Note: Descriptions are shown in the official language in which they were submitted.
5:~L3
ANTIBIOTIC DERIVATIVES
m i~ invention relates to a semisynthetic 1-
substituted derivati~es o~ kanamycin A or B, said compounds
being prepared by acylating the 1-amino~function of kanamycin
A or ~ wlth a ~-amino-a-hydroxypropionyl or ~-amino-a-hydroxy-
valeryl moiety.
me kanamycin~ are known antibiotics described in
Merck IndexJ 8th Edition, pp. 597-598. Kan~mycin A is a
compound havin~ the formula
HO CH2-NH2
HO ~ \
HO
NH2
H2
Kanamycin B i.s:a compound ha~ing the formula :
CH2-NH2
~1
HO ~
NH2 ~ N~I2
H~
HO ~ / ~ NH2
H2N~/
Ib
. ~ :
~ .
.,~
6S~3
The compounds of the present invention have the
formula
6' .
H o 4' C~I2-NH?
~ .
H ~ 1' 1 2
~ ~H-R
ln which R3 is CH or NH2 and R iæ L~ -amino-~-hydroxypro-
pionyl or L~ amino-a-h~droxyvalèryl; or nontoxic pharmaceu-
tically acceptable acid addition saltæ thereo~ The compounds are
named l-~L~ mino-a-hydroxypropionyl~kanamycin A, l-[L-(~
amino-a-hydroxypropionyl~kanamycin B, l-~L~ amino-a-hydroxyval-
eryl~kanamycin A and l-tL~ -amino-~-hydroxyyaleryl]kanamycin B.
, ~ The compoun~ de~ignated l-~L-(-)-y-a~ino-a-hydroxy-
butyryl~-kanamycin A ~BB-K8~ i8 de~cribed in the Journal of
Antibiotics, 25 (12), pp. 695-731 (December9 1972).
For the purpoAe of this di~clo~ure, the term "nontoxic,
ph~rmaceutically acceptable aci~ addltion salt" shall mean a
mono, di-, tri~, tetra- or pentasalt formed by the lnteraction ~:
of one molecule o~ compound I~ ~ith 1-5 moles o~ a nontoxic,
pharmaceutically acceptable acid. Included among these acids
are acetic, hydrochloric, sul~uric, maleic, pho~phoric, nitric,
hydrobromic, ascorbi~ malic and citric acid, and those other
acid~ commonly used to make salt~ o~ amine containing pharmaceu-
tical~.
5~3
The compounds o~ the present invention are prepared
by the following dlagramatic scheme (whiCh illu8tratea the
preparation of the ~-amlno-a-hydroxypropionyl- substituted
kanamycins).
1) Kanamycin A (Ia) or N-(Benzyloxycarbonyloxy)
Kanamycin B (Ib) Succinimide >
R
HO~NH-C-O-CH2-C6H5
HO ~ \
R ~ 2 II
HO ~ H ~ NH2
H2N~ /
HO ~
2) Compound II N-Hydroxysuccinimide ester o~ L-
(-)-~-benzyloxycar~onyl-amino-~-
hydroxypropionic acld
HO CH2-NH-C-O-C~2-c6H5
~L_o
HO ~
H ~ HO ~ NH
H2N~ C-O
HO-CH o
CH2-NH-b-O-CH2-c6H5
III-l
~LO~ 3
3) Compound III-l H~/Pd/C
> ~ .
H ~ 12-NH2
HO ~
R3 ~ NH2
H2~ ~ !=0
H HO-CH
~2
~2
IV
A preferred embodiment of the present invention i5 the
compound having the fo~mula
HO~Rl
H ~ \
R ~ . :~ :
HO CH20H HO ~ NH-R2
H ~
V
in which Rl i~ H or C6H5-CH2-0-~-, R2 is Hg L-(-)-~-amino-~-
hydroxypropionyl or L-(-)-~-amino-a-hydroxyvaleryl and R3 is ~H
or NH2,wherein Rl or R2 m~t be other than H; or a nontoxlc
pharmaceutically acceptable acid addition ~alt thereof.
Another pre~erred embodiment is the compound o~ form~la
V in which R2 i~ L-(~ -amino-~-hydroxypropionyl or L~
~mino-~-hydroxyvaleryl, ~1 is hydroeen and R3 is CH or NH2.
- 4 -
s~3
A more preferred embodiment is the compound o~
formula V wherein Rl i~ H~ R2 i L~ -amino-~-hydroxypropionyl
or L-(-)-~-amino-a-hydroxyvaleryl and R3 i~ CH; or a nontoxic
pharm~ceutically acceptable acid addition salt thereof.
Another more preferred embodiment is the compound of
formula Y wherein Rl is H, ~2 is L-(~ -amino-~-hydroxypropionyl
or L-(-)-6-amino-~-hydroxyvaleryl and R3 is NH2; or a nontoxic
pharmaceutically acceptable acid addition salt thereof.
Other preferred embodiment~ are the sulfate3 hydro-
chloride, acetate, maleate, citrate, ascorbate, nitrate or
phosphate salt~ of compound V.
Another more preferred embodiment i5 the monosulfate
s~lt o~ co~pound V.
