Note: Descriptions are shown in the official language in which they were submitted.
~2717~4
The present invention relates to the preparation
of N-substituted derivatives of aminoglycosides which are useful
as antibiotics or as intermediates in the preparation of amino-
glycoside antibiotics.
Microorganisms are known to frecluently acquire
resistance to aminoglycoside antibiotics by a mechanism known in
the art as "R~Factors". Very generally an r'R~Factor" is the
extrachromosomal genetic capability of biochemically modifying
the antibiotics in such a way as to interfere with its antîbacterial
action, thereby enabling the organism to grow,
The phenomenon of bacterial resistance to amino-
glycoside antibiotics has fostered many new developments in the
area of semi-synthesis and chemical modification leading to second
and third generation aminoglycosides, The latter class comprise .
N-acylated and N-alkylated analogs that exhi~it much broader
antibacterial activities than the parent antibiotics~ Improved
methods for the selective N~acylation of aminoglycosides are in
demand as current procedures are confined mostly to the more
accessible sites, or are based on relative basicities,
. In a broad aspect the present invention provides
a method for the preparati.on of N-substituted derivatives of amino-
glycosides which comprises the steps of (a) complexing an am:Lno-
glycoside with divalent metal ion ~e1.ected .E~om t~le ~,roup con~q1st:irlg
of the cations of F~, Co, Ni., Cu, Pd, Ag and Pt to form a prot:ected
species having at least one amino group bound as a meta~ ion chelate
and at least one unbound amino gro~lp, (b) reacting the protected
species with an N--acylati.rlg reagent selected from the group con-
sl~ti.ng of carboxylic and sulfollic acid anhydrides, halides and
active esters to acylate at least one unbound amino group, and
'~ '
i 1/ -1-
~ 71744
(c) removing the amino-protecting chelate groups.
Included among the aminoglycosides useful in the
invention are kanamycin A, kanamycin B, kanamycin C, sisomicin,
gentamicin B, tobramycin, ribostamycin, butirosin A, butirosin B,
neomycin B, paromomycin I~ lividomycin A and lividomycin B.
Broadly speaking, suitable acylating reagents
include the anhydrides, halides and active esters of carboxylic
and sulfonic acids,
The term halide is taken to include azide in view
of the pseudo-halide reactivity of the anion _ N3.
Suitable carboxylic acid anhydrides, halides and
active esters include those of the formulae (R-CO) 2; R-COX and
R-COORl wherein R is
r~ _ N ~ ; - N ~ ; ~no ~ Q 2
O O
NO2
Preferred N~acylating reagents include the following:
(a) acetic anhydride O O
Il 11
CH3 - C - O - C - CH3
(b~ p-nltrophenylacetate O
Ct~3 - C - O ~ ~ - NO~,
(c~ N-acetoxy-5~norbornene-2,3-dicarboxamide
O O
- O - C - CH3
o
jl/ ~2_
"` 1271744
(d) N-benzyloxycarbonyloxy succinimide
O O
CN O C O C H 2 ~
te) N~ benzyloxycarbonylaminobutyroxy) succinimide
O O O
CN O C ( CH 2 ) 3 NH I - O CH 2 ~3
Il
o
(f) benzyl p~nitrophenyl carbonate
~ CH2 O C O ~NO2
(g) N-acetoxy succinimide
1l
CN - O C CH3
o
(h) N-benzyloxycarbonyloxy-5-norbornene-2, 3-dicarboxamide
O O
,- ~. ," 11
¢~ N o c o C ~1 2 ~
(i) benzyl pentachlorophenyl carbonate
C~ ~1 0
Cl ~ O - C - O - C~
Cl Cl
and
~ 3-
44
(j) benzyl-2,4-dinitrophenyl carbonate
O N ~2
CH2 - O - C ~ O ~ - NO2
Using the readily available kanamycin A as an
