Note: Descriptions are shown in the official language in which they were submitted.
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
3,6-DISUBSTITUTED PENAll'VII SULFONE DERIVATIVES AS ANTIBACTERIALS
FIELD OF INVENTION
The present invention is directed to ~i-lactamase inhibitors useful in
combination with /3, lactam antibiotics. More specifically, the present
invention is
directed to novel 3,6-disubstituted penam sulfone derivatives which
demonstrate
potent ~i-lactamase inhibitory activity.
BACKGROUND OF THE INVENTION
Penicillins and cephalosporins are the most frequently and widely used (3-
lactam
antibiotics. However, the development of bacterial resistance to these
antibiotics has
had a damaging effect on maintaining the effective treatment of bacterial
infections.
The most significant, known mechanism related to the development of bacterial
resistance to the (3-lactam antibiotics is the production of Class A and Class
C serine
~-lactamases. These J3-lactamases compounds degrade the ~3-lactam antibiotics,
resulting in a loss of antibacterial activity. It is known that Class A serine
(3-
lactamases, which have molecular weights of about 29 kDa, preferentially
hydrolyze
penicillins, whereas Class C serine (3-lactamases which have molecular weights
of
about 39 kDa, preferentially hydrolyze cephalosporins, Bacterial resistance to
these
antibiotics could be greatly reduced by administering, the ~i-lactam .
antibiotic in
combination with a compound which inhibits these enzymes.
A number of ~3-lactamase inhibitors are known in the art. For example, U.S.
Patent
No. 4,234,579 discloses ~penicillanic acid l,l-dioxide (i.e., Sulbactam)
useful for
enhancing the activity of a number of /3-lactam antibiotics. U.S. Patent No.
4,562,073
discloses a penicillin derivative (Tazobactam) useful as a ~i-lactamase
inhibitor.
Likewise, U.S. Patent Nos. 4,287,181 and.5,637,5.79 disclose penicillanic acid
derivatives
useful in enhancing the effectiveness of (3-lactam antibiotics. However, the
compounds
disclosed in these patents are active only towards Class A serine ~i-
lactamases and
demonstrate poor activity against Class C serine ~3-lactamases.
CA 02355738 2001-06-20
WO 00/43399 PCT/IJS00/01630
-2-
Two positions can. be modified in the penam sulfone base structure:_ the 3 (3-
methyl group and the 6-position. In the compounds discussed above, only one of
the
3 or 6 positions is modified. Thus far, such a singular modification on the
penam
S sulfone structure has not produced the desired activity against both Class A
and Class
C serine /3-lactamases. Accordingly, it can be seen that there is a need for
compounds
having such dual activities.
SUMMARY OF THE INVENTION
The present invention provides compounds having ~i-lactamase inhibitory
activity
against both Class A and Class C serine ~3-lactamases, which compounds have
the
formula (A):
Y
(A)
wherein -Y is
~R3
or
(i.e. A has one of the following formulae I and II:
R2 O,~ O R2 O
~S' R3
n R
N w N
O
COOR1 COOR1
I B )
wherein
n is 0 or 1 and
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-3-
when n=1, R is a 5 or 6 membered heterocyclic ring, hydroxy, halogen, oxo,
carbamoyl, alkoxy, disubstituted amino, or
when n=~, R is an ester, cyano or amide group;
X is CH or NH;
R3 is cyano, methoxy or acetarnido; and
R, is hydrogen, alkyl, a negative charge, a cation selected from the group
consisting
of sodium, potassium and tetraalkylammonium and acyloxyalkyl, or alkoxy-
carbonyloxyalkyl; and
RZ is hydrogen, acyl, trisubstituted silyl, carbamoyl, di-(C,~,
alkyl)aminocarbonyl or an
amino acid residue; or
pharmaceutically acceptable salts thereof.
DETAILED DESCRIPTLON OF THE INVENTION
Heterocyclic rings for R can be aromatic or non-aromatic and have one to
three heteroatoms, the same or different, selected from oxygen, nitrogen and
sulfur.
Examples include 1,2,3-triazole, isoxazole, imidazole or pyridine.
Examples of R when disubstituted amino are di-(Cm alkyl)amino where the
alkyl groups are the same or different. Examples of R when an ester are alkyl
esters
where the alkyl group has 1 to 4 carbon atoms. Examples of R when an amide are
CONHZ or mono- or di-(C'» alkyl)-amide, where the alkyl groups are the same or
different.
Examples of RZ w:hen trisubstituted silyl are tri-(C,.~ alkyl)silyl where the
alkyl groups are the same or different. Examples of R2 when acyl are (C»
alkyl)carbonyl. Examples of amino acid residues for R2 are glycyl, alanyl,
leucyl,
isoleucyl and valyl.
Preferably, the compounds of the present invention are those of the formula
set
forth above where n is l and R is 1,2,3-triazole, isoxazole, imidazole or
pyridine; R, is
sodium; and Rz is hydrogen; or pharmaceutically acceptable salts thereof.
