Note: Descriptions are shown in the official language in which they were submitted.
- ` 1068'~Z6
NOV~ ~MINOG~YCOSIDE - ~MINOC`Y(~LI'~O~ DERIVATIVES AND PROCESS
.
FOR PREPARI~ T~E S~lE~
This invention relates to aminocyclitol derivatives
and is concerned with novel aminoglycoside-aminocyclitol
derivatives having pharmacological activity and with a process
for preparing the same.
~ he aminoglycoside-aminocyclitol derivatives with
which the invention i~ concerned can be represented by the
` general formula:
" 10
:: c~l 0~
2 ox
.,'., I~ ~
:. ~ I
' ~21 ~~ ~ Z 1ll2
"', ' ~ ' '
Ho OH
wherein Rl and R2, which are different, represent hydrogen or
j 20 CH~NH~ and R is sclected f.rom the gro~p consis-ting of:
,~, l~i,
~` 110 --~ ~0
~ o _ ~ , N112 2 ~ 1~12
~, H2N o ~~~\ 112N o~~\
\ ~ N112 \ ~ N~12
.,~
A 3 and B = R5
30 wherein R~ represents NH2 or OH, and R4 represent~ CHNH2 or
~, CH3
i~ 1
CHNHCH3 wherein R5 represents hydrogen or methyl.
.~j
' 1 -
` ~i
~ - ,
~068Z26
The pharmaceutically acceptable acid addition salts
of these derivatives are also included within the scope of
t;he invention.
~he compounds of formula I can exist in the form of
~pimers and of mixtures of the said epimers.
Such epimers and mixtures thereof are included
within the scope of the present invention.
~he compounds of tne present invention have chemical
structures which are similar to that of neomycin or paromomycin.
~or this reason~ compounds of formula I will be designated
hereinafter as neomycin or paromomycin derivatives as follows:
6-Deoxyneomycin ~ when Rl represents hydrogen, R2
represents CH2~H2 and R represents the group A wherein R3
represents NH2.
; 6-Deoxyneomycin C when Rl represents CH2~H2, R2
represents hydrogen and R represents the group A wherein R3
represents NH2.
The mixture of these two epimer~ as obtained by the
process described herein will be referred to hereinafter as
-,
~; 20 ~-deoxyneomycin.
6-Deoxyparomomycin 1 when Rl represents hydrogen,
R2 represents CH2NH2 and R represents the group A wherein R3
represents OH.
6-Deoxyparomomycin II when Rl represents CH2NH2,
R2 represents hydrogen ~nd R represents the group A wherein
R3 represents OH.
he mixture of these two epimers as obtained by the
; process described herein will be referred to hereinafter as
6-deoxyparomomycin.
.J
~he mixture of 3~, 4~-dideoxyneomycin and its deri-
vatives to designate the unseparated combination of compounds
wherein Rl and R2~ which are different, represent hydrogen
~! .
3 ~ --2--
!
~ 068Z;~6
or C1~2NH2 and ~ represents the group ~, thé said mixture being
obtained by the process described hereinafter.
"Neomycin" will be used herei~after to designate
the mixture of neomycin ~ and neomycin C which is obtained
when cultivating Streptom~ces fradiae
. . .
Similarly, "paromomycin" will be used hereinafter
to designate the mixture of paromomycin I and paromomycin II
which is obtalned wnen cultivating StrePtomyce~ rimosu~ forma
paromom~cinus.
Another object o~ the invention is to provide a
pharmaceutical or veterinary composition containing a~ an -
essential active ingredient at least one of the epimers of
formula I or a pharmaceutically acceptable acid addition salt
thereof or a mixture of the said epimers or of their pharma-
ceutically acceptable acid addition salts, in association with
, a pharmaceutical carrier or excipient therefor.
Yet another object of the present invention is con-
cerned with a process for preparing pharmaceutical or vete-
rinary compositions comprising the association of at least
one of the epimers of formula I or a pharmaceutlcally accep-
table acid addition salt thereof or a mixture of the saidepimers or of their pharmaoeutically accep~able acid addition
salts with an appropriate carrier or excipient therefor.
As will be demonstrated further on, the aminogly-
co~ide-aminocyclitol derivatives of the invention have been
: ~s ~
; found to present raluable antibiotic aotivity which is likely
~-s~ to~ render them useful for -the treatmen-t of disea3cs provoked
by~the growth of pathogenic bacteria such a~ for e~ample:
Escherlchia coli, Proteus mira~ , Staph.Yloooccus aureus.
~ ~ Sarcina lutea, Klebsiella edwardsii, Shi~ lla sonnei, S~lmo-
` nella ty~ ri;um.
...
