I89 C(decO)O
~,1 ' E

-, kan ~
i A mixture vf 218 mg (0.912 m mole) of N-Cbz-;' L-isoserine,163 mg (0.912 m mole) of N-hydroxy S-norbornene-2,3-dicarboximide (HONB) and 188 mg , tO~912 m mole) of DCC (dicyclohe~ylcarbodiimide) i~ 10 ml of THF was kept to stand at S C overnight and then ~iltered. The fil~rate being added to a solution of 441 mg to.8g2 m mole) of 6'-N-methyl kanamycin A in 20 ml- of 50% aq THF~ the mixture was :, , , . -37- .

., .
~' ' ,. . .

;`:
;

~5Z7 ', .
.. ~ .
stirred at room tempe~ature for S hours and con-centrated under reduced pressure to about 2 ml.
~he concentrate was adsorbed on a column of Amberlite CG SO (NH4~, 26 ml~, which was washed with 40 ml of water and eluted with 500 ml of 0.1 N NH40~. The eluate was collected in lO ml xactions. Fractions 11-16 wexe combined and evapora~ed in vacuo to give 231 mg of the N-acylated product. From the 0.3 N NH40H eluate was re~coYered 175 mg (40~) of the starting material, BB-~ 25.
To a solution of the acyl derivative in 20 ml of 50% aq EtQH was added 130 mg of 10~ Pd on char- :
coal and the mixture was h~drogenated under ordinary pressure at room temperature. The catalyst was 11 ered off and the filtrate was evaporat~d to remove the organic solvent. The resulting aqueous .
~olution was sub~e~ted to column chroma~ography on CG-50 (NH4~ 25 ml). The column was eluted uccessively with 40 ml o~ water, 245 ml o 0.~ N
NH~OH~ 500 ml of 0.2 N NH40H, 300 ml of 0~4 N
NH40H and the eluate was collerted in 10-ml frac-tions. ~he bio-active fractions were combined and evaporated in vacuc ~o afford 127 mg of the crude.
product, which was re-chromatographed on a column CG,50 (tetramine copper type, 4 ml) and eluted .

; , ~ -3~-~.
: '' ' , .,.. , . . :
``' .~SZ7~10 , with ~00 ml vf 003 ~ N~40H, 300 ml of O . 5 N NH40H
:~ and finall~ with 300 ml of 1 N NHqO~I~ The eluate was collected in 10-ml fractions.
~5 q~ube No Eluant .~ount R~ ~, 4 003 N NH40}i' 27 mg 0.33 inactive .. 19-24 0,5 N NH40H 38 mg 0.33 inactivet probably a position ~svmer .. 50-54 1.0 N N~40H 35 mg 0.37 . tha desired produ Tube nos. 50 through 54 were combined and - evaporated into dryness to give 35 mg (6.8~) o~ the desired bio-active product, BB-K 16~; m.p.
178-185 C IdecO). v~Bo 1630 cm 1, , , .
: . . .
Exam~l~ 5 i N-lL(-)-~-amino~ hydroxy~aleryI]~6~-N-methy~

A mixture of 94 mg ~0.353 m mole) o~ N-Cbz-L-y-amino-~-hydroxyvaleric acid, 64.5 mg (0.353 m mole) of HONB and 74.5 mg ~0.3S3 m mole~ of DCC
in 5 ml o~ dry TH~ was kept to stand at 4 C
overnight and filtered. The filtrate was added to a solutlon of 175 m~ (0.351 m mole) of 6'- .
N-met~ylkanamycin A in 10 ml of 50% a~ THF and the mixture was stirred at room temperature ~o* 5 hours. ~he reaction mixture was hydrogenated . I , .

!
.1 .

. 1.

~ ~ . , . I
~S27~

at room temperatuxe in the.presence of 70 mg o 10% palladium on charcoal. The catalyst was filtered off and the filtrate ~was evaporated to re~ove the TH~ The resulting aqueous concentrate was ch~omatographed on a column of Amberlite.
C~-50 ~NH4~type, 20 ml), which was eluted successively with water ~50 ml), 0.1 N NH40~ ~250 ml), 0.~ N NK40 (450 mll and O.5 N NH40H ~400 ml). The eluate was collected in 10-ml ~ractions. The bio-active fractions were combined a~d evaporated into dryness to a~ford 40 mg of the crude product, which was re-chromatographed on a column of CG-50 (tetramine coppsr type~ 105 ml~ and eluted with 10 ml of water and 100 ~1 of 0.5 N NH40H. The eluate was collected in 5 ml fractions.

