Note: Descriptions are shown in the official language in which they were submitted.
356~3
This invention relates to a novel process for the
preparation of aminoglycoside antibiotics and with novel inter-
mediates for use in such process, and is particularly concerned
with a process for ~he preparation of l-N-substituted-kanamycin
derivatives and with selectively N-protected kanamycin deriva-
tives as intermediates for use in the process.
Examples of such l-N-substituted kanamycin derivatives
are described in co-pending Canadian Patent
~PC 5643) Application 238,246 others are known compounds such as l-N-
[4-amino-2-hydroxybutyryl]-kanamycin A (BB-K8) described in
British Patent Specification Number 1401221. In order to pre-
` pare such compounds from the readil~ available fermentation pro-
.
` duct kanamycin, it is desirable to protect some, or all, of the
amino groups other than the 1-amino group. Substitution may
then be effected preferentially on the amino group on the
l-position and isolation of the final l-N-substituted product
is thereby simplified. It is an object of this present inven-
tion to provide a process for the preparation of l-N-substituted
` kanamycin derivatives by providing such selectively N-protected
intermediates.
Thus, according to the invention there is provided a
process for preparing compounds of the formula:
`~ `
`
, '
--2--
.
,.
. . ~ "
.: - . . . -, .
:, -, :. : . , ; ,~ : , . .
, .; ~
s~;~
- ` ~
Ho ~' IH2NH2
H~ ~ ,
~'] ~1'
a ~ NE12
HO
~HO 1~ ` /
1 ... (I)
where R may be an amino or hydroxyl group and Rl may be a lower
alkyl or lower aliphatic alkanoyl group either of which may
optionally be substituted with hydroxyl and/or amino groups;
which process comprises acylation or alkylation of a compound of
the formula:
CH 2NHR3
H
H ~
~EIR
' ~ ~ ,
HO~
` ' ~H20H /\~H2
. ~ /
R N ~ /
...(II)
where R2 is a hydrogen atom or a benæyl group; R3 is a labile
amino-blocking acyl group as herein defined; and R~ is a hydr-
oxyl group or a group NHR3; to produce a cornpound oE the ~ormula:
--3--
: ,.......... .. :
, : -
, . ; ,
3~
Ho~R3
HO
R4~ 2
C'~
Hn ~ . \
HO ~ / ~ HR
R3N ~ ... (III~
wherein Rl to R are as previously defined; removal of the groups
; R2 ~if benzyl) and R3; and isolation of the compound of Formula
(I).
In this specification the term lower alkyl or lower
aliphatic alkanoyl indicates that such groups contain from 1 to
6 carbon atoms and may be straight or branched chain. The
labile amino-blocking acyl group R3 is an acyl group which may
be selectively removed from the compound of Formula (III) by
conventional techniques. In the case where Rl is a lower
aliphatic alkanoyl group, R3 may be a haloacetyl group, prefer-
; ably a trifluoroacetyl group, which may be removed by hydrolysis
under mild conditions, for example, with dilute ammonium hydr-
oxide solution, such that the alkanoyl group R is not affected.
R may also be a formyl or mono- or dichloro-acetyl group.
When Rl is a lower alkyl group, R3 may additionally comprise an
; acetyl group, a benzoyl group substituted in the aromatic ring
with, for example, a nitro group or one or more halogen atoms
or a lower alkoxy-carbonyl group, e.g., an etho~ycarbonyl or
methoxycarbonyl group. The~e groups require more vigorous
_ a~ -
,~ . . ....
IV~S~
hydrolysis c~n~itions for their removal and are not ~hereore
suitable when Rl i5 a lower alkanoyl group. Preferred labile
amino-blocking acyl groups for use in the process of the inven-
tion are the acetyl and trifluoroacetyl groups.
