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(11) | EP 0 000 800 A1 |
| (12) | EUROPEAN PATENT APPLICATION |
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| (54) | Selective N-protection of aminoglycosides and novel, selectively protected aminoglycoside intermediates |
| (57) This invention relates to the selective N-protection of aminoglycosides and to novel,
selectively protected aminoglycoside intermediates. Modifications in aminoglycoside
molecules are usually accomplished by multi-stage reactions whereby in most cases
amino groups need to be protected in order to avoid side reactions. To obtain 1-N-alkyl-
and 1-N-acyl- substituted- 4,6-di-O- (aminoglycosyl) -1,3-diaminocyclitols which are
very potent antibiotics, ideally all other amino groups are protected before introduction
of the 1-N-substituent into the 4,6-di-O- (aminoglycosyl) -1,3-diaminocyclitol. A
process is provided for the selective protection of the 3"-amino function in a 1,3"-di-N-
unprotected-poly-N- protected -4,6-di-O- (aminoglycosyl) -1,3-diaminocyclitol, wherein
the 6-0-aminoglycosyl has an amino function at the 3"-position and hydroxyl functions
at the 2" and 4" positions. Also, the respective 1-N-unprotected-poly-N- protected
-4,6-di-O- (aminoglycosyl) -1,3-diaminocyclitol intermediates are described. Selective protection of the 3"-amino function is accomplished by reaction with no more than one molar equivalent of available 1-Z-imidazole wherein Z is an amino protecting group. |
[0001] This invention relates to a process whereby 1,3"-di-N-unprotected-poly-N-protected-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols are selectively protected at the 3"-amino function and to novel 1-N-unprotected-poly-N-protected-4,6-di-o-(aminoglycosyl)-1,3-diaminocyclitols. The compounds are useful as intermediates in the preparation of 1-N-substituted-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols. very potent antibacterial agents such as netilmicin (1-N-ethylsisomicin).
[0002] U.S. Patent No. 4,002,742 describes a multistep
method whereby 4,6-di-O-(aminoglycosyl)-1,3-diaminocyciitols are converted to certain 1-N-unprotected-poly-N-protectmd-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols which are disclosed as intermediates in the preparation of 1-N-alkylated derivatives.
[0003] French Patent No. 74-23811 describes the per-N-formylation of an aminoglycoside which is further reacted to produce a 1-N-unprotected-poly-N-formylated aminoglycoside which can be further reacted to produce a 1-N-substituted aminoglycoside.
[0004] By this invention, a novel chemical process was developed whereby a 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol, also known herein as an aminoglycoside, is converted to a 1-N-unprotected-poly-N-protected aminoglycoside, many of which were heretofore unknown and could not be prepared by prior art processes.
[0005] The process of this invention resides in the concept of the selective protection of a 3"-amino function in a 1,3"- di-N-unprotected-poly-N-protected-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol, wherein the 6-0-aminoglycosyl has an amino function at the 3" position and hydroxyl functions at the 2" and 4" positions, and is characterised in that the 1,3"-di-N-unprotected-poly-N-protected-4,6-di-O-(aminoglycosyl)-l,3-diaminocyclitol is reacted in a solvent with no more than one molar equivalent of available 1-Z-imidazole, wherein Z is an amino protecting group selected from lower alkanoyl, lower alkoxycarbonyl, aralkoxycarbonyl, trichloroethoxycarbonyl and N-carbonylimidazole. Thereby are produced 1-N-unprotected-poly-N-protected-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols, said compounds being useful as intermediates in the preparation of 1-N-substituted aminoglycosides.
[0006] As used herein, the term "aminoglycoside" refers to a 4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitol, "per-N-protection" refers to protection on all the amino functions of an aminoglycoside, and "poly-N-protection" refers to protection on all amino functions unless otherwise excluded as, for instance, the terminology 1,3"-di-N-unprotected-poly-N-protected.
[0007] The novel process for the selective 3"-amino protection of 1,3"-di-N-unprotected-poly-N-protected aminoglycosides to produce 1-N-unprotected-poly-N-protected aminoglycosides was found in an effort to provide another process for the production of 1-N-substituted aminoglycosides, especially 1-N-ethylsisomicin, whereby side products/ reactions are minimized.
