Structure-based design of β-lactamase inhibitors. 2. Synthesis and evaluation of bridged sulfactams and oxamazins

Article abstract of DOI:10.1021/jm9800245

A series of bridged monocyclic β-lactams activated by various groups on the β-lactam nitrogen (X = OCH₂CO₂H, OSO₃H) has been synthesized and evaluated. Among them, the bridged sulfactams (X = OSO₃H) were found t

Full text of DOI:10.1021/jm9800245

3972
J . Med. Chem. 1998, 41, 3972-3975
Str u ctu r e-Ba sed Design of â-La cta m a se In h ibitor s. 2. Syn th esis a n d Eva lu a tion
of Br id ged Su lfa cta m s a n d Oxa m a zin s
Christian Hubschwerlen,* Peter Angehrn, Klaus Gubernator,† Malcolm G. P. Page, and J ean-Luc Specklin
Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4070 Basle, Switzerland
Received J anuary 14, 1998
A series of bridged monocyclic â-lactams activated by various groups on the â-lactam nitrogen
(X ) OCH2CO2H, OSO3H) has been synthesized and evaluated. Among them, the bridged
sulfactams (X ) OSO3H) were found to be effective â-lactamase inhibitors. They inhibit both
class A and class C â-lactamases.
In tr od u ction
Compound 16 was reacted with pyridine-SO3 com-
plex to give the protected sulfactam 2a . The BOC group
was removed by treatment with TFA, and the interme-
diate betaine was reacted with the activated side chain
18 to give compound 2b. The intermediate 16 was
treated with benzyl bromoacetate affording the bridged
oxamazin 17 which was hydrogenolyzed to give 3a .
The development of â-lactamase-mediated resistance
to â-lactam antibiotics is becoming a major threat to
these antibiotics. Bridged monobactams 1 have been
found to be potent inhibitors of class C â-lactamases and
to display useful synergy with cephalosporins.1,2 The
nature of the substitution on the pyrrolidine ring was
found to play an important role both in the efficacy of
the compounds as â-lactamase inhibitors and in their
ability to protect the cephalosporin in the presence of
intact bacteria.1 It was also of interest to study other
activating groups attached to the â-lactam nitrogen,
which are known to confer antibacterial activity in other
classes of â-lactam antibiotics, and show their effect on
the efficacy of the bridged compounds. In this paper
we describe the synthesis and the evaluation of bridged
sulfactams 2 and oxamazins 3.
Resu lts a n d Discu ssion
The bridged oxamazin 3a was not a â-lactamase
inhibitor (Table 1) possibly due to insufficient activation
of the â-lactam nitrogen. In contrast, the bridged
sulfactams 2a ,b were potent inhibitors and were more
active than the corresponding bridged monobactams
1a ,b. This was particularly true against class A â-lac-
tamases, where the latter were inactive. The IC50 is a
composite value of intrinsic affinity (KS), rate of acyla-
tion (kon), and rate of deacylation (koff). An analysis of
the kinetic parameters revealed that the low IC50 values
of the bridged sulfactams stem primarily from a high
affinity and a high rate of acylation (Table 2). However,
the deacylation rate was also high, and therefore the
steady-state occupancy of the enzyme, which is set by
the ratio of kon/koff, was not as high as with the bridged
monobactams. This incomplete inactivation was re-
flected in the in vitro data of the combination with
ceftriaxone, where only a weak synergy was observed
(Table 1).
Ch em istr y
The bridged sulfactams evidently have a higher
chemical reactivity than the bridged monobactams (high
acylation rates achieved), but the active site of the class
C â-lactamase is less tightly blocked by the sulfonyloxy
group than by a sulfonyl group.
The key intermediate 16 was synthesized as described
in Scheme 1. The alcohol 6 was obtained from the
acetylenic derivative 53 by condensation of its anion with
formaldehyde. Selective reduction into the allylic alco-
hol 7 was achieved with LAH in the presence of sodium
methoxide. Sharpless epoxidation4 followed by oxida-
tion with ruthenium oxide led to the formation of the
acid 9. Opening of the epoxide with ammonia and in
situ protection with di-tert-butyl dicarbonate gave the
amino acid 10 which was reacted with O-benzylhy-
droxylamine to give the amide 11. Miller5 ring closure
gave the â-lactam 12 which was transformed in a two-
step process into the mesylate 14. The bicyclic lactam
15, obtained after treatment with NaH, was deprotected
by hydrogenolysis over Pd/C to give the key intermedi-
ate 16.