This process for the preparation of compounds of
Formu~a (I) comprises as an initial step acylation or alkyla-
tion of a compound of Formula (II) in order to introduce the
substituent Rl onto the amino group at the 1-position. Such a
reaction may be performed in a number of ways well known to those
skilled in the art. For example, acylation may be achieved
using an activated derivative of a lower-aliphatic alkanoic acid,
e.g., the N-hydroxysuccinimide ester, or by using the acid
chloride or anhydride. Alkylation may also be achieved by con-
ventional reactions, for example by reductive alkylation using
an appropriate aldehyde or ketone or an aldehyde derivative such
(PC 5756) as described in our Canadian SN 272,613 filed Feb. 24, 1~77,
or by reduction of the corresponding acylated derivative (e.g.,
with diborane). Naturally in the case where a compound of
Formula (II) is used wherein R2 is a hydrogen atom, reaction
will also take place on the 3-N-position bùt if only a slight
excess of reagent is used the required l-N-substituted isomer
; can comparatively easily be separated from the 3-N-substituted
isomer and from the 1,3-di-N-substituted product by conventional
methods, for example, by ion-exchange chromatography. This may
25 be done at this stage of the process or more conveniently after
removal of the amino-blocking groups.
The second step of the process comprises removal of
the amino-blocking groups R3, from the 2'-amino group, if
present, and the 6' and 3"-amino groups and also the benzyl
group, if present, from the 3-amino group. In some instances
.
:
~ ~ ' ', . .
.
.
wher~ the l-N-substituent itself be~rs an amino substituent
group it may be desirable to protect this yroup during -the course
of the process and it will then be necessary to remove this amino-
blocking group as well in the final step of the process~ There
:~ 5 are various conditions for completely removing amino-blocking
groups, well known to those skilled in the art, and they will
naturally depend on the nature of the protecting group employed
and the environment of the protected amine, and will, as already
mentioned, need to be chosen having regard for the substituent
; 10 on the N-l position. The medium employed may be anhydrous or
aqueous and in particular instances it may be acidic or basic to
various strengths. For example, the benzyl group, when present,
can be removed by catalytic hydrogenolysis in a conventional
manner in the presence of a palladium catalyst. Some acyl groups
may be removed by hydrolysis under mild basic conditions, for
example, the trifluoroacetyl group may be removed by treatment
with l-N ammonium hydroxide at room temperature for 24 hours,
while the acetyl, benzoyl and ethoxycarbonyl groups require more
vigorous conditions for their removal, e.g., heating with 5N
sodium hydroxide for several hours at 60 - 80C. The product (I)
may finally be purified, if desired, by conventional techniques,
for example, by crystallization or by chromatography.
The process of the invention is exemplified by the prep-
aration of l-N-[(S)-4-amino-2-hydroxybutyryl]-kanamycin A (BB-K8)
from 3-N-benzyl-3", 6'-di-N-trifluoroacetyl-kanamycin A. The
acylation reaction in this case is conveniently performed using
` the N-hydroxy-succinimide ester of (S)-4-benzyloxycarbonylamino-
2-hydroxy-butyric acid. The reaction is suitably carried out with
the reactants dissolved in an inert organic solvent, for example
tetrahydrofuran, and is conveniently performed by adding a 501u-
--6~
,
:
,;~ :. , :
.: . . :
..
.33S~
tion of the active ester to a solution of ~he k~namycin deriva-
tive at 0C. The reaction can be moni-tored by thin layer chroma-
tography and more active ester added if desired to ensure com-
plete reaction. The reaction is conveniently allowed to proceed
at room temperature and we have found that under these conditions
acylation is substantially complete within 48 hours. The product
is isolated by evaporation of the solvent and the product may be
purified at this stage, if desired, by conventional techniques
(e.g., crystallization or chromatography) but is more convenient-
ly used in crude form in the next step of the process.