[0008] Chemical transformation on an aminoglycoside wherein the site of reaction is to be one of the amino functions should ideally be carried out on intermediates wherein every other amino function is selectively protected by a blocking group; otherwise, mixtures of various mcno-N-and poly-N-derivatives are formed which require tedious separation techniques (usually one to several column chromatographies) to isolate the desired transformation product. However, by the prior art methods, it is not always possible to prepare the ideal selectively blocked intermediate, so that with any of the amino functions unprotected, multiple products are produced, and the desired.transformation product is therefore obtained in low yields.
[0009] The present invention provides such ideally protected intermediates for the production of 1-N-substituted aminoglycosides with increased yields.
[0010] In U.S. Patent 4,002,742, which relates to the production of 1-N-substituted aminoglycosides, there is described a multistep method for preparing a 1-N-unprotected-poly-N-protected aminoglycoside, the sequence of steps being as follows:
Sisomicin is converted to:
a) Penta-N-carbobenzyloxysisomicin
b) 1,3',2',6'-tetra-N-carbobenzyloxy-3"-N-4"-O-carbonylsisomicin →
c) 3"-N-4"-O-carbonylsisomicin→
d) 2',3',6'-tri-N-t-butoxycarbonyl-3"-N-4"-O-carbonylsisomicin.
[0011] The aforementioned process produces the product in inherently low yields. Furthermore, this process cannot produce the N-alkanoyl protected compounds claimed in this invention.
[0012] The French Patent No. 74-23811 describes a method whereby, for example, kanamycin B is formylated to 1,3,2',6',3"- penta-N-formylated kanamycin B, thence treated with base to produce 3,2',6',3"-tetra-N-formylated kanamycin B. The aforementioned process is, however, restricted to the preparation of formyl derivatives, and is such that the product is produced in low yields (15 - 25%).
[0013] In view of the heretofore inherent problems in blocking all the amino groups, regardless of configuration, other than the 1-N-position in an aminoglycoside, it has been the practice to block some of the amino functions (keeping 1 unblocked), then introduce the 1-N-substituent. Of course, inherent in this is the possibility of getting a multiplicity of compounds wherein substitution has taken place at every unblocked amino function.
[0014] The process of this invention utilizes as starting compounds 1,3"-di-N-unprotected-poly-N-protected aminoglycosides which are prepared by using a transition-metal (e.g. copper) complexing process,said process being described in German Offenlegungsschrift (DOS) 27 26 712..
[0015] Heretofore, if the aforementioned 1,3"-di-N-unprotected-poly-N-protected-aminoglycosideSwere further acylated by the prior art methods, it would be expected that the 1-amino function would be preferentially acylated, not the 3"-amino function since the 1 amino function is primary and less hindered than the 3"-amino function, the latter having neighboring 2" and 4"-hydroxyl functions, and in the case of aminoglycosides such as sisomicin and gentamicin, being a secondary amine. Indeed this is illustrated in Example 3 wherein 1,3"-di-N-unprotected-3,2',6'-tri-N-(trichloroethoxycarbonyl) sisomicin is reacted with N-acetoxysuccinimide, deblocked at the 3,2',6'-amino functions to produce 1-N-acetylsisomicin.
[0016] By this invention it was found that in a 1,3"-di-N-unprotected-poly-N-protected aminoglycoside, the 3"-N-position can preferentially be protected by means of 1-Z-imidazole, this reaction taking place cleanly and in good yields. The 1-Z-imidazole is a compound wherein Z is an amino protecting group which is lower alkanoyl (acetyl, propionyl, butyryl, trifluoroacetyl), lower alkoxycarbonyl (ethoxycarbonyl, t-butoxycarbonyl, trichloroethoxycarbonyl), aralkoxycarbonyl (benzyloxycarbonyl) and N-carbonylimidazole. In this invention, 1-Z-imidazole is preferentially 1-lower alkanoyl-imidazcle, 1-acetylimidazole being the most preferred.
[0017] The 1-Z imidazoles .are a known class of compounds. Those which are not readily available are conveniently prepared by reaction of imidazole with the appropriate acyl chloride or chloroformate.
