Note: Descriptions are shown in the official language in which they were submitted.
~3~3Z7~
~ ,
This invention rela-tes to antibacterial agents and
is particularly concerned with a class of novel antibacterial
2-deoxystreptamine aminoglycosides and with methods for the
preparation of such compounds.
Many naturally-occurring 2-deoxystreptamine amino-
glycosides have in common a three ring structure which may
be represented by the general formula:
4, ~ _ O ~ 2
~ H
where the ring A is the skeleton of a hexopyranose group hav-
ing an amino group in the 2' and/or 6'-positions, the ring B
is the 2-deoxystreptamine group and the ring C represents a
glycosyl group attached by a glycosidic linkage to either the
5- or the 6-position of the streptamine ring, the other posi-
tion being occupied by a hydroxyl group.
The novel antibacterial agents of the invention are
a series of 2-deoxystreptamine aminoglycosides having a ~-
hydroxy-~-aminoalkyl-substituent on the l-amino group and
having a glycosyl group attached to the 5- or 6-posi.tion o~
the streptamine ring B. Such compounds are effec~ive in
-2-
~ 3~7~
trea~ing a variety o~ gram-pos~tlve or gram-negative bacterial
infectlons, including urinary tract in~ect~ons, ln anlmal~,
including humans and possass advantages :Ln uae over 2-deoxy-
~treptamine amlnoglyco~ides havlng an un~ub~tituted amino
group in the l-positlon of the 2-deoxy~tr~pt~m~ne r~ng ~,
~uch a~ the naturally-occurrlng kanamycin A and ~, neomycln~
and ribostamycln.
According to the invention, therefore, there i~
provided a proces.~ for preparing novel 2--deoxy~treptamine-
10 aminoglycosides of the general formul~: :
~H2NHRl H
HO ~ O ~ N~CH2CH(CH2)nNH2
HO~R2 R30~'oR
...(I)and their pharmaceutically-acceptable ~alts;
wherein Rl represents a hydrogen atom or an alkyl
group having from 1 to 4 carbon atoms;
R2 represents an amino or a hydroxyl group;
one o~ R3 an~ R4 represents a hydrogen atom, while
the other represents a glycosyl group; and
n is 1, 2 or 3;
wherein R3, when it is a glycosyl group, is o~ the
20 formula: ~O-CH2 ~
HO-CH ~ or ~ ~ ~ OX
HO OH ~
H~ N%2
and R , when it is a glycosyl group, is of the ~orm~la:
~ -3- :
, ,~
~3~2t7~
~o
HO-~ < ~ _ CH2
H2~ OH
whl~h ~omprla~s reducing a compou~d of the ~ormulao
C~2NHRl NH~
HO ~ O ~ NN~-CN~cN2)n 1XN~2
... ~II) :
where Rl to R4 and n are a~ previou~ly defined and X 19 CH2
or CO, and isolating the compound of Formula (I).
The term lower alkyl group means a group contain-
ing from 1 to 4 carbon atoms whlch may be ~traight or branch-
-3a-
~ t~
ed chain.
One particular group of compounds according to the
invention comprises the group in which Rl is a hydrogen atom
and n is 1 or 2.
A preferred class of compounds according to the in-
vention comprises compounds in which R3 is a hydro~en atom
and R4 is a 3~amino-3-deoxy-a-D-glucopyranosyl group, i.e.,
derivatives of kanamycin A and B. Also preferred are com-
pounds in which the ~-hydroxy-~-aminoalkyl group at the l-N
lG position has the (S) configuration and n is 2 or 3. Rl is
preferably a hydrogen atom or a methyl group.
Particularly preferred individual compounds accord-
ing to the invention include l-N-[(S)-4-amino-2-hydroxy-
butyl~-kanamycin A and l-N-~(S)-5~amino-2-hydroxy-pentyl]-
kanamycin A.
Pharmaceutically-acceptable acid addition salts of ~
the compounds of the invention are those formed from acids ?
which form non-toxic acid addition salts containing pharma-
ceutically-acceptable anions such as the hydrochloride, hydro-
bromide, hydroiodide, sulphate or bisulphate, phosphate or
acid phosphate, acetate, maleate, fumarate, oxalate, lactate,
tartrate, citrate, gluconate, saccharate, p-toluene sulphon-
ate and carbonate salts.
The novel compounds of Formula (I) may be prepared
according to the invention from compounds of the formula:
(~H2NHRl H2 ~ ~'
H ~ O ~ NH-~-CH(CH~) YNH
H R R O OR
.