Still another preferred embodiment is the disulfate
salt of compound V.
m e ob~ecti~es of the present in~ention haYe been
achieved by the pro~islonJ according to the pre~ent invention,
Or the proce~ for the preparation o~ compounds having the
formula
CHz-NH2
HO
HO ~
HO ~ HO ~ NH-R2
H2N ~
in which R2 i8 L~ -amino-a-hydroxypropionyl or L~ amino-
a-hydroxyvalerylg dnd R3 is CH or NH2i or a nontoxic pharmaceu-
tically accept~ble acid addltion ~alt thereof; which process
compri~es the consecutiYe steps of
A) reacting k~namycin A or kanamycln B with an agent
~elected from the compounds having the ~ormulas
S~L3
~4 1 ~
R5 ~ CH2-O~C-0- ~ R ~ -CH2-0-C-X
X~3~
CH3_1_0_C_N3 , 02N - ~ h , ~ -C-0-C-CH3
CH3 ~2 CH3
C--X , X-C~2~CI-X X CH ~
(or a carbodiimlde addltion compound thereo~ or~LC-CH2-CH2-C02H
(or a carbodiimide addition co~npound ~ereof), in whlch ~4 and R5
are allke or di~ferent and each is H, F, Cl~ Br, NO23 aH9 (lower)-
alkyl or (lower)alkoxy~ X 1~ ~hloro, bromo or iodo, or wi~h a `
~unctionally equi~alent agent; in a r~tio of one mole or less of ~ ;
agent per mole Or kanamycin A or B in a ~ol~ent, preferably . .:
~elected ~rsm the group comprised o~ dime~hyl~or~mide, dimethyl~
acetamide, tetrahydro~ur~n, dioxane, l,2-dl~ethoxyethane, meth~nol,
ethanol, water, a~etone, pyridine, N-(lower)alkylpiperidine, or .
m~xtures thereo~ but pre~erably dimethyl~ormamlde-~ater, preferably
at a temperature below 50C. and most preferably below 25C., to
produce the compound having the.formula
H0 ~ CH2-NH-Y
H0 ~
R3 ~ NH2
- CH2oH ~ ~ \
~0 ~ / `~
~"\ /
. H0 ~ II
~ ~ .
in which R3 is as defined abo~e and Y i~ a radical o~ the
formula
3 ll r \ ~
RY ( H3 ~N02
~1
~ C X-CH2-C , or ~--C-CH2-CH2-C-
I
~' ''
in which ~ and R5 are as defined above;
B) acylating compound II with an acylating agent
having the formula
- ~R
W-NH-(CH2)n~CH-C-M
VII
in which n is:l or 3 and W i8 a radical selected from the group
comprising
2--- C~3-~ 0-0- , 02N
--~ X-CH~-C 3 or ~ C-CH2-CH2-C-
f~
~
ut preferably ~ CH2-0-C- , in which R4 and R5 are
R
as defined above, and M is a radical selected ~rom the group
compri31ng
o~ ~Lo~6S~3
_o~ 0 ~ N2 ~ - ~ N2 J
O N02
-o-r ~ - ~ but preferably -O-N ~ ,
or with its functlonal derivative as an acylating agent, in a ratio
of at least 0 5 mole of compound VII per mole of compound II, but
preferably in a ratio of about 0.5 to about 1.4, and most preferably
in a ratio of about o.8 to about 1.1, in a solvent preferably
selected from the group comprising a mixture of water and ethylene-
glycol dimethyl ether, dioxane, dimethylacetamide, dimethylformamide~
tetrahydrofuran, propyleneglycol dimethyl ether or the like, but
preferably water-tetrahydrofuran or water-ethylene glycol dimethyl
ether, to produce a compound of the formula
HO~-NH-Y
HO
R3 ~ -
HO ~ \
H ~ CH2CE / ~ NE
H2N ~)~ O
HO ~ / HO- ~
( ~2)n
~H
III W
in which n is 1 or 3,and R3, Y and W are as described above; and
C) if desired, remo~ing the blocking groups W and Y
from compound III by methods commonly known in the art, and pre-
ferably when W and Y are radicals of the ~ormula
- 8 -
6~3
R4 0
~C~2-0~
R5
by hydrogenating compound III with hydrogen in the presence
of a metal catalystJ preferably selected from the group com-
prising 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 sioxane,
tetrahydrofuran, ethyleneglycol dimethyl ether, propyleneglycol
dimethyl ether, of the like, but preferably 1:1 water-dioxane
(preferably in the presence of a catalytic amount of glacial
acetic acid) or 1:1 water-tetrahydrofuran to produce the compound
of formula IV.
It should be apparent to those knowledgeable in the
art that other agents can be used in the process above to react
wi~h and protect the amine functions o~ the intermediate
compounds of the instant invention. mis disclosure is meant
to include all such agents that produce labile amine blocking
groups, said labile blocking groups commonly employed in the
synthesis of peptides. me labile blocking groups must be
readily removable by methods commonly known in the art. Examples
o~ said labile blocking groups and their removal can be found
in the review of A. Kapoor, JO Pharm. Sciences, 59, pp. 1-27
(1970). Functionally equivalent agents would include corres-
ponding carboxylic chlorides, bromides, acid anhydrides, including
mixed anhydrides and particularly the mixed anhydrides prepared
from stronger acids such as the lower aliphatic monoesters of
carbonic acid, of alkyl and aryl sulfonic acids and of more
hindered acids such as diphenylacetic acid. In addition, an
acid azide or an active ester of thioester (e.g., with p-
nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetlc acid)
may be used or the free acid it~elf may be coupled with the
1~65~L3
kanamycin derivative (II) after first reacting said free
acid with N7N~-dimethylchloroforminium chloride tc~. Great
Britain 1, oo8, 170 and Novak and Weichet, Experientia XXI/6,
360 ( 1965 ) ] or by the use of enzymes or of an NJ N'-carbonyl-
diimidazole or an N,N~-carbonylditriazole [cf. Sheehan and
Hess, J. Amer. Chem. Soc., 77, 1067, (1955) ] or of alkynylamine
reagent ~cf. R. Bui~lle and H. G. Viehe, Angew. Chem., Inter-
national Edition 3, 582 (1964)~, or of a ketenimine reagent
tc~. C. L. Stevens and M. E. Monk, J. Amer. Chem. Soc., 83,
1010 (1961)]. Another equivalent of the acid chloride is a
corresponding azolide, i.e. an amide o~ the corresponding acid
whose amide nitrogen ls a member of a quasiaroma~ic five
membered ring containing at lea~t two nitrogen atoms, i.e.
imidazole, pyrazole, the triazoles, benzimidazole, benzotria-
zole and their substituted derivatives. As an example of the
general method for the preparation of an azolide, N,N~-carbonyl-
diimidazole i8 reacted with a carboxylic acid in equimolar pro-
portlons at room temperature in tetrahydrofuran, chloroform,
dimethylformamide, or a similar inert solvent to form the car-
boxylic acid imidazolide in practically quantitative yield with
liberation of carbon dioxide and one mole of imidazole. Dicar-
boxylic acids yield diimidazolides. m e by-product, imidazole~
precipitates and may be separated and the ~mida~olide isolated,
but this is not essential. m ese reactions are well-known in
the art (cf. U. SO Patent Nos. 3~079,314, 3,I17,126 and 3,129,224
and British Patent Nos. 932,6443 957,570 and 959,054).