[0018] By this process the 1,3"-di-N-unprotected-poly-N-protected aminoglycoside is allowed to react with a 1-Z-imidazole in an inert organic solvent, wster and/or lower alkanol at ambient temperatures for a period of from about 0.5 - 24 hours. Using separation techniques described hereinafter, the 1-N-unprotected-poly-N-protected aminoglycoside is obtained. For example, 3,2',6'-tri-N-acetylsisomicin is reacted with acetylimidazole in a water/tetrahydrofuran mixture at room temperature for about 2 hours, then isolated utilizing known techniques to obtain 3,2',6',3"- tetra-N-acetylsisomicin.
[0019] The inert organic solvents employed in this process are usually water miscible and will not take part, in the reaction, e.g. tetrahydrofuran, dioxane dimethoxyethane, dimethylformamide and acetonitrile. In the instances where inert organic solvents are utilined alone, the ratio of the imidazole to the aminoglycoside is about equimolar. When the reaction is carried out in a solvent containing water and/or lower alkanol (wherein the lower alkanol has up to 4 carbon atoms), the molar ratio of the imidazole- to the aminoglycoside should be up to 2:1, as the water/ lower alkanol solvents will partially react with the imidazole compounds.
[0020] The ambient temperature utilized in this reaction can be from 5°C to about 40°C. The reaction is generally a rapid one, from about 0.5 - 2 hours but can be run over a longer period of time, up to about 24 hours.
[0021] When, in this process 1-Z-imidazole is utilized wherein Z is lower alkancyl, the lowar alkanoyl protects only the 3"-N-position. However, when Z is other than lower alkanoyl, the protecting group will bridge the 3"-N and 4"-O-positions to become a 3",4"-N,O-carbonyl moiety. When gentamicin A, Antibiotic 66-40B, kanamycin A, kanamycin B, 3',4'-dideoxykanamycin B and tobramycin are utilised, a mixture will be obtained wherein bridging will occur not only at the 3"-N and 4"-O-positions, but also at the 3"-N and 2"-O-positions.
[0022] A preferred group of starting materials are 1,3"-di-N- unprotected-pcly-N-protected sentamicin A, gentamicin B, gentamicin B1, gentamicin C1, gentamicin C1a, gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin X2. Antibiotic G-52, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic G-418, Antibiotic JI-20A, Antibiotic JI-20B, kanamycin A, kanamycin B, 3',4'-dideoxykanamycin B, verdamicin, sisomicin, tobranycin and the 5-epi, 5-deoxy, 5-epi-fluorc-5-deoxy analogs of the foregoing. 3,2',6'-tri-N-acetylsisomicin and 3,6'-di-N-acetylkanamycin A are specifically useful compounds.
[0023] The,5-epi-, 5-deoxy-; 5-epi-fluoro-5-deoxy- analogs are best prepared prior to the introduction of amino protecting groups. The preparation of 5-epi-aminoglycosides is described in U.S. Patent 4,000,261 and the preparation of 5-deoxy-aminoglycosides is described in U.S. Patent 4,053, 591.
[0024] The 5-epi-fluoro-5-deoxy aminoglycosides (e.g. 5-epi- fluoro-5-deoxysisomicin) are prepared by reacting an aminoglycoside having a 5-hydroxyl function (e.g. sisomicin) and having all its amino and hydroxyl functions protected, other than the 5-hydroxyl function, with a dialkylamino- sulfur trifluoride (e.g. diethylaminosulfur trifluoride) in an inert organic solvent in the temperature range of from -100°C to about -50°C, followed by removal of the protecting groups (c.f. Example 4).
[0025] The preparation of 2-N-substituted aminoglycosides, for which the compounds or this invention are useful as intermediates, are desorlbed in U.S. Patent 4,002,742. It has been found, however, that when utilizing a 1-N-unprotected-poly-N-acylaminoglycoside (e.g. 3,2',6',3"- tewra-N-acetylsisomiein) for conversion to a 1-N-ethyl- aminoglycoside, (e.g. 1-N-stnylsisomicin), a convenient hydride conor reducing agoht to be employed is sodium bore- hydride.
