Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 802~
This invention relates to pseudotrisaccharides, to
methods for their production, to pharmaceutical formu-
lations~and to methods for their use as antibacte~ial
agents.~
More speci~ically, this invention relates to 5-epi-,
5 epi~amino 5~deoxy and to 5 epi-azido-5-deoxy derivatives
of 4,6-di-0-(aminoglycosyl)-2-deoxystreptamine anti-
bacterial agents including the gentamicins, sisomicin,
verdamicin, tobramycin, the kanam~cins, Antibiotics G-418,
66-40B, 66 10D, JI-20A, JI-20B and G-52 and the l-N-
alkyl derivatives thereo.
' ~ - ' - ' " ' '" ~
~nown in the art are broad spectrum antibacteri~l agents
which may be classified chemically as 4,6-di-0-(amino-
glycosyl)-1,3-diaminocy~litols. Valuable antibacterial
agents o~ this group are those wherein the aminocyclitol
is 2qdeoxystreptamine~ Partiçularly valuable antibac-
terials o the 4,6~di-_~(aminoglycosyl)-2-deo~ystrepta-
mi~es axe those whe~ein the aminoglycosyl group at the
6-position is a garosaminyl radical. Within the class
o~ ~-0-aminoglycosyl-6-_-garosamLnyl-2-deoxystreptamines
axe antibiotics such as gentEmicins B~ Bl, Cl, Cla, C2,
C2a, C2b, and X2; verdamicin, sisomicin, Antibiotic
G-418, Antibiotic G-52, Antibiotic JI-20A and Antibio-
tic JI-20B.
~ .
2 ~
. .
~C~4~3CI12~
Also known in the art are l-N-alkyl derivatives of the
; ` aforementioned 4,6-di-O-(aminoglycosyl)-1,3-diaminocycli-
tols which, in general, exhibit broad spectrum antibacte-
rial activity and possess enhanced activity against com-
pounds resistant to the l-N-unsubstituted antibacterial
agent.
The novel pseudotrisaccharides of the present invention
are derivatives of the 4,6~di-O-(aminoglycosyl)-2-deoxy-
streptamines gentamicin Cl, gentamicin Cla, gentamicin C2,
10 gentamicin C2a, gentamicin C2b, verdamicin, Antibiotic
G-52, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic
JI-20A, Antibiotic JI 20B and sisomicin, wherein the 2-
- - deoxy streptamine moiety is replaced by a 1,3-diaminocyli-
~: tol of the formula
. . - ~H2
: 15 ~ ~ I
: ---O, ~
wherein, in the formula, R is hydrogen or a group -CH2Y
; wLth Y being hydrogen, lower alkyl or lower aminoalkyl;
and the pharmaceutically acceptable acid addition salts
thereof.
The compounds.are defined by the following structural
formula X:
~ ' .
- -- 3 --
i'~
`
~L04~02
,., , ~
.'.',' ' , ` ~
~ AG O ~
., . . X
,., , X
.. . HO ~s~'~O
. : 4~ ~ N~C~
. C~
OH
. wherein X and R are as hereinabove defined for formula I
and wherein AGl is an aminoglycosyl function selected from
the group consistlng of
~- CH3 ~ ~FH2NH2 ~~`~--
tamicin Cl)~ entamicin C
CH ~ OEI 2
~ H + ~H2
~ ~ tamicin C2) ~ ta~icin C2~)
~H2 - 2
fH2MHCH3
r o\ (gentamicin C2b)
\_/~
.
~:
- . . ; i
.
. ~
~04~ZO
. . CH3
H2~I2 H ¦ NEI
~omiCin) ~verdamicin)
~H2 . cH 2
C H2~H2 H~ ~H2
/~ (Antibiotic . ~\ (Antil:~iotic:~
HO~ I:IO\~
~H2
. C1~21~ICH3 ' ' .
~_ D~ io:io ~--52
~2
derivatives of Antibiotic 66-40D by following formula XII:
CH2~2 ~2
~5 (~
--/ , , , .1~ ' .
. . ~H2 ¦ XII
r ~ '1
OH
f~
~ 8~D2q~
wherein X and R are as hereinabove defined for formula I;
and derivatives of Antibiotic 66-40B by the following
formula XIII:
~H2
AG3 - O ~ XIII
~0
HO
0~
wherein X and R are as hereinabove defined for formula I
and where~n AG3 is
~o2
The derivatives of the 4,6-di-D-~aminoglycosyl)-2-deoxy-
.strept~mines o this invention as defined in formula I .
or by formulae ~ - XIII are characterized ~y being white
amorphous powders.
8~20
Also included within the composition-o~matter aspect of~
this invention are pharmaceutically acceptable acid addi-
tion salts o~ the derivatives of the 4,6-di-0-tamino-
~ ~lycosyll-2-deo~ys~xeptamines such a~ defined abotre,
which salts are m~de according to known procedures suc~
as by neutralizing the free base with the appropriate acid,
usually to about pH 5~ Suitable acids for this purpose
includ~ acids such as hydrochloric~ sulfuric, phosphori~,
hydrobromic and the like. The physical embodiments o~
the acid addi~ion salts of the derivatives of the 4~6-di-
O-(aminoglycosyl)-2-deox~streptamines are characterised
by being white solids which are soluble iD water and
. insoluble in most polar and non-pola~ organic solven~s,.
The compounds of this invention such as deined by
ormulae X- XIIT~particularly those wherein the 6~0_
aminoglycosyl is 6.-0-~arosaminyl, and their non-toxic,
pharmaceutically acceptable acia addition salts, in
general, exhibi~ broad spectrum antibac~erial acti~ity
and possess an improved antibact~rial spectrum c~mpared
2~ to that of the parent antibio~ics.
In d uded among the substituents contemplated for the
group -C~2Y in ~ormula I are straight and branched
chain alk~l groups.such as ethyl, n-propyl, ~-~utyl,
~ethylpropyl, n- pentyl, ~methy.lbutyl, ~-methyl-
butyl and ~ dimPthylpropyl; n-hexyl, ~methylp~ntyl,
~-ethylbutyl, ~ethylbutyl~ n-heptyl, ~-methylheptyl,
-- 7
~)4~0;~t~
~-ethylpentyl, ~-ethylpen~yl~ ~-ethylpentyl, 1r-prop~l~
butyl, n-octyl, ~so-octyl, ~-ethylhexyl, ~-ethylhexyl,
-ethylhexyl, ~-pxopylpentyli lf~prPylPPntYl;amino
substituted straight and branchcd chain alkyl groups such as
~-aminopentyl, ~ -aminopropyl,1v-aminopropyl, ~-aminobutyl,
~-amino-r-methylbutyl and W-aminooctyl.
In a pre~erred group o~ compounds R in formula ~ and in
formula X is hydroyen or alkyl ha~ing up to four carbon
atoms (especially 2-4 carbon atoms) with hydrogen and
ethyl being most pre~erred and propyl being also preferred.
; ~ ~ particularly preferred gro~p of compounds em~races
the derivatives of the 4-0-aminoglycosyl-6-0-garosaminyl-
2-deoxystreptamines gentamicin Cl, gentamicin Cla,
verdamicin, and sisomicin, wherein R in formula I
is hydroge~ and X is aæido or amino; and the pharma;
ceutically acceptable acid addition salts thereof.
- '' ,
~nother particularly preferred group of compounds em-
brances the deriva_ive~ of the 4~0-aminoglycosyl-6-o_
garosaminyl-2-deoxy~treptamines gentamicin Cl, genta-
2~ micin Cla, gentamicin C2 and verdamicin, wherein R informula I is ethyl or hydrogen and X is hydroxy; and the
pharmaceutically acceptable acid addition salts thereof.
.
-- 8 --
"
- :
~8020
. The process for preparing above compounds comprises a
procedure selected from the following procedures A to C:
,,
A) subjecting a derivative of one of the above named
4,6-di-0-(aminoglycosyl)-2-deoxystreptamines, wherein the
2-deoxystreptamine moiety is replaced by a 1,3-diamino-
cyclitol of the formula
' ~ ' ' ' t~ , .' ,
-O ~
' - ' ~' '1.,
:
- i
:~ ~herein, in the formula, R is as above defined and X' is
hydro~y or azido; and which derivative is per-~-protected
and O-protected in all positions other than postion S .
to removal of the protecting groups, and, if a deriv~ive
of a 4,6-di-O~(aminoglycosyl)-2-deoxystreptamine is desired
wherein substituent X is amino, to reduction of the azido
group in position 5 either before or af~er remo~al of the
1~ protecting groups;
- .
B) for the preparation of compounds of formula I~ wher~in
X is hydroxy; treating a derivative of one of the above
named 4,6-di-0-(aminoglycosyl)-2-deoxystreptamines, wherein
the 2-deoxystreptamine moiety is replaced by a 1,3-diamino-
23 cy~litol of the formula
,~
' - :
10~8~
R
,'
wherein, i~ the formula, R i~ as above de~ined and Xi i~
unsub~tituted or subQtituted hydrocar~onsulfonyloxy; and
wherein the hydroxyl and amino group3 in the 4,6-di-0-
(aminoglycosyl)-2-deoxy~treptamine derivative are protected.
by groups ~usceptible to reductive cleavage or to ba~ic or
mild acid hydrolysi~; with dimethylformamide at temperatu~e~
in ~he range o~from about 80 C to about 155 C; and removing
the protecting groups in the resulting product;
' ' ' '
: 10 ~C) for the prepara~ion o~ compounds of ~ormula I, wherein
X is hydroxy; reacting a derivative o~ one of the a~ove
.
~ ~named 4,6-di-0-(aminoglycosyl)-2-deoxys~rep~amines, wherein
,
: : the 2-deoxystreptamine moiety is replaced by a 1,3-diamino
~ cyclitol of the ~ormula
. ~ . . .
` 1 2
' fl--~ N}IR ' - ' - ,
. / \1
' ' ~
. . ' .
-- 10 -- .
,,~''-' -
.
o
wherein, in the formula, R and X are as above defined;
and wherein the amino and hydroxy groups other than the
5-hydroæy group in the 4,6-di-O-(aminoglycosylj-2-deoxy-
streptamine derivative are protected by groups susceptible
to redu~ive cleavage.or to basic or mild acid hydrolysis;
wit~ an oxidizing agent; reacting the resulting ~-protected-
O-protected 5-d~hydro-4,6-di-O-(aminQglycosyl)-2-deoxystrep-
tamine with an alkali metal borohydrid and remo~ing tho
protecting groups in the resulting pxoduct, said procedure~
A) to C) being followed by the optional step o~ alkylating
a compound wherein R i~ hydrogen, to obtain a compound,
wherein R is the group -C~2Y with Y being as defined above;
and isolating the derivative as suah.or as a pharma~eutically
acceptable acid addition salt.
: 15 In procedure A), when a final compound is desired having
a 5-epi-azido group the protecting groups must be such
that the reaction conditions employe1for removal thereof
do not interfere with the.azido group. Thus, the M- and
~ O-protecting groups should, in general, be susceptible
to cleavage by basic or mild acid hydrolysis if the
azido group is desired in the final compound. In such
a case, removal of the protecting groups may be effected
by treatment of a compound, wherein the 1,3-diaminocycli-
tol moiety is of formula II with X' being azido, at ele-
vated t~mperatures with aqueous base and, when acetals orketals are present, by treatment also with aqueous mild acid.
-- 11 --
d~
~ , .
2CII
When a ~inal compound is desired, wherein X is hydroxy
or amino, startiny campounds are used, w~er~in X is
hydroxy or azido, respectiYely~ In such cases also
protecting groups may be present removal of which is
advantageously e~fected by reductive cleavage.
,
For example, ~n a 5-epi-azido-5-deoxy intenmediate wherein
the 1,3-~*~mino~ycl-itol moiet~ is o~ formula I~-
and wherein all tha 0- and N-protecti~g groups are sus-
ceptible to cleavage by base (e.g. as in 1,3,2'~6'-
tetra-~-benz~loxycarbonyl-5-epi-azido-5-deoxy-2"-0-
benzoyl 3",4"-E,0-carbonylsisomicin) treabment thereof
at 100C-with aqueous sodium hydroxide will pxoduce an
antibacterially active 5~epi~azido 5-deoxyaminoglycoside
of this invention (e.g. 5-epi-azido-5-deoxysisomicin).
When the 5-epi-azido-5-deoxy~per-~-and 0-protected inter-
me~iates also contain ace~al and ketal unc~ions which
are cleaved by acid (e.g. as in 1,3,2',6'-~etra-~
benzylox~car~onyl~5-epi-azido-5-deoxy-3'~4'-0-benzylid~le -
2"-0-~enzoyl-3"7~ ,0-car~onyl-Antibiotic JI-20A), ~fter
treatment with base as described hereinabove, the result-
ant product is trea~ed with mild aqueous acid followed
by neutralization of said aqueous acid mixture with mild
bas~ (eOg. aqueous ammonium hydroxyide)~ Purification
of the resulting product by known tech~iques, usually
by chromatography, yields an antibacterially active
5-epi-azido~5_deoxy am~noglycoside a~tibacteria~ agent o~
this invention ~.g. ~-epi-azido-5~deo~y-~nti~iotic ~I-20A)~
. .
- 12 -
':':j ~ ,,!;
1~80Z0
If final compounds are desired having a 5-epi-amino group
in the molecule, the starting compound having a 5-epi-
: azido group i.s subjected to removal o~ protecting groups,
and the azido group is converted into amino. Conversion
: S of the azido gxoup is usually effected ei~hex with hydxogen
in the presbnce of a catalyst or with an alkali metal in
liquid ammonia~ ;
.
~eduction with hydrogen in the.presence of a catal~st is .
preferred ~hen reducing.5-epi-azido-4,6-di~O (aminoglycosyl~-
2,5~dideoxystreptamine-N-pxotected-O-pr~ected intermediates
devoid of unsaturation such as the S-epi-azido-5-deoxy-0-
and N-protected derivatives of gentamicins Cl, Cla, C2, C2a,
C~b.and of Antibiotics JI-20A and
. JI-20B. On the other hand, when reducing 5-epi-azido-~-
protectad-0-protected-4,6-di-_-(aminoglycosyl~-2,5-dideox~-
streptamine intermediates in which double bonds are present
such as in derivatives of sisomicin, verdamicin, Antib;.otic
G-52, and A~tibiotics 66-40B, 66-40D, xeduction by mea~s
o an alkali metal in liquid ammonia is preferable in
. 20 order to avoid redu~tion o~ the double bond.
.
. When reducing a 5-epi-azido S-deoxy-intermediate with
hydrogen in the presence of a catalyst, the catalysts
most frequently e~Ployed are platinum, palladium9 and~ pre-
ferably palladium on charcoal.
13 -
i ~ . .
.
J
8~)ZO
The hydr~genation is usually carried out at room tempera-
tures in lower alka~oic acids, preferably acetic acid,
although other solvents such as lower alkano~ may be used.
- ~he hydrogenation is continued until there is no ~urther
.
S discernible drop in hydrogen pressure and the 5-epi-amino- -
5-deoxy derivati~e thereby formed i~ usually.isolated by
removing the solvent such as by distillation, and thence
tr ating the 5-epi-amino-5-deoxy residue thereby formed
with base and acid, i~ necessary, ~o remove any remaining
N-protecti~g and Q-protecting groups. WhQn the process is
carried out with 5:~epi-aæido S-deoxy intermediates having
per-N-ben~y1oxycarbonyl amino protecting groups and 3",4"-
~,0-carbonyl protecting groups, the benzyloY.ycarbonyl p*o-
tecting ~roups are advantageously removed durins the hydro-
genation.procedure and the resulting 5~epi~amino-5-deoxy-
2~-0-hydrocar~oncarbonyl-3"~4~ ,0-~arbo~yl derivative.
need only be trea~ed with base (e.gO with 2~ sodium hydro-
( xide) ~o remo~e the.acyl radi~a~s. The resul~ing anti-
baterial~y active S-epi-amino-S-deox~aminoglycoside is
then isolated and purified utilizin~ known techniques~
Thus, in a typical mode of carrying out the hydrogenation
o~ a 5-epi-azido-5-deoxy per-~-pro~ected~per~0-pxotected
intermediate, the 5-epi-azido-5-deox~ intermediate (e.g.
1,3,2',6'-tetra~-benzyloxycarbonyl-5- epi-a7~ido-S~-deoxy~
2"-O-benzoyl-3"~4"-N~o-carbonylgent~micin Cla~, dissolved
in_acetic acid, is hydrogenated at room temperat~re at 4
abmospheres starting hydrogen pressure in the presence
of 30% palladi~m on charcoal ca~alyst. ~en no ~urther
{" ` 3 ~
'` ` ~ ~i
~L04~0'~
drop in hydrogen pressure is discernible, the ca~alyst is
removed hy ~iltration, and the solvent removed by distill-.
ation in vacuo to produce a residue comprising 5-spi_amino-
5-deoxy-2"-0-benzoyl-3",4"-~,0-carbonylgentamicin Cla
which, upon treatment with 2~ sodium hydroxid~ at elevated
~emperature~ ~e.g,100C) ~ollowed by neu~xalization with
a~etic acid, removal o~ any insolubles by filtration, con~
cen~ration of the reaction solutio~ to a small volume and.
thence ~romatography thereo~ on Amberlite IRC-50 resin
- 10 (H+ form), followed by elution with ammonium hydroxide,con- .
centration and lyophilization o the eluate gives S-epi-
amino-5~deoxygentamicin Cla~ a n~vel antibacterial agent
of this invention.
~ .
Any acetal or Xetal protecting groups in the starting S-
epi~azido-5~deoxy-pex-_-protected-per-0-prote~ted amino- -
glycoside may be removed a~t~r hydrogenation and treatment
C. o~ the product thereby ~ormed with base, by treatment wi-~h
mild a~ueous a~id, e.g. with dilute mineral acidst or with
tri~luoroacetic acid, or usually with dilute alXanoic
a~ids such as acetic acid . ~ .
,
.
When reducing a 5-epi-azido-5-deoxy-per-~-protected-per-0-
protected aminoglycoside having a double bond by reaction
thereo~ ~ith an alXali metal (e.g. po~assi~m, li~hium a~d,
preferably sodium) in liquid ammonia, the 5-epi-azido-5-
deoxy~intermediate (e.g. 1,3,2',6'-tetxa~-benzyloxy~
- 15 -
,
~0~8~2(~
c~rbonyl-5-epi-azido-5-aeoxy~ 0-benzoyl-3",4"-N,0-
carbonylsisomicinl is usually dissolved in a mixture o~
a cosolvent such as tetrahydrofuran and liyui~ ammonia to
which the alkali metal.(e.g. sodium) is added slowly and
S ~he reaction mixtureiss~ed.for a ew hours. The ammonia
is allowed to evàporate and any remainin~ 0- and N-protec~-
ing groups are removed by addition of water to the reaction
mixture af~ording sodium hy~roxide and heating at elevated.