Con clu sion s
The fact that the bridged oxamazins 3 are devoid of
any â-lactamase inhibitory activity seems to indicate
that the structural requirements for â-lactamase inhibi-
tion are very different from those for antibacterial
activity. Despite their lower IC50 values, the bridged
sulfactams 2 are less attractive â-lactamase inhibitors
than the bridged monobactams 1 because of their high
chemical reactivity and their less stable acyl-enzyme
complexes.
Exp er im en ta l Section
Gen er a l Meth od s. All reagents were obtained from com-
mercial sources and used without further purification. Sol-
* To whom correspondence should be addressed.
† Present address: CombiChem Inc., 9050 Camino Santa Fe, San
Diego, CA 92121.
S0022-2623(98)00024-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/15/1998

Design of â-Lactamase Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3973
MICb (µg/mL) ceftriaxone:inhibitor ) 1:4
Ta ble 1. Enzyme Inhibition and in Vitro Synergy with Ceftriaxone
a
IC50 (nM)
C. freundii
P. aeruginosa
E. coli TEM-3
C. freundii
P. aeruginosa
E. coli
compd
1982 (class C)
18 SH (class C)
(class A)
1982
18 SH
TEM-3
refc
1a d
2a
128
8
4
0.25
1
128
8
32
16
16
4
16
8
225
20
53
12
26000
900
1500
28
217
26
185000
800
>1000000
165
9000000
580
1000000
15
1bd
2b
4
128
>16
>32
17
>16
>16
taze
8
0.25
a
b
Inhibition of the isolated enzymes. Against the strains that produce the â-lactamases. c MICs of ceftriaxone in the absence of any
inhibitor. Values from ref 1. e Values obtained with tazobactam, reference inhibitor.
d
Sch em e 1a
Ta ble 2. Detailed Kinetic Parameters of the Inhibitors 1a ,b
compd
KS (µM)
kon (s-1
)
koff (10-6 s-1
)
1a
2a
127
600
12.5
20
<0.8
120000
extraction were washed with water and brine and dried over
MgSO4. The solvent was evaporated under reduced pressure
with a water bath temperature below 35 °C. Flash chroma-
tography was performed using Merck silica gel 60 (particle size
40-63 µm), the fractions containing the substance of interest
were pooled, and the solvent was evaporated. TLC was
performed on Merck TLC plates (silica gel 60 F254), and spots
were visualized with aqueous KMnO4. Proton NMR spectra
were recorded on a Bruker FT AC-250 spectrometer. Chemical
shifts (δ) are reported in ppm relative to Me4Si as internal
standard; values are given in Hz. As many derivatives were
mixtures of rotamers and/or diastereoisomers (THP ethers),
signals are not assigned. IR spectra were recorded on a Nicolet
FT IR 170 SX spectrophotometer as KBr pellets (unless
indicated otherwise). Ion spray mass spectra were recorded
on a Finnigan MAT SSQ 7000 instrument and EI mass spectra
on
a Perkin-Elmer Siex API III instrument. Elemental
analyses are indicated by the symbols of the elements; analyti-
cal results were within 0.4% of the theoretical values. Optical
rotation measurements were performed on a Perkin-Elmer 241
polarimeter at 20 °C. Melting points were determined with a
Buchi 510 melting point apparatus and are uncorrected.
2-(3-Bu tyn yloxy)-tetr a h yd r o-2H-p yr a n (5). A solution
of 3-butyn-1-ol (105.1 g, 1.5 mol) in CH2Cl2 (0.5 L) was reacted
at 0 °C with p-toluenesulfonic acid (0.2 g) and 3,4-dihydro-
2H-pyran (163 mL, 1.8 mol, 1.2 equiv). The reaction mixture
was stirred for 48 h at room temperature and then poured into
2% aqueous NaHCO3 (0.1 L). The organic layer was worked
up, and the residue was distilled under vacuum affording 223.7
g (96%) of 5, bp4
) 52-53 °C (lit.5 bp12
) 90 °C).
