2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-β-lactamases

Article abstract of DOI:10.1016/j.bmcl.2012.08.012

Bacterial resistance to β-lactam antibiotics caused by class B metallo-β-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.

Full text of DOI:10.1016/j.bmcl.2012.08.012

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6229–6232
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors
of metallo-b-lactamases
Pinhong Chen a, Lori B. Horton b, Rose L. Mikulski a, Lisheng Deng a, Sandeep Sundriyal a,
Timothy Palzkill a,b, Yongcheng Song a,
⇑
a Department of Pharmacology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, United States
b Department of Molecular Virology and Microbiology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacterial resistance to b-lactam antibiotics caused by class B metallo-b-lactamases (MBL), especially for
certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We
report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure
activity relationship (SAR) and molecular modeling studies were performed and implications for further
inhibitor design are discussed.
Received 22 May 2012
Revised 24 July 2012
Accepted 1 August 2012
Available online 10 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Metallo-b-lactamase
Inhibitor
Rational design
Molecular modeling
The development of b-lactam antibiotics over the past 60 years
has led to the availability of drugs to treat a wide range of Gram-
positive and Gram-negative bacterial infections. However, during
the past few decades, populations of drug resistant bacteria have
increased significantly.1 One important mechanism bacteria use
to resist b-lactam antibiotics is the production of b-lactamases,
which can hydrolyze the 4-membered b-lactam ring and render
the drugs inactive. There are two mechanistically distinct types
of b-lactamases: serine b-lactamases and metallo-b-lactamases
(MBLs).2 The class A, C and D b-lactamases belong to the first type
and use a serine –OH group as a nucleophile to attack and hydro-
lyze the amide bond in the b-lactam ring. In contrast, the class B
b-lactamases are Zn2+ dependent metalloenzymes, with one or
two Zn ions at the active site.3,4 MBLs are able to hydrolyze essen-
tially all b-lactams, including imipenem, which have the widest
antibacterial spectrum and are one of the very few effective drugs
to treat some Gram-negative bacterial infections, such as Pseudo-
monas aeruginosa. The clinical significance of these enzymes has
only recently been recognized, when plasmid-encoded, transfer-
able MBLs were found to spread quickly among many species of
Gram-negative bacteria around the world and confer resistance
to imipenem and extended-spectrum cephalosporins.5–7
identified, such as thiol-containing compounds,9,10 trifluoromethyl
ketones,11 tetrazoles,12 and succinates.13 However, none of these
compounds have been used in clinical trials to sensitize b-lactam
antibiotics due to chemical instability, a narrow spectrum of activ-
ity or side effects. For example, captopril (Fig. 1) was discovered to
be very active against IMP-1 from P. aeruginosa, one of the most
prevalent forms of transferable MBLs.5–7,14 It is also used in the
clinic to treat hypertension as a potent inhibitor of angiotensin-
converting enzyme. There is, therefore, a pressing need to find
potent, drug-like inhibitors that have a new scaffold for further
development. Here, we report the discovery and structure activity
relationships (SAR) of 2-substituted 4,5-dihydrothiazole-4-carbox-
ylic acids as a new class of MBL inhibitors.
N
N
N
N
HS
O
S
OPh
O
H
N
H
N
N
COOH
SH
O
COOH
O
CF3
captopril
MCI
There has been much interest in discovering and developing
MBL inhibitors during the past decade.8 As representatively shown
in Figure 1, a number of structurally diverse inhibitors have been
N
N
N
NH
N
S
Bu
N
O
HOOC
COOH
⇑
Corresponding author. Tel.: +1 713 798 7415; fax: +1 713 798 3145.
E-mail address: ysong@bcm.edu (Y. Song).