[0026] In its product aspect, this invention relates to novel
aminoglycosides. partion- larly, this invention relates to novel 1-N-unprotected-poly-N-R-3"-N-Z'-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols. selected from
3;2'-di-N-R-3''-N-Z'-gentamicin A,
3,6'-di-N-R-3"-N-Z -gentamicin B,
3,6 -di-N-R-3"-N-Z'-gentamicin B1,
3.2',6'-tri-N-R--3"-N-Z-gentamicin C1,
3.2',6'-tri-N-R-3"-N-Z'-gentamicin C1a,
3,2'.6'-tri-N-R-3''-N-Z'-gentamicin C2,
3,2-,5 -tri-N-R-3"-N-Z-gentamicin C2a,
3,2'5-tri-N-R-3"-N-Z'-gentamicin C2b,
3,2-di-UN-R-3"-N-Z'-genamicin X2,
3,2',5'-tri-N-R-3"-N-Z'-Antibiotic G-52,
66-4OE,
3,2',6'-tri-N-R-3"-N-Z'-Antibiotic 66-40D.
3,2'-di-N-R-3"-N-Z'-Antibiotic G-418,
3,2',6'-tri-N-R-3"-N-Z'-Antibiotic JI-20A,
3,2',6'-tri-N-R-3"-N-Z'-Antihiotic JI-20B,
3,6'-di-N-R-3"-N-Z'-kanamycin A,
3,2',6'-tri-N-R-3"-N-Z'-kanamycin B,
3,2',6'-tri-N-R-3"-N-Z'-3',4'-dideoxykanamycin B,
3,2',6'-tri-N-R-3"-N-Z'-verdamicin,
3,2',6'-tri-N-R-3"-N-Z'-sisomicin,
3,2',6'-tri-N-R-3"-N-Z'-tobramycin, and
the 5-epi, 5-deoxy, 5-epi-fluoro-5-deoxy analogs of the foregoing, wherein R is lower alkanoyl, aroyl, lower alkoxycarbonyl, trichloroethoxycarbonyl, aralkoxycarbonyl, and Z' is lower alkanoyl with at least two carbon atoms.[0027] Of these the derivatives of gentamicins B, B1 and C1a, kanamycin A and sisomicin represent a preferred range of compounds.
[0028] The definitions of the protecting groups have baen described hereinabove in more detail. Specifically, preferred is that both R and Z'be lower alkanoyl, preferentially acetyl.
[0029] Particularly valuable compounds of this invention are 3,2', 6',3"-tetr-N-acetylsisomicin, which compound is used obtain
and 3,6',3"-tri-N-acetylkanamycin A.
[0030] The term "lower" as usea herein for defining protecting groups R, Z and Z' comprises such radicals having up to six carbon atoms.
EXAMPLE 1
1-N-Unsubstituted-poly-N-protected-4,6-di-O-(aminagly- cosyl)-1,3-diaminocyclitols
A. 3,2',6'3"-tetra-N-acetylsisomicin
[0032] To a solution of 3 g (5.2 mmol) 3,2',6'-tri-N-acctyl sisomicin in 150 ml of water, add 572 mg (5.2 mmol) of 1-acetylimidazole in 8 ml of tetrahydrofuran. Stir the solution at room temperature for 30 minutes and then add 572 mg (5.2 mmol) of acetylimidazole. Stir the solution a further 2 hours and then evaporate off the solvent and chromatograph the residue on 150 g of silica gsl, eluting with the lower phase of a chloroform-methanol-15% ammonium hydroxide (2:1:1) solvent mixture to obtain 3,2',6',3"- tetra-N-acetylsisomidin (1.96 g, 61% yield)
+ 204° (H2O):δ(D2O) 1.05 (rotamers, C-CH3), 2.25, 2.05, 1.95 (12H, COCH3), 3.1, 3.0 (3H, rotamers, N-CH3), 4.85 (1H, m, H-4'), 5.3 (1H, m, H-1", J = 4 Hz) and 5.5 ppm (1H, d, J = 2 Hz, H-1').
B. 3,6',3"-Tri-N-acetylkanamycin A
[0033]
(1) Dissolve 300 mg of kanamycin A and 1.9 g of cupric acetate hydrate in 13.5 ml
of dimethylformamide and 4 ml of water and stir the mixture at 25°C for 30 minutes.
This add 0.17 g of acetic anhydride in 2 ml of dimethylformamide and stin for 1 hour
at 25°C. Bubble hydrogen sulfide through the solution, filter off the solids and wash
with dimethylformamide. Evaporate the filtrate to dryness and chromatograph the residue
on a silica gel column (110 x 2.3 cm, asing chloroform-methanol-3.5 % ammonium hydro-
mide (1:2:1) as the eluant to obtain 3,6'-di-N-acetylkanamycin A (130 mg, 37% yield);
+ 94.6° (H2O); [θ]290 + 1002 (TaCu); δ(D2) 2.05 (3H, s, NAc), 2.08 (3H, s, NAc), 5.13 (1H, m, H1") and 5.41 ppm (1H, m, H1').
