4,5-Carbamates of fortimicin B

Fortimicin B-4,5-carbamates represented by the formula: ##STR1## wherein R is hydrogen or an amine-protecting group; R.sub.1 is hydrogen or an amine-protecting group; R.sub.2 is hydrogen or loweralkoxycarbonyl; and R.sub.4 is hydrogen or loweralkoxycarbonyl. The compounds are useful as intermediates in the preparation the 2-epi-fortimicins.

BACKGROUND OF THE INVENTION 
Aminoglycoside antibiotics have proven to be a valuable class of 
antibiotics which include the streptomycins, kanamycins, neomycins, 
gentamicins, tobramycins, amikacin and the more recently discovered 
fortimicins. As with other classes of antibiotics, chemical modification 
of the parent antibiotics has been found to advantageously alter either 
the pharmacological properties or the antibacterial properties of many of 
the naturally produced aminoglycoside antibiotics either by increasing 
their antibacterial spectrum, increasing their intrinsic activity, 
increasing their activity against resistant strains or providing compounds 
which are less toxic than the parent antibiotics. 
Chemical modification has been found to be of value in the fortimicin 
family of antibiotics as well. See for example, U.S. Pat. Nos. 4,091,032; 
4,207,314; 4,124,746; 4,192,867; 4,169,198; 4,183,920; 4,176,178; 
4,187,296; 4,187,298; 4,187,297 and 4,187,299. 
Another valuable modification is disclosed in commonly assigned, co-pending 
application Ser. No. 25,236 filed Mar. 29, 1979, now abandoned, which 
claims 2-epi-fortimicin A, 2-epi-fortimicin B and 2-epi-4-N-acyl and 
alkylfortimicin B derivatives. The present invention provides 
intermediates useful in the preparation of the 2-epi-fortimicins A and B. 
SUMMARY OF THE INVENTION 
The present invention provides 4,5-carbamates of fortimicin B. The 
compounds are useful as intermediates in the preparation of the antibiotic 
2-epi-fortimicin B. 
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The 4,5-carbamates of fortimicin B of the present invention are represented 
by the formula: 
##STR2## 
wherein: R is hydrogen or an amine-protecting group; R.sub.1 is hydrogen 
or an amine protecting group; R.sub.2 is hydrogen or loweralkoxycarbonyl; 
and R.sub.3 is hydrogen; and when R,R.sub.1 or R.sub.2 are hydrogen, the 
salts thereof. 
The term "an amine protecting group" refers to monocyclicaryloxycarbonyl 
groups such as benzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 
2-chlorobenzyloxycarboxyl, etc. The preferred amine protecting group is 
benzyloxycarbonyl. 
The term "loweralkoxycarbonyl" refers to such groups having from 1 to 6 
carbon atoms in the alkoxy radical, ie. methoxycarbonyl, ethoxycarbonyl, 
etc. 
The preferred group is methoxycarbonyl. 
The preparation of the intermediates of this invention is detailed in the 
following examples and summarized in the following reaction schemes. 
Generally speaking, the carbamates of this invention are used in the 
preparation of 2-epi-fortimicin B as follows. A 4,5-carbamate of 
fortimicin B is converted to the corresponding 2-O-methanesulfonate. 
Solvolysis of the latter in aqueous 1,2-dimethoxyethane in the presence of 
ammonium acetate yields the protected 2-epi-carbamate and bis-carbamate in 
equimolar mixtures. 
Alternatively, when solvolysis of the 2-O-methanesulfonate derivative is 
carried out in a mixture with aqueous tetrahydrofuran and sodium 
bicarbonate, the 2-epi-oxazoline is formed. When the latter is heated 
under reflux in a solution prepared from ammonium acetate and aqueous 
1,2-dimethoxyethane, an approximately equimolar mixture of 
2-epi-4,5carbamate and the 2-epi-bis-carbamate is formed. 