Figure 1. Representative MBL inhibitors.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.012

6230
P. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6229–6232
tested for their inhibitory activities against recombinant IMP-
1,18,19 together with captopril as a positive control, which was
found to be a good inhibitor with an IC50 value of 5.0 M (Table
1), or a Ki value of 2.4 M. Another control is ethylenediaminetetra-
acetic acid (EDTA), a non-competitive, broad inhibitor of MBLs,
which acts as a metal chelator depleting Zn2+ from the active site
of the metalloenzyme. EDTA was found to inhibit the activity of
High-throughput screening (HTS) has been widely used to find
inhibitors of enzymes, including MBLs, in the pharmaceutical
industry. However, due to the high costs, HTS is often not available
in many academic institutes, where alternative methods have to be
used. For metalloenzymes, a coordination chemistry based ap-
proach has been successfully applied, by us15 and other groups,16,17
to discover new inhibitors. In order to find novel MBL inhibitors,
we synthesized or purchased the following compounds:
l
l
IMP-1 with an IC50 value of 27.9
lM. Compound 1, 2-benzylthiaz-
S
H
N
N
COOH
COOH
N
OH
N
O
O
COOH
COOH
N
1
OH
N
N
N
N
OH
OH
O
OH
O
N
OH
O
OH
OH
OH
O
H
N
N
COOH
OH
N
O
N
N
OH
O
N
OH
O
N
H
O
OH
O
O
P
O
P
O
OH
OH
OH
OH
OH
P
HO
O
N
P
N
OH
HO
OH
COOH
Each of these compounds features a different potential Zn2+-binding
group. In addition, they also contain a hydrophobic group, such as a
phenyl or benzyl, that could potentially extend to the lipophilic
pocket of MBLs to enhance the binding. These compounds were
ole-4-carboxlic acid was identified to be a novel IMP-1 inhibitor
having an IC50 of 34.7 M.
l
Since compound 1 potentially represents a new class of MBL
inhibitors, we performed a medicinal chemistry study in an effort
to find compounds with improved activity as well as structure
activity relationships (SAR). First, compounds 2–7 (Fig. 2) were
synthesized to examine the effects of related core structures as
well as those of phenyl versus benzyl at the 2-position. The inhib-
itory activities of these compounds against IMP-1 are listed in Ta-
ble 1. For the fully aromatic thiazole ring, 1 with a 2-benzyl is more
S
S
S
S
N
N
N
N
COOH
COOH
COOH
COOH
S
2
3
4
1
Table 2
Structures and activities of compounds 8–17
S
R
N
S
S
HN
6
COOH
IC50 for IMP-1
HN
N
R-
IC50 for Bla2
COOH
COOH
COOH
8
9
10
11
12
13
4-Cl-phenyl
4-Br-phenyl
3-NH2-phenyl
3-acetamido-phenyl
3-BOCNH-phenyl
2-OH-phenyl
>200
>200
77.0
>200
>200
>200
>200
>200
4.9
>200
74.1
>200
5
7
Figure 2. Structures of compounds 1–7.
Table 1
H
N
MBL inhibitory activities (IC50 in
lM) of 1–7
14
>200
>200
Compound
IC50 for IMP-1
IC50 for Bla2
O
H
Captopril
5.0
25.8
MeO
N
EDTAa
27.9
34.7
>200
>200
5.5
>200
>200
>200
1060
>200
>200
>200
>200
>200
>200
>200
15
16
>200
>200
>200
>200
O
1
2
3
4
5
6
7
H
N
O
O
H
O
N
17
>200
>200
O
a
with a 12-hour incubation.

P. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6229–6232
6231
active than 2 with a phenyl (IC50 >200
l
M). However, for the par-
replacing 2-phenyl of 4 with an lipophilic amide or carbamate
group. These groups were designed to mimic the sidechain of pen-
icillin, one of the substrates of IMP-1. However, as can be seen in
Table 2, none of these compounds show activity against IMP-1.
Molecular modeling (docking) was used to rationalize the above
observed SARs. The crystal structure10 (PDB code: 1DD6) of IMP-1
in complex with the thiol inhibitor MCI (Fig. 1) was chosen to be
the docking template, which was prepared, according to our previ-
ous method,15,20,21 by removing only the inhibitor using the pro-
gram Glide22 in Schrödinger (version 2010).23 The two Zn2+ ions
were treated as an integrated part of the protein. Compound 4
was built, energy-minimized using OPLS_2005 force field in Schrö-
dinger, and docked into the prepared IMP-1 structure using Glide.