:~3~
where Rl to ~ and n are as previously defined and X is CH2
or C0, by reacting with a reducing agent in a suitable
solvent in order to effect reduction of the amide link at
N-l and X in the case where X is a carbonyl group.
This process entails as an optional initial step,
formation of a suitable acid addition salt in order to render
the compounds of Formula ~II) soluble in organic solvents.
Such a reaction may be performed, for example, by dissolving
the compound of Formula (II) in anhydrous trifluoroacetic
acid, the latter being used in excess at a temperature gen-
erally between room temperature and 0C. Excess acid is re-
moved by evaporating to dryness under vacuum. The salt is
then dissolved in an anhydrous, reaction-inert, organic
solvent, for example, tetrahydrofuran or dimethoxyethane and
treated with the reducing agent, for example, diborane, con-
veniently added as a solution of diborane in tetrahydrofuran,
generally in excess at a temperature generally between room
temperature and reflux temperature, depending on the nature
of the particular reactants and solvent employed. In the
case where X is C0, sufficient reducing agent is naturally
used to ensure reduckion of both amide carbonyl groups.
The reaction i9 substantially complete within 24 hours when
it is performed in tetrahydrofuran at 50 with an excess
o~ diborane; the product is then conveniently isolated by
the addition of water to destroy unreacted diborane and re-
- moval of the organic solvent by evaporation under vacuum.
The pH of the remaining a~ueous solution is adjusted to pH 5
and the crude product may then be purified from unreacted
starting material and bi-products by a conventional
chromat~graphic technique.
-5-
~ ~3~Z'7~ ~
Many of the compounds of Formula (II) where X is
CH2 are known antibiotics previously disclosed, for example
N-1-(4-amino-2-hydroxy-butyryl) kanamycin A, which is also
referred to as BB-K8, is disclosed in United States Patent
3,781,268. Other examples are described in United States
Patents Nos. 3,781,268, 3,541,078 and 3,860,574 and in
published West German Patent Application Nos. 2,350,203 and
2,322,576. The l-N-(5-amino-2-hydroxy-valeryl) and 1-N-~3-
amino-2-hydroxy-propionyl) derivatives of kanamycin A and B ;
are described in published West German Patent Application
No. 2,408,666 and in J. Antibiotics 1974, 27, 851. 6'-N-
Alkyl derivatives are described in published West German
Patent Application No. 2,350r169 and in J. Antibiotics,
1975, 28, 483.
Compound~ of Formula (II) where X is CO may be
derived by acylation of the l-amino group of 2-deoxy-
streptamine aminoglycosides by methods analogous to those
used in the preparation of compounds of Formula (II) where
X is CH2 but using as acylating agent a reactive derivative
of an acid of the formula:
OH
HOOC CH(CH2)n_lcONH2
~he novel compounds of Formula (I) according to ~;
the invention may exist in various conformational forms and
the invbntion is not limited to any one such form thereof.
Generally the rings A and B are each in the "chair" form and
each of the moieties R2, oR3 and oR4 and the amino and
hydroxyl groups is disposed equatorially with respect to the
rings A and B. Furthermore, the glycosidic linkage between
khe hexopyranosyl ring A and the 2-deoxystreptamine ring B is
-6-
., ~ . . .
. . . .
,: . . . .
~, :
~ 3~
more usually an ~-linkaye with respect to the former, par-
ticularly when the compounds o~ Yormula (II~ are derived
from naturally-occurring 2-deoxystreptamine aminoglycosides.
Additionally the ~-hydroxy-~aminoalkyl group at N-l may
exist in the S or R configuration or may be present as a
mixture of both optical isomers.
The ln vitro evaluation of the compounds of the
invention as antibacterial agents has been performed by
determining the minimum inhibitory concentration (M.I.C.~ of
the test compound in a suitable medium at which growth o~ the
particular micro-organism fails to occur. In practice~ agar
plates, each having incorporated therein the test compound at
a particular concentration are inoculated with a standard
number of cells of the test micro-organism and each plate is
then incubated for 24 hours at 37C. The plates are then
observed for the presence or absence of the growth of bacteria
and the appropriate M.I.C. value noted. Micro-organisms used
in such tests have included strains of Escherichla coli,
Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas
aeruginosa, Staphylococcus aureus and Stre~ococcus faecalis.
