N-Heterocyclic dicarboxylic acids: Broad-spectrum inhibitors of metallo-β-lactamases with co-antibacterial effect against antibiotic-resistant bacteria

Article abstract of DOI:10.1016/j.bmcl.2012.06.074

In an effort to identify novel, broad-spectrum inhibitors against the metallo-β-lactamases (MβLs), several N-heterocyclic derivatives were tested as inhibitors of MβLs CcrA, ImiS, and L1, which are representative enzymes from the distinct MβL subclasses. Three N-heterocyclic dicarboxylic acid derivatives were competitive inhibitors of CcrA and L1, exhibiting K _i values ≤2 μM, while only 2,4-thiazolidinedicarboxylic acid (1b) was a competitive inhibitor of ImiS. Two 2-mercapto-1,3,4-thiadiazole derivatives were noncompetitive inhibitors of CcrA and ImiS, exhibiting K _i values <7 μM; however, these same compounds did not inhibit L1. Two 2-mercapto-1,3,4-triazole derivatives were shown not to inhibit any of the tested MβLs. The N-heterocyclic derivatives were tested for antibacterial activity by examining the MIC values for existing antibiotics in the presence/absence of these derivatives. Consistent with the steady-state inhibition data, the inclusion of three N-heterocyclic dicarboxylic acid derivatives resulted in lower MIC values when using Escherichia coli BL21(DE3) cells containing the CcrA or L1 plasmids or Klebsiella pneumoniae (ATCC 700603), while 1b was the only dicarboxylic acid derivative to lower the MIC value of E. coli cells containing the ImiS plasmid. Inclusion of the 2-mercapto-1,3,4- thiadiazole derivatives resulted in lower MIC values for E. coli cells containing ImiS or L1 plasmids; however, these derivatives did not alter the MIC values for K. pneumoniae or E. coli cells containing the L1 plasmid. None of the N-heterocyclic derivatives affected the MIC of two methicillin resistant Staphylococcus aureus (MRSA) strains. Taken together, these studies demonstrate that N-heterocyclic dicarboxylic acids 1a-c and pyridylmercaptothiadiazoles 2a,b are good scaffolds for future broad-spectrum inhibitors of the MβLs.

Full text of DOI:10.1016/j.bmcl.2012.06.074

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5185–5189
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Heterocyclic dicarboxylic acids: Broad-spectrum inhibitors of
metallo-b-lactamases with co-antibacterial effect against antibiotic-resistant
bacteria
Lei Feng a, Ke-Wu Yang a, , Li-Sheng Zhou a, Jian-Min Xiao a, Xia Yang a, Le Zhai a, Yi-Lin Zhang a,
⇑
Michael W. Crowder b,
⇑
a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069,PR
China
b Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to identify novel, broad-spectrum inhibitors against the metallo-b-lactamases (MbLs), several
N-heterocyclic derivatives were tested as inhibitors of MbLs CcrA, ImiS, and L1, which are representative
enzymes from the distinct MbL subclasses. Three N-heterocyclic dicarboxylic acid derivatives were
Received 17 May 2012
Revised 20 June 2012
Accepted 25 June 2012
Available online 1 July 2012
competitive inhibitors of CcrA and L1, exhibiting Ki values 62
acid (1b) was a competitive inhibitor of ImiS. Two 2-mercapto-1,3,4-thiadiazole derivatives were non-
competitive inhibitors of CcrA and ImiS, exhibiting Ki values <7 M; however, these same compounds
lM, while only 2,4-thiazolidinedicarboxylic
l
Keywords:
did not inhibit L1. Two 2-mercapto-1,3,4-triazole derivatives were shown not to inhibit any of the tested
MbLs. The N-heterocyclic derivatives were tested for antibacterial activity by examining the MIC values
for existing antibiotics in the presence/absence of these derivatives. Consistent with the steady-state
inhibition data, the inclusion of three N-heterocyclic dicarboxylic acid derivatives resulted in lower
MIC values when using Escherichia coli BL21(DE3) cells containing the CcrA or L1 plasmids or Klebsiella
pneumoniae (ATCC 700603), while 1b was the only dicarboxylic acid derivative to lower the MIC value
of E. coli cells containing the ImiS plasmid. Inclusion of the 2-mercapto-1,3,4-thiadiazole derivatives
resulted in lower MIC values for E. coli cells containing ImiS or L1 plasmids; however, these derivatives
did not alter the MIC values for K. pneumoniae or E. coli cells containing the L1 plasmid. None of the
N-heterocyclic derivatives affected the MIC of two methicillin resistant Staphylococcus aureus (MRSA)
strains. Taken together, these studies demonstrate that N-heterocyclic dicarboxylic acids 1a–c and pyr-
idylmercaptothiadiazoles 2a,b are good scaffolds for future broad-spectrum inhibitors of the MbLs.