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-4-
Preferably the asymmetric centres have the 2S, 3S, SR, 6R configuration.
Most preferably, the compounds of the present invention are:
(2S, 3S, SR, 6R)-6-Hydroxymethyl-3-methyl-4,4,7-trioxo-3-[1,2,3]triazol-1-
ylmethyl-
4-lambda(6)-thia-1-aza-bicyclo[3.2.0)]heptane-2-carboxylic acid;
(2S, 3R, SR, 6R)-6-Hydroxymethyl-3-(methoxyimino-methyl)-3-methyl-4,4,7-trioxo-
4-lambda(6)-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid;
(Z)-(2S, 3S, SR, 6R)-6-Hydroxymethyl-3-methyl-3-(3-nitrilo-propenyl)-4,4,7-
trioxo-
4-lambda(6)-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid; or
(E)-(2S, 3S, SR, 6R)-6-Hydroxymethyl-3-methyl-3-(3-nitrilo-propenyl)-4,4,7-
trioxo-
4-lambda(6)-thia-1-aza-bi.cyclo[3.2.0]heptane-2-carboxylic acid.
As used herein the teams "alkyl" and "alkoxy" are used to represent straight
or
branched carbon chains of I-6 atoms. The term "halogen" is used to represent
chlorine, bromine, fluorine and iodine.
The compounds of this invention contain asymmetric carbon atoms and thus
give rise to optical isomers and diastereomers. While shown without respect to
stereochemistry, the present invention includes such optical isomers and
diastereomers; as well as the racemic and resolved, enantiomerically pure R
and S
stereoisomers; as well as other mixtures of the R and S stereoisomers and
pharmaceutically acceptable salts thereof. It is recognized that one optical
isomer,
including diastereomer and enantiomer, or stereoisomer may have favorable
properties over the other. 'Thus when disclosing and claiming the invention,
when one
racemic mixture is disclosed, it is clearly contemplated that both optical
isomers,
including diastereomers and enantiomers, or stereoisomers substantially free
of the
other are disclosed and claimed as well.
The compounds of th.e present invention may also be used in the form of a
pharmaceutically acceptable acid addition salt having the utility of the free
base.
Such salts, prepared by methods well known in the art, may be formed with both
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-5-
inorganic or organic acids, e.g., fumaric, benzoic, malefic, ascorbic, pamoic,
succinic,
bismethylenesalicylic, methanesulfonic, ethanolsulfonic, acetic, oxalic,
propionic,
tartaric, salicylic, citric, gluconic, lactic, malic, mandelic, annamic,
citraconic,
aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic,
benzene-
sulfonic, hydrochloric, hydrobromic, sulfuric, cyclohexylsulfamic, phosphoric,
and
nitric acids.
This invention also provides a process for preparing compounds of formula A
which comprise one of the following:
a) deprotecting a compound of formula III:
OR2
OSOY
N
O
cooQ
(III)
wherein Y, R2, Z and n are as defined above, and Q is a suitable protecting
group
such as benzhydryl, to give a corresponding compound of formula A; or
b) resolving a mixture (e.g racemate) of optically active isomers of a
compound
of formula A to isolate one enantiomer or diastereomer substantially free of
the other
enantiomer or diastereomers; or
c) acidifying a basic compound of formula A with a pharmaceutically acceptable
acid to give a pharmaceutically acceptable salt.
Removal of protecting groups, as illustrated by process a) can be carried out
by processes known in the art to provide the compound of formula A.
With regard to process b) standard separation techniques may be used to
isolate particular enantiomE:ric or diastereomerie forms. For example a
racemic
mixture may be converted to a mixture of optically active diastereoisomers by
reaction with a single enantiomer of a 'resolving agent' (for example by
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-6-
diastereomeric salt formation or formation of a covalent bond). The resulting
mixture
of optically active diastereoisomers may be separated by standard techniques
(e.g
crystallisation or chromatography) and individual optically active
diastereoisomers
then treated to remove the 'resolving agent' thereby releasing the single
enantiomer of
the compound of the invention. Chiral chromatography (using a chiral support,
eluent
or ion pairing agent) may also be used to separate enantiomeric mixtures
directly.
The compounds of formula A may be isolated in the form of a salt of a
pharmaceutically acceptable. acid, e.g. an organic or inorganic acid by
treatment with
an acid such as described above."
The compounds of the present invention may be prepared by any suitable
method which will be recognized by those skilled in the art. However, the
present
compounds may be advantageously prepared according to the Schemes set forth
below.