exemplary substrate because of the clinical importance of its
derivatives and the manipulative difficulties associated with
selective N-acylations of aminoglycosides eontaining multi amino
functions, the following derivatives can be prepared by seleetive
N-aeylati.on, according to the following reaction scheme, subsequent
to protection of vicinal amino aleohol functions as eopper(II)
ehelates by eomplexing kanamycin A with Cu ions:
~ UO
O ~ lating agent ~ ~
~ OH I NHR"
~---~~1~~~-- ~O ~ H
Scheme
1 R = aeetyl; R',R" ~ 11
2 R = benzyloxycarbonyl; R', R" = H
3 R,R',R" = acetyl
4 R,R~,R" - benzyloxycarbonyl
R,R" = acetyl; R' ~ H
6 R,R' - aeetyl; R" = H
`` 1~7~744
7 R ~ benzyloxycarbonyl; R',R1' - acetyl
8 R = benzyloxycarbonyl; R',R" = 4-benzyloxycarbonylaminobutyryl
9 R = H; R',R" = 4-aminobutyryl
R = H; R',R" = 2-hydroxy~4-benzyloxycarbonyLaminobutyryl
- The above derivatives are named as follows:
1 6'-N-acetyl kanamycin A,
2 6'-N-benzyloxycarbonyl kanamycin A,
3 1,3,6'~tri~-acetyl kanamycin A,
4 1,3,6'~tri-N_benzyloxycarbonyl kanamycin A,
1,6'-di~N-acetyl kanamycin A,
6 3,6'-di~N-acetyl kanamycin A,
7 6'-N-benzyloxycarbonyl_1,3~di-N-acetyl kanamycin A,
8 6'-N-benzyloxycarbonyl_1,3-di-N-(4-benzyloxycarbonylaminobutyryl)
kanamycin A,
_ 1,3-di~N-(4_aminobutyryl) kanamycin A, and
L0 6'-N-benzyloxycarbonyl-1,3~di-N-(2-hydroxy-4-benzyloxycarbonyl-
aminobutyryl) kanamycin A.
Derivative 2, i,e. 6'~N-benzyloxycarbonyl kanamycin
A, is a particularly important species in that it is a key inter-
mediate in the preparation of the antibiotic amikacin which is
taught in Kawaguchi et al U,S, Patent No. 3,781,268 issued December
25, 1973.
While Scheme 1, above, ilLustratcs chelation of
h~ o ~ ~1
A the 3" amino group with the 4" ~ Froul~ :Lt w:Lll be app~rent
to those ski.l:led ln t:lle art that all equal:l.y pr:Lmary chelation sit:e
~ ra~ I
is provided by the 3" amino group and the vicinal 2" h~dre:c~
group and that complexing wil:L undo~lbtedly occllr at both sites.
Kanamyc:l.n A has, as menti.oned previously, been
used as an exemplary am:inoglycoside both because of its ready
availability and the clinical importance of a number of its
jl/ _5~
~X~7:1~44
derivatives. It should however be understood that the
simple, selective N-acylation afforded by the present
invention as a consequence of temporarily protecting
certain amino groups as divalent metal ion chelates is
applicable to a broad range of aminoglycosides.
The primary chelation site(s) vary dependent
on the aminoglycoside, or the type of aminoglycoside,
employed.
n the case o~ tobramycin, an aminoglycoside
o pseudo trisaccharide of the kanamycin type, the primary
chelation sitets) are the same as in kanamycin A, i.e.
the complexing will involve the 3~ amino group with
either the 2" or 4~ hydroxyl group.
In the case of pseudotrisaccharides in the
4,5-disubstituted 2-deoxystreptamine series, for example
ribostamycin or its 6'-oH analog, butirosin A and
butirosin s, the primary chelation sites involve the C-
2'/C-3' and C-l/C-6 substituents.
pseudotetrasaccharides of the neomycin type,
for example neomycin B and paromomycin I, primarily
chelate at the C-2'/C-3' substituents, although
chelation is also prevalent in the D ring. The primary
chelation site of lividomycin A, another
pseudotetrasaccharide of the neomycin type, is at the
vicinal amino hydroxyl groups of the D ring.