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibitors
Metallo-b-lactamases
Antibiotic resistance
b-Lactam-containing antibiotics are the most widely used clin-
ical agents for the treatment of bacterial infections.1 However,
overuse of antibiotics in the clinical setting has resulted in a large
number of bacteria that produce metallo-b-lactamases (MbLs), and
the resulting bacteria are resistant to the most commonly-used
antibiotics including penicillins, cephalosporins and carbapen-
ems.2,3 These antibiotic resistant bacteria exhibit their activity by
using MbLs to hydrolyze the b-lactam ring of b-lactam-containing
antibiotics. The b-lactamases are divided into class A, B, C, and D.4
The B class enzymes, called the MbLs, are Zn(II)-dependent and
hydrolyze nearly all known b-lactam-containing antibiotics. There
are no known clinical inhibitors of the MbLs.1 The MbLs are further
divided into B1, B2, and B3 subclasses.4 Both B1 and B3 subclasses
have a broad-spectrum substrate profile including penicillins,
cephalosphorins and carbapenems. In contrast, the B2 subclass
enzymes have a narrow substrate profile including carbapenems.
Given the enormous biomedical importance of MbLs, there has
been a large amount of effort in identifying novel inhibitors of
these enzymes.1,5 Succinic acid derivatives were shown to be
inhibitors of IMP-1 with IC50 values in the nanomolar range.6
Lienard et al. reported that D
-captopril inhibits L1 and CcrA,7 and
Abbreviations: IPTG, isopropyl-b-thiogalactopyranoside; LB, Luria-Bertani; MH,
Mueller Hinton; MIC, minimum inhibitory concentration; MRSA, methicillin-
resistant Staphylococcus aureus.
thiomandelic acid was tested as an inhibitor for nine different
MbLs.8 Picolinic acid derivatives were shown to inhibit CphA with
Ki values in the micromolar range,9 while hydroxamic derivatives
inhibit Fez-1 but not L1, IMP-1, BcII, or CphA.7 A propionic acid-
based inhibitor of IMP-1 was reported that exhibited covalent,
⇑
Corresponding authors. Tel./fax: +86 29 8830 2429 (K.-W.Y.); tel./fax: +1 513
529 7274 (M.W.C.).
E-mail addresses: kwyang@nwu.edu.cn (K.-W. Yang), crowdemw@muohio.edu
(M.W. Crowder).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.074

5186
L. Feng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5185–5189
O
O
O
H
N
H
O
O
H
N
O
N
HO
HO
HO
OH
OH
OH
S
O
1a
1c
1b
2,4-Oxazolidinedicarboxylic acid 2,4-Thiazolidinedicarboxylic acid 2,5-Pyrrolidinedicarboxylic acid
N
N
N
N
SH
SH
N
S
S
N
2a
2b
5-(2-pyridyl)-2-mercapto-1,3,4-thiadiazole
5-(4-pyridyl)-2-mercapto-1,3,4-thiadiazole
N
N
N
N
SH
SH
N
N
N
H
H
N
3a
5-(2-pyridyl)-2-mercapto-1,3,4-triazole
3b
5-(4-pyridyl)-2-mercapto-1,3,4-triazole
Figure 1. Structures of the synthesized N-heterocyclic derivatives.