(2) To a solution of 100 mg of 3,6'-di-N-acetylkanamycin A in le ml of (1:1) tetrahydrofuran-water,
add 29.1 mg of acetylimidazole and stir the mixture at 25°C for 23 hours. Evaporate
the solution to dryness and chromatograph the residue on a silica gel column (110
x 1.5 cm) using chloroform-methanol-3,5% ammonium hydroxide (1:2:1) as the eluant
to obtain 3,6',3"-tri-N-acetylkanamycin A (41 mg, 38% yield)
+ 104.7° (H2O); δ (D2O) 2.02 (3H, s, NAc), 2.07 (3H, s, NAc), 2.09 (3H, s, NAc), 5.17 (1H, d, J1", 2", 3.5 Hz, H1"), and 5.41 ppm (1H, d, J1', 2' 3.0 Hz, H1').
1-N-unsubstituted-poly-N-acetyl-4,6-di-O-(aminogly- syl)-1,3-diaminocyclitols
[0034]
stmilar to Example lA, by utilizing as starting
a) 3,2'-di-N-acetylgentamicin A,
b) 3,6'-di-N-acetylgentamicin B,
c) 3,6'-di-N-acetylgentamicin B1,
d) 3,2',6'-tri-N-acetylgentamicin C1,
e) 3,2',6'-tri-N-acetylgentamicin Cla'
f) 3,2',6'-tri-N-acetylgentamicin C2,
g) 3,2',6'-tri-N-acetylgentamicin C2a,
h) 3,2',6'-tri-N-acetylgentamicin C2b'
i) 3,2'-di-N-acetylgantamicin X2,
j) 3,2',6'-tri-N-acetyl Antibiotic G-52,
k) 3,2',6'-tri-N-acetyl Antibiotic 66-40B,
1) 3,2',6'-tri-N-acetyl Antibiotic 66-40D,
m) 3,2'-di-N-acetyl Antibiotic G-418,
n) 3,2',6'-tri-N-acetyl Antibiotic JI-20A,
o) 3,2',6'-tri-N-acetyl Antibiotic JI-20B,
p) 3,2',6'-tri-N-acetylkanamycin B,
q) 3,2',6'-tri-N-acetyl-3',4'-dideoxykanamycin B,
r) 3,2',6'-tri-N-acetylverdamicin,
s) 3,2',6'-tri-N-acetyltobramycin, and
t) the 5-epi, 5-deoxy, 5-epi-fluoro-5-deoxy analogs of the foregoing and of 3,2',6'-tri-N-acetylsisomicin and 3,6'-di-N-acetylkanamycin A there are obtained respectively:
a) 3,2',3"-tri-N-acetylgentamicin A,
b) 3,6',3"-tri-N-aeetylgentamicin B,
c) 3,6',3"-tri-N-acetylgentamicin B ,
d) 3,2',6',3"-tstra-N-acatylgentamicin C1,
e) 3,2',6",3"-tetra-N-acetylgentamicin C1a,
f) 3,2',6',3"-tetra-N-acetylgentamicin C2,
g) 3,2',6',3"-tetra-N-acetylgentamicin C2a,
h) 3,2',6',3"-tetra-N-acetylgentamicin C2b,
i) 3,2',3"-tri-N-acetylgentamicin X2,
j) 3,2',6',3"-tetra-N-acetyl Antibiotic G-52,
k) 3,2',6',3"-tetra-N-acetyl Antibiotic 66-40B,
1) 3,2',6',3"-tetra-N-acetyl Antibiotic 66-40D,
m) 3,2',3"-tri-N-acetyl Antibiotic G-418,
n) 3,2',6',3"-tetra-N-acetyl Antibiotic JI-20A,
o) 3.2',6',3"-tetra-N-acetyl Antibiotic JI-20B,
p) 3,2',6',3"-tetra-N-acetylkanamycin B,
g) 3,2',6',3"-tetra-N-acetyl-3',4'-dideoxykanamycin B,
r) 3,2',6',3"-tetra-N-acetylverdamicin,
s) 3,2',6'.3"-tetra-N-acetyltobramycin, and
t) the 5-epi, 5-deoxy, 5-epi-fluoro-5-deoxy analogs of the foregoing and of 3,2',6',3"-tetra-N-acetylsisomicin, and of 3,6',3"-tri-N-acetylkanamycin A.