As will be understood by those skilled in the
art, chelation should be carried out with appropriate
divalent cation/ligand concentrations. In general a low
divalent cation/ligand ratio is preferable to avoid
undue complexing of functions which are to be N-
acylated. stated differently, selectivity i9 relative
to availability, such that chelation will occur at
secondary or tertiary etc., sites when the primary sites
are chelated.
lX7174~
As stated previously, divalent cations of Fe, Co,
Ni, Cu, Pd, Ag and Pt are suitable for complexing with the amino-
glycoside to form the protected species which is subsequently
N-acylated. The preferred cation is Cu+ , for reasons including
availability and cost, although it will be obvious to those
skilled in the art that other divalent cations can be used in the
invention with equal efficacy.
The diva]ent cations are provided by dissolution
of suitable metal salts in a solvent which already contains an
aminoglycoside or which may be combined with an aminoglycoside
containing solution or to which may be added an amount of an
aminoglycoside.
Particularly preferred salts include CuS04.5H20;
Cu(CH3COO) 2 ,H20; Cu(NO3~2 and CuC12.
Deprotection of the aminoglycosides, that is
removal of the protective chelate following selective N-acylation~
can be con~eniently accomplished, in the case of product recovery
by column chromatography, by utilizing a basic eluent such that
deprotection and product recovery are effected in a single step.
The cations removed from the aminoglycoside tend to adsorb at the
top of the column. Other procedures for chelate removal will be
obvious to those skilled in the art.
The following non-lLTnl~t:Lve ~xalllples Ll:LIl~tra~e
the present inverltlorl.
Jl/ -7-
~2717~4
E X A M P L E
6'-N-Acetyl Kanamycin A
Kanamycin A free base (484 mg, 1 mmole) was
dissolved in 20 ml of distilled water, A solution of cupric
acetate monohydrate (150 mg, 0~75 mmole) in 5 ml distilled water
was then added dropwise and the resulting purple colored solution
was stirred for 5 minutes, p-Nitrophenylacetate (182 mg, 1 mmole)
in THF (25 ml) was added to the above solution, which immediately
turned green in color. After stirring for 23 h at 25, TH~ was
removed under reduced pressure at 25 and the solution poured into
acetone (300 ml). The green precipltate thus obtained was filtered,
washed with acetone and air~dried, The powdery material was then
suspended in 5 ml of ~ CH30H:0.05N ~H40H (1:3.2:2) and 28%
NH40H (0.1 ml) was added to obtain a clear deep blue solution.
This was then applied on a silica gel column (3.5 cm x 8.0 cm).
C J~C~3
The column was first eluted with CHC~,:CH30H:0,05N NH40H (1:3.2:2)
~c/3
(one bed volume) and then with-C~ECL~:CH30H:28% NH40H (1:3:2)
(10 ml fractions). The product was found in fractions 26-33,
which were combined and coevaporated with ethanol and dried in
vacuo to give colorless powder (430 mg, 82%), m,p, 213~215C,[~]D5 +
94.8 (Hzo), The material was analyzed by mass spectrometry and
found to have the correct 6tructllrc~.
7~744
E X A M P L E _ _
6~-N-Benzyloxycarbonyl Kanamycin A
. . .
Kanamycin A free base (250 mg, 0,515 mmole)
was dissolved in 5 ml of distilled water. To thls was added,
with stirring, a solution of cupric acetate (160 mg, 0.4 mmole~ in
5 ml distilled water, 3 ml of THF (tetrahydrofuran) was added
to the resulting purple solution, followed by the addition of a
solution of benzyl p-nitrophenylcarbonate (2.3 mg, 0.778 mmole)
dissolved in 13 ml THF, After stirring at room temperature for
12 h, the reaction mixture was slowly poured into acetone ~200 ml)
with stirring, The fine blue precipitate was col.lected by
filtration, washed with acetone followed by ether and air drled.
