Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
The present invention relates to novel compounds,
to their preparation and to pharmaceu~ical compositions
containing them.
The compounds o the formula (I):
CH3
I
~.~~
HO3SO ~Ll ~ x NH.CO.CH3 (I)
O ~ CO H
and salts thereo~ wherein X is a trans -SO.CH=CH-,
trans -S.CH=C~- or -S-CH2-CH2~ group are disclosed in
Belgian Patents Nos. 827331, 827332 and 839324 where they
are designatèd MM 4550, MM 13902 and MM 17880 respectively.
These compounds are named respectively:
MM 4550 (5R,6R)-3-(2-Acetamidoethenylsulphinyl)-6-
~(lS)-l-sulphonatooxyethy~ -7-oxo l-azabicyclo
L3.2.0~ hept-2-ene-2-carboxylic acid.
MM 17880 (5~,6R)-3-(2-Acetamidoethylthio)-6- [(lS)-l-
sulphonatooxyethyl]-7-oxo-1-azabicyclo
[3.2.0~ hept-2-ene-2-carboxylic acid.
MM 13902 (5R,6R~-3~(2-Acetamidoethenylthio)~6-
~(lS)-l-sulphonatooxyethy~ -7-oxo-1-azabicyclo
L3.2.0] hept-2-ene-2-carboxylic acid.
,~
DJ
It has now been found that the esters of the compounds
of the formula (I) are antibacterial agents. In addition
the esters of the compounds of the formula (I) are also
able to enhance the effectiveness of penicillins and
cephalosporins against ~-lactamase producing strains of
various gram-positive andgram-negative bacteria such as
Staphylococcus aureus, Klebsiella aero~enes, Escherichia coli,
Proteus mirabilis and other similar organisms.
Accordingly the present invention provides the salts
of the compounds of the formula (II):
CH
X - NH,CO,CH3 ~IIj
co2~
wherein R is an organic group o up to 16 carbon atoms
and X is as defined in relation to formula (I).
Suitably X in the compounds of the formula (II)
is a trans -âO-CH=CH- group. Suitably X in the compounds
of the formula (II) is a trans -S-CH=CH- group. Suitably
X in the compounds o~ the formula (II) is a -S CH2-CH2-
group.
,,
3~
Suitably the group R is selected from aryl, alkyl,
aralkyl and alkenyl groups which may be substituted by
halogen, or by hydroxyl alkoxy, acyloxy, aryloxy, aralkoxy,
alkylthio, arylthio, aralkylthio, nitro, cyano, carboxamido,
acetamido, sulphonamido or phenylsulphonyl groups. Alkyl
and alkenyl groups are suitably straight or branched
chained but straight chained groups are generally more
convenient.
More suitably R will contain up to 12 carbon atoms.
Thus R may ~e a methyl, ethyl, propyl, butyl or benzyl group
or a benzyl group substituted by one or two halogen atoms
or Cl 4.alkoxy or nitro ~-roups. The benzhydryl.group and
the benzhydryl group substituted by one or two halogen atoms
Cl 4 alkoxy or nitro groups are also suitableO
Particularly suitable esters include those wherein
the ester group C02R is of the ~ormula C02Rl wherein the
group C02Rl is readily hydrolysed in the human body, ~or
example in tissues or blood, to yield the parent acid or
its salt, or in equivalent mammalian tissues.
- Other particularly suitable esters include those
wherein the ester group C02R is as in an ~xample herein.
L"3~
Suitably Rl is a group of the sub-formuia (a~ - (c~:
- CH - O - CO R ~a)
O
- CH~ R ~c)
wherein R2 is a hydrogen atom or a methyl group; R3 is a
Cl 4 alkyl, Cl 4 alkoxy, phenyl or benzyl group; and R
is a hydrogen atom or a methoxy group.
Suitably R2 is a hydrogen atom.
Suitably R3 is a methyl, t-butyl, phenyl or ethoxy
group~
Most suitably Rl is a group of the sub-formula (a)
or (b) and is especially suitably of the sub-formula (b).
-- 5 --
Preferably Rl is a phthalidyl group as the resulting
compounds have particularly good activity when administered
by injection.
Thus preferred compounds of this invention include
the pharmaceutically acceptable salts of the compounds
of the formula tIII):
I~H3
H03SO ~ X - NH - CO - CH3 (III~
CO - O
~r~
wherein X is a trans -SO-CH=CH~, trans -S-CH=CH or
-S-CH2-CH2- group.
A particularly suitable value for Xl is the trans
-S-CH=CH- group.
A further ~avoured vàl~ue~for X is the.~S-C~2-CH
group.
Those compounds of the invention wherein R contains
a chiral centre may be presented as pure optical isomers
(for example the R isomer or the S- isomer) or a mixture
thereo (for example the R,S- form). Thus for example the
R- and S~ phthalidyl esters are included within this invention
as well as mixtures thereof such as the R,S- phthalidyl esters.
- 6 -
3~3
Suita~le ~alts of the compounds of the formula (II)
include the sodium, potassium, calcium, magnesium,
~mmonium or su~stituted ammonium, for example trimethyl-
ammonium, trimethylamine, 2-hydroxyethylamine,
bis-(2-hydroxyethyl)-amine, tri-(2-hydroxyethyl)-amine,
benzathine, procaine, bicyclohexylamine, dibenzylamine,
N,N-di~enzylethylenediamine, l-ephenamine, N-ethylpiperidine,
N-benzyl-~phenethylamine, pyridine, collidine, quinoline,
or a quaternary ammonium salt such as tetramethylammonium.
Most suitably the salt will be a sodium, potassium
or quaternary ammonium, for example tetra-Cl 6 alkyl
ammonium such a~ tetra-n-bu~yl ammonium and tetra-n-hexyl
ammonium.
Preferably the salt will ~e a sodiumor potassium salt
and most preferably the sodium salt.
'rhe present invention also provides a process for
the preparation of the compounds of the formula (II) by
esterification of the corresponding compound of the
formula (I) or salt thereof.
Suitable methods of ester formation are those
conventionally used to prepare esters of ~-lactam
containing compounds and include:
(a) the reaction of a di-salt of a compound of the
formula (I) with a compound of the formula (IV):
R - Q (IV)
wherein R is as defined in relation to formula (II) and
Q is a readily displaceable group; and
(b) the reaction of a mono-salt of a compound of the
formula (I) with a diazoalkane.