mm
mm
(R,S)-5-(Tetr a h yd r op yr a n -2-yloxy)p en t-2-yn -1-ol (6). A
solution of 5 (15.4 g, 0.10 mol) in absolute THF (0.15 L) was
reacted at -78 °C with dipyridyl (2 mg) and, dropwise, with a
1.6 N n-BuLi solution in hexane (72 mL, 115 mmol). The deep
red solution was reacted for 1 h at -30 °C with gaseous
formaldehyde.7 The formaldehyde gas was transferred into
the reaction flask via a 1-mm diameter Teflon tube. The
mixture was allowed to reach 0 °C before quenching with
saturated NH4Cl solution (25 mL) and dilution with Et2O. The
organic phase was separated, worked up and chromatographed
(hexane/AcOEt, 75:25) affording 15.6 g (85%) of 5. IR (film):
3418, 2943, 2872, 2300, 2240 cm-1. MS (EI): 153 (M - CH2-
OH). NMR (CDCl3): 1.54-1.76 (6H, m), 2.20 (1H, b), 2.52 (2H,
m), 3.49-3.90 (2 × 2H, 2m), 4.24 (2H, m), 4.63 and 4.81 (1H,
2t, J ) 3.5).
a
Reagents: (a) 2,3-dihydropyran; (b) CH2O (gaz); (c) LAH,
(E)-(R,S)-5-(Tetr ah ydr opyr an -2-yloxy)pen t-2-en -1-ol (7).
A suspension of LAH (9.6 g, 253 mmol) in absolute THF (0.8
L) was reacted with NaOMe (27.4 g, 0.508 mol). After 10 min,
the mixture was treated dropwise, over 45 min, with 6 (15.6
g, 84.67 mmol) in THF (70 mL) and refluxed for 1 h. The
reaction mixture was sequentially treated at 5 °C with water
(9 mL), 3 N NaOH (9 mL), and water (26 mL). The slurry
was stirred for 1.5 h at room temperature and filtered. The
MeONa; (d) Sharpless epoxidation, (L)-diethyl tartrate; (e) RuO2/
NaIO4; (f) NH4OH then (BOC)2O; (g) BnONH2, EDC; (h) DEAD/
P(Ph)3; (i) pTSA; (j) MsCl/TEA; (k) LiN[Si(CH3)3]2; (l) H2, Pd/C;
(m) pyridine‚SO3; (n) TFA.
vents were dried by filtration through Al2O3 (neutral, Brock-
mann number 1) when necessary and stored over a bed of
molecular sieves (3 Å). All the organic solutions obtained after

3974 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Hubschwerlen et al.
filtrate was evaporated and distilled affording 13.3 g of 7 (84%),
bp0.11 mm ) 83-85 °C. NMR (CDCl3): 1.42-1.90 (6H, m), 2.34
(2H, m), 2.54 (1H, t, J ) 5.5), 3.43 (2H, m), 3.70-3.95 (2H,
m), 4.06 (2H, b), 4.59 (1H, t, J ) 3), 5.70 (2H, m).
1:1 Mixtu r e of (2R,3S)-1-(Ben zyloxy)-4-oxo-2-[[(R)- a n d
(S)-tetr a h yd r op yr a n -2-yloxy]eth yl]a zetid in -3-ylca r ba m -
ic Acid ter t-Bu tyl Ester (12). A solution of 11 (1.74 g, 3.97
mmol) and triphenylphosphine (1.15 g, 4.37 mmol) in absolute
THF (25 mL) was reacted with diisopropyl azodicarboxylate
(0.88 g, 4.37 mmol) for 2 h at room temperature. The solvent
was evaporated; the residue was chromatographed (hexane/
Et2O/CH2Cl2, 5:4.5:0.5) affording an oil which was crystallized
from Et2O/hexane (820 mg, 50%), mp 104-08 °C. [R]D -195°
1:1 Mixtu r e of (2R,3S)-3-{2-[(R)- a n d (S)-Tetr a h yd r o-
p yr a n -2-yloxy]eth yl}oxir a n -2-ylm eth a n ol (8). Molecular
sieve (3 Å) was activated at 120 °C under high vacuum for 12
h and finely pulverized before use. A suspension of molecular
sieve (20 g) and L-(+)-diisopropyl tartrate (3.4 mL, 16.25 mmol)
in absolute CH2Cl2 (0.25 L) was reacted at -30 °C with
tetraisopropyl orthotitanate (3.8 mL, 13 mmol). After 15 min
the suspension was treated with tert-butyl hydroperoxide in
isooctane (3 M; 108 mL, 325 mmol) for 30 min. A solution of
7 (25 g, 130 mmol) in absolute CH2Cl2 (50 mL) was added at
-20 °C and further stirred for 4 h. The reaction mixture was
quenched at this temperature with a solution of NaOH (1 g)
and NaCl (1 g) in water (10 mL). After 30 min at 10 °C, the
reaction mixture was diluted with CH2Cl2. The organic
solution was worked up and chromatographed (AcOEt/hexane,
1:1) giving 18.5 g (70%) of 8. [R]D ) -22.9° (c 1, CHCl3). NMR
(CDCl3): 1.52-1.90 (6H, m), 1.85 (2H, m), 2.60 (1H, 2t, J )
6), 3.00 (1H, m), 3.10 (1H, m), 3.49-3.92 (2 × 3H, 2m), 4.60
(1H, t, J ) 3).
