Note: Descriptions are shown in the official language in which they were submitted.
1::1L32588
~et~iled Description of the Invention
The present invention relates to the preparation
of 3-de-0-methylfortimicins A and B and 4-N-acyl- and 4-
N-alkyl 3-de-0-methylfortimicin B derivatives which are
useful as antibiotics or as intermediates for preparing
other useful derivatives having antibacterial activity.
The novel compounds of this invention have the following
structural formula
IH3
CHNH2 H2N\ /OH
\~0 ~
--O J O {
NH2 HO N-R
. CH3
wherein R is hydrogen, acyl, amin~acyl, N-monoloweralkylamino-
acyl, N,N-diloweralkylaminoacyl, hydroxy-substituted æmino-
acyl, alkyl, aminoalkyl, N-monoloweralkylaminoalkyl, N,N-
diloweralkylaminoalkyl or hydroxy-substituted aminoalkyl and
the pharmaceutically acceptable salts thereof, for ex~mple,
salts formed from hydrochloric,sulfuric , and phosphoric
acids.
The naturally occurring fortimicins are produced in
several forms by cultivation of a strain of Micromonos~ora
.
,
. . .
5813
olivoast:erospora in a suitable nutrient medium as taught
in U.S. Patent 3,931,400 issued January 6, 1976 and U.S.
Patent 3,976,768, issued August 24, 1976.. The structure of
two of these forms is represented by the following formula
7'CHg
6'CHNH 2 H 2 N OH
~ ~ - O ~ ~ ~ OCH3
~< ~
NH 2 HD N-R
CH3
In this formula, when R is hydrogen the structure illustrated
is fortimicin B. When R i5 glycyl the structure of
fortimicin A is shown. As denoted in the formula above,
the fortimicin compounds consist of two cyclic moieties
referred to respectively as purpurosamine and fortamine.
The positions of the purpurosamine ring are indicated by
primed numbers while the positions on the aminocyclitol
moiety, fortamine, are indicated by unprimed numbers.
According to the method of this invention, in
performing the 3-0-demethylation reaction, fortimicin B,
4-N-(B-aminoethyl) fortimicin B prepared as taught in
Canadia.n Patent Application No. 287,269 filed September
22, 1977 or other appropriate derivative containing the
fortamine moiety are reacted
bm ~D
.
. 113~58~3
wlth excess metallic lithium in an amine solvent such as
ethylamine or ethylenediamine. The re~ctants are admixed
in the solvent and the reaction allowed to proceed at a
suitable temperature for the desired period~ The resulting
3-de-0-methylfortimicin B, 4-N-(B-aminoethyl)-3-de-O-methyl-
fortimicin B, or other derivatlve is isolated by conventional
column chromatographic methods.
The 3-de-0-methylfortimicin B prepared above is re-
acted with N-(benzyloxycarbonyl)-succinimide to prepare 1,
2', 6'-tri-N-benzyloxycarbonyl-3-de-0-methylfortimicin B.
The product formed in the above reaction is isolated by column
chromatography and 4-N-acylated by treatment with suitable
N-benzyloxycarbonyloxy protected amino acids. The
benzyloxycarbonyl-3-de-O-methyl-4-N-acyl fortimicins prepared
as above are conveniently reduced to the corresponding
4-N-alkyl derivatives with diborane. After isolation by
column chromatography the benzyloxycarbonyl groups of both
- the 4-N-acyl and 4-N-alkyl derivatives are conveniently
removed by catalytic hydrogenolysis and the products may be
isolated as the hydrochloride salts.
bm~
1~3258~3
The 3-de-0 me~hylfortimicin B prepared above is
reacted with N-(benzyloxycarbonyloxy)succinimide to prepare
1, 2', 6'-tri-N-benzyloxycarbonyl-3-de-0-methylfortimicin
B. The product formed in the above reaction is isolated
by column chromatography and 4-N-acylated by treatment
with suitable N-benzyloxycarbonyloxy amino acids. The
benzyloxycarbonyl groups of the products are conveniently
removed by cataly~ic hydrogenolysis and the product may
be isolated as the hydrochloride salt.
The following examples more clearly illustrate
the invention but are not intended to limit the scope of
the invention to the examples described.