The mixture of 2-epi-mono and biscarbamates may then be separated into the 
pure components by chromatography. Alternatively, the mixture can be 
heated under reflux with a mixture of sodium bicarbonate and methanol and 
the monocarbamate converted the the bis-carbamate which may then be 
isolated by chromatography. 
Hydrogenolysis of the resulting 
1,2'-di-N-benzyloxycarbonyl-2-epi-fortimicin B-bis-carbamate with 5% Pd/C 
in the presence of 0.2 N-methanolic hydrochloric acid gives 
2-epi-fortimicin B dihydrochloride which in turn is an intermediate in the 
preparation of 2-epi-fortimicin A and 2-epi-formimicin B derivatives as 
disclosed in co-pending commonly assigned patent application Ser. No. 
25,236 filed Mar. 29. 1979, now abandoned. 
Briefly, 2-epi-fortimicin B is converted to 
1,2',6'-tri-N-benzyloxycarbonylfortimicin B with 
N-benzyloxycarbonyloxysuccinimide, following the general procedure 
described in U.S. Pat. No. 4,091,032. Catalytic hydrogenation of the 
resulting produts, 
tetra-N-benzyloxycarbonyl-2-O-[N-benzyloxycarbonylglycyl]-2-epi-formticin 
A and tetra-N-benzyloxycarbonyl-2-epi-fortimicin A, with 5% Pd/C in 0.2 N 
methanolic hydrochloric acid yields 2-O-glycyl-2-epi-fortimicin A and 
2-epi-fortimicin A, respectively, isolated as their perhydrochloride 
salts. 
Alternatively, fortimicin B is converted to tetra-N-acetylfortimicim B by 
known methods. Selective hydrolysis of the latter with sodium bicarbonate 
in aqueous methanol gives 1,2', 6'-tri-N-acetylfortimicin B which is 
converted to the corresponding 4-N-ethoxycarbonyl derivative which is then 
readily cyclized to the 4,5-carbamate in a refluxing suspension of sodium 
bicarbonate in aqueous methanol. Treatment of the carbamate with 
methanesulfonic anhydride in pyridine results in the 2-epi-1,2-oxazoline 
derivative of the carbamate and hydrolysis of the latter with aqueous 
hydrochloric acid in tetrahydrofuran results in 
1,2',6'-tri-N-acetyl-2-epi-fortimicin B-4,5-carbamate. The latter is 
converted to the 2-O-benzyl ether with benzylbromide and barium hydroxide. 
Hydrolysis of the latter with aqueous sodium hydroxide resulted in 
2-O-benzyl-2-epi-fortimicin B. Treatment of the latter with 
N-benzyloxycarbonyloxysuccinimide results in 
1,2',6'-tri-N-benzyloxycarbonyloxy-2-epi- fortimicin B. Treatment of the 
latter with the N-hydroxysuccinimide ester of N-benzyloxycarbonylglycine 
results in tetra-N-benzyloxycarbonyl-2-O-benzyl-2-epi-fortimicin A which 
is converted to 2-epi-fortimicin A tetrahydrochloride as described above. 
The preparation of the intermediates of this invention are summarized in 
the following reaction schemes: 
##STR3##

The following examples further illustrate the present invention. 
EXAMPLE 1 
1,2',6'-Tri-N-benzyloxycarbonylfortimicin B (1) 
To a stirred solution of 2.0 g of fortimicin B, 30 ml of water and 60 ml of 
methanol, cooled in an ice bath, is added 4.44 g of 
N-(benzyloxycarbonyloxy)succinimide. Stirring is continued at 0.degree. C. 
for 3 hours and then at room temperature for 22 hours. The major portion 
of the methanol is evaporated under reduced pressure and the residue is 
shaken with a mixture of chloroform and water. The chloroform solution is 
washed with water and dried over anhydrous magnesium sulfate. The 
chloroform is evaporated and the residue is chromatographed on silica gel. 