"
Tu~e ; No~ ~ Amount Rf Remark

7-80.5 N ~H40H S ~g . 0~21 inactive 90.5 ~ NH40~ 9 ~g 0.21, 0.29 a mixture o~ ~wo .
. components 10-140.5 N NH~OH 24 ~g 0.29 th~ desired pxoduct, BB ~ 163 ~ube nos~ 10 throu~h 14 were combined and evaporated into d~yness to give 24 mg ~11%~ of the .' desired bioaative product, BB-X 163; m~p~ 167-~l I7~ C ~dec~j. v~O 1620 cm lo .
.1, 1 ;.
':
o _ . ' ~ ,'.
. ~ .

`1 ~Q5Z7~

. . .
Example 6 69-N-Meth~ylkanamycin s! BB-K 140 To a suspens~ian of 2.0 ~ ~3.25 m moles) of 6'-N-Cbz-kanamycin B in lOO ml of dry pyridine were added 20 ml of trimeth~lsilyl chloride and 50 ml of hexamethyldisilazane at 70 C. under ~tirring and the mixture was continued to s~ir overnîght at the same temperature~ The precipitate was removed by filtration and washed with d~y THF. The filtrate was combined with the washings and evaporated into dryness. The oily residue was dissolved in 80 ml of dry THF. The solution was added dropwise to a stirred suspension of 4.4 g of lithium aluminum hydride in 2ao ml o~ ~ :
-dry THF and~the mixture heat2d under.reflux over-night. After cooling~ the reaction mixture was :
treated caut~iously with ice water, adjusted to pH 7 w1th 2 N HCl and evaporated to dryness under reduced pressure. After washing with ethanol thoroughly, the residue ~2.7 g) was dissolved in 5 ml o water and chromatographed on a column of Amberlite CG 50 (NH4~,40 ml). The column was washed with 300 ml of water and~eluted with 0.1 N
ammonia. The eluate was collected with 10-ml fraotions and monitored with ni~hydrin spot test, disc assay (B, -subtilis PCI 219) and TLC (silica . . . .

- 4 1 - ~

.f,~" ~

~L~S~7~ Ql .
gel plate, CHC13-C1l30H-2B~ NH40~-~20 =
1:4:2:1~. Practions 70 - 134 which showed a ninhydrin-positive and bio-active spot at - Rf 0.47 by TLC were combined, evaporated under .; S reduced pressure to give 889 mg (55 %) of BB-X 140; m~p. 177 -,181 C ~dec.)~
N~R (D20): 2~74 ppm (3H, s, N-CH3).
Analysis Calc'd. for Cl~H39N501o-H2~03 C~ 42~93; ~, 7g39; N, 12.52 - 10 F.ound: C, 42093; ~, 7.1~; N~
. 11.86.
., ~,~. ~
. .
`;i ~ ~

, 15 A mixture of ~53 mg ~l m mole~ o~ N-Cbz-L-`,' . y-amino-~hydroxybu~yxic acidg 115 m~ ~1 m mole) ~, .
`;~ of ~-hydroxysuccinimide (~OSu) and 206 mg (1 m mole) ' o~ DCC wais stirred ~ox 3 hours at 5Co 8nd filtered ..
to remove the precipitate which separated during . the reaction. The resulting actlve ester ~olution -, wa~ added to a solution o~ 497 mg ~1 m mole) o~
i 6~-N-m~thylkanamycin B ~BB-K 140) in 10 ml of water ., and the mixture stirred overnigh at xoom tempera-.1 tu~e. The reaction mixture was filtered and ;-i25 eYapora~ed in vacuoO ~he residue was dissolved ..
`.$ in S ~1 o water and adsorbed on a ~olumn o~ CG-50 '.
.j , , ' ' . ':
-42- :
; .