~` Removal of the 3" and 6'-N-trifluoroacetyl groups is
achieved by mild base hydrolysis and this may be performed by
simply dissolving the product from the first step of the process
in lN ammonium hydroxide and allowing the solution to stand for
several hours (e.g., overnight) at room temperature. Finally the
benzyl and benzyloxycarbonyl groups may be removed together by
catalytic hydrogenolysis. This is conveniently performed by dis-
solving the product from the previous step in a suitable solvent,
e.g., a mixture of methanol, water and acetic acid and subjecting
the mixture to a conventional hydrogenation e.g., at 50 p.s.i.
and 40C. in the presence of a palladium catalyst. We have ~ound
that under these conditions deprotection is substantially com-
plete within 14 hours. The product is isolated, after filtration,
by evaporation of the solvent. Purification may then be achieved,
if desired, by for example ion-exchange chromatography, to give
the required product in pure form.
The process may be performed in an exactly analogous
manner, but starting with 311,6'-di-N-trifluoroacetyl-kanamycin A.
In this case the l-N-substituted product is formed, together with
the 3~N-substituted derivative and the l,3-N-disubstituted
--7--
. . . . . :: : ,. : . ~ . .
. .
... .. . , . . . . . ~ -
.:.. .. :. - -, . ., : ,.. : .: ~,.... .: . .
.::: .::: :, ,.- ,
3S6;~
product. However, the desired l-N-substituted product c~n be
readily separated from the other by-products, for example, by
the final ion-exchange chromatography step, although naturally
the product in this instance is obtained in lower yield.
Si~ilarly 2',3",6'-tri-N-trifluoroacetyl kanamycin B may be used
in the process to provide l-N-substituted kanamycin B derivatives.
The process of the invention is also exemplified by the
preparation of l-N-~(S)-4-amino-2-hydroxybutyl]-kanamycin A. In
this case the protected kanamycin intermediate (II) is first
alkylated, for example, by reductive alkylation with an aldehyde
[PC5756] derivative, such as described in our Co-pending Patent Applica-
tion 272,613 and the N-blocking groups are then removed and the
required product isolated. Thus when 3-benzyl-6-[S]-dihydroxy-
methyl-tetrahydro-1,3-oxazin-2-one is used in the reductive alkyla-
tion with 3",6'-di-N-acetyl kanamycin A, subsequent base
hydrolysis to remove the acetyl groups and hydrogenolysis to re-
move the benzyl group yields the required compound of Formula (I)
in which R is a hydroxy group and Rl is an (S)-4-amino-2-hydroxy-
butyl group. The reductive alkylation may conveniently be
achieved with the reagents dissolved in a suitable organic
solvent, e.g., dimethyiformamide using sodium borohydride and the
reactlon is generally complete within several hours at 30CC.
Removal of the acetyl groups is achieved by hydrolysis with 3N
sodium hydroxide at 80 for 4 hours and the benzyl group is remov-
ed by catalytic hydrogenation at 60C. and 60 p.s.i. for 16 hours.
The required product is then separated from the co-formed 3-N-
substituted isomer by chromatography.
The compounds of Formula (II) are themselves novel com-
pounds according to the invention. They may be prepared by a
selective 0~ N acyl miyration reaction. Thus ln one process for
--8--
'
.' ' . , '
- ~Vi5;3~;8
.
their preparation according to the invention an acid addi-
tion salt of kanamycin A or B or 3~N-benzyl-kanamycin A is first
treated with an excess of acylating agent under acidic conditions
such that initially only the hydroxyl groups are acylated.
Secondly, the acid addition salt o~ the C-acylated product, dis-
solved in an inert organic solvent is neutralized. Under these
conditions intramolecular acyl migration can take place onto any
amino group having an acyloxy group on an adjacent ring position,
i.e., the 6' and 3" amino groups and the 2' amino group in
kanamycin B. The remaining O-acyl groups are then removed in the
usual manner e.g., by hydrolysis or alcoholysis and the product
may be purified if desired e.g., by chromatography.