EXAMPLE 1
3 De-0-methylfortimicin B
To a solution of 2.0 g of fortimicin B free base
in 50 ml. of freshly distilled ethylamine is added 40 ml. of
ethylamine containing 0.859 g. of lithium wire freshly cut
into small pieces. The dark blue reaction mixture is stirred
under reflux for 2 hours, then methanol is slowly added to
consume excess lithium. The solvents are removed under
_5_
bm:~
1~3258~3 -
reduced pressure and the resulting or~anic products are
separated from the lithium salts by column chromatography
on silica gel prepared and eluted with the lower phase of
a mixture of chloroform-methanol-concentrated ammonium
hydroxide (l:l:lv~/v). Fractions enriched in 3-de-0-methyl-
fortimicin B are collected and rechromatographed on a column
of a cation exchange resin, acrylic type, such as Bio Rex@3
70, 100-200 mesh, NH4 form. Elution with a gradient of
water to lN NH40H gave fractions containing pure 3-de-O-
methylfortimicin B. Lyophilization gave 0.267 g of color-
less material: [~] 2D4 ~41.4 (c 1.02, CH30~); IR 3370,
1585 cm~l; PMR (D2O) ~ 1.5 ~C6 -CH3, J6',7' )~
4 3 ( 1 ~ Jl',2' = 3.8); Mass spec Mt 334 222
Calculated for C14E30N45 334-2216-
EXAMPLE 2
-, 2', 6'-Tri-N-benzyloxycarbonyl-3-de-O-methylforti~icin B
To a stirring, ice-bath cooled solution of 3-de-
O-methylfortimicin B free base (1.59 g) in 24 ml. water and
48 ml. methanol is added 3.55 g of N-(benzyloxycarbonyloxy)-
succinimide. The reaction is stirred at ice-bath temperature
for 4 hours and then at room temperature for 22 hours. The
reaction is concentrated under reduced pressure and poured
into 400 ml. water to which is added 200 ml. chloroform.
The organic layer is separated and washed with water and dried
~MgSO4). The chloroform is evaporated and the residue chromato-
graphed on silica gel prepared and eluted with a solvent
--6--
,, . I ~.
1~;3258~3 -
system consisting of chloroform-methanol-concentrated am-
monium hydroxide (23.4:1. 4 o.lvhlv). Fractions containing
pure 1, 2~, 6'-tri-N-benzyloxycarbonyl-3-de-0-methyl-
fortimicin B are collected and evaporated to dryness to giv~
1.70 g of product: [~]2D3 ~ 19.4D (c 1.0, CH30H); IR 3437,
3350, 1705, 1505 cm 1; PMR (CDC13) ~ 0.99 (C6'-CH3, J6'
5-0), 2.27 (C4-N-CH3), 7.27 (Cbz).
Analysis: Calculated for C33H48N4011: C, 61.94; H, 6.57;
N, 7.60, Found: C, 61.83; H, 6.74; N, 7.51
Example 3
Tetra-N-benzyloxycarbonyl-3-de-0-methylfortimicin A
To a stirred solution of 0.80 g of 1, 2', 6'-
tri-N-benzyloxycarbonyl-3-de-0-methylfortimicin B in 5.35 ml
of tetrahydrofuran is added 0.399 g of N-hydroxysuccinimidyl-
N-benzyloxycarbonylglycine Stirring is continued for
22 hours at room temperature. The reaction is concentrated
to dryness under reduced pressure and the resulting product
chromatographed on a column o~ silica gel with a solvent
system consisting of benzene-methanol-95% ethanol-concen-
trated ammonium hydroride (23.5:1.4:2.0:0.2~/v). Fractions
containing the desired product are taken to dryness to gi~e
0.488 g of tet-a-N-benzyloxycarbonyl-3-de-0-methylfortimicin
A as a colorless glass: [~]2D4 + 45.2~ (C 1.03, CH30~
3425, 1705, 1645, 1500 cm 1; PMR (CDC13) ~ 1.15 (C6,-CH3),
2.9 (C4-~-CH3), ~.28 (Cbz).