Figure 3A shows the 20 docking structures of 4 with the lowest
energies, which can be basically classified into two binding confor-
mations A and B. Conformation A represents 7 tightly clustered
docking structures with a lower average energy, and there are 13
docking poses in conformation B, as representatively shown in Fig-
ure 3B and C. The common feature of conformations A and B is that
one O atom of the carboxylate group binds to both Zn2+ ions,
behaving similarly as the S atom of MCI. The other O atom of 4
in conformation A also interacts with the tightly bound Zn1 (coor-
dinated by three imidazoles of His77, 79 and 139), while that of B
binds to the Zn2 (which is bound to IMP-1 via coordination by
Asp81, Cys158 and His197 with less affinity). None of the N atoms
of 4 in conformations A and B are predicted to bind to Zn2+, with
the distances being >3 Å. It is of interest that the binding of the
2-phenyl group of 4 in conformations A and B is different. The phe-
nyl of A is located almost in the same position as the phenyl of MCI,
and the mainly hydrophobic pocket is tightly surrounded by Val25,
Val31, Glu23 (the hydrophobic carbon skeleton), Ser21, Pro32,
Phe51 and His197 (Fig. 3b). The phenyl group of conformation B
is situated in a much larger and longer channel that holds the
two aromatic rings of MCI (Fig. 3c). Our SAR results suggest the
conformation A better mimics the real binding structure of com-
pound 4 to IMP-1, because the pocket shown in Fig. 3b could only
accommodate a phenyl group favorably. Any additional substitu-
ent, even as small as a –Cl or –OH, could result in an unfavored ste-
ric and/or hydrophobic interaction. In addition, our SAR and
docking studies could provide useful implications for future inhib-
itor design. For example, derivatives of compound 4 that possess
an additional group that extends to the other binding site of
IMP-1 could be more active.
tially saturated 4,5-dihydrothiazole ring, compound 3 with a 2-
benzyl group is inactive, while compound 4, (R)-2-phenyl-4,5-
dihydrothiazole-4-carboxylic acid, was found to be a good IMP-1
inhibitor with an IC50 value of 5.5
lM (or a Ki of 3.3
l
M), ꢀ6Â more
active than 1. However, its enantiomer 5, (S)-2-phenyl-4,5-dihy-
drothiazole-4-carboxylic acid, was found to be inactive. In addi-
tion, none of compounds 6 and 7 having a thiazolidine ring
possess inhibitory activity against IMP-1.
We next synthesized compounds 8–13 shown in Table 2 to
investigate the SAR for a substituent on the 2-phenyl ring of com-
pound 4. Compounds 8 and 9 with a 2-(4-chlorophenyl) and 2-(4-
bromophenyl), respectively, were found to completely lose the
inhibory activity. Compound 10 with a 3-NH2 substituent was ob-
served to have a weak inhibitory activity with an IC50 of 77.0 lM.
Its derivatives 11 and 12 containing an additional acetyl and tert-
butoxylcarbonyl (BOC) group, respectively, are inactive. Compound
13 with a 2-OH substituent on the phenyl ring has also no activity.
Compounds 14–17 (Table 2) were synthesized to find the effects of
Finally, we tested the inhibitory activities of newly synthesized
compounds against another MBL, Bacillus anthracis Bla2,24 in order
R
S
COOH
SH
i
+
RCN
N
H2N
COOH
3 - 5, 8 - 13
O
O
O
S
N
S
iv
R
iii
R
N
H
ii
H
N
H2N CN
R
N
H
CN
N
COOMe
COOLi
14 - 17
R
S
COOH
SH
v
+
RCHO
HN
H2N
COOH
6, 7
Figure 3. (A) Stereoview of the 20 lowest-energy docking structures of compound 4
in the active site of the crystal structure of IMP-1:MCI complex, with the protein
shown as an electrostatic potential surface and Zn2+ as a light blue sphere; (B) The
lowest-energy docking structure of conformation A of 4 (the ball and stick model in
green) in the active site, superimposed with the crystal structure of MCI (in orange);
(C) The lowest-energy docking structure of conformation B of 4 superimposed with
the structure of MCI.
Scheme 1. General synthesis for compounds 3–17. Reagents and conditions: (i)
NaHCO3, phosphate buffer (pH 6), MeOH, 66 °C, 72 h; (ii) RCOCl or (BOC)2O, Et3N,
CH2Cl2; (iii) L-Cys-OMe, Et3N, MeOH; (iv) 0.9 equiv 1 M LiOH, 0 °C to room
temperature, 1 h; (v) EtOH, H2O, room temperature, 18 h.