This precipitate was dissolved in 2 ml of a solvent mixture
(CHClg:CH30H 28~NH40H; 10:7:2.5~, applied on a silica gel column
(silicAR~ GF 254) and the product separated using the above
solvent system under mild suction from an aspirator, 6'~N-benzyloxy-
carbonyl kanamycin A free base (250 mg, 0,406 mmole) was isolated
in a 78.8% yield as an amorphous colorless powder; m.p. 204_212
(dec)C (lit:204-212(dec)C)l, [~]D5 + 115.6C (c 0.92, H20) .
(lit: + 116),
1. H, Kawaguch:L, T~ Naito, S. Nalcagaw~ ~nd K. Fu~ saw;.l, J. Anti-
biotlcs 25, 695 (.1.972),
lZ71744
E _~ M P L E 3
1,3,6'-t _ N ~cetyl Kanamycin ~
Kanamycin free base (242 mg, 0,5 mmole) was
dissolved in 10 ml of distilled water. Sodium bicarbonate (420 mg,
10 equivalent) was added followed by copper sulfate pentahydrate
(3.75 g, 15 mmole) in 50 ml cf distilled water, Tlle evolution of
carbon dioxide was allowed to cease and acetic anhydride (1 ml,
20 equivalent) added. After stirring overnight the mixture was
treated with 4 ml of acetylacetone and stirred for five minutes.
The copper acetyl-acetone complex was extracted with chloroform
(200 ml), then the aqueous layer treated with two drops of 28%
ammonium hydroxide and the solution extracted with chloroform
(100 ml). The inorganic material was precipitated by adding
absolute ethanol to the aqueous solution and filtered off. The
filtrate was treated with 1:1 ethanol:light petroleum ether
(b.p. 30-60C) to give crude 1,3,6'-N~acetyl kanamycin (300 mg3.
This was purified on a silica gel column using CHCL3:CH30H:0,05N
NH40H (1:3.2:2) as the solvent system to give 251 mg (82.5~) of
chromatographically pure product, m,p, 235-237C [a]D5 -~ 88,7
(c 1.06, H20); t.i.c, (CHCl3:CH30H:0.05N NH40H, 1:3.2:3), ~f 0 ~9,
Mass spectra (as N,0-permethyl derivatives) M~ 778 (molecular ion).
Spectroscopic data (l9Cmr) was in accord with the structure.
~ ~Z7~L744
E X A M P L E 4
1,3,6'-tri-N-Acetyl K namycin A
A solution of kanamycin free base (2.00 g, 4.13
mmole) in 200 ml of water was added to a stirred solution of
copper sulfate pentahydrate (1,03 g, 4.13 mmole) in 100 ml of
water, The resulting complex was treated with acetic anhydride
(3.9 ml, 4,22 mmole) and the solution stirred at room temperature
f~r five hours. The solution was extracted with ether and the
aqueous layer treated with hydrogen sulfide gas. The insoluble
black precipitate was filtered off over C,elite~ and the yellow
colored filtrate decolorized with activated charcoal, basified
with Dowex~ 1 x 8( OH~ resin, coevaporated with ethanol and dried
in vacuo (2,37 g), Chromatography of the residue over silica
gel with CHel9:CH30H:0.05N NH40H (1:3:1) 'as e].uent solvent afforded
~h~ 60.4~ yield as amorphous powder identical to the material
obtained in Example 3.
" 1~7~744
E X A ~ P L_E 5
1,3,6'-tri-N-Benzyloxycarbonyl ~Canamycin A
Cupric acetate (1.0 g, 5 mmole) in 20 ml distilled
water was added to a solution of kanamycin A free base (242 mg,
0.5 mmole) and sodium hydrogen bicarbonate (126 mg, 1,5 mmole) in
~rbo~7 d,ox ;1~
10 ml distilled water, The slow evolution of ~Lbv.ldl~xl~e was
allowed to cease, the mlxture treated with N-(benzyloxycarbonyl)
succinimide (623 mg, 2.5 mmole) in 25 ml tetrahydrofuran and the
solution sitrred at room temperature for ten hours. The mixture
was concentrated at 25C to 30 ml under reduced pressure and the
precipitated material filtered, washed with 100 ml acetone and air
dried (605 mg). Chromatography of this green powder over silica
~ o, 7; ~.s
gel with CHCl3:CH40H:28%:NH40H (10l ,5-~ -) as eluent gave
1,3,6'-tri-N-benzyloxycarbonyl kanamycin A (393 mg~ 85.8%), m,p.