1132588
Analysis: Calculated for C48H57N5O14: C, 62-13; H, 6.1g;
N, 7.55; Found: C, 61.80; H, 6.31; N, 7.64
Example 4
Tetra-N-benzyloxycarbonyl-3-de-0-methylfortimicin A
To a stirred solution of 0.525 g of 1, 2', 6'-
tri-N-benzyloxycarbonyl-3-de-O-methylfortimicin B, 0.199 g
of N-benzyloxycarbonylglycine and 0.228 g of l-hydroxy-
benzotriazole monohydrate in 3.0 ml tetrahydrofuran is added
0.88 g of N,N'-dicyclohexylcarbodiimide dissolved in 1.5 ml
tetrahydrofuran. An additional 1.5 ml of tetrahydrofuran is
used to rinse all the N,N'-dicyclohexylcarbodiimide into
the reaction vessel. Stirring is continued for 22 hours at
ambient tem~erature. Insoluble dicyclohexylurea is re-
moved by filtration. The filtrate is concentrated to dry-
ness under reduced pressure to yield a yellow froth. The
froth is chromatographed on a column of silica gel using a
solvent system consisting of benzene-methanol-95~/~ ethanol-
concentrated am~onium hydroxide (23.5:1.4:2.0:0.2 v/v/v/v)
Fractions containing the majority of the product are taken
to~~ryness and rechromatographed on a column o~ Sephade~
LH20 prepared and eluted with 95% ethanol. Fractions con-
taining pure product are collected and the solvent removed
under reduced pressure to give 0.105 g of tetra-N-benzyl-
oxycarbonyl-3-de-O-methylfortimicin A identical in all
respects with the same material prepared in Example 3.
11;3~2S8~3
Examp'le 5
Tetra-N-benzyloxycarbonyl-3-de-O-methyl-4-N-sarcosyl-
fortimicin B
To a stirred solution of 0.298 g of 1, 2', 6'-
tri-N-benzyloxycarbonyl-3-de'-O-methylfortimicin B, 0.113 g
of N-benzyloxycarbonylsarcosine and 0.129 g of l-hydroxy-
benzotriazole' in 3.0 ml of tetrahydrofuran is added 0.107 g
of N,N'-dicyclohexylcarbodiimide in 1.5 ml tetrahydrofuran.
An additional 1.5 ml of tetrahydrofuran is used to rinse all
the ~,N'-dicyclohexylcarbodiimide into the reaction flask.
Stirring is continued for 16 hours at room temperature.
Insoluble dicyclohexylurea is removed by filtration and
the filtrate concentrated to yield a pale yellow solid.
The solid is chromatographed on a column of silica gel
using a solvent system consisting of benzene-methanol-
95% ethanol-concentrated ammonium hydroxide (23.5;1.4;2.0;
0.2~/v~u). Fractions containing homogeneous material are
taken to dryness. Other fractions containing a minor second
component are rechromatographed on a column of silica gel
using a 'solvent system consisting of benzene-methanol-con
centrated ammonium hydroxide (85:15:1~1v/v). Homogeneous
fractions are combined with material obtained in the first
column to give 0.709 g of tetra-N-benzyloxycarbonyl-3-de-
O-methyl-4-N-sarcosyl-fortimicin B as a glass: ~]2D4
42.9~ (c 1.01, CH30H); IR 3435, 1703, 1635, 1500 cm 1;
PMR (CDC13) ~ 1.17 (C6,-CH3), ~ 2.9 (broad) (Sarcosyl-N-
C~3) 2-99 (C4-N-CH3), 4.83 (Hl " Jl',2' )~
_g_
1~3Z5813
Analysis: Calculated for C4gHsgNsO14 C ~ 62.48; H, 6.31;
N, 7.43; Found: C, 62.35; H, 6.65; N, 7.57
Example 6
3-De-0-methylfortimicin A tetrahydrochloride
Tetra-N-benzyloxycarbonyl-3-de-O-methyl*ortimicin
A (0 .14 g) in 25 ml 0. 2 N hydrochloric acid in methanol is
hydrogenolyzed for 4 hours under 3 atmospheres of hydrogen
in the presence of 0.1 g of 5a/o palladium on carbon. The
catalyst is removed by filtration and the filtrate con-
centrated to dryness under reduced pressure. Excess
acid is removed by co-distillation with methanol under
reduced pressure to give 0. 071 g of 3-de-O-methylfortimicin A
tetrahydrochlQride: [~]2D3 ~ 79-4 (c l.0~ CH30H); IR 3410,
2930, 1639, 1595, 1483 cm 1; PMR (D20) ~ 1.81 (C6,-CH3,
J6t7 = 6.5) ~ 3.62 (C~-N-CH3), 5.79 (Hl~ J Jl~ 2~ = 3-5)i
Mass spec. M 391.2414, Calculated for Cl6H33NsO6 391.2431,
Example 7
3-De-O-methyl-4-N-sarcosylfortimicin B tetrahydrochloride
Tetra-N-benzyloxycarbonyl-3-de-O-methyl-4-N-
sarcosylortimicin B (Q.125 g) in 25 ml 0.2 N hydrochloric
acid in methanol is hydrogenolyzed for 4 hours under 3
atmospheres of hydrogen in the presence of 0.13 g of 5%
palladium on carbon. The catalyst is removed by filtration
and the filtrate concentrated to dryness under reduced
pressure. Excess acid is removed by co-distillation with
- 10 -
1:~3~588
methanol under reduced pressure to give 0.073 g of 3-de-
O-methyl-4-N-sarcosylfortimicin B tetrahydrochloride:
[~]2D4 + 8~.5(c 1.01, CH30H); IR 3420, 2930, 1635, 1485
cm~l; PMR (D2O) 1.8 (C6,-CH3, J6'~7'
(Sarcosyl-N-CH3), 3.6 (C4-N-CH3), 5.79 (Hl,, Jl' 2~ =
3.5); Mass Spec M + 405.2614, Calculated for C17H35N5O6
405.2587.