~;~ . , , ,, ~
0 ~ 8 ~ ~ ~
~ furt;hcr object of ~he pr~sent inven'ion is therefore
concerned with a m~thod for inhibiting the grow.h of pathogenic
bacteria in a host in need of such inhibition comprising the
administration to said host o~ at least one of the epimers
of formula I or a pharmaceu-tically acceptable acid addition
salt thereof or a mixture of -the said epimers or of their
pharmaceutically acceptable acid addition salts.
It has been observed, for some years, that certain
bacterial strains have de~eloped a re~istance to most of the
commercialized antibiotics with the result that the therapeutic
value of the latter has been reduced by more than half, while
certain bacteria are now even strong enough to withstand all
the known antibiotics.
It is thus of prime necessity and of general m tere~t
to create new antibiotics capable of destroying organisms
which ha~e become resistant to antibiotics currently used.
It is kno~m that this inactiv.--tion of antibiotics is
dus to the d~struction or alteration of the a~tibiotic mole-
;~ cule by enzymes ~hich attack the latter at certain vulnerable
polnts.
In particular, the aminogl~coside-aminocyclitol
antibiotics which
`~ :
` 1068Z26
are being employed on an increasin~ scale are prone to inactivation by
resistant bacteria. The resistance developed by some or~anisms normally
susceptible to these aminoglycoside-aminocyclitol antibiotics is due to
phosphorylating, acylating or adenylylating enzymes which attack the
frcc hydro~.yl and amino ~roups of the antibiotic (hnn. Rev. Biochem.,
~2, l~7, 1~3~
Removal of the hydroxyl groups will render the antibiotic immune
to inactivation at these sites. It is, thus, desirable to produce anti-
biotics with less unessential hydroxyl and amino ~roups.
In the course of trials carried out with the compounds of the
invention, the mixture of ~'~ 4'-dideoxyneomycin and its derivatives
was found to present marked antibiotic activity against organisms which
are resistant to other amino~lycoside-aminocyclitol antibiotics and more
particularly neomycin and gentamicin.
Thus, it was demonstrated that the mixture of 3~, 4~-dideoxyneomycin
and its derivatives i6 active against Escherichia coli strains which are
kno~ln to be resistant to eentamicin and neomycin by virtue of the 2"-
adenylylating enzyme and the 3'-phosphorylatin~ enzyme respectively
For this reason, the mixture of 3~ 4~-dideoxyneomycin and its
derivatives, referred to above, is the antibiotic covered by formula I
which is the preferred antibiotic of the invention.
It is known from U S Patent No. 3,669,838 that mutants of micro-
or~anisms known to produce antibiotics containin~ an aminocyclitol
subunit, such as, for example neomycin or paromomycin, can be formed
which lack the capacity to biosynthetize the aminocyclitol subunit
because of a defect in the aminocyclitol pathway so that no antibiotic
can be produced.
~ owever, such aminocyclitol-negative mutants have the capacity to
utilize a suitable aminocyclitol molecule when the latter is added to
the nutrient medium so that an antibiotic can be formed.
It is also known that this technique can be utilized by substitutin~
a pseudodisaccharide to the aminocyclitol subunit.
However, from the results disclosed in ex1stin~ publications, it is
not possible to predict whether a ~iven diaminocyclitol or a pseudodi-
saccharide ~lill be incorporated into an antibiotic by the aforesaid
bioconversion technique
". ~
--4--
,
1068ZZf~
Therc are, in fact, numerou~ instances described
in the literature where this method o~ producing new antibiotics
has proved to be ineffective.
The procedure hereabove described cannot, therefore,
be generalized for producing new semi-synthetic or totally
s~n thetic antibiotics .
This fact is confirmed, for example, in "The Journal
of Antibiotics", Vol. XXVI, No~ 10, p. 551-561 (1973) w~ere a
description iS given of trials carried out with 29 different
aminocyclitols for incorporation into an antibiQtic by the
method of the abo~e-cited U.S. Patent.
Amongst the ~9 aminocyclitols so tested it was found
that only strepta~ine and 2-epistreptamine were incorporated
by means of the 2-deoxystreptamine-negati~e (D ) mutants
of Streptomyces fradiae, of strePtomyces rimosus forma Paromo-
mycinus and of Streptomyces Xanamyceticus used for this
purpose.
i
~ ikewi~3e, the diaminocyclitols cited in ~rench
Patent No. 2,203,808 failed to be incorporated into an anti-
,~ 20 biotic using the same D mutants o~ S~ fradiae and of S. rimosus
for~3a paro~omycinus a9 those utilized in the aforesaid
reference. ~ -
In "Biochemistry", Vol. 13, No. 25, p. 5073-5078
(1974), it is disclosed that no success was achieved when
attempts were made to incorporate the pseudodisaccharides
nown as neamine, paromamine or 6-kanosaminido-2-deoxystrep-
tamine~using D mutants of S. fradiae, of S rimosus forma
L~ Y~ and of S. kanam~ceticus.