irreversible inhibition.10 Penicillin-based inhibitors have been
shown to be micromolar inhibitors of L1, BcII, and Bla2,11 and some
N-arylsulfonyl hydrazones are inhibitors of IMP-1.5 A series of
pyrole-based inhibitors of IMP-1 have recently been reported.12
However, most of the inhibition reports have involved studies on
one or two of the MbLs, and to the best of our knowledge, only
three classes of inhibitors, thiomandelic acid,8 thiols,7 and
mercaptophosphonate compounds,13 have been reported to be
broad-spectrum inhibitors of the MbLs. Our goal is to develop
broad-spectrum inhibitors of MbLs and to use these inhibitors as
drug/inhibitor combinations to combat bacterial infections in
which the bacteria produces a MbL.
Towards this goal, N-heterocyclic dicarboxylic acid derivatives
2,4-oxazolidinedicarboxylic acid (1a), 2,4-thiazolidinedicarboxylic
acid (1b), 2,5-pyrrolidinedicarboxylic acid (1c), and thiol com-
pounds 5-(2-pyridyl)-2-mercapto-1,3,4-thiadiazole (2a), 5-(4-pyri-
dyl)-2-mercapto-1,3,4-thiadiazole (2b), 5-(2-pyridyl)-2-mercapto-
1, 3,4-triazole (3a) and 5-(4-pyridyl)-2-mercapto-1,3,4-triazole
(3b) were synthesized and characterized (Fig. 1).14–17 These com-
pounds were tested as inhibitors against purified MbLs CcrA, ImiS,
and L1, which are representative enzymes belonging to the B1, B2,
and B3 subclasses of b-lactamases, respectively.4 Furthermore,
in vitro antimicrobial activities of these inhibitors in combination
with existing antibiotics against antibiotic resistant strains were
evaluated.
O
O
O
O
O
NaOH, H2O
Cbz-Cl
H
HO
OH
OH
HO
OH
N
Cbz
O
NH2.HCl
6
4
5
AcOK, H2O
O
Pd-C, H2
O
HS
OH
OH
O
NH2.HCl
H
N
O
O
O
7
HO
OH
OH
S
N
H
1b
1a
Scheme 1. Synthesis of 2,4-oxazolidinedicarboxylic acid (1a) and 2,4-thiazolidin-
edicarboxylic acid (1b).
protecting group benzylamine was removed with Pd-C/H2 to afford
the methyl ester 11, which was hydrolyzed to give 2,5-pyrrolidin-
edicarboxylic acid 1c.
5-Substituted-2-mercapto-1,3,4-thiadiazoles
2 and 5-substi-
tuted-2-mercapto-1,3,4-thiadiazoles 3 were synthesized as shown
in Scheme 3, as previously described.19,20 Carboxylic acid 12 was
esterified to afford 13. The pyridinyl hydrazides 14 were obtained
by reaction of the esters 13 with hydrazine in ethanol. Treatment
of 14 with CS2 and KSCN gave the corresponding compounds 15
and 16, which were treated with cold concentrated sulfuric acid
and 2 N NaOH, respectively, to provide the target compounds
2a,b and 3a,b.
To test whether compounds 1a–c, 2a,b, and 3a,b are broad-
spectrum inhibitors of the MbLs, we tested their inhibition proper-
ties against a representative enzyme from each of the distinct MbL
subclasses: CcrA (B1), ImiS (B2), and L1 (B3). The data shown in
Table 1 summarize Ki values and modes of inhibition for the
compounds.21
2,4-Oxazolidinedicarboxylic acid 1a was prepared using the
method shown in Scheme 1. A mixture of the glyoxylic acid and
L-serine in 2 N NaOH was allowed to react overnight at 0 °C. Cbz-
Cl was added dropwise at 0 °C, and the mixture was stirred for
12 h, diluted with water, and extracted with ether to offer com-
pound 6. The protecting group Cbz was removed with Pd-C/H2
and the mixture was filtered and purified by ion exchange chroma-
tography to afford compound 1a.