Scheme 1
r ~TMS r
MeCN B~~~~~ iaMOs
.-->
~r
OzBH COsBH COzBH
1 2
1. t-BuAAgCI. -78°C
2. Anh. CH:OIfHF Hd ~ ~ Bu~.SnH
-------> ~ >
i
s
3 COzBH
H H
1. m-cresol
2. NaHCCa
11
OzBH ~ OsNa
4 Ex 1
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
_7_
Scheme 2
s Bs
B~~~ 5 5
Ta~ He
s~H ;COOBH COOBH OOOBH ~COOBH
s ~ ~ ,
S BS TBS TBS
SAT
CICH,CO vH
HseT/ OCOCHZCI _2a
~ W.
a ~s ~ T ~
, H ~o ~H » ~H a COOBH
BS ~ TBS
NHIf HF m~mso~
CN ~ CN ~ ~ CN
COOBH ~IICOOBH ~COpBH ;CCONa
~a a ~s
BH: Benzhydryl
BT: 2-Banzothiazolyl
Scheme 3
Q
dNe Q a
M ~ON H: NH. F.Hf m.asdh
a
V
;CDOBH COOBH ~CppgH COONa
m ~s tT cs a
INTERMEDIATE 1
A solution of (:2S,SR)-6,6 dibromo-3,3-dimethyl-4,7-dioxo-4-thia-1-aza-
bicyclo(3.2.0)heptane-2-carboxylic acid benzhydryl ester (2.42 g, 4.47 mmol;
prepared in accordance with Heterocycles, 32:1505 (1991)) in acetonitrile (20
ml)
was prepared. TMS-triazole (prepared in accordance with Chem. Pharm. Bull.,
45(7):1140 (1997)) was added and the reaction mixture was heated to lI0
°C for 4
hours. It was then cooled to room temperature and diluted with methylene
chloride.
The organic solution was washed with water, dried over magnesium sulfate and
evaporated to dryness to give 0.63 g (23.8%) of Intermediate I after flash
chromatography (30% ethyl acetate/hexane).
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
_g_
NMR(CDC13) 8: 7.86(1H, s), 7.75(1H, s), 6.90(1H, s), 5.92(1H, s), 5.01(1H, s),
4.86(1H, d; J= 14.4 Hz), 4,.42(1H, d; J= 14.4 Hz), 1.17(3H, s). FAB High
Resolution:
M+H =590.9694 (Om= 0.7 mDa)
INTERMEDIATE 2
A solution of Intermediate 1 (1.0 g , 1.6883 mmol) in methylene chloride (12
ml) and acetic acid (2.4 ml) was prepared. Potassium permanganate (640 mg,
4.05
mmol) was added and the reaction mixture was heated at 38 °C for 3.5
hours. It was
then cooled to ice bath temperature, diluted with methylene chloride (12 mI)
and
water (6 ml). Excess permanganate was destroyed with aqueous sodium
metabisulfite.
The organic phase was separated, washed with water, saturated sodium
bicarbonate,
dried over magnesium sulfate and evaporated to dryness to give 935 mg (89%) of
Intermediate 2.
NMR(CDCl3) 8: 7.74(1H, d; J= 0.9 Hz), 7.72(1H, d; J= 0.9 Hz), 7.3(lOH, m),
6.99( 1 H, s), 5.09( 1 H, d; J= 4.8 Hz), 5.03( 1 H, s), 4.79( 1 H, s),
1.08(3H, s).
INTERMEDIATE 3
Intermediate 2 (624.3 mg, 1 mmol) was dissolved in , anhydrous tetrahydro-
furan (2 ml) at room temperature. The solution was cooled to -78 °C
under nitrogen.
The 2M i-butyl magnesium chloride solution in ether (550 wl, 1.1 mmol) was
added
dropwise maintaining the temperature at -78 °C. The light yellow
solution was stirred
at -78 °C for 45 minutes and the cold anhydrous formaldehyde solution
in THF
(excess) was added maintaining the temperature below 66 °C. Stirring
continued for
minutes at -78 °C and the solution was quenched with HOAc (57 p,l) in
THF (0.5
25 ml). The reaction mixture was allowed to warm to room temperature, then was
diluted
with ethyl acetate and filtered. The filtrate was evaporated to dryness to
give after
flash chromatography (50% ethyl acetate/hexane) 165 mg (28.7%) of Intermediate
3.
NMR(CDC13) 8: 7.?3(1H, s), 7.70(1H, s), 7.38(lOH, m), 6.99(1H, s), 5.13(2H,
q),
4.84(1H, s), 4.81(1H, s), 4.18(1H, m), 4.71(1H, m), 2.52(1H, q, OH), 1.08(3H,
s).
30 FAB High Resolution: M+H = 575.0609 (Dm= -0.9 mDa).
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-9-
INTERMEDIATE 4
Intermediate 3 (513 mg, 0.892 mmol) was dissolved in methylene chloride (S
ml). 1,1'-Azabis(cyclohexar~ecarbonitrile) (5 mg) and tributyltin hydride (240
~.1,
0.892 mmol) were added and the reaction mixture was refluxed for 30 minutes
and
then evaporated to dryness to give, after flash chromatography, 344 mg (78 %)
of
Intermediate 4.