Similarly, "~he Journal fo Antibiotics", Vol. XXVIII,
' ~ 3Q ~ ~ No. 8, p. 57~-579 (1975) reports that gentamines, which are
r ~ ~ pseudodisaccharides, were not incorporated into antibiotics
-1 using a D mutant of Micromonospora inyoensis.
10~8'~
It has also b~en fo~ that 5-0-~D-ribopyranosyl-2,
4-dideo~ystreptamine is incapable o~ producing a~tibiotics
using D mutants of S. fradiae and of S. rimosus forma paro-
momycinus in accordance with the a~i~e3aid procedure.
Prior publications have also demon~trated that
diaminocyclitols or pseudodisaccharides which are present in
already known antibiotics, such as stxeptomycin, bluensomycin
and hygromycin, cannot be incorporated into antibiotics using
the above-cited mutants.
~or example, it is disclosed in "The Journal o~
~ntibiotics", Vol. XXVI, No. 10, hereabo~e cite~, that bluen-
samine, streptidine, bluensidine,
:
10~8226
hyosamine and actinamine are not incorporated into antibiotics by the
D mutants of S. fradiae, S rimosus forma paromom~cinus and S.
ka~amyceticlls when these mutants are used for this purpose.
Similarly, neamine and paromamine, which are found in neomycin and
~aromomycin rcspcctive~y, werc not incor~orated into antibioticF. u-;inG
a D mutant of S fradiae ("Biochcmistry" : refcrence hereabove-cited).
Furthermore, it has also been observed that a diaminocyclitol or a
pseudodisaccharide, which can be incorporated into an antibiotic using
a mutant strain in accordance with the process of the above-cited U.S.
Patent, will not necessarily be incorporated into an antibiotic by a
different mutant strain. Examples are given in the aforesaid reference
from "The Journal of Antibiotics", Vol. XXVI, No. 10, which sho~s that
a D mutant of S. rimosus forma paromomycinus with 2-epistreptamine does
not produce any antibiotic while a D mutant of S. fradiae and of
S kanamyceticus are capable of incorporating this subunit into anti-
~ . .
~iotics
Furthermore, the same D mutant of S. kanamyceticus with streptaminedoes not provide any antibiotic while the sa~e D mutant of S fradiae
and of S. rimosus forma paromomycinus incorporates this diaminocyclitol
into antibiotics.
~ Jith respect to neamine, this pseudodisaccharide can provide
ribostamycin wllen a D mutant of S. ribosidificus is used as reported
in "The Journal of Antibiotics", Vol. XXVI, No. 12~ p. 784-785 (1973)
while neamine is incapable of bein~ incorporated into an antibiotic when
a D mutant of S. fradiae, for example, is employed.
Finally, prior publications also show that the structure of a
hypothetical antibiotic cannot be predicted, in any event,-when a
diaminocyclitol or a pseudodisaccharide is used together with a mutant
strain. This fact is clearly demonstrated in "The Journal of Antibiotics",
Vol. XXVI, No. 12, cited above where it is disclosed that the antibiotics
obtained when a D mutant of S. kanamyceticus is used together with
1-N-methyl-deo~ystreptamine or myo-inosa-1,3-diamine, are " not the
expected products " but other compounds. Likewise~neamine and a D
mutant of M. inyoensis provide sisomicin but this mutant together with
paromamine, a different pseudodisaccharide, a~so produces sisomicin
(I'The Journal of Antibiotics"~ Yol. XXVIII, llo. 8, cited above).
--6--
. .
.
-~ \
10682Z6
It has also been demonstrated that a D mutant of S. ribosidificus
in the presence of neamine only provides ribostamycin but not an analog
of neomycin as could be expected ("~he Journal of Antibiotics", Vol.
XXVI, No. 12, nbove-cited).
It ir, clear from the fore~oin~ that no valid prediction can be
made re~arding the possible incorporation of a particular diaminocyclito1
or pscudodisaccharide into an antibiotic using a given mutant strain in
accordance with the procedure of the above-cited U.S. Patent. In no case,
i8 it possible to prcdict whether this method will be valid or what will
be the exact structure of the antibiotic which it is hoped to produce.
Therefore, the allegation made in J. Or6. Chem.~ Vol. ~, No. 4,
p. 456-lt61 (1975) that 2,4-dideoxystreptamine might be incorporated into
neomycins, paromomycins and ribostamycin by a bioconversion tcchnique
must be considered as excessively vague, indefinite and devoid of any
reasonable and valid foundation.
It has now been quite unexpectedly found, in accordance with the
present invention~ that 2~4-dideoxystrep~amine can be incorporated into
new antibiotics by D mutants of S. fradiae and of S rimosus forma
~aromo~ycinus and that a mixture of pseudodisaccharides i.e. a mixture
of gentamines can also be incorporated into new antibiotics by a D
mutant of S. rimosus forma paromomycinus.