This process for the preparation of compounds of Formula
(II) has been found to be particularly effective for the prepara-
tion of the compounds wherein R4 is a hydroxyl group and R3 is a
trifluoroacetyl group. In this case kanamycin A or 3-N-benzyl
kanamycin A is dissolved in trifluoroacetic acid and treated at
O. with excess trifluoroacetic anhydride. Reaction is substan-
tially complste after several hours at OC., (e.g., overnight)
and the per-O-trifluoroacetyl-kanamycin derivative as its tri-
fluoroacetate salt may be isolated by evaporation of the solvents
under vacuum. The product is dissolved in an inert organic
solvent, preferably tetrahydrofuran, and neutralized by treating
with a base, for example, by stirring the solution with sodium or
potassium carbonate. We have found that under these conditions
the O-~N acyl migration reaction proceeds rapidly and is substan-
` tially complete within 20 minutes at room temperature. The re-
maining O-trifluoroacetyl groups are removed in a conventional
manner, e.g., by methanolysis, and the 3",6'-di-N-trifluoroacetyl
- 30 product may then be i~olated by evaporation of the ~olv~nt and
, _g_
.
.
. .. . ~ ~ . ~ , . .. .
- : ,. . :
"... , ., :
: . .. . ~. : , ,
~33S6~
puri~ied if desired by conventional colurnn chrom~tography.
As an alternative method of preparation, kanamycin A or
3-N-benzyl ~anamycin A is first treated with a reagent to intro-
duce selectively removable amino-bloc~ing groups. Suitable block-
ing groups are for example the t-butyloxycarbonyl group or the
benzyloxycarbonyl group. The ~ully N-protected product is then
O-acylated by known techniques for example by treatment with an
acid anhydride or chloride, e.g., acetic anhydride in pyridine or
with an alkyl chloroformate e.g., ethyl chloroformate, and the
amino-blocking groups are then removed (e.g., the t-butyloxycarb-
onyl groups are removed by treatment with trifluoroacetic acid
and the benzyloxycarbonyl groups are removed by catalytic hydro-
genolysis)0 The O-7N acyl migration can then proceed as before
and the remaining O-acyl groups are removed and the product
isolated as previously described.
The process may also be applied to kanamycin B, acyl
migration in this case additionally proceeding from the 3'-
hydroxyl group to the adjacent 2'-amino group to give a tri-N-
acylated intermediate.
The compounds of Formula (II) according to the inven-
tion, as well as those of Formula ~I) and (III), may exist in
; various conformational forms, and the invention is not lim ted toany one such ~orm thereof. Generally the rings are each in the
"chair" form, and each of the substituent groups, is disposed
~5 equatorially with respect to the ring. Furthermore, tha glycosidic
linkages between the hexopyranosyl rings and the 2-deoxystrept-
arnine ring are more usually X-linkages with respect to the ~ormer.
3-N-Benzyl-kanarnycin A is itself a novel compound. It
may be prepared by reductive alkylàtion of kanamycin A with benz-
aldehyde under carefully controlled pH conditions. We have dis-
--10--
. .
.... .. .. : - ......... .
" ' ' ' ' ', ', . :, ' ,
- .: . . . : ,: ,:
. ., ~ . . . .. .
. . : . - : : .. :
.: -:: .. , : . :
, .:-. , ~ : .
S68
covered that when kanamycin A in aqueous solution i5 subjected
to reductive alkylation at room temperature or below, with a
slight excess of benzaldehyde in the presence of sodium cyano-
borohydride and the pH of the solution is carefully adjusted to
6, then the major product from the reaction is 3-N-benzyl-
kanamycin A. Naturally minor amounts of the other N-substituted
isomers and poly-substituted products are also produced in the
reaction but these may be mainly separated ~y conventional ion-
exchange chromatography. The main fraction isolated from the
column by elution with ammonium hydroxide is 3-N-benzyl-kanamycin
A contaminated with a minor amount of the l-N-benzyl isomer. In
practice, this product is sufficiently pure to use directly in
; the process of the invention although naturally the l-N-benzyl
isomer present will lead, after acylation or alkylation and
deprotection, to the formation of the 3-N-substituted isomer as
a minor component together with the required l-N-substituted
product of Formula (I). It can, however, then be readily separat-
ed by the final chromatography step described.
In the following Examples, Example I describes the
preparation of 3-N-benzyl-kanamycin A. Examples 2 to 5 describe
the preparation of novel compounds of Formula (II) according to
the invention. Examples 6 to 9 illustrates the novel process of
the invention for preparing compounds of Formula (I).