6232
P. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6229–6232
to see whether our inhibitors have a broad spectrum of activity. B.
anthracis is the causative agent of anthrax and expression of Bla2
can result in resistance to b-lactam antibiotics. Therefore, inhibi-
tors of this enzyme have potential therapeutic value. The results
are shown in Tables 1 and 2. Captopril was also found to be a weak
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.
012.
inhibitor of Bla2 with an IC50 value of 25.8
lM, or a Ki value of
17.9 M, while EDTA is almost inactive against this enzyme (IC50
l
References and notes
>1 mM). Although compound 4 is a good IMP-1 inhibitor, it was
observed to have essentially no activity against Bla2 with an IC50
1. Leeb, M. Nature 2004, 431, 892.
2. Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Chem. Rev. 2005, 105, 395.
3. Crowder, M. W.; Spencer, J.; Vila, A. J. Acc. Chem. Res. 2006, 39, 721.
4. Wang, Z.; Fast, W.; Valentine, A. M.; Benkovic, S. J. Curr. Opin. Chem. Biol. 1999,
3, 614.
5. Walsh, T. R.; Toleman, M. A.; Poirel, L.; Nordmann, P. Clin. Microbiol. Rev. 2005,
18, 306.
of >200
stituent showed improved inhibitory activity against Bla2 as com-
pared to captopril, with an IC50 value of 4.9 M (Table 2) or a Ki
value of 5.1 M. All other compounds, except for compound 12
being a weak inhibitor (IC50 = 74.1 M), are also inactive against
lM. However, analogous compound 10 with a 3-NH2 sub-
l
l
l
6. Bebrone, C. Biochem. Pharmacol. 2007, 74, 1686.
7. Walsh, T. R. Clin. Microbiol. Infect. 2005, 11, 2.
Bla2. Nevertheless, the activity of compound 10 shows that it is
also possible to develop 4,5-dihydrothiazole-4-carboxylic acids to
find a compound that have a broad activity against other MBLs.
General methods for synthesizing compounds 3–17 are illus-
trated in Scheme 1.25 For 4,5-dihydrothiazole compounds 3–5
and 8–13, an aromatic nitrile, 1.2 equiv amount of L-cysteine (or
D-cysteine) hydrochloride and NaHCO3 was refluxed in methanol
and phosphate buffer (pH 6) for 72 h,26 to give these compounds
in 40–70% yield. However, this method does not work for making
analogous compounds 14–17. Rather, the methyl esters of these
compounds were synthesized using a similar procedure,27 which
were carefully undergone a mild hydrolysis using 0.9 equiv of LiOH
to afford compounds 14–17 in 70–80% yield. Excess amount of
LiOH caused decomposition of the products. Thiazolidine com-
pounds 6 and 7 were prepared in ꢀ80% yield by reacting benzalde-
hyde or phenylacetaldehyde with L-cysteine at room temperature
in a mixture of ethanol and water.28
8. Toney, J. H.; Moloughney, J. G. Curr. Opin. Investig. Drugs 2004, 5, 823.
9. Garcia-Saez, I.; Hopkins, J.; Papamicael, C.; Franceschini, N.; Amicosante, G.;
Rossolini, G. M.; Galleni, M.; Frere, J. M.; Dideberg, O. J. Biol. Chem. 2003, 278,
23868.
10. Concha, N. O.; Janson, C. A.; Rowling, P.; Pearson, S.; Cheever, C. A.; Clarke, B. P.;
Lewis, C.; Galleni, M.; Frere, J. M.; Payne, D. J.; Bateson, J. H.; Abdel-Meguid, S. S.
Biochemistry 2000, 39, 4288.
11. Walter, M. W.; Felici, A.; Galleni, M.; Soto, R. P.; Adlington, R. M.; Baldwin, J. E.;
Frère, J.-M.; Gololobov, M.; Schofield, C. J. Bioorg. Med. Chem. Lett. 1996, 6, 2455.
12. Toney, J. H.; Fitzgerald, P. M.; 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.
13. Toney, J. H.; Hammond, G. G.; Fitzgerald, P. M.; Sharma, N.; Balkovec, J. M.;
Rouen, G. P.; Olson, S. H.; Hammond, M. L.; Greenlee, M. L.; Gao, Y. D. J. Biol.