This discovery is rendered still more surprisin~ when it is consi-
dered that trials carried out with 2,4-dideoxystreptamine in the presence
of a D mutant of S. kanamyceticus did not produce any antibiotic and
that a D mutant of S. fradiae was incapable of incorporatin~ the mixture
of gentamines in question intoi~ antibiotic.
All thë results obtained with 2,4-dideoxystreptamine and the mixture
of gcntamines render the invention completely unexpected with respect to
the state of the art.
The compounds of the invention can be prepared by cultivating a
deoxystreptamine-negative mutant of S eptomyces in an appropriate medium
containing a soluble carbohydrate, a source of assimilable nitrogen,
essential mineral salts and : i~
~ --7--
.. .. .
,~
,. ~ . ~ , . . . .. . ~ . . ... .
1068ZZ6
a) either 1D-(1, 3, 5/2)-1,5-diamino-2,3-cyclohexanediol or 2,4-
dideoxystreptamine of the formula :
2 :
~10 ~\ "'
\ . \ II ~`
HO NH2
or an acid addition salt thereof, for example the dihydrochloride.
b) or a mixture of gentaminès represented by the general formula :
R4
~ 0
III
NH2 ¦ NH2
~ HO ~ ~ NH2
i ~ .
: -8
, ,
.
1068Z26
or an acid addition salt thereof, wherein R4 has the same
meanings as in group B of formula I.
When u~ing the process described herein the antibiotic
obtained is in free base form, regardless of whether the dia-
minocyclitol of formula II or the mixture of gentamine~ of
formula III is in ~ree base form or in the form of a salt. `~
If it is desired to utilize the resulting antibiotic as a
salt, it is sufficient to react it with a suitable organic or
inorganic acid such as, for example, sulphuric acid, to obtain
a pharmaceutically acceptable acid addition salt.
The microorganisms which are used in the present
invention are D StrePtomyces fradiae ~CC21401 and D
Streptom~ces rimosus forma paromom.ycinus ATCC21484 both des-
cribed in U.S. Patent No 3,669,838: D strePtomyces fradiae
A~CC21401 is utilized to produce 6-deoxyneomyoin of the
invention and D Streptomyce~ rimo~us forma ~aromomycinus
ATCC21484 is employed for the preparation of the 6-deoxy-
paromomycin and the mixture of 3',4'-dideoxyneomycin and its ~-
derivative~ of the invention. -
i
~ 20 In accordance with known techniquest the mioroorganism
~; :
is grown in a nutrient medium having the appropriate pH value
and containing for example gluoose, casein hydrolysate, (NH4)2
HP04, NgS04 .7H20, FeS04 .7H20, CuS04 .5H20, CaC03 and then
~ adae~ to another growth medium containing the diaminocyclitol
d'', ~ o~formuIa II or the mixture of gentamines of Yormula III both
in the; form of~a~free base or of an acid addition salt and,
or example~oyabean meal, yea~t extract, NaCl, CaC03 and
glucose, again at~ ~he appropriate pH value The culture medium
~is~incubated~at~a temperature of about 28 to ~0C with shaking
dj,~ and~ good aeration during at least 5 days to achieve the optimal
production of the required 6-deoxyneomycin, 6-deoxyparomomycin
~d ~ 9 _
~068ZZ6
or the mixture of 3',4~-dideoxyneomycin and its derivatives.
~-Deoxyne`omycin B and ~-deoxyneomycin C as well as
6-dcoxyparomomycin I and 6-deoxyparomomycin II will be obtained
from 6-deoxyneomycin and 6-deoxyparomomycin respectively, by
conventional procedures, for example by separating them by
means of paper chromatography.
~he isomers constituting the mixture of 3',4~-
dideoxyneomycin and it~ derivative~ can also be 3eparated by
conventional procedures. -
The diaminocyclitol of formula II is a known compound
having been described in British Patent No 1,445,675. It can
be obtained tollowing the method described in the aforesaid
British Patent namely by hydrogenating in the presence of a
catalyst, for example Raney's nickel, lD-tl, 3, 5/2)-1,5-
diazido-2,3-cyclohexanediol obtained from 1~-1,5-di-0-tosyl-1, ~-
2, 5/~-cyclohexa~tetrol and sodium azid~.
With regard to the mixture of gentamines of formula
III, this can be obtained from commercial gentamicin sulphate
by the method of COOPER et al. desoribed in J.Chem. Soc. (c)
, 20 1971, 960.