Thin layer chromatography was performed on silica plates
using the solvent system stated. The spots were visualized after
drying the piates by spraying with a 5% solution of t-butyl-
hypochlorite in cyclohexane, drying the plates at 100 for 10
minutes in a ventilated oven, cooling and spraying with starch-
potassium iodide solution~
~` 30 Temperatures are given in C. "~mberlite" is a Registered
- . , . : . . . .
'' ' ' :'' ' , ......... ' ', ~, ', ' .
,
.
1~3SÇ;~
Trade Mark.
EX~MPLE 1
Kanamycin A sulphate (24.3 g., 0.03 mole) was dissolved
in water (150 ml.) and the pH adjusted to 6 by the dropwise addi-
tion of 5-N-hydrochloric acid. Sodium cyanoborohydride (1.95 g.,
0.03 mole) was added and the mixture was cooled to OC. and
stirred while a solution of benzaldehyde (3.61 g., 0.033 mole)
dissolved in methane (15 ml.) was added slowly over the course of
2-1/2 hours. The mixture was allowed to warm to room temperature.
After 16 hours the pH of the solution was adjusted to 5.5 by the
addition of l-N-hydrochloric acid and the solution was filtered
and added to a column of Amberlite CG-50 ion-exchange resin in
the ammonium-ion form. Elution firsk with water and then with a
gradient of ammonium hydroxide of increasing concentration from
0 - 0.7N gave as major product 3-N-benzyl-kanamycin A contaminat-
ed with some l-N-benzyl derivative (5.0 g., 28%) Rf 0.44 in
methanol, chloroform, 17~ ammonium hydroxide 4:1:2. (Kanamycin
A gave an Rf value of 0.15).
A sample was converted to the volatile tetra-N-acetyl-
hepta-O-trimethylsilyl derivative by treatment with acetic
anhydride in methanol at room temperature for 24 hours followed
by reaction with a 2:1 mixture of hexamethyldisilazane and tri-
methylchlorosilane at room temperature for 24 hours. m/e found
C54H106N4l5Si7 requires m/e 1246.
The positlon of substitution was confirmed by the
following sequence o reactions: (a) treatment with t-butyloxy-
carbonyl aæide gave a compound containing three t-butyloxycarb-
onyl groups as well as the benzyl group (from n.m.r.), (b) hydro-
genation to remve the benzyl group, (c) acylation with N-[(S)-4-
benzyloxycarbonylamino-2-hydroxy-butyryloxy]-suacinimide, and
-12-
- . , .:: ................. , : .
'''. '' '.' ' ': ', :
B3$~8
(d) ~:emoval of the N-protecting yroups by hydrogenakion ~ollowed
by treatment with trifluoroacetic acid gave, as major product,
3-N-[(S)-4-amino-2-hydroxybutyryl]-kanamycin A (BB-K29) identical
to a sample prepared according to the procedure of Naito eti al.,
(J. Antibiotics, 1973, 26, 297).
EXAMPLE 2
Trifluoroacetic anhydride (5.0 ml.) was added slowly to
a stirred solution of kanamycin ~ (1.0 g.) in trifluoroacetic acid
(40 ml.) at O. The solution was allowed to stand at O - 4~. for
20 hours. The solvent was then evaporated under vacuum and the
residue treated with toluene (10 ml.) and evaporated to dryness.
The trifluoroacetate salt was taken up in dry tetrahydrofuran and
neutralized by slowly adding to a stirred suspension of excess
anhydrous potassium carbonate in tetrahydrofuran. The mixture
was stirred at room temperature for 20 minutes and the suspension
was then filtered and the filtrate evaporated to dryness. The
product was taken up in methanol (20 ml.) and kept at room tem-
perature for 30 minutes. The solvent was evaporated under reduc-
ed pressure and the residue was chromatographed on silica, eluting
with a solvent gradient of chloroform, methanol (3:1) to chloro-
form, methanol, 17% ammonium hydroxide (8:4:1) to give
3",6'-di-N-trifluoro-acetyl-kanamycin A hydrate (0.52 g.~ dS a
white hygroscopic solid. Rf 0.7 in methanol, chloroform, 17%
ammonium hydroxide 4:1:1 (kanamycin A gave an Rf of 0.05).