Suitable di-salts of the compounds of the formula (I~
which may be reacted with compounds R - Q include
alkali metal salts such as the sodium or potassium salts
or other conventional salts such as the quaternary
ammonium salts.
Suitable groups Q include those atoms or groups known
to be displaceable by carboxylate anions and include chlorine r
bromine and iodine atoms, sulphonic acid esters such as
OSO2CH3 or OSO2C~H4CH3 and other conventional groups
displaceable by nucelophiles.
A particularly suitable compound of the formula (IV)
is phthalidyl bromide. Other phthalidyl derivatives are
also favoured.
~ _
- , :
.
This displacement reaction is normally carried out
in an aprotic organic solvent such as dimethylformamide,
dimethylsulphoxide, hexamethylphosphoric triamide,
acetone, dioxane or acetonitrile optionally in the presence
of a crown ether andthe like solvents and at a non-extreme
temperature such as -5C to 100C. ~e have found that
the reaction may suita~ly be carried out in anhydrous
~ dimethylformamide at ambient temperature.
Those compounds in which the group R contains a
chiral centre are generally inltially formed as the R,S- form.
Separation into the R- and S- isomers may ~e achieved
by conventional methods such as chromatography, s~eding
out and crystallisation and the like.
The reaction of a mono~salt o~ a compound of the
formula (I) with-a diazocompound is a mild method of
making alkyl, aralkyl or substituted alkyl or aralkyl esters.
The diazotization reaction may be performed under
conventional reaction conditions, for example at a non-
extreme temperature and in a conventional solvent~ Such
reactions are normally carried out at between -5C and
100C and conveniently at ambient temperature. Suitable
solvents fQr this reaction include lower alkanols such as
methanol and ethanol and solvents such as tetrahydrofuran,
dioxane and the like. These solvents will normally be
present with a small amount of water which aids the
solubility of the compounds of the formu1a (I) in the solvent.
3~
-
Aqueous ethanol has-proved a Particularly useful
solvent ~or this reaction.
T~e mono ester mono ~uaternary ammonium salts of the
compounds of the formula (IIl may also conveniently be
prepared by the dissolution of a water soluble salt of
a compound of the f~rmula ~ in water ollowed by the
mixing of this solution with a solution of a quaternary
ammonium halide in a water immiscible inert organic solvent.
The organic solvent is t~en separated and evaporated to
yield the quaternary ammonium salt. Suitably the organic
solvent will be a halogen substi~uted hydrocarbon, such
as methylene dichloride or chloroform, and the mixing of
the two solutions will take place between 1C and 100C,
for example between 1C and 30C. The two solutions
will normally be mixed by shaking or stirring.
Salts of the compounds of the formula ~ may be
interconverted in conven~ional manner, for example by
using ion exchange resins, Metal ion salts useful for this
purpose include lithium, sodium and potassium salts~
Salts within this in~ention, e.g. of the compounds
of the formula (III~ may be obtained crystalline,
for example by crystallisation from inert organic solvents
such as ethyl acetate, cyclohexane or their mixtures.
- la -
In a further aspect this invention provides a
pharmaceutical composition which comprises a salt of a
compound of the formula (II~ as hereinh~ore defined
together with a phar~aceutically acceptable carrie~,
Such compositions may be in a form suitable for
oral, topical or parenteral use. For example; tablets,
capsules, syrup, reconstitutable powders and more suitably
sterile forms suitable for injection or infusion may be
used. Such compositions may contain conventional
pharmaceutically acceptable materials such as diluents,
binders, colours, flavours, preservatives, disintegrants
and the like in accordance with conventional pharmaceutical
practice in a manner well known to those skilled in the
formulation of antibiotics such as penicillins and
cephalsporins. Preferred compositions are adapted for
injection, for example as a sterile solution in water.
The salt of the compound of the ~ormula (II)
may be present in the co~positIon o~ the inyenti~n
as sole therapeutic agent or it may be present together
with a ~-lactam antibiotic. Suitable ~-lactam antibiotics
include those known to be susceptible to ~-lactamases
and also those having some intrinsic resistance to
~-lactamases. Such ~-lactam antibiotics include ampicillin,
amoxycillin, benzylpenicillin, phenoxymethylpenicillin,
propicillin, cephaloridine, cefoxitin, cephalothin,
cephalexin, carbenicillin, ticarcillin, and in-vivo
hydrolysable esters of such compounds such as the phenyl,
~ 3
tolyl or indanyl esters of car~enicillin or ticarcillin
or the acetoxymethyl, pivaloyloxymethyl or phthalidyl
esters of ampicillin, ~enzylpenicillin, amoxycillin,
cephaloridin~, cephaloglycin and the like.
The ratio o the salt of the compound of ~he. fonmula (II
to ~-lactam anti~io~ic is normally between 2Q:l
and 1:20 and more usually between 10 r 1 and 1:10, for
example between 3:1 and 1:3.
The total quantity o~ compound of this invention
present in any unit dosage form will normally be between
50 and 1500 mgs and will usually be between lOG and 1000 mgs~
Preferred unit dosage compositions according to this
invention may be administered one or more times a day~
for example, 2 to 4 times a day, in the treatment of
diseases of the urinary tract, respiratory trac~, soft
tissues and the like. Thus the compositions may be used
in the treatment of such- diseases:às bronchitis, gonorrhea,
otitus media, mastitis and the like~ -
3~
The following Examples illustrate the invention:
Example 1
The disodium szlt of MM 13902 (0.60 g, 1.37 mmol)
was treated with methyl iodide (0.53 g, 4.17 mmol) in
dimethylformamide (10 ml) at room temperature for 2h.
The solvent was removed in vac o and the product chromatographed
on silica gel using chloro~orm-ethanol mixtures as eluant.