Elution with a solvent system composed of chloroform-methanol-ammonium 
hydroxide [234:1.4:0.1(v/v/v)] gives 1.05 g of product (1): 
[.alpha.].sub.D.sup.25 -16.5.degree. (c 1.0, CH.sub.3 OH); IR(CDCl.sub.3) 
1712 and 1507 cm.sup.-1 ; NMR(CDCl.sub.3) .delta.1.03 (C.sub.6' 
--CH.sub.3,J.sub.6'7' =6.0 Hz), 2.32(C.sub.4 --NCH.sub.3), 3.41(OCH.sub.3 
). 
Analysis Calcd. for C.sub.39 H.sub.50 N.sub.4 O.sub.11 : C,62.39; H, 6.71; 
N,7.46; Found: C,62.16; H, 6.76; N,7.43 
EXAMPLE 2 
4-N-Ethoxycarbonyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B(2) 
To a magnetically stirred solution of 3.02 g of 
1,2',6'-tri-N-benzyloxycarbonylfortimicin B(1), 130 ml of methanol and 60 
ml of a solution of 3.02 g of sodium bicarbonate in 72 ml of water is 
added 0.90 ml of ethyl chloroformate. Stirring is continued at room 
temperature for 3 hours. The major portion of the methanol is evaporated 
under reduced pressure and the residue is shaken with a mixture of 200 ml 
of chloroform and 200 ml of 5% aqueous sodium bicarbonate. The chloroform 
solution is separated and washed with 200 ml of water. The aqueous 
solutions are washed in series with four 100 ml portions of chloroform. 
The chloroform solutions are combined, and the chloroform is evaporated 
under reduced pressure leaving 3.36 g of white glass. The latter is 
chromatographed on 250 g of silica gel packed and eluted with 
benzene-methanol[85:15(v/v)] to yield 2.57 g of the desired product: 
NMR(CDCl.sub.3) .delta.1.15d(J=6.4 Hz)(C.sub.6' --CH.sub.3); 1.27 t(J=7.2 
Hz)(OCH.sub.2 CH.sub.3); 3.02(NCH.sub.3); 3.43(NCH.sub.3); IR(CDCl.sub.3) 
3555, 3437, 1707, 1658 cm.sup.-1. 
EXAMPLE 3 
1,2',6'-Tri-N-benzyloxycarbonylfortimicin B-4,5-carbamate(3) 
A solution of 13.0 g of 
4-N-ethoxycarbonyl-1,2',6'-tri-N-benzyloxycarbonylfortimicin B(2), 5.3 g 
of sodium bicarbonate and 370 ml of methanol is heated under reflux for 
1.5 hours. The methanol is evaporated under reduced pressure, and the 
residue triturated with chloroform. The chloroform suspensions are 
filtered. Evaporation of the chloroform from the filtrate leaves 12.1 g of 
product. The latter is chromatographed on 850 g of silica gel using a 
solvent system prepared from benzene-ethanol[9:1(v/v)] to yield 10.9 g of 
pure product(3):[.alpha.].sub.D.sup.22 +2.5.degree. (c 1%,CH.sub.3 OH); 
NMR(CDCl.sub.3).delta.0.98d(J=6.0 Hz)(C.sub.6' 
--CH.sub.3),2.83(NCH.sub.3),3.44(OCH.sub.3); IR(CDCl.sub.3)3562, 3468, 
3320, 1759, 1706 cm.sup.-1. 