Compound IVa-l, l-[L-(~ -amino-a-hydroxypropionyl]-
kanamycin A~ Compound IVb~ L~ amino-~-hydroxypro-
pionyl~-kanamycin B, Compound IVa-2, l-~L~ amino-a-hydr
valeryl~kanamycin A and Compound IVb-2, l~ -amino-a-
hydroxyvaleryl~kanamycin B, possess excellent ant1bacterial
activity that appears superior in some respects against certain
pathogenic micro-organisms than kanamycin A or B respectively.
- 10 --
S~3
Illustrated below are two tables showing the mini-
mal inhibitory concentration~ (~IC16) of ka~amycin A compared
wlth compounds IVa-l (BB-K101) and IVa-2 (BB-K23) and
kanamycin B compared with compounds rVb-l (BB-K122) and IVb-2
(BB-K33) against a varlety of gram-positive and gram-negative
bacteria as obtained by the Steers agar-dilution method.
Mueller-Hinton agar medium was used in the study o~ both
tables.
... .
f65~3
TABLE I (MrC mcg ./ml . )
BP,- K101 B~-K23Kanamycin
~fICRO-ORGANTS~I ~ ~ A
.
E. coli ~IHJ 1.6 1.6 o.8
" Juhl A15119 1.6 1.6 1.6
" A15169 1.6 1.6 1.6
K~I-R A20363 1.6 1.6 ~100
" A9844 0,8 106 0.8
KM-R A20365 004 .4 100
K--12 1.6 1.6 o.8
" " KM-R A20664 1.6 6.3 6.3
" ~ 11 KM-R A20665 0.8 0.8 100
" W677 A20684 1.6 1.6 o.
ll JR/W677 A20683 1.6 3.1 ~100
K. pneumoniae D-ll Q.4 o.8 0~2
Type 22~3038 A20680 1.6 3.1 ~100
S. marcescens A20019 1.6 3.1 1.6
P, aeruginosa D-15 3.1 6.3 12.5
~' H9 D-113 ~-R 6.3 25.0 ~loo
A9923 603 6 ~ 3 50
A9930 0.4 0 O 8 12.5
tl A15150 6.3 12.5 .100
" A15194 3.1 6 3 ~5 : . -
It GM-R A20717 6.3 1205 5 .
GM-R A20718 6.3 12.5 5 .
P. ~ulgaris A9436 0.4 00~ 0.4 ~;
~l A9526 0.4 o.8 0.4
P . mlr2bilis A9554 o . 8 106 o ~ 8
A9900 o .8 1 ~ 6 o .8
-
.
,
3~4tjS~3
TABL~; I (MIC mcg /ml.)~Continued~
BB-K101BB-K23 ~anamycin
~ C~o-OR&AMISh (IJa-l~ (IYa-2) A
P, morganii A9553 ~ o.8 o.8
" A20031 o.8 o.8 o.8
S. aureus Smith 0.4 0.4 .4
" 209P S~L-R 1.6 3~1 1.6
~I-R A20239 1.6 3.1 100
Mycobacterium 607 oO8 1.6 -4
" KM-R ~100 ~100 >100
" K~,S~ R >100 ~100 >100
" phlei 0~4 1.6 0.4
" ranae o . 81~ 6 0.4
' ~
KM-R is kanamycin resistant.
G~-R is gentamicin resistant.
SM-R is streptomycin resistant. ;~
'' ,
" ~
;~' ' , ~ .
' ~
.. . .
,.
- 13 - ~
~' .
~ ' .
.... , ~ , ~
. ~ ,
.
: . .. '.. : .. : ..
~:14~S~3
o o o o o o o o U~ o o o
O 1~) N O
~m
~:
~4
~r ~ ~ ~ ~r~ o
~O~O ~O ~D~O O ~ ~ ,~
N
m
m
N ~J~0 ~0 ~ oe~r ~o~) 0~ ~0 10
~) I
X 5~~Ir-l ~ ~~1 0 ~1~O Or~l N O
m ~H ~1
m--
N
,1 ~ ~ ~ ~ ~ ~ ~ ~ ~1 ~ ~7 0
K
m
m
.
:~.
H~) ~ ~O
N
~o ~ u~ ~~o r~~o ~o ~ o ~
K N U~ ::
H l
H m
~ m :~:
m
1~1 :~ '`.. .
~ ~ C~ co co N er'$' N d ~0 N . I
N ~1 . , . . . . . . . . . . :
-1 1 O O O O O O O O o o ~1 o ' :
I ~ ~ ~ :
m H
m-- ~ ~ .
a~ ~ ~ ' ~o' ~o ~o~ ~O I '
~r ~o ~ o ~ o o~o o C~
~i ~1 ~f ~ ~t~, ~) N~, NK N t` N ~ N
U~ In 1~') 0 0~1 0 ~1 i¢
H 1~~I r~ ; N CJ~, N I ~O p~
Z H ~ K ~K ~ K
O .,1 ~ ,' .
p; o Q~ ~:
H
F~ - - K
, ' : `
5~3
~1
O O ~D O 111 U) Ll~ ~1 011'\ 0
O O ~
~ ~ ~ ~ ' ' '.
K
~r
o Ln ~ I
K ~ ~1 In ~ ~ ~
m :
m
~ ~ -
I L~ ) O ~D ~1 ~ N t~
0 -1 -J ~ ~ ` :
mH .-
O ~r (~)~1~1 u~ ~ ~ u~ ~o' :
U ~
L~ ~ ~ ~ ~ :
mm ~ ~ '
` :,
t~) ~) In ~ ::.. ..
lQt~l . . . ! . . ~ , .