The mono-methyl ester mono-sodium salt of MM 13902 was
obtained as a white solid (0.36 g, 61~);
~max (H20) 323 and 228 nm
vmax (KBr) 1760, 1685, r620 and 1220-1280 br cm 1
n.m.r. (DMSO-d6) 1.33 (3H, d, J, 6 Hz, CH3.CH), 1.92 (3H,
s, CH3), 2.92 (lH, dd~ J 19, Jl 10 Hz, Ha of ABX system) 7
ca. 3.5 (lH, m, Hb of ABX system), 3.75 (4H, s ~ m,
CH3O2C and CH.CH.CH), 4.20 (lH, m, Hx of ABX system),
4.38 (lH, dq, J 10, J' 6 Hz, CH3CH.CH), 5.82 (lH, d,
J 14 Hz, CH:CHNH), 7.07 (lH, dd, J 14, J' 11 Hz, CH:CHNH),
and 10.45 ~ br (lH, d, J 11 Hz, NH.CH:3.
The compound was also prepared as $ollows:
The disodium salt of MM 139Q2 ~1~1 g, 2~52 mmol~ was stirred
at room temperature ~ith methyl iodide ~1~5 g, 10.56 mmol)
in NN-dimethylformamide C2a mll for 2 hours. The solvent
was removed in vacuo and the residue chromatograp~ed on
silica gel (30 g~ employing a gradient elution from chloroform
to chloroform:ethanol ~1:1). Fractions containing the
product were combined and evaporated in vacuo. Ethyl acetate*
was added to the residue and then removed in vacuo to afford
- 13 _
the monomethyl ester monosodium salt of MM 13902 as a
white solid (0.68 g, 63%)~
* Other solvents which Will azeotropically remove
ethanol (e.g. CH3CN, toluene) may be employed at this stage.
Example 2
The disodium salt of MM 13902 (0.50 g, 1.15 mmol~ was
treated with benzyl bromide (1.00 g, 5.85 mmol~ in
dimethylformamiae tlO ml) for 2 hours at room temperature.
Work up and chromatography as in Example 1 afforded the
mono-benzyl ester mono-sodium salt of MM 13902 (0029 g, 50%);
A max (H20) 325 and 226 nm. v max (KBr) 1765, 1685, 1620
and 1210 - 1280 br cm
Example 3
The di-sodium salt of ~M 13902 (0.970 g, 2.22 mmol)
was treated with ~-~romobenzyl bromide (1.7 g, 6.80 mmol)
in DMF (17 ml) for 2.5 hours at room temperature. Work-up
and chromatography as described in Example 1 afforded the
mono p-bromobenzyl ester mono-sodium salt of MM 13902 as
a white solid (0.73 g, 56%);
A max (H20) 325 and 226 nm. v max (KBr) 1760, 1675, 1620
and 1210 - 1280 br cm 1.
- 14 -
.
3~
Example 4
The di-sodium salt of MM 13902 (1.67 g, 3.83 mmol)
~as stirred at room temperature with p-nitrobenzy~
bromide (2.48 g, 11.6 mmol~ in dimethylormamide (30 ml).
Work-up and chromatography after 2 hours as described in
Example 1 gave the p~nitrobenzyl ester mono sodium salt
of MM 13902 as a pale yellow solid (1.27 g, 6Q%);
~ max (EtQH) 325, 266, 225 sh and 220 nm.
v max tKBr) 1760, 1680, 1620, 1210 - 1280 br cm
~ tDMSO - d6) 1.36 (3H, d, J 6 Hz, CH3CH), 1.92 (3H, s,
CH3CO), 2.96 (lH, dd, J 19 and 10 Hz, CHA of ABX),
3.52 (lH, dd, J 19 ana 9 Hz, CHB of Asx)~ 3.67 (lH, dd,
J 10.5 and 6 Hz, CH.CH.CH), ca. 4.20 (lH, m, CHX of ABX),
4.42 (lH, dq, J 10.5 and 6 Hz, CH.CH3), 5.22 and 5.42 (each
lH, d, J 14 Hz, CH2Ar), 5.80 (lH, d, J 14 Hz, CH:CH.S~,
7.04 (lH, dd, J 14 and 10.5 Hz, CH:CH.NH), 7.63 and
8.17 (each 2H, d, J 8 Hz, aromatic protons) and 10.38
(lH, d, J 10.5 Hz, NH).
Found: C, 42 94; H/ 3.77; N, 7.21; 5, 11.54; Na, 4.41%,
C20H20N3S2OloNa ~H2O requires C, 43-01; H~ 3-76;
N, 7.54; S, 11.47, Na, 4.12%.
- 15 -
.
3~
Example 5
The di-sodium sal~ of MM 13902 (0.30g, 0.69 mmol) was treated
with p-iodoben~yl bromide (0.90g, 3.03 mmol) in dimethylformamide
(9 ml) at room temperature. Work-up and chromatography after
5 h as described in Example 1 a~forded the mono p-iodobenzyl
ester mono-sodium salt of ~M 13902 as a white solid (0.15g, 34%),
~ max (H2O) 325 and 232 nm~
v max (Ksr) 1765, 1675, 1620 and 1210-1280 br cm 1.
Example 6
The di-sodium salt of MM 13902 (0.18g, 0.41 mmol) was treated
with o-iodobenzyl bromide (0.55g, 1.85 mmol) in DMF (5ml) for
6 h at room temperature. Work-up and chromatography as
described in Example 1 afforded the mono o~iodoben~yl ester mono-
sodium salt of ~M 13902 as a buff-coloured solid (0.07g, 26%)
v max (RBr) 1760, 1680, 1620 and 1210-1280 br cm 1,
Example 7
The di-sodium salt of MM 13902 (0.25g, 0.57 mmol) was stirred
at room temperature with p-bromophenacyl bromide (0.80g, 2.~8 mmol)
in dimethylformamide (5 ml). After 1.5h, work-up and chromatogxaphy
as described in Example 1 afforded the mono p-bromophenac~l
- 16
.. ~
ester mono-sodium salt of MM 13902 as a cream-coloured solid
(0.21g, 60%)
~ max (EtOH) 327 and 257 nm
v max (KBr) 1765, 1690, 1620 and 1210 1280 cm
Found: C, 39.90; H, 3.64; N, 4.09; S, lOolO; Br, 12.55%
C21H20N2BrOgS2Na.H2O requires C, 40.06; H, 3.50: N, 4.45;
S, 10.17; Br, 12.72%.
Example 8
The di-sodium salt of MM 13902 (0.30g, 0.69 mmol) was treated
wlth bromophthalide (0.50g, 2.35 mmol) in DMF (5ml)~ Work-up
and chromatography after 1.5h as described in Example 1 afford~d
the phthalide ester mono-sodium salt of MM 13902 as a buff-coloured
solid (0.08g, 21%).