C tempera~ure (e.g. 100C). Puri~ication of the resulting
product is usually carried out via chromatographic te~h-
niques to o~tain an an~ibactqriall~ active 5-epi-amino-5-
deoxy-aminoglycoside of this invention, e.g. 5-epi amino-
5-deoxysisomicin.
.
If, in the final compouna, it is desired to have a 5-epi-
~15 amino group, it.is also possible to prepaxe the correspond-
ing final compound having a ~-epi-azido group by removi~g
all protecting gxoups and then to convext the 5-epi-aæi
.
group into the.S-epi-amino.~roup by applying essen~ially
the same condi~ions as-described above.
. .
2a When a final compound is desired w'nerein, in formula I, X
is hydroxy, a starting compound.wherein, in formula II, ~'
is hydroxy, is subjected ~o removal o~ the protecting
groups present in the molecule. The protecting groups are
removed by reaction with aqueous base or, when protecting
yroups susceptible to re~uctive cleavage are present, b~
reaction with a reducing agent (such as hydrogen in the
- 16 -
:` ~
~)4~30Z(~
. presence o~ a catalyst or an alkali metal in liquid
ammonia) followed by treabment with aqueous base; thén,
when acetals or ketals are present, by treabment with
aqueous acid. In general, the conditions of reaction are
the same as descri~ed above for the case where a 5-epi-
amino derivative is ob ained.
The starting compounds in the~a~ove process, whe~ein the
~,3-diaminocyclitol moiety is of formula II, and which
contain protecting groups, are novel compounds and may be
described as follows thereby using the 5-epi-azido (X'-N3
in formula II~ ¢ompounds as an example.
:, ' ' ' '
. Although any amino protecting group may be used, partic-
. ularly useful amino protecting groups (designated ~y "Z"
: - . .
. in Formulae XIV to XXI shown hereinbelow) ~or the starting
lS compounds o~ the process of this invention include lower
alkoxycarbonyls (prefera~ly having up to 8 carbon atoms,
.g. inethoxycarbonyl, ethoxycarbonyl, _-pro~yloxycarbonrl,
so-prop~loxy~arbonyl, n-butyloxy~arbonyl, tert- butoxy-
carbonyl, octyloxycarbonyl and the like), substituted
benzyloxycarbonyl :(including o, m ana ~methoxybenzyloxy-
carbonyl, and the like) and, preferably, benzyLox~arbonyl,
Lower alkanoyls preferably having up to 8 carbon atoms
(e.g~ acet~l, propionyl, valeryl, caprylyl) are~also use-
ful amino protecting groups t~)~ particularly for compoun~s
derived from antibacterials which cannot fonm a 3",4"-N,
car~onyl de~ivative ~e.g.compounds not having a 6-0-garo-
- 17 -
~1~4 !31)ZO
saminyl substituent such as derivatives of gentamicin A,
and the kanamycins)O
The foregoing amino prot~cting groups are removable by
treatment with b~se (e~g. with sodium hydroxide) or, in
S the case of benzyloxyaarbonyl~ by reductive cleavage me~h-
ods known i~ the art. ~enzyloxycarbonyl is a pre~erred
amino protecting group because it is removed under the
reducing condition~ w~ereby a 5-epi azido-5-deoxy-per~-
. ' protected per-_-protected intermediate is treated with
hydrogen in the presence of a catalyst (preferably pallad- .
ium? or with an alkali metal (e.g. sodium or potassium) in
: liquid ammonia to produce a 5-epi amino-5-deox~amino-
glycoside of this invention. In addition to the ~oregoing,
~enæylox~carbonyl is a preferred amino protec~ing group
1~ for ~he starting ~ompounds of this proaess sin~e, in amin~-
.
glycosides having a hydroxyl unction aajacent an amino
: function, such as at posi~ions 3" and 4" in the 6-o--gar
aminyl radical o~ the aminoglycosiaes o~ fonmula X, the N-
. benzyloxycarbonyl derivative te~ the 3"~ enzylo~ycar-
: bonyl derivatives of compounds of ormula X)
when subjected to basic conditions (such as with sodium
hydride in dimethylformamide) foxms an oxazolidinone with
the adjacent hydrox~l function (e.g. a 3",4"~N,O-carbonyl
derivative of compounds of formula ~) with concomitan~
elimination o benzyl alcohol. Similarly, N-alkoxy carbonyl
derivatives will ~orm oxazolidinones with an adjacent
hydroxyl function. Additionall~, when a starting compound
has a l~N-C~2Y substituent which has a hydrox~l group
- 18 ~ .
~ 20
alpha or beta to an amino protecting group, Z, ~Ihich is
benzyloxycarbonyl or alXoxycarbonyl, the hydroxyl gxoup
together with said protecting group æ will form an oxazo-
lidinone or a tetrahydro-1,3-oxazin-2--one, respectively.
,
Hydrox~l functions in the starting compounds of this pro-
cess are convenien~ly protected by 0-acyl radicals o~
hydro~arboncarboxylic acids pre~erably having up to 8
car~on atoms (said radicals being designated as "æl" in
Formulae XIV to XXI hereinbelo~Y)or by 0-hydrocar~onylidene
radicals of ketones and aldehydes prefera~}y having up to
8 car~on atoms to form ketal~ and acetals, respectively~
- including cyclic ketals and ace~als (sai~ hydxocarbonyli-
~ dene radicals being designated as "W" in formulae XIV to
- ~ XXX hereinbelow), although any other hydroxy protecting
1~ group may also be used to protect hydroxyl functions.
:; ' ' ' ~ ' ~'
In general, neighboring hydrox~l groups in the amino-
glycoside precursors o~ the starting compounds o~ ~his
proce~s are conveniently protected by cyclic ~etals or -
a~e~als. By "neighboring hydroxyl gxoups" are contem-
plated vicinal and non-vicinal hydroxyl groups which are
situated so th~ together they ~orm a c~clic ke~al or
cyclic acetal function with ketones and aldehydes or their
derivatives, respec~ivel~ E}:emplary of such"neiyhb~ring
hydrox~l gro~s" are the 2',3'-.hydroxyl groups in genta-
micins B and Bl and in ~anamycin A (~hich form 2',3~-o
hy~rocarbonylidene deri~atives), ~he ~',6'-hydxo~yl groups
- 1-9 -
.,.
.
~5)48(~ZO
in gentamicins A and X2 and in An~ibiotic G-41~ ~which
form 4',6'-0-hydrGcarbonylidene derivatives) the 3',4'-
hydro~yl groups in Antibis~ics ~I-20A ànd JI-20B and in
kan~mycin ~ (which form 3',4'-0-hydrocarbonylidene deriva-
tives) and the 4"06"-hydrox~l groups in tobramycin, kana-
mycins A and.BJ. and 3',4'-dideoxykanamycin B twhich form
4"~6"-0-,hydrocarbonylidene deri~atives).
..
The cyclic ketal and acetal der~vatives of said neighboring
hydroxyl groups,include 0 alkylidene (e.g. 0- so-propyli-
~0 dene~0 0-cycloalkylidene (e~g.0-cyclohex~lidene), and 0-
arylalkylidene (eOg.0-benæylidene) derivatives, all of
which are removable upon,treatment with dilute aqueous mild: -
- acia ~e.g~ by 50 to 8~o acetic acid~ The nature of ~he
hydrocarbon or substituted hydrocarbon "ylidene" radicals
o~ the cyclic ketals and acetals is immaterial since they
act only as "blocking groups", do not enter into th~ pro
cess of the inven~ion,,and are subsequently~,removed so that
the free hydroxyls are regenera~ed in their original ~ormO
,.
In the startin~ compounds o~ the processes of this invent-
ion, isolated hydroxyl groups other ~han the 5-hydroxyl
group, such as the 2"-hydro~y present in all the amino-
gly~oside precursors o~ this invention, the 4'-hydroxy
in tobramycin, and the 4"-hydroxy in Antibiotic 66-4oB
, and,in gentamicin A as well as other hydroxyl groups
whi~h are not prote ted by cyclic ketal or acetal functions
~e.gO the 3'-hydroxy in gentamicin A and ~he ~' and 4'-
- 20 -
1~4~ZI~
hydroxy in kanamycin A) are conveniently protected by
h~drocarboncarbonyl radicals.(designated as "Zl" in
formulae XIV to XXI hereinbelow), said hydrocarbon pre~er-
ably having up to 8 carbon ato~ls. Useful hydrocarbon-
carbonyl radicals are acyl radicals derived from lower
alkanoic acids having up to 8 carbon atoms including
: acetyl, propion~l, n-butyryl, valeryl, an~ caprylyl, as
well as acyl radicals derived ~rom aralkanoic acids such
as phenylacetyl and fro~ arylcarbo~ylic acids su~h as o,
. m and ~-toluoyl, mesitoyl, and preferably benzoyl. .
.
Included among the 5-epi-azido-5-deoxy-pex-~-pro~ected-
per-_-protected intermediates are compounas which are .
! exemplified by the ~ollowing formulae: 1,3,2!,6'-tetra~N-
Z-5-epi-azido-5-deoxy-2"-0~ 3",4"-~,0-caxbony1-Antibiotic
66-40D of the following formula XIV
NHZ ~HZ
~0 ,L ~ Z
'~= I XIV
~0\, 0
\~U
Zl
wherein R' is as defined hereinabove for R but whe~ein any
amino func~ion is subs~itu~ed by a group Z, and any hydroxy
function i5 convertea to an ester Zl~ or, when said h~droxyl
group is alpha or beta to an amino protecting group Z,
- 21 -
.,
~(~4~2~
which is benzyloxycarbonyl or alkoxycarbonyl, the hydroxyl
group together with said protecting group ~ is converted
to an oxazolidinone or a tetrahydro-1,3-oxazin-2-one,
respectively, Z and Zl being as defined hereinbelowj
and 1,3,2',6'-tetra-~-Z-5-epi-azido-2"-0--Z1-3'''4''-~'Q-
carbonyl-4,6-di-~aminoglycosyl)-2,5-dideoxystreptamine~
o~ following ~onmula XV: ~z
R Z
AG4 _ o ~
~,.
. - ' .
wherein R' is as hereinabove de~ined; and Z is.a me~oer
selected from the gxoup consisting of benzyloxycarbonyl,
substituted benzyloxycarbonyl,and alkoxycarbonyl, and Zl
is a hydrocar~oncarbonyl, said hydrocar~on having up to 8
carbon atoms; and AG4 is an aminoglycosyl function select-
ed from the group consisting of: . -
.
- 22 -
~4~Z~
~,
I C~3
3N + d CH2NH2
~ (in S-epi-azido-5- ~ o (in 5-epi-azido-
< ~ ~ ~entami~in Cl ~ eoXy-gentemic~A
~ - NHZ
O (in 5-epi-azido-5- / (in 5-epi-azido--
~ ~ en~amiCin ~ Xy qentamicin~
:, ~Z , ~ ' -
~--CH3 C ~ ~zH3
in S-epi-azido-S- // \ (in 5-epi-azido-5--
/ \deoxy-gentamicin // \deoxy~ntibi~tic
2b) ~ G-52)
-- ,
~Z ' ' `:
: CH3
I~NHZ ~ ~23~IZ
~~ (in 5-epi-Rzido-5- ~ \ (in S-epi-azidQ-5-
damicin) ~ ~ or~y-9isoml~in)
~HZ
wherein Z is as hereinabove defined.
zo
1,3,2',6',3"-panta~-Z'-5-epi-aæido-5-deoxy-2",4"-di-0-
Zl-Antibiotic 66-40B of the following formula XIX
'~ CH2~HZ ' ~HZ '
~ ~J;
Z' 3 0
Zl~
z
-whexein R'~ Z' and Zl are as hereinabove deined;
: and 1,3,2',6'-tetra-N-Z-5-epi-azido-5-aeoxy-3',4'-0-W-2"-
0-~1-3"~4"-N,O~carbonyl derivatives of AntLbiotics JI-20A
and JI-20B of following formula XXI :
.
IZ , .
R ' Z
AG8
E13C~
Z
-
- 2~ -
2~
wherein R', Z and Zl are as hereinabove defined and AG8
. is an aminoglycosyl function selectea from the group
consisting of: 3
H ¦. ~HZ
CH2NEZ
~ (in 5-epi-azido-5-deoxy ~ (in 5-epi-azido 5-
/0 \ Antibiotic JI-20A) / O \ deox~-Antibiotic
f JI -20B )
NH~ ~HZ
: 5 Z and W being as hereinabove deined.
The requisite, novel starting compounds ror this ~rocess
~f the invention, i.e. the 5-epL-azido-4,6-di-0-(amino- . -
glycosyl)-2,5-dideo~ystreptamines having hydroxyl and
amino protecting groups, are prepared by treating~the
-- 10 corresponding 5-0-hydrocarbonsulfonyl (or substituted
hydrocarbonsulfon~ 4,6-di~O-(amino~lycosyl)-2-deoxy
streptamine (i.e. compounds o~ ~ormulae XIV-XXI devoid
of the 5-epi-azido moiety and having a ~-O-hydrocar.'oon-
sulfonyl group) with an alkali me~al azide in an organic
solvent.
.
Organic solvents suitable ~or this process are orga~ic
solvents in which the 5-0-hydrocarbonsul~onyl (or sub- -
stituted hydrocarbonsulfonyl)-per-~-protected-per-D~-
protected 4,6-di-O~(aminoglycosvl)-2-deoxystrepta~ine
and the alkali metal. azide reagent are soluble and are
solvents which will not react with the reagen~- so ~hat
. .
~ - 25 -
1~4802~11
the possibility of competing ~ide reactions is minimized.
Suitable organic solvents most useful in this process are
solvents such as dimethylformamide~ d~methylacetamide
and hexamethylphosphorictriamide. Dimethylformamide is
S frequently con~eniently used.
Sodium azide is usually employed when converting a 5-O-
hydrocarbonsul~on~l intermediate of this invention to the
corresponding 5-epi-aziao-5 deoxy intermediate o~ formulae
~IV-XXI~ however, other alkali metal azides may be used,
such as potassium azide and lithium azide.
: :
The 5-O-hydrocar~onsulfonyl and 5-O-su~stituted h~dro-
car~onsul*onyl ester intermecllates useful in this proc~ess
and also useful as star~ing materials in another ~r~cedure
of this invention are those derived from hy~rocar~onsul- -
15 foni~ acids having up to 8 carbon atoms including e~han~-
sulfonic acid, benzenesulonic acid, ~-toluenesul~onic
a2id, and preferabl~, -
methanesulfonic acid; also those derived from nitro-
ben ene sulfonic acids (e.g. oO m, and p-ni~robenzene
sulfonic acids) and those derived ~rom halogenohydro-
carbonsulfonic acids (e.g. trifluoromethanesulfonic acid,
~-chlorobenzenesulfonic acid, o or p-bromobenzenesulfonic
~acids and the li~e). The 5-O-hydrocarbonsulfonyl and
5-o-subs~ituted hydrocarbonsulfonyl intermediates axe
prepared from the corresponding p~r-N-protected-per-O-
26
.,
1~480Z~)
protected-S-hydroxy-aminoglycosides (i.e.compounds of
ormulae XIV-~XI devoid o~ t~e 5-epi-~3-
moiety and having a 5-hydroxyl ~unction which compounds
-are used as starting materials in yet another ~oce~ure.of
this inven~ion) by treatment thereo~ with a hydrocarbon-
sulfonyl hali~e (preferably methanesulfonylc~loride)in.a.
tertiary amine (usually trlethylamine))~
When converting a 5-0~-hydxocarbonsulronyl ~or 5-0-s~b-
stituted hydrocarbonsul~onyl)-4,6-di-Otaminoglycosyl?-2-
-10 deoxystreptamine (having amino functions and all other
hydroxyl functions protected by groups susceptib1e to
!~ reductive cleavage and/or to basic or mild acid h~dro~y-
sis) to the corresponding 5-epi-azido-5-deoxy intermediate
(such as defined by ~ormulae XIV-XXI) by this process
the starting 5-0-hydrocarbonsulonyl derivative (e.g. 1,
3,2',6'-tetra~N-~enzyloxycarbonyl-5-_~methanesulfonyl-2"-
0-be~zoyl-3",4"-~,0-carbonylsisomicin and 1,3,2',6'-
tetra~ enzyloxycarbonyl-5-D~ethanesulfonyl-2"-0-
benzoyl-3",4"-~,0-carbonylgentamicin Cl) is usually diss.-
: olved in dimethylformamide to whic~ at least an e~uivalent
and usually an excessi~e molar quantity (with re~erence
to the molar quan~ity of aminoglycoside) of sodium azide^-
is addad under an atmosphere o~ argon.- The reaction
mixture i5 heated (usually over 100C) until the 5-_-
methanesulfonyl intermediate is no longer present as
- 27 -
. .
~34~
evidenced by thin layer chromatographic analysis. The
resulting product is isolated usually by concentrating
the reaction mixture, dissolving the residue in an acid~
.~ free organic solvent, washing the organic solution with
water, thence evaporating the washed organic solution to
a residue comp~ising a 5-epi-azido-S-deoxy intermediate
(e.g. 1,3,2',G'-tetra-N- benzylo~ycarbonyl-5-epi-azido-
5-deoxy~2"{)-}~enzoyl-3"~4"-N,O-carbonylsisomicin and
l,3~2',6'-te~ra~N-benzyloxycarbonyl-S-epi-aæido-5-deoxy-
. 10 . 2"-O-benzoyl-3",4"-~,O-carbonylgentamicin Cl),
The per-_-protected-per-O-protected-5-O-h~drocarbon-
sulfonyl-4,6qdi-O-(amino~lycos~l)-2-aeoxystreptamine~
which are ~recursors for the corresponding 5-epi-azido_
- ; 5-d~oxy intermediates and also staxting material~ ~or an-
other-~rocedure~o~ this invention, are derived rom
k~own, unprotected-4,6-di~ (aminog1ycosy1)-2-deoxy-
streptamines including 4-O aminoglycosyl-6-O-~arosaminyl-
2-deoxystreptamine antibiotics such as gentamLcin Cl, .
gentamicin Cla-, gentamicin C2, gentamicin C2a, gentamicin
C2b, sisomicin, verdamicin, Antibiotic JI-20A, Antibiotlc
JI-20B and Antibiotic G-52; and 4,6-di-O-(aminoglycosyl)-
2-deoxystreptamines such as Antibiotic 66-40B, Antibiotic
66-40D~ of the foregoing, preferred precursors are genta-
l la~ C2, C2a, C2b, Antibiotic 66-40D, verdamic1n
Antibiotic G-52 and sisomicin, all of which are most easi-
ly converted to preferred compounds of this invention,
i.e. to the corresponding 5-epi-a~ido-5-deoxy and the
- 28 -
.
4~0~0
5-epi-amino-5-deoxy-derivatives and the 5-epimers.