(c 1, CHCl3). IR: 3343, 1784, 1691, 1531 cm-1
. MS (FAB):
421.3 (M + H)+. NMR (CDCl3): 1.42 (9H, s), 1.50-1.98 (8H,
m), 3.30-4.00 (5H, m), 4.30-4.80 (2H, m), 4.90-5.1 (2H, m),
5.5-5.75 (1H, 2d, J ) 10), 7.40 (5H, s). Anal. (C22H32N2O6)
C, H, N.
(2R,3S)-1-(Ben zyloxy)-2-(2-h ydr oxyeth yl)-4-oxoazetidin -
3-ylca r ba m ic Acid ter t-Bu tyl Ester (13). A solution of 12
(5.1 g, 12.13 mmol) and p-toluenesulfonic acid (0.5 g) in EtOH
(0.1 L) was warmed to 80 °C for 2 h. The solvent was
evaporated, and the residue, dissolved in CH2Cl2, was worked
up and crystallized from Et2O/hexane (3.54 g, 87%), mp 102-
103 °C. [R]D +123.3 ° (c 1, CHCl3). IR: 3429, 3355, 1765, 1680,
1536 cm-1
.
MS (EI): 337.8 (M + H)+, 281.8 (M - (CH2d
C(Me)2)). NMR (CDCl3): 1.42 (9H, s), 1.71 (2H, dd, J ) 7 and
12), 3.67 (2H, t, J ) 7), 3.75 (1H, dt, J ) 5), 4.75 (1H, dd, J )
6 and 8), 4.99 (2H, ab, J ) 10), 5.75 (1H, bd, J ) 8), 7.40 (5H,
s). Anal. (C17H24N2O5) C, H, N.
1:1 Mixtu r e of (2R,3S)-3-[2-[(R)- a n d (S)-Tetr a h yd r o-
p yr a n -2-yloxy]eth yl]oxir a n e-2-ca r boxylic Acid (9).
A
solution of 8 in MeCN (0.2 L) and CCl4 (0.2 L) was diluted
with water (0.4 L) and reacted with RuO2 (0.2 g) and NaIO4
(80.2 g, 0.375 mol) for 4 h. The temperature of the reaction
mixture was kept below 30 °C by occasional cooling, and the
pH was adjusted to pH 4.5 by addition of 2 N Na2CO3. The
reaction mixture was diluted with CH2Cl2. The aqueous layer
was saturated with NaCl and extracted with CH2Cl2 (3×). The
combined organic layers were worked up leaving an oil which
was dissolved in ether and filtered over Dicalite. The filtrate
was evaporated giving a foam (15.11 g, 76%). MS: 215 (M -
H). NMR (CDCl3): 1.52-2.00 (6H, m), 1.88 (2H, m), 3.33 (2H,
m), 3.40-3.53 and 3.80-3.90 (2 × 2H, 2m), 4.60 (1H, m), 7.30
(1H, b).