~ max (H2O) 333 and 233 nm
v max (KBr) 1775, 1680 br., 1620, 1210 and 1275 cm 1
Example 9
The di-sodium salt of MM 13902 (0.30g, 0.69 mmol) was treated
with N-chloromethylben~oxazolone (0.50g, 2.72 mmol) in DMF (6 ml~
at room temperature or 2 h. Work-up and chromatography as
described in Example 1 aforded the mono N-benzoxazolonyl-
methyl ester mono-sodium salt of MM 13902 (0.155g, 40%)
~ max (H2O) 330, 267 and 225 nm
v max (KBr) 1775 br., 1690, 1620 and 1220-12180 br cm 1
Exa ple 10
The di-sodium salt of ~M 17880 (0.3 g, 0.68 mmol)
in N,N-dimethylformamide (4 ml) was treated with p-bromo-
benzyl bromide (0.75 g, 3 mmol) and the mixture was
stirred for 2~ hours. The sol~ent was removed on a
rotary evaporator and the residue chromata~raphed on
silica gel, eluting with chloroform/ethanol mixtures
(up to 3:2). Evaporation of the fractions containing
the ester (followed by evaporation of the residue from
toluene) gave the mono ~bromobenzyl ester mono-sodium
salt of MM 17880 (0.195 g, 48%~;
v max (KBr) 1765, 1690, 1635, 1220 cm
max (H20) 320 nm;
~ (DMSO - d6) 1.37 (3H, d, J 6 Hz, CH3CH), 1.81 (3H, s,
CH3CO~, 2.8 - 3.6 (6H, m, CH2.C.SCH2CH2N), 3.69 (lH,
dd, J 11 and 6 HZ, CH.CH.CH), 4.05 - 4.6 (2H, m, CH.CH.CH)/
5.08 and 5.26 (each lH, d, J 14 Hz, CH2Ar), 7. 36 and
7.57 (each 2H, d, J 8~5 Hz, aromatic protons) and
8.10 (lH, broad, NH).
~ 18 -
Ex~ple ll
The di-sodium salt of MM 4550 (816 mg) in N,N-dimethyl-
formamide (10 ml) was treated with iodomethane (0.9 ml).
The mixture was stirred for 2 hours. The solvent was
then removed on a rotary evaporator and the residue
chromatographed on silica gel (40 g), eluting initially
with chloroform, and then ~ith chloroform/ethanol (3:2).
The monomethyl ester mono-sodium salt of MM 4550 was
isolated as a solid (260 mg~ after com~ination and
evaporation in vacuo of the fractions con~aining the ester
followed by evaporation in vacuo of the residue from
toluene.
v max (KBr) 1775, 1710, lZ20, 1260 cm
~ max 297 and 245 nm;
The n.m.r. spectrum in (CD3)2SO ~howed inter alia
signals at ~ 1.36 (3H, d, J = 6 Hz), 2.0 (3H, s~,
3.77 (3H, s), 6.22 (lH, d, J - 15 Hz), 7.38 (lH, dd,
Jl = 15 Hz, J2 = 10 Hz).
Exam~le 12
The di-sodium salt of ~ 4550 (500 mg) in dimethyl-
formamide (5 ml) was treated ~ith p-bromobenzyl bromide (1.5 g)
and the mixture was stirred ror 3 hours. Work-up as in
Example 11 gave the mono p-bromobenzyl ester mono-sodium
salt of MM 4550 (350 mg).
v max (KBr) 1780, 1710, 1620, 1260 - 1220 cm
~ max (H2O~ 305, 244 (sh~, 228 nm. The n.m.r. spectrum in
D2a showed signals inter aiia at ~ 1.43 (3H, d, J = 6 Hz),
2.00 (3H, s), 6.23 (lH, d, J = 15 Hz), 6.96 (2H, d, J = 8 Hz),
7.14 (2H, d, J = 8 Hz), 7.32 (lH, d, J = 15 Hz).
1l3
Example 13
The di-sod~um salt o~ MM 4550 (800 mg) in N,N-dimethyl-
formamide t8 ml? ~as treated with benzyl bromide (1.0 ml).
The mixture ~as stirred for 3 hours, and then worked up
as in Example 11. The mono~enzyl ester mono-sodium salt
of MM 4550 was obtained as a solid (420 mgl.
max (KBr) 1780, 1705, 1620/ 1220 - 1260 cm
max (H2O) 306, 246 nm;
~ [(CD3)2SO] inter alia 1.36 (3H, d, J 6 Hz, CH3CH),
2.01 (3H, 5~ C~3CO), 5.26 (2Hr broad s, OCH2Ph), 6.24 (lH,
d, J 14 Hz r CH=CH )
Example 14
The di-sodium salt of MM 4550 (400 mg) in N,N-dimethyl-
formamide (4 ml~ was treated with p-nitrobenzyl ~romide (1.2 g).
The mixture was stirred for 2.25 hours and then worked up
as in Example 11 to give the mono p-nitrobenzyl ester mono-
sodium salt of ~ 4550 (234 mg).
v max ~KBr) 1780, 1710, 1620r 1260 - 1220 cm 1;
~ max 250, 296 (sh) nm. The n.m.r. spectrum in D20 showed
inter alia signals at about ~ 1~53 (3H, d, J = 6 Hz)~
2.1 (3H, s), 6.4 (lH, d, J - 15 Hz), 7.33 (lH, d, J = 15 Hz)~
7.38 (2H, d, J = 8 Hz) ~ 8.03 (2H, d, J = 8 Hz).
Example 15
The di-sodium salt of MM 4550 (670 mg) and p methoxy-
benzyl bromide (1058 g) were stirred together in N,N-dimethyl
formamide (10 ml) for 3 hours. Work-up as in Example 11
gave the mono p-methoxybenzyl ester mono-sodium salt of
MM 455~.
- 20 -
'', ., :
Example 16
The di-sodium salt of MM 4550 (50 mg) and o~iodobenzyl bromide
(190 mg) were stirred together in N,N-dimethylformamide
(2 ml) for 6 hr. The solvent was evaporated off and the
residue was triturated under toluene. After centrifuging the
residue (92 mg) was collected and then chromatographed on cellulose,
eluting with i propanol/water (9:1). The requisite fractions were
combined and evaporated down ~n vacuo to give the mono o-iodobenzyl
ester mono-sodium salt of MM 4550. v max (C~C13) 1785, 1710,
1620, 1260-1220 cm~l.