The aforementi.oned 4,6-di-0-(aminoglycosyl)-2-deoxy-
streptamine antibiotics are known. The starting compound
referred to herein as gentamicin C2b, having the struc-
S tural formula shown herein, is named in some prior artas gentamicin C2a.
When preparing the l-N-CE2Y-derivative of this invention
via the corresponding starting materials, the l-N-CH2Y-
per N-protected-per-0-protected-5-0-hydrocarbonsulfonyl-
4,6-di-0-(aminoglycosyl)-2~deoxystreptamlne precursors
are also derived from the li~.-CH2Y derivatives of the
afoxedescribed l-~-unsubstituted-4,6-di-0-(aminoglycosyl)-
2-deoxystrep~amines. These compound~ are known in th~
art. -
Wh2n preparing the per-~prot~cte~;per-0-protected-S-0-
hydrocarbonsulfonyl-4,6-di-0-(aminoglycosyl)-2-deoxy-
~trep~amine ~ompounds, the amino groups are protected
first by formation o~ amides susceptible to reductive
cleavage or basic hydrolysi~, For the proce~es o~ thiS
--20 invention it is preexred to protect the i~mino groups
b~ forming N-benzyloxycarbonyl derivativas thereo~ ~e.g.
1,3,2',6',3"-~enta-N-benzyloxycarbonylgentamicin Cl).
.j :.
.;. .,~
10~
The per-N-protected aminoglycosides thereby formed which.
have a garosaminyloxy radical at C-6 are then treated with
an alkali metal hydride, usually sodium hy~ride in
dimethylformamide, whereby the 3"-~-hydrocarbonyloxy-
carbonyl protecting group is cyclized with the 4"-hydroxy
. function to ~orm an oxazolidinone derivative, i.e. a 3",.
- 4"-~,O~carbonyl derivative. In'aminoglycoside~ wherein
other amino group~ are adjacent to a hydroxyl group (such
a.q the 6'-amino and 4'-hydroxyl in gen~amicin Bt, othex
,O-~arbon~l derivatives will be fo~med~ Thus, upon rea
ction of 1,3,2',6',3"-penta-N-benzyloxycarbonylgentamicin
Cl with sodium hydride in dimethylformamide, there are
formed oxazolidinone derivatives, i.e. 1,3,2',6'-tetra-
N-benzyloxycarbonyl-3",4"-N,O-carbonylgentamicin Cl.~
Usually next are protected adjacent hydroxyl groups which
will form a ketal or acetal group upon treatment with a
ketone or aldehyde or derivative thereo~ in dimethylforma-
mide in the presence of catalytic amounts of a strong acid
such as ~-toluenesulfonic acid utilizing known technlques.
Finally, any isolated hydroxyl functlons (except the 5-hy-
droxyl group) remaining in the partially pr~tected aminogly-
coside derivatives are converted to the corresponding hydro-
car~oncar~onyl- derivative3 by treatment thereo~ with an
acid chloride of the hyarocarboncarboxylic acid in a
tertiary amine (preferably pyridine?0 the mo~ar quantity
of acid halide reagent being based upon the number o~
- hyaroxyl groups to be esterified~ If only one hydroxyl
- 30
~ ~ .
,
: group o~her th?.n the 5-hydrox~ remains in the molecule
. (e.g. the 2"-hydroxy) an e~uivalent quantity of acid
halide to the molar quantity of aminoglycoside is used;
if two hydroxyl groups remain to be pro.tected~ two molar
equivalen~s of acid halide are used per mole of amino-
glycoside. Acyl halides o~ hydrocarboncarboxylic acids
having up to 8 carbon atoms are preferentially usedO
: in~luding acid chlorides.of lower alkanoic acids such
as ace~ic, propionic, valeric and caprylic acids; of
10 aralkanoic acids such as phenylacetic acid and aryl- .
~arboxylic acids such as toluic and, preferably benzoi~
acids. Thus, each of the a~orenamed intermediates of
gentamicins Cl and B upon treatmen~ with equimolar
- quantities of benzoylchloride in pyridine, yields the
~5 ~ corre~pondi~g 2"-O-benzoyl deriva~ive, i.e~l,3,2',6'-
tetra~N~benzyloxycarbonyl-2"-O-be~zoyl-3",4"i~,0- .
carbonylgentamicin Cl which, upon treatment with methane-
sulfonyl chloride in triethylamine yields the corre~ ~
sponding S-O-methanesulfonyl derivatives. The compounds
(4,6~di-O-(aminoglycosyl)-2-deoxystreptamines) belng per-
N-protected and O-protected in all positions-other than
position 5, are also used as starting materials in one
process of this invention.
The other requisite, novel starting compounds for a
: process of the invention, i.e. compounds wherein the
1,3-diaminocyclitol moiety is of the formula II, wherein,
- 31 -
,.f .
~0~8(~ZO
in the formula, X' is hydroxy (5-epi-4,6-di-0-(aminogly-
cosyl)-2-deoxystreptamines) and which compounds are per-
N-protected and 0-protected in all positions other than
position 5 are prepared by processes described hereinbelow.
Procedure B) is carried out in dimethylformamide alone or
preferably also in the presence of a tetraalkylammonium
alkanoate. The treatment of a 5-0-hydrocarbon-
sulfonyl intermedia~e with dimethylformamide alone is
often carried out at reflux tempera~ure (i.e. about 155C)
since the rate o~ reaction is usuall~ greater than when .
the reaction is carried out at lower te~peratures; wnen
; carried ou~ in the pre~ence of a tetraalXylammonium
alkanoate, ho~eve~ ~he reaction proceeds well at lower.
temperatures (e.g. 100-140C) to produce good yields o~
a purer produc~ Tetra-n-butylammoniu~ acetate is usually
the reagent o~ choica, but o~her tetraalkylammonium
` alkanoates may be used, e.gO tetraethylammonium acetate~
~etramethylammonium acetate, tetraethylammonlum ~ormat~,
tetra-~-butylammonium formate, and the like. The molar
:
20 quantity of tetraalkylammonium alkanoate per mole of
.
aminoslycoside is usually from about 1.5 to about 5 moles.
. ' ' . , ' .
- The intermediates produced upon reaction o~ the ~-protec~
ed~0~-protected-5-0-hydrocarbonsulfonyl (or substituted
hydrocarbonsulfonyl)-4,6-di-0-(aminoglycosyl)-2_d20xy-
- 32 -
. , .
~6~48~Z~
s~reptamine with dLmethylformamide upon hydxolysis pro~
duces a 5-epi ~ompound o this invention~ ~hen tetra-n-
- butylammonium acetate is used toyether with dimethyl-
. formamide, the int~rmediate produced is the corresponding
~-protec~ed-O-protected-S-epi-~-acetyl derivative which,
upon hydrolysis, produces a S-epi-compound o~ this inven-
tion~ -
.
Protecting groups susceptible to reauctive c-eavage are
frequently preferentially used in carrying out the pro-
ess since they can be removed with ease via reduc~ivetechniques after epimerization at the 5-position. Other
protecting groups will remain after-the reduction step,
however, elg. N,O~arbonyl groups which are removed upon
treatment with aqueous base at elevated temperatures.
Additionally9 to remove acetals or ketals, acid hydrolysis
-~ is required~
., ` ` , ' '
When removing prote~ting groups suscepti~le ~o redu~tive
c}eavage r~m the intermediates pxoduce~ in this process,
- : reduction with hydrogen in the presence of a eatalyst is
preferred when the 5-epi-4,6-di-O-(amin~glycosyl)-2-
deoxystreptamine-~-protected-O-prote~ted intermediates
produced are devoid of unsaturation, such as the inter-
- mediates derived by treatment with dimethylfoxmamide of
O- and N-protected derivatives of gentamicins Cl, Cla, C2,
C2a, C2b and Antibiotics JI-20A and JI-20B. On the other
hand, when removing protecting groups susceptible to
~'~ ,
~ ,
- 33 -
~8~)Z~
reducti~e cleavagefrom N-protected-O-protected 5-epi-4,6-
di-O-(aminoglycosyl)-2-deoxystreptamine intermediates in
which a double bond is presentf such as those derived from
sisomicin, verdamicin, Antibiotic G-52, Antibiotic 66-40B
and 66-40D, reduction by means of an alkali metal in liquid
ammonia is preferable in order to avoid reduction o~ the
double bond.
When deblocking an intermediate with hydrogen in the pre-
sence of a catalyst, the catalyst most frequently employed
is palladium, preferably palladium on charcoal.
The hydrogenolysis of protecting groups is usually carr-
- ied out at room tempera~ure in lower alxanQic acids, pre-
æerabl~ acetic acid, although other solven~s such a~
; iow~r alkanols may be used... The hydrogenation is contin- `
ued until there is no urther discernible drop in hydko-
. . gen pr ssure and the 5-epl-4,6-di-O-(~minvglycosyl)-2- -
deoxystrqpt2mlne o~ this invention is then usually isola-
ted by removing the solvent such as by distillation, and.
thence treating the ~- and O-pro~ectëd-5-epi-2-deoxy-
streptamine intenmediate thereby formed with base, and
when acetals or ketals a.~e a~so present, with aqueous acid
to remove the remaining protec~ing groups.
In a typical mode o~ carrying out this process, a 5-O-
hydrocarbonsulfonyl-per-N~-pro~ected-per-_-protected-
intermediate similar to tho~e of foxmulae XI~-XXI but
\
- 34 -
~6~4i3~20
~ OS0 -hydro-
having a hydrocarbonsul~onyloxy group at C~S \5 2 carbon
rather than an epi~ azido group ~3 e.g. 1,3 9
2',6~-~etra N-benzyloxycarbonyl-5-O~methanesulfonyl-2"-
: 0-benzoyl-3",4"-N,0-carbonylgentamicin Cl, is dissolved
- S ~i~ dimethylfo~mamide and heated at re~lux temperature for
18 hours, then the solution is evaporated to a residue
of a S-epi~-protected-0-protected-4,6-di-0-(amino-`
glycosyl)-2~aeoxystreptamine intermediate which is diss-
olved in acetic acid and hydrogenated at ro~m temperature
a~ 4 abmospheres starting hydrogen pressure in the pre-
sence of 30% palladium on charcoal catalyst. When no
further ~rop in hydrogen pressuxe is discernible, the
- catalyst is removed by filtration, and the- sol~ent
removed by distillation in vacuo to produce a residue
15 which, upon treabment with 2N sodium hydroxide at eleva~
tea temperatures (e.g. 100C) followed by neutxalization
with acetic acid, thence isolation and purification
- utilizing known techniques yields 5-epigentamicin Cl, a
novel an~ibacterial agent of this inventionO
' " ' , .
In another preferred mode of carrying out this process a
5~-hydrocarbonsul~onyl-per-N-protected~per-0-pro~ected
- intermediate, e.g. 1,3,2',6'-te~ra-N-benzyloxycarbonyl-
5-0-methanesulfonyl-2"-0-benzoyl-3",4"-~,Q-carbonyl-
sisomicin and l-N-ethyl-l,3,2',6'-te~ra-N-~enzylo~y-
~arbonyl-5-O~methanesulfonyl-2"-0-benzoyl-3",~ ,Q-
.
- 35 -
~ 6~48~31ZO
~arbonylsisomicin, in dimethylformamide to which tetra-
n-butylammonium acetate has been add~d, is heated at
120C ~or 16 hours and ~he solution evaporated to give
~ the corresponding S-epiacetyl derivative, e.g. 1,3,2',6'-
tetra-~-benzylo~ycarbonyl-5-epi-0-acetyl-2"-0-benzoyl-
3",4"-N,0-carbo~lsisomicin and the corresponding 1-~
e~hyl derivative, which, upon treatment with aqueous
potassium hydroxide followed by neutralization, thence
.. ~ . .
isolation and puriication via chromatographic techniques
yields a 5-epi-compound e.g. 5-episi~o~icin and l-N-
ethyl-S-episisomicin.
Any acetal or ketal protecting groups in the in~ermediates
are removed af~er remo~al of the N-protecting groups ~y
treatment with dilute aqueous acid, e.g. ~ith ailute miner-
lS al acids, dilute trifluoroacetic acid, or usually wi~h
~ilute alkanoic acids, such as acetic acid.
!
When removing carbobenz~loxy protecting gxoups ~rom a per-
~-protected-per-0-protected aminoglycoside intermedia~e
having a double bond (e.g-. the:intermediate deri~ed fr~m
1~3~2'~6~tetra-~-benzyloxycarbonyl-2~-o-benz~yl-3"s4"
E,0-carbonylver~amicin, upon ~reatment with dLmethyl-
formamide) by rea~tion thereof with an alkali metal (e.g.
potassium, lithium, and-preferably, ~odium) in liquid
ammonia, the intermediat`e is usually dissolved in a mixt-
- 36 -
8~20
uxe of a co-solvent such as te~rahydro~uran and liquid
amm~nia to which the alkali metal (e~y. sodium) is added
and the xeaction mixture stirred for a few hours. After
allowing the ammonia to evapore, any remaining 0- and ~-
prot~cting groups ~such as the 3",4"~ carbonyl and
the 2"-0-ben20yl group) are removed by addition of water
to the reaction mixture affording so~ium hydroxide and
heatins at elevated temperatures (e.g. 100C). Puri-
fication of the resulting product is ef~ected via c~roma-
tographic technioues to obtain an antibacterially active
- S-epi-aminoglycoside of this invention, e.gO 5-ePiVer-
damicin.
.
~ Al~ernatively, the protecting groups may be removed
; rom the N- and 0-protec~ed intermediate proauced upon
:~ 15 ~ treatment with dimethyl~oxmamide of an N- and 0-pro-
tected-S-0-hydrocarbonsul~onyl-4,6-di-0-(aminogl~cosyl)-
2~deoxystreptamine, by reaction thereo~ with base at; .
elevated temperatures and~, when acetals or ketals are
pr~sent, by treatment with a~ueous acid.
.
-- 37 --
ff~,
.
~()4~0ZO
In procedure C~ starting compounds are used which are
per-~-protected and per-0-protected with the exception
of ~he extremely hindered hydroxy group in position 5,
and which are obtained by introducing blocXing groups
as described above.
In the firs~ step of this reaction the oxidizing agen~
pre~erably employed is sele~-ted from ruthenium tetroxidet
chro~ic acid in acetone and chro~i~m ~rioxide-pyridin~
complex in methylene chloride. The resultant 5-dehydro
compound difers ~rom the compounds o ormulae XIV-x$I
only i~ that a group is substituted for th~ 5-epi-
', - ~ ,
aæido group ~ .
~ 3
The o~idation step o this process is usually carried
~ ; out in an oxganic solven~ such as acetone when chromic
acid is used, or a halogenated hydrocarbon, preerably
methylene chloride, when chxomium trioxide-p~ridine~
camplex o~ ruth~nium tetroxide i9 used as oxidizing
agent, at temperatures in the range o from about 0C
to about 40C, preerably at 20C to 40C.
In the second step o this process where~y the per~W
and _~protected-5-dehydro-4,6-di-0-(amino~lycosyl)-
intermediabe,is reduced to produce the corresponding
5-epi-intermediate, hindered alkali metal borohydrides
are preferred reagents, e.g. sodium, potassium or
-lithium tri-sec-butyl borohydrides, although any alkali
- 38 -
,1 ,
~48~ZO
metal borohydride may be used, e.g. s~dium or potassium
borohydride. The reaction is usually carried out in a
lower alkanol (e.g.methanol) or ether (e.g. dioxane or
preferably tetrahydrofuran) at temperatures in the range
of ~rom abou~ oC to about 50C (preferably at 0C to
ZSC) under conditions known in the ar~ ~or ~arryin~ out
alkali metal borohydride reductions.
In a typical mode of carrying out this process aspect
of the invention a per-~-protected-0-protected 4,6-di-o_
(aminoglycosyl)-2-deoxystreptamine (e.g. 1,3,2',6',3"- -
penta-N-benzylox~arbonyl-2"-0-acety1gentamicin Cla),
dissolved in methylene chloride ;s treated with chrvmium
trioxide-pyridine complax at room temperature until the
xeaction is complete as determined by thin layer chroma-
tograp~i~ analysis of an aliquo~ o the reaotion mixtùre
- (usually about 28 hours reaction tLme)~ The resul~ing
5-keto intermediate, i.e. the 0-protected-per-N-protect-
ed-5-dehydro-4,6-di-0-~amin~slycosyl)-~-d~o~ystreptamine,
e.g. 1, 3, 2 ', 6 ', 3 " -penta~enzyloxy~arbonyl-5~ehydro- `
2"-0-acetylgentamicin Cla, is convenientIy isolated b~
extraction with ether, evaporating the solvent, and
purifying the residue via chromatographic technigues~
The 5-keto-intermediate is then dissolved in a lower
alkanol or ether, preerably tetrahydro~uran and an
alkali metal hydride ~e.g. lithium tri-sec.- bu.yl
borohydride) added (usually 2 to a moles of metal bora-
hydride is used per mole o~ aminoglycoside intermediate)
.
- 39 -
~..,,j
~4~ 20
and the reac~ion mixture stirred at room temperature for
about 20 hours. The ~-pro~ected-O~-protected-5-epi-
aminoslycoside thereby formed is usually isolated by
- adding saline to the reaction mixture extracting with
eth~l acetate~ thence evaporating ~he solvent. The N-
and O;pro~ec~ing groups in the 5-epi-aminoglycoside
- intermediate are then removed as described above,~or
example, by treatmen~ with sodium in iiquid ammonia ~o
remove ~he benzyloxycarbon~l group~ ollowed by treat-
ment with sodiu~ hydroxide at elevated temperatures.
.
.
Any compound (5-epi-azido-, S-epi-amino or 5-epimers)
bbtain~ by the above described processes of the invent- -
i ~ ~ ion and wherein, in ~he 1,3-dla~inocy~litol-~i~ty,-~he-
amino group in position 1 is unsubstituted, may be-
alkylated ac~ording to methods known~in the art in
..
or~er to introduce the group -CH~Y with Y being as above
aefined into position l of the molecule.
One alkylation process comprises treating the compound
which may have amino-protecting groups at any position
o~her than position 1, with an aldehyde o the ~ormula
Y' - CHO
with Y' being a group as defined for ~ above, wher~in
an~ amino or hydroxy group present may be pro~ec~e~; in
the presence of a h~aride donor-reducing agent, and, if
required, removing all protecting groups present in the
molecule.
- 40 -
~ .
~o~z~
- This process, whereby the l-amino function in a 1-~-
unsubstituted derivative of a 4~6-di-o-taminog
1,3-diaminocyclitol antibacterial agent is selecti~ely
condensed with an aldehyde and concomitantly reduced in
situ to ~orm a 1~-C~2Y-derivative of the 4,6-di-o-
(aminoglycosyl)-1,3-diaminocyclitol ant~acterial age~t, -
is usually carried out at room temperature ln the pxesence
o~ air, although it may be advantageously carried out
.
under an inert atmosphere (e~g. argon or nitrog~n).