(2R,3S)-1-(Ben zyloxy)-2-[2-(m eth ylsu lfon yloxy)eth yl]-
4-oxoa zetid in -3-ylca r ba m ic Acid ter t-Bu tyl Ester (14). A
solution of 13 (3.44 g, 10.2 mmol) and triethylamine (1.13 g,
11.2 mmol) in CH2Cl2 (50 mL) was treated dropwise with
MeSO2Cl (1.28 g, 11.2 mmol) at 0 °C. The reaction mixture
was stirred for 1 h at room temperature, then diluted with
CH2Cl2, and washed with ice water and a cooled 10% NaCl
solution. The organic layer was dried over MgSO4, filtered,
and evaporated, leaving an oil which was crystallized from
AcOEt/hexane. The yield was 4.2 g (99%), mp 148 °C. [R]D
+83.2° (c 1, CHCl3). IR: 3324, 1777, 1683, 1533 cm-1. MS
(ISP): 437.3 (M + Na), 415.3 (M + H). NMR (CDCl3): 1.43
(9H, s), 1.87 (2H, m), 2.98 (3H, s), 3.75 (1H, dt, J ) 3 and 5),
4.19 (2H, m), 4.70 (1H, t, J ) 5), 4.95 and 5.02 (2H, ab), 5.20
(1H, d, J ) 5), 7.41 (5H, s). Anal. (C22H34N2O7) C, H, N.
(1R,5R)-6-(Ben zyloxy)-7-oxo-2,6-diazabicyclo[3.2.0]h ep-
ta n e-2-ca r boxylic Acid ter t-Bu tyl Ester (15). A solution
of 14 (4.14 g, 10 mmol) in absolute THF (40 mL) was treated
dropwise at -10 °C with 1 M lithium bis(trimethylsilyl)amide
in THF (11.5 mL, 11.5 mmol). After stirring at room temper-
ature overnight, the reaction mixture was quenched with
saturated NH4Cl solution (25 mL) and extracted with AcOEt.
The organic layer was worked up, chromatographed (hexane/
AcOEt, 6:4) and crystallized from Et2O/hexane (819 mg, 25%),
mp 88-90 °C. [R]D -195° (c 0.9, CHCl3) (lit.8 [R]D -214.9° (c
1, CHCl3)). IR: 1767, 1687, 1421 cm-1. MS (EI): 290 (M -
CO). NMR (CDCl3): 1.46 (9H, s), 1.52 (1H, m), 1.75 (1H, dd,
J ) 6 and 14), 3.10 (1H, dt, J ) 6 and 10), 3.80 (1H, bt, J )
10), 4.09 (1H, b), 4.75 and 5.00 (1H, b), 4.96 (2H, s), 7.40 (5H,
s). Anal. (C17H22N2O4) C, H, N.
1:1 Mixtu r e of (1S,2S)-1-Ca r boxy-2-h yd r oxy-4-[(R)- a n d
(S)-tetr a h yd r op yr a n -2-yloxy]bu tylca r ba m ic Acid ter t-
Bu tyl Ester (10). A solution of 9 (2.58 g, 11.9 mmol) in MeOH
(10 mL) was treated with KHCO3 (1.31 g, 13.1 mmol) in water
(20 mL). The solvents were evaporated. The residue was
taken up in concentrated NH4OH (40 mL), placed in a sealed
tube, and warmed to 50 °C for 20 h. Excess ammonia was
evaporated, and the residue was dried under high vacuum. It
was dissolved in dioxane/water (60 mL; 2:1) and treated with
(BOC)2O. After stirring for 3 h, the organic solvent was
evaporated and the aqueous layer was extracted twice with
AcOEt. The combined organic extracts were discarded. The
pH of the aqueous layer was adjusted to pH 3.5 with 1 N HCl
and extracted with AcOEt (3×). The combined organic phases
were worked up affording 2.62 g (66%) of 10. NMR (CDCl3):
1.45 (9H, s), 1.50-1.98 (8H, m), 3.50-4.05 (4H, m), 4.13 (1H,
m), 4.36 (1H, m), 4.55 (1H, m), 5.65 (1H, d, J ) 9).