As mentioned hereabove the amino~lycoside-aminocyclitol
derivatives of the invention ha~e been found to present v~lu-
able antibiotic activity.
his antlbacterial activity against various test
organisms is lllustrated by the following data:
a) ~ tibacter]al activit~ of 6-deox~neom~cin and
6-deox~aromom~cin
he~e aminoglyco~ide-aminocyclitol derivatives of
the invéntion were tcsted for antibiotic activity by adding
3~ ~
measured quantitles o~ the compound under study in sterile
water to nutrient agar.
--10--
.~ , .
lQ~8'~
Thc agar cont~.irling increasing concentrations of
the compound to be tested was thcn poured into ~etri dishes to
e~ch of which se~er~l different or~nisms, prevlously gro-~n
in an appropriate medium and stored a-t - 20C in a 1 ~o 1
mi.-~ture of growth medium and glycerol were applied mechani-
cally with a multiinoculator. The dishes were i~cubated at
37C .for 1~ hours and then inspected for growtn.
The concentration of antibiotic whi~h just
i.nhibited growth oE the bacteria was then recorded and was
r~ferred to as "min~mum inhibitory concentration`' or M.I.C.
The results regi~tered in this test are listed
in ~able I in comparison with neomycin and paro~omycin both
in sulphate form.
``` lQ68ZZ6
The figures shown in Tables I and II correspond to the quantity
of ~ree base.
TABLE I
:
Organism (a) : ~g/ml required to prevent growth
neomycin 6-deoxy- paromomycin 6-deoxy-paro-
: : : neomycin: : momycin
:
Escherichia coli W 3110
i.e. ATCC 27325 2 ~ 5 1.25 5.0 40
Proteus mirabilis i.e.
: NCIB 11355 : 5~ : 5 1.25 : 20
: Staphylococcus aureus
i.e. ATCC 9144 2.5 3.75 1.25 20
Sarcina lutea i.e.
NCIB 11356 2.5 2.5 5.0 40
Klebsiella edwardsii 7.5 10 2.5 4
: Shigella sonnei C63 1978
i.e. NCIB 11357 2 - 5 3.75 2.5 40
Salmonella typhimurium
' LT2 i.e. ATCC 19585 5.0 5.0 2.5 40
, . .
(a) ATCC, American Type Culture Collection ; NCIB, National Collection
of Industrial Bacteria (Great Britain)
These results show that the removal of the hydroxyl group àt the 6th
position of the aminocyclitol moiety of neomycin, which corresponds to
6-deoxyneomycin does not significantly alter the overall antibacterial
activity of neomycin. Attention may be drawn to the antibacterial acti-
vity of 6-deoxyneomycin against Escherichia coli W 3110 i.e. ATCC 27325
which is superior to that of neomycin.
b)~Acti~ity of 6-deoxyneomycin B and 6-deoxyneomycin C.
The~an~t~ibacterial activity of 6-deoxyneomycin B and 6-deoxyneomycin C
`~ wa~tested~by the same method as that described above using D.S.T. agar
a&-~nutrlent edlum and tho~M.I.C. wa~ regiotered in comparison with
neomycin~and 6-deoxyneomycin :
`~
.' ,: .
l ~:
~ . . ... .. ..
`~` 1068226
...... .. ,.. ~ .. ........................................
~ O fC~ fLr~ fLr~ N C~.'f : ~
, '.
..
Of ~ ~
~ o m N flt~ N ~
h .~f .. .. .. .. . . .. .. . . .. .. .. .. . . .. . . .. .. .. . . ..
~C h u~ ~
h ~ f C~ f~ ~ f :` .
s~ `d .........................................
~ .~ .'
~ O V fXf O o ..
H O ~
~ h v .. .. .. . .. .. .. .. .; .. .. .. .. .. .. .. .. .. .. ..
': f~f ~f ~ ~f C~f ~
~; ~ h ;Q O f~ O Cf ~f
f~ S
~ :: C) ~ U~ C~l ~ ` .
~ ~ ~1 ~ Ll~
O O r-i O N ..
: : ~ .
~ ~ .'
f`~ ~ f ~f V
I V ,~ ff~,_ff
:f~ Q . t fU~f E~ f fE-~
a~ ~ff ,~ ff Frl ~ h
~ ~ ~ O ~
: ,` ~ f~f ~ ;~ f ~; O ~`f ~ O,f
:, ~ ~ C) ~ ~ f~ - ` O '~I rl ~f
7.:, ~ : : f~ - ~rl ff--l rl fl~f a~ ff~ )
~ ~ ~ :hff :af ~ a) ~ fl~ ,~ ~ O ~ f~
~ o ~ h Qh ~qff ~f ~
~ m ~; ~f ~I ff~l ftf~f f~ ff J~ U~ El f~l
:~7~ 12- ` ~
f: :~
lQ~8'~
~ rom th~se results i~ c~n b~ concluded that 6-deoxy-
neumycin B is somewha~ s po-tent th<~n neomycin B but that
6-deoxyneomycin C is more potent t~lan neomycin ~.
c) ~ctivity of the mixture of ~,4'-~id~ox~neom~cin and its
_______ _____________________ _________ ____ ___________
derivati~es
___________
The antibacterial activity oY the mixture of 3~,4'-
dideoxyneomycin and its derivatives was determined in accor-
d~lce with the procedure described hereunder.
