Penicillin-derived inhibitors that simultaneously target both metallo- and serine-β-lactamases

Article abstract of DOI:10.1016/j.bmcl.2003.12.037

The synthesis and β-lactamase inhibitory activity of four 6-(mercaptomethyl)penicillinates and the four corresponding 6-(hydroxymethyl) penicillinates are described. These penicillins include both C6 stereoisomers as well as the sulfide and sulfone oxidation states of the penam thiazolidine sulfur. All compounds were evaluated as inhibitors of representative metallo- and serine-β-lactamases enzymes. Selected (mercaptomethyl)penicillinates are shown to inactivate both metallo- and serine-β-lactamases and to display synergism with piperacillin against β-lactamase producing strains.

Full text of DOI:10.1016/j.bmcl.2003.12.037

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1299–1304
Penicillin-derived inhibitors that simultaneously target both
metallo- and serine-ꢀ-lactamases
John D. Buynak,a,* Hansong Chen,a Lakshminaryana Vogeti,a
Venkat Rao Gadhachanda,a Christine A. Buchanan,b Timothy Palzkill,c
Robert W. Shaw,d James Spencere and Timothy R. Walshe
aDepartment of Chemistry, Southern Methodist University, Dallas, TX 75275-0314,USA
bDepartment of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA
cVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA
dDepartment of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA
eDepartment of Pathology and Microbiology, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
Received 30 October 2003; revised 4 December 2003; accepted 5 December 2003
Abstract—The synthesis and b-lactamase inhibitory activity of four 6-(mercaptomethyl)penicillinates and the four corresponding
6-(hydroxymethyl)penicillinates are described. These penicillins include both C6 stereoisomers as well as the sulfide and sulfone
oxidation states of the penam thiazolidine sulfur. All compounds were evaluated as inhibitors of representative metallo- and serine-
b-lactamases enzymes. Selected (mercaptomethyl)penicillinates are shown to inactivate both metallo- and serine-b-lactamases and
to display synergism with piperacillin against b-lactamase producing strains.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
bitors needed to overcome several differences between
the metallo and serine b-lactamases.7 Serine b-lactamase
inhibitors usually involve formation of a stable acyl-
enzyme. The metallo-b-lactamases utilize active site zinc
as a Lewis acid to catalyze the hydrolysis of the b-lac-
tam in a single step.8 Inhibitors of the zinc b-lactamases
are tight binding reversible inhibitors, often with a
strongly coordinative zinc ligand. Commercial serine-b-
lactamase inhibitors are substrates of the metallo-b-lac-
tamases.9 Additionally, the general architecture of the
active sites of serine and metallo-b-lactamases are dis-
similar. However, these two classes of hydrolase share
the property of being able to specifically hydrolyze
b-lactam antibiotics. Thus, we employed a penicillin
scaffold to ensure recognition of both classes of b-lacta-
mase. These inhibitors are designed to form a stabilized
acyl-enzyme to inhibit the serine b-lactamases, but also
incorporate a ligand to inhibit the metallo-b-lactamases.
The most common form of bacterial resistance to the
b-lactam antibiotics involves the ability to produce one
or more types of b-lactamase.1 More than 300 b-lacta-
mases have been characterized2 and divided into four
molecular classes, A–D. Classes A, C, and D are serine
enzymes, while class B are zinc metalloenzymes.
Although strains expressing class A serine b-lactamases
are the most clinically relevant, increasing numbers of
strains are developing resistance through classes B, C,
and D enzymes. Class B metalloenzymes have a broad
substrate profile3 and metallo-b-lactamase producing
strains may also possess a serine enzyme, thus combin-
ing the protective effects of both a metallo- and a serine-
b-lactamase. While metallo-b-lactamase inhibitors are
known,4 none are clinically useful. Current commercial
b-lactamase inhibitors are only useful against strains
producing class A enzymes. The Buynak Group has
reported new inhibitors that simultaneously inactivate
class A, C, and D b-lactamases.5 We now report the
synthesis and evaluation of inhibitors which inactivate
both metallo- and serine-b-lactamases.6 Such dual inhi-
Several antibiotics and b-lactamase inhibitors possess a
hydroxyalkyl side chain a to the b-lactam carbonyl.