O~U~O O ~ O O O O ~i . ~ ',
~U~ ~ O O ~ O O O O ~ ,~
H I
~m
H - . :~,
H ~ ~ ~ ;
m ~
.".-, ~ . .': ..
,; ;-
:~ .
K o "~
~o co ~1 ~ O I
tn~ u,u~ o ~ ~ ~ (d ~ ~~ 1-- ~; ~ ~
H O~: OU~ ~5) ~ U~ 10 15~ 1 0 1 0 ~r
i t~l ~D ~ O ~ C~ O
~1
O ~ ~ 5
l ~ 4 h ~ ~1
o
H . . .
K u~ ~: ~ ~,
~~15-
-: , .
.. :
. ~-:
.
6~3
.,,
o o o o o o o o ,~ o o ~ ,~
m ,~ ,~
K
~D ~ r~ D O O
K ,~ ,~ o o
n A i~
m
~D r~ r~
~~ ~ ,~
,~ m
_ :~
o
_~ ~ ,~
Ki ~ O ~ o o
u~ , ~ . ,, . ~
H m ,~
K g o o o o o o oo o o o o o ~ .
m H ~J r~l - ' :
m~
, .
K~
K a~ o rl a)
~ ~ In o In o ~
U~ In U) O~ O ~ no
H ~ ~n a) ~n ~ t~lE~ ~
R ~ K
o t~ "
tY; g .~ ~0 ~ Q
H p~
- 15a -
. . ,
..
-
'~ ' ' ' '
13
The above MIC data show that compounds IVa-l, IVa-2,
IVb-l and IVb~2 are substantially more active against a number
the test organisms~ particularly the pseudomonas and other
kanamycin resistant organisms.
The compounds IV are valuable as antibacterial agents,
nutritional supplements in animal feeds, therapeutic agents
in poultry and animals, including man, and are especially
valuable in the treatment of infectious diseases caused by
Gram-positive and Gram-negative bacteria,
me compounds IV when administered orally are
useful as an ad~unctlve treatment for preoperative sterilization
of the bowel. Both aerobic and anaerobic flora~which are
suseptible to these drugs are reduced in the large intestine.
When accompanied by ade~uate mechanical cleansing, they are
u~eful in preparing for colonic ~urgery.
The compounds IV are effective in the treatment of
sy~temic bacterlal lnfections in man when administered parent- !
erally in the dosage range of about 250 mg. to about 3000 mg.
per day in divlded doses three or four times a day, Generally
. .
the compounds are effective when administered at a dosage of
about 5.0 to 7,5 mg./kg~ of body weight every 12 hoursO
- 16 -
13
EXAMPLES
Example 1
Pr~ara~ion of L-~-Benzyloxycarbonylamino
hydrox~pro~ionic Acid (VI-l)
L-~-~mino-~hydroxypropionic ~cid* (8.2 g., o,o78
mole) was dissolved in a solution of 6.56 g, (0,0164 mole)
o~ sodium hydroxide and in 60 ml. of water. To the stirred
solution was added dropwise 14.7 g. ~o.o86 mole) of carbo-
benzoxy chloride below 5 C. ~he mixture was stirred for
an hour at room temperature, washed with 60 ml. of ether and
ad~usted to pH 2 with dilute HCl. me precipitate was collected
by filtration, washed with water and air-dried to give 9~65 g.
(52%) o~ VI-l. The filtrate was extracted with five 100-ml.
portions of ether. me ethereal solution was washed with water,
dried over sodium sul~ate and ev~porated to dryness in vacuo to
give additional 2.0 g. (11~) of VI-l. A total of 11. 65 g. of
VI-l was crystallized ~rom 500 ml. of benzene-ethyl acetate (4:1)
to gi~e 9.36 g. (5~) o~ pure VI-l, m.p. 12805-129.5 C. Infrared
( ) (KBr): Yc=o 1745, 1690 cm~l, ~a~D5 ~2 9 (c 5 o th
Nuc'ear Magnetic Resonance Spectra CNMR (DMS0-d6) ]: ~ (in ppm)
3.05-3.45 (2Hg m, CH2N), 4,os (lH, d-d, -0-CH-C0-), 5.03 (2H, s,
CH2Ar) 7.18 (lH, broadJ NH), 7.36 (5H, s, ring H).
Analysi~: calculated for C H N0 : C, 55 23; H, 5 48;
N, 5. 86. 11 13 5
Found: C, 55,34; H, 5.49; N, 5.87.
* K. Freudenberg, Ber., 47, 2027 (1914) .
Example 2
N-Hydroxysuccinimide Ester of L-~-benzyloxycarbonyl~
amino-a-hydroxypropionic Acid (VII-l)
To a chilled and stirred solutlon of 478 mg. (2 m.
moles) o~ VI-l and 230 mg. (2 m. moles) of N-hydroxysuccinlmide
- 17 -
6S13
in 10 ml. of tetrah~drofuran (I~IF) was added 412 mg, (2 m,
moles) of dicyclohexylcarbodi~mide. The mixture was stirred
for an hour at 0-5 C., for two hours at room temperature and
then filtered to remove the NgN1-dicyclohexylure~. me
filtrate containing VII-l was used for the next reaction
without isolation.