D Streptomyc~s rimosus forma paromomycinus ATCC
21484 was incubated at 30C on a nutrient agar plate containing
50 ~g/ml of a mixture of gcntamines of formula ~II. After
three days, the plate was overlaid with agar seeded with a
test organism ~d the production of antibiotic was shwon
by a zone of inhibition around tne StrePtomyc~s. This zone
of inhibition was measured an~ compared with that of a control
conætituted ~y nutrient agar alone.
As a comparlson, a similar ~est was also carried out
wit~ g/ml of ne~nine as a supplement to nutrient agar,
in place of the mixture ol g~ntamines o~ formula III and also
using D StrePtomyces rimosus ~orma ~aromomyclnus ATCC 21484.
. 'rhe antibiotlc so produc~d ~as found to be neomycin~
~ iO~8Z26
The results obtained are given in the following Table :
Table III
.
Zone of inhibition (in mm) when nutrient
: : agar is supplemented with
Organism Nothing Neamine Mixture of genta-
: : : : mines of formula :
III
:
Eschérichia coli PT2 0 O 21
Escherichia coli ML
: 1629 i.e. NCIB11354 : O : : 25
: Escherichia coli ML
1410 i.e. NCIB11353 O O 20
Escherichia coli JR
66 i.e. NCIB 11352 0 0 25
'' :
The6e figures show that the mixture of 3~,4'-dideoxyneomycin and its
derivatives of the invention i8 active against orgar.isms which are -`-
re~i6tant to neomycin. It may be added that the orga~ism E. coli PT2
i8 also resistant to gentamicin.
It will be appreciated that for therapeutic use the compounds of the
invention will normaly be administered in the form of a pharmaceutical
or veterinary composition in a dosage unit form appropriate to the requi-
red mode of administration, the composition comprising as active ingre-
dient at least one compound of the invention in association with a phar-
maceutical carrier or excipient therefor. For oral administration, the
n ~ composition may take the form of, for example, a coated or uncoated
tablet, à hard- or soft gelatin capsule, a suspension or a syrup. The
composition may alternatively take the form of a suppository for rectal,
or~vàginal administration, a solutîon or suspension for parenteral
àdministration, or a cream or ointment for topioal administration.
The fol`Iowing Examples illustrate the preparation of the compounds
of~t~e~invention~
XAMPLE 1
paration of~6-deoxyneomycin baFie and its ~ulphate
à)~6_deoxyneomycin in ~free base form
In~a~flask, D Streptomyces fradiae ATCC 21401 was grown for 48 hours
on~the~following medium previouæly adjusted to pH 7.2-7.3 :
Glucose
Casein~hydrolysate
( 4 2 4
` -13-
~0~8ZZ6
so4.7ll2 05
Fe S04,7H20 0.005 -~
Cu S04.5H20 0.005
CaC03 1 ~- ' '
Wnt;er to 00 ml
This culture was shaken and maintened at 280C during the indicated
period of time, A 10% inoculum of the culture was then added to the fol-
lowing growth medium previously adjusted to pH 7,2 to 7,3 :
':
Glucose
Soyabean meal 2,5
Yeast extract 0.5
NaCl '5
CaC0~ 0,2
2,4-Dideoxy-streptamine dihydrochlQride 0,025
Water to 100 ml.
Cultures were incubated in Erlenmeyer flasks at 280C to 30C on a
rotary shaker and with good aeration,
The production of 6-deoxyneomycin started after 2 days and was
optimal after 5 days.
Control cultures containin~ no 2,4-dideoxystreptamine dihydrochloride
did not produce any 6-deoxyneomycin.
~ ~ The culturè medium was then centrifuged and the supernatant liquid
! ~ poured through a column containing a weakly acidic~ carboxylic (polymetha-~
~ crylic) type cation exchanGe resin of medium porosity (Amberlite IRC-50,
,~ .
, Amberlite is a registered Trade Mark) in the ammonium form.
This operation was undertaken twice to extract the 6-deoxyneomycin and
unchanged 2,4-dideoxystreptamine dihydrochloride, The column was washed
with~water~and the 6-deoxyneomycin was eluted with a 2N ammonium hydroxide
solut~on in water. The~eluate was concentrated under vacuum on a rotary
evaporator~and then~ applied~to a Sephadex G-10 column. Elution with a
0~ U-ammonium hydroxide solution gave 6-deoxyneomycin in the first frac-
tions snd~2,l~-dideoxystreptamine dihydrochloride in the subsequent fractions
By~this method a quantity of 6-deoxyneomycin in free base form was
;produc~d wh1ch repre6ented o3 units (1 unit = 1~g/ml neomycin) of activity.
