3-Mercapto-1,2,4-triazoles and N-acylated thiosemicarbazides as metallo-β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.10.116

The production of β-lactamases is an effective strategy by which pathogenic bacteria can develop resistance against β-lactam antibiotics. While inhibitors of serine-β-lactamases are widely used in combination therapy with β-lactam antibiotics, there are no clinically available inhibitors of metallo-β-lactamases (MBLs), and so there is a need for the development of such inhibitors. This work describes the optimisation of a lead inhibitor previously identified by fragment screening of a compound library. We also report that thiosemicarbazide intermediates in the syntheses of these compounds are also moderately potent inhibitors of the IMP-1 MBL from Pseudomonas aeruginosa. The interactions of these inhibitors with the active site of IMP-1 were examined using in silico methods.

Full text of DOI:10.1016/j.bmcl.2011.10.116

Bioorganic & Medicinal Chemistry Letters 22 (2012) 380–386
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Mercapto-1,2,4-triazoles and N-acylated thiosemicarbazides
as metallo-b-lactamase inhibitors
Faridoon a,b, Waleed M. Hussein a,c, Peter Vella a, Nazar Ul Islam b, David L. Ollis d, Gerhard Schenk a,e
,
Ross P. McGeary a,f,
⇑
a The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Qld 4072, Australia
b Institute of Chemical Sciences, University of Peshawar, Peshawar 25120, Pakistan
c Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, Egypt
d Australian National University, Research School of Chemistry, Canberra, Act 0200, Australia
e National University of Ireland-Maynooth, Department of Chemistry, Maynooth, Co. Kildare, Ireland
f The University of Queensland, School of Pharmacy, Brisbane, Qld 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The production of b-lactamases is an effective strategy by which pathogenic bacteria can develop resis-
tance against b-lactam antibiotics. While inhibitors of serine-b-lactamases are widely used in combina-
tion therapy with b-lactam antibiotics, there are no clinically available inhibitors of metallo-b-lactamases
(MBLs), and so there is a need for the development of such inhibitors. This work describes the optimisa-
tion of a lead inhibitor previously identified by fragment screening of a compound library. We also report
that thiosemicarbazide intermediates in the syntheses of these compounds are also moderately potent
inhibitors of the IMP-1 MBL from Pseudomonas aeruginosa. The interactions of these inhibitors with the
active site of IMP-1 were examined using in silico methods.
Received 27 September 2011
Revised 25 October 2011
Accepted 31 October 2011
Available online 6 November 2011
Keywords:
Antibiotic resistance
3-Mercapto-1,2,4-triazoles
Thiosemicarbazides
Inhibition assays
Ó 2011 Elsevier Ltd. All rights reserved.
Metallo-b-lactamases
Many pathogenic bacteria have developed resistance against
b-lactam antibiotics via mechanisms such as reduced cell-wall per-
meability, efflux of antibiotics and drug degradation mediated by
b-lactamases. b-Lactamases are enzymes that inactivate b-lactam
antibiotics by hydrolysing the key four-membered lactam ring of
these drugs.1 Class B b-lactamases are zinc-containing metalloen-
zymes (metallo-b-lactamases, MBLs) which use a metal-bound hy-
droxyl group as the nucleophile2 and are able to promote the
hydrolysis of a broad range of antibiotics, including penicillins,
cephalosporins and carbapenems.3 While clavulanic acid effec-
tively inhibits serine b-lactamases,4 there are no clinically available
inhibitors of MBLs. Therefore, there is an urgent need to develop
such compounds since multi-drug resistant pathogens such as
Pseudomonas aeruginosa and Acinetobacter spp. produce clinically
relevant levels of MBLs.
biotic resistance has also been observed in clinical isolates of Serra-
tia marcescens, Klebsiella pneumoniae, and Citrobacter freundii,6
which arises because mobile genetic elements allow such resis-
tance to spread to unrelated bacterial species.