NMR(CDCl3) b: 7.71(2H, d; J= 3.18 Hz), 7.38(IOH, m), 6.98(1H, s), 5.12(2H, q),
4.71 (1H, s), 4.70(IH, d; J= 4.6 Hz), 4.25{3H, m), 2.10(1H, OH), 1.06(3H, s).
ESI MS: 497.3(M+H).
EXAMPLE 1
(2S,3S,SR,6R)-6-Hydroxymethyl-3-methyl-4,4,7-trioxo-3-[1,2,3]triazol-1-
ylmethyl-4-lambda(6)-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.
A solution of Intermediate 4 (298.5 mg, 0.6 mmol) in m-cresol ( 1.8 ml) was
prepared and heated at 47 °C for 3.5 hours. It was then cooled to room
temperature,
diluted with ether, filtered and washed with ether. The solid material was
then
dissolved in water {5 ml) containing sodium bicarbonate (50.4 mg) and
filtered. The
filtrate was washed with ether and freeze-dried to give 109 mg (51.5%) of
product as
a white solid.
NMR(D20) 8: 8.12(1H, s), '7.85(1H, s), 5.37(1H, d; J= 15.4 Hz), 5.16(1H, d; J=
15.4
Hz), 5.08(1H, d; J= 4.6 Flz), 4.57(1H, s), 4.38-4.31(1H, m), 4.28-3.98(2H, m),
1.41 (3H, s).
INTERMEDIATE 5
A solution of (2S,5R)-6,6-dibromo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-
(3.2.0)heptane-2-carboxylic acid benzhydryl ester (10.0 g; 0.019 mole;
prepared in
accordance with J. Org. Chem., 52:3659 (1987)) in anhydrous THF (60 ml) was
prepared under nitrogen and cooled to -77°C. To the cold solution was
added with
good stirring 2M t-butyl magnesium chloride in an ether solution (11 ml;
0.0209 mol)
in such rate that the intern~~l temperature remained under -65°C. The
cold reaction
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
- 10-
mixture was stirred for 4:5 minutes. To this solution was then added via
syringe a cold
dry monomeric formaldehyde solution in anhydrous THF ( 100 ml) maintaining the
temperature below -65°C. The monomeric formaldehyde/T fiF solution was
prepared
as follows: Paraformalde;hyde (30 g. 1.0 mol) and p-toluenesulfonic anhydride
(4.9 g,
15 mmol) were placed in a three-necked flask, and tetrahydrofuran ( 1.0 L) was
added
thereto. The mixture was heated and a slow distillation of the solvent was
maintained.
Under a weak stream of nitrogen, a solution of anhydrous monomeric
formaldehyde
in THF was collected in a dry flask maintained at -78 °C. The solution
was stored at
-78 °C. The reaction mixture was finally stirred at -77 °C for
30 minutes and quenched
with glacial acetic acid ( 1.2 ml) in THF ( 10 ml).
The reaction mixture was allowed to warm to room temperature and was
diluted with ethyl acetate ( 100 ml) and water (50 ml). The organic phase was
removed, washed with water, dried over sodium sulfate and evaporated to
dryness to
give, after flash column chromatography (30% Ethyl Acetate/ Hexanes), 3.2 g.
(36%)
of Intermediate 5 as a mixture of a,(3 isomeric alcohols (75% ~3 and 25% a).
(3-alcohol: NMR(CDC13) b: 7.31 (lOH, m), 6.93 (1H, s), 5.56 (1H, s), 4.61 (1H,
s),
4.23-4.06 (2H, m), 2.25 (1H, t, OH),1.58 (3H, s), 1.26 (3H, s).
a-alcohol: NMR(CDC13) b: 7.33 ( l OH, m), 6.98 ( 1 H, s), 5.66 ( 1H, s), 4.56
( 1H, s),
4.27-4.06 (2H, m), 2.24 ( 1H, t, OH),1.58 (3H, s), 1.29 (3H, s).
High Resolution MS: Calcd. for M+H = 476.0531, measd. = 476.0351 (Dm = 0.0
mDa).
INTERMEDIATE 6
To a solution of Intermediate 5 (12.7 g; 26.65 mmol) in anhydrous methylene
chloride (160 ml) under nitrogen tributyl tin hydride (7.16 ml; 26.65 mmol)
and 1,1'-
Azabis(cyclohexanecarbonitrile) (159 mg) were added. The resulting solution
was
refluxed for two hours and then evaporated to dryness. Oily residue was
treated with
50% ethyl acetate/Hexanes and the crystals isolated by filtration to give 9.8
g (92.9%)
of Intermediate 6.
NMR(CDC13) 8: 7.35(10H, m), 6.92(1H, s), 5.5(1H, d; J=4.3 Hz). EI HIGH
RESOLUTION 397.1341
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-11-
INTERMEDIATE 7
A solution of Intermediate 6 (ll.Sg; 28.9 mmol) in anhydrous methylene
chloride (290 ml) was cooled to -20°C under nitrogen. Hunig's base (7.2
ml; 41.3
mmol) was added followed by addition of t-butyl-dimethylsilyl triflate (8.6
ml; 37.5
mmol). The resulting light yellow solution was stirred at -20°C for 30
minutes. It was
then washed with two 75 ml portions of water, dried over MgS04 and evaporated
to
dryness to give l3.Sg (91.3% ) of chromatographed Intermediate 7 (30% Ethyl
acetate/Hexanes).