~ydride-donor reducing agents include dialkylaminoboranes
(e~g. dimethylaminoborane, diethylaminoborane and prefer-
ably mo~pholino~orane), tetraalkylammonium cyanoborohy-
drides~e.g, tetrabutylammonium cyanoborohydride), al~ali
metal borohydrides(e.g. sodiu~n borohydride) ~nd preerably,
alkali metal cyanoborohydrides(e.g. lithium-cyano~oro
hydride and sodium c~anoborohydride).
: ' :
- This process is conveniently c~rried out in an inert
- solvent~ Al~hough anhydrous aprotic solvents may some-
times a~vantageously be employed in this process~ (such as
tetrahydrofuran when utilizing morpholinoborane as
h~dride-donor reducing agent~ this process is usuall~
carried out in protic solvents e.g. in a lower alkanol
or, pre~erably, in water or in an aqueous lower alkanol -
(a.g~ aqueous methanol, aqueous ethanol) 9 although other
water~iscible co-solvent systems may be employed such as
aqueous dimethylformamide, aqueous hexamethylphosphor-
. . .
~ ~ .
~(~4W~
amide, a~ueous tetrahydro~uran and aqueous ethylene
glycol dimethyl ether~
The p~ocess is conveniently carried out at a p~ in the
range of from l to 11, pre~erably ~r~m 2 to 5 and pro-
ceeds best in the range of from ~.5 to 3~5. The
acidic mediu~ which is pre~erred may be obtained by
adding an o~ganic acid such as acetic acid, tri~luoro--
acatic aci~, or ~-toluenesulfonic acid or an inorganic
acid such as hydrochloric acid, sul~uric acid, phos- -
phoric acid, or nitric acid ~o the aminocyclitol deriva- -
tive. Ther~by acid addition salts are form~d and it
- is usually most convenient to use the addi~ion salts
aerlv d from sulfuric acid~ Optimum resul~s are achie-
~ ved when all amino groups pxesent in the molecule are
- 15 ~ully neutralized, It is usuall~ ~onvenient to pre-
- pare the requi~ite acid addition sal~ starting compound
- ~n si~ by adding the desired acid (e.g. sulfuric acid)
to a solution or suspension o~ the derivative o~ the
~ ~,6-di-0-(aminoglycosyl~-1,3-diaminoc~cli~ol in a pro~ic
solvent (e.ga water) until the pH o~ the solution is
adjusted ~o the desired value.
In oraex to minimise competing side reactions when an
aminoaldehyde is used as a reagent, it is pre~erable to
protect the amino function in the aldehyde, e.gO with
_.
an acyl blocking group ~uch as acetamido, phthalimido,
- or the liXe, prior to carxying out this process, and
.
- 42 -
.
11)~80Z~
thence removing the ~-protecting ~roup in the resultant
product. It may also be ad~antageous to protect the
hydroxyl group in hydroxyl-containing aldehydes when
carrying out thi~ process; however, i~ is not gensrally
necessary.
,
Alternatively, partially M-pro~ected intermediate~ may
be utilized. Thus, or example, one may u~ilize a liN-~
unsubstituted derivative-wherein ~he amino function at
the 6'-carbon is ~-protected e.g. the SU1LUriC acid
addition salt of 6'-~-t-butox~carbonyl-5-epi-azido-5-
deoxysiso~icln- or a l-~-unsubstituted de~ivative
wherein the amino functions at C-2' and C-3 are N-pro-
tected (e.g. the suluric acid addition salt of 2',3-
di-N-trifluoroacetyl -S-epigentamicin Cl) and tnere will
be formed the corresponding partially N~-pro~ected-l~-
.
~ alkyl derivative (eig.l-~-ethyl-6'~Lt~buto~yca~bonyl-
- 5-epi-azido~5-deoxy~is~micin and 1-~-ethyl~2',3-di-~-
trifluoroacetyl-5qepigentamicin Cl~respectively)which
upon removal of the N-protecting groups, according to
known methods, ~ields 1-~-alkyl-5-epi compounds o this
invention, e.g. l-~-ethyl-5-epi-amino-5-deoxysiso~icin
and l-~-ethyl-5-epigentamicin Cljrespectively
'' ;
Additionally, the l-N-CH2Y-derivatives o the 4,6-di-o-
(aminoglycosyl)-1,3-diaminocyclitols are prepared by
reducing a Schifr base deri~ative of the l~amino function
in a partially ~-protected derivative of a 4,6-di-0-
(aminoglycosyl)-1,3-diaminocyclitol followed by removal
; ~
02~
o the ~-p~otecting groups. Thus, for example, 2',3-di-~-
tri~luoroace~yl-5~epigentamicin Cl upon reaction with an
aldehyde (e.g. benzaldehyde, phenylacetaldehyde or acetal-
dehyde) is converted to the corresponding 3",4" oxazolidine-
l-ylidene Schif~ base, whic~ upon reduction wit~ sodium
borohydride and methanolic sodium methoxide yields the
corresponding l-~-CH2Y-3",4"-oxazolidine,-which upon treat-
ment with acid yields a l~-CH2Y-5-epi compound of this in-
vention, (e.g. l-N-benzyl-5-epigentamiCin C~ phenethyl-
S-epigentamicin Cl and l-~-ethyl-5 cpigentamicin Cl, respec-
tively).
In these processes, suitable as ~-protecting groups are
those ~roups known in the art to be easily removable a~er
.
preparation o~ the l-N-CH2Y-5-epi compounds without effecting
~ . .
the l-~CH2Y substituents therein. Exemplary of such amino
protecting groups are 2,4-dinitropher.yl; acyl groups such
as acetyl, propionyl and be~zoyl; alkoxycarbonyl groups such
- as methoxycarbonyl, ethoxycarbonyl, 2,2,2-~richloroethoxy_
carbonyl, t-~utox~carbonyl and 2-iodoethoxycarbonyl; and
arylalkoxycarbonyl groups such as benzyloxycarbonyl and 4-
methoxybenzylox~carbonyl groups.
- : . .
Another alkylating process for the preparation of a com-
pound wherein, in formula I, R is a straight chain
alkyl having up to five carbon atoms, comprises treating
a l-N-unsubstitute~ compound, which contains amino-
protecting groups at any position other than position 1,
-
and wherein the l~amino group may be in an acti~ated
sta~e, with an alkylating agent containing the straight
44 -
16~4~Z4~ 1
chain alkyl gxoup having up to 5 carbon a~oms and a lea~-
ing group; and removing the protecting groups, ana, if
required, the activating group or groups present in the
molecule.
S Examples o~ alkylating agents advantageously used in this
process ara alkyl iodiae, alkyl bromide, dialkyl sulfate,
alkyl fluorosulphona~e and alkyl ~-toluenesul~onate
wherein the alkyl group is the required straight chain
alkyl group having up to S carbon atoms. Other alkylati~g
lQ agents, wherein the alkyl group preferably has one or two
carbon atoms,are trialkylanilinium hydroxide, trialkyloxon- ~
ium fluoroborate, trialkylsulfonium fluoro~orate, or tri- -
alkylsulfoxonium fluoroborate. All o~ these alkylating
agents contain a good leaving group, such a-s Br , I ,
1~ OS02F , dialkylanillne-or dialkylether.
~'' - ''
The ~mino ~roup in position 1 of the derivative o~ the 4,6-di-o
.
(aminoglycosyl)-1,3-diaminocyclitol can be ~ree or activa-
ted. An example o~ an activating group is trifluoro-
methylsul~onyl. These a~tivating groups may be introduc-
ed into the molecule by reacting-a derivative of the 4,6-
; di-(aminoglycosyl)-1,3-diaminocyclitol, which possesses
amino-protec~ing groups at any posi~ion other than posit-
ion 1, with a c~npound providing the activating group,
such as trifluoromethylsulonyl chloride~
.
:
~,
,1
)
80Z()
The l-amino gxoup can also be alkylated by way o~ the
corresponding di-(2-cyanoethyl)-derivative which is
deriv~d by treatmen~ with acrylonitrile of thé aerivative
o* a 4~6-di-(aminoglycosyl)-1,3-diamino~yclitol which
possesses amino protecting groups at any position other
than position 1. ~he 1-N-di~2-c~anoethyl)-derivative -
thus prepared is t~en alkylated with one o~ the above
listed alkylating agents followed by removal o the
cyanoethyl groups. . --
.
The process of the invention is carried out under condi-
tions similar to those employed in the wellknown direct
alkylation pxoc~dures o~ amines.
- ~
Yet another alkylation process for the preparation o~ a
~ compound wherein X in ~ormula I is hyd~oxy or amino,
~omprises trea~ing the compound, which may have amino
protecting groups at any position other ~han position 1,
. with an acylating agent selected ~rom an acid of the
formula
.
HO ~ - Y'
with Y' being a group as defined for Y hereinabove,
wherein any amino group present may be protected; in
the presence of a carbodiimide, and a reactive derivative
of above said acid; removing all protecting groups present
in the molecule; and treating the resultant l~ acyl
derivative with an amide-reducing hydride reagent.
3 - 46 -
~4~132al
Reduction of the l-~-ac~l compound is usually ef~ected
in a non-reactive organic solvent in which the l-~-acyl
deriva~ive and the amide-reducing reagent are soluble and
which will not react with the reagant so there is prod~ c
uced a minimum of ~ompeting side reactions. Non-
xeactive organic solvents which are most useful àn ~his
reduction process are ethers such as diox~ne, tetrahydro- -
furan, diethyleneglycol dLmèthyl ether, nd the like.
.
Preferred amide-reducing hydride reagents are aluminum
hydrides and borohydrides including lithium aluminum
hydride, lithium trimethoxy aluminum hydride~ aluminum
hydriae, diborane, di~isoamylborane, and 9-BBN (i.e. 9-
borabicyclo~3.3.1]nonane).
In general, diborane is preferably used as the amide-
. 15 reducing agent except when the starting compound posse~s- - es a double bond, which compounds are conve~ien~l~ re-
: .
duced by means of lithium aluminum hydride.
The preparation of the l-N-acyl intermediates is achieved
by reacting an aminoglycoside which may have amino pro-
tecting groups at any position other than position l; with
an acylating agent selected from an acid of the formula
O
HO - C - Y'
with Y' being a group as defined for Y above, wherein
any amino group present may be ~rotected~ in the pre- -
: . - 47
~ .~
o~
sence of a carbodiimide, such as dicyclohexylcarbodiimide,
and a reactive derivative of above said acid.
Amino protecting groups useful in this process must be
-' - removeable under conditions which will not a~fect the
l-N-acyl group, preferred protecting groups being tri-
' fluoroacetyl, t-butovycarbonyl and benzyloxycarbonyl.
The starting compounds of this process may have free
amino groups or protected amino,groups. .I~ a~ino groups
are pro~ected in starting c~mpounds having a 5'-CH2~l2
.lO gxoup, it is usually the 6'-amino group being protected,
`I
Derivatives of gentamicin Cl may be protected at posit:~
. , ions 2' and 3. The starting compounds may be used as
free nitrogen base (with or withou~ ~-protecting
~roups) or, as a compound which is partially neutralised
: 15 by ~ormation o~ an acid addition salt.
C ~As used herein, the term "partially neutralized by-
formation of an acid addition salt" means that each mole
of 4,6-di-(aminoglyc~syl)-1~3.-diaminocyclitol has ass~- -
ciated ~herewith less than the stoichi~metric number of
.
moles of acid that is re~uired to form the per acid
addition salt. Furthermore, this term means that each
mole of ~,6-di (aminoglycosyl)-1,3-diaminocyclitol has
~- at least one mole o~ acid associated therewith.
. - 48 -
~48~Z~I
For example, one equivalen~ ~f 5-epi-gentamicin Cl
having ~ive amino grou~s would re~uire five equivalents
o acid to ~orm the per acid addition salt.- This pro-
cess îs effected on an acid addition salt of S-epi-
~- 5 gentamicin Cl having less than five equivalents and at
leas~ one equivalent of acid ~e.g.4.5, 4.0, 3.5, 30o,
2~5, 2~0, 1.5 or 1.0 equivalents o~ acid)~
In the preferred feature of this process the starting
compound is neutralized with (n-l) equivalents o~ acid,
n being the num~sr of amino groups in -the molecule~
Thus, (n-l) amino groups are neutralized by formation
of an acid addition salt. It will, however, be under-
stood that ~he process also can advantac;eously be
carried out on partially neutralized s~arting compounds9
wherein more or less than ~n-13 equivalents of acid give
rise t~ ormation o the acid addition salt within the
limits given a~ove. In terms or pH- ranges the process
is carried ou~ in a range of from 5.0 to ~0, pre~erabl~
,
~rom 5.0 to 8Ø The most preferred range of the re-
action medium is a pH of from 605 to 7~5, especially
6.8 to 7.2,
The term "acid addition salt" embraces such salts as
may be formed between the basic antibiotic and an acid
withou~ regard to whether the acid may ~e termed in-
organic or organic. Exemplary of acids embraced by the
term are sulfuric, hydrochloric, phosphoric, nitric,
trifluoroacetic or the like~
If i~ is desired to use as a starting material an acid
addition salt, wherein (n-l) amino ~xoups are protonated,
this compound is advantageously produced _n situ thereby
reacting a "per" acid addition salt with an equivalent
~ strong base, ~.g. trie~hylamine.
In ~eneral, the use of reacti~e derivatives o~.the acid
. HO-C-Y' as ac~lating agents is pre~erred. Reacti~e deri-
.
vatives of the acid comprise esters, azides, imida~ole
derivatives or anhydrides. In those instances wherein
~10 Y' is unsubs~ituted, one of the pre~erred reacti~e deri-
vatives i5 the anhydride o~ the requisite acid. In
~ther instances it may be prefera.ble to use the N- -
hydroxy-succinimidyl ester of ~he~acid.
,
-
~When carrying ou~ ths process where~y a reactive derivi-
~5 tive o an a~id containing an amino ~unction is used,
: it is pre~erable to protect the amino function pxior to
.~arrying o~t the-process and then removing the ~-~xo-
tecting group in the compound there~y formed. It may
also be advantayeous to pro~ect a hydroxy group presen~
~0 . in the acylating agent, howa~er, it is not generally
necessary.
'. - ' '
~he following Examples illustrate the invention.
- 50 -
.
. - `
. ..~ .
1~4~30Z0
~
P~R-N~BE~ZYLO,XY~ARBO~Y~A~T~OGLYC~SIDE-S
A. 1,3,2',6'.3,"~Pen,ta-N-benzyloxycarbon~qentamici ~ a-
Dissolve 40 gms o~ gentamicin Cla in 200 ml o~ methanol
and 20 ml. of saturated sodium bicarbo~ate and cool t~e
solution to 0C. While stirring the solut~on, add dro~-
wise over a period o~ 2 hours, 88 ml o~ carbobenzyloxy
chloride kee~ing the reaction t~peratur~ between 0C to .
. ~ 5 &. stir the mixture overnight while allowing the
reactlon temperature to come to roo~ temperature. Add
500 ml o~ chloro~orm to the reaction mi~ure w~ich will
then separate into 2 layers~ Wash ~he organic pha~
with 4 x 100 ml o~ water and c~ry over 100 sms Q~ so~iUm
sulfate- Evaporate the organic phase under vacuum at
a temperature of less than 40 C. Dissol~e ~he resul~ant
crude product in 100 ml chlorofonm and add dropwise to
250 ml of 75% hexaneje~her. ~ er the resulta~ pre- -
cipita~e and wash wi~ 100 ml ~exane and air dry to
ob~ain 87 gms (8P~o) o:E 1,3,:2',6',3'`--Penta-N-benzylQx~-
car~onylgent~micin Cla, melting poin~ = 185-190C,
. [a~D ~ 71.2 (C~30~), In~rared (IR~ tKcl): 3300
- 3500 cm 1, PM~.(CDC13):c~1~2 (C~e), 3.0 (~Me),
7,25 taromatic H~o
B ~. 1, 3 1 2 ' ~6 ' . 3 " -P_ta -N -benzyloxyc~rbonyl s i soll~i cin
Dissolve 25 gm~ of sisomis::in and 13 gms o:f sodiu~ car--
bonate in 6Z5 ml of water~ ~ile stirring the solution
a~d 100 ml o~ carbobenzyloxy chloride at 25 C~ Stir
- 51 - -
~ .
~ .
~4~ Z~
the mixture for 16 hours and then ~ilter of~ the solid,
washing thoroughly wi~h wa~er. Dry the solid in vacuo
and then wash with hexane and air dry to obtain 62 gms
0~ 1 J 3,2',6' 9 3 11 -pent~ benzy10xycarbony1sisomicin.
. Melti~ point = 165~173 C; ~a[D ~ ~602 (CH30H?, In-.
. ~raxed (IR) = ~r max (C~C13), 3600, 1720~ 1515, 1215,
1050, 695 cm l; PMR S(CDC13) 1.03 (3H, broad sing-
1et, 4"-C~C~3),. 3.02 (3H, broad singletr 3"~ C~3),
5002 (10~, br~a~ ~ingl~-CH2C6~5), 3~2~, 3.30 pp~.
. (25~, broad singlets~ -C~2C6~5).
,
- E ~ ~PL~ 2
-
- PE~--BE~ZYLOXYCARBONXI.--3" ,4~ o~-cARBo~ oGLycosIl:)Es
Ao 1~3~2',6,'.-~etra-~-benzvLoxvcarbon~1-3",4"-~,O~ar-l
- bonylqentamicin c~a_ . - -
~o a stirred mixture of 6~ m~ sodium h~dride in 5 ml o~
dry d~methy1~ormamide aad a so1ution of-2 gms o~ the pr~
uct o Example 1~ in 5~ ml o~ dry dimethylormamlde over
1/2 hour at room temperature under nitro~en. Stix the
reaction mixture fox Z hours and then filter off insolu-
bles. To the ~iltrate add 100 ml of chloroform and wash
the organic phase with 3 x 50 ml of water. Dry the
organi~ phase ove~ 25 gm sodium sulfate and then evaporate
under reduced pressure. Dissolve the resultant residue
: in 15 ml o chloroform and add dropwise to ?5~ hexane~
ether (lS ml). Filter the precipitate and wash with
25 ml he~ane to obtain 1.82 sm (~ 95~') o~ 1,3,2',6'-
. tetra~N-henzyloxycarb~nyl-3~ N,o-carbonylsentamicin Cla D
52 -
10480Z0
. Melting point = 215C (dec.) [a]D ~ 6304 I~frared (IR)-~ '
(K~l) = 3300, 3500, 1680~ 1545, P~ (CDCI3) dr1.28
(C~Me), 2.58 (~Me), 7r25 (aromatic H).
B. 1,3.? ' ~ ~ JTetra~N-Benz~loxvcarbon~tl-3"~4"-~,0-Car-,
S b ~ .