Although no inhibitors of MBLs are clinically approved, a num-
ber of MBL inhibitors have been reported, including phthalic acid
derivatives,7 maleic acid derivatives,8 succinic acid derivatives9
and trifluoromethyl ketones.10 Irreversible thiol-containing inhibi-
tors of MBLs have also been described.11
We recently reported the discovery, by fragment-based screen-
ing of a 500 compound Maybridge™ library, of several new classes
of lead inhibitors against the IMP-1 MBL. All of these compounds
displayed Ki values of around 1 millimolar.12 Of the small frag-
ments identified in that work, we considered that 4-methyl-5-(tri-
fluoromethyl)-4H-1,2,4-triazole-3-thiol (1) was the most
promising for further study as kinetic assays indicated that its
mode of inhibition was purely competitive, that is, showing no
component due to uncompetitive inhibition. Herein, we report
our efforts for elaborating this ring system in attempts to improve
the potency of this compound, and our finding that intermediates
in these syntheses, N-acylated thiosemicarbazides, are also potent
inhibitors of the IMP-1 MBL.
The imipenemase (IMP-1) MBL from P. aeruginosa has been
identified in many reported cases of antibiotic resistance in medi-
cal facilities world-wide, leading to diseases such as pneumonia,
bacteriemia, urosepsis and wound infections.5 MBL-mediated anti-
⇑
Corresponding author. Tel.: +61 7 3365 3955; fax: +61 7 3346 3249.
E-mail address: r.mcgeary@uq.edu.au (R.P. McGeary).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.116

Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 380–386
381
O
Me
N
H
N
H
N
H
N
H
N
Me
Me
a
F3C
SH
N
H
Me
H2N
Me
Me
S
S
5
N
N
6
1
, Kic = 0.97 0.60 mM
Scheme 2. Reagents and conditions: (a) CMe3COCI, pyr, THF,
D, 3 h, 44%.
1,2,4-Triazole-3-thiols 4 were prepared in most cases from acyl-
ated thiosemicarbazides 3 on treatment with strong base, either
aqueous sodium hydroxide solution or sodium ethoxide in ethanol.
Acylated thiosemicarbazides 3 were prepared by a variety of meth-
ods, either direct thermal condensation of thiosemicarbazide 2
with carboxylic acids or by acylation of thiosemicarbazide 2 with
acid chlorides or acid anhydrides. In some cases the intermediate
acylated thiosemicarbazides 3 could not be isolated, and the
1,2,4-triazole-3-thiol products (4o and 4p) instead formed directly
(Scheme 1).
Me
Me
Me
Me
H
N
H
N
SH
SR1
a
Me
Me
N
N
N
N
7a, R1 = Me
4f
7b, R1 = Hex
Scheme 3. Reagents and conditions: (a) R1-X, Et3N, MeCN,
(7b).
D
, 2 h, 37% (7a); 37%
4-Methyl-1-pivaloylthiosemicarbazide 6 was prepared by the
H
N
H
N
acylation reaction of 4-methylthiosemicarbazide
Scheme 2.
5 shown in
Me
Me
SH
a
Two S-alkylated 1,2,4-triazole-3-thiols, 7a and 7b, were pre-
pared by chemoselective alkylation of 4f with the appropriate alkyl
halide and base in hot acetonitrile (Scheme 3). Desulfurisation of
4a gave 3-methyl-4H-1,2,4-triazole (8) according to Ainsworth’s
general method13 (Scheme 4).
N
N
N
N
4a
8
Scheme 4. Reagents and conditions: (a) NaNO2, HNO3, 45 °C, 30 min, 36%.
A number of substituted benzoic acids were required for this
work. Their syntheses are outlined in Schemes 5–7. Starting with
4-benzoylbenzoic acid 9, hydrogenolysis gave 4-benzylbenzoic
acid 10 (Scheme 5).
O
a
Ph
Ph
4-Benzoylbenzoic acids 13, were prepared form the appropriate
substituted benzoic acids 11 by regioselective Friedel–Crafts acyl-
ation of toluene promoted by phosphorus pentoxide adsorbed onto
silica, as described by Zarei et al.,14 followed by benzylic oxidation
of 12 using Jones reagent in hot acetic acid (Scheme 6).