NMR(CDC13) s: 7.3(lOH, m), 6.93(1H, s), 5.44(1H, d; J=4.2 Hz), 4.44(1H,
s),4.09-
3.81 (3H, m), 1.63(3H, s), 1.25(3H, s), 0.88(9H, s). CI HIGH RESOLUTION (M+H)
512.2249.
INTERMEDIATE 8
Intermediate 7 (1.26 g; 2.24b mmol) was dissolved in methylene chloride (7
ml). Formic acid (88%, 0.36 ml) was added followed by addition of 35% hydrogen
peroxide (0.43 ml). The resulting reaction solution was heated at 35 °C
for 30
minutes. Water (1.8 ml) was then added and the solution was stirred for
another 15
minutes. The organic phase was removed, washed with water, saturated NaHC03,
water and evaporated to dryness to give, after flash chromatography (20% ethyl
acetate/Hexanes), 908 mg (70 %) of Intermediate 8 {a mixture of a,~i-isomers).
NMR(CDC13) 8: a-isomer: 7.3(lOH, m),6.99(1H, s), 4.69(1H, s), 4.67(1H, d;
J=3.9
Hz), 4.4( 1 H, t; J=7.2 Hz), 4.02( 1 H, m), 3.91 ( 1 H, dd; J=5.4 Hz).
-isomer: 7.35(IOH, m), 6.97(1H, s), 4.62(1H, d; J=3.6 Hz), 4.39(1H, s),
4.08(2H, d;
J=4.5 Hz), 3.95(1H, m), 1.4(3H, s), 1.1(3h, s), 0.91(9H, s).
INTERMEDIATE 9
A solution of Intermediate 8 (9.658, 18.28 mmol) and 2-
mercaptobenzothiazole (3.12 g, 18.28 mmol) in anhydrous toluene (64 ml) was
heated
at reflux for 1.5 hours using a Dean-Stark apparatus. Heating was then
removed. The
reaction solution was cooled to room temperature and evaporated to dryness to
give
12.37 g of Intermediate 9 which was used 'as is' for the next step.
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-12-
NMR(CDC13) S: 7.86-7.20(14H-m}, 6.99(1H, s), 5.52(1H, d; J=5.4 Hz), 5.1(2H, d;
J=14.1), 4.86(1H, s), 4.14(2H, m), 3.78(1H, m), 1.96(3H, s), 0.94(9H, s),
0.08(6H, d).
HIGH RESOLUTION NiS: calcd. for M+H= 677.1998, measd.: 677.2016 (0m=1.8
mDa.
INTERMEDIATE 10
To a solution of :intermediate 9 (6.1 g; 9.02 mmol) in anhydrous methylene
chloride (40 ml) was added silver acetate (3.05 g, 18.17 mmol) followed by a
solution
of chloroacetic acid (36.6 g; 388 mmol) in anhydrous methylene chloride (94
ml) via
addition funnel. The reaction mixture was stirred at room temperature for 48
hours
and then filtered through celite. The filtrate was evaporated to dryness and
the crude
residue was purified by flash chromatography giving l.Sg (27.7%) of
Intermediate 10.
NMR(CDC13) 8: 7.26( l OH, m), 6.86( 1 H, s), 5.48 ( 1 H, d; J=4.1 Hz), 4.71 (
1 H, s), 4.25-
3.75(7H, m), 1.2(3H, s), 0.81(9H, s), 0.015(6H, s).
HIGH RESOLUTION MS: calcd. for M+H= 604.1956, measd.= 604.1907(Om=4.9
mDa).
INTERMEDIATE 11
A solution of Intermediate 10 (l.Sg; 2:48 mmol) in methylene chloride (12.5
ml) was prepared. To the solution was added acetic acid (3.7 ml) and potassium
permanganate ( 1.05 g, fi.65 mmol). The reaction mixture was stirred at room
temperature for 3 hours. The mixture was then cooled in ice bath and the
excess
potassium permanganate was destroyed with an aqueous solution of sodium
metabisulfite. The methylene chloride phase was separated, washed with water,
dried
over magnesium sulfate and evaporated to dryness. The crude product was
purified by
flash chromatography (2,0% ethyl acetate/ hexanes) to afford 1.38g (88%) of
Intermediate 11.
NMR(CDC13) b: 7.28(10H, m), 6.89(1H, s), 4.67(2H, dd; J=12.0 & 17.4Hz),
4.54(2H, d; J=12.6Hz), 4.33(1H, t; J=9.9 Hz), 4.09(1H, m), 4.0(2H, d; J=0.6
Hz),
3.89(1H, dd; J=6.9 & 7.lHz), 1.01(3H, s), 0.81(9H, s), 0.08(6H, s).