Example 3
Preparation of l-[L-(-)-~-amino-a-hydroxypropionyl~-
kanamycin A (IVa-l, BB-Kl01)
To a stirred solution of 1,48 g. (2.4 m. moles) of
6~-carbobenzoxykanamycin A (IIa) in 25 ml, of water-THF (4:1)
was added dropwise the solution of VII-l at 5 C. ~he mixture
was stirred for two hours at room temperature and filtered to
remove a small amount of ~nsoluble material. The filtrate was
hydrogenated overnight with 300 mg. of 10~ palladium on
charcoal at room temperature at atmospheric pressure and then
filtered to remove the catalyst. me filtrate was concentrated
in vacuo to remo~e most of the organic solvent. me resultant
aqueou~ solution wag adjusted to pH 7 with dilute HCl and
passed through a column of Amberlite CG-50 (NH4, 80 ml.)**,
whlch was waæhed with 200 ml. of water and then-eluted with
400 ml. of 0~1 N, 870 ml. of 0.2 N, and 430 ml~ of 0.5 N NH~CE~
The eluate was collected in 10 ml. fractionæ. Fraction number~
95-106 which showed activity against Pseudomonas aeru~inosa
A9843 and Rf values at 0,25 and 0.41 (desired ~roduct) by thin
layer chromatography (TLC) on a silica gel plate (S-llO,
ninhydrin)*** were pooled, evaporated in vacuo and freeze-dr~ed
to give 170 mg, of crude IVa-l.
The crude IVa-l (162 mg,) was adsorbed on a column of
CG-50 (cupro-ammonium form*, 15 ml.), which was washed with
50 ml. of water and eluted with 1 liter of 0.2 N~ 700 ml. of
~(~4~5~3
0~5 N and finally 400 ml. of 1.0 N ~I4CH The eluate was
collected in 7-ml. fraction. Tube numbers 269-282 which
showe~ a single spot at R~ 0.41 was combined, evaporated in
vacuo and lyophilized to give 79 mg~ of copper complex of IVa.
The copper wa~ removed by column chromatography on CG-50
(NH4, 5 ml.) using 0~2 N NH40H as an eluent. m e eluate was
collected in 10-ml. fraction. Tube numbers 2-10 were combined,
evaporated in vacuo and lyophilized to gi~e 26 mg. of rVa-l
(2% based on VI-l), m.p. 200-205 C. IR (B r): 16409 1540 cm~l.
Analysis: calcu~lated for C2lH4lNsol3-2H2co3-3H2o:
C, 36.84; H9 6.84; N, 9.34.
Found: C,36.93; H, 6.oo; N, 9.62.
* The cupro-ammonium form of CG-50 was prepared in the
fo~lowing way: to a stirred ~uspension of CG-50 (NH4) in water
Wa8 added 10% cupric sulfate solution to give copper salt of
.. : . : . ' ' ......................... .. ~ ! , . ,
CG-50 which was filtered. The resin was washed several times
.
with water, then treated with lN NH4CH under stirring, filtered
and washed se~êral times with w~ter to give deep blue, cupro-
~, :
ammonium form of CG-50. ~ ~
." . .
** Amberlite CG-50 is the trademark for ~he chromatographic ~
.
grade o~ a-weakl~ acidic cationic exchange resin of a carboxylic-
polymethacrylic type.
*** TLC: ~ilica gel plateJ CHC13-MeCH~28~ MH4CH-H20 (1:4:2:1).
:
Example 4
Preparation of N-(Benzyloxycarbonylox~)succinimide
.
- N-Hydroxysuccinimidel (23 g., 0.2 mole) was dissolved
in a solution Or 9 g, (0.22 mole) of sodium hydroxide in 200
~- -ml~.-of~water.= T~-the-stirred solution was added dropwise 34 g.
(0.2 mole) of carbobenzoxy chloride with water-cooling and then
the mixture was ~tirred at room temperature overnight to
~eparate the carbobenzoxy deri~ative which was collected by
'~,3 ' ' ~
- lg ~
. .
~'" " ;
;5~3
filtration, washed with water and air-dried~ Yield 41.1 g.
(82%). Recrystallizatlon from benæene-n-hexane (10:1) gave
colorless pri~m~ melting at 78-79 C.
1. G, W. Anderson et al~ J J. Am. Chem. Soc., 869
1839 (1964).
Example 5
Preparation of 6~-Carbobenzoxykanamycin A (IIa)
A solution of 42.5 g. (90 m.moles) of kanamycin A
~ree base in 450 ml. of water and 500 ml. of dimethyl~ormamide
(DMF) was cooled below 0 C. and stirred vigorously, To the
solution was added dropwise over a period of about two hours a
solution o~ 22.4 g. (90 m.mole) of N-(benzyloxycarbonyloxy)-
succinimide in 500 ml. of DMF. me mixture was stirred at -10
to 0 C. overnight and then at room temperature for one day.
The reaction mixture was evaporated under reduced pressure below
about 50 C. The oily residue was dissolved in a mixture of
500 ml. water and 500 ml. butanol> the mixture being filtered
to remo~e insolu~le material and separated into two layer~.
The butanol and aqueous layers were treated with butanol-saturated
water (500 ml. x 2) and water-saturated butanol (500 ml. x 2)9
respectively, uslng a technique similar to counter current
distribution. me three aqueous layers were combined and
evaporated to dryness under reduced pressure to give an oily
residue, a part of which crystallized on ~tanding at room
temperature, To the residue including the crystals was added
about 100 ml. o~ methanol, which dissolved the oil and separated
it from the crystals, After ad~ing about 300 ml. of ethanol,
the mixture was kept at room ~emperature o~ernight to give a
crystalline ma~s which was collected by ~iltration. It weighed
44 g. ~he product contained a ~mall amount of kanamycin A as
indicated by thin layer chromatography using n-propanol-pyridlne
acetic acid-water (15:10:3:12) as the solvent system and
ninhydrin a~ the spray reagent.
- 20 -
~6S~3
The crude product was dis~olved in 300 ml. o~
water and chromatographed on a column (30 mm. diameter) of
CG-50 ion-exchange resin (NH4 type, 500 ml,). me column
was irrigated with Ool N ammonium hydroxide solution and the
eluate was collected in lO~ml. fraction. m e desired product
was contained in tube numbers 10-100, while kanamycin A
recovered from slower-moving fractions and the position
isomer(s) of the product seemed to be contained in the faster-
moving fractions. The fractions 10-110 were combined and
evaporated to dryness under reduced pressure to give 24.6 g.
(45%) of a colorles~ product 6'-carbobenzoxykanamycin A (IIa)
~6~-Cbz-kanamycin A~, which began to melt and color at 204 C.
and decomposed at 212 C. with gas evolution. ~]D +106
(c=2, H20).