D. 3,2',6'-Tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N- acetylgentamicin C1a
[0035] Dissolve 500 mg of 3,2',6'-tri-N-(2,2,2-trichldroethoxy- carbonyl)=gentamicin C1a in 20 ml (1:1) tetrahydrofuran and water. Add 84.6 mg of acetyl imidazole dissolved in
ml tetrahydrofuran. Stir the solution at room temperature for 2 hours. Evaporate the tetrahydrofuran and decant off the remaining water. Dissolve the resultant residue in a small amount of chloroform and wash with water. Evaporate the chloroform and azeotrope the resultant residue with benzene. Chromatograph the azeotroped resultant residue on a silica gel column (30 x 3 cm) eluting with 10% methanol/ chlorofrom. Combine the fractions to obtain 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-acetyl gentamicin Cla (428 mg, 82% yield); [α]26D + 69.9° (CHC3); νmax. (KBr) 1720, 1620 cm.-1; δ(CDCl3) 1.08 (3H, s, 4"-CH3), 2.16 (3H, s, NAc), 3.12 (3H, s, 3"-N-CH3) and 4.76 ppm (6H, s, CO2CH2CCl3).
E. 3,2'.6'-Tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N, 4"-O-carbonylqentamicin C1a
[0036]
(1) Dissolve 20 g of imidazole in 180 ml of tetrahydrofuran; cool the resultant solution to 0°C. To this solution, add 31 g of 2,2,2-trichloroethylchloroformate in 180 ml of tetrahydrofuran dropwise over a period of 1 hour. Then stir the mixture at 25°C for 1 hour, filter the mixture and concentrate the filtrate to dryness. Wash the resultant solid with water and dry to obtain 1-N-(2,2,2-trichloroethoxycarbonyl)-imidazole: m.p. = 80°C: νmax. (CSCl3) 3000, 1780, 1400, 1310, 1280, 1235, 1165, 1015 cm -1; δ(CDCl3) 5.03 (2H, s, -CH2CCl3), 7.13 (1H, m, H4), 7.48 (1H, m, H5) and 8.20 ppm (1H, s, H2).
(2) To a solution of 500 mg of 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-gentamicin
Cla in 10 ml of anhydrous tetrahydrofuran, add 624 mg of l-N-(2,2,2-trichloroethoxycarbonyl)-imidazole
and then allow the solution to remain at 25°C for 24 hours. Evaporate the solution,
dissolve the residue in ethyl acetate and wash with water, then dry the ethyl acetate
layer over MgSO4. Filter the ethyl acetate layer, evaporate to dryness and chromatograph the residue
on a silica gel column (110 x 2.5 cm) using 4% methanol in chloroform as the eluant
to obtain 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin
C1a (290 mg, 56% yield)
+ 78.7° (CHCl3), max. (KBr) 3375, 2940, 1730, 1520 cm-1; δ (CDCL3) 1.38 (3H, s, 4"-CH3), 2.98 (3H, s, 3"-NCH3), 4.77 (6H, broad s, -CH2CCl3), 5.08 (1H, m, H1"), and 5.43 ppm (lH, m, H1').