Thls~oorresponds to~the incorporatlon in the 6-deoxyneomycin
~` '
;~ : :
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thu~ obtained of approximately 22% of the 2,4-dideoxystreptamine dihydro-
chloride initially employed.
This repre6ents the maximum activity obtained and further incubation
did not result in any increase in 6-deoxyneomycin.
As an alternative process, the crude 6-deoxyneomycin was purified
by paper chromatography on Whatman No 3 MM paper developed with a 4/1
methanol/ammonium hydroxide solution. The location of the 6-deoxyneomycin
wa6 visualized using 0.25% sodium hypochlorite in water, then absolute
ethanol followed by lX soluble starch and 1% pota6sium iodide in water with
air-drying between each application. -
In this assay, the Rf of the 6-deoxyneomycin was found to be 0.30,
For comparison purposes, it may be mentioned that the Rf of neomycin~
of Z-deoxyotreptamine and of 2,4-dideoxystreptamine with the ~ame 6y~tem
of ~ol~ents are :
Rf
Neomycin 0.26
2-Deoxystrèptamine o.58
2,4-Dideoxystreptamine 0.62
b) 6-Deoxyneomycin ~ul~hate
The 6-deoxyneomycin obtained as described above was converted to
its 6ulphate salt by reaction with the equivalent quantity of dilute
sulphuric `acid.
t~a3~ ~of 6-deoxyneomycin sulphate ~ +430(c = 1.0)water)
c) Structure of 6-deoxyneomycin
The structure of the antibiotic compound of formula I in free base
form obtain~ed hereabove, wa~ determined by comparison with the natural
antlbiotio, neomycin, formed when 2-deoxystreptamine i~ incubated in the
appropri~ate medium.
-15-
10~82Z6
Thc presumed 6-deoxyneomycin is rcferred to hereinafter as
Compound X.
}lethanolysis of both neomycin and Compound X with a 10~ methanolic
hydrogen chloride solution for two hours under reflux, gave two major
prodllct , one of which corIcsponds to methylncobiosamini~c and was pro-
duced from both compounds.
The other product referred to hereinafter as Z was different in the two
antibiotics, the Z compound from neomycin migratin~ less than the Z
product from Compound X in the chromatographic assay usin~ a 4/1 methanol/
ammonium hydroxide solution as elution solvent.
Confirmation of the structure of each Z wus obtained by mass spectro-
metry of the per-trimethyl-silyl derivatives.
Although the molecular ions were not obtained~ the spectra of the/v~r
Z compound from neomycin and that of the Z compound from Compound X t~ere/
similar except that two peaks from the spectrum of the former (m/e 343
and It60) were shifted 88 mass units lower in the latter (m/e 255 and 372).
This corresponds to a loss of OSi(C~13)3 and replacement by hydrogen
The structure of Z from neomycin thus corresponds to neamine and the
structure of Z from Compound X corresponds to 6-deoxyneamine.
~ Acetylation of both Z compounds in methanol followed by acid hydro-
i lysis with a 3N hydrochloric acid solution under reflux for 10 hours~ ~ave
in each case two maJor products
~- On paper chromatography~ one of these products appeared to be the same
in both cases whether obtained from Z issued from neomycin or Z issued
from Compound X and must be presumed to be 2,6-diamino-2,6-dideoxy-~-
glucose. The other two compounds w~re shown to ~e chromatographically
identical to 2-deoxystreptamine from neamine and to 214-dideoxystreptamine
f~rom~deoxyneamine.
hc6e results lead to the conclusion that Compound X corresponds
bo 6-d~eoxyneomycin.
.,:
, . . ,.. , . - ` .-. - ~ ~ . .. , . ; .. , . `
~0~82Z6
EXAMPIE 2
Separation of 6-deoxyneomycin into 6-deoxyneomycin B and 6-deoxy-
neomycin C
The 6-deoxyneomycin obtained following the method described in
Example 1 was separated into 6-deoxyneomycin B and 6-deoxyneomycin C
by paper chromatography usine a 3/16/6/1 mixture of tert~utanol/buta-
none/ 6.5N ammonium hydroxide solution/methanol as 60lvent.
6-Deoxyneomycin B and 6-deoxyneomycin C were detected on the paper
using the same method ~s that dc6cribed hereabove.
U6ing aR ~olvent system, methanol and ammonium hydroxide in the
proportion of 4 to 1 on 3MM Whatman chromato~raphy paper the ~f values
of 6-deoxyneomycin~ B and C were deter~ined in comparison with the
correspondin~ epimers of neomycin and the following re~ult6 were obtained
6-Deoxyneomycin B 0.29 `
6-Deoxyneomycin C 0.21
Neomycin B 0,25
Neomycin C 0.165
~ .