These include the carbapenems (1), penems (2), oxape-
nems (3), and the 6-hydroxyalkylpenicillins (4, n=0 or
2) shown in Figure 1. Penams 4 are reported to have the
ability to inhibit serine b-lactamases.10 We hypothesized
that substituting the hydroxyl group with a thiol would
* Corresponding author. Tel.: +1-214-768-2484; fax: +1-214-768-
4089; e-mail: jbuynak@mail.smu.edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.037

1300
J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1299–1304
treatment with cesium thioacetate produced thioester
17. Selective cleavage of thioacetate 17, deprotection
with m-cresol,12 and neutralization produced 5a.
Compound 5b was prepared from the same mixture 14
by reduction with tributylphosphine13 as shown in
Scheme 3. Conversion of 19 to mesylate 20, treatment
with cesium thioacetate and cleavage of thioester 21
with NaOMe produced 22 which was deprotected with
m-cresol.
Figure 1. Representative hydroxyalkyl and mercaptoalkyl antibiotics
and b-lactamase inhibitors.
produce a compound 5 with the dual ability to inhibit
both serine and metallo-b-lactamases.
Attempts to oxidize 17 to the corresponding sulfone
with excess mCPBA surprisingly produced only a mix-
ture of epimeric sulfoxides 23. The requisite oxidation
to 24 could be achieved using excess KMnO4. However,
the subsequent cleavage of the thioester produced an
inseparable 2:1 mixture of epimeric 6-(mercapto-
methyl)penicillinates 25 and 26 (Scheme 4).
As shown in Scheme 1, either compounds 5, or com-
pounds 6, having one less carbon, would also be capable
of bidentate chelation of zinc subsequent to enzymatic
hydrolysis of the b-lactam. We thus targeted four com-
pounds of structure 5 and four compounds of structure 6
as shown in Figure 2. For comparison, we also prepared
the corresponding 6-(hydroxymethyl)penicillinates, 4,
using procedures of Kellogg10a and of Mobashery.10e,j
Obviously, sulfone 24 (and/or the corresponding mer-
captans 25 and 26) was less configurationally stable (at
C6) under these basic conditions than were sulfides 17
and 18. Thus we replaced the thioester with a more
readily removable protecting group. As shown in
Scheme 5, we protected the thiol of 18 with Troc before
oxidation of sulfide 27. Subsequent KMnO4/AcOH
2. Results
2.1. Synthesis
As shown in Scheme 2 and 3, 5a and 5b were prepared
from 6-APA. 6,6-Dibromopenicillanic acid (12) was
prepared using the procedure of Volkmann11 and
esterified. This dibromide, 13, was converted into
bromoalcohols 14 and stereoselectively reduced to 6b-
(hydroxymethyl)penicillinate 15 using the procedure of
Kellogg.10 Conversion to mesylate 16, followed by
Scheme 2. Synthesis of 5a. (i) NaNO2, H2SO4, Br2, CH2Cl2/H2O
(85%); (ii) Ph2C¼N2, acetone, rt, 14 h, (90%); (iii) t-BuMgBr, ꢀ78 ꢁC;
(iv) CH2O, ꢀ78 ꢁC to rt, (36% for steps iii and iv); (v) (n-Bu)3SnH, cat
AIBN, CH2Cl2, 89%; (vi) MeSO2Cl, DMAP, CHCl3, 3 h, 74%;
(vii) AcSCs, MeCN, rt, 15 h (77%); (viii) NaOMe, THF, MeOH
ꢀ78 ꢁC to ꢀ40 ꢁC (85%); (ix) m-cresol, 48 ꢁC, 6 h; (x) NaHCO3 (40%
for steps ix and x).
Scheme 1.
Scheme 3. Synthesis of 5b. (i) Bu3P, MeOH, rt, 1.5 h, 85%; (ii) MsCl,
DMAP, CHCl3, rt, 1.5 h, 85%; (iii) AcSCs, MeCN, rt, 2.5 h, 85%;
(iv) NaOMe, THF–MeOH, ꢀ78 ꢁC to ꢀ40 ꢁC, 89%; (v) m-cresol,
50 ꢁC, 6 h; (vi) NaHCO3 (41% for steps v and vi).
Figure 2. b-Lactamase inhibitors prepared and tested.

J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1299–1304
1301
oxidation produced an 85:15 mixture of the 6b and 6a
epimers of sulfones 28 in 90% yield. Separation and
removal of the Troc group with zinc/copper couple14
and subsequent removal of the benzhydryl group pro-
duced 5c. A similar strategy produced 5d, as shown in
Scheme 6.
topenicillin sulfone 6c. Upon treatment of sulfone 39
with zinc/copper couple, the C6 b-isomer was epimer-
ized to the 6a isomer 41. Given the mild conditions, we
decided the 6b-isomer was not isolable. The 6a epimer,
6d, was prepared without further difficulty. As shown in
Scheme 9, the 6-(hydroxymethyl)penicillinates were
prepared utilizing reported procedures.10
6-Mercaptopenicillinates 6a and 6b were prepared as
shown in Scheme 7. 6-APA was converted to 6a-hydroxy-
penicillanic acid and esterified to produce the benz-
hydryl ester 31. Compound 31 was then converted to
triflate, 32, treated with cesium thioacetate to produce
thioacetate 33 and subsequently cleaved with NaOMe
to produce thiol 34, which was deprotected to carbox-
ylate 6a. The epimer, 6b, was produced by treating 6-
diazopenicillinate 35 with thiolacetic acid in the pre-
sence of BF3–OEt2. Cleavage of the thioacetate 36 and
removal of the benzhydryl group produced 6b.
As shown in Scheme 8, the Troc group was again
employed in an attempted synthesis of the 6b-mercap-
Scheme 7. Synthesis of 6a and 6b. (i) NaNO2/aq HClO4; (ii)
Ph2C¼N2; (iii) Tf2O, Et3N; (iv) AcSCs, MeCN; (v) NaOMe, THF–
MeOH, ꢀ78 ꢁC, 8 h, 80%; (vi) m-cresol, 50 ꢁC, 6 h; (vii) NaHCO3
(30% for steps vi and vii); (viii) i-pr-ONO, cat. TFA; (ix) AcSH, BF3–
OEt2, rt, 15 min (40%); (x) NaOMe, THF–MeOH, ꢀ78 ꢁC to ꢀ40 ꢁC,
2.5 h, 71%; (xi) m-cresol, 50 ꢁC, 6 h; (xii) NaHCO3 (41% yield for
steps xi and xii).
Scheme 4. Initial attempt to prepare 5c. (i) 4 equiv mCPBA, CH2Cl2,
pH=6.4 buffer; (ii) xs KMnO4, AcOH/CH2Cl2=1/2; (iii) NaOMe/
MeOH, ꢀ78 ꢁC to ꢀ40 ꢁC.
Scheme 5. Synthesis of 5c. (i) Troc-Cl, DMAP, CH2Cl2, 0ꢁC to rt, 3 h,
83%; (ii) KMnO4/HOAc, CH2Cl2, rt, 14 h, 90%; (iii) Zn–Cu/HOAc,
THF–MeOH, rt, 1 h, 54%; (iv) m-cresol, 50 ꢁC, 2.5 h; (v) NaHCO3
(53% for steps iv and v).
Scheme 8. Synthesis of 6d. (i) Troc-Cl, DMAP, 0 ꢁC to rt, 3 h, 73%;
(ii) KMnO4/HOAc, rt, 15 h, 78%, 39:40=1:1; (iii) Zn–Cu, HOAc,
rt, 30 min; (iv) KMnO4/HOAc, rt, 15 h, 60%; (v) NaOMe, 78%;
(vi) m-cresol, 50 ꢁC, 4 h; (vii) NaHCO3, (60% for step vi and vii).
Scheme 6. Synthesis of 5d. (i) Troc-Cl, DMAP, CH2Cl2, 0ꢁC to rt, 3 h,
86%; (ii) KMnO4/HOAc, CH2Cl2, rt, 14 h, 90%; (iii) Zn–Cu/HOAc,
THF/MeOH, rt, 1 h, 54%; (iv) m-cresol, 50 ꢁC, 2.5 h; (v) NaHCO3
(67% for steps iv and v).
Scheme 9. Synthesis of 4a–d. (i) m-cresol; (ii) NaHCO3; (iii) xs
KMnO4, AcOH, CH2Cl2.

1302
J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1299–1304
2.2. Inhibitory activity
2.3. Microbiological activity
The activity of the newly prepared molecules as inhibi-
tors of representative serine and metallo-b-lactamases is
presented in Table 1. For comparison, the activity of the
6-(hydroxymethyl)-penicillinates (4a–d), and the com-
mercial serine b-lactamase inhibitor, tazobactam (46),
and pyridine 2,6-dicarboxylic acid (47) as a standard
metallo-b-lactamase inhibitor were also measured (Fig. 3).
The new inhibitors were assayed for synergism with
piperacillin against b-lactamase-producing strains. Data
against serine-b-lactamase producing strains is shown in
Table 2. The MIC values shown in Table 2 were
obtained while maintaining the inhibitors constant at 4
mg/mL and varying the concentration of piperacillin. In
Table 3, 6-(mercaptomethyl)penicillinates
5
were
screened as inhibitors of metallo-b-lactamase producing
strains, including one E. coli strain producing the IMP1
metallo-b-lactamase, and three clinical isolates of P.
aeruginosa strains carrying either blaSPM-1, blaVIM-1 or
blaVIM-7 as well as an AmpC serine b-lactamases. It is
noteworthy that the P. aeruginosa carrying blaVIM-7 also
carries blaOXA-45, an unusual class D enzyme. This
initial screen for activity was performed at a concen-
tration of 128 mg/mL while maintaining a 1:1 anti-
biotic:inhibitor ratio. These inhibitors were tested both
in the presence of the serine b-lactamase inhibitors,
tazobactam (46) and DVR-II-183 (48), and, in the case
of inhibitor 5c, without an additional b-lactamase inhi-
bitor. We have previously reported the class A and class
C serine b-lactamase inhibition and in vitro synergism
(with piperacillin) of 48 against Gram-negative b-lacta-
mase-producing strains.6d All metallo-b-lactamase pro-
ducing strains were resistant to piperacillin and to the
co-administration of piperacillin with tazo or with 48.
Table 4 shows the MIC values of 5c (in combination
with piperacillin) against a number of metallo-b-lacta-
mase producing strains. 5c was tested alone and in
combination with tazo and also in combination with 48.
Figure 3. Standard b-lactamase inhibitors.
Table 1. b-Lactamase inhibitory activity (IC50, mM) against repre-
sentative class A (TEM-1), class C (P99), and class B (metallo)
enzymes15
Compd
TEM-1
(Class A)
Serine
P99
(Class C)
Serine
L1
(Class B)
Metallo
BCII
(Class B)
Metallo
tazo
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6d
47
0.122
752
275
0.65
14.6
601
53.2
409
96.2
3.9
10.0
0.10
3.75
10.5
7.5
>200
>200
>200
72.3
>200
32.1
10.9
0.10
0.30
41.4
36.9
7.6
>200
>200
>200
>200
>200
2.9
Table 3. Synergy of compounds 5 with piperacillin and other b-lac-
tamase inhibitors against bacteria producing metallo b-lactamases
(S=susceptible; R=resistant)
648
6.8
1.7
1.4
51.7
>1000
988
12.0
17.8
17.2
106.2
21.8
Antibiotic/
E. coli P. aeruginosa P. aeruginosa P. aeruginosa
(VIM-7)
42.3
7.9
1.4
b-Lactamase (IMP-1)
inhibitors:
1:1:1 at concn
(SPM-1)
(VIM-1)
69.2
NT
NT
7.8
of 128 mg/mL
pip
pip/tazo
pip/48
pip/tazo/5a
pip/tazo/5b
pip/tazo/5c
pip/tazo/5d
pip/48/5c
pip/5c
R
R
R
S
S
S
S
S
S
R
R
R
R
R
S
R
S
S
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
Table 2. Synergy of 4, 5, and 6 with piperacillin against bacteria
producing representative serine b-lactamases
Minimal inhibitory concentration (mg/mL)
Antibiotic/
Inhibitor
E. coli
ATCC 35218
(TEM-1)
E. coli
P99
(AmpC)
Staphylococcus
aureus
ATCC 29213
R
(Class A, Serine) (Class C, Serine)
Table 4. Synergy of compound 5c with piperacillin and other b-lac-
tamase inhibitors against bacteria producing metallo b-lactamases
Piperacillin
Pip/tazo
Pip/4a
Pip/4b
Pip/4c
Pip/4d
Pip/5a
Pip/5b
Pip/5c
Pip/5d
Pip/6a
Pip/6b
Pip/6d
>128
1
>128
>128
1
4
0.5
4
8
1
4
8
1
2
2
4
2
4
8
8
8
Minimal inhibitory concentration (mg/mL)
4
0.125
0.25
2
Antibiotic/
b-Lactamase
inhibitors: 1:1:1
E. coli P. aeruginosa P. aeruginosa P. aeruginosa
(IMP-1)
8
(SPM-1)
(VIM-1)
(VIM-7)
>128
>128
8
2
pip
pip/tazo
pip/tazo/5c
pip/DVRII183/5c
pip/5c
>128
>128
>128
>128
64
>128
>128
>128
128
>128
>128
>128
>128
>128
0.25
0.5
2
2
2
32
8
8
8
>128
>128
>128
64
64
>128

J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1299–1304
1303
3. Discussion
4. Conclusion
Table 1 indicates that although nearly all compounds
display some inhibition of the two serine b-lactamases,
only the thiols have the ability to inhibit the metallo-b-
lactamases. Tazobactam displays no inhibition of either
of the two metallo-b-lactamases. Data involving the
inhibition of the representative class A and class C ser-
ine b-lactamases by 6-(hydroxymethyl)penicillinates 4,
parallel results obtained by Wyeth researchers.10h,i Both
studies observed greater (serine) b-lactamase inhibition
by sulfones 4c and 4d than by the corresponding sulfides
4a and 4b. However, it should be noted that the C6-
hydroxyalkyl penicillin sulfides are also known to be
b-lactamase inhibitors and have been very effectively
utilized to elucidate the direction of approach of the
hydrolytic water molecule to the intermediate acyl
enzyme.16,17 Table 1 shows that all of the new 6-(mer-
captomethyl)penicillins, 5, did, in addition to inhibiting
serine b-lactamases, also inhibit the two metallo-b-lac-
tamases. Sulfones 5c and 5d were good inhibitors of the
L1 metallo-b-lactamase, while sulfides 5a and 5b and
sulfone 5c were all good inactivators of the BCII
hydrolase, implying different chemical mechanisms of
inhibition of these penams against the two metalloen-
zymes. As in the case of the hydroxymethylpenicillinate
sulfones (4c and 4d), the corresponding 6-(mercapto-
methyl)penicillinate sulfones (5c and 5d) displayed good
activity against both serine b-lactamases. By compar-
ison with the (mercaptomethyl)penicillinates (5), the 6-
mercapto-penicillinates (6) were relatively inactive as
inhibitors of either the metallo- or serine-b-lactamases.
The synergy data against the representative serine-b-
lactamase producing strains presented in Table 2 shows
a close parallel between the mercaptans and the corres-
ponding alcohols. This involves both the oxidation level
of the thiazolidine sulfur as well as the stereochemistry
at C6. Thus, in both cases, the 6-(hydroxymethyl)-
penicillin sulfones 4c, 4d and the corresponding 6-(mer-
captomethyl)penicillin sulfones 5c, 5d produce the
better synergy against these strains, with the a stereo-
chemistry at C6, compounds 4c and 5c, being the two
most potent inhibitors.
In conclusion, new compounds, 5, simultaneously
inhibited representative metallo- and serine-b-lacta-
mases and had the ability to differentially inhibit the
two metallo-b-lactamases, with the two sulfides (5a and
5b) being more active against the BCII enzyme than
against the L1 enzyme and sulfone 5c displaying inhibi-
tion of both metallo-b-lactamases. These new com-
pounds were also active in vitro, displaying synergy with
piperacillin against b-lactamase-producing strains,
including a strain of P. aeruginosa.
Acknowledgements
We thank the Robert A. Welch Foundation (J.D.B.,
R.W.S.) and the Texas Higher Education Coordinating
Board (J.D.B.) for support.
References and notes
1. (a) Dax, S. L. b-Lactam Antibiotics. In Antibacterial
Chemotherapeutic Agents; Chapman and Hall: London,
1997; Chapter 3, p 74. (b) Rice, L. B.; Bonomo, R. A.
Drug Resis. Updates 2000, 3, 109. (c) Bush, K. Curr.
Pharm. Des. 1999, 5, 839. (d) Matagne, A.; Dubus, A.;
Galleni, M.; Frere, J.-M. Nat. Prod. Rep. 1999, 16, 1. (e)
Bradford, P. A. Curr. Infect. Dis. Rep. 2001, 3, 13. (f)
Livermore, D. M. Clin. Microbiol. Rev. 1995, 8, 557. (g)
Philippon, A.; Dusart, J.; Joris, B.; Frere, J.-M. Cell. Mol.
Life Sci. 1998, 54, 341. (h) Majiduddin, F. K.; Materon,
I. C.; Palzkill, T. G. Int. J. Med. Microbiol. 2002, 292,
127.
2. Bush, K. Clin. Infect. Dis. 2001, 32, 1085.
3. (a) Felici, A.; Amicosante, G.; Oratore, A.; Strom, R.;
Ledent, P.; Joris, B.; Fanuel, L.; Frere, J.-M. Biochem. J.
1993, 291, 151. (b) Felici, A.; Amicosante, G. Antimicrob.
Agents Chemother. 1995, 39, 192. (c) Murphy, T. A.;
Simm, A. M.; Toleman, M. A.; Jones, R. N.; Walsh, T. R.
Antimicrob. Agents Chemother. 2003, 47, 582. (d) Doc-
quier, J.-D.; Lamotte-Brasseur, J.; Galleni, M.; Amico-
sante, G.; Frere, J.-M. J. Antimicrob. Chemother. 2003,
51, 257.
4. (a) Payne, D. J.; Hueso-Rodriguez, J. A.; Boyd, H.; Con-
cha, N. O.; Janson, C. A.; Gilpin, M.; Bateson, J. H.;
Cheever, C.; Niconovich, N. L.; Pearson, S.; Rittenhouse,
S.; Tew, D.; Diez, E.; Perez, P.; de la Fuenta, J.; Rees, M.;
Rivera-Sagredo, A. Antimicrob. Agents Chemother. 2002,
46, 1880. (b) Bounaga, S.; Galleni, M.; Laws, A. P.; Page,
M. I. Bioorg. Med. Chem. 2001, 9, 503. (c) Fitzgerald,
P. M. D.; Wu, J. K.; Toney, J. H. Biochemistry 1998, 37,
6791. (d) Toney, J. H.; Fitzgerald, P. M. D.; Grover-
Sharma, N.; Olson, S. H.; May, W. J.; Sundelof, J. G.;
Vanderwall, D. E.; Cleary, K. A.; Grant, S. K.; Wu, J. K.;
Kozarich, J. W.; Pompliano, D. L.; Hammond, G. G.
Chem. Biol. 1998, 5, 185. (e) Greenlee, M. L.; Laub, J. B.;
Balkovec, J. M.; Hammond, M. L.; Hammond, G. G.;
Pompliano, D. L.; Epstein-Toney, J. H. Bioorg. Med.
Chem. Lett. 1999, 9, 2549. (f) Payne, D. J.; Bateson, J. H.;
Gasson, B. C.; Khushi, T.; Proctor, D.; Pearson, S. C.;
Reid, R. FEMS Microbiol. Lett. 1997, 157, 171. (g) Payne,
D. J.; Bateson, J. H.; Gasson, B. C.; Proctor, D.; Khushl,
T.; Farmer, T. H.; Tolson, D. A.; Bell, D.; Skett, P. W.;
Marshall, A. C.; Reid, R.; Ghosez, L.; Combret, Y.;
Marchand-Brynaert, J. Antimicrob. Agents Chemother.
Tables 3 and 4 show that the mercaptomethyl inhibitors
5 also display synergy with piperacillin against strains
producing metallo-b-lactamases, including an E. coli
producing IMP-1, as well as several Pseudomonas aeru-
ginosa strains producing either VIM b-lactamases or the
recently characterized8c SPM-1 metallo-b-lactamase.
While a number of the inhibitors displayed synergy
against the IMPproducing strain, only 5c showed
activity against SPM-1 and the VIM-1 producing strain.
None displayed synergy against the VIM-7 producing
strain, which also possesses a Class D b-lactamase. An
additional serine b-lactamase inhibitor was not needed
to support the synergy of 5c against the SPM-1 produ-
cing strain. P. aeruginosa represents a particularly chal-
lenging microorganism, due to the reduced permeability
of its outer membrane and the overexpression of multi-
drug efflux systems. This is the first report of synergy
against a metallo-b-lactamase producing P. aeruginosa
strain.

1304
J. D. Buynak et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1299–1304
1997, 41, 135. (h) Mollard, C.; Moali, C.; Papamicael, C.;
1997, 7, 11. (g) Mourey, L.; Miyashita, K.; Swaren, P.;
Bulychev, A.; Samama, J. P.; Mobashery, S. J. Am. Chem.
Soc. 1998, 120, 9382. (h) Bitha, P.; Li, Z.; Francisco,
G. D.; Rasmussen, B. A.; Lin, Y. I. Bioorg. Med. Chem.
Lett. 1999, 9, 991. (i) Bitha, P.; Li, Z.; Francisco, G. D.;
Yang, Y.; Petersen, P. J.; Lenoy, E.; Lin, Y. I. Bioorg.
Med. Chem. Lett. 1999, 9, 997. (j) Golemi, D.; Maveyr-
aud, L.; Vakulenko, S.; Tranier, S.; Ishiwata, A.; Kotra,
L. P.; Samama, J. P.; Mobashery, S. J. Am. Chem. Soc.
2000, 122, 6123. (k) Maveyraud, L.; Golemi-Kotra, D.;
Ishiwata, A.; Meroueh, O.; Mobashery, S.; Samama, J. P.
J. Am. Chem. Soc. 2002, 124, 2461.
Damblon, C.; Vessilier, S.; Amicosante, G.; Schofield,
C. J.; Galleni, M.; Frere, J.-M.; Roberts, G. C. K. J. Biol.
Chem. 2001, 276, 45015. (i) Siemann, S.; Clarke, A. J.;
Viswanatha, T.; Dmitrienko, G. I. Biochemistry 2003, 42,
1673. (j) Boerzel, H.; Koeckert, M.; Bu, W.; Spingler, B.;
Lippard, S. J. Inorg. Chem. 2003, 42, 1604.
5. (a) Buynak, J. D.; Vogeti, L.; Doppalapudi, V. R.; Solo-
mon, G. M.; Chen, H. Bioorg. Med. Chem. Lett. 2002, 12,
1663. (b) Buynak, J. D.; Doppalapudi, V. R.; Rao, A. S.;
Nidamarthy, S. D.; Adam, G. Bioorg. Med. Chem. Lett.
2000, 10, 847. (c) Buynak, J. D.; Doppalapudi, V. R.;
Adam, G. Bioorg. Med. Chem. Lett. 2000, 10, 853. (d)
Buynak, J. D.; Rao, A. S.; Doppalapudi, V. R.; Adam,
G.; Petersen, P. J.; Nidamarthy, S. D. Bioorg. Med. Chem.