Example 8
Tetra-N-benzyloxycarbonyl-3-de-O-methyl-4-N-(~-aminoethyl)
fortimicin B
To an ice cold stirred solution of 0.3 g of tetra-N-
benzyloxycarbonyl-3-de-O-methylfortimicin A in dry tetrahydro-
furan (6 ml.) is added 1.0 ml of a 1 M solution of diborane
in tetrahydrofuran. The reaction mixture is stirred for
3 hours under a nitrogen atmosphere and then treated with an
additional 1.0 ml. of the diborane solution. After stirring
for an additional 2 hours under nitrogen water is added and
the solvents evaporated under reduced pressure. Purification
by column chromatography on silica gel prepared and eluted
with a solvent system consisting of chloroform-methanol-
concentrated ammonium hydroxide (23.4:1.4:0.1v/v/v) gave pure
tetra-N-benzyloxycArbonyl-3-de-O-methyl-4-N-(~-aminoethyl)
fortimicin B.
Example 9
3-de-O-methyl-4-N-(~-aminoethyl)fortimicin B tetrahydrochloride
Tetra-N-benzyloxycarbonyl-3-de-O-methyl-4-N-(~-aminoethyl)
fortimicin B (0.10 g) in 25 ml. 0.2 N hydrochloric acid in
1132588
methanol is hydrogenolyzed for 4 hours under 3 atmospheres of
hydrogen in the presence of 0.11 g of 5% pallidium on carbon.
The catalyst is removed by filtration and the filtrate con-
centrated to dryness under reduced pressure. Excess acid is
removed by co-evaporation with methanol under reduced pressure
to give 3-de-O-methyl-4-N-(~-aminoethyl) fortimicin B
tetrahydrochloride.
Example 10
3-De-O-methyl-4-N-(~-~minoethyl) fortimicin B
To a solution of 1.0 g of 4-N-(~-aminoethyl)fortimicin
8 in 25 ml of freshly distilled ethylamine is added 20 ml. of
ethylamine containing 0.430 g of lithium wire freshly cut into
small pieces. The dark blue reaction mixture is stirred under
reflux for 2-16 hours, then methanol is cautiously added to
consume the excess lithium. The solvent is evaporated under
reduced pressure and the residue chromatographed on silica gel
prepared and eluted with the lower phase of a mixture of chloro-
form-methanol-concentrated ammonium hydroxide (1:1:1 ~ /v).
Fractions containing the desired product are collected and re-
chromatographed on a column of a weakly acidic, carboxylic
(polymethacrylic) type, cation exchange resin in the ammonia
form, for example, Biore ~70, 100-200 mesh. Elution with a
gradient of water ~o 1 N NH40H gave fractions containing pure
3-de-O-methyl-4-N-(~-aminoethyl)fortimicin B.
113~S88
Examples 11-13
In Vitro, Antibiotic Activities of 3-De-O-methyl-fortimicins
B and A and 3-De-O-methyl-4-N-sarcosylfortimicin B
The in vitro antibiotic activities of the
following 3-de-0-methylfortimicins
(11) 3-De-O-methylfortimicin B
(12) 3-De-O-methylfortimicin A tetrahydrochloride
(13) 3-De-o-methyl-4-N-sarcosylfortimicin B
- tetrahydrochloride
are listed in Table 1.
The in vitro antibiotic activities were determined
by a two-fold agar dilution method using Mueller-Hinton agar,
10 ml per Petri plate. The agar was inoculated with one
loopful (0.001 ml loop) of a 1:10 dilution of a 24 hour brvth
; culture of the indicated test organism and incubated at 37 C
for 24 hours. Appropriate fortimicins were used as control
antibiotics. The activities are listed in Table 1. Minimum
inhibitory.concentrations (MIC) are ex~ressed as mcg/ml.)
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