1:1 Mixtu r e of (1S,2S)-1-(Ben zyloxyca r ba m oyl)-2-h y-
d r oxy-4-[(R)- a n d (S)-tetr a h yd r op yr a n -2-yloxy]bu tylca r -
ba m ic Acid ter t-Bu tyl Ester (11). The pH of a solution of
10 (2.62 g, 7.85 mmol) and BnONH2‚HCl (1.63 g, 10.2 mmol)
in THF/water (0.1 L; 1:1) was adjusted to pH 4.5 with 1 N
NaOH. The solution was reacted with a solution of 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (2.85
g, 15.71 mmol) in water (10 mL) at pH 4.5 (kept by the addition
of 1 N HCl). After the mixture stirred for 4 h, the pH was
adjusted to 6.7, and the reaction mixture was diluted with
AcOEt, the organic phase separated, and the aqueous layer
extracted with AcOEt. The combined organic phases were
worked up and chromatographed (hexane/ethyl acetate, 3:7)
affording 1.74 g (51%) of 11, mp 87-88 °C (hexane). [R]D
-9.4°(c 1, CHCl3). MS (ISP): 461.4 (M + Na), 439.4 (M + H).
NMR (CDCl3): 1.43 (9H, s), 1.50-1.98 (9H, m), 3.45-4.10 (6H,
m), 4.55 (1H, m), 4.91 (2H, 2m), 5.5-5.7 (1H, m), 7.35 (5H, s),
9.50 (1H, 2s). Anal. (C22H34N2O7) C, H, N.
(1R,5R)-6-Hydr oxy-7-oxo-2,6-diazabicyclo[3.2.0]h eptan e-
2-ca r boxylic Acid ter t-Bu tyl Ester (16). A solution of 15
(769 mg, 2.41 mmol) in EtOH (15 mL) was hydrogenated over
10% Pd/C. The catalyst was filtered off, and the filtrate was
evaporated under reduced pressure and crystallized giving 0.48
g (87%) of 15, mp 150-52 °C (AcOEt/n-hexane). IR: 3437,
1754, 1721, 1697, 1404 cm-1. MS (EI): 211 (M - OH). NMR
(DMSO-d6): 1.40 (9H, s), 1.68 (1H, m), 2.0 (1H, dd, J ) 6 and
12), 3.09 (1H, m), 3.81 (1H, dd, J ) 9 and 12), 4.39 (1H, t, J )
4.5), 4.8 (1H, broad), 10.32 (1H, s). Anal. (C10H16N2O4) C, H,
N.
Sod iu m [(1S,5R)-2-(ter t-Bu t oxyca r b on yl)-7-oxo-2,6-
d ia za bicyclo[3.2.0]h ep ta n -6-yl]su lfa te (2a ). A solution of
16 (114 mg, 0.5 mmol) in pyridine (2 mL) was reacted with
C5H5N‚SO3 complex (95 mg, 0.6 mmol) and molecular sieve (4
Å). The reaction mixture was filtered, and the filtrate was
evaporated. The residue was dissolved in EtOH (3 mL) and
precipitated by the addition of Et2O. The first oily crystals

Design of â-Lactamase Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3975
were removed by filtration, and the filtrate was further treated
with Et2O. The crystals were filtered, affording the pyridinium
salt of 2a , 160 mg (82%). NMR (DMSO-d6): 1.40 (9H, s), 1.65
(1H, m), 2.15 (1H, dd, J ) 6 and 12), 3.15 (1H, m), 3.80 (1H,
t, J ) 9 and 12), 4.60 (1H, t, J ) 4.5), 4.80 (1H, broad), 8.03
(2H, m), 8.54 (1H, m), 9.90 (2H, m).
The pyridinium salt (160 mg, 0.413 mmol) was treated with
NaHCO3 (34.7 mg, 0.413 mmol) in water (10 mL). The solution
was concentrated under reduced pressure to about 3 mL and
purified by gel filtration (MCI gel; water then 10% MeOH in
water) affording 52 mg of 2a (38%) after lyophilization. IR:
1775, 1701 cm-1. MS (ISN): 307.2 (M - Na). NMR (DMSO-
d6): 1.40 (9H, s), 1.68 (1H, m), 2.15 (1H, dd, J ) 6 and 12),
3.15 (1H, m), 3.78 (1H, dd, J ) 9 and 12), 4.62 (1H, t, J ) 4.5),
4.85 (1H, broad). Anal. (C10H15N2O7SNa) C, H, N.