To a stirred solution o 5 gms o~ the product o ~xample
1~ in 50 ml. o dImethylforMamide add 250 mg o~ sodium
hydrideO Stir ~he reaction mixture under argon ~or 2
hours at room temperature. Filter and add 2 ml glacial
acetic acid to the filtrate. Concentrate the ~iltrate ~
in vacu~ and ex~ract the residue with 200 ml of chlorofoxm
- (purified by p ssage through basic alumina). Wash the
-chloxoorm extracts with water and dry over sodium sul~ate
~ a~d evapora~e to o~tain 3.5 gms of 1,3,2'~6'-tetra-~-
15 . benzylox~carbony} 3"~4"~,0~~arbonylsiso~icin. Melting
:~ p~int - 210~213 C; ~D ~ 6808 (c 0022) In~rared ~IR)
max (~ujol) 3550, 1760, 1580 cm 1 p~ ~(C~Cl~)
1.34 t3~, singlet-4"-C~3), 2.68 (3~, sLn~le~-3"-~-CH3),
: 5004 (8H, broad singlet-C~zC6~5). :
E~oe~ E 3
PER-M-BE~ZYI OXYCARBONYI -2 " -O -HYDROC~BO~CARBO~ 3 ", 4" - ;
N,O{~ARB0 ~yhaklT~7oGL~rcosII)ES
A" 1,3 ~2 ',6 ' -Tetra~-benz~x~arbon~1~2" {)-Benzoyl-~
3" ,~" -N,0-carbonyl~amic~in C~ a_
To a s~irred solution of 10 gm of 1,3,2',6'-tetra-~-
ben~yloxycarbo~yl-3" ,4" -N~0-carbonylgentamicin Cla in
5 3 -- -
~ .
1~480Z0
50 ml o~ dry pyridine add dropwise over a period o~ 10-15
minutes under an a~mosphere of nitrogen~ 2 ml benzoyl
chlorideO Stir the reaction mixture ~or 1/2 hour and
then remove the pyridine via a xotary evaporator keeping
the bath temperature at less than 30C~ Dissolve the
residual pale yellow oil.in 100 ml of chloro~orm~
Wash ~his organic phase with 3 x 50 ml wa~e~ and ~hen
dry ov~r 25 gm sodium sul~ate. Evaporate the chloro-
form under vacuum9 ~riturate the residual yellow foam
with a small volume o~ ethes to obtain lloO gms (~ 95%)
1,3,~l~6'-tetra~-benzyloxycarbonyl-2"-O~ben~oyl-3",4"-
. ~,0-carbonylgentamicin Cla~ Meltin~ point a l 20-123C
- 1]D ~ 73.8
. B. 1~3,2'~,6'JTetra-~-B nzYlo.~vcar~on~a-2"-O-Benzoyl-
- ~o a stirred solution o~ 3 gms of the p~o~uct o Ex2mple
2~ in 20 ml o~ dr~ pyridine a~ 25 C under an atmosphere
o~ a~gon add 1.7 ml of benzoylchloride over a 10 minute :
period~ Stir at room temperature un~i all ~he startin~
material r~acts (monitor by thin layer chromatography),
Evapora~e the ~ixture at room temperatur~ under ~igh
vacuum; extract the solid residue with 100 ~1 chloro~orm
tpreviously passed through ~asic aluminaj. Wash the
chloroform ex*racts with 5% aqueous sodium bicarbonate,
water and then dry over sodium sulfate. Evapo~ate the
solvent to obtain 2.8 gms 1,3',2',6l-~etra~ enzyloxy-
carbonyl-2"-0-~enzoyl-3",4"~,0~~arbQnylsisomicin~
54 -
,- .~
. . ., . . l
,1
104~3020
melting point 157-160 C, ta~D ~86,~c 0.2) In~ared (IR)
~max (nujol) 3325, 1780, 1680, 1560 cm PMR ~ (CDCl~)
1-35 (4"-C-CH3), 2.i4 (3"-~-C~3), 5.03 (CH2-C6H5)~
.
C. 1,3~2'~6'_tetra-H~-benzyloxycarbonvl-2"~0-acetyl-3",~-
2=~c3b9~ol9u~ ~a
(1) 1~3~2~',6'/3"-penta-~-benzyloxYcarbonY1-2"-o-,
a~etyl~ntamicin C~a
To a stirred solution of 10 gms. of 1,3,2',6',3"-penta~
benzyloxycarbonylgentamicin Gla in 50 ml. o~ dry pyridin0,
add dropwise over a period of 10-15 minutes ùnder an at-
~- mosphere o~ nitrogen, 1.4 ml. of acetic anhydride.~ Stir
the reaction mixture for 1/2 hour and then remove the py-
- ridine via a rotary evaporator, keep bath temperature at
less than 30 C. Dissolve the resultan~ residue in 100 ml.
o~ acid-~ree chloroform. Wash this organic solution with
3 x 50 ml. water, *hen ary over sodium sul~ate and evapor-
ate in vacuo. Puri~y the resultant residue by tritur-
ation with small volumes of ether to obtain 1,3,2',6',3"-
penta-~-benzyloxycarbonyl-2"-0-acetylgentamicin Cla.
(2) 1~3~2~6'-tetra-N-benzYloxycarbonyl-2
acetyl-3",4"-~0-carbonyl~entamicin Cl~
In a manner similar to that described in Example 2A treat
1,3,2',6',3"-penta-~-benzyloxycarbonyl-2"-0-acetylgenta-
micin Cla in dry dimethylformamide with sodium hydride.
Isolate and purify the resultant product in a manner simi-
lar to that described in Example 2A to obtain 1,3,2',6l-
- 55 -
,i .:
. ~ , .
~ ` ~
~4S(~Z6~
tetra-N-benzyloxycarbonyl-2"-0-aCetyl-3",4"-N,O-carbony1-.
gentamicin Cla.
D. 1,3.2i~6'-tetra-N-benzYloxys~arbonvl-2~l-o-acetyl-3ll~4
LIl~L:.be3Y~ ~ n
(1? In a manne~ similar. to that described in Exam-
p~e 3C(l) treat 1,3,2',6',3"-penta-~-benzyloxycarbonyl-
sisomicin with ac~tic anhydride in pyridine. Isolate and
purify the resultant product in a manner similar to that
described in Example 3C(l) to obtain 1,3,2',6'-tetra-N--
benzyloxycarbonyl-2"-0-acetylsisomicin.
.
(2) In a manner similar to that descri~cd in
1~ ` .
Example 2B treat 1,3,2',6',3"-penta-N-ben~yloxycarbonyl-
2"-0-acetylsisomicin with sodium hydride in dimethyl-
formamide~ Isolate and puri~y the product in a manner
similar to that described in Example 2B to obtai~ 1,3,~
6'-te~ra-N-benzyloxycarbonyl-2'!~0-acety1-3",4"-~,Q-carbonyl-
sisomicin.
ExamPle 4
PER-~-BE~ZYLoXYCARBONYL-2"-0-HYDROCARBONCA~BONYL-
5-0-HYDROCARBONSULFO~YL-3",4"-N,O-CARBO~YIAMI~o-
GLYCOSIDES
A 1,3,2' 6'~tetra-N-benzvloxYcarbonYl-5-O-Methanesul-
,. , . . . _
fonyl-2'1-O-benzov1-3"~4~'~ ~ carbonYlqentamicin Cla
Cool a solution o~ 1 gm. of 1,3,2',6'-tetra-~-benzyloxy-
carbonyl-2"-0-benzoyl-3",4"-~0-carbonylgentamicin Cla in
- 56 -
1~48~2~
5 ml~ o~.triethylamine and 15 ml. tetrahydro~uran to below
0C. Stir ~he solution and to it add, over a period o~ lS
minutes, a solution o~. 1 ml. o~ methanesulfonyl chloride-
in 5 ml.. o:E tetrahydrofur~an. Stir t~e reaction mixture
for 2 hou~s at oC. Pour the reaction mixture into 25 m~
o~ water and 2S ml o~ chloroform. Wash the organic phase
with 2 x 15 ml o~ water and then dry the organic pha~e ove~.
sodium sulfateLi~ Evaporate.the chloro~orm and trituxate ~ :
the resulting yellow ~oam with small amounts of e~ er to .
obkain 1.~ gm (~ 95%) of the 1~3,2',6'-tetr.a-~-benzyloxy- :
carbonyl-5-0-me~hanesulfonyl-2"-0-benza~1-3",4"-N;O-carbonyl-
gentamicin Cl Melting point = 130C LaJ26 ~ 53 4 (CHCl )
PMR (C~Cl~ ~ L35 tC-Me), 2.74 (~-Me), 2.99 (OS02CE3), ~.28
~4xCbz and benzoyl).
.
In similar manner ~reat 1,3,2',6'-tetr~-N~benzyloxycarbonyl-
.2"-_-ace~y1-3"/4"- O-carbon~lgentamicin Cla in triet~yl-
amine and tetrahydrouran wit~ methanesulfonylchloride to
obtain 1,3,2',6'-tetra-~.benzyloxycarbonyl-5-0-methane-
sulfonyl-2"-0-a~etylr3" ,4"-~l,O-c2rbonylgentamicin ~là' ~ ~:
. .
- 57 -
R`~
., ~
,21~
B. ~
sulfonyl~2"_0-Benzoyl-3_~4"=N,O-CarbonYlsisomicin
Dissolve 2.5 ~m5 . of the product of Example 3C in 15 ml.
of dry pyridine. Cool the solution to 10C and add 4 ml
of methanesul~onyl chloride over a period of 10 minutes,
allow the reaction mixture to stand overnight, then con-
- centrate the reaction mixture undcr vacuum at 25C. Ex-
tract th~ resi & e with 150 ml. of acid-~re~ chloroform. .
Wash the chlor~form extract with water and dry over sodium
sulfate. Evaporate the chloroform to give 2.4 gm. o
1,3,2',6'-tetra-N-benzyloxycarbonyl-5-0-methanesul~onyl-
2"-0-benzoyl-3",4"-~,0-carbonylsisomicin; melting point
84-88 C, LJD + 21.3 (C 0.29) Infrared (IR)-~ max
--1 . . .
(nujol) 3325, 1750, 1540 cm ; P ~ ~(CDC13) 1.32 (4"-C-
CH3), 2.68 (3"-M-CH3)~ 3.04 (5.0-~ ) 5.00( -C~2C6~5).
.~ , . . .
.
'
: Exam~la ~
~ . 5-EPI-AZIDO-5-DEOXY-2"-0-BENZOYL-PER-N-
- . _ ._
BE~ZYhOXYCARBO~LAM ~OGLYCOSIDES
~- A. 1,3,2',6'-tetra-~-benzYloxYcarbonyl-s-epi-azido-5-
deoxv-2"-0-benzoYl-3",4"-N,O-carbonyl~entamicin Cla
Heat 12 gm. 1, 3, 2 ', 6 ' -tetra-~-benzyloxycar~onyl-5-o-
methanesulfonyl-2"-0-benzoyl-3",4"-N,O-carbonylgentamicin
Cla and 2 gm. sodium azide in 30 ml. of dimethylformamide
together at 120 C for 24 hours. Cool the reaction mixture
and remove the sol~ent in vacuo at 60 C~ Dissolve the
- 58 -
~0~L80Z0
residue in 50 ml. water and 100 ml. chloro~orm. Wash the
organic phase with 2 x 50 ml. water and dry over 25 gm. of
sodium sulfate. Evaporate the solvent to give a white so-
lido Dissolve the solid in a small volume of chloroform
and chromatograph over 200 gm. of silica gel. Elute the
column with CHC13/3% MeOH to give 6 gm. o~ the resultant
1,3,2',6'~tetra-N~benzylOxycarbonyl-5-epi-aZidO-5-deoxy-
2"-0-benzoyl-3",4"-~ carbonylgentamicin Cla. Melting
POint = 195-200C 1]D6 + 88.9 (CHC13) 0
- 10 B. 1,3~2', 6 ~ -Tetra-N-BenZY1OXYCarbOnV1--5-EPi-AZ1dQ-5
Deoxv-2"-O-Benzovl 3',~ O-Carbony~Lsisomicin
! 1) DLssolve 2 gm. of ~he pxoduct of Example4B-in lS ml.
of dry dimethylformamide.. Stir the mixture and add 1.5 gm~
of sodium azide. Keep the reacticn mixture under argon at
.15 120 C overnight. Concentrato the solution under high
: vacuum. Extract ~he residue with 200 ml. of acid-free
chloroform. Wash the chloroform extract. wi~h water and
dry over sodiu~ sulfate. Evaporate the solvent to give
1,3,2',6'-tetra-~-benzyloxycarbonyl-5~epi-azido-5-deoxy-2'`-
0-benzoyl-3",4"-~,0-carbonylsisomicin. Infrared (nujol)
~f max 2100 cm 1
2) In a similar manner subject to ~he foregoing process
an equivalent quantity of a corresponding product to ob~
tain 1,3,2',6'-tetra-~-benzyloxycarbonyl-5-epi-azido-5
~5 deoxy-2"-0-benzoyl-3",4"-~,0-carbonylverdamicin.
r~
~IL048(~
Eæam~le
5-EPI AMI~0-5-DEo ~ I~OGLYCOSIDES
A. 5-epi-amin~-5-deoxvqentamicin C
la .
Hydrogenate over 1 gmO. 3~0 palladium~ca~.bon at 4 atmos--
pheres at room temperature a solution of 6 gm. o~ 1,3,2',-
6'-tetra-~-ben~ylox~arbonyl-5-epi-azido-5-deoxy-2"-0-
. benzoyl-3",4"-~,0-carbonylgentamicin Cla in 50 ml. of
; acetic acid. Remove the solvent and cat~lyst tto obtain
.. . .
a gummy residue) then hea$ the resultant residue i~ 25 ml.
! . . .
2N sodium hydroxide at 100 C for ~ hours. Cool the mixture
and neutr~liza with acetic acid. Filter off the resulting-
..
- . precipitate and concentrate the filtr~te to 10 ml. Pass
the concentrated filtrate through a column oE IRC-50 (H
form) resin. Wash the co~umn wi~h 200.ml. o~ water and
then elute the column with 100 ml. o lN ammonium hydrox- -
. ide. Concentrate ~he eluate to dr~ne~s and lyophilize the
`- re~idue to obtain 1 gm. 5-epi-amino-5-deoxygentamicin Cla.
Melting point 112-116 C [~D ~ 167.0 (H20), PMR (100 M~z~- :
D20 )
-' ' .
- 60 ~
.
~ '
~[)4802~ I
1.21 . (3H,S,C-CH3)
2.00 ~IH, dt, H-2e~)
2.50 ~ (3H, S, ~-CH3)
2.61 (lH,d, J-10 Hz,H-3")
3.39 ~ ~lH, d,~=12 Hz,~-5"ax).
3.8} (lH,q,H-2")
3.82 - (lH,d,J=12 Hz,H-5"eq3
4.94 (lH,d,J=3Hz,H-l')
5.06 : . ~ (IH,d,J=3~5 Hz,H-l")
. ~
B. In a similar.manner, proceed according to the pro- -
cess described in Example 6A and isolata the resultant
. product~ to obtain respectively, 5-epi-amino-5-deoxy- -
gentamic.in Cl, mOp. 95- 98C, taJD6 ~ 150.7 ~ 0.64,H20),
5-epi-amino-5-deo~ygentamiqin C2,
~15 S-epi-amino-5-deoxyge~tamicin C2a,
5-epi-amino-5-deoxygentamicin C2b, . ~ -
-ethyl-5-epi-amino-S-d oxygentamicin C
~ ethyl-5-epi-amino-5-deoxygentamici~ C~
l~N-ethyl~5-epi-amino-5-deoxygentamicin C~, - .
20 1-E-ethyl-5-epi-amino-5-deoxygenta~icin C2a and
l-N-e~hyl-5-epi-amino-5-deoxygentamicin C2b,
:
C, 5-Epi-Amino-5-Deoxvsisomicin
1) Dissolve the product of Example ~5B~l in a mixture
of 10 ml. o~ tetrahydrofuran and 50 ml~ of liquid amm~nia.
Slowly add 2 gm. of sodium to the stirred mixture and
continue to stir at -40 C for 2 hours~ Allow ~he ammonia
- - 61 -
~8alZi~
to evaporate at room temperature overnight. Dissolve the
re.qultant residue in 25 ml~ of water and heat to 100C
overnight. Cool the solution and adsorb on Amberlite
rRC-50 (H ) ~e~in and elute the product with 500 ml. o~
LN ammonium hydroxide. Concentrate the ammonium hydroxiae
eluate under high vacuum to give-an oily product. Chroma~
togr~ph this material on 50 gm. of silica gel using chloxo-
form/methanol/15% ammonium hydroxide (2~1:1? to give 102 mgO
o~ 5-epi-amino-5-deoxysisomicin; melting point = 110-116 C,
lo ~]D6 ~ 185.2 (c 0.32)~
,
2) In a similar manner proceed according ~o the fore-
~ going process and obtain respectively,
::; 5-epi-amino-5-deoxy-Antibiotic G-52,
.~5-epi-amino-S-deoxy-Antibiotic 66-40D,
; 15 5-epi-amino-5-deoxyverdamicin,
5-epi-amino-S-deoxy-~ntibiotic 66-40~, .
l-~-ethyl-5-epi-amino-5.-deoxy-antibioti~ G-52,
l-~-ethyl-S-epi-amino-5.-deoxy~Antibiotic..66-40D, :
~ ethyl-5-epi-amino-5-deoxysisomicin,
1-E-ethyl-5-e~i-amino-5-deoxyverdamicîn,
l-~-ethyl-5-epi-amino-5-deoxy-Antibiotic 66-40B,
l-~-propyl-5-epi-a~ino-5-deoxysi omicin,
1-~-(~-butyl)-5-epi-amino-5-deoxysisQmicin,
l-~-(~-aminobutyl)-5-epi-amino-5-deoxysisom-cin,
1-~ -aminopropyl)-5-epi-amino-5-deoxysisomicin,
l-N-(~-methylpropyl)-5-epi-amino-5-deoxysisomicin,
-pentyl)-5-epi-amino-5-deoxysisomicin,
-- 62 --
~ .....
3L048QZ~D
l-N~ meth~lbutyl)-5-epi-amino-5-deO~y:3isomicinf
l-N-(~-methylbutyl)-5-epi-amino-5-deoxysisomicin,
l-N~ -dime~hylpropyl)-5-epi-aminO-5-deOXy3isomicin,
l-N (~-ethylbutyl)-5-epi-amino-5-deoxysisomicin,
l-N- ~-oc~yl)-5 epi-amino 5-deoxyQisomicin,
l-N-(~-aminoethyl)-5-epi-amino-5-deoxysisomicin.
.