Example 17
The di-sodium salt of MM 4550 (185 mg) in N,N-dimethylformamide
(2 ml) was treated with pivaloyloxymethyl bromide ~80 mg) in
N,N-dimethylformamide (1 ml). The mixture was stirred at room
temperature for 4~ hr., and then in a refrigerator for 2 days.
The solvent was evaporated off, and then the residue placed on
a very short silicagel chromatography column and eluted off with
ethanol/chloroform (3:2). The mono pivaloyloxymethyl ester
mono-sodium salt of MM 4550 was obtained rom the eluate as a
solid, v max (KBr) 1780, 1705, 1610, 1260 cm 1. The. u.v.
spectrum showed a maximum at 296 nm (in H2O).
Example 18
The di-sodium salt of MM 4550 (300 mg) and ~l-chloromethylbenzoxa-
zolone (360 mg) were stirred together in DMF (5 ml) for 3 hr.
The solvent was then evaporated off and the mixture was worked
up as in Example 11 to give the mono-N benzoxazolonylmethyl ester
- 21 -
3~
mono-sodium salt of MM 4550,vmax (KBr) 1780, 1720-17109 1620,
1260 cm 1. ~ max (H20) 306, 247, and 226 nm. ~(D20, HOD internal
standard at ~4.6) inter alia. 1.40 ~H, d, J 6E-Iz,, cH3c~ll 2,00
(3H, s, CH3CO) 5.90 (2H, broad s, OCH2N), 6.25 (1H, d, J l~Hz,
CH = CH), 7.0 - 7.25 (4H, m, ArH) 7 7.36 (1H,d, J 14 Hz, CH=CH).
The di-sodium salt of MM 13902 (60mg) was suspended in ethanol
(5ml) and water was added until dissolution was complete. The
solution was cooled to 0 (ice bath) and heated with a cooled
(0) solution of p-toluenesulphonic acid (90 mgs) in ethanol
(6 mls). The resulting solution was stirred for ca. 5 seconds
and treated with excess ethereal diazomethane. After 20 minutes
at room temperature the solvents were removed in vacuo. The
residual semi-solid was triturated wi-th ether, filtered and
washed with ether.
Chromatography of the solid on silica gel, eluting with butanol
ethanol-water (16:4:7), gave the monomethyl ester mono-sodium
salt as an amorphous solid.
Example 20
The di-sodium salt of MM 4550 (500 mg) was dissolved in water and
passed down a column o~ DOWEX 50W-X8 ion exchange resin which
had been converted to its calcium form by treatment with an
aqueous slurryof calcium carbonate. Evaporation in vacuo o~ the
relevant fractions gave the calcium sal-t o~ MM 4550, vmax (KBr)
1755, 1690, 1620, 1260-1220cm 1.
The calcium salt so obtaine~ was taken up in N,N-dimethyl-
formamide (9 ml) and p-bromobenzyl bromide (1.4g) was added.
* Trade Mark
-- 22
3~
The mixture was stirred for 3 hr., and the solvent was then
evaporated. The residue was chromatographed on silica gel
eluting with chloroform/ethanol mixtures to give the mono-~-
bromobenzyl ester of MM 4550 calcium salt (180 mg) v max
(CHCl3/Me2NCHO) 1790, 1720, 1620, 1260 cm
Example 21
The mono-sodium salt mono-p-bromobenzyl ester of MM 13902 (191 mg)
in water (6 ml) was shaken with a solution of tetrahexylammonium
bromide (138 mg; Q.95 equivalent) in methylene chloride (10 ml3.
The organic layer was separated, filtered through cotton and
evaporated to give the product as a foam in almost quantitative
yield; v (CH2Cl2) 3200-3400 (broad), 1775 (~-lactam C-O), 1695
(broad, ester and amide carbonyls), 1620 cm 1 (C=C); n.m.r.
(CDC13) showed inter alia 6.Z0 (lHr d, J 14Hz, -S C~=CH-NH),
7.55 (5H, m, -S-CH=CH-NH- and aromatic protons), 10.06 ~ (lH,
d, J 10Hz, SCH=CHNH).
Example 22
The monomethyl ester, mono-sodium salt of MM 13~02 (0.05g) was dis~
solved in waterj and the solution stirred vigorously with a solution
of tetrabutylammonium hydrogen sulphate (0.04g) in CH2C12 (5 ml).
After 5 min. the organic layer was separated and dried (MgSO4).
Evaporation of the solvent gave the crude tetrabutylammonium
salt (II, R=(C4Eig)~N~ Rl=CH3) (0.61g). Rapid chromatography
on silica (CE~Cl3-EtOH as eluant) afforded a pure sample of the
mono tetrabutylammonium salt monomethyl ester of MM 13902 ~o.0~ g);
v max (CHCl3) 1775 (~ lactam C~0),
- 23
r
1695 (unsaturated ester and enamide C=0)~ 1625 and 1200-1280
br cm 1 (sulphate).
Example 23
The mono-p-nitrobenzyl ester mono so~ium salt of ~q 13902--(0.05g)
was ~reated - with tetrabutylammonium hydrogen sulphate--
as described in Example 22. The mono tetrabutylammonium salt
mono-p-nitrobenzyl ester of MM 13902 was obtaine-d as a gum (0.05 g);
v max (CHC13) 1775 (~-lactam C=0~, 16gO (unsat. ester and
enamide C=0), 1625 and 1200-1280 br-cm l(sulphate).
.