High resolution MS: calcd. For M+H= 636.1854, measd.= 636.1847(~m= 0.7 mDa).
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-13-
INTERMEDIATE 12
A solution of Intermediate 11 (636.24 mg, 1 mmol), and thiourea (229 mg, 3
mmol) in dry DMF ( 1.2 ml) and anhydrous pyridine (480 p,l) was prepared and
stirred
at room temperature for 3 hours. The resulting suspension was diluted with
ethyl
acetate, filtered and washt:d with ethyl acetate. The combined ethyl acetate
solutions
were evaporated to dryness. The residue was dissolved in methylene chloride
and
washed with water. The organic phase was dried over magnesium sulfate and
evaporated to dryness to give 542 mg, 97% of Intermediate 12 as a white solid.
NMR(CDC13) S: 7.27( 10H, m), 6.9( 1 H, s), 5.18 ( 1 H, s), 4.48( 1 H, d; J=4.8
Hz),
4.32(1H, t; J=10.02& 9.93 Hz), 4.09(1H, m}, 4.01 & 3.97(1H, dd; J=3.6 & 3.54
Hz},
3.87(1H, dd; J=7.2 Hz), 3.69(1H, dd; J=10.7 Hz), 2.62(1H, dd; J=3.7 Hz),
0,91(3H,
s), 0.81 (9H, s).
INTERMEDIATE 13
1 S Pyridinium chlorochromate ( 1.07 g; 4.8 mmol) and silica gel ( 1.5 g) were
slurried in anhydrous methylene chloride (12 ml). To this mixture was added
with
good stirring a solution of Intermediate 12 (559.7 mg; I mmol) in anhydrous
methylene chloride (4 ml). The resulting reaction mixture was stirred at room
temperature for 16 hours. It was then diluted with methylene chloride,
filtered and
washed with methylene chloride. The filtrate was washed with 30-50 ml portions
of
water, dried over magnesium sulfate and evaporated to dryness to give the
aldehyde
Intermediate 13 (382 mg; 68.4%):
NMR(CDCl3) b: 9.61 ( 1 H, s), 7.25 ( l OH, m}, 6.92( I H, s), 5.41 ( 1 H, s),
4.58( 1H, d;
J=4.8 Hz), 4.29(IH, t; J=9.9 Hz), 4.15(1H, m), 3.88(1H, dd; J=7.2 Hz), 1.2(3H,
s),
0.81 (9H, s).
TNTERMEDIA,TE 14
Cyanomethylenetriphenylphosphorane (387.2 mg; 1.286 mmol; prepared in
accordance with Chem. Ber-94:578 (1961) and lithium perchlorate (137.6 mg;
1.29
mmol) were slurned in anhydrous methylene chloride (3.2 ml) at -20C1C. To the
cold
suspension was added dropwise a solution of Intermediate 13 (640 mg; 1.147 ml)
maintaining the temperature at -20~C. The cold bath was then removed and the
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-14-
reaction mixture was stirred at room temperature for 5 hours. The resulting
green
solution was evaporated to dryness and the two isomers were separated by flash
column chromatography yielding Intermediate 14 (cis:) 302.2 mg; 45.4% and
(traps:)
158 mg; 27.2%).
NMR(CDCl3) 8: cis isomer : 7.36(lOH, m), 6.96(1H, s), 6.44(1H, d; J=12.3 Hz),
5.88(1H, d; J=12.2 Hz), 4.9(IH, s), 4.71(1H, d; J=4.9 Hz), 4.39(1H, t; J=9.96
Hz),
4.2(1H, m), 3.96(lH,dd; J='7.26 Hz), 1.66(3H, s), 0.89(9H, s), 0.07(6H, s).
traps isomer: 7.23(lOH, m), 6.98(1H, s), 6.77(1H, d; J=16.5 Hz), 5.28(1H, d;
J=16.5
Hz), 4.75(1H, s)> 4.6(1H, d; J=4..9 Hz), 4.39(1H, t; J=6.9 & 9.99), 4.2(1H,
m),
3.95(1H, dd; J=7.29 Hz), 1.22(3H, s), 0.88(9H, s), 0.07(6H, s).
INTERMEDIATE 15
Ammonium hydrogen difluoride (106 mg, 1.86 mmol) was added to a solution
of Intermediate 15 (cis isomer) (135 mg, 0.2324 mmol) in dry DMF (0.88 ml) and
N-
methyl-2-pyrrolidinone (0,.325 ml). The resulting clear light yellow solution
was
stirred at room temperature for 5 hours and filtered. The filtrate was
evaporated to
dryness and chromatographed to give Intermediate 15 (cis isomer) {95 mg, 88%).