. . .: .
.:~

'. ' '' , . ' :' .
ion-exchange resin (NH4 + 1:ype, 45 ml), which .` . was elu~ed successively wit:h 200 ml of water, 1500 ml of O.OS N ammonia and 500 ml of 0.3 N

~mmonia. The eluate was collected in lO-ml fractions. The desired intermediate derivati~e .,: .
~as contained in ~xactions 58 through 72 which were eluted with 0.05 N ammonia, while 163 mg - .
33%) of BB-K 140 was recovered from fractions .- -eluted with 0.3 N a~monia.
- A solution of the in~ermediate derivative in r, 20 ml.of 50% THF was hydrogenated overnight under ordinary pressure in the presence of 30 mg of 10% palladi~m on carbon. The reaction mixture was filtered and concentrated to about 4 ml.
The concentrate was adsorbed on a column of ~mberlite CG-50 (NH4 type, lO ml), which was .
washed ~ith~150 ml of water and eluted successively with 350 ml o~ 0.05 N NH4OH7 300 ml of O.l N NH40H~ 150 ml of 0,~ N NH40H and 300 ml of 0.5 ~ 40Hb The eluate was ~ollected in lS-ml fxactions.~ Fractions 67 and 68 which showed a bio-active spot at Rf 0.22 on TLC ~silica gel plate, CHC13-CH3OH-28~NH4OH-H20=l;~:2Ol) were combined and re-chromatographed on a column of CG-50 (the lowex part,~:NH4 type lml; the upper part, tetramine , . , ,; : . ' , ~f~
~ -43- . ` ;;

, _ .

:

5~81D
.~ copper type 2 ml). The column was eluted ~ith . 100 ml of 0.5 N NH~OH and then with 5~ ml of 1.0 ., N NH~OH and the eIuate was collected in 5-ml fractionsO Fractions 15 through 33 were combined and evaporated i~ vacuo to give 29.4 m~ tS%) of the -desired hio-active product, BB-X 142; m.p. 188-192 C
^ (dec.). IR(KBr): vC=O 1640 cm ~ -- Analy~is Calo'd. for ~3~46N6ol2-2H2cO3 r:, ~' C, 41.5S; H, 6.97; N, 11~63.
Found: C, 41~52; H, 6.72; N, 10.95.
A small sample of BB-K 142 was heated at 100 C
l ~ for 1 hour i~ 0.5 N NaOH to afford 6'-N-methylkanamycin .-.~ B ànd 1-HABA which were confirmed by TLC. . .:
;`, - , ~ ., ~ l-N-fL~ Amino--h _ x~

! ' ~ e ~ L ~
A stirred mixture of 120 mg (0.5 m mole) of ~' ~ N~Cbz-L-isoserine, 58 mg ~Q.S m mole) of HOSu and 103 mg t0.5 m mole) of DCC in S ml of THF was kept to stand at 5 C overnight. The mixture being ,,, iltered, the filtrate was added to a solution o~ /.
,.J 248 mg (0.5 m mole) of BB-K 140 in 5 ml of water and Stirred overnight. A~ter removing ~F, the aqueous 801ution 'was adsorbed on a column of ~mberlite CG-50 ;.
~NH4+ type, 10 ml)O Elution was carried out with 300 ~1 of 0.5 N.NH40H followed by 300 ml of ~:
', . , '` "~,~

~ 44 "' ~: :
f ' `' ~;
s~
;~ :

;, O.l N N~14C~ and lO-ml fractio~s were collected.
` The desired intermediate dexi~ative (68 mg) was .: ;~
. obtained by evapora~ion of ractions 18 through .-, . , , ~ .
31 which were eluted with 0.05 N NH4CII~ while 69 ~g (28%1 of BB-K 140 was recovered from frac-tions eluted with O.l N ammonia.
i - A solu~ion of the intermediate derivative in :
',J''~ 10 ml of water was hydrogenated overnight in the .-, presence of 20 mg of 10% Pd~=C and the reaction :--, mixture ~as filtered and concentrated to about 5 ml. The concentrate was chrom~togxaphed on a ;~ column of Amberlite CG-50 ~NH4 , 8 ml~ and eluted - successively with 2~0 ml o~ 0.05 N NH4CH~ 340 ml ~. .. ..
o O.l N NH40H and 200 ml of 0.~ N N~40H. The desired bio-active component ~35 mg~ was obtained from fractions elut~d with 0.2 N NH4QHo The pxoduct which still showed two to three ninhydrin positive.
spots was purified by re-chromatography on a column of:CG-50 (the lower part, NH4~ typb 1 ml; The upper part, tetramine copper type 2 ml), which eluted `~
~uccessively with 60 ml of 0.3 N NH40H, llO ml of 0.5 N NH40H and lO0 ml of l;0 N NH40H. The desired pxoduct, BB-R 148, was obtained from fractions .. . . . .
.. i - ~ 40-50 which were eluted with 1.0 N NH40H.
;, ~.