EXamPle~;~
5-EPI-AZID0-5-DEOXY~MINOG~YCOSIDES
A. 5-EPi-Azido-5-DeoxY~entamicin C
la
Re~lux a solution o~ 1 gm. 1,3,2',6'-tetra-N-benzyloxy-
f carbonyl-5-epi-a~ido-5-deoxy-2"-0-benzoyl-3",~'~N,O-carbonyl-
gentamicin Cla in 25 ml. 1:1 dioxane/water and 25 ml. 1~/~
sodium hydroxide for 24 hours. Evaporate the solution to
dryness, dissolve ~he residue in 10 ml. wa-ter and neutra-
lize with acetic acid. Evaporate thQ solution, take up
the re~idue in 5 ml; water and pas~ throug~ 20 gm. o~ an
Amberlite IRC-50 (H form) resin column, wash the column
with 200 ml. water and then with 100 ml~ mmonium hy-
droxide. Collect the ammonium hydroxide eluate and evapor-- -
ate to a residue. Freeze dry the resiaua, (to produce a
pale brown solid) then chromatograph on a ~S, gm
.
silica gel column, eluting with chloro~orm:methanol-7%
ammonium hydroxide (2:1:1) to obtain 186.4 mg. of 5-epi-
azido-5-deoxy-gentamicin Cla. Melting point = 115-121C
[ID ~ 133.9.
- 63 -
~ '
i~048~
B. In a similax manner, proceed according to the proc-
ess de~cribed in Example 7A and isolate the resultant pro-
ducts to obtain respectively,
5-epi-azido-5-deoxygen~amicin Cl, m.p. 95-98C,[a]
129.5 ~ 0.46, N20),
5-epi-azido-5-deoxygentamicin C2,
S-epi-azido-5-deoxyg~ntamicin C2a,
5-epi-azido-5-deo~yg~ntamicin C2b,
.... . ..
5-epi-azido-5-deoxywAntibiotic G-52,
5-epi-azi.do-5-deoxy-Antibiotic 66-40D,
eth~1-5-epi-azido-5-deoxygentamicin Cla,
l-l~-ethyl-5-epi~azido-5-deoxygentamicin C
-ethyl-5-api-azido-5-deoxyg~ntamicin C2,
:- . l-~-ethyl-5-epi-azido-5-deoxyg~ntamicin C2~,
: .. . .
~ 15 - 1-~-e~hyl-5-epi-azido-5-deoxygentamicin C2b, ~ .
.
: l-N-ethyl-5-~pi-azido-5-deoxy-Antibiotic G-52 and
~ ethyl-5-epi-azido-5-deoxy-Antibiotic 66~40~.
._~ C... 5-Epi-Azido,-5-DaoxYsisomicin '
In a similar manner proceed according to the process de-
scribed in Example 7A and isoiate each o~ the resultant
products to obtain 5-epi-azido-5-deoxysisomicin. M.P. -
165-170 C (dec.) Mass Spectrum (M) m/e 472; Monosaccha--
- - rides m/e 160, 127; 2-Deoxystreptamines m/e 2L6, 198rl88
170; Disaccharides m/e 342, 314, 296 m/e 347, 375,329
- 64 -
.~ '' ?.
.~3 .
48~
CMR (D20): ~
PPM: 149.2, 102.4,98.0, 97.2, 84.8, 7~.7,73.1,69.9,68.6,
63.9 (2c),48.g,47.7, 47.1,42.9, 37.7,36~2,25.8,~2.4
: 5-epi-azido-S-deoxyverdamicin,
. 1-~ethyl-5-epi-~zido-5-deoxysisomicin,
l-~-ethyl-5-epi-azido-5-deoxy~erdamicin,
l-~-propyl-S-epi-azido-5~deoxysisomicin,
-butyl)-5-epi-azido-5-deoxysisomicin,
aminobutyl)-5-epi-aæido-5-deoxyQisomicinf
1-~ aminopropyl)-5-epi-azido-5-deox~sisomicin,
methylpropyl)-5-epi-azido-5-deoxy~isomicin,
~ g-pentyl)-5-epi-azido-S-deoxysisomicin,
l-~-(~-methylbutyl)-5-epi-azido-5-daoxysisomicin,
j..... .
l-N-(~-methylbutyl)-5-epi-aziclo-5-deoxysisomicin,
1~ -dimethylpropyl~-5-epi-azido-5-aeoxy~isomicin, .
.
: l-E~ ethylbutyl)-5-epi-azido-5-deoxysisomicin,
-octyl)-5-epi-azido-5-d~oxy i~omicin,
l-N~ aminoethyl)-5-epi-azido-5-deoxysisomicin.
.
- 65 -
~ ,,,
10~8~
. Exam~le,~
5-EPIGE~TAMICI~ Cl
A. Add 2 gms. of 1,3,2',6'-tetra~ enzylo~carbonyl-
5-0-methane~ulonyl-2~-0-benzoyl-3!~4~'-N,0-carbonylgenta-
micin CLJto 15 ml. of dimethylformamide, heat at re1ux.
temperature for 18 hours then evaporate the solution to a
re~idue comprising an E-protected-0-protec~ed interm~diate,
Bo Dissolve this re~idue in acetic acid, add 500 mg.
o 3~0 palladium-on-charcoal and hydrogenate at.room tem-
perature u ing.4 atmosphe~es starting hydrogen pres~u~.
Remove the catalyst by filtration.and evaporate the filt~-
: ate to a residue. Dissolve the residue in ~5 ml; o~ 5%
. sodium hydroxide and he~t at 100 C for 4 hours. Cool ~he
solu-tion and pass through an IRC-50 (H form) column, wash
the resin columr.. well with water, then elute the product
with 200 ml. o~ 1 N ammonium hydroxide. Concentrate the
ammonium hydroxide eluate to a re~idue comprising 5-epi-
g~ntamicin Cl. Purify the product by chromatographing
on a silica gel column.elu~ing with the lower pha~e o~ a
chloroform:me~hanol:l5% ammonium hydroxide (2:1:1) solven~
system. Combine the like eluates as determined by thin
layer chromatography and lyophilize to a xesidue -to ob-
tain 5-epigentamicin Cl as a white solid; m.p. 115-120C~
~a~D ~ 136.5 ~0.32 water); Mass Spectrum: (M) m/e 477,
(~ ~ m/e 478:;
Monosaccharides m/e 157 - purpurosamine A ion
m~e 160, 142 -garosamine ion
- 66 -
~)4~
m/e 191,173,163,145-5-epi-2-
deoxys~reptamine ion.
Disaccharides 350, 322, 304,
347, 319, 301
~ d~ (100 ~Z, D20)
5.08, d, J=3.8Hzl
. ~ H'-l' and ~-1"
4.9g, d, J-3 HzJ
4.39, broad singlet E-5
3.93 d, ~=12~5Hz H-5" eq
3.77, dd, ~=ll,J=~ 3.6Hz H-2"
3.30, d, J=12.5Hz H-S" ax
2.66 d, ~=10.5 Hz H-3"
2.53, singlet ~-CH3(3")
2.33, singlet ~-CH3(6')
- 2.05, m . H-2 eq :
1.23, s C-CH3 ~4")
1.04, d, J-7Hz C~-CH3~6')
C. : Alternatively, the ~pro~ecting and 0-protecting
group~ in ~he in~ermediate~ prepared in Example gi are re
moved by heating the intermediate with 1 to 2 ~ sodium
hydroxide at 100 C until thin layer chromatographic analy-
sis of aliquotg!-of the reaction mixture indicate the pro-
tecting groups have been removed (usua~ly 24 to 48 hours~.
Isolate and purify the resultant product in a manner simi-
lax to that described in Example 8-B.
.
~,
.. ~ .
~.,~p ..... , .
J
~o~o~o
D. Alternatively, the ~-protecting and O-protecting
groups may be removed ~rom the intermediate prepared as
de cxibed in Example 8-A~ in the ~ollowing manner. Dissolve
the product of E~ample-8Ain a mixture of 10 ml. of tetra-
hydrofuran and 50 ml. of liquid ammonia. Slowly add 2 gm~.
of sodium to the stirred mixtur~e and continue to ~tir for
2 hours. Allow the ammonia to evaporate by warming to
room temperatur~ overnight. Dis~olve the resultant resi-
due in 10 ml. of 5% sodium hydroxide and heat at 100C for
4 hours. Cool and pass the solution through IR~-50 (E )
resin. Wash the resin well with water and elute the
product with 100 ml. of 1 ~ ammonium hydroxide. Concentr-
ate-the ammonium hydroxide eluate to a residue and purify
this residue in a manner similar to that described in
Example :8Bto obtain 5-epigentamicin Cl.
' ' . -~ ' ' -:
OT~ER 5-EPI-4,6-DI-o- 1AMI~OGLYCOSY~) -
2-DEoXYSTR~PTAMI~ES
A, 1 In a manner similar to that described in Exampley
8A and 8s treat each o~ the ~ollowing aminoglycoside deri-
vatives with dimethylformamide at reflux temperature,
thence h~drogenate each of the resulting 0-protected-~-
protected intermediates ~hereby formed in acetic acid in
the presence of palladium on-charcoal, and finally treat
each of the resulting 2"-0-benzoyl-3",4"-~,0-carbonyl-5-
epiaminoglycosides with sodium hydroxide at 100C:
o~~ - 68 -
~ ~. .
:~4~0ZO
1) 1,3,2',6'-tetr~-N-benz~loxycarbonyl-5-0-methane-
- sul~onyl-2"-0-benzoyl-3",4"-~,0-~arbonylgentamicin
Cla '
2) 1,3,2',6'-tetra-~-benzyloxycarbonyl-5-O-methane-
~5 sulfonyl-2U.-O-benzoyl-3~4~ ,O-carbonylgentamicin
C2,
3) 1,3,2',6'-tetra-~-benzyloxycarbonyl-5-0-methane-
sulfonyl-2"-0-benzoyl-3'i,4'!-N-O-carbonylgentamicin
~2a'
4) 1,3,2',6'-tetra-N-ben2yloxycarbon~1-5-O-methane- :
sulfonyl~2"-0-benzoyl-3",4'`-~,0-carbonylgentamicin- .
C2b ' ' .,
Isolate and-purify each of the resulting products in a
manner similar to that described in Example~g to obtain,
respectively, 5-epigentamicin Cla,~-epigentamicin C2, 5-
epigentamicin C2a, and 5-epigent~micin ~2b.
2. Alternativel~,after treatment of each o~ ~e star~-
ing material~ of Example 9A. with dimethylformamide at
re~lux temperature the protecting groups in each o the
intermediates thereby formed may be removed b~ treatment
with sodium hydroxide according to th~ procedure of Example
BC or by reduction with sodium in ammonia followed by
treatment wi.th sodium hydroxide in the manner of Example
8D~
69 =
,' J
B. In a manner similar to that described in ~.xample~;~A
and 8~ treat each of ~he following aminoglycoside deriva-
tive~ wi~h dimethyl~ormamide at re~lux temperature, then
treat ~h~ resulting ~-protected-0-protected intermediatQ
with sodium ln li~uid a.mmonia.followed.by treatment wi~h
sodium hydxoxide at..100C: -
1) 1,3,2',6'-tetra-~-benzyloxycarbonyl-5-0-methane-*
sulfonyl-2"-0-benzoyl-3",4"-N,0-carbonylsisomicin,
2) 1,3,2',6'-~etra-~-benzyloxycarbonyl-5-0-methane-
sulfonyl-2"-0-benzoyl-3",4"-N,0-carbonylveraamicin,
3) 1,3,2',6'-tetra-N~benzyloxycarbonyl-5-0-methane-
sulfonyl-2"-0-benzoyl-3",4"-~,0-carbonyl-Antibio~ic
G-52
4) 1,3,2',6'-tetra-~-benzyloxycarbonyl-5-0-methane-
sulfonyl-2"-0-benzoyl~3",4"-N,0-carbonyl~Antibioti.
66-40D,
5) 1,3,2',5',3"-p~nta-~-benz~loxycarbonyl-5-0-methane~
sulfonyl-2",4"-di-0-benzoyl-Antibiotic 66-40B,
Isolate and puri-fy each of the resultant products in a
manner similar to that described in Example 8B to obtain,
~espectively,
5-episisomicin m.p. 135-138C tdec,)
5-epiverdamicin m.n. =110-113C, [~3 ~ ~ 159.7 (H20)
5-epi-Antibiotic G-52,
5-epi~Antibiotic 66-40D,
5-epi-Antibiotic 66-40B.
~ .
- 70 -
~L~48020
2. Alternatively, ater treatment with dimethyl~ormami-
de at re~lux tempexature the protecting groups in each of
the intermediates thereby formed may be removed by treat-
m~nt with sodium hydroxide in the mann~r o~ Example 8C to
~ o~tain the corresponding 5-epiaminoglyco3id~,
C. In a similar manner obtain
-5-epi-Antibiotic JI-20B, NMR: (D20 ext. ~MS): 5.17 (d.J=4~z,
. -~ 1"-M); 5.13 (d.J=3Hz, l'-H),
2 6? (~-Me); 1.20 ~d. J-Hz,
C "-CH3), 1.20 ~ tC4, CH3)
CMR: (D2U, diox, ref.): 102 (Cl,);
96.4 (Cl"); 85.8 and 80.5 ppm
~: ' '' ~ ) ' '
- Exam ~el~
5-EPISISOMICI~ A~D l-N-ALKY~L-5-EPISIS~Q~
~ ~ A. 5 EPisisomicin
- 1. Add 1.2 gm. o~ 1,3,2',6'-tetra-~-benzylo~ycarbonyl-
5-0-methanesulfonyl-2"-0-benzoyl-3" ,4ll-N~o-carbonylsisomicin
- and 1.0 gm. of tetra-n-butylammonium acetate to 10 ml. of
dimethylformamide. Heat at 120 C for 16 hours, evaporate
: to a residue and extract the residue with chloro~orm. Wa~h
the chloroform solution with water, dry over sodium sulfate,
- then evaporate in vacuo to a residue comprising 1,3,2',6-
tetra-N-benzyloxycarbonyl-5-epi-0-acetyl-2"-0-benzoyl-3",-
4"-N,O-carbonylsisomicin.
.
2 . Dissolve the residue obtained in above Example lQA~l)
in 10 ml. o~ dime-thylsulfoxide Add a solution o~ 2 gm.
`b
- 71 -
~ 480Z~3
o po~a~siu~ hydroxide in 4 ml o~ water and heat at loo&
for 24 hours. Cool the ~eaction mixture, add 80 ml
of A~berlite IRC-50 (~), stir the mixture ~or ~ hour,
then separate the resin, wash with water, then elute
: 5 with 10 ~ ammonium.hydroxideO Concentrate the comb~ned
ammonium hydxoxide-eluates in ~ ana chr~matog~aph
the resultant residu~ over 25 gm o~ silica gel eluting
with ~he low~r phas~:of a 2:1:1 chlorofor~:~e~hanol-:10
ammoniu~ hydro~ide solvent system. Combine the like .
10 eluates containing 5-episisomicin as determined by thin
layer chromatograp~yand evaporate the c~mbined eluates
to a residue of 5-episisomicin. ~.P. 135 - 138 C
(dec.); ~a]D ~ 187,3 (D20); Mass Spectrum ~M)
mfe 447, (M ~ 1) m/e 448~
Monosaçchariaes m/e 160,- 127. -
2-Deoxy-S-epistrèptamines m/e 191,173, 163, 145.
.. .. .
. ~ m/e 317, 289, 271
~/e 350, 322, 304
PMR (Cf ? D20: -
5.1~ . - d~ J=2.5 H2 1'~
5~07 a, J=4.0 Hz li~
4.89 broad single~ 4'-~
4.37 broad singlet 5-~
3~94 d, J~12.5 Hz 5"e~
3.77 q' 2"-E
3O39 d, J=12.0 Hz S"a-H
3.21 ~2~) broad singlet 6'-~
2.65 d, J=ll Hz 3"-H
- 72 -
~8020
2.S2 singlet 3"-N-CH3
l.Z3 singlet . 4"-C-C~3
CMR (D20):
PP~: ~ 150~3, 10~.6, 97.1 (2C), 85.8, 80.~, 73.3,
70.3, 6907 68.5, 64.0, 48.1, 4702, 47.1, 43.2,
37.7, 3604, 25-.6, 2~.40
B. I ~ :
. (1) The xeguisi~e intermediate, i~e. l~N-ethyl-1,3,2',6~-
tetra-~-benzyloxy~arbonyl-5-O~me~hanesul~onyl-2"-0_ :
benzoyl-3",4"iN~0-carbonylsisomicin is prepared by
reactLng l-~-eth~lsis icin according to the procedures
of Examples I to 4.
In a mann~r similar to that de~,cribed in Example ~Q~.
trea~ eth~1-1,3,2",6'-tetra~N~benzyloxy~arbonyl-5~
0-methanesulfonyl-2"-0-benzoyl 3",4"~,Q-car~o~ylsisomi~ln
dimethyl~or~amiae in the presence o~ tetra-n~butylw
ammonium acetate at 120 & for l6 hours. Isolate the
re~lting 5-epi ~ cetate aeri~ative i~ tha manner
described in Example L0~ , then treat this derivative
:~ 20 with agueous potassium hyd~oxide in the ma~ner of Example
lb~(2) followed by purification via chromatographic
techniqueslas described to obtain l~Nethyl-5~episiso-
micin., M.P~ 118-12~ C (dec.); Mass Spectrum (M)+
m/e 475, (M + 1)~ mJe 4760
Monosaccharide~ m~e 160~ 127.
~ 73 -
~04~ ZO
l-N-ethyl-2-deoxy-5-epistreptamines . m/e 219, 201, 191, 173.
D~.sacc!laride~ m/e- 345, 317, 299~
m/e -378, 350, 322.
P~lR (J~) D20: , , ,
5.14 . d, J=2.5 Hz 1'-~
5000 d, ~34.1 Hz l"-H
.9 broad singlet 4'-H
4.38 broad s.inglet 5-H -
3,93 ~, J=12.5 Hz 5"e-H
3078 q. : 2"-~
3.38 ~, J-12.5 Ez 5"~-H
3.21 (lH) broad singlet . 6'-H
2.6s. . d, J=ll.O Hæ 3"-~
2.52 singlet 3"~-CH3
1. 22 singlet ~"-C-CH3
1.07 - triplet 1-~-CH2~CH3
CMR 1D2):
PPM: ~ 149.8, 102.9, 97.4t 9700, 83.9, 80.5, 73.2,
- 70.1, 69.6~ 6~5, ~3.g, 54.5, 47~1, 47.0, 43.1
~0.8~ 37.:5,. 33.0, 25.6, 22.4, 14.6.
.. ..
C. Other l-N-Al~;vl-5-Epi2mino~lycos~ Deriv~tives,
(1) In a manner similar to that aescribed in Example lQ-A
obtain via the corresponding 5-epi-O~acet~l dexivative,
respectively,
- 74 -
~ .
~, - -- .