2,4-Thiazolidinedicarboxylic acid 1b was prepared using a pre-
viously described method as shown in Scheme 1.4 A mixture of gly-
oxylic acid, L-cysteine, and potassium acetate in water/ethanol was
stirred for 1 h at room temperature and then stirred for 1 h at
90 °C. After cooling, the precipitate was collected and recrystallized
from H2O–ethanol to offer the pure 1b. 2,5-Pyrrolidinedicarboxylic
acid 1c was synthesized as shown in Scheme 2.18 Adipic acid 8 was
brominated to afford dibromodimethyladipate 9, which was
converted to intermediate 10 by benzylamine treatment. The
The 2,4-oxazolidinedicarboxylic acid 1a is a competitive inhib-
itor, exhibiting Ki values <2 lM, against CcrA and L1, which contain
two Zn(II) ions;3 however, there was no inhibition observed when
using ImiS, which contains a single Zn(II) ion.22 The 2,4-thiazolid-
inedicarboxylic acid 1b is a competitive inhibitor of all three MbLs
tested, with the greatest inhibition against the dinuclear Zn(II)-

L. Feng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5185–5189
5187
O
O
O
Br
benzylamine
1,SOCl2;
2,Br2
K2CO3,CH3CN
O
O
COOH
COOH
COOMe
COOMe
N
3,CH3OH
reflux
Br
10
8
9
NaOH,
CH3OH/H2O
O
O
O
O
Pd-C,H2
O
HO
OH
N
N
H
H
1c
11
Scheme 2. Synthesis of 2,5-pyrrolidinedicarboxylic acid (1c).
O
O
O
NH2NH2, H2O
reflux
CH3OH, H2SO4
reflux
R
NHNH2
R
R
O
OH
12
14
13
S
H
N
H2SO4, 0-5ć
S
CS2
HS
R1
R
N
SK
H
N
N
KOH,CH3CH2OH
O
O
15
2
S
H
N
2N NaOH
reflux
H
N
KSCN, HCl
HS
R2
R
N
H
NH2
N
N
3
16
1
2
R1= R2=
2a, 3a:
R = R =
2b, 3b:
N
N
Scheme 3. Synthesis of 5-substituted-2-mercapto-1,3,4-thiadiazoles (2a,b)–triazoles (3a,b).
Table 1
Inhibition constant Ki values (
against metallo-b-lactamase L1, CcrA, and ImiS
results were recently reported by Faridoon et al. using IMP-1 and
a series of 3-mercapto-1,2,4-triazoles.24
l
M) and inhibition mode of the synthesized compounds
The in vitro antibacterial activities of the compounds 1a–c,
2a,b, and 3a,b were investigated by determining the minimum
inhibitory concentrations (MIC) of existing antibiotics in the
presence and absence of 1a–c, 2a,b, and 3a,b.20,25 Six antibiotic-
resistant bacterial strains (Klebsiella pneumoniae (ATCC 700603),
Escherichia coli BL21(DE3) containing the pMSZ02-CcrA, pET-26b-
ImiS and pET26b-L1 plasmids, MRSA1, and MRSA2 were used to as-
sess these inhibitors (Table 2). While this strain of K. pneumonia
does not produce a metallo-b-lactamase, it is known to produce ex-
tended spectrum b-lactamases (ESBL’s);26 therefore, the use of this
strain in the MIC experiments allowed for us to evaluate the
biological activity of the N-heterocyclic derivatives toward other
b-lactamases. Similarly, MRSA1 and MRSA2 do not produce metal-
lo-b-lactamases; however, their use in the MIC experiments
allowed for us to evaluate the biological activity of the N-heterocy-
clic derivatives towards these clinically-imporant strains.
Compound
CcrA (B1)
ImiS (B2)
L1 (B3)
1a
1b
1c
2a
2b
3a
3b
1.1 0.1/C
0.64 0.09/C
0.73 0.06/C
5.4 0.3/NC
5.9 0.3/NC
NI
NI
1.9 0.2/C
1.8 0.2/C
0.69 0.03/C
NI
NI
NI
NI
7.1 0.7/C
NI
3.5 0.2/NC
6.8 0.4/NC
NI
NI
NI
NI: No inhibition, C: competitive inhibition, NC: noncompetitive inhibition.
containing CcrA and L1. Like with 1a, the 2,5-pyrrolidinedicarboxy-
lic acid 1c is a competitive inhibitor of CcrA and L1, exhibiting Ki
values <1
l
M; however, this compound exhibited no inhibition of
ImiS at concentrations up to 50
lM.
Steady-state kinetic studies indicated that both pyridylmerca-
ptothiadiazoles 2a,b are noncompetitive inhibitors of CcrA (Ki
<7 lM) and ImiS (Ki values of 3.5 and 6.8 lM, respectively). It is
not clear why 2a,b did not inhibit L1, although it is possible that
the thiol group on 2a,b (and 3a,b) may have reduced the disulfide
in L1, and the inhibitors did not bind to the active site.23 The
related pyridylmercaptotriazoles 3a,b exhibited no inhibitory
activity against any of the MbLs tested, even at concentrations up
MIC data indicate that the inhibitor 1a and 1c increase the anti-
microbial effect of antibiotics on Gram-negative K. pneumoniae K6
(ATCC 700603) and E. coli BL21(DE3) containing the CcrA and L1
plasmids; however, these compounds showed no effect on Gram-
positive MRSA1 and MRSA2 or on E. coli BL21(DE3) containing
the ImiS plasmid (Table 2). The N-heterocylic compounds 1a–c
apparently affect the activity of the ESBL’s in K. pneumoniae. The
results on MRSA strains are not surprising since neither strain is
thought to express a MbL.4 Inhibitor 1b lowered the MIC values
of b-lactam containing antibiotics on all tested Gram-negative
to 50
lM, suggesting that the sulfur in the azole ring may play
an important role in binding of these compounds (2a,b). Similar

5188
L. Feng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5185–5189
Table 2
Antibacterial activities (MIC,
lg/ml) of the inhibitors 1a, 1b, 1c, 2a, 2b, 3a, and 3b
Compound
E. coli-CcrAa
E. coli-ImiSb
E. coli-L1a
ATCC 700603a clinical
MRSA1 clinical
cefazolin vancomycin
MRSA2 clinical
cefazolin vancomycin
Inhibitor blank
2
1
1
0.25
1
1
2
2
4
4
0.5
4
2
2
4
4
2
1
1
64
16
16
16
64
64
64
64
>128
>128
>128
>128
>128
>128
>128
>128
2
2
2
2
2
2
2
2
>128
>128
>128
>128
>128
>128
>128
>128
4
4
4
4
4
4
4
4
1a
1b
1c
2a
2b
3a
3b
0.125
2
2
2
2
The used antibiotic was cefazolin (a) and imipenem (b), respectively
11. Buynak, J. D.; Chen, H.; Vogeti, L.; Gadhachanda, V. R.; Buchanan, C. A.; Palzkill,
T.; Shaw, R. W.; Spencer, J.; Walsh, T. R. Bioorg. Med. Chem. Lett. 2004, 14, 1299.
12. Mohamed, M. S.; Hussein, W. M.; McGeary, R. P.; Vella, P.; Schenk, G.; Abd El-
Hameed, R. H. Eur. J. Med. Chem. 2011, 46, 6075.
13. Lassaux, P.; Hamel, M.; Gulea, M.; Delbruck, H.; Mercuri, P. S.; Horsfall, L.;
Dehareng, D.; Kupper, M.; Frere, J. M.; Hoffmann, K.; Galleni, M.; Bebrone, C. J.
Med. Chem. 2010, 53, 4862.
14. General information. All purchased chemicals and reagents are of highest
commercially-available grade, and solvents were purified and dried before use.
Column chromatography was used for routine purification of the products, and
the column output was monitored with analytical thin-layer chromatography
(TLC). Melting points were measured on a Yanaco melting point apparatus. 1H
NMR spectra were recorded on a VARIAN INOVA 400 MHz spectrometer in
CDCl3, DMSO-d6, or D2O using tetramethylsilane as an internal reference. MbL
L1 from S. maltophilia, ImiS from Aeromonas veronii bv. sobria and CcrA from
Bacteroides fragilis were over-expressed and purified as previously
described.22,27,28 All kinetic studies were conducted on an Agilent 8453 UV–
Vis diode array spectrophotometer at 25 °C. The buffer used in kinetic studies
was 50 mM Tris, pH 7.0, and the substrate was imipenem (for ImiS) and
cefazolin (for L1 and CcrA). Working stock solutions of imipenem and cefazolin
were prepared by dissolving them in 10 ml of 50 mM Tris, pH 7.0, and filtering
through a 0.45-mm syringe filter to remove any undissolved particulates.
15. Experimental procedure for the preparation of N-heterocyclic dicarboxylic acids
bacteria, and the largest effect was observed with E. coli BL21(DE3)
containing the ImiS plasmid. It is not clear why the MIC value for
E. coli containing the ImiS plasmid was so low given the higher Ki
value of 1b, as compared to 2a and 2b, when used in inhibition
studies with ImiS. The lower MIC value may be due to the different
mode of inhibition (competitive vs noncompetitive). Experiments
to obtain a crystal structure of ImiS bound to 1b and 2a/2b are
underway.
Inclusion of compounds 2a,b resulted in a 50% reduction in MIC
for E. coli BL21(DE3) containing the CcrA and ImiS plasmids, but
none of the other strains were affected by 2a,b. In agreement with
the steady-state inhibition studies, the inclusion of compounds
3a,b did not lower the MIC values for any of the tested strains.
The MIC data nicely correlate with the steady-state inhibition data
that are shown in Table 1 as those compounds that exhibited
inhibition of the MbLs also showed increased antibiotic activity
in the bacterial strains.
Taken together, these studies demonstrate that N-heterocyclic
dicarboxylic acids 1a,c and pyridylmercaptothiadiazoles 2a,b are
good scaffolds for future broad-spectrum inhibitors of the MbLs.
These compounds, once optimized using structure/function stud-
ies, could be given in combination with existing b-lactam contain-
ing antibiotics as a treatment for infections caused by antibiotic
resistant bacteria.
(1a–c): NaOH (2 N, 5 ml) and glyoxylic acid (0.74 g, 10 mmol) were added to L-
serine (1.05 g, 10 mmol) in a round-bottom flask at 0 °C, and the solution was
stirred at 0 °C for 7 h. To this solution was added NaOH solution (0.5 g in 1.4 ml
H2O), and the resulting solution was stirred for 15 min. Then, benzyl
chloroformate (12 mmol) was added at room temperature, and the mixture
was stirred for 3 h, diluted with water, and washed with ether. HCl (20%) was
added to the resulting aqueous layer, which was then extracted with ethyl
acetate. The organic layer was dried, and the solvent was removed to give pure
6.
A solution of compound 6 (2 g, 6.8 mmol) in MeOH (20 ml) was
hydrogenated in presence of 10% Pd-C (0.25 g) for 24 h, the catalyst was
removed by filtration through celite, and the pad was washed with MeOH
(5 Â 10 ml). The solvent was removed under vacuo to offer pure 2,4-
oxazolidinecarboxylic acid (1a) as white solid. Yield 0.9 g (77%). 1H NMR
(400 MHz, D2O): 3.75 (t, J = 8.8 Hz, 1H), 3.84–4.14(m, 2H), 5.30(s, 1H). 2,4-
Thiazolidinedicarboxylic acid (1b) was prepared as previously described.29 The
recrystallized 1b was obtained as a white solid, mp 180 °C. Yield 93%, 1H NMR
(400 MHz, DMSO-d6) d: 2.73 (t, J = 9.8 Hz, 1H), 3.30 (dd, J = 10.1 Hz, 6.4 Hz, 6H),
3.82 (dd, J = 9.5 Hz, 6.4 Hz, 1H), 4.89 (s, 1H). Dimethyl 2,5-dibromohexane-1,6-
dioate 9 was synthesized as previously described: 1H NMR (CDCl3): d 2.00–2.05
(m, 2H), 2.28–2.35 (m, 2H), 3.80 (s, 6H), 4.24–4.26 (t, 2H).18 A mixture of
compound 9 (3.33 g, 10 mmol), benzylamine (1.12 ml, 10 mmol), potassium
carbonate (1.67 g, 12 mmol), toluene (7 ml), and water (3 ml) was refluxed for
24 h and cooled, the resulting organic layer was separated, and the aqueous
layer was extracted with hexane (2 Â 100 ml). The organic layer was combined
and washed with brine (2 Â 100 ml) and dried over Na2SO4. Evaporation of
solvent afforded dimethyl-1-benzylpyrrolidine-2,5-dicarboxylate 10: Yield,
2.54 g (87%). 1H NMR (CDCl3): d 2.02–2.07 (m, 4H), 3.41–3.45 (t, 2H), 3.56 (s,
6H), 3.91 (s, 2H). A solution of 2 g of 10 in 20 ml absolute methanol was
hydrogenated under 20 atm of H2 in the presence of 0.2 g of 10% Pd-C. The
reaction was complete in about 2 h. The catalyst was removed by filtration, and
the methanol was evaporated under reduced pressure. The oily residue was
distilled to afford 11 with yield of 94%. To a solution of NaOH and MeOH was
added 1 g compound 11, and the solution was stirred for 24 h at room
temperature. The reaction mixture was washed with ether, the aqueous layer
was passed through a cation exchange resin, and product was lyophilized to
give 2,5-pyrrolidinedicarboxylic acid (1c) as a white powder: Yield, 0.75 g
(88%). Mp 260–261 °C. 1H NMR (400 MHz, D2O): d 2.13 (m, 2H), 2.40 (m, 2H),
4.35 (t, J = 6.1 Hz, 2H).
Acknowledgements
This work was supported by grants (to K.W.Y.) from National
Natural Science Fund of China (20972127), Doctoral Fund of China
(200806970005), Natural Science Fund of Shaanxi Province (2009
JM2002) and Key Fund for International Cooperation of Shaanxi
Province (2010KW-16) and from the National Institutes of Health
(GM093987 to M.W.C.).
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7. Lienard, B. M. R.; Garau, G.; Horsfall, L.; Karsisiotis, A. I.; Damblon, C.; Lassaux,
P.; Papamicael, C.; Roberts, G. C. K.; Galleni, M.; Dideberg, O.; Frere, J. M.;
Schofield, C. J. Org. Biomol. Chem. 2008, 6, 2282.
8. Mollard, C.; Moali, C.; Papamicael, C.; Damblon, C.; Vessilier, S.; Amicosante, G.;
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45015.
16. Experimental procedure for the preparation of 5-substituted-2-mercapto-1,3,4-
thiadiazoles (2a,b). Compounds 2a,b were prepared as previously described:
2a: 1H NMR (400 MHz, DMSO-d6), d 7.58 (m, J = 4.9 Hz, 1H), 8.34 (m, 2H), 8.74
(m, J = 4.6, 1.2 Hz, 1H), 8.94 (d, J = 1.6 Hz, 1H). Compound 2b: 1H NMR
(400 MHz, DMSO-d6), d 7.91 (d, J = 5.8 Hz, 2H), 8.85 (d, J = 6.1 Hz, 2H).19
9. Horsfall, L. E.; Garau, G.; Lienard, B. M. R.; Dideberg, O.; Schofield, C. J.; Frere, J.
M.; Galleni, M. Antimicrob. Agents Chemother. 2007, 51, 2136.
10. Kurosaki, H.; Yamaguchi, Y.; Higashi, T.; Soga, K.; Matsueda, S.; Yumoto, H.;
Misumi, S.; Yamagata, Y.; Arakawa, Y.; Goto, M. Angew. Chem., Int. Ed. 2005, 44,
3861.