Example 24
The mono-p-iodobenzyl ester mono sodium salt of MM 1~902 (0.22g)
wàs dissolved in water (2 ml) and the solutiorl treated with an
aqueous solution of S-benzyl-iso-thiouronium chloride (0.100 g)
at 5. An immediate white precipitate was formed. Chloroform
was added and the product was extracted into the organic layer,
which was dried (MgSO4) and evaporated to afford the mono
S-benæylisothiouronium salt mono p-iodobenzyl ester of MM 13902
(0.15 g); v max tCHC13) l780, 1690, 1670 and 1625 cm 1,
Example 25
20 The mono-p-bromophenacyl ester mono sodium salt of MM 13902
(0.055 g) was treated with S-benzyliso-thiouronium chloride (0.04 g)
in the manner described in Example 24. The mono S-benzyl-
isothiouronium salt, mono p-bromophenacyl ester of MM 13902
was obtained as a gum (0.047 g); v max (CHC13) 17~0, 1695,
--1
25 1670 and 1625 cm
-- 24 --
3~ `
Example 2 6
The mono p-nitrobenzyl ester mono sodium sal t OI MM 13902 (0 .11 g) was
treated with- S-benzylisothiouronium chloride (0.075g) as described
in Example 24 The 5-benzyltsoth~ouron~um s~lt ~f ~he .
p-nitrobenzyl est~r of MM 13~02 wa~ o~tained as a gum ~0.080 g1;
v max (CHCl3) 1775, 1690 ~r, and 162a cm l
.
Example 27
~M 4550 monobenzyl ester mono-sodium salt was dissolved in water
and a chloroform solution containing Aliquat*336 (methyl trioctyl-
ammonium chloride from General Mills Chemical Inc., Kanakee,
Illinois, U.S.A.) was added, the mixture was shaken thoroughly,
the layers separated and the chloroform layer was evaporated
to give MM 4550 monobenzyl ester monomethyl-trioctylammonium
salt.v max (CH2C12) 1785, 1720, 1620, 1240-1220 cm 1.
Example 28
Antibacterial and Synergistic Activity of the Mono-Pivaloyloxymethyl
Ester Mono-Sodium Salt of MM 4550 (el) and the Mono-Phthalide
Ester Mono-Sodium Salt of MM 13902 (e2)
The above two esters (el) and (e2) were tested in parallel with
20 MM 13902 and MM 4550 against 5 ~-lactamase producing organisms
both alone and in combination with ampicillin against 3 of these
organisms. The results are shown in Tables 1 and 2.
* Trade Mark
- 25
.
Table 1
Antibacterial Activity of (el) and (e2) expressed as minimum
inhibitory concentra~ion (MIC3 in ~g/ml _ _
~-lactamase I
ompound S~aphylococcus Klebsiella Proteus E.coli E.coli
aureus Russell aerogenes mirabilis JT39 JT10
E70 C889
_ _ _ _
Di--Sodium
MM 4550 12.5 6.25 25 12.5 25
Di-Sodium
MM 13902 0.2 0.8 002 0.~ 0.4
el 125 125 125 8.0 250
e2 31 15.6 15.6 31 62
_ ._ .
able 2
Synergistic Activi~y of (el) and (e2) with ampicillin and
cephaloridine expressed as minimum inhibitory concentration
(MIC) in ~g/ml
_ _ _ _
ompound Conc. of Staphylococcus Proteus Klehsiella
compound aureus Russell mirabilis aerogenes
in ~g/ml C889E70
_ ~-
Ampici-
llin _ 250 >20001000
el 1.0 250 > 500>500
4 15.6 5~0
e2 1.0 4 15.6 >500
c 0.5 1.0 250
_ _ _
- 26 -
In~vivo Activity o~ the mono-phthalide ester mono-sodium
salt of MM 139Q2 (e2) and the mono-phthalide ester of the
mono-sodium salt o:~ MM 4550 (e3).
5 The above two esters (e2) and (e3) were tested in parallel
with MM 13902 and MM 4550 by administration by su~-cutaneous
injection i~ aqueou~ pH 6.6 phosphate ~u~fer solution~
In this test four doses were applied at 1~ 2, 3 and 4 hou~
post intra peritoneal infection by E-coli 8.
The results were as follows:
Test compound CD50
MM 4550 Di-sodium3.9 mg/kg x 4
(e3) 22.0 mg/kg x 4
MM 13902 Di-sodium10.5 mg/kg x 4
(e2) . 4.4 mg/kg x 4
In a similar test against St~hylococcus aureus Smith
the following results were ob tained
Test Compound CD50
MM 13902 Di-Sodium 23.5 mg/kg x 4
(e2) 7.8 mg/kg x 4
-- 27
L39
Exam~le 30
MM 4550 di-sodium salt (1.05g) and bromophthalide (625mg)
were stirred together in ~rN~dimethyl~ormamide (19ml) for
2 hours at ambien-t temperature. The solvent was then
removed on a rotary evaporator and the residue chroma-
tographed on silica gel (50g) eluting with a 1:1 mixture
o~ chloroform and e~hanol. The fractions containing the
ester were combined and evaporated on a rotary evaporator,
toluene was added to the residue and evaporated of~ in
the evaporator to give the mono-ph-thalide ester mono
sodium salt o~ MM 4550 as a solid ~max ~H20) 306 and
230-235 nm. v max (KBr) 1785, 1720-1700 (br.), 1620, 1260,
and 980 cm 1 ~ [(CD3) 2S0] (inter alia) 1.33 (3H, d, J
ca 6Hz), 1.98 (s) and 2.01 (s) (3H, COCH3 o~ the two
epimers), 6.20 (d, J = 14Hz) and 6.26 (d, J = 14Hz)
(1H, S-CH = C, o~ the two epimersl.
The di-sodium salt of MM 4550 (200mg) in water (2ml) was
shaken with cetylbenzyl dimethyl ammonium chloride
(~50mg, 2 eq.) in dichloromethane (2ml). The dichloro-
methane layer was dried (MgS04) and evaporated down to
leave the di-quaternary ammonium sal-t of MM 4550 as a
gum (397mg). v max (CH2C12) 1765, 1690, 1620 cm 1.
Methyl chloroformate (33mg) in dichloromethane (5ml) was
cooled to -~0 and the diquatenary ammonium sal-t of
MM 4550 in dichloromethane (8ml) containing one drop o~
- 28
dimethylbenzylamine was added drop-wise. The mixture
was stirred at -30 to -10 for 45 minutes after addition
was complete, when the infra red spectrum indicated that
the mixed anhydride had formed ~v max (CH2C12) 1820, 1790,
1620 cm 1], With the temperature at ~3O ethanol (1 ml)
was added and the mixture was then stirred at -10 for
15 minutes, then at 0 for ca 10 minutes, then at 10 for
45 minutes and finally at room temperature for 1 hour.