~1~4~ZO
propyl-5-epi~isamici~,
(n-butyl)-5-episis~micin,
a~inobutyl) 5-apisis~icin,
~ r-amLnopropyl)-5-episisomicin,
1~ ~methylpropyl)-5-episisomici~,
l-N-(n-pentyl)-5~episisomicin,
l-N-(~r-methylbutyl)-5-epi~is~micin,
methylbutyl)-S-episis~micLn,
dimethylpropyl) -5 -episisomicin,
-10 1~ ethylbutyl3-S-episisonicin,
l ~ w(n-octyl)-~5-episiscmicin,
~mLnoethyl)-5-episisomicin,
l-N-ethyl-5-epigentamicin Cla,
N-e~hyl-5-epigentamicin Cl,
1-N-ethyl-5-epigentamicin C2,
l-N-ethy -S-epigentamicin C2a,
. l-~-ethyl-5-epigentamicin C2b,
l-_-eth~l-S-epi-~n~i~iotic G-52,
~ ethyl-5-epiverdamicin~
l~-ethyl 5-epii~ntibiotic 66-4OD,
- 75 -
C
~9L8(~ZU
. . -- .
S~ DIA ~CIN Cla_
~ la~
To a solutio~ of 1.2 g~s of 1,3~2',6'-tetra~benz~loxy-
~arbonyl 2"~0-benzoyl d "~4"~N,~-carbonylgentami~in Cla'
in 5 ml of aceton~ at ~0C add over a 20 minute period
Jones ~eagent prepared from 1 gm of chr~mium trioxi~e
. in l ml of concentrated sul~uric acid and 1 ml o~ water.'
10 , Continue stirring the solution at room temperature ~or
16 hours khen extract with chlorofonm, wash the chloro-
~orm extract with water, dry over sodium sulfate, and
, . evapoxate the solution to a residue (0.619 g) c~mprising
1,3,2',6'~te~ra-~benzyloxycarbonyl~2"-0-benzoyl-3"~4"-
E~0-car~onyl 5-dehydrogentamicin-Cla.
' ~.
B . 1 2 3,2',6'-tetra~N ~anz~loxv~arbon~1-2"-0-benzoyl-
3",4"~ c,a,rbony~ 5-eDi~en amicin ~ a-
Dissolve ~he ~-protected-0-protected-5-aehydro~enta~i~in
,Cla prepared in Example 11~ in 10 ml of metha~ol,
Add 100 mg~ o~ sodium borohydride and warm the mixture
at ~oC or 4 hours. Cool the solution, remove the
solvent ln va~uo and extract the resultant residue with
chloro~oxm. Wash the combined chloro~orm extracts wi~h
water, ~r~ over sodium sulfate and evaporate the solven~
to a residue compri.sing 1,3,2',6'-tetra~N~benzyloxy~ar-
bonyl 2"-0 benzoyl 3",4"-N,0-carban~1-5-epigentamicin Cla.
~"`3 - 76 -
8(~Z0
C. 2"~ benzovl-3" ! 41~ -N,0-car}:lony~_,-,epi~e~tamicin.,,cla_
Di~solve the product obtained in Example li-~3 in 15 ml o:
acetic acid and h~ ogenate over 200 mg o:E 30~0- palladium-~
~n-charcoal at rowa ternperatu:re for 18 hours at 4 atm~
S phere~ starting pressure. Fil~er arld evaporat~ th~2 -
:Eiltrat~ in vacuo to a residues corQpris~g :2"-_-benzoyl--
3" ,4" ~,0-carhonyl -5-~epigentaF~icin Cla.
D. ~Q~SLI~la-- -
Dissolve the product of Example 11~ :Ln 10 ml c-~ 5%
sodium hydroxide and heat at 100 C for 4 hours, cool
and pa~s the solution through IRC-S0 (E ) resin~ wash
- - the resin well with-water, then elute the prodNct wit}~
- 100 ml o:E 1 N a~noniu3~ hydroxicle. Evaporate the
a$u;lonium hydroxide eluate- to a residu~ comprising 5- -
epigen~am;ci~ Cla. Puri~y by chromato~raphing on a
silica gel column eluting with th~ lower pha~3 o;E a
chloroform:methanol:l5% amwoniu~ hydroxide t2~ ) solv~n~
sy~tam. C~m~ine ~he like eluates a~ determined b~ thi~
lay2r chrcma~ograp~yand evaporate the com~ined eluate~ to
a resiaue comprising 5-epigentamicin Cla. M.P. 145-152 & ,
~a]D ~ 149C (c~ 0~55~ ~2~
E. 1,3~2',6',3"~Penta-N-~en2yloxyca~bonyl-2"-o-acet
5-dehy~roq~tamicin C~a--
To a solution of lQ gms of 1,3,2',6',3"-penta-~benzyloxy-
carbonyl-2"-0-acet~lgentamicin Cla in 600 ml of methylene
- 77 -
~ 4 ~ 2
chloride under an abmosphere of argo~ add 11~2 gm~ o~
chromium trîoxide-pyridine complex. Heat the resulting
slurry at re~1ux tempera~ure. Aad 1201 ~ms 0~ addi-
tional ~hromium txioxide-pyridine complex a~ter 22 hours
and 11~2 gm~ of additional chromium trioxide~p~ridine
co~plex a te~ 25 hours. When the.reaction is compl~te~
a~ indicated by ~hi~ layer chrcmatography(usually about
28 hour~) evaporate about 500 ml.of the solvent ~n vacuo
add 600 ml o~ eth~r~`to the resulting solution, decant
the ethereal solution ~rcm the resulting tarry . pre--
cipitate and wash the precipitate with 200 ml o~ et~er.
Wash the ~om~ined ethereal solutions with saturated
I sodium bicar~onate solution-~ t~mes) with 1 ~ h~dro_
~hloric acid (3 times) and then wi.th water (2 times).
: 15 Dry over sodium sulfate and evaporate in vacuo to a
residue (8.2 gma) comprisin~ 1,3,2',6',3"-pentaiW-
benzyloxy~arbon~l-2"-0-acetyl-5-dehyd~ogentamici~ Cla,
Further puri~y by chromatographin~ on a 700 gm silica
gel "dry" column. Develop the column with 60~o ethy
acetate/40~0 chloroform, then elute tha produ~t with
ethyl ace~ate and evaporate the c~mb~ned eluates to
a residue (4.6 gms) comprisi~g the 1,3,2'~6'l3"-penta-~-
benzylox~car~onyl~ acetyl-5-dehydrogentamicin Cla;
NMR: (CDC13-CD30D, (3:1)); dr 2.93 (N-C~3), 1~90 (CH3C0~),
1.0:4 (C-C~3) PP~, CMR: (CDC13-CD30~ (3:1)); 201 PPM
(C=O) ,.
- 78 -
104~3()Z0
In a manne~ simil~r ~o that descxibed hereinabove, ~reat
l~3~2~6~-tetra-N-benz~loxy~arbonyl-2"-o-acetyl-3"~
N,0-carbonyl-~antamicin-Cla with chromium trioxide-pyridLne.
complex. Isolate and purify the resultant pr~ducts in
a manner sL~ilar to that describ~d to obtain 1,3,2'96'-
te~ra~N~benzylo~y~arbonyl-2"~0-acetyl-3",4"-~,O~carbonyl--
5-dehyarog~ntamicin C
. . . .
F. To a solution of 2.1 gms o~ 1,3,2'~',3"-pentai~-
benz~loxy~a~bon~l-2"-Oqace~yl-5-dehydrogentami~in Cla
lb in 40 ml o dry te~rahydro~uran under an atmosphere o~
argon add 8 ml of 1 M "L-Selectride" tlithium tri-sec-
bu~yl boroh~ride in tetrahydro~uran). Stir ~he mix~ura
under an abmosphere o nitrogen at room temperature for
20 hours, pour intG 400 ml o~ aqueous sodium chlori~e
and extract with 3 x 80 ml portion~ of ethyl acetate.
Wash ~he cambined eth~l aceta~e ex~ract 3 t~me~ with water
tcontaining some soaium chloride). Dry over sodium
sul~ate and ~vaporake in va~uo to a residue (2.2 gm~)
~ampri5ing a 1,3,2'D6'-tetraiY-benzyloxycarbonyl-3",4"-
N,0-carbonyl derivative o gentamicin Cla, whîch is
used without further puriication in th~ proce~ure of
Example 11-&. Alternatively, the above procedure
may ~e carried out on 1~3,2',6'-tetra-W-benzylo~carbon~l-
2"-O-acetyl-3"~4"-w~o-carbonyl-5-deh~drogentamicin Cla
to ob~ain ~he same product obtained in the p~ragraph
above~
79 -
~80~
G~ 3-1 4"~ 0-carhon~1-5-e i entamicin C
la-
Dissolve the pxoduct (2.2 gms) obtained in the ~irst
paragraph o above Example ll-F in 2Q ml of dry tetra-
h~drofuran, cool the solution ~o -75 to -85C and
condensa 300 ~1. o ammonia into the reaction vessel~
Add 2.2 gms o~ sodium metal and stir the reaction:
mixtura vigorously ~or 2.5 hours. Slowly add 20 ml.
o water to the mix~uxe and allow the ammonia ~o evapo--
rate with warmLng ~o ro~m tc~perature. A~sorb the
residual solution ~ a BioRex 70 cation exchange resin
(100 ml, ~ fon~).... Wash the neutral impuri~ie~ o~
wi~h water t4Q0 ml) then elu~e the product wi~h 1.5 ~
ammonium hydroxide. Combine.the like am~onium hy ~ oxids
. eluate~ as determined by thin layer chromat~graphy .
and evaporate in vacuo to a residue.comprising 3",~
N,0-car~onyl-5-epige~amicin C~a, which is used without
further purification in the procedure o Example~
'-
DissolYe 143 m~s of 3",4"-~,0-carbonyl-5-epigenta~icin
Cla prepared as aescr~bed in Example ll-G in 20 ml. of
2 ~ sodiu~ hydroxide. Heat the solution at reflux
~emperature for 4 hours then cool to room temperature
and place on a BioRex 70 cation exchange resin column
(100 ml, H~ form). Wash off the neutral salts with
25 - 200_ml of water, then elute with 200 ml of 1~5 N
ammonium hydroxide~ Concentrate the combined ammonium
- 80 - -
, . . ~
.
3L048~2~
hy~roxide eluate in vacuo to a re~idu~ comprising 5-epi-
~gentamicin Cla, yield 139 mg. Puri~y by chromato_
graphing on a 33 gm silica gel column elu ing with ~he
lowar pha~e of a d loro~orm:methanol:concen~rated
ammonium hydroxide solvent system (1:1:1). Combine .
the like fraction~ a~ determLned by thin layer chroma_
~ography and evapora~e in vacuo to give puri~ied 5-
epigenta~ici~ Cla~. ~ass Spectrum: m~e 450 (M ~ H)~;
32~, 304, 16~, 129~
'
EXAMPLE 12
': ' . .
. In a manner similar to that de~cr~bed in pre~l~us~-Examples
: 15 trea~ l~N-acetyl-r-epi-amino-S-deoXygen~amiein Cl or
l~N-acet~l-5-epigentamicin Cl with diborane in tetra-
h~dro~uran~ Isolat~ and purif~ the resultant produc~s
in a ma~ner similar to that describea inprev~ous.~x~mples
to obtain l~ th~1-5-epi-amino-5-deo~ygentamicin Cl ~r
. 2fl 1-E-e~hyl-5-epigentamicin Cl.
- 81 -
1()480ZQ
(2) In the manner similar to that desc.ribed obtain,
respectively, l-N-e~hyl-5-epi-amino-5-deoXygentamiCin Cla,
l-N-ethyl-5-epi-amino-5-deoxygentamicin C2, l-N ethyl-5-
epi-amino-5-deoxygentamicin C2a, 1-N-ethyl-5-epi-amino-
5-deoxygentamicin C2b, 1-N-ethyl-5-e~i-amino-5-deoxy-
Antibiotic JI-20A, l-N~ethyl-5 epi-amino-5 deoxy-Antibiotic
JI-20B, l-N-ethyl 5-epigentamicin Cla, l-N-ethyl-5-epi-
gentamicin C2, 1-N-ethyl-5-epigentamicin C2a, l-N-ethyl-
5-epigentamicin C2b, 1-N-ethyl-5-epi-Antibiotic JI-20A,
1-N-ethyl-5-epi-~ntibiotic JI-20~.
(3) l~N-Ethyl-5-e~ mino-5-Deo~ysisomicin
Suspend 1 gm o~ l~NQace~yl-5~epi-amino-5 deoxy~isomicin.
- . in loO ml of tetrahy~rofuranO. Add 1 gm of lithium
aluminum hydxide, then stir ~he re~ul~ant suspension
15 . at re~lux temperature for 24 hours under an atmosphere ~.
o~ nitrogen. Cool and decompose the excess hydrid~
by ca~e~ul addi~ion of e~hyl acetate. Evapora~e the
- reac~ion mix~ure to a small volumQ and dilu~e with
water. Separate ths insoluble solid by ~iltration
20 - an~ wash well wi~h acetic acid~ Evaporate the c~mbined
filtrate and washings and dissolve thQ resultan~.residue
in water. Adjust the p~ of the aqueous solution to
about 7 by addition o am~onium hydroxide. Pass the
solution ~hrough a column o~ IRC-50 resin in the a~moniu~
cy~le and wash the column well wi~h wQterO Elute with
0.5 ~ ammonium hydroxiae ~ evaporate the eluate, and
chromatograph khe resultant residue over 20 ym of silica
82 -
~2~ ~
~ 04~020
gel eluting ~ th the low~r phase o a 2:1:1 chloroform:
methanol:concentrated ammoniu~ h~droxide ~olvent system.
Combine and evaporate the like fractions as determined
b~ thin layer chrcmatographyto obtain l-~-e~hyl-5-epi-
amino-S-deoxy~isomicin.
(4) In ~he mannsr ae~cribed in the procedurQ o~ ~xample
12-A(3) obtain, respec~ively, 1~-ethyl-5~epi_amino-
5-aeo~yverdamic~n, 1~-ethyl-5-epi~amino-5-deoxy- .
An~ibiotic 66-40B, l~N-ethyl-5-epi-amino-5-deoxy-
Antibiotic 66~AOD, l~N~-ethyl-5-epi-amino-5-deoxy .
Anti~ioti.c G-S2, li~-ethyl-5q2piverdamici~, .1~-
ethyl-5-epi~-~ntibiotic 66-40B, li~-ethyl-5-epii~nki-
biotic 66-40D, li~-ethyl-5-epi~ntibiotic G-52.
'
A, l~N-Eth~1-5-ePiverdamicin
To a solution o~ 5 gm o~ 5-epi~erdamicin in 250 ml o~
- water add l N sulfuric acid until the p~ o~ the solution
is adju~ted to about 5. To tho solution of 5-epiverda
micin sulfuric acid addition salt thereby formed, add
2 ml o~ acetald~hyde, stir for 10 m~nute~, then a~d
0.85 $m of sodiu~ cyanoborohydride~ Continue stixring
at room temperature ~or 15 minutes, then concentrate
the solutio~ in vacuo t~ a vo~ume o~ about 100 ml,
treat the solution with a basic ion exchange resLn
--25 ~e.g. Amberlite IRA ~OlS (0~ )), then lyvphilize to a
resi~ue comprising l-N-ethyl-5-epivexdamicin~
- 83 -
~o~oz~
Puri~y by chr~ma~ographing on 200 sm of silica gel,
eluting with lower phas~ o~ a chloroform:methanol:7%
aqueous ammonium hydroxide ~2:1Ol) system. Combine the
like eluate~ as de~ermined by thin layer chroma~ogr~phy
an~ concentra~ the cc~bined eluate3 o~ ~he major ccm- -
pcnent i vacuo to a residue comprising 1~N~ethyl-5-
epiveraamicLn~ Further purify by again chramato~raphing
on 100 g~.of silica gel eluting wi~h a chloroform:~e~hao
nol:3~5% ~m~onium hydsoxide (~:2:1) sy~te~ Pa~3 th~ .
combined~ lik~ eluates-(as determined by thin layer
chromatography) through a column o basic ion exch~nge
resin and lyophilize the eluate to obtain l~ thyl-5-
epiverdamicin
:- B. In the ~rocedure o~ Example. 1-3~ substitu~
equivalen~ guantitie~ of other 5-epi-~inoglycosides
and 5~epi_aziao (and 5-epi-amino)-5~deo ~ aminoglyco~ide~
to ob~ain, respectively, l~N-athyl S-epi-amino-5- :
deox~gentamicin CI~, l~N~etXyl-5-epi~amino-5-deoxy- -
: ~ ~ gentamicin C~ N-ethyl~5~epi-2mino-5-deoxygentamicin
C2, li~-ethyl-5-epi-amino-5~deox~gentamicin C2a, 1-~-
ethyi-5-api-amino-5-~eoxygen~a~icin C~b~ l~Nqethyl-5- :
epi-amino S-aeo~ysisomicin, ~-N-eth~1-5-epi-amino-S- . .
deoxy~ntïbioti~ G-52, li~qethyl-5-epi-amino-5~deox~-
':- Antibiotic 66-40D, l-N~e~hyl~5-epi~amino-5~deoxyver-
2~ dami~in, l~N-e~hyl-5-epi-amino-5-aeoxy-~ntibiotic 66-40B,
- 84 -
~L048~Z(~
l-N-ethyl-5-epiamino-5-deoxy-Antibiotic JI-20A, l-N-ethyl-
5-epi-amino-5-deoxy-Antibiotic JI-20B, l-N-ethyl-5-epi-
azido-5-deoxygentamicin Cla, l-N-ethyl-5-epi-azido-5-
deoxygentamicin Cl, l-~l-ethyl-5-epi-azido-5-deoxygentamicin
~5 C2, 1-N-ethyl-5-epi-azido-5-deoxygentamicin C2a, l-N-ethyl-
5-epi-a~ido-5-deoxygentamicin C2b, 1-N-ethyl-5-epi-azido-
5-deoxysisomicin, 1-N-ethyl-5-epi-azido-5-deoxy-Antibiotic
G-52, 1-N-ethyl-5-epi-azido-5-deoxy-Antibiotic 66-40~
l-N-ethyl-5-epi-azido-5-deoxyverda~icin, 1-N-ethyl-5-epi-
azido-5-deoxy-Antibiotic 66-40B, l-N-ethyl-5-epi-azido-5-
deoxy-Antibiotic JI-20A, l-N-ethyl-5-epi-azido-5-deoxy-
- Antibiotic JI-2OB, l-N-ethyl-5-epigentamicin Cl, l-N-ethyl-
- 5-epigentamicin C2, 1-N-ethyl-5-epigentamicin C2a, l-N-
1-~ ethyl-5-epigentamicin C2b, 1-N-ethyl-5-epi-Antibiotic G-52,
1-N-ethyl-5-epi-Antibiotic 66-40D, l-N-ethyl-5-epi-Anti-
~iotic JI-20A, l-N-ethyl~5-epi-Antibiotic JI-20B.
- .