L. Feng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5185–5189
5189
17. Experimental procedure for the preparation of 5-substituted-2-mercapto-1,3,4-
triazoles (3a,b). Compounds 3a,b were prepared as previously described: 3a: 1
(ATCC 700603), MRSA1, and MRSA2 on LB agar plates were transferred to 5 ml
of MH liquid medium and were grown at 37 °C overnight. The bacterial cells
were collected by centrifugation (4000 rpm for 10 min). The supernatant was
discarded, and the remaining cells were re-suspended in MH medium and
diluted to an O.D600 of 0.5. MIC values were determined by using the Clinical
and Laboratory Standards Institute (CLSI) macrodilution (tube) broth
H
NMR (400 MHz, DMSO-d6), d 8.21 (m, 1H), 8.35 (m, 1H), 8.75 (m, 1H), 9.14 (m,
1H). Compound 3b: 1H NMR (400 MHz, DMSO-d6), d 8.31 (m, 2H), 9.09 (m,
2H).20
18. Ozasa, N.; Tokioka, K.; Yamamoto, Y. Biosci. Biotech. Biochem. 1905, 1995, 59.
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method.30 Antibiotics were dissolved in ddH2O to prepare 1280
l
g/ml stock
solutions and the compounds 1a, 1b, 1c, 2a, 2b, 3a and 3b were dissolved in dd
H2O respectively to prepare 320 g/ml stock solutions. MH solution (0.9 ml)
l
21. Inhibition studies. Solutions of compounds 1a–c were directly prepared in
50 mM.
and 0.1 ml inhibitor stock solution were added to a series of sterile test tubes,
with an additional 0.5 ml MH solution and 0.1 ml inhibitor stock solution
Tris, pH 7.0, while the solutions of 2a,b and 3a,b were dissolved in a small
volume of DMSO and then diluted with 50 mM Tris, pH 7.0. The inhibition
studies were conducted at 25 °C using imipenem as substrate of ImiS and
cefazolin as substrate of L1 and CcrA. The substrate concentrations were varied
added to the first tube. Antibiotic stock solution (400 lL of 1280 lg/ml) was
added to the first test tube, and a series of twofold dilutions were prepared by
transferring 1 ml to successive tubes. Cultures (5 ml) of six bacterial strains:
E. coli BL21(DE3) containing the pMSZ02-CcrA, pET-26b-ImiS and pET26b-L1
plasmids, and K. pneumoniae (ATCC 700603), MRSA1, and MRSA2 were grown
to an OD600 of 0.5. The concentration of bacterial strains was diluted to 106
colony forming units (CFU) per ml with MH medium, and 1 ml bacterial
solution was added to each tube containing different concentration of
antibiotic. The final concentration of bacterial strains was 5 Â 105 CFU per
ml. These cultures were incubated at 37 °C for 24 h. Each measurement was
performed in duplicate.
between 40 and 120
lM, and inhibitor concentrations were varied between 0
and 4 M. The enzyme and inhibitor were preincubated for 30 min before
l
starting the kinetic experiments. The mode of inhibition was confirmed by
generating Lineweaver–Burk plots and Ki values were determined as
previously described.27
22. Crawford, P. A.; Sharma, N.; Chandrasekar, S.; Sigdel, T.; Walsh, T. R.; Spencer,
J.; Crowder, M. W. Protein Expr. Purif. 2004, 36, 272.
23. Crowder, M. W.; Yang, K. W.; Carenbauer, A. L.; Periyannan, G.; Seifert, M. A.;
Rude, N. E.; Walsh, T. R. J. Biol. Inorg. Chem. 2001, 6, 91.
24. Faridoon; Hussein, W. M.; Vella, P.; Islam, N. U.; Ollis, D. L.; Schenk, G.;
McGeary, R. P. Bioorg. Med. Chem. Lett. 2012, 22, 380.
25. Determination of MIC values. Single colonies of E. coli BL21(DE3) containing the
pMSZ02-CcrA, pET-26b-ImiS and pET26b-L1 plasmids and of K. pneumoniae
26. Rasheed, J. K.; Anderson, G. J. Antimicrob. Agents Chemother. 2000, 44, 2382.
27. Crowder, M. W.; Walsh, T. R.; Banovic, L.; Pettit, M.; Spencer, J. Antimicrob.
Agents Chemother. 1998, 42, 921.
28. Yanchak, M. P.; Taylor, R. A.; Crowder, M. W. Biochemistry 2000, 39, 11330.
29. Pellegrini, N.; Refouvelet, B.; Blacque, O. Chem. Pharm. Bull. 1999, 47, 950.
30. MO7-A8, C. Clinical and Laboratory Standards Institute 2009, 29, 1.