EXAMPLE 3
;~ Preparation of 6-deoxyparomomycin and its I and II epimers
In a fla~k, D- Streptomyces rimosus forma paromomycinus ATCC 21484
was grown for two to three days on the following medium previously
adjusted to pH 7~5 :
F
Soyabean meal
Ca~ein hydrolysate 0,25
CaCO, 0 5
G;luco~e
NaCl ~ ~ ~-5
NH4Cl 0.167
Water to 100 ml
,.
~,
1 y-
. .
.
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This culture was shaken and maintained at 28OC during the
indicated period of time. A 10X inoculum of this culture was then
added to a medium identical to that given hereabove but containing in
addition 0.025g of 2,4-dideoxystreptamine dihydrochloride.
Culturcs were incubated in Erlenmeyer flasks at 280 to 30C on
a rotary shaker and with good aeration.
The subsequent operations of preparation and purification of the
6-deoxyparomomycin were exactly the same as those described in
Example la hereabove.
Purification by paper chromatography under the fiame conditions
as those described in the foregoing Example 1 showed that the Rf of
6-deoxyparomomycin was 0,30.
For comparison purpo6es it may be mentioned that the Rf of paromo-
~ mycin in an identical chromatographic assay is 0.27.
.j :
Using as solvent sy6tem, methanol nnd ammoniu~ hydroxide in the
proportion of 4 to 1 on 3~1M Whatman chromatography paper the Rf values
of 6-deoxyparomomycin8 I and II were determined in comparison with the --
correspondine epimers of paromomycin and the followin~ results wer~
obtained :
, :
6-Deoxyparomomycin I 0.34
6-Deoxyparomomycin II 0.275
Paromomycin I 0 3
Paromomycin II 0.22
EXAMPLE 4
Preparatlon_of the mixture-of 3',4'-dideoxyneomycin and its derivntives
a) Mixture of gentamines of formula III
, ~
;A mixture~of gentamineæ of formula III was first prepared from
commercially available genta~icin sulphate; This ~alt was first convertel-
to its;free ~base and evaporated to obtain an oil. A solution of hydro-
ch]orlc acid in methanol, prepared by addin~ hydrochloric acid to dry
m~etha~nol, was adde~d to this oil and the solution so obtained wa~ refluxed
, ~
'il ~
` : :
1068ZZ6
for two hours. The methanolic hydrochloric acid was removed under
vacuum and the resultant oil wa6 taken up in a little water and pas6ed
through a column of ~mberlite I.R.A. 400 to ~rovide the free bafic of
tho dcsired Gentaminc~. The difrcrerlt fraction~ 60 collectcd were
evaporated to dryness and separated into methyl garo~aminide and
mixed gentamines by chromatography on silica gel using a 1~1/1 methanol/
chloroform/ammonium hydroxide mixture as 601vent. In this chromatographic
separation methyl garosaminide showed a Rf of o.8 and the mixture of
gentamine6 a Rf of 0.2.
-.
b) Mixture of 3',4'-d'ideoxyneomycin and its derivatives
In a flafik~ D- Streptom~e~ rimo~us forma paromomycinus ATCC 21484
was grown for two to three days on the following medium previously
adjusted to pH 7.5 :
.,: .
Soyabean meal
Casein hydroly~ate 0.25
CaC03 .5
',~ Glucose
NaCl .5
NH4Cl 0.167
This culture was shaken and maintained at 280C durin~ the
indicated period of time. A 10% inoculum of this culture was then added -'
to a medium identical to that given hereabove adjusted to pH 7.2 but
,~ containing in addition 50 ~g of the mixture of gentamines previously -` -
~ obtained.
`~ Cul~ure6 were incubated in Erlenmeyer fla~s at ~0C on a rotary
baker~and with good aeration during 5 days.
The crude mixture~of 3~4~-dideoxyneomycin and its derivative6 so obtained
was purified by paper chromatography on Whatman No 3l:~ paper developed ~ -
with~a 4/1 met~hanol/ammonium hydroxide solution. The location of the
mixture of 3';4'-dideoxynecmycin and its derivatives was visualized using ~ ,
~ ~ ,
1068ZZ6
0,25% sodium hypochlorite in water, then absolute ethanol followed by
1% 601uble 6tarch and 1% potassium iodide in water with air-drying
between each application.
In this assay, the Rf of the mixture of 3',4'-dideoxyneomycin
and its dcrivatives was found to be 0.25.
For comparison purposes, it may be mentioned that the Rf of the `~
mixture of gentamines of formula III in an identical chromatographic
assay is from 0.5 to .?.
20-