~i . . , ' :~' -45 ~

~' ' '' ' ' ;'',.
, ' , . .

.

.' , ' :
S~7~
,, Yield 8.1 mg. ~3 %); m.p. l90- .

198 C. (dec.). IR(KBr): vc=0 1630 cm l.

..
. . ' ~ ~
.` ~ =~ ` i ,:
Substitution in the procedure of example 5 ; . . . . . :
for the 69-N--methylkanamycin A used therein of an e~uimolar quantity of 6'-N-methylkanamycin B
,; , ~
, produces the title product. .-.` ~
, ~mberlite CG-50 is the tradename ~or the ., chromatogr~phic grade of a weakly acidic cationic - exchange resin of a carboxylic-polymethacrylic type.
., ' ~;~.:, - ', was prepared in the following way: to a stirred ' . suspension of CG-50 ~JH4 ) in wa~er was added j lO~cupric suIfate solution to give the copper salt of CG; 50,which was filtered. The resin was washed several t~mes with water/ then treated with lN NH40H undex stirring~ ~iltered and washed several times with water to give deep blue~ cupra-ammonium form.of CG-S0. :~

~.

.
~;~
, `: ~os~
Exam~le 10 P~ ~, dro ~ ylk B
- One mole of l-lL~ y-amino-a-hydroxybutyryl]-. 6'-N-methylkanamycin A or B is dissolved in 1 to 3 ~.:
. liters of water~ The solution is filtered to remove any undissolved solidsO ~o the chilled and stirred ~olution is added one mole of su~furic acid dissolved in ~00 ml of water. The mixture is ., .
allowed to stir for 30 minutes, following which cold ethanol is added to the mixture till preci-p~tation occurs. The solids are collected by fil-tration and are determined~to be the des7xed mono- -,........................ . . .
~ulfate salt.

. xample 11 .

.

~ or B.
, Thirty-~ive grams o~ 1-[~ ~nino-a-hydroxy-propionyl]-6'-N-methylkanamycin A or B i~ dissolved .: in 125 ml. o~ deionized waterv The p~ is lowered '~ .
,~ to 7-7.5 with 50% v./v. sulfuric acid.
i Eight and one-hal~ grams of Darco G-60 (activàted charcoal~ is added an~ the mixtur~ is ,,; .

. ' `;, .

: -47-''' .

:~ `

iifff~fft ffff~f~ff~f . . .
slurried at ambient room emfperature for 0. 5 hour.
, ~ , fTfhe caxbon is rf~noved by suiltablff~ filtration and .. ~ washed with 4 0 ml . of water O fIffhe water wa~h is added to the ~iltrate.
The combined filtrateo~wash above is adjusted to pH 2-2.6 w~th 50% v./v. ~ulfuric acid. A
Lfff~rge amount of carbfon dioxide is evolved. f~fh . ~olution is le~t a~ house vacuum wi~h stirring c~r 21fO, minutes to expel additional carbon dioxide.
Eif~ht and one h~ rams of Darco fffff;-60 are added to the degassed sctflution~ The mixture i~ ~:
~lurried for 0 0 5 hour at ambient room temperature .
The carbon is rfemoved b5~ suitablfe filtration and washed with 35 ml. of deioniæed wa~er. f-Tfhe wash is added to the f iltrate.
. The combined fil~rate-wash is adjus~f~ to .. .
p~ 1 lffff 3 .with S0~ v./v. sulfuric acid. This olutiofn iS added with rap ff d ~tirring over a lfflf minute perlod to 600-8lDffffff ml. of ~nethanol t3-4 Yolumes of methanol). The mixture is s~lrred ~f iEox 5 minutes at pH 1-1.3,, pa~sed through a : .
- 100 mesh screen, stirred fox 2 minutes and allowed to settle ~or 5 minutes. ~Sost of the Eiu~ern~tant is decanted,. ~he remaining slurry ~s suitably filtered, washed with 200 ml~ o 2Itethanol and vacuum dried at 50 C. for t2f4 hours to yield the t ff tle disulfate salt~

*Trade Mark t fff f-- .