TLC R_ Value
(silica gel Fa64; ninhydrin) 6l-Cbz- Kanamycin
Solvent S~stem Kanam~cin A A
n-propanol-pyridine-acetic
ac1d-H20 0.42 ~ 0.04
(15:10:3:12) (main) minor
Acetone-acetic-acid-H20 0.24 0.14
(20:6:74)
CHC13-methan~l-c~NH40H-H2o 0.76 0.50
(1:4:2:1)
Methyl acetate-n-propanol-
C.NH4oH O~22* 0.04*
(45:105:60)
*Detected by anthrone-sulfuric acid.
The final product was found to be accompanied by two
minor components by TLC with one of the solvent systems tested,
However, the final product wa~ used without further purification
for the preparation o~ BB-K101 (IVa-l).
_ 21 -
5~3
Exam~le 6
Preparation of 6l-Carbobenzoxykanamycin B (IIb)
To a chilled solution of 8.1 g. (0.0168 mole) of
kanamycin B ln 120 mlO of water and 80 ml. of 1,2-dimethoxy-
ethane was added dropwise with stirring a solution of 4.2 g.
(0.0168 mole) of N-(benzyloxycarbonyloxy)succinimide in 40 ml.
of l,2-dimethoxyethane. The reaction mixture was stirred
o~ernight and evaporated under reduced pressure. The residue
was dissolved in 100 ml. of water and shaken twice with 50
ml. of water-saturated n-butanol. m e aqueous layer was
~eparated aind adsorbed on a column of 100 ml. of CG-50 (MH4
+type). The column was wa~hed with 200 ml. of water, eluted
with 0.05 N NH4CH. me eluate was collected in 10-ml. fraction.
Fractions 121 to 180 were collected, evaporated and freeze-dried
to give 1.58 g. (15~) of the desired product. Fractions 1 to
120 were evaporated and re-chromatographed on CG-50 (NH4 ) to
give 1.21 g. (12~) of the product (IIb). M.p. 151-152 C.
(dec.). ~a~24 +104 (C. 2.5, H20)- yc=01710 cm.
Analysis: calculated for C26H43N4012 C, 50-56;
H, 7.02; N, 11.34.
Found: C, 50.71; H, 7.38; N, 11.48.
TLC (3ilica gel F2s4)~ RF O.03 in n-propanol-pyridine-
acetic acid-H20 (15:10:3:12); R~ 0.16 in acetone-acetic acid-
H20 (20:6:74)~
Example 7
Preparation of l-~L~ -Amino-~-hydroxypropionyl~-
kanamycin B ~BB-K122 (IVb-l)]
To a stirred solution of 1.23 g. (2.0 m.moles) of
IIb in 20 ml. of water was added dropwise a solution of VII-l
prepared from 478 mg. (20 m.moles) of VI-l in 20 mlO of THF at
room temperature. The mixture wa~ stirred overnight and then
- 22 -
- - ~
hydrogenated overnight o~er 300 mg. of 10~ palladium on
charcoal at room temperature at atmospheric pressure. The
hydrogenated mixture was filtered and the filtrate was con-
centrated in vacuo to remove most of the organic solvent,
The resultant aqueous solution was ad~usted to pH 7 with 1
N hydrochloric acid and adsorbed on a column of CG-50 (NH4~,
40 ml.) which was washed with 80 ml. of water and then eluted
with 900 ml. of 0.1 N and 1.2 L of 0.2 N NH4CH. The eluate
was collected in 10-ml, fraction, monitored by ninhydrin spot
test, TLC with S-110 system and disk assay using Pseudomonas
aeruæinosa A9843, and cut into the following appropriate
fractions~ Each fraction was evaporated in vacuo and freeze-
dried.
Tube
Fraction No, NH4CH(N) Weight Identity
1 91-113 0.2420 mg. kanamycin B
2 121-143 0.2253 mg. BB-K122 (IVb-l) +
BB-K123 + kanamycin B
3 157-196 0.266 mg. BB-K124 ~diacylated
compoun )
Fractlon 2 (250 mg.) in 30 ml. o~ wat~er was ad~usted
to pH 7 with 1 N hydrochloric acid and passed~through a column
of CG-50 (cupro-ammonium form, 14 ml.), which was washed with
50 ml. of water and eluted with 2.5 liters of 1.0 N NH4QH and
1.5 liters of 1.5 N NH4CH. The eluate was collected in 10-ml.
fraction and cut into the appropriate fraction~ on the basis of
the Rf values of TLC and activity against Pseudomonas aeruginosa,
Tube
Fraction No. NH4CH(N) Weight Identity
1l191-240 1.0126 mg, copper complex of
B:B-K123
2~331-360 1.580 mg. copper complex of
the desired BB-K122
3'361-385 10541 mg, copper complex of
kanam~cin B
~0~65~3
Chromatography of fraction 2' (80 mg.) in order
to remove copper afforded 35 mg~ (3~) o~ BB-K122 (IVb-l).
In the same way 27 mg. (2.4~) of BB-K123 was
obtained ~rom 126 mg. of ~raction llo
The physico-chemical data of these compounds are
given in the following Table.
BB-K Rf
No. IR (cm l)_(M.p. (C.) (_-110, ninhydrin~
122 1640~ 1570 192-196 0ol~l
123 1640, 1570 177-180 0.35
124 1650, 1550 179-185 0.21 (main), 0.31
Microanal~is data
BB-K122: Anal~sis: calculated for C21H42N6012-2H2C3
C, 39.77; H, 6.67; N, 12.10.
Found: C,39.84, H, 6.23; N,11.66,
BB-K123: Analysis: calculated for C21H42N6013-3~12C3
: G, 38.10; H, 6.39; N, 11.11.
Found: C, 36.84; H, 6.33; N, 11.26.
BB-K124: Analysis: calculated ~or C24H4 N7014-3H2C03:
C, 38.43; H, 6.33; N, 11.62.
Found: C, 38.28; H~ 5.95; N~ 11.43.