Vc=o 1665 cm~
A sample was converted to the volatile di-N-acetyl-
hepta-O-trimethylsilyl derivative as described in Example 1. m/e
found 1264- C47H94N4015F6si7 requires m/e 126~-
EXAMPLE 3
-30 Trifluoroacetic anhydride (0.7 ml., 5 mmole) was added
-13-
.
" . : . . .. ..
,
~, ; ; , .
: ~ ,
~ ~3S~
slow'~y to a solution of 3-N-b~nzyl-kanamycin ~ (0.23 g., 0.4
mmole) in trifluoroacetic acid ~15 ml~) at 0. The solution was
kept at -4~. for 20 hours. The solvent was then evaporated and
the residue treated with toluene (10 ml.) and evaporated to ~ry-
ness. The product was dissolved in tetrahydrofuran (20 ml.) andslowly added to a stirred suspension of excess potassium carbon-
ate in tetrahydrofuran. The suspension was stirred at room tem-
perature for 30 minutes, filtered and the fil~rate evaporated to
dryness under reduced pressure. The residue was taken up in
methanol (20 ml.) and allowed to stand at room temperature for
30 minutes. The solvent was then removed under vacuum to yield
3-N-benzyl-3",6'-di-N-trifluoroacetyl-kanamycin A Rf O.S in
methanol, chloroform, 8~ ammonium hydroxide, 4:1:0.1 (3-N-benzyl-
kanamycin A gave an R~ value of 0.01).
EXAMPLE 4
(A) A solution of 1,3,3",6'-tetra-N-benzyloxycarbonyl
kanamycin A (Bull. Chem. Soc. Japan, 1965, 38, 1181) (189.4 g.)
_
in pyridine (568 ml.) and acetic anhydride (189 ml.) was stirred
o~ernight at room temperature and then poured into water (1.9
liters). The aqueous solution was extracted with chloroform
(1 x 1.8 liters and 1 x 1.0 liters) and the organic extract was
evaporated to dryness under reduced pressure. Trituration of
the residue with ether gave penta-0-acetyl-1,3,3",6'-tetra-N-
benzyloxycarbonyl kanamycin A (224.8 g.) which was filtered and
dried under vacuum. The product had m.p. 223-229; Rf 0.55 in
chloroform, industrial methylated spirit (12.1), ~ 1.8 - 2.05
(15 proton multiplet, 5 acetyl groups) and 7.4 (20 proton
singlet, 4 phenyl groups).
(B) A solution o~ penta-0-acetyl-1,3,3",6'-tetra-N-benzyl-
30- oxycarbonyl kanamycin A (53 g.) in ethyl acetate (260 ml.) con-
-14
. .
~...................................... . . .
- ' ~
-~ . . . .
" ~ . ' ' . ,.,., . ' ~ ' ' .
- , , . . :
. .
3s~
.
ta1ning glaclal acetic aci~ (260 ml.) was hydroyenated over 5
palladium on carbon (15 y.) at 60 and 50 p.s.i. for 7 hours.
The solution was filtered and the ~iltrate was evaporated to
dryness under reduced pressure. The residue was triturated with
ether and the product pe nta-O-acetylkanamycin (32.9 g.~ was
collected and dried under vacuum, m.p. 97 - 105, Rf 0.0 in
chloroform, industrial methylated spirit tl2:1) compared to an
Rf of 0.55 for the starting material. The proton magnetic
resonance spectrum showed a complete absence of aromatic protons.
tC~ A solution oE penta-O-acetyl kanamycin A (139.2 g.) in
methanol (1.4 liters) saturated with ammonia was allowed to stand
overnight at room temperature and then evaporated to dryness
under reduced pressure. The residue was dissolved in methanol
(140 ml.) and the crude product was precipitated with chloroform
(2.5 liters), filtered and dried in vacuum. The crude solid was
slurried with industrial methylated spirit (400 ml.~ and the
product 3",6'-di-N-acetylkanamycin A (91.9 g.) was collected by
filtration, washed with ether and dried under vacuum, m.p. 150 -
180, Rf 0.77 in methanol, 0.880 ammonium hydroxide 1:1. It
gave a 13C n.m.r. spectrum and proton n.m.r. spectrum in full
agreement with the required structure.
