Note: Descriptions are shown in the official language in which they were submitted.
7~
This invention relates to antibacterial agents
and is particularly concerned with novel antibacterial
2-deoxy-streptamine aminoglycosides and with methods for
the preparation of such compounds.
According to our Co-pending Application Number
238246, there are provided novel compounds having the general
formula~
CH2NHRl NH2
N~lcEl2cH(c~l2)n 2
HO R2 R30 oR4 .~.(I)
; .
where
R1 represents a hydrogen atom or a lower alkyl
group;
R2 represents an amino or hydroxyl group;
~, one of R3 and R4 represents a hydrogen atom,
; while the other represents a glycosyl group as hereinafter
defined; and
n is 1, 2 or 3;
wherein R3, when it is a glycosyl group, is of ~he
formula:
O~
HO-CH ~ ~
2 l l or
~'1 HO OH
HO-CH2
~' H N-CH~ : ~ O ~
.~0
0~0 0~
HO ~ -2- b~
:.~' ' ' ' ," .'. :,': :, ", ", " ' " . :' ',', '. " ' :' :"". .' ' .,, . ''. '. " . "' "'' . ' . ' '. ,. ' ',' " ' , '.: , ' . ,."' ' '
,
i ~ ~
75;, -
and R9, when it is a glycosyl group, is o the formula:
~0 ,
' `. HO~CH2-OH
~ 2N b}~
and their pharmaceutically-acceptable acid addition salts.
When R4 represents a glycosyl gro~p such group may
be a ~ingle hexopyranosyl group, prefera~bly containing an
amino group, for example, a 3-amino-3-deoxy--D-glycopyranosyl
gr~up as found in kanamycin A and B. When R3 represents a
glycosyl group such group is generally a pentofuranosyl group,
optionally iinked to a further hexopyranosyl group by a further
glycosidic linkage. For example, R may be a ~-D-ribofuranosyl
group as found in ribostamycin. The term lower alkyl group
means a group containing from 1 to 4 carbon atoms which may
be straight or branched chain.
-~acording to the present invention, which is an
improvement or modification. of the invention the subject
of the above-mentioned Application Number 238,246, there are
~ow provided a process ~or the production of~novel compounds
of the general formula:
'' C~12NEIR
il~O ~i~gCH2CH(CH2)nilH
HO R OR ...~
where R represents a hydrogen atom or a lower alkyl group;
R2 represents an amino or hydroxyl group; ~
one of R and R represents a hydrogen atom, while
~;the other represents a glycosyl gsoup as herein definèdj and
n is 4, 5 or 6; ~
wherein R , when it is a glycosyl group, is of the
formula: HO-C~2
il2ilCI~
HOCH~ ~ ~ ; or ~ ~ ~ ~ ' :
2~
ana R4, when it is a glycosyl group, is o~ the formul2l~-
r
-2a-
~5C39~5
~o
HO ~ ~ CH2-OH
H2N H I
and their pharmaceutically acceptable acid addition salts,
which comprises reducing a compound of the formula:
CH2NHRl \2
H ~ _ O ~ _ NHC-~H~CH2)n_lxNH2
HO 2 oR4 ...(II)
where Rl to R4 and n are as previously defined and X is
CH2 or CO, and isolating the compound of E`ormula (I).
Pharmaceutically-acceptable acid addit:ion salts
' of the compounds of the invention are those formed from
acids which form non-toxic acid addition salts containing
pharmaceutically-acceptable anions, such as the hydrochloride,
hydrobromide, sulphate, or bisulphate, phosphate or acid
phosphate, acetate, maleate, fumarate, lactate, tartrate,
citrate, gluconate, succinate, p-toluene sulphonate and
carbonate salts.
The compounds of the invention may be prepared by
the methods described in the complete Specification of the
above-mentioned Application Number 238246, for example, by
` reduction o a compound of the formula:
CH2N ~ NH2~ O OH
HO ~ O _ ~ NH -CH(CH2)nNH2
, HO R oR4 ................................. (II)
where Rl to R4 are as previously defined and n is 4 to 6, by
,
~ 3- ::
.'' ~,~i. ~
' :'
~5~975
reacting with a reducing agent in a suitable solvent in
order to effect reduction o the amide link at N~l.
Many of the compounds of Formula (II) are known
compound~ previously disclosed, for example, l-N-(6-amino-
2-hydroxy~hexanoyl)-kanamycin A is described in J Anti-
biotics, 1974, 27, 851. Other compounds may be prepared
by analogous methods to those disclosed therein.
; Generally, the rings are each in the "chair"
form, and each of the substituent groups is disposed equa-
torially with respect to khe ring. Furthermore, the glyco-
sidic linkage between the hexopyranosyl ring and the 2-de-
oxystreptamine ring is more usually an -linkage with
respect to the former, particularly when the compounds of
Formula ~II) are derived ~rom naturally-occurring 2-deoxy-
streptamine aminoglycosides. Additionally, the ~-hydroxy-
~aminoalkyl group at N-l may exist in the S- or R-form or
may be present as a mixture of both optical isomers.