The~e three compounds regenerated isoserine and
kanamycin B by hydrolysis with 0.5 N NaCH at 100 ~or an hour.
The identities of the compounds BB-K123 and BB-K124
were not established although it is known that BB-K123 is a
monoacylated derivative o~ kanamycin B and BB-K124 i a di-
acylated derivative o~ kanamycin B. 'Ihe compounds have weak
antibacterial activity as compared to kanamycin B and BB-K122
(IVb-l). See Table II~
- 24 -
~ 5~ 3
Example 8
Preparation of the Monosulfate Salt of l-~L~
amino-~-h~droxypropion~l~kanamycln A or B
One mole of l-[L-(~ -amino-G-hydroxypropionyl~- -
kanamycin A or B i8 dissolved in 1 to 3 liters of water, The
solution i~ filtered to remove any undissolved solids. `To
the chilled and stirred solution is added one mole of sulfuric
acid dissolved in 500 ml. o~ water. The mixture i~ allowed to
stir for 30 minutes~ following which cold ethan d is added to
the mixture till precipitation occurs. The solids are
collected by-filtration and~are determined to be the desired
monosulfate salt.
~ Example 9
Preparation of the Disulfate Salt of l-~L-(~
amino-a-hydroxypropion~l~kanamycin A or B.
Thir~y-five grams of l-[L-(~ -amino-a-hydroxy-
propionyl~kanamycin A or B (as the monobicarbonate trihydrate)
i8 dissolved in 125 ml. of deionized water. m e pH is adjusted
to 7-7.5 with 50% V~V sulfuric acid.
Eigh* and one half grams of Darco G-60 (activated
- charcoal) i8 added and the mixture i~ slurried for 0.5 hour
at ambient room temperature. m e carbon is removed by suitable
filtration and washed with 35 ml. of deionized water. The
water i~ added to the ~iltrate.
m e combined filtrate-wash i8 ad~usted to pH 1-1.3
with 50% V/V sulfurlc acid. This solution $s added with
rapid stirring over a ten minute period to 600-800 ml. of
methanol (3-4 volumes of methanol)~ The mixture i~ st~rred
for five minutes at pH 1-1.3, passed through a 100 mesh screeng
~t~rred ~or two minutes and allowed to settle for five minute~.
Nost o~ the ~upernatant iB decanted. The remaining slurry i~
suitably filtered, washed with 200 ml. of methanol and vacuum
drled at 50 C. ~or 24 hour~ to yield the appropriate di3ulfate
~alt.
3 *Trade Mark - 25 -
., ~
5~L3
Exam~le_10
Preparation of L-~-Benzyloxycarbon~lamino a-
hydroxyvaleric acid (VI-2).
To a stirred solution of 400 mg. (3.0 m.moles) of
L-~-amino-a-hydroxyvaleric acid* and 250 mg. (6.5 m,moles) of
sodium hydroxide in 25 ml. of water was added dropwise 580
mg, (3.3 m,moleæ) of carbobenzoxy chloride over a period of
30 minutes at 0-5 C. The mixture was stirred for an hour at
5-15 C., washed with 25 ml. of etherJ ad~usted to pH 2 with
hydrochloric acid and extracted with three 30-ml. portions of
ether, The combined ethereal solutlon wa~ shaken with 10 mlO
of a saturated sodium chloride solution, dried~over anhydrous
sodium æulfate and evaporated in vacuo to give~crystals which
were recrystallized from benzene to yield 631 mg. (78~) of
VI-2, mp 110-111C.; infrared spectrum [IR(KBr)~: 3460, 3350,
1725j 1685, 1535, 1280, 730, 690cm~l. Nuclear magnetic
resonance spectrum tN~R(acetone-d6)~: ~(in ppm) 1.70 (4H, m)
4.14(2H, q, J--4.5Hz), 4.19(1H, m), 4.82(2H, s), 6.2(3H, broad),
7.25(5H, s). t~2D5+ 1.6 (c 10, methanol).
Analysis: calculated for Cl Hl NO : C, 58.42;
3 7 5
H, 6,41; N, 5.24.
Found: C, 58.36; H, 6.50; N, 5.27.
*S. Ohshiro et al., Yakugaku Zasshi, 87, 1184 (1967).
Example 11
N-Hy r xy uccinimide ester of L-~-benzylox~arbonyl-
a-hydroxyvaleric acid tVII-2).
.
To a stirred and chilled ~olution of 535 mg. (2.0
m.moles) of VI-2 and 230 mg. (2,0 m.mole~) o~ N-hydroxysuccin-
imide in 55 ml. of ethyl acetate was added 412 mg. (2.0 m.moleæ)
of N,N~-dicyclohexylcarbodiimide (DCC). The mixture was stirred
for 3 hours at room temperature and filtered to remove precipi-
tated N,N~-dicyclohexylurea. The filtrate was evaporated in
vacuo to yleld 780 mg. (100~) o~ viscou~ 8yrup (VII-2). IR(Neat):
~C=O 1810~ 1785J 1725 cm~l
- 26 -
~4~.t3
Example 12 f
1-~L (~ amino-~-hydroxyvaler~1]-
kanamycin A, ~B-K 23, IVa-21.
To a stir~ed solution of 1.24 g. (2.0 m.moles) of
kanamycin A (IIa) in 12 ml. of water and 60 ml. of 1,Z-di-
methoxyethane (DME) wa~ added dropwise 780 mg. (2.0 m.moles)
of VII-2 in 10 ml. o~ DME. The mixture was stirred overnight
and then evaporated to dryness in vacuo. The resldue was
treated with 20 ml. o~ water and shaken with two 30-ml. portions
of water-saturated butanol.~ The combined butanol layer was
evaporated in acuo to give 1.36 g. of solid, which was dissolved
in 10 ml. of water, 10 ml. of dioxane and 1 ml. of acetic acid,
and hydrogenated overnight with 200 mg. of 10% palladium on
charcoal at atmospheric pressure at ordinary temperature. m e
hydrogenated mixture was filtered and the ~iltrate evaporated
to dryness in ~acuoO The residue was dissolved in 20 ml. of
water. The solution was passed through a column of Amberlite
CG-50** (NH4+, 15 ml. )J which was washed with 80 ml. o~ water
and then eluted with 760 ml. of O.lN and 880 ml. of 0.2 N NH4CH.
m e eluate was collected in 10-ml. fraction. ~Tube Nos. 109 to
172 which upon thin layer chromatography (TLC)* showed R~ 0.18
(S-llO, ninhydrln) were combinedg evaporated in vacuo and freeze-
dried to give 330 mg. (28%) o~ IVa-2, mp 185 (dec.) IR(KBr):
1635, 1570 cm~l,
Analy~ calculated for C23H45N5013 H20:
C, 43.57, Hy 7~16, N, 10.59.