The mixt;ure was then evaporated in vacuo and the
residue was taken up iXl a mixture of water (pH 7.03 and
ethanol and passed down an Amberlite*120 (Na) ion exchange
resin . Evaporation of the- eluate, and chromatography
o the residue on silica gel (10 g) eluting with chloroform/
ethanol (3:2) gave the mono ethyl ester mono sodium salt
of M~ 4550.
~ (D2O) (inter alia) (HOD internal standard ~ 4.6 ppm)
1.26 (3H, -t, J 8 Hz, CH3CH2), 1.44 (3H, d, J 6 Hz, CH3CH),
2.04 (3H, s, CH3CO), 6.34 (lH, d, J 14 Hz, CH=CEI),
7.62 (lH, d, J 14 Hz, CH=CH).
2~ * An alternative method for conversion of a mono ester
mono quaternay ammonium salt to the mono ester mono sodium
salt i5 to shake a dichloromethane solution of the ester
with water containing a sodium salt such as sodium tetra-
fluoroborate or sodium iodide. Separation of the layers
and evaporation of the aqueous layers gives the mono ester
* Trade Mark
- 29
3~
mono sodium salt of MM 4550. This ~Toxks ~?ro~7ided the.
~ùat~rnary a~nonium iodl.de or ~etrafllloro~or~te is
relatively insolu~le in ~ater.
~3~
The mono methyl ester mono sodium salt o:E MM 4550 (30mg3
in water (2ml) was treated with chloroform (3ml) contain-
ing cetylbenzyl-dimethylammonium chloride (2'7mg). After
shaking the chloro:Eorm ].ayer was separated, dried (MgS04)
ard evaporated in vacuo to give the mono methyl ester
mono cetylbenzyldimeth~lammonium salt of MM 4550 (ca 80%
yield). v max ~HCl3~ 1785, 1705, (broad), 1620 cm 1.
Example 33
The di-sodium salt of MM 4550 (105mg) in water (2ml) was
treated with cetylbenzyldimethylammonium chloride (184mg)
in dichloromethane (2ml). A~ter shaking the dichlorometh-
ane layer was separated, dried (:M~S04~ and evaporated in
v_cuo to leave the di (Cetylbenzyldimethylammonium) salt
o~ ~1 4550, v max 1775, 1695, 1625 cm 1.
The diquaternary ammonium salt o~ MM 4550 was redissolved
in dichlorome-thane (2ml) and methyl iodide(0.5ml) was
addedO After stirring the mixture for ca 2 hr. the
solvent and excess methyl iodide were removed in vacuo.
Chromatography.of -the residue on silica gel (10g) eluting
-- 30
. . .
~ 3~
with chloroform/ethanol mixtures (gradien-t elution from
CHC~3 to GHCl3/EtOH 3:2) gave the mono methyl ester mono
cetylbenzyldimethylammonium salt of ~M 4550. (ca 30
yield from MM 4550l.
Alternati~ely the esteri~ication o~ the di (cetylbenzyl-
dimethylammonium) salt of MM 4550 can be accomplished in
solution in dry 1,2-dimethoxyethane; after evaporation
to 2 low volume ce-tylbenzyldimethylammonium iodide
crystallises out, leaving the mono methyl ester mono cetyl~
benzyldimethylammoniu~ 5alt o~ ~M 455Q in the mother li~uors~
Confirmation of the structure of the ester can be obtained
by conversion to the mono sodium salt by two methods;
a) sodium iodide(1 equivalent) is added to a concen-trated
solution of the mono methyl ester mono cetylbenzyldimethyl
ammonium salt of MM 4550 in 1,2-dimethoxyethane. Cetyl-
benzyldimethyl ammonium iodide crystallises ou-t leaving
the sodium salt in the mother liquors. After chromato-
graphic purification the mono methyl ester mono sodium
salt of MM 4550, identical to that described in Example
11 is obtained.
b) A solution of -the mono methyl ester mono cetylbenzyl-
dimethylammonium salt,of ~M 4550 in dichloromethane is
shaken with water containing sodium tetrafluoroborate
3~
(1 equivalent), separation and evaporation of the
aqueous layer, followed by chromatography of the residue
on silica gel gives the mono methyl ester mono sodium
salt of MM 4550.
Example ~.
.
The mono p-nitrobenzyl ester mono sodium salt of MM 4550
(100mg) and ce-tylbenzyldimethylammonium chloride (73mg)
were dissolved in water (5ml) and chloroform (5ml). After
shaking the layers were separated and the chloroform
layer dried (MgS04) and evaporated in vacuo to give the
mono p-nitrobenzyl ester mono cetylbenzyldimethylammonium
salt of ~M 4550 (_ 80% yield~ vmax (CHC13) 1785,
1710 (broadt, 162Q cm l.
Example 35
The mono methyl ester mono sodium salt of MM 4550 (52mg)
and benzyltriphenylphosphonium chloride (44 mg) were
shaken together in water (5~1) and dichloromethane (5ml).
On separation the dic~loromethane layer was dried (MgS04)
and evaporated in vacuo to leave the mono methyl ester
mono benzyltriphenylphosphonium salt of MM 4550 as a
semi-solid foam (76mg) ~max (CH2Cl2) 1790, 1710 (broad)
1610 cm 1.
The di-sodium salt of MM 17880 (492 mg3 in DMF (5 ml)
- 32
3 ~
was treated with p-ni-trobenzylbromide (1.Og) and the
mixture was stirred for 1~ hr. Work-up as in Éxa~ple 1
gave the mono p-nitrobenzyl ester mono sodium salt of
MM 17880 t200 mg).
Example 37
The di-sodium salt of MM 13902 (O.2g, 0.46mmol) was
treated with methyl tosylate (o.Zsg~ 1.47mmol) in
dimethylformamide (5ml) ~or 3 h at room temperature.
Work-up and chromatography as described in Example 1
af~orded the mono methyl ester 9 mono sodium salt of
MM 13902 as a white solid (o.06g~ 30%).
Example 38
To a suspension o~ the disodium salt of MM 13902 ~0.3g,
o.69 mmol) in acetonitrile (10ml) was added 15-crown-5
(3 drops) and benzyl bromide (0.35g, 2.05 mmol). After
stirring at room temperature for 3 h, the mixture was
treated as in Example 1. The mono-benzyl ester, mono
sodium salt of MM 13902 was obtained as an off white
solid (0.145g,42%).