Claims (16)

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 the formula (IV) in which R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl, L-(-)-.beta._aminco-.alpha.-hydroxypropionyl or L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl, and R3 is -OH or -NH2' which cpmrises the consecutive steps of A) acylating the compound having the formula II

in which R3 is -OH or -NH2 with an acylating agent:
having the formula (VII) in which W is a radical selected from the group comprising , , or M is a radical selected from the group consisting of , , , and in which n is an integer of 1 to 3; in a ratio of about 0.5 to about 1.4 mole of the compound VII per mole of the compound II, in a solvent selected from the group comprising a mixture of water and ethyleneglycol dimethyl ether, dioxane, dimethylacetamide, dimethyl-formamide, tetrahydrofuran and propyleneglycol dimethyl ether, to produce the compound having the formula III

in which n, R3 and W are as above; and B) Removing the blocking group W from compound III to produce a compound of Formula IV.

2. A process as claimed in Claim 1, where Step A
is carried out with an acylating agent of the formula , in which W is a radical of the formula and M is or in which n is an integer of 1 to 3, in a ratio of about 0.8 mole of acylating agent to about 1.1 mole of compound VII in aqueous tetrahydrofuran.

3. A process according to claim 1, wherein the blocking group W is a radical of the formula and is removed in Step B by hydroqenation.

4. A process according to Claim 3, wherein the hydrogenation is carried nut with hydrogen in the presence of a metal catalyst in a water-water miscible solvent system.

5. A process according to Claim 3, wherein the hydrogenation is carried out with hydrogen in the presence of palladium, platinum, Raney nickel, rhodium, ruthenium or nickel in a mixture of water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether or propyleneglycol dimethyl ether.

6. A process according to Claim 3, 4 or 5, wherein the hydrogenation is carried out with hydrogen in the presence of Palladium on charcoal in a 1:1 water-dioxane solvent system and in the presence of a catalytic amount of glacial acetic acid.

7. A process for the preparation of a compound of the formula (IV) in which R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl, L-(-)-.beta.-amino-.alpha.-hydroxypropionyl or L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl, and R3 is -OH or -N2, which comprises the consecutive steps of A) acylating the compound having formula II

II

with an acylating agent having the formula or in which n is an integer of 1 to 3 in an aqueous tetrahydrofuran or aqueous dimethylformamide-acetone solvent system at about room temperature to produce the compound having the formula III
in which n is an integer of 1 to 3; and B) hydrogenating compound III in aqueous tetrahydrofuran in the presence of palladium on charcoal at about atmospheric pressure to produce compound IV.

8. A process according to Claim 7, wherein the acylating agent of Step A is generated in situ by mixing a compound of the formula with equimolar quantities of N-hydroxy-5-norbornene-2,3-dicarboximide and dicyclohexylcarbodiimide.

9. A process according to Claim 1, 2 or 7, wherein R3 is OH and R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl, L-(-)-.beta.-amino-.alpha.-hydroxypropionyl or L-(-)-.delta.-amino-.alpha.-hydroxyvalervl.

10. A process acorrding to Claim 1, 2 or 7, wherein R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl and R3 is OH.

11. A process acorrding to Claim 1, 2 or 7, wherein R is L-(-)-.beta.-amino-.alpha.-hydroxypropionyl and R3 is OH.

12. A process acorrding to Claim 1, 2 or 7, wherein R is L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl and R3 is OH.

13. A process acorrding to Claim 1, 2 or 7, wherein R3 NH2 and R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl, L-(-)-.beta.-amino-.alpha.-hydroxypropionyl or L-(-)-.delta.-amino-.alpha.-hydroxyvalervl.

14. A process acorrding to Claim 1, 2 or 7, wherein R3 NH2 and R is L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl.

15. A process acorrding to Claim 1, 2 or 7, wherein R3 NH2 and R is L-(-)-.beta.-amino-.alpha.-hydroxypropionyl.

16. A process acorrding to Claim 1, 2 or 7, wherein R3 NH2 and R is L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl.