The synthesis and evaluation of 2-substituted-7-(alkylidene)cephalosporin sulfones as β-lactamase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00094-9

A series of 2-substituted-7-(alkylidene)cephalosporin sulfones were prepared and evaluated as β-lactamase inhibitors. Compound 11c showed excellent activity as an inhibitor of the class C β-lactamase derived from Enterobacter cloacae, strain P99. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00094-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 847±851
The Synthesis and Evaluation of 2-Substituted-7-
(alkylidene)cephalosporin Sulfones as ꢀ-Lactamase Inhibitors
John D. Buynak,* Venkata Ramana Doppalapudi, A. Srinivasa Rao,
Sirishkumar D. Nidamarthy and Greg Adam
Department of Chemistry, Southern Methodist University, Dallas, TX 75275-0314, USA
Received 20 August 1999; accepted 7 February 2000
AbstractÐA series of 2-substituted-7-(alkylidene)cephalosporin sulfones were prepared and evaluated as b-lactamase inhibitors.
Compound 11c showed excellent activity as an inhibitor of the class C b-lactamase derived from Enterobacter cloacae, strain P99.
# 2000 Elsevier Science Ltd. All rights reserved.
The hydrolytic destruction of b-lactam antibiotics,
mediated by the b-lactamase enzymes, represents the
most common mechanism of bacterial penicillin-resis-
tance.1 The b-lactamases are divided into serine (A, C,
and D) and metalloenzyme (B) classes.2 Successful
treatment of such resistant infections can often be
e€ected by the co-administration of an antibiotic and a
b-lactamase inhibitor.2 Established inhibitors of the
serine b-lactamases include clavulanate (1), which is co-
administered with the antibiotic amoxicillin in the form
of the product Augmentin1 and tazobactam (2), co-
administered with piperacillin in the product Zosyn1
These inhibitors target the class A b-lactamases, which
have, historically, been the most clinically important.
penicillin series, relatively few cephalosporin-derived
b-lactamase inhibitors are known.9 We reported that 3
is a good inhibitor of the Class C b-lactamase, derived
from E. cloacae, strain P99, while 4 inhibits the class A
enzyme, TEM-1.7 We recently undertook a project to
design modi®cations of these inhibitors with the 3-fold
goal of increasing their potency, broadening their
spectrum, and furthering our understanding of the
mechanism of inhibition. Our discovery of the enhanced
activity and spectrum of the 20-substituted-6-alkylidene-
penams, 5, has already been described.8 In contrast to
the penams (which possess few easily functionalized
positions), the cephalosporin nucleus provides an excel-
lent opportunity to create modi®cations at both the C-2
and C-3 positions on the six-membered ring. We now
report the e€ect of selected C-2 modi®cations on the
biological activity of 3 and 4.
Recently, however, the class C enzymes (AmpC) have
become an important clinical threat. De®ned as cepha-
losporinases that are not inhibited by clavulanate,4 the
chromosomally encoded class C b-lactamase is present
in 10 to 50% of patients infected with Citrobacter
freundii, Enterobacter cloacae, Serratia marcescens, and
Pseudomonas aeruginosa. Furthermore, plasmid-encoded
class C b-lactamases5 have also been described in iso-
lates of Klebsiella pneumoniae, Klebsiella oxytoca,
Escherichia coli, and Salmonella senftenberg.6
Synthesis
Three separate 7-aminocephalosporin derivatives (6),
were converted to the corresponding 7-oxocephalospor-
inates (7), utilizing our earlier protocol.10 Reaction of
these with either (20-pyridylmethylene)triphenylphos-
phorane or (tert-butoxycarbonyl)methylenetriphenyl-
phosphorane generated ®ve new compounds 8 (all
exclusively of the Z-geometry, as shown). The reaction
of compound 7c with (20-pyridylmethylene)triphenyl-
phosphorane was unsuccessful.
Our research group has recently described several new
b-lactamase inhibitors, including cephalosporin-derived
compounds (3 and 4),7 as well as the 6-alkylidene-20-
substituted penams, 5.8 Especially in comparison to the
These sul®des were then oxidized to the corresponding
sulfones and treated with Eschenmoser's Salt11 to
produce the 2-exomethylidene cephems,12 10a±10e. The
*Corresponding author. Tel.: +1-214-768-2484; fax: +1-214-768-
4089; e-mail: jbuynak@mail.smu.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00094-9

848
J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 10 (2000) 847±851
30-chloride of 9e was replaced (via the intermediate
iodide) by N-methylthiotetrazole to produce 9f, which
was subsequently converted to 10f. Lastly, the sulfone
9c was treated with CS2 in the presence of base and
subsequently treated with methyl iodide to generate the
dithiomethylidene analogue 10g. All of these com-
pounds were deprotected to generate the corresponding
carboxylate salts. In the case of tert-butyl esters 10b and
10d, the deprotection also produced substantial quan-
tities of the corresponding bis(carboxylate) salts 11h and
11i, respectively.
Our initial attempts to incorporate a methyl (or other
alkyl) group at C-2 by generation of the allylic anion,
followed by alkylation, were unsuccessful. In cases
where some alkylation product could be isolated, it
appeared that the alkylation had occurred at C-4, with
allylic transposition of the double bond. An alternative,
and successful, strategy is shown below. Reaction of the
2-methylidene compound 10c with 9-BBN, followed by
treatment with acetic acid, produced a 2:1 mixture of
the b:a isomers of 12, respectively.12 These compounds
were then deprotected to the corresponding carboxylate
salts, 13.
Alternatively, we sought to incorporate single-bonded
substituents at C-2. Introduction of methyl, for exam-
ple, might make the cephem more penam-like and
thus broaden its spectrum as an inhibitor of the class A
penicillinases. We also desired to explore the activity
of compounds with potential leaving groups at this
position.
We also explored the incorporation of heteroatom sub-
stituents at C-2. Reaction of compound 9c with NBS
in the presence of Et3N produced bromide 1413 which
was converted to the thiotetrazole 1514 by reaction with
5-mercapto-1-methyltetrazole. This was successfully
deprotected to produce carboxylate 16.

J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 10 (2000) 847±851
849
Results and Discussion
behave as a leaving group subsequent to enzyme acyla-
tion. In fact, Pratt has shown16 that, upon enzymolysis
of typical cephalosporin antibiotics, loss of the 30-leaving
group results in the formation of a stabilized acyl-
enzyme (leading, for example, to transient inhibition of
the PC1 lactamase). In the present case, however,
incorporation of a 30-acetate, as exempli®ed by com-
parison of 11a and 11c, decreased the inhibitory
potency.17 The data indicate that addition of the
2-methylidene group, while resulting in improved inhi-
bitory activity of the 7-(200-pyridylmethylidene)cephems
toward the class C P99 b-lactamase, degraded their
As shown in Table 1, these new compounds were eval-
uated in cell-free systems as inhibitors of the two class A
b-lactamases TEM-1 and PC1, derived from Staphylo-
coccus aureus, as well as the class C enzyme, P99,
derived from E. cloacae. Both 11a and, especially, its
corresponding 30-desacetoxy analogue 11c, display
improved inhibition of the class C enzyme, relative to
the C-2 unsubstituted compound 3. The 30-acetate is
known to activate the b-lactam carbonyl toward
nucleophiles15 (such as the active site serine) and to
Table 1. Inhibition of three representative serine b-lactamases19
IC50 (mM)
Compd
Type
R1
R2
R3, R4
R5
P99
51.9
0.498
0.165
0.039
0.51
3.9
29.0
6.56
1.55
TEM-1
PC1
2.57
402
733
1860
NT
102
844
977
NT
NT
NT
230
NT
2
3
Tazo
II
I
I
I
II
II
II
I
I
I
Ð
CH2OAc
CH2OAc
CH3
CH3
CH3
Ð
20-pyr
Ð
Ð
Ð
H
Ð
Ð
0.29
34.8
99.2
91.1
173
74.1
31.6
0.03
0.932
56.5
1.13
498
11a
11c
11g
13
16
4
11b
11d
11f
11h
11i
20-pyr
H, H
H, H
SCH3, SCH3
Ð
20-pyr
20-pyr
Ð
20-pyr
CH3
SC2H3N4
H
CH3
20-pyr
Ð
Ð
CH2OAc
CH2OAc
CH3
CH2SC2H3N4
CH2OAc
CH3
CO2-t-Bu
CO2-t-Bu
CO2-t-Bu
CO2-t-Bu
CO2Na
CO2Na
H, H
H, H
H, H
H, H
H, H
Ð
Ð
Ð
Ð
48.6
2.38
2.49
I
I
Ð
36.8
277

850
J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 10 (2000) 847±851
Scheme 1.
Scheme 2.
ability to inhibit both of the class A enzymes. This trend
is also evident in comparing inhibition of the 7-[(tert-
butoxycarbonyl)methylidene]cephems 4, 11b, 11d and
11f.18 In the latter series, however, the extremely poor
overall activity of the desacetoxy cephem 11d indicates
a mechanistic requirement for a leaving group at the
30-position, in contrast to the 7-(pyridylmethylidene)-
cephem series.
Chemical Society, for support of this research. The b-
lactamases were generously provided by Dr. Osnat
Herzberg, Dr. Natalie Strynadka, and Dr. Timothy
Palzkill. We also thank Wyeth-Ayerst Research for
helping to provide biological assays, Dr. Stan Lang
(Wyeth) for his continued support.
References and Notes
In the case of the penicillin sulfones (5), good inhibitors
have been prepared with R=20-pyridyl,20 and also with
R=COONa8b (with selected 20-position modi®cations
improving activity in both series).8a While the inhibitory
activity of the corresponding 20-pyridylmethylidene
cephalosporin (3), is also high, that of the 7-position
carboxylate is not.7 Similarly, in the present 20-methyl-
idene series, the 70-carboxymethylidenes 11h and 11i
lack signi®cant inhibitory activity.
1. (a) Matagne, A.; Dubus, A.; Galleni, M.; Frere, J. M. Nat.
Prod. Rep. 1999, 16, 1. (b) Medeiros, A. A. Clin Infect. Dis.
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tors of elastase: (a) Doherty, J. B.; Ashe, B. M.; Argenbright,
L. W.; Barker, P. L.; Bonney, R. J.; Chandler, G. O.; Dahlgren,
M. E.; Dorn, C. P.; Finke, P. E.; Firestone, R. A.; Fletcher,
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The present data imply that the 7-(pyridylmethylidene)-
and the 7-[(tert-butoxycarbonyl)-methylidene]cephalos-
porins, which are established inhibitors of the class C
and class A b-lactamases, respectively, operate by dif-
ferent inhibitory mechanisms. The penicillin sulfones,
including sulbactam and tazobactam, represent the clo-
sest available mechanistic analogy of the present cepha-
losporin sulfones. In the former case, it is believed that
inhibition results from the series of chemical transfor-
mations shown in Scheme 1.21
An analogous mechanism for the cephalosporin sulfones is
shown in Scheme 2. We anticipated that a ring opening of
the six-membered ring might be promoted by the presence
of a C-2 methylidene group. We are currently engaged
in crystallographic and kinetic studies to clarify the nature
of the chemical transformations leading to inhibition.
Acknowledgements
We thank the Robert A. Welch Foundation and the
Petroleum Research Fund, administered by the American

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2-methylidene cephems was examined. We found no sig-
ni®cant hydrolysis (NMR) of these materials upon standing in
bu€er for a 3 h period.
12. Wright, I. G.; Ashbrook, C. W.; Goodson, T.; Kaiser, G.
V.; Van Heyningen, E. M. J. Med. Chem. 1971, 14, 420.
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19. Assay method involves 4 min incubation of a solution of
inhibitor and enzyme, followed by transfer of an aliquot into
a dilute solution of the substrate nitroce®n. Hydrolysis is
monitored spectrophotometrically at 480 nm for 1 min. The
rate is constant throughout this period. Error is ‹10%, based
on multiple experiments. The purity of all compounds and
intermediates was veri®ed by NMR.
20. Chen, Y. L.; Chang, C. W.; Hedberg, K.; Guarino, K.;
Welch, W. M.; Kiessling, L.; Retsema, J. A.; Haskell, S. L.;
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