Su lfu r ic Acid (1S,5R)-2-(4-Ca r ba m oylp h en ylca r ba m -
oyl)-7-oxo-2,6-d ia za bicyclo[3.2.0]h ep ta n -6-yl Ester So-
d iu m Sa lt (2b). TFA (0.9 mL) was added to 2a (0.46 g, 1018
mmol) at 0 °C. After 1 h at room temperature, Et2O was added
and the crystals were filtered and washed with Et2O affording
308 mg (81%) of intermediate sulfuric acid mono [(1S,5R)-7-
oxo-2,6-diazabicyclo[3.2.0]heptan-6-yl] ester. NMR (DMSO-
d6): 1.75 (1H, m), 2.58 (1H, dd, J ) 6 and 15), 3.20 (1H, m),
3.59 (1H, dd, J ) 6 and 12), 4.75 (1H, t, J ) 3.5), 4.91 (1H, d,
J ) 3.5).
A solution of this betaine (0.13 g, 0.46 mmol) in water (4
mL) was reacted at 0 °C with NaHCO3 (82 mg, 0.98 mmol)
and a solution of 4-H2NCOC6H4NHCO2NSu2 (18; 142 mg, 0.51
mmol) in MeCN (5 mL). The reaction mixture was filtered,
the solvent evaporated, and the residue, dissolved in water,
purified by gel filtration, affording 47 mg (26%) of 2b. IR:
1772, 1660 cm-1. MS (ISN): 369.2 (M - Na). NMR (DMSO-
d6): 1.75 (1H, m), 2.35 (1H, dd, J ) 6 and 12), 3.25 (1H, m),
4.0 (1H, dd, J ) 9 and 12), 4.70 (1H, t, J ) 4.5), 7.17 (1H,
broad), 7.55 (2H, d, J ) 8), 7.8 (3H, m), 8.80 (1H, s).
according to a standard method.10 The compounds were
incorporated, following a serial 2-fold dilution pattern, into
Mueller-Hilton agar (Difco Laboratories, Detroit, MI). An
inoculum of approximately 104 test organisms was applied to
the agar surface using a mulipoint inoculator. The MIC of a
compound or a combination was defined as the lowest concen-
tration that prevented visible growth of bacteria after incuba-
tion at 35 °C for 18 h.
Ack n ow led gm en t. The authors would like to ex-
press their thanks to Drs. Arnold, W. Huber, and S.
Mueller, Mr. W. Meister, and A. Bubendorf for spectral
data and microanalysis. We also wish to thank E. Bald,
M. Kania, J . Nell, I. Pfister, and H. Straub for their
valuable technical assistance.
Refer en ces
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(2) Livermore, D. M.; Chen, H. Y. Potentiation of â-Lactams against
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(1S,5R)-[2-(ter t-Bu toxycar bon yl)-7-oxo-2,6-diazabicyclo-
[3.2.0]h ep ta n -6-yloxy]a cetic Acid (17). A solution of 16
(74.4 mg, 0.326 mmol) in THF (2 mL) was reacted at 0 °C with
DBU (53 µL, 0.36 mmol) and benzyl bromoacetate (57 µL, 0.35
mmol) for 1 h. The reaction mixture was diluted with AcOEt,
worked up, and chromatographed (AcOEt/hexane, 4:6), afford-
ing 127 mg of 17 (100%). IR (CH2Cl2): 1783 cm-1
.
(1S,5R )-6-[(Be n zyloxyca r b on yl)m e t h oxy]-7-oxo-2,6-
d ia za bicyclo[3.2.0]h ep ta n e-2-ca r boxylic Acid ter t-Bu tyl
Ester (3a ). A solution of 17 (127 mg, 0.325 mmol) in EtOH
(3 mL) was hydrogenated over Pd/C. The catalyst was filtered
off, the filtrate evaporated, and the residue crystallized from
Et2O/hexane giving 57 mg of 3a (62%). IR (CH2Cl2): 1783
cm-1. Anal. (C12H18N2O6) C, H, N.
En zym e In h ibition . â-Lactamases were purified to ho-
mogeneity, and their hydrolytic activity was measured using
standard methods.9
In Vitr o An tiba cter ia l Activity. The minimal inhibitory
concentrations (MICs) of the test compounds were determined
J M9800245