NMR(CDCl3) b: 7.36(10H, m), 6.97(1H, s), 6.5(1H, D; J=12.3Hz), 5.9(1H,D;
J=12.3Hz), 4.95(1H, s), 4.;~8(1H, d; J=4.59 Hz), 4.3-4.1(3H, m), 1.6(3H, s).
HIGH RESOLUTION MS: calcld. For M+H= 467.1277, measd. :467.1260 (0m=1.7
mDa).
The same conditions were followed for the preparation of the traps isomer.
NMR(CDCl3) 8: 7.3(lOH, m), 6.98(1H, s), 6.84(1H, d; J=16.5 Hz), 5.374.84(1H,
s),
(1H, d; J=16.5 Hz), 4.74(1H, d; J=4.56 Hz), 4.3-4.1(3H, m), 1.2(3H, s).
CA 02355738 2001-06-20
WO 00/43399 PCTNS00/01630
-15-
EXAMPLE 2
(Z)-(2S, 3S, 5R, 6R)-b-Hydroxymethyl-3-methyl-3-(3-nitrilo-propenyl)-4,4,7
trioxo-4-lambda(ti)-this-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid.
and
S (E)-(2S, 3S, 5R, 6R)-6-Hydroxymethyl-3-methyl-3-(3-nitrilo-propenyl)-4,4,7-
trioxo-4-lambda(6)-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid.
A solution of Intermediate 15 (cis isomer) (226 mg, 0.4845 mmol) in m-cresol
(1.5 ml) was heated for 4 hours at 47 °C and then cooled to room
temperature. It was
diluted with ether and a ;solution of sodium bicarbonate (40.7 mg, 0.4845
mmol) was
added. The aqueous phase was separated, washed with ether and freeze-dried to
give a
mixture of the title cis isomer. (123.5 mg, 79%).
NMR(D20) b: 6.68(1H, d; J=12.4 Hz), 6.16(1H, d; J=12.4 Hz), 5.25(1H, d; J=4.7
Hz), 4.36(1H, rn), 4.22(1:H, dd; J-8.73 Hz), 4.08(1H, dd; 8.16 Hz), 1.94(3H,
s).
HIGH RESOLUTION M'S: calcd. For M-H=299.0338, measd. = 299.0343 (0m = -0.5
mDa).
The same conditions were followed to obtain the traps isomer. NMR(D20)
8: 7.08(1H, d; J=16.5 Hz;), 6.07(1H, d; J=16.5 Hz), 5.2(IH, s), 4.36{1H, m),
4.21(1H,
dd; J=8.10 Hz), 4.07(1H, dd; J=8.10 Hz), 1.64(3H, s).
INTERMEDIATE 16
A solution of Intermediate 13 (381.5 mg; 0.684 mmol) and methoxylamine
hydrochloride (58.3 mg; 0.684 mmol) in anhydrous methylene chloride (1.5 ml)
and
anhydrous pyridine (83 p.l; 1.026 mmol) was srirred at room temperature for
7.5
hours. The reaction mixture was then diluted with methylene chloride and the
solution
was washed three times with water (40 ml portions). The organic phase was
dried
over magnesium sulfate and evaporated to dryness to give, after flash column
chromatography (20% ethyl acetate/hexanes), 192 mg of Intermediate 16 in 47%
yield.
NMR(CDC13) b: 7.5(1H, s), 6.96(1H, s), 5.08(1H, s), 4.67(1H, d; J=4.5 Hz).
4.4(1H,
t), 4.1( 1H, m), 3.97(3H, s.), 3.9(1H, dd), 1.33(3H, s), 0.8(6H, d).
High Resolution MS: M+H=587.2226 (0m=2.lmDa).
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
- 16-
INTERMEDIATE 17
A solution of Intermediate 16 ( 185 mg, 0.3153 mmol) and ammonium
hydrogen difluoride (72 mg, 1.26 mmol) in dry (mol sieves) DMF (1.2 ml) and 1-
S methyl-2-pyrrolidinone (0.49 ml) was stirred at room temperature for 24
hours. The
reaction mixture was filtered and evaporated to dryness to give, after flash
column
chromatography (20-50% ethyl acetate/ hexanes}, 114.5 mg of Intermediate 17 in
76.5% yield.
NMR(CDCl3) 8: 7.48(1H, s), 6.96(1H, s), 5.2(1H, s), 4.7((1H, d; J=4..SHz),
4.3(1H,
m), 4.15(2H, m), 3.98(3H, s), 1.33(3H, s).
High Resolution MS: M+H=473.1393 (Dm=-1.1 mDa).
EXAMPLE 3
(2S, 3R, 5R, GR)-6-Hydroxymethyl-3-(methoxyamino-methyl)-3-methyl-4,4,7-
trioxo-4-lambda(6)-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic a~d.
A solution of Intermediate 17 ( 107.5 mg, 0.2275 mmol) in m-cresol (0.7 ml)
was heated at 47 °C for 4 hours. The solution was then cooled to room
temperature
and diluted with ether. A solution of sodium bicarbonate ( 19.1 mg, 0.2275
mmol) in
water (10 ml) was added and the organic phase removed. The aqueous phase was
washed with ether and freeze-dried to give 47.8 mg of product in 64% yield.