EXAMPLE 4 
2',6'-Di-N-benzyloxycarbonylfortimicin B-1,2:4,5-bis carbamate(4) 
To a solution prepared from 1.02 g of the compound of Example 3 in 20 ml of 
dry N,N-dimethylformamide, magnetically stirred, under a nitrogen 
atmosphere and cooled in an ice bath, is added 0.280 g of 57% oily sodium 
hydroxide. Stirring is continued for 4 hours with ice bath cooling. Acetic 
acid(0.8 ml) is then added to the cold suspension. The resulting solution 
is shaken with a mixture of 100 ml of chloroform and 200 ml of 5% aqueous 
sodium bicarbonate. The chloroform solution is separated and washed with 
200 ml of water. The aqueous solutions are washed in series with three 100 
ml portions of chloroform. The chloroform solutions are combined and dried 
over magnesium sulfate. The chloroform is evaporated under reduced 
pressure and residual N,N-dimethyformamide is removed by co-distillation 
with toluene under reduced pressure leaving 1.05 g of a white glass. The 
latter product (1.01 g) is dissolved in 20 ml of pyridine and 2.0 ml of 
acetic anhydride is added. The resulting solution is kept at room 
temperature for 24 hours. The resulting solution is then shaken with a 
mixture of 200 ml of chloroform and 200 ml of 5% aqueous sodium 
bicarbonate. The chloroform solution is separated and washed with 200 ml 
of water. The aqueous solution is washed with three 100 ml portions of 
chloroform. The chloroform solutions are combined and the chloroform 
evaporated under reduced pressure. Residual pyrydine is removed by 
co-distillation with toluene under reduced pressure leaving 1.04 g of a 
white glass. The latter (1.01 g) is chromatographed on a column of 100 g 
of silica gel packed and eluted with a solvent system composed of 
1,2-dichloroethane-ethyl acetate [14:16(v/v)]. Initial fractions yield 
0.161 g of 1,2',6'-tri-N-benzyloxycarbonyl-2-O-acetylfortimicin 
B-4,5-carbamate. Further elution of the column yields 0.594 g of a white 
class which is rechromatographed on a column of 40 g of silica gel packed 
and eluted with a solvent system composed of methylene chloride-ethyl 
acetate [3:3(v/v)] to yield 0.398 g of product (4):[.alpha.].sub.D.sup.21 
-2.33.degree. (c 1%, CH.sub.3 OH); NMR(CDCl.sub.3) .delta.1.16d(J=7.0 
Hz)(C.sub.6' --CH.sub.3),2.85(NCH.sub.3),3.52(OCH.sub.3); IR(CDCl.sub.3) 
3440, 3300, 1750, 1697 cm.sup.-1. 
EXAMPLE 5 
1-N-Methoxycarbonyl]-2',6'-di-N-benzyloxcarbonylfortimicin-4,5-carbamate(5) 
A magnetically stirred suspension of 0.8200 g of 
2',6'-di-N-benzyloxycarbonylfortimicin B-1,2:4,5-bis-carbamate(4), 0.5158 
g of sodium bicarbonate and 42 ml of methanol is heated under reflux for 2 
hours. The methanol is evaporated under reduced pressure and the residue 
shaken with a mixture of 200 ml of 5% aqueous sodium bicarbonate and 100 
ml of methanol. The methanol solution is separated and washed with 200 ml 
of 5% aqueous sodium chloride solution. The aqueous solutions are washed 
in series with two 50 ml portions of chloroform. The chloroform solutions 
are combined and dried over magnesium sulfate. Evaporation of the 
chloroform under reduced pressure leaves 0.8610 g of 
1-N-methoxycarbonyl-2',6'-di-N-benzyloxycarbonylfortimicin 
B-4,5-carbamate(5):[.alpha.].sub.D.sup.24 -7.8.degree. (c 1%, CH.sub.3 
OH); NMR(CDCl.sub.3).delta.1.12d(J=6.5 Hz)(C.sub.6' --CH.sub.3). 
2.84(NCH.sub.3), 3.45(OCH.sub.3), 3.63(OCOCH.sub.3); IR(CDCl.sub.3) 3563, 
3438, 1755, 1706 cm.sup.-1. 
EXAMPLE 6 
2',6'-Di-N-benzyloxycarbonylfortimicin B-4,5-carbamate(6) 
A magnetically stirred mixture of g of 
2',6'-di-N-benzyloxycarbonylfortimicin B-1,2:4,5-bis-carbamate(4), 10 ml 
of 2 N aqueous sodium hydroxide and 30 ml of 1,2-dimethoxyethane is heated 
under reflux for 2.7 hours. The resulting mixture is shaken with a mixture 
of 150 ml of chloroform and 200 ml of a saturated sodium chloride 
solution. The chloroform solution is separated and triturated with 200 ml 
of saturated sodium chloride solution. The aqueous solutions are washed 
with three 100 ml portions of chloroform. The chloroform solutions are 
combined and dried over magnesium sulfate. Evaporation of the chloroform 
under reduced pressure leaves 0.887 grams of product as a glass. 