~ E~
A sample of crude (ca 60% pure) disodium salt of MM 17880
- 33
iL3~
(lg) was treated with me~yl iodide (1ml) in DMF (15ml).
After 2 h the solvent was removed in vacuo, and the
residue chromatographed o~ silica-gel (30g) using a
gradient elution from chloroform to chloroform/ethanol
(2~3). The fractions containing the produc-t were combined
and evaporated in vacuo. Ethyl acetate was added to the
residue and then removed in vacuo to afford the monomethyl
ester mono-sodium salt of MM 17880 as a light-yellow
solid (0.25g); Amax. (H20) 314nm ~ max. (KBr) 1765,
1700~ 1660br and 1220-1270br cm 1. ~(DMS0-d63 1~35
(3H, d, J 5Hz, CH3CH), 1.80 (3H, s, CH3co)~2.8-3.8 (7H,
m,CH.CH.CH.CH2 C.SCH2CH~N), 3.65 (3H,s,C02CH3) 3.95 -4.6
(2H,m,CH.CH.CH and 8.0 (1H, br NH).
The mono p-bromobenzyl, mono sodium salt of MM 13902
(0.05g, 0.086mmol) was ~issolved in water (5ml) and the
solution shaken with one of cetylbenzyldimethylammonium
chloride ~0.032g, 0.08mmol) in dichloromethane ~5ml).
The organic layer was separated, dried (M~504~ and
evaporated in vacuo to a~ford the mono p-bromobenzyl ester,
mono cetylbenzyldimethylammonium salt of MM 13902 as a
~oam (0.074g,93%); vmax. (CH2C12) 1780, 1700, 1685sh and
1625cm 1
Example 41
A solution of the mono methyl ester, mono sodium salt of
_ 34
3~
MM 13902 (0.10 g, 0.23 mmol~ in water (5ml) was treated
with a solution of cetylbenzyldimethylammonium chloride
(0.09 g, 0.23 mmol) in CH2C12 (5 ml) as described in
Example 40. The mono methyl ester, mono-cetylbenzyldimethyl-
ammonium salt of MM 13902 was obtained as a gum (0.156 g,
87%) v max (CHC13) 1780, 1695 and 1630 cm 1.
Example 42
The di-sodium salt of MM 13902 (1.0 g, 2.29 mmol)
was treated with bromophthalide ~0.60 g, 2~82 mmol) in
dlmethylformamide (15 ml) at room temperature. After
1.5 hours the dimethylformamide was removed in vacuol
and the residue chromatographed on silica gel (30 g) using
a gradient elution (CHC13 to CHC13:EtOH, 1:1).
The first few fractions which contained the mono-
phthalide ester of MM 13902 (by thin layer chromatography~
slowly deposited white crystals. These were obtained by-
filtration (0.03 g). The mother liquors and the remaining
fractions wère combined and evaporated in vacuo. Ethyl
acetate was added and the solvents again removed in vacuo
to afford the mono-phthalide ester, mono sodium salt of
MM 13902 as a cream-coloured amorphous solid (0O62 g, 50~).
~ max (H2O) 333and 233 nm;
v max ~KBr) 1775, 1680 br, 1620, 1210 and 1275 cm 1;
- 35 -
The white crystals also consisted of the required
mono-phthalide ester of MM 13902, ~(DMSO-d6) 1.32 (3H,
d, J 6 Hz, CH3CH), 1~92 (3H, s, CH3CO), 2.98 (lH, dd,
J 20 and 10 Hz, HA of ABX), 3.45 - 3.75 (2H, m, HB of ABX
and CHCHCH), 4.05 - 4.55 (2H, m, CHCHCH~, 5.80 (lH, d,
J 14 Hz, CH=CH.S), 7.05 (lH, dd, J 14 and 11 Hz, N~.CH=CH),
7.49 (lH, s, CO2CHO), 7.55 - 8.0 (4H, m, aromatic protons).
Example 4 3
Using the I50 determination method of Belgian Patent
No. 827926, the sodium saltsof esters of this invention
have been found to have the following I50 values.
- 36
- -- ---~ ~ ~--
~D ~ ~ ~ ~ ~ ~ ~D ~D
~ ~ o o ~ ~ o o o ~ o s~
~ r----- _ _ _ ~
~ ~I ~ ~o r~ c~ ~O ~9 ~ ~ a)
Il~ O V O O O O O O O N
__ _ __ _ _ __
~0~ O ~ _~ ~ O O O ~ O
1~ A A
_ _ _ _ _ ~0 '
~ o o o ~ ~r o~ o o Ir) rl
O ~ _~ O A O O O O O C~
__ _ __ _ ~
~ ,4 0~ O O O O O O O O O ~ N
O (d O o o o o o o o o .,1 a)
S~ V ~
O _ ~ N ~D _ ~O O O O O
;~ O A _~ G~ O t`') O O O O O
1~ o~ _ _ _ _ _
~ * ~ ~ ~1~
Q~ O O O Oo O O O ~0 ~ ~D rl
;~:1 U O O O o O O o o v E o
_ __ I;~
~ 1~
~ Y a ~ ~ 3'
*
-- 37 --
3~ _ _ _ _
.=~ _ . .u~,
~r ~ tn o l o
~ _ U~ (d K ~ .
0~ t`~ O 0~ Cto~ G:) ~r ~r
o ~ o o o o
. _ _ _ ~ ___
O ~ L~ ~r O ~ _i ~
o ~ o A o o
_ ~ ~ ~
R ~o l o q o o ~ ~ ~ ~
~0~ ~0~ A A
. , =~-1 .-- . ---
~ o lo` ~ ~ ~
~oO O o ~,QCO O ~r o o
O~c~ o o O~c~ o o ~ o
_ _ ~ .r _
O O A O _~ A A A
h ~ _ _ _
~ ~D i r~ ~0 ~
~0 a) r-l O Q (D O O O O
C ~1 O O C -1 VO A A
_ _ _ _ r
N h ~J ~C
~ s~ a) a) ~ ~ ~
~i a), ,4 ~ ~ o ~ .
O ~ h ~i ~i 3 ~1
~1 m u~ N O ~ N
Q~ ~ ~1:1 '~ ~ _
. _ _ ,
-- 38 --