258-263(dec~C*, [~]D5 + 83.9C (60%aq, THF); M. 1054 (on the
corresponding per-N,O-methyl derivative),
* T. Naito, S. Nakagawa, Y~ Abe, S, Toda, K. Fuiisawa, T~ ~iyaki,
fs'~,z) Q9~C~,`
H, }Coshigawa, H. Ohkuma and H, Ka~7agu~1i-a, J. Antibiotics 26,
297 (1973)-
" ~27~7~4
E X A M P L E 6
1,3,6'_tri~N-Benzyloxycarb =
A solution containing 6'-N-benzyloxycarbonyl
kanamycin A (205,5 mg, 0.33 mmole), from Example 2, in 10 ml of
distilled water was treated with cupric acetate monohydrate
(200 mg, 1 mmole) in 5 ml distilled water, To -this was added,
over thirty minutes, N-(benzyloxycarbonyloxy)-succinimide(164.5 mg,
0.66 mmole) in 10 ml tetrahydrofuran and the solution stirred at
room temperature for ten hours. Tetrahydrofuran was removed by
evaporation at 25C under reduced pressure and the mixture filtered,
washed with distilled water, acetone and finally with ether.
1,3,6'-tri~N-benzyloxycarbonyl kanamycin A (2~0 mg, 92.6%) was
isolated by silica gel column chromatography using CHCl3:CH30H:25%
NH40H (10:3:0.5) as eluent solvent. The physical properties proved
identical to those of the 1,3,6'-tri_N-benzyloxycarbonyl kanamycin
A of Example 5,
~27~744
E_AMPLE 7
Synthesis of 1,3,6'-tri-N-Acetyl Kanamycin A:1,6'-di-N
Acetyl_Kanamycin A and 3,6' -dl-N-Acetyl Kanamycin ~
A solution of copper sulfate pentahydrate
(75.2 mg, 0.3 mmole) in 2 ml of water was added to a
stirred solution of kanamycin A free base (192 mg, 0.4
mmole) in 8 ml water The resulting purple chelate was
treated with a solution of N-acetoxy-5-norbornene-2~3
dicarbo~amide (353.6 mg, 1.6 mmole) in water:
tetrahydrofuran (4:1, 4 ml) and the solution stirred at
room temperature for 1 1/2 hours. The copper chelate
was precipitated with acetone (100 ml) and filtered to
give a green solid. chromatography of this over silica
get with CHC13 : CH3OH : 0.05N NH40H (1:3.2:2) isolated
the following products:
Tetra-N-acetyl kanamycin A (14 mg, 6.2%), m p. 222-
225 (dec)C, Rf 0.71*;
1,3,6'-tri-N-acetyl kanamycin A (108 mg, 44.2~),
m~p. 235-237(de c)C, Rf 49* [~q 25 88
H2O);
1,6'-di-N-acetyl kanamycin A (56 mg, 24.6%), m.p.
228-231C, ~C]D + 92.1 (c 1.06" H20), Rf 0.37*
3l6~-di-N-acetyl kanamycin ~ (43 mg, 18.9%), m.p.