Found: C, 43.25; H, 7.12; N, 9.83.
** Amberlite CG-50 is the trademark ~or the chromatographic
grade of a weakly acid~c cationic exchange resin o~ a
carboxylic-polymethacrylic type.
* TLC: ~ilica gel plate, CHC13-methanol-28% NH4CH-H20
(1:4
'.~. ~3
- 27 -
.;
~ 3
Exam~le I3
Preparation of l-~L~ amino-~-hydroxyvaler~
kanamycin B, ~BB-K 33 (IVb-2~].
To a stirred solution of 618 mg, (1 m.mole) of IIb
in 30 ml. of water-DME (1:2) was added 364 mg. (1 m,mole) of
VII-2 in 10 ml, of dry DME in one portion. The mixture was
stirred overnight at room temperature and evaporated in vacuo.
me aqueous concentrate was extracted with two 20-ml. portions
of water-saturated n-butanol, me butanol extracts were
evaporated to dryness to give 530 mg. of solid, which was
diæsolved in 40 ml. of water-DME (1:1) and hydrogenated over-
night with 150 mgO of palladium on carbon at atmospheric
pressure at room temperature, me catalyst was removed by
filtration, Thè ~iltrate was evaporated in vacuo to remove ~`
most of the organic solyent. The aqueous solution was charged
on a col D of Amberlite CG-50(NH4, 14 ml.), which was washed
with 140 ml. o~ water and then eluted with 410 ml. of 0 1 N,
760 ml. of 0.2 N, 650 ml. of 0.5 N and 510 ml. of' 1.0 N NH4CE~.
Ihe eluate wa~ collected in 10-ml, fraction. Tube Nos. 127 to
141 which showed Rf 0.22 by TLC on silica gel plate (S-llO,
ninhydrin) were pooled, ev~porated in vacuo and lyophilized to
give 103 mg. (17~) of BB-K33, mp 185-190C.(dec). IR~KBr):
Yc=o 1635 cm 1.
Analy~ calculated for C23H46N612 2H2C3
C, 41.55; H, 6.97; C, 11,63.
Found: C, 41.44; H, 7.09; C, 11.75.
Tube Nos. 195 to 216 which showed Rf 0.11 (S-llO, ninhydrin)
were comgined, evaporated in vacuo and lyophilized to give 42
mg. (7%) of BB-K34, mp 180-185C. (dec). IR(B r): Yc=o 1640 cm
Analy~is: calculated for C28H55N7014 2 3
C, 39.96; H, 6.60; N, 10.19.
Found: C, 39.57; H, 6.63; N, 10.64.
_ 28 -
5~ ~
m e identity of the compound BB-K34 wa~ not
established although it is known that it i8 a diacylated `
derivative of kanamycin B possessing weak antibacterial
activity as compared to kanamycin B and BB-K33 (IVb~.
Example 14
Preparation of the Monosul~ate Salt of l-CL-(-)-~-amino-
a-h~droxyvalel~l]kanam~cin A or B.
One mole of l-~L-(~ -amino-a-hydroxyvaleryl~-
kan~mycin A or B is dissolved in 1 to 3 liters of~water.
me solution i8 filtered to remove any undissolved solids.
To the chilled and stirred solution is added one mole of
sulfuric acid dlssolved in 500 ml. of water. m e mixture is
allowed to stir for 30 minutes, following which cold ethanol
is added to the mixture until precipitation occurs. The solids
are collected by ~lltration and are determined to be the
de81red monosulfate salt.
~,~ . .
Example 15
Pre~aration of the Disulfate Salt of l-~L~ amino-
a-hydroxyvaler~l~kanam~cin A or B
.
mirty-five grams of l-~L~ amino-~-hydroxy-
valeryl~kanamycin A or B (as the monobicarbonate trihydrate)
is dissolved in 125 ml. of deionized water.
The pH i~ adjusted to 7 7~5 with 50% V/V sulfuric
acld, Eight and one half grams of Darco G-60 (activated
charcoal) is added and the mixture is slurried at ambient room
temperature for O.5 hour. The carbon is removed by suitable
filtration and washed with 40 ml, of water. m e water wash is
added to the filtrate.
The combined filtrate-wafih above is ad~usted to pH
2-2.6 with 50~ V~V sulfuric acid. A large amount of carbon
- 29 -
5~l3
dioxide evolves, The solution is left at house vacuum with
stirrin~ for 20 minutes to expel additional carbon dioxide.
Eight and one half grams of Darco G-60 is added
to the degassed solution, me mixture is slurried for 0O5
hour at ambient room temperature, The carbon 1s removed
by suitable ~iltration and washed with 35 ml, of deionized
water, m e water is added to the filtrate,
me combined filtrate wash is ad~usted to pH
1-1,3 with 50~ V~V sulfuric acidO This solution is added
with rapid stirring over a 10 minute period to 600-800 ml.
of methanol (3-4 volumes of methanol). The mixture is
stirred for 5 minutes at pH 1-1.3, passed through a 100 mesh
screen, stirred for 2 minutes and allowed to settle for 5
minutes, Most of the supernatant is decanted. The remaining
slurry i~ suitable filtered, washed with 200 ml, of methanol
and vacuum dried at 50Co for 24 hours to yield the appropriate
disulfate salt.
30 -