F. 1-N-unsubstituted-3"-N-4"-O-carbonvl-poly-N-(2,2,2-trichloroethoxycarbonyl)-4,6-di-O-(aminocrosyl)-1,3-diaminocvclitols
[0037] In a manner similar to Example 1E there are obtained the following compounds, respectively:
a) 3,5'-di-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin B,
b) 3,6'-di-N-(2,2,2-trichloroethoxycarbonyl)-3"N-6"-O-carbonylgentamicin B1,
c) 3,2',6-using-N- (2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin C1,
d) 3,2',6'-tri-N-(2,2,2-trichlorethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin C2'
e) 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin C2a,
f) 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin C2b,
g) 3,2'-di-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonylgentamicin X2,
h) 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonyl Antibiotic G-52,
i) 3,2',6-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonyl Antibiotic 66-40D,
j) 3,2'-di-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonyl Antibiotic G-418,
k) 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonyl Antibiotic JI-20A,
1) 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-O-carbonyl Antibiotic JI-20B,
m) 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4"-0-carbonylsisomicin,
n) 3,2',6-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N-4'-O-carbonylverdamicin, and
the 5-epi, 5-deoxy, 5-epi-fluoro-5-deoxy analogs of the foregoing and of 3,2',6'-tri-N-(2,2,2-trichloroethoxycarbonyl)-3"-N,4"-O-carbonylgentamicin Cla.EXAMPLE 2
1-N-Substituted-4,6-di-O-(aminoglycosyl)-1,3-diminocycli- tols
A. 1-N-Ethylsisomicin
[0038] To a stirred solution of 40.5 g of 3,2',6',3"-tetra-N-acetylsisomicin in 500 ml of isopropanol, add 6.5 ml of acetaldehyde and 2.3 g of sodium borohydride in 100 ml of isopropanol, continue stirring for 3 hours at.room temperature. After 3 hours, add water and then remove the isopropanol by evaporation. Add 150 ml of 50% sodium hydroxide to the residue and heat at 100°C for 18 hours in an argon atmosphere. Cool the solution, adjust the pH to 6 and treat the solution with 2.15 liter of IRC-50 (NH4+) ion exchange resin, wash the resin with water and elute with 2N aqueous ammonium hydroxide. Concentrate the ammoniacal eluant to dryness and dissolve the residue in the lower phase of a chloroform-isopropanol-14% ammonium hydroxide (2:1:1) solvent mixture and chromatograph on a 300 g silica gel column utilizing the same solvent to obtain 1-N-ethylsisomicin (25 gm, 80% yield).
like eluates and evaporate and pass the residue through a column of IRA-401S (OH-) ion exchange resin, eluting with water. The eluate was lyophilized to give 1-N-acetylsisomicin identical with authentic material. (0.9 gm - 60% yield).
EXAMPLE 4
5-Epi-fluoro-5-deoxysisomicin
[0039] A. To a stirred solution of 4 g of 1,3,2',6'-tetra-N-benzyloxycarbonyl-2"-O-benzoyl-3",4"-N,O-carbonylsiso- micin in 60 ml of dry methylene chloride at -78°C under an atmosphere of argon, add 3 ml (6 eq.) of diethylaminosulfur trifluoride (DAST). Stir the reaction mixture at -78°C for two hours and then allow to warm to 0°C. Add 50 ml of 5% sodium bicarbonate and separate the organic phase. Wash this organic phase with 50 ml water and then dry over 25 gm sodium sulfate. Evaporate the methylene chloride under vacuum. Triturate the residual yellow foam with a small volume of ether to obtain 3.6 g of 5-epi- fluoro-5-deoxy-1,3;2',6'-tetra-N-benzyloxycarbonyl-2"-O-benzoyl-3",4"-N,O-carbonylsisomicin as a white solid; m.p. = 225°C.
D. 1-N-Ethyl-4,5-di-D(amince)-1,3-diaminocyclitois
[0040]
1. In a manner similar to Example 1A, by utilizing the products of Example 1A, B and C there are obtained the corresponding 1-N-ethyl-4,6-di-O-(amincglycosyl)-1,3-diaminocyclitols.
2. In a manner similar to Example 2A, by utilizing the products of Example 1E and F there are obtained the corresponding 1-N-ethyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols.