In vlvo evaluation of the compounds has also been
carried out for the more active compounds, by administering
the compounds subcutaneously to mice which are exposed to a
strain of Escherichia coli. Each compound is administered at
a series of dosage levels to groups of mice and its activity
is determined as the level at which it gives 50% protection,
against the lethal effect of the Escherichia coli organism
over a period o~ 72 hours.
For human use, the antibacterial compounds of the
0 invention can be administered alone, but will generally be
--7--
7~
administered in admixture with a pharmaceutical carrier
selected with regard to the intended route of administration
and standard pharmaceutical practice. ~or example, they may
be administered orally in the form of tablets containing such
excipients as starch or lactose, or in capsules either alone
or in admixture with excipients, or in the form of elixirs or
suspensions containing flavoring or coloring agents. They
may be injected parenterally, for example, intravenously,
intramuscularly or subcutaneously. For parenteral administra- `
tion, they are best use~ in the form of a sterile aqueous
solution which may contain other solutes, for example, enough `
salts or glucose to make the solution isotonic.
Fo~ administration to human patients, it is expect-
ed that the daily dosage level of the antibacterial compounds
o~ the invention will be comparable with that of aminoglyco-
side antibacterial agents currently in use, e.g., from 0.1 to
50 mg./kg. (in divided doses~ when administered by the
parenteral routes, or from 10 to 100 mg./kg. (in divided
doses) when administered by the oral route. Thus tablets or
capsules of the compounds can be expected to contain from
0.1 to 1 g. of active compound for administration orally up
to 4 times a day, while dosage units for parenteral admin-
istration will contain from 10 to 500 mg. of active compound.
The physician in any event will determine the actual dosage -
which will be most suitable for an individual patient and it
will vary with age, the weight and response of the particular
patient. The above dosages are exemplary of the average
host. There can, of course, be individual cases where higher
or lower dosage ranges are merited and such are within the
scope of this invention.
--8--
The following are Examples of the preparation of
novel compounds according to the invention. Temperature~ are
given in C; I'Amberlite" is a registered trade mark.
EXAMPLE 1
l-N-[(S) 4-Amino-2-hydroxybutyryl]-kanamycin A
~BB-K8, prepared as described in United States Patent
3,781,268) (150 mg.) was dissolved in anhydrous trifluoro-
acetic acid (10 ml.) at 0C. The solution was evaporated to
dryness in vacuo and dried under high uacuum at 20C., for
15 minutes to yield a glassy solid. This was taken UE~ in dry
tetrahydrofuran (5 ml.) and a lM solution of diborane in
tetrahydrofuran (20 ml.) was added in portions, under an
atmosphere of nitrogen. The resulting clear solution was
heated at 50 for 3 hours, allowed to stand at room tempera- :
ture for 16 hours and heated for a further three hours at
50. The excess diborane was destroyed by the cautioùs addi- ~
tion of a few drops of water and the organic solvent was `~.
then removed by evaporation under reduced pressure. The
residue was taken up in water (10 ml.) and basified with
N/10 aqueous sodium hydroxide. The pH of the resulting solu-
tion was adjusted to 5 by the addition of 2N hydrochloric
acid. The solution was then chromatographed on a column con-
taining Amberlite CG 50 ion-exchange resin (50 ml.), in the
ammonium ion form, eluting in turn with distilled water to
remove inorganic solids and then with a gradient of aqueous
ammonium hydroxide of increasing concentration from 0.1 to
l.ON. Fractions containing the product (as monitored by
thin layer chromatography) were combined and evaporated ln
vacuo to give l-N-l(S)-4-amino-2-hydroxybutyl~ kanamycin A
(75 mg., 50% yield).
_g_
~, .
~3~
Thin layer elec-trophoresis. Rf = 0.6
(The electrolyte was an equipart mix~ure of acetic
and formic acids, giving a pH value of 2 and a potential
difference of 900 volts was applied across the ends of the
S 20 cm. silica coated plate for 45 minutes. Detection was
performed by drying the plate, spraying with a cyclohexane
solution of tertiary-butyl hypochlorite and then drying, cool-
ing and developing the plate with starch~potassium iodide
solution~ Under these conditions the r~ference standard
BB-K8 gave a~ Rf value of 1.0 and kanamycin A an Rf value
of 0.9).
Infra-red spectrum confirmed the loss of the amide carbonyl
absorption band observed in BB-X8 at 1635 cm~l.
Optical Rotation [~]D25 ~ 73~ (c 1.0, H2O).
Mass spectrometry (field desorption) showed a strong P~l peak
at m/e 572.