EXAMPLE 5
Trifluoroacetic anhydride (3.6 mls.) was added slowly
` to a stirred solution of kanamycln B (960 mg., 2 mmole) in tri-
- 25 1uoroacetic acid (50 ml.) at 0. The solution was allowed to
stand at 0 - 4. for 20 hours. The solvent was then evaporated
under reduced pressure and the residue treated with toluene
(10 ml.) and evaporated to dryness. The trifluoroacetate salt
was dissolved in tetrahydrouran (30 ml.) and added slowly to a
~o stirred solution o excess triethylamine in tetrahydro~uran. The
-15-
,~ ~ .,':" ',, '
: ,; :: .
: ; - ' . ' ' . . .
' ~ ' ''' ~' - ' " ' :
.: - .
-,:
~ 3S~;8
soiut.ion was allowed to stand at room temperatur~ ~or 40 minutes
and the solvent was then evaporated under reduced pressure. The
residue was dissolved in methanol to hydrolyze the remaining O-
trifluoroacetyl groups and after 30 minutes at room temperature
the solvent was evaporated un~er reduced pressure and the product
was chromatographed on silica eluting with a solvent yradient of
chloroform, methanol (3:1) to chloroform, methanol, 17% ammonium
hydroxide (20:10:1) to give 2',3",6'-tri-N-trifluoroacetyl-
kanamycin B (452 mg., 29%~ as a glass. Rf 0.70 in methanol,
chloroform, 8% ammonium hydroxide 4:1:0.1 (kanamycin ~ gave an
Rf of 0.0~.
The struc~ure was confirmed by the following sequence
of reactions: (a3 acetylation with acetic anhydride in methanol
for 20 hours at room temperature followed by treatment with lN
ammonium hydroxide for 18 hours to remove the trifluoroacetyl
groups gave a product containing two acetyl groups. m/e (field
desorption~ found 568, C22H41N5O12 requires M ~ 1 568; (b) Treat-
; ment with deuterioacetic anhydride in methanol at room tempera-
ture for 24 hours followed by reac~ion with a 2:1 mixture of
hexamethyldisilazane and trimethylchlorosilane at room tempera-
ture for 24 hours gave the volatile tri-N-deuteroacetyl-di-N-
acetyl-hexa-O-trimethylsilyl derivative. m/e Found 1134,
C46H86N5O15DgSi6 requires m/e 1134. Diacetylation was shown to
have occurred on the 2-deoxystreptamine ring from the fragmenta-
tion pattern, thereby confirming that trifluoroacetylation hadinitially taken place on the 2',3" and 6' positions in kanamycin B.
EXAMPLE 6
3",6'-Di-N-trifluoroacetyl-kanamycin A (prepared from
1.0 g. kanamycin by the method of Example 2~ in tetrahydrofuran
~n (40 ml.) was treated with N-~S]-4-benzyloxycarbonylamino~2-
-16-
.
.
, . . . , ~ ,
., ~ ~ . . . . . . .
- .,
-
- ': '
~153$6;~
hydroxy-butyryloxy) succinimide (1.08 y., 3.1 rnmoles) in tetra-
hydrofuran (50 ml.). The solution was allowed to stand at room
temperature for 24 hours, then a further 0.54 g. of N-([S]-4-
benzyloxycarbonylamino-2-hydroxy-butyryloxy) succinimide was
added and the solution was kept at room temperature for a
further 24 hours. The solvent was evaporated under vacuum and
the residue was dissolved in lN ammonium hydroxide and allowed
to stand at room temperature for 20 hours. The solution was
concentrated under vacuum and the product taken up in a mixture
of dioxan, water and acetic acid (55 ml., 5:5:1) and hydrogenated
over 5% palladium on charcoal catalyst at 30. and 50 p.s.i. for
6 hours. The mixture was filtered and the filtrate evaporated.