CO2H
CO2H
9
10
Scheme 5. Reagents and conditions: (a) H2/Pd/C, AcOH, 65 °C, 44 h, 64%.
O
H
H
H
N
N
N
NH2
NH2
R
SH
4a-p
a
b
H2N
R
N
H
S
S
N
N
2
3a-n
c
Cl
O
O
a
Me
Et
e
f
HO2C(CH2)3-
CMe3
i
m
n
O
b
j
Me
O
O2
N
c
g
Ph
k
l
o
CF3
CO2H
O
d
HO2C(CH2)2-
h
p
CH3(CH2)5-
O2N
O2
N
Scheme 1. Reagents and conditions: (a) (i) RCO2H,
(3f); 71% (3g); 43% (3h); 76% (3i); 55% (3j); 26% (3k); 50% (31); 41% (3m); 39% (3n); (b) (i) Na, EtOH,
76% (4c); 56% (4d); 72% (4e); 37% (4f); 88% (4i); (c) RCO2H, , 6 h, 24% (4o); 17% (4p).
D
, 2 h, 60% (3a); 53% (3b) or (ii) (RCO)2O,
D
, 2 h, MeCN, 36% (3c); 66% (3d); 92% (3e) or (iii) RCOCI, pyr, rt, 24–72 h, 25%
, o/n, 73% (4a); 63% (4b); 50% (4g); 47% (4h) or (ii) 10% aq NaOH, , 3 h,
D
D
D

382
Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 380–386
MeO2C
CO2Me
HO2C
CO2Me
CO2Me
O
a
CO2H
a
R
R
14
15
Me
b
11a-c
12a-c
O
O
b
CO2H
c
O
17
16
13a-c
R
Scheme 7. Reagents and conditions: (a) NaOH, MeOH, rt, 18 h, 83%; (b) PhMe, P2O5/
SiO2, , 3 h, 35%; (c) NaOH, H2O, , 2 h, 80%.
CO2H
D
D
a, R = 2-Cl b, R = 3-NO2 c, R = 4-NO2
The inhibitory effects of 1,2,4-triazole-3-thiols 4, 7 and 8
against the IMP-1 MBL were performed as previously described.12
Briefly, kinetic studies were performed using the chromogenic sub-
strate CENTA (see Supplementary data)16 and a 96-well plate read-
er at pH 7.00 and monitoring the development of the chromophore
4-nitrothiophenolate at 405 nm. The results are shown in Table 1.
In examining structure–activity relationships for the structural
elements of the starting 4-methyl-5-(trifluoromethyl)-4H-1,2,4-
triazole-3-thiol (1), a number of general features are apparent:
Scheme 6. Reagents and conditions: (a) PhMe, P2O5/SiO2,
(12b); 66% (12c); (b) Jones reagent, AcOH,
D
, 3 h, 51% (12a); 63%
D
, 4 h, 30% (13a); 64% (13b); 60% (13c).
The final benzoylbenzoic acid 17 used in this study was pre-
pared as shown in Scheme 7. Dimethyl isophthalate 14 was hydro-
lysed to the monoacid 1515 which was then subjected to the
Friedel–Crafts acylation with toluene and phosphorus pentoxide
on silica gel to yield the ester 16. Finally, saponification of 16 gave
the required carboxylic acid 17.