Chem. 2001, 276, 31913.
14. Watanabe, M.; Iyobe, S.; Inoue, M.; Mitsuhashi, S. Antimicrob. Agents Chemother.
1991, 35, 147.
15. Deng, L.; Sundriyal, S.; Rubio, V.; Shi, Z.; Song, Y. J. Med. Chem. 2009, 52, 6539.
16. Jacobsen, F. E.; Lewis, J. A.; Cohen, S. M. J. Am. Chem. Soc. 2006, 128, 3156.
17. Jacobsen, F. E.; Lewis, J. A.; Cohen, S. M. ChemMedChem 2007, 2, 152.
18. Brown, N. G.; Horton, L. B.; Huang, W.; Vongpunsawad, S.; Palzkill, T.
Antimicrob. Agents Chemother. 2011, 55, 5696.
In summary, this work is of interest for several reasons. First,
using rational compound screening followed by medicinal chemis-
try, 2-phenyl-4,5-dihydrothiazole-4-carboxylic acid (4) was identi-
fied to be a novel inhibitor of P. aeruginosa MBL IMP-1 with an IC50
19. The enzyme assay was performed using 2 nM IMP-1 (or 10 nM Bla2) and
25
l
M nitrocefin in 50 mM HEPES buffer (pH 7.0) containing 20
lg/mL BSA and
0.01% triton in a final volume of 300
lL. For inhibition assay, compounds were
pre-incubated with the enzyme for 20 min at room temperature, before
initiation of the reaction by adding nitrocefin. The increasing absorbance at
482 nM was monitored using a Beckman DU640 UV spectrometer. The initial
velocities for different concentrations of an inhibitor were imported into Prism
(version 5.0, GraphPad, La Jolla, CA). The IC50 as well as Ki values were
calculated by using a standard dose response curve fitting in the software.
20. Deng, L.; Endo, K.; Kato, M.; Cheng, G.; Yajima, S.; Song, Y. ACS Med. Chem. Lett.
2011, 2, 165.
21. Deng, L.; Diao, J.; Chen, P.; Pujari, V.; Yao, Y.; Cheng, G.; Crick, D. C.; Prasad, B. V.
V.; Song, Y. J. Med. Chem. 2011, 54, 4721.
22. Glide, version 5.5, Schrödinger, LLC., New York, NY, 2010.
23. Schrödinger Suite, version 2010, Schrödinger, LLC, New York, NY, 2010.
24. Materon, I. C.; Queenan, A. M.; Koehler, T. M.; Bush, K.; Palzkill, T. Antimicrob.
Agents Chemother. 2003, 47, 2040.
25. Experimental Section can be found in Supplementary data on-line.
26. Lu, Y.; Li, C.; Wang, Z.; Chen, J.; Mohler, M. L.; Li, W.; Dalton, J. T.; Miller, D. D. J.
Med. Chem. 2011, 54, 4678.
27. Merino, P.; Tejero, T.; Unzurrunzaga, F. J.; Franco, S.; Chiacchio, U.; Saita, M. G.;
Iannazzo, D.; Piperno, A.; Romeo, G. Tetrahedron: Asymmetry 2005, 16, 3865.
28. Lu, Y.; Li, C.; Wang, Z.; Ross, C. R.; Chen, J.; Dalton, J. T.; Li, W.; Miller, D. D. J.
Med. Chem. 2009, 52, 1701.
value of 5.5 lM. Second, docking studies were explored to rational-
ize the structure activity relationships of this class of compounds
and provide implications for future inhibitor design. Third, 2-(3-
aminophenyl)-4,5-dihydrothiazole-4-carboxylic acid (10) was
identified to be an inhibitor of B. anthracis MBL Bla2
(IC50 = 4.9 lM), showing the promise of further developing this
class of compounds in the context of overcoming b-lactam
resistance.
Acknowledgments
This work was supported by a Grant (R21AI090190) from Na-
tional Institute of Allergy and Infectious Disease (NIAID/NIH) to
Y.S. and a Grant (R01AI32956) from NIAID/NIH to T.P., L.B.H. was
supported by a training Grant (T90DK070109) from NIH/NIDDK.