- The residue was chromatographed on Amberlite CG-50 ion-exchange
resin (NH4 ~ form) eluting with a gradient of ammonium hydroxide
of increasing concentration from O - 0.5 N, to give BB-K8
(0.11 g., 9O2~ from kanamycin A) identical to a reference sample.
EXAMPLE 7
3-N-Benzyl-3"-6'-di-N-trifluoroacetyl-kanamycin A
(prepared from 0.23 g. 3-N-benzyl-kanamycin A as described in
Example 3) was treated dlrectly with a solution of N-[S]-4-benzyloxycarb-
onylamino-2-hydroxy-butyryloxy) succinimide (.017 g., 0.5 mmole)
in tetrahydrofuran (15 ml.) at 0. The solution was allowed to
` stand at room temperature for 24 hours. A further 0.35 g. of
the acti~e ester in tetrahydrofuran was then added and the solu-
tion kept for a further 20 hours at room temperature. The solu-
tion was concentrated under vacuum and the residue taken up in
a mixture of methanol, water and acetic acid (30 ml., 10:10:1)
and hydrogenated over palladium on charcoal catalyst at 40 and
50 p.s.i. for 13.5 hours. The suspension wa5 filtered and the
filtrate evaporated. The product was purified by ion-exchange
-17-
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chromatography on Amberlite CG-50 lNH4 ~ form) as be~ore to
yield BB- K8 (84 mg., 36~ from 3-N-benzyl kanamycin A) identical
to a reference sample.
EXAMPLE 8
2',3",6'-Tri-N-trifluoroacetyl-kanamycin B is reacted
with N-([S]-4-benzyloxycarbonylamino-2-hydroxy-butyryloxy) suc-
cinimide in a similar manner to that described in Example 6, to
give, after deprotection and purification l-N-([S]-4-amino-2-
hydroxybutyryl) kanamycin B (BB-K26~.
EXAMPLE 9
.
A solution of 3",6'-di-N-acetylkanamycin A (2.84 g.)
and 3-benzyl-6-(S)-dihydroxymethyl-tetrahydro-1,3-oxazin-2-one
(1.305 g.) in dimethylformamide (28.4 ml.~ was heated at 60
for one hour and then cooled to 30. Sodium borohydride (0.189 g.)
was added and the mixture was stirred for a further one hour.
Water (1.0 ml.~ was added, the mixture was allowed to stand over-
night and the sol~ent was then removed under reduced pressure.
The residue was heated with 3N sodium hydroxide solution
(28.4 ml.) at 80 for 4 hours and, after cooling, the pH of the
reaction mixture was adjusted to 5.7 with concentrated hydro-
chloric acid. The crude solution of l-N-[(S)-4-benzylamino-2-
hydroxybutyl] kanamycin A and 3-N-[(S)-4-benzylamino-2~hydroxy-
butyl] kanamycin A was passed down a column of Amberlite
CG-50 ion-exchange resin (NH4 ~ form) eluting first with water
2S to remove inorganics and then with 0.15M ammonia to isolate the
crude aminoglycoside mixture. The required column fractions
were evaporated and the residue was dissolved in a mixture of
methanol (15 ml.), acetic acid (15 ml.), and water (15 ml.) and
hydrogenated over 30% palladium on carbon catalyst at ~0 and
3r- 60 p.s.i. fox 16 hours. ~he solution was filtered and the
-18-
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solvent removed under reduaed pressure. rrhe product was purified
by ion-exchange chromatoyraphy as previously described to yield
l-N-[(S)-4-amino-2-hydroxybutyl] kanamycin A identi~al to a
reference sample.
-19-
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