Table 1
Inhibitory activities of 1,2,4-triazole-3-thiols against IMP-1
Compound
Structure
Inhibition % (1000
51
l
M)
Inhibition % (100
—
lM)
Inhibition % (10
—
lM)
Me
N
F3C
SH
SH
1
N
N
H
N
Me
Et
4a
4b
33
32
—
—
—
—
N
N
H
N
SH
N
N
H
N
SH
4c
—
7
0
N
N
N
CO2H
H
N
HO2C
SH
4d
4e
—
—
10
5
10
0
N
H
N
SH
HO2C
N
N
Me
H
N
Me
Me
SH
4f
32
0
—
—
—
—
N
N
H
N
4g
SH
N
N
H
N
4h
—
—
Insoluble
SH
O2N
N
N

Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 380–386
383
Table 1 (continued)
Compound
Structure
Inhibition % (1000
l
M)
Inhibition % (100
lM)
Inhibition % (10 lM)
O
H
N
4i
—
44
10
SH
N
N
H
N
F3C
SH
4o
4p
45
—
—
—
—
N
N
H
N
SH
Me
Insoluble
N
N
Me
Me
H
N
SMe
7a
7b
10
—
—
Me
N
N
N
N
Me
Me
H
N
S
Me
—
0
—
—
Insoluble
—
Me
H
N
Me
8
N
N
— Not determined.
1. The N-methyl group of 1 is not necessary for potency, as com-
pound 4o, lacking this group, has very similar activity.
to the corresponding diaryl methane resulted in minimal decrease
in potency (compare 3i with 3j), suggesting that the carbonyl
group was unimportant for potency.
2. The thiol is a requirement for activity. Compound 8, in which
the thiol group has been removed, has no activity. Compound
7a, in which the thiol group has been methylated, has some-
what lower activity than its non-methylated analogue 4f.
3. Alkyl groups can substitute for the trifluoromethyl group of 1
without significant loss of activity. The 5-methyl- (4a) and 5-
ethyl- (4b) derivatives retain much of the potency of 1, as does
the bulky 5-tert-butyl-derivative 4f, although anionic alkyl side
chains (4d and 4e) exhibited diminished activity.
4. While simple 5-aryl-derivatives such as 5-phenyl (4g) and 5-(2-
carboxyphenyl)- (4c) derivatives showed low inhibitory activi-
ties, a significant improvement in potency was observed for
the 5-(4-benzoylphenyl)-derivative 4i.
Based on the encouraging findings of the compounds listed in
Table 2, several inhibitors were selected for more careful kinetic
analyses to determine Ki values and their modes of inhibition
(competitive versus uncompetitive). These results are summarised
in Table 3. For comparison, the Kic value for the known competitive
MBL inhibitor L
-captopril17 is included in Table 3.
The results in Table 3 indicate that the 1,2,4-triazole-3-thiol (4i)
and the acylated thiosemicarbazides (3) exhibit mixed inhibition
(both competitive and uncompetitive inhibition modes were ob-
served). This mixed inhibition has been observed previously by
us for small fragments binding to IMP-1 MBL,12 and also for inhib-
itors of another binuclear metallohydrolase, purple acid phospha-
tase.18 We interpret this mixed inhibition mode to indicate that
these inhibitors are capable of both binding in the active site of
IMP-1 (competitive inhibition) and also of forming a ternary en-
zyme–substrate-inhibitor (ESI) complex which inhibits hydrolysis
of the substrate (uncompetitive inhibition). Both of these possible
binding modes may offer insights for the future design of more po-
tent inhibitors.
Although analysis of the structure–activity relationships of the
compounds listed in Table 1 was instructive, it nonetheless did
not lead to any large improvements in potencies relative to the
starting triazole-thiol 1. A fortuitous discovery was that many of
the acylated thiosemicarbazide synthetic precursors 3 (Scheme 1)
of the triazole-thiols 4 did themselves possess high potencies
against the IMP-1 MBL (Table 2)
The structure–activity data in Table 2 shows that acylation of
thiosemicarbazide with the bulky pivaloyl group led to derivatives
with no activity (compounds 3f and 6) whereas anionic alkyl side
To gain insight into the possible binding modes of these inhib-
itors, in silico docking of the most potent inhibitor, 3l (Kic
11
4 lM), in the active site of the IMP-1 MBL was examined using
Molegro Virtual Docker.19 The lowest energy binding orientation of
3l is shown in Figure 1. While we had anticipated that the sulfur
atom of 3l would bind to one or both of the metal ions in the active
site, modelling unexpectedly suggested that the oxygen atoms of
the nitro group were interacting with the zinc ions, twisting the ni-
tro group out of planarity with the aromatic ring to allow oxygen–
metal distances of 3.3 Å (Zn1) and 2.0 Å (Zn2) to be attained. The
nitro-aromatic ring of inhibitor 3l makes a close contact with the
indolic side-chain of Trp 64 on the flexible loop of IMP-1 (Fig. 1),
chains gave modest inhibition at 10 lM (3d and 3e). A sharp in-
crease in potency was observed when the thiosemicarbazide was
acylated with aromatic groups. With the exception of the 2-carbo-
xylbenzoyl compound (3c) which exhibited no inhibition at 10
all other aromatic substituents showed strong inhibition at 10
l
l
M,
M.