Lett. 1999, 9, 1997.
6. Presented in part: Buynak, J. D.; Chen, H.; Vogeti, L.;
Gadhachanda, V. R.; Shaw, R. Abstracts of Papers, 224th
National Meeting of the American Chemical Society,
Boston, MA, Aug. 18–22, 2002; American Chemical
Society: Washington, DC, MEDI-048.
11. Volkmann, R. A.; Carroll, R. D.; Drolet, R. B.; Elliott,
M. L.; Moore, B. S. J. Org. Chem. 1982, 47, 3344.
12. Torii, S.; Tanaka, H.; Taniguchi, M.; Kameyama, Y.;
Sasaoka, M.; Shiroi, T.; Kikuchi, R.; Kawahara, I.; Shi-
mabayashi, A.; Nagao, S. J. Org. Chem. 1991, 56, 3633.
13. (a) Chern, J. W.; Huang, M.; Tien, J. H.; Pai, S. H. Het-
erocycles 1988, 27, 1349. (b) Ishiwata, A.; Kotra, L. P.;
Miyashita, K.; Nagase, T.; Mobashery, S. Org. Lett. 2000,
2, 2889.
7. Frere, J.-M.; Dubus, A.; Galleni, M.; Matagne, A.; Ami-
cosante, G. Biochem. Soc. Trans. 1999, 27, 58.
8. (a) Wang, Z.; Fast, W.; Valentine, A. M.; Benkovic, S. J.
Curr. Opin. Chem. Biol. 1999, 3, 614. (b) Wang, Z.; Ben-
kovic, S. J. J. Biol. Chem. 1998, 273, 22402.
9. Prosperi-Meys, C.; Llabres, G.; de Seny, D.; Paul Soto,
R.; Hernandez Valladares, M.; Laraki, N.; Frere, J.-M.;
Galleni, M. FEBS Lett. 1999, 443, 109.
10. (a) Kellogg, M. S. Ger. Offen. DE 3039504, 1981. (b)
Knight, G. C.; Waley, S. G. Biochem. J. 1985, 225, 435.
(c) Chen, Y. L.; Hedberg, K.; Barrett, J. F.; Retsema, J. A.
J. Antibiot. 1988, 41, 134. (d) Chen, Y. L.; Hedberg, K.;
Guario, K.; Retsema, J. A.; Anderson, M.; Manousos,
M.; Barrett, J. J. Antibiot. 1991, 44, 870. (e) Miyashita,
K.; Massova, I.; Taibi, P.; Mobashery, S. J. Am. Chem.
Soc. 1995, 117, 11055. (f) Czajkowski, D. P.; Reddy,
A. V. N.; Setti, E. L.; Phillips, O. A.; Micetich, R. G.;
Kunmugita, C.; Maiti, S. N. Bioorg. Med. Chem. Lett.
14. LeGoff, E. J. Org. Chem. 1964, 29, 2048.
15. (a) For serine enzymes: Assay involved 4 min incubation
of inhibitor and enzyme (100 mL volume), followed by
transfer of an aliquot into a dilute solution of the sub-
strate nitrocefin (1 mL final volume). IC50 is reported as
concd of inhibitor at 100 mL volume in incubation solu-
tion. Error was ꢂ10%, based on multiple experiments.
(b) For metallo enzymes: Assay involved 4 min incubation
of a solution of inhibitor and enzyme in 970 mL volume,
followed by addition of a concd soln of nitrocefin to pro-
duce 1 mL final volume.
16. Golemi, S.; Maveyraud, L.; Ishiwata, A.; Tranier, S.;
Miyashita, K.; Nagase, T.; Massova, I.; Mourey, L.;
Samama, J.-P.; Mobashery, S. J. Antibiot. 2000, 53,
1022.
17. Maveyraud, L.; Massova, I.; Birck, C.; Miyashita, K.;
Miyashita, K.; Samama, J. P.; Mobashery, S. J. Am.
Chem. Soc. 1996, 118, 7435.