EXAMPLE 3
1-N-Acetylsisomicin
[0041] To a stirred solution of 3.0 g of 3,2',6'-tri-N-(tri- chloroethcxycarbonyl) sisomicin in 75 ml of methanol and 15 ml of water, add 0.55 g of N-acetaxysuccinimide. Stir the reactants for 20 hours at room temperature, then add 7 ml of acetic acid and 6 g of zinc dust and heat the mixture for 2.5 hours. Cool and filter the reaction, washing with methanol. Evaporate the filtrate and dissolve the residue in 30 ml of water, add a solution of 3 g sodium carbonate in 600 ml of water, and heat to boiling. Filter the formed salts and wash the salts with water. Evaporate the filtrate and reflux the residue with 150 ml of isopropanol. Filter, evaporate the filtrate and chromatograph the residue on 70 g of gilica gel, eluting with chloroform, methanol, concentrated ammonia (3:1:0.15). Combine B. To a solution of 4 g of 5-epi-fluoro-5-deoxy-1,3,2', 6'-tetra-N-benzyloxycarbonyl-2"-O-benzoyl-3",4"-N,O-carbo- nylsisomicin in 5 ml of tetrahydrofuran and 60 ml liquid ammonia slowly add 2 g of sodium with stirring. Continue to stir for two hours, then add 15 ml of methanol dropwise and allow the ammonia to evaporate by warming to room temperature overnight. Dissolve the resultant residue in 10 ml 5% sodium hydroxide, and heat at 100°C for two hours under an atmosphere of argon. Cool and pass the solution through IRC-50 (H+) resin. Wash the resin with water and elute the product with 100 ml of 1N ammcnium hydroxide. Concentrate the ammonium hydroxide eluates to a residue comprising 5-epi-fluoro-5-deoxysisomicin. Purify the product by chromatographing on a silica gel column eluting with the lower phase of a chloroform-methanol-15% ammonium hydroxide (2:1:1) solvent system. Combine the like eluates as determined by thin layer chromatography and lyophilize to a residue to obtain 750 mg 5-epi-fluoro-5-deoxysisomicin as a white solid; m.p. 100°C - 102°C;
+ 200° (MeOH); PMR (100 MHz) (D2O), 5.1 (1H, d, J=57.0 Hz, 5-H), 5.13 (1H, d, J=2.5 Hz, 1'-H), 5.03 (lH, d, J=4.0 Hz, 1"-H), 4.88 (1H, br, s, 4'-H), 3.85 (lH, d, J=12.5 Hz, 5 "-H), 3.36 (lH, d, J= 12.5 Hz, 5a''-H), 2.56 (lH, d, J=10.5 Hz, 3"-H), 2.47 (3H, s, 3"-N-CH ), 1.17 (3H, s, 4'-C-CH3).
3,2'-di-N-R-3"-N-Z'-gentamicin A,
3,6'-di-N-R-3"-N-Z'-gentamicin B,
3,6',di-N-R-3"-N-Z'-gentamicin B1,
3,2',6'- tri-N-R-3"-N-Z'-gentamicin Cl,
3,2',6'-tri-N-R-3"-N-Z'-gentamicin C1a,
3,2',6'-tri-N-R-3"-N-Z'-gentamiein C2,
3,2',6'-tri-N-R-3"-N-Z'-gentamicin C2a,
3,2',6'-tri-N-R-3"-N-Z'-gentamicin C2b,
3,2'-di-N-R-3"-N-Z'-gentamicin X2,
3,2',61-tri-N-R-3"-N-Z'-Antibiotic G-52,
3,2',6'-tri-N-R-3"-N-Z'-Antibiotic 66-40B,
3,2',6'-tri-N-R-3"-N-Z'-Antibiotic 66-40D,
3,2'-di-N-R-3"-N-Z'-Antibiotic G-418,
3,21,61-tri-N-R-3"-N-Z'-Antibiotic JI-20A,
3,2',6'-tri-N-R-3"-N-Z'-Antibiotic JI-20B,
.3,6'-di-N-R-3"-N-Z'-kanamycin A,
3,2',6'-tri-N-R-3"-N-Z'-kanamycin B,
3,2',6'-tri-N-R-3"-N-Z'-3',4'-dideoxykanamycin B,
3,2',6'-tri-N-R-3"-N-Z'-verdamicin,
3,2',6'-tri-N-R-3"-N-Z'-sisomicin,
3,2',6'-tri-N-R-3"-N-Z'-tobramycin and
the 5-epi, 5-deoxy, 5-epi-fluoro-5-deoxy analogs of the foregoing wherein R is lower alkanoyl, aroyl, lower alkoxycarbonyl, trichloroethoxycarbonyl or aralkoxycarbonyl, and Z' is lower alkanoyl with at least two carbon atoms.
3,6'-di-N-R-3"-N-Z'-gentamicin B,
3,6'-di-N-R-3"-N-Z'-gentamicin B
3,2',6'-tri-N-R-3"-N-Z'-gentamicin C1a,
3,6'-di-N-R-3"-N-Z'-kanamycin A, and
3,2',6'-tri-N-R-3"-N-Z'-sisomicin
wherein R and Z' are as defined in claim 10.