C. In the procedure of Exam~les 13q~ and B by substi- -
tuting for acetaldehyde equivalent quantities o~ other .
aldehydes, e.g. propena~, butanal and fi-acetamidobutanal,
~o there are obtained the corresponding l-N-propyl, l~N-
butyl and l-N-~ -acet2midobutyl derivatives o the 5-
epiamin~glycosides~. 5-epi~amino-5-deoxy- and 5-epi-
azido-5-deoxy aminoglycoside~ listed therein. Treat-
ment of the l~N-( ~ -acetamidobutyl) derivatives with
base yields the corresponding 1~ aminobuty~ deriva-
tives.
- 85 -
~ .
A, ? ', 3 ~ 6 ' ~rri ~-~u~oxycarbon~l -3 " v4" ~, Q-carbon~
epiver~damici,n
- Dissolve 25. 5 gm o~ 5-epiverdamicin and 13 gm o:i~ sodium
~ carbonate in 625 ml of distilled water. Add 100 ml o~
car~sbe~zo~ychloride to the stirred solution at 25C
and stir the mix~ure for sixteen hours. Filter of~ the
soli~, w~sh thoroughly with water, d~y in vacuo,, and
then w~sh with hexane to o~tain pentai~car~obenzox~-5-
1~ epiveraamicin as à colorless amorphous solidDis-
solve 51 gm thexe~f in 50 ml o~ dimethylformamide, add
: 250 mg of sodiu~h~ide to the s~irred solutàon, and
stir the reac~ion mixt~re-under argon at xoom temperatuxe
~ ~or t~o hours. Filter and aad glacial acetic acia
15~ ~2ml) to the filtrate which i5 then concentrat~d in
acuo. Ext~act-the residue with chloroorm (200 ml,
previously passad through ~a~ic alum~na), Wa5~ thQ
extract:with water and ~ over sodiumsulfate. The
solution is evaporated to give ~etraiN-carbobenzoxy~ -
3" ,4"i~,Q-carbonyl--5--epiverdamicin as an amorphous
powder.
: . .
-- 86 --
.
To a . -. solution of 1,3,~',6'-tetra~ enzyloxy~ ..
carbonyl 3",4"~jO-carbonyl-5-epive~damicin (10.2 gm).
in tetrahy~rofuran ~00 ml) add 1 litr~ o~ liquia ammoni~
(redistilled fr~m sodium). To the stirred solution
add 6 gm o~ sodium in small pieces~ After stixring
for 3 hours de-~troy the excess sodium by addition of
ammonium chlorideO Allow the solvents to evaporate
under a stream of ni~rogen. Dissolve the residue in
water and pass through a medium of Amberlite IRC-50
resin (E~ form) and wQsh the resin well with wat~r-
then elute the product with 2N ammonium hydroxide
solution. Evaporate the ammonia elua~e in vacuo to
gi~e 3",4"-~,0-carbonyl-5-epiverdamicin.
' ' '
Dissolve 3"~ ,0-carbo~yl-5-~pivexdamicin ~1~4 gm) in
10 ml of 5~/~ a~ueous me~hanol ~ontaining triethylamin~
(3.5 mmole~)c With sti~ri~g~ ad~ t~bu~oxycarbonyl
azide (3.5 mmoles) dropwise. Stir the mixture for tw~
days at ra~m tempera~ure. Add 5 ml of AmberLite. IRA-i.
401S (0~ ) ion exchange resin along with 5 ml methanol
and stir for 1/2 hours. ~emove the resin by filtra-
tion and wash with methanol. Concentrate the *iltra~e
and chromatograph the residue on a col~mn of silica
- 87 -
~ .
104l302~)
gel (60-1~0 mesh, 2000 gm) using chloro~orm:methanol:am-
monium hydroxide ~30:10:0.4) as the solvent sy3tem.
..- Pool the homoyen~ou~ ~ractions containing the title.
material ana remove the solvent.by evaporation in vacuo
Dissolve the residue in methanol and precipita~e wi~h
excess ether. Isolate the solid produc~:hy filtration
,
and dry.
B. ~ 2~0~=~=5S~Y~ 5l~
(1) Dissolve 3',4"~N9~-carbonyl-2',3,6'-tri~N~t~butox~-
10 carbonyl-5-epiverdamicLn.(0.77 gm) in te~rahydrofuran
(20 ml) and cool in an ice~bath. Add ethyl fluoro- .
. sulphonate (0.14 gm) and allow to warm tc room temper-
- ature. R~move the solvent and dissolve the residue in
tri~luoroacetic acid. After :Eive minutes at ro~m
temperature remove the triEluoroacetic acid in vacuo
and treat the residue with lO~o potassium hydroxide
solution at 100 & ~or 5 hours.
.
Pass the cooled solution down a colu~n o~ Amberli~e
I~C-50 (~+) ion-exchange re~in and elut~ with 2~
aqueous ammonium hydroxi~e. Concen~rate~the eluant
.
and lyophilize to obtaIn the crude title product-..
- 88
'. '
, .~'~ ,
- lQ4~ ZO
Chro~a~ograph the crude ma~erial on silica gel in the
low~r phase o~ a chloroform:~ethanol:7% ammonium hy-
~roxiae (2~1:1) solv~nt mixture to o~tain l-~-ethyl-
5~epiv~rdamicin.
(2) Trea~ 2~3~6~-rriiN-t~bu~oxycarbon~l-3~4~-E~o-
carbonyl-S-epi~erdamicin (0.77 g~) in T~E (25 ml) wq~h
,~- m~hyla~ine (lOl ~g) and tri1uoromethylsulphonic an-
hyaride ~290mg) at 0 C for l8 hour-~. Reduce the
solution to ~r~nes~ an~ dissolve the residue in DMF
. ~10 ml) and sti~ with ethyl iodide (330 mg) and potassium
car~onate (130 mg) for a fur~her 18 hours. Remove th~
solvent by evaporation and treat the residue with 10%
aqueou pota~siu~ hydroxide at 100 C for 1~ hou~s~ Pass
the caoled ~olu~ion through a column of Am~erlite IRC So
(~ ) ion exchange resin. The crude produc is elute~ -
with 2 ~ aqueous ammonium h~roxide. ~he comb~ned
; eluant is reduced to dr~nes~ in vacuo and the residue
chr~matograph~d on silica gel (200 gm) in the low~r phas~
o~ a chlorofon~:me~hanol:7~0 ammonium hydroxide (2
solvent syste~ to give 1-~-ethyl-5-epiverdamicin.
(3) Dissolve 2',3,6'JTri-_-t-butox~car~onyl-3",4"~,0-
carbonyl-5~epiverdamicin (0.77 gm) in dichlor~methane
tlOO ml) with acrylonitrile to.24 gm) and leave at roam
temperatu~e ~or 24 hoursO Remove the solvent in vacuo
.
to leave a residue which is aissolYed in dImethylforma~ide
and treat with e~hyl iodid t2~0 mg) at 50C for 12 hour~
- 89 -
'
. ~4802~
Remove the solven~, and treat the residua with lO~o
aqueous potassium hydroxide at 100 fo~ 8 hours. The
cooled solution is pas3ed down a column o~ Amberlite
IRC-50 (~ 3 ion exchange resin and th~ crude product is
eluted with 2~ aqueous ammonium hy*roxide~ The combined
eluant is reduced to a~yne_s Ln vac~o ana ~he re.Qidue
~hromatographed on siIica ge} (200 gm) in the lower phaso
o~ a chlorofo~:~e~hanol:7% ammonium ~ydroxide (2 1 1?
solvent system to give l~W-ethyl-5-epiveraamicin.
'10 EX~MP~E. 15
rc~h
A. 5ulfate Salts (sulfuric acid addition salts)
.
: Dissolve 5.0 gm of 5-epigenta~cin Cl or 5-epi-amino
5Ddeoxygentamici~ Cl in 25 ml o~ water and adjus~ the
i5 p~ o~ the solu~ion to 4.5 wi~h 1 N~sul~uric acid~ -
Pour Lnto about 300 ml o~ methanol w~th ~igorou~ agita- -
tion, contin~e the agitation ~ox about 10-20 minutes
and filter. Wash the precipitate with me~hanol and
dry at about 60 & in vacuo to obtain 5-epigen~amicin C
. 20 sul~ate or 5-epi~amino-5-deoxygen~amicin Cl sulfate.
.
B. Hydrochloride Salts
Dissolve 5.0 gm of 5-epigentamicin Cla or S~epi~nino-
5-deox~gentamicin Cla in 25 ml of water. Aciaify with
2 N hyarochloric acid to p~ 5. Lyophilize to obtain 5-
epigentamicin Cla hydrochloride or 5-epi-amino-5_deoxy-
gentamicin Cla hydrochloride~
-- 90 --
~he compound~ o~ this invention and the non-~oxic,
pharmaceutically acceptable acid addi~ion salts there-
of are broad spectrum antibacterial agents which,
advantageously, exhibit activity against many organisms
~ which are resistant to their 5~hydrox~ precursors~
Thus, the compounds o~ this invention can be used
alone or in combination with other antibiotic agents
to prevent the growth or reduce the nu~ber o~ bacteria
in various environments. They may be used, for
example, to disin~ect laboratory glassware, dental
and medical equipment contaminated with 93~.~Y~a~3y~
aureus or other ~acteria inhibitcd by the aminoglyco
sides o~ this invention. The activit~ of the compounds
of this invention against gram negative bacteria renders
~ them useful for combating in~ections caused by gram
negative organisms, e.g. species o~ Proteus and
PseudomonasO The compounds, e~g. 5-epi-azido- or
.
5-~pi-amino-S-deoxysisomicin, 5-epi-azido- or 5-epi-
amino-S-deoxyverdamicin, 5-epigentamicin Cl and S-epi-
gentamicin Cla have veterinary applications~particularlyin the treatment of mastitis in cattle an~ Salmonella
induced diarrhea in domestic animals such as the dog
and the cat.
. -- 91 --
~ .
~04~
The improved spectrum o the compounds o~ this invention
consists o~ enhanced potency against many or~anisms
resistant to the parent campound~ Thus, ~or example,
compounds o~ this invention, e.g. 5-epi-4-0-~inoglyco-
syl-6-0-garosaminyl-2-deoxystrepamines, or 5-~pi~amino-
4~0-aminoglycosyl-6-0-garosaminyl-2,5 dideoxystreptamines~
are more active against many organisms which inactivate
the paren~ antibiotics by acetylation of the 3-amino
group and/or by adenylylation o~ the 2"-hydroxyl group.
Of these, some also exhibit anti-proto20al, anti-amoebic
and anthe~mintic properties~ The l~-alkyl derivatives
o~ this invention, particularly the l-~-ethyl-S-epi-
4-O~aminogly~osyl-6~0-garosaminyl-2-deoxystreptamines
and the l-~-ethyl-S-epi~amino-4-O_amin~lycosyl-6-o-
garosami~yl-2,5-dideoxystreptamines also exhibit Im--
pro~ed spectru~against Pseuaamonas as compared to their
l-~-unsubstituted precursors having the normal con~ig-
~uration at ~-5.
- ' : - ' ,
Particularly valuable ~ompounds o~ this invention are
S-epi~4-O-aminoglycosyl-6-_-garosaminyl-2-deoxys~re~pta-
mines, particularl~ the S-epi- derivatives o~ gentami-
cin Cl, gentamicin Cla, gentamicin C2, gentamicin C2a,
- 92 -
1)20
gentamicin C2b, verdamicin, Antibiotic G-52, and Antibiotic
66-40D; and 5-epi_amino-and 5-epi-azido-4-_-aminoglycosyl-
` 6-0-garosaminyl-2,5-dideoxystreptamines, particularly the
derivati~es o ~entamicin Cl7 gentamicin Cla, gentamicin
C2~ gentamicin ~2ar gentamicin C2b, sisomicin, verdamici~,
Antibiotic G-52 and An~ibiotic 66-40D. These compounds
are broad spectrum antibacterial agents, being active
against gram positive bac~eria (e.g StaPh~lococcuQ 3~
and gram negative bacteria (e~g. ~39~e ~ .coli~and
: 10 Pseudomonas aeruqinosa)as determined ~y standard ~ilution
tests, including bacteria resistant to the paren~ compounds~
Additionally, the 1~ ethyl derivatives of the foregoing
amînoglycosides possess im~roved potency against Pseudomonas
, . .. . .
~n general, the dosage administered o the derivatives o~
the 4,6-di-0~-taminoglyc.~syl)-2.-deoxystreptamines will be
dependent upon the age and weight of the animal species
being treated, the mode of administration, and the t~pe
and severity o~ bacterial infection ~aing prevented or re-
auced. In general~ the dosage of the derivati~es o~ the
4,6-di-0-~aminoglycosyl)-2~deox~streptamines employed to
combat a given bacterial infection will be similar to the
dosage requirements of the corresponding 4~6-di-o-(amin
glycosyl)-2-desxystreptamines.
., ' . .
The derivatives o~ the 4,6-di-0-~aminoglycosyl)-2-deox~-
streptamines and the pharmaceutically acceptable acid
addition salts thereo~ may be a & inistered orally. The~
~ .. . . . . . . .... .. . . . . .. . . ..
may also be applied topically in the ~orm o~ ointments,
. - 93 -
:, .
~L04~02all
both hydrophilic and hydrophobic, in the form of lotions
which may be aqueous, non-aqueous or of the emulsion t~pe
or in the ~orm o~ creams~ Pharmaceutical carriers useful
in the preparation of-such formulations will include, for -
example, such substances as water, oils, greases, poly-
esters, polyols and the like. - --
For oral a&~inistration the compounds of this invention
may be c~mpounded in the form o~ tablets, capsules, elixi~s
or the like or may even be admixed with animal ~eed. It
is in these dosage forms that the antibacteriaIs are mos~
effective for treating bacterial infections of the gastro-
intestinal tract, which in~ections cause diarrhea.
~,, . , . ~ .
In genaral~ the topical preparations will contain ~rom
about 0.1 to about 3.0 gms. of active ~ redient per 100 gms.
of ointment, creams or lotio~. ~he topical preparations
are usually applied gently to lesions ~om a~out 2 tQ about
5 times a day.
~ .
The antibacterials o this invention ma~ be utilized in
li~uid fonm such as solutions, suspensions and the like
20~ for otic and optic use and may also be administered parent-
erally via intramuscular injection. The injectable solu~-
ion or 6uspension will usually be a~miniskered at ~rom
about 1 m~. to about 10 mgs. of antibacterial per kilogra~
of body wei~ht per day divided into about 2 to about 4
doses. The precise dose depends on the stage and severit~
of the infection, the susceptibilit~ of the infecting
~ 94 -
organism ko the antibactarial and the individual charac~
teristics o~ the animal species being treated.
~he following formulations are to exempli~ some of the
aosage fonm~ in which ~he antibacterial agents o~ this
inven~ion may be employed:
. . Formulàtion 1,
Tablet. 10 mq.Tab~ 2S m~.Tab.~ 100 ~T~b~
5-epigentamicin Cl: 10.5* mg.26.25* mg.105.0* mg.
~ Lac~ose, ~mpalpable197.50 mg~ 171~25 mg~ 126.00 ms.
powder
Corn Starch25.00 mg.~25.00 mg. 35.00 mg.
Polyvinylpyrrolidon~ 7.50 mg. 7.50 ~g. 7~50 mg.
: Magnesium Stearate 2.50 mg. 2.5Q mgO 3.50 mg.
*5% excess ~ : .
15. In the above fonmulation the active ingredient may ~e re-
placed hy the same amount of S-epi-amino-5-deoxygen~amicin
la'
:
Procedure
Prepare a slurry consisting of the 5-epigentamicin Cl (or
5-epi_amino-5-deoxysentamicin Cla) lactose and polyviny~- -
p~rrolidone~ Spray dry the slurry~ Add the corn starch.
and magnesium s~earate. Mix and compress into tablets,
- 95 -
!,~ U
`` ~7
~L~11480Z0
Formulation 2
Ointment
5-eplgentamicin Cla 1.9 gm
Methylparaben U.S.P, O.S gm
Propyl paraben U.. S,P. 0.1 ~tm
Petrolatum to 1000 gm
.
Pxoceaure . :
(1) ~elt the petrolatumO
(2) Mix the 5-epigentamicin Cla, methylparaben and propyl-
. 10 . paraben with about 1~/~ o~ the molten pe~rolatum, -
(3) Pass the mixture through a colloid mill~ .
(4) Ada the remainder o~ the petrolatum with agitation and
cool the mixture until it becomes semi-solid. A~
this stage the proauct may be put into suitable con-
lS tainers.
.
Ointments of other compounds of this invention are pr~-
pared by substi utLng an equivalant quantity o such
compoundO e~g. 5-epi~azido-5-deoxygentamicin Cla,~or
5-epigentamicin Cla in the ~oregoing example
and by following substantially the procedure o the
exampleO
,'
- 96 -
:, ,
l.
9~
Formulation 3
!3~3_ a~LlY~ Per 2,.o ml.,vial Per 50 Liters
5-epigentamicin Cl sulfate 84 * mgs 2100* gms
Methylparab~n, U.S.P.3.6 mgs. 90.0 gm5
Propyl paraben, U.S.P.094 mgs10.0 gm5
Sodium bi~ul~ite, U.S.P.6~4 mg~16000 gms
Disodium Ethylenediamine
tetraacetate dihydrate, R~a. 0.2 mgs - 5,0 gms
Water~ U.S~P. q~s. ~ 2.0 ml50.0 ~it~r
* Includes a 5% manu~acturing overcharge
Procedure: For a 50 0 liter batch
Charge approximately-3~ liters of wQter for injection to a
: :- suitable stainless steel jacke~ed vessel and heat to about
70 &. Charge the methyl paraben and propyl paraben to
. the heated water for injection and dissolve wi~h agitation,
When the para~ens are completely dissolved, cool the con
tents of the tank to 25-30C by circulatin~ cold wa~er
through the tank jacket. Sparge tha solution with nitro
; . gen gas for at least 10 mi~ute~ and keep covered with
nitrogen during subsequent processing. Charge and dis-
solve the disodium EDTA and sodium bi5ulfite. Charge and
dissolve the 5-epigentamicin Cl sulfate. Bring the
batch volume up to 50.0 liters wikh water for in~ec~ion
- and agitate until homogeneous.
':
Under sterile conditions~ filter the solution through a .
suitable bacteria retentive ~ilter collecting the filtrate
in a filling tank~
- 97 -
~ .
~L~4~30Z~
Fill the filtrate aseptîcally into sterile pyrogen-free
multiple dose vials, stopper and seal.
In like manner, injectable solutions o other compounds
o~ this inventi~n and especially acid addi~ion salts of
such compounds may be prepared by substituting an equiva-~
lent quantity o~.such compounds, e.g. 5-epi-amino-5-deoxyr-
gentamicin Cla sulfate, for 5-epigentamicLn Cla sul~a~e .
- and by following the procedure set forth above.
, , ' ' ' ' . '
' , ' ' ~'
: . ' .
,
.
' '. ' ' ' ' ,
'
.
.
- 98 -
,r~
~r., ,,~