The most potent compounds in this series included 4-(ben-
zoyl)benzoyl derivatives, particularly 3i and 3k–n. Removal of
the oxygen atom of the linking diaryl ketone group by reduction

384
Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 380–386
Table 2
Inhibitory activities of acylated thiosemicarbazides 3 against IMP-1
Compound
Structure
CO2H O
Inhibition % (10
0
lM)
H
N
NH2
3c
N
H
S
O
H
N
NH2
3d
3e
3f
13
8
HO2C
HO2C
N
H
S
O
H
N
NH2
N
H
S
O
H
Me
Me
N
NH2
NH2
0
N
H
Me
S
O
H
N
3g
22
N
H
S
O
H
N
O2N
NH2
3h
3i
30
34
N
H
S
O
S
H
N
N
H
NH2
NH2
O
O
S
H
N
3j
26
45
N
H
O
O
O2N
S
H
N
3k
N
H
NH2
NH2
O
O
S
H
N
3l
45
51
O2N
Cl
N
H
O
S
H
N
3m
N
H
NH2
O
O
O
H
N
NH2
N
3n
6
38
0
H
S
Me
O
H
N
H
Me
Me
N
N
Me
H
Me
S

Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 380–386
385
Table 3
Kinetic data for selected compounds against IMP-1
Compound
Structure
Kic
(lM)
Kiuc (lM)
O
O
H
N
NH2
3g
19
13
8
30
8
7
N
H
S
S
S
H
3i
4
26
N
N
H
NH2
NH2
O
O
H
N
3j
18 10
37 15
N
H
O
O
O2N
S
H
N
3k
16
11
14
7
4
4
15
20
5
5
N
H
NH2
NH2
O
O
S
H
N
3l
O2N
Cl
N
H
O
S
H
N
3m
3n
4i
13
20
2
4
N
H
NH2
O
O
O
O
H
N
NH2
N
41 24
H
S
Me
H
N
SH
75 30
56 10
N
N
O
Me
HS
N
12.5 2.412
—
L
-Captopril
CO2H
O
— Indicates result >2 mM.
and two N-H bonds on the terminal thiourea group of 3l form
hydrogen bonds to the carbonyl oxygen on the backbone of Tyr
227 (not shown).
of the IMP-1 enzyme, formed by methylene groups of the side-
chains of Lys 224 and His 263.
In conclusion, we have generated a number of analogues of our
lead compound 1 and conducted structure–activity studies of these
derivatives against the metallo-b-lactamase IMP-1. While a num-
ber of structural features of these 1,2,4-triazole-3-thiols have been
shown to be important for strong binding, only modest improve-
In contrast, modelling of inhibitor 3g (Kic 19
8 lM), in the ac-
tive site of the IMP-1 MBL indicated that the sulfur atom of the thi-
osemicarbazide moiety was binding to the two metal ions in an
expected manner, with sulfur–metal distances of 2.3 Å (Zn1) and
2.0 Å (Zn2) (Fig. 2). This compares well with crystallographic data
from a thiol inhibitor in complex with IMP-1, which shows sulfur-
zinc lengths of 2.4 Å