1VMR(D20) 8: 7.68(1H, s), 5.18(1H, d; J=4.5 Hz), 4.88(1H, s), 4.35(1H, m),
4.2(1H,
t), 4.08(1H, dd), 3.97(3H, s), 1.66(3H, s).
High Resolution MS: M-H:=305.0443 (Dm = -1 mDa)
The activity of the present compounds is demonstrated by the following
pharmacological test procedures.
Representative compounds according to the present invention were tested in a
microbiological assay in combination with the penicillin antibiotic
piperacillin. The
enhanced combined synergestic antibacterial activity is representative of the
(3-
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-17-
lactamase inhibitory properties of the compounds of the present invention. In
these
tests, minimum inhibitory properties (MIC's) are determined in broth using
several
two-fold dilutions with a 1:1 ratio of piperacillin to inhibitor (N. A. Kuck,
N. V.
Jacobus, P. J. Peterson, W. J. Weiss, and R. T. Testa, Antibiotic Agents and
Chemotheranv, 33:1964--1969 (1989)). The results are set forth in Table 1.
Representative compounds of the present invention were also tested against a
comercally available inhibitor, tazobactam, to determine inhibition of the
AmpC and
TEM-1 (3-lactamase enzymes. ICso values were determined spectrophotometrically
using a 10 minute preincubation of enzyme with inhibitor at 25°C before
addition of
nitrocefin as a substrate. 'The results are also set forth in Table 1.
Table 1
ICso (nM) MIC (p.g/ml; 1:1 d)
o un TEM-1 E. colic S. marcescensb
Amy
Example 1 6 360 2 16e
Example 2 {cis)19 270 4 8
Example 2 (traps)74 280 16 4
Example 3 64 280 8 16
Tazobactam 60 47,700 2 32
Piperacillin >64 32
eGC6265, TEM-1 (Class A); bGC4132, AmpC (Class C); °GC2847, TEM-1
(Class A);
dpiperacillin:inhibitor ratio; °GC2894; AmpC (Class C).
When the present. compounds are employed for the above utility, they may be
combined with one or more pharmaceutically acceptable carriers, e.g.,
solvents,
dilutents, etc., and may be administered parentally in the form of sterile
injectable
solutions or suspensions containing from about 0.05% to about 5% suspending
agent
in an isotonic medium. Such pharmaceutical preparations may contain from about
0.05% to about 90% of the active ingredient in combination with the carrier,
more
usually between about 5% and about b0% by weight.
The effective dosage of active ingredient employed may vary depending upon
the particular compound used, the mode of administration and the severity of
the
condition being treated. However, in general, satisfactory results are
obtained when
the compounds of the invention are administered at a daily dosage of from
about 2 to
CA 02355738 2001-06-20
WO 00/43399 PCT/US00/01630
-18-
100 mg/kg of animal body weight, preferably given in divided doses two to four
times
a day. In general, the total daily dose is from about 100 mg to about 750 mg,
preferably from about 100 to about S00 kg. Dosage forms suitable for internal
use
include from about 1(>n to 750 mg of active compound in intimate admixture
with a
5 liquid pharmaceutically acceptable carrier. This dosage regimen may be
adjusted to
provide the optimal therapeutic response. For example, several divided doses
may be
administered daily or the dose may be proportionally reduced as indicated by
the
therapeutic situation. A practical advantage is that the present compounds may
be
administered by intravenous, intramuscular or subcutaneous rates. Liquid
carriers
10 include sterile water, polyethylene glycols, non-ionic-surfactants and
edible oils such
as corn, peanut, and sesame oils as appropriate. Adjuvants normally employed
in the
preparation of pharmaceutical preparations may be used, such as vitamin E,
ascorbic
acid, BHT and BHA.
15 The present compounds may be administered parentally or intraperitoneally.
Solutions or suspension of these active compounds can be prepared in water
suitably
mixed with a surfactant such as hydroxypropyicellulose. Dispersions can be
prepared
in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under
ordinary
conditions of storage and use, these preparations contain a preservative to
prevent the
20 growth of microorganisms.
The pharmaceutical forms suitable for injection use include sterile aqueous
solutions and sterile powders for the preparation of sterile injectable
solutions or
dispensors. In all cases, the forms must be sterile and must be fluid so that
easy
25 syringability exists. It must be stable under conditions of manufacture and
storage
and must be preserved against the contaminating action of microorganisms. The
earner may be a solvent or dispension medium containing, e.g., water, ethanol,
apolyl
(e.g., glyceol, propylene glycol and liquid polyethylene glycol) and vegetable
oils.
30 The present invention may be embodied in other specific forms without
departing from the spirit and essential attributes thereof and accordingly,
reference
should be made to the .appended claims, rather than to the foregoing
specification, as
indicating the scope of the invention.