The in vitro evaluation of compounds of the in-
.
vention as antibacterial agents is performed by determining
the minimum inhibitory concentration (M~I~Co) of the testcompound in a suitable medium at which growth of the parti
cular microorganism 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 microorganism and each plate is
then incubated for 24 hours at 37~C. 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
Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,
.~ .
9~7~
; Pseudomonas ~ , ~ us aureus ~nd
' coccus faecalisO
_ vivo evaluation is carried out by administering
the compounds subcutaneously to mice which are exposed to a
strain of Escherichia coliO 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 of 72 hours.
For human use, the antibacterial compounds of
the invention can be administered alone, but w:ill generally
be administered in admixture with a pharmaceut:ical carrier
selected with regard to the intended route of administration
and standard pharmaceutical practice. For example, they may
; 15 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 administration, they are best used in the form of
a sterile aqueous solution which may contain other solutes,
~ for example, enough salts or glucose to make the solution
;~ isotonic.
` 25 For administration to human patients, it is
expected that the daily dosage level of the antibacterial
compounds of the invention will be comparable with that of
aminoglycoside antibacterial agents currently in use, e.g.,
~rom 0.1 to 50 mg/kg (in divided doses) when administered
i,
by the parenteral routes, or from 10 to 100 mg/kg (in
-5-
; :
' ~ '
. .
~5~75
divided doses) when administered by the oral route. Thus
tablets or cap~ules of the compounds can be expected to con-
tain from 0.1 to 1 g of active compound for administration
orally up to 4 times a day, while dosage units for parenteral
administration will contain from 10 l:o 500 mg of active
~ compound. The physician in any event will determi.ne 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. ThPre can, of course, be individual
cases where higher or lower dosage ranges are merited, and
such are within the scope of this invention.
; The ~ollowing Example describes the preparation
of one particular compound according to the invention.
Temperatures are given in C; "Sephadex" is a registered
trade mark.
E X A M P L E
l-N~(S)-2-hydroxy-6-amino-hexanoyl]-kanamycin A
dicarbonate ~1.0 g, 1.36 mmole) was dissolved in anhydrous
trifluoroacetic acid ~10 ml) and the solution evaporatod to
dryness under vacuum to yield a viscou~ gum. The residue
was treated with a 1 molax solution of diborane in tetra-
hydrofuran (75 ml) under an atmosphere of dry nltrogen, and
the resulting ~olution heated at 50-55 for five hours.
Evaporation of the organic solvent under reduced pre$~ure
yielded a gum which was taken up in 2N hydrochloric acid
~10 ml). After 10 minutes the solukion was basified to p~
with 5N sodium hydroxide solution and finally taken
to p~ 6 with 2N hydrochloric acid. The solution was then
chromatographed on a column of Sephadex CM-25 in the
--6
` ammonium-ion form (3.5 x 90 cm) eluting with water and a
gradient of ammonium hydroxide of inc:reasing concentration
from 0 to 0.6N. Fractions containing the product, as
monitored by thin layer chromatography were combined and
- 5 evaporated under vacuum to yield l_N~ e~L~_
amino-hexyl]-kanamycin A (0.64 g, 63~)o
Thin layer electrophoresis. Rf = 0.85
- (The electrolyte was an equipart mixture of
acetic and formic acids, giving a pH value o 2, and a
~- 10 potential difference of 900 volts was applied across the
ends of the 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, cooling and developing the plate with starch-
potassium iodide solution. Under these conditions the
. .
starting material gave an Rf value of 1.0)~
Mass s~ectrometry
. .
~i, A sample was converted to the volatile penta-N-
acetyl-octa-O-trimethyl~ilyl derivative by treatment with
,
acetic anhydride in methanol at room temperature for 24
hours followed by reaction with a 2:1 mixture of hexamethyl-
di~ilazane and trimethylchlorosilane at room temperature for
, 24 hours. /e found 1385.C58H123N5O17Si8 requires m/e 1385.
Results of the testing of the compound of the
.l 25 Example for antibacterial activity in vitro by the methods
;,Z previously described are given in the folIowing Table.
-¦ TABLE: In vitro activ ty
,, .
1 M.I.C.'s (~ug/ml)
} Klebsiella Proteus Pseudomonas Sta~ylococcus
E. Coli pneumoniae mirabilis aeru~inosa aureus ~ -
6.2 6.2 3.1 3.1 1.6
,
. ,
,
; : . . . ~ . . ::: .. : :
~5l9~75
It will be appreciated from the foregoing that
.. what we wlll claim may comprise any novel feature described
herein, for e~ample:
(1) Novel 2 deoxystreptamine am.inoglycosides of
Formula ~I) as previously defined;
~ 2) Pharmaceutical compositions comprising a novel
aminoglycoside of the Formula (I) as a pharmaceutically
acceptable acid addition salt;
(3) A method of treating gram-positive and gram-nega-
tive antibacterial infections in animals, includinq humansby administration of novel aminoglycosides of Formula (I)
.in therapeutic dosages;
(4) Processes for the preparation of novel aminoglyco-
sides of Formula ~I) as previously described. :
i, .
. ~'.
. .