25206-208C, [C]D + 91.8 (c 0.98, H20), Rf 0.25*
The structures of these products were proved
by mass spectrometry and 13Cnmr spectrometry
*CHC13:MeOH:0.05N NH40H (1:3.2:2)
X
127:174~
E X A M P L E 8
6'-N-Benzyloxycarbonyl-1,3-d_-N-Acetyl Ka amycin A
A solution of copper sulfate pentahydrate (2.475 g,
9.9 mmole) in 30 ml of distilled water was added to a stirred
solution containing 6'~N-benzyloxycarbonyl kanamycin A (205.5 mg,
0.33 mmole), from Example 2, and sodiumbicarbonate (277,2 mg,
3.3 mmole) in 15 ml distilled water. The resulting slow evolution
of carbon dioxide was allowed to cease and the mixture heated with
acetic anhydride (0.66 ml, 6.6 mmole) and stirred at room temper-
ature for ten hours. The insoluble matter was filtered and dis~
carded and the filtrate evaporated to dryness under reduced
pressure. Chromatography of the residue on silica gel using
CHCl3:CH30H:0.05N NH40H (1:3,2:2) as eluting solvent afforded
A 189 mg (81,7~o) of 6'-N-benzyloxycarbonyl~1,3-di-N-acetyl kanamycin A,
m.p. 173-177(dec)C. The structure of the compound was confirmed,
using 13 Cmr spectroscopy, by the diagnostic ~-protonation shifts.
- l5 -
127~ 7d~
E X A M P L E 9
6'-N-Benzyloxycarbonyl~1,3-di-N-(4-Benzyloxycarbonylaminobutyryl)
Kanamycin A
A solution of 6~-N-benzyloxycarbonyl kanamycin A
(789.2 mg, 1,28 mmole), from Examp]e 2, in 10 ml o~ distilled water
was treated with cupric acetate monohydrate (255,5 mg, 1.28 mmole)
in 25 ml of distilled water and then diluted with 10 ml of -tetra-
hydrofuran. To this was added, over thirty minutes, N-hydroxy-
succinimide ester of 4-benzyloxycarbonylaminobutyric acid (479 g,
1.28 mmole) in 15 ml tetrahydrofuran and the solution stirred at
room temperature for ten hours. Ammonium hydroxide ~28%) was
added dropwise to the reaction mixture until a clear blue colored
solution was obtained. The product (Rf 0.44) was separated by
/0,,~ '~
A thick layer chromatography using CHCl3:CH30H:28% NH40H (~ }-~)
as solvent system. 6'-N-benzyloxycarbonyl-1,3-di-N-(4-benzyloxy-
carbonylaminobutyryl) kanamycin A was isolated (213 mg, 15.6%) as
an amorphous powder, m.p. 192 195(dec)C, M. 1252 (on corresponding
N,0-permethyl derivatives), The structure was further confirmed
from mass spectral and 13 Cmr spectroscopic studies.
~.~7~744
E X A M P L E 10
1,3-di-N-(4-Amino _t~ Kanamycin _
6'~N-ben7yloxycarbonyl 1,3-di-N-(4-ben~yloxy
carbonylaminobutyryl)kanamycin A (168 mg, 0.16 mmole), from
Example 9, in 20 ml of 60%aq tetrahydrofuran was brought to pH 4
with 3%aq hydrochloric acid. To this, 10% palladised charcoal
(100 mg) was added and the mixture hydrogenated at room temperature
for three hours. The mixture was filtered, concentrated under
reduced pressure and applied on a Dowex~ 1 x 8 ( OH) column.
/yo~ t'~
A The product was eluted with water and cyophiliscd'(97 mg, 91.0%),
m.p. 168-74C. The product was a chromatographically homogeneous
amorphous solid,
~:7~7~
E X A M P L_E 11
6'-N-Benzyloxycarbonyl-1,3-di-N-~2~Hydroxy-4~Benzyloxycarbonyl-
aminobutyryl) Kanamycin A _ _ _ _
Using the procedure of Example.9, but employing
N-hydroxysuccinimide ester of 2~hydroxy~4_benzyloxycarbonyl_
aminobutyric acid in place of N-hydroxysuccinimide ester of 4-
benæyloxycarbonylaminobutyric acid, there is obtained 6'~N-
benzyloxycarbonyl-1,3-di-N-(2-hydroxy-4-benzyloxycarbonylamino-
butyryl) kanamycin A.
Modifications within the true broad spirit and
scope of the invention will be obvious to those skilled in the art~