Chromatography of the latter(0.824 g) on a column of silica gel packed and 
eluted with a solvent system composed of chloroform-methanol-ammonium 
hydroxide(conc.) [17.9:1.2:0.1(v/v/v)] gives 0.410 g of 
product(6):NMR(CDCl.sub.3) .delta.1.16d(J=6.5 Hz)(C.sub.6' 
--CH.sub.3),2,82(NCH.sub.3), 3.46(OCH.sub.3); IR (CDCl.sub.3) 3568, 3446, 
1752, 1702 cm.sup.-1. 
EXAMPLE 7 
Fortimicin B-4,5-carbamate trihydrochloride 
1,2',6'-Tri-N-benzyloxycarbonylfortimicin B-4,5-carbamate(3)(400 mg) in 35 
ml of 0.2 N hydrochloric acid in methanol is hydrogenated under 3 
atmospheres of hydrogen for 4 hours in the present of 400 mg of 5% 
palladium on carbon. The catalyst is removed by filtration and the 
methanol evaporated under reduced pressure. Residual hydrochloric acid is 
removed by co-distillation with methanol under reduced pressure leaving 
244 g of product as a glass:[.alpha.].sub.D.sup.22 +12.degree. (c 
1%,CH.sub.3 OH); NMR(D.sub.2 O).delta. 1,79d (J=6.4 Hz), 
3.33(NCH.sub.3),4.00(OCH.sub.3), 6.12d(J=4.8 Hz)(C.sub.1 --Hz); IR(KBr) 
1744 cm.sup.-1. 
EXAMPLE 8 
2',6'-Di-N-benzyloxycarbonylfortimicin B-1,2;4,5-Biscarbamate (4) 
A solution prepared from 5.36 g of 
4N-ethoxycarbonyl-2',6'-di-N-benzyloxycarbonylfortimicin B-1,2-carbamate, 
5.61 g of 1,5-diazabicyclo [5.4.0] undecene-5, and 250 ml of benzene was 
heated under reflux for 6 hours and then allowed to stand overnight at 
ambient temperature. Water (100 ml) was added and the resulting solution 
was stirred at room temperature for 1 hr. The resulting mixture was shaken 
with 300 ml of CHCl.sub.3. The organic phase was separated and washed with 
two 200-ml portions of saturated NaCl solution and dried (MgSO.sub.4). 
Evaporation of the solvent under reduced pressure left 5.19 g of crude 9. 
Chromatography of the latter on a column of 450 g of silica gel prepared 
and eluted with a solvent system prepared from 1,2-dichloroethanemethanol 
[10:1 (v/v)] gave 1.49 g of 2',6'-di-N-benzyloxycarbonylfortimicin 
B-1,2;4,5-biscarbamate identical with that prepared in example 4. 
EXAMPLE 9 
1-N-Methoxycarbonyl-2',6' -di-N-benzyloxycarbonylfortimicin B-4,5-carbamate 
(5) 
To a magnetically stirred solution of 0.2645 g of 
2',6'-di-N-benzyloxycarbonylfortimicin B-4,5-carbamate (6), 0.463 g of 
NaHCO.sub.3, 14 ml of CH.sub.3 OH, and 2 ml of water is added 0.12 ml of 
methoxycarbonyl chloride. Stirring is continued at ambient temperature for 
3.5 hr. The resulting suspension is shaken with a mixture of CHCl.sub.3 
and 5% aqueous NaHCO.sub.3. The CHCl.sub.3 solution is separated and dried 
(MgSO.sub.4). Evaporation of the CHCl.sub.3 under reduced pressure leaves 
0.2665 of 5 identical with that prepared as described in example 5 above.