A sample was converted to the volatile penta-N-
acetyl-octa-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 hexamethyl-
disilazane and trimethylchlorosilane at room temperature for
24 hours. M~ found 1357.C56HllgN5O17Si8 reqUire
Analysis:- Found: C, 40.1; M, 6.7; M, 9.6%
22 45N5O12.2 1/2H2CO3 requires: C, 40.5; H, 6 9; N 9 6%
EXAMPLE 2 -
Butirosin (l-N~(S)-4-amino-2-hydroxybutyryl]ribo- ;
stamycin), as free base, (100 mg.) was dissolved in anhydrous
trifluoroacetic acid (5 ml.) at room temperature. Excess
acid was removed by evaporation to dryness under vacuum to
yield the trifluoroacetate salt as a glass. This was taken -
~
--10--
: ~ . .
~39~
up in dry diethylene glycol dimethyl ether (diglyme) (10 ml.)
and a lM solution of diborane in tetrahydrofuran (lO ml.) was
added to give a clear solution, which was allowed to stand
for 18 hours at room temperature. A further 5 ml. diborane
solution was added and the solution kept at room temperature
for a further 24 hours. Excess diborane was destroyed by
the cautious addition of a few drops of water and the organic
solvents were removed under vacuum at 50. The residue was
basified with a few drops of 2N sodium hydroxide solution and
the pH adjus~ed to 5 by the addition of 2N hydrochlor:ic acid.
The product was isolated by ion-exchange chromatography on
Amberlite CG 50 resin as described in the foregoing Example.
Fractions containing the product in pure form were combined
and evaporated under vacuum to give l~N-[(S)-4-amino-2-
hydroxy butyl]ribostamycin.
Thin layer electrophoresis. Rf = 0.5
(The conditions were as previously described,
butirosin was used as the reference standard with an Rf value
of l.O).
EXAMPLE 3
l-N-[(S)-5-Amino-2 hydroxyl-valery]-kanamycin A
(0.35 g.) was converted to the trifluoroacetate salt, re-
duced and chromatographed as described for Example l to give
l-N-[(S)-5-amino-2-hydroxy-pentyl]-kanamycin A. (0.12 g. 35~).
Th n layer electrophoresis. Rf = 0.7
(Conditions as described for Example l, the start-
ing material was used as the reference standard with an Rf
of l.O).
EXAMPLE 4
l-N-(3-Amino-2-hydroxy-propionyl)-kanamycin A
--11--
: .
(0.15 g.) was similarly reduced by the method of Example 1 to
give l-N-t3-amino-2-hydroxy-pro~x~kanamycln A. (0.04 g, 27%).
Thin layer electrophoresis. Rf = 0.6
(Conditions as described for E,xample 1, the start-
ing material was used as the reference standard with an Rf
of 1.0).
EXAMPLE 5
l-N-[(S)-4-Amino-2-hydroxy-butyryl]-kanamycin B was
similarly reduced by the method of Example 1 to yield l-N-
[(S)-4-amino-2-hydroxy-butyl]-kanamycin B.
EXAMPLE 6
6'-N-Methyl-l-N-[(S)-4-amino-2-hydroxy-butyryl]-
kanamycin A ~prepared as described by H. Umezawa et. al~,
in ~. Antibiotics, 1975, 28, 483) was reduced as described ?
for Example 1 to yield ~'-N-methyl-l-N-[tS)-4-amino-2-hydroxy-
butyl]-kanamycin A.
Thin layer electrophoresis. Rf = 0.7
tConditions as described for Example 1, the start-
ing material was used as the reference standard with an Rf
of 1.0 and kanamycin A gave an Rf value of 1.03).
Results of the testing of the compounds of the
Examples for anti-bacterial activity ln vitro by the methods
previously described are given in the following Table:-
-12-
, . . .
. .
7~
TABLE: In vitro activity
Ex- M.I.C.~s ~g./ml.
ample . .
No.
E. KlebsieLla Proteus Pseudomonas ~ lo-
Coli pneumonlae mirabl- eru~inos3 coccus
lis aureus
1 6.2 3.1 3.11.6 1.6
2 6.2 6.2 2512.5 12.5
3 6.2 3.1 12.53.1 1.6
412.5 6.2 12.53.1 3.1
Additionally the compound of Example 1 has been
tested for ln vivo activity by the methods previously de-
scribed. The PD50 against E. Coli in mice was 3.8 mg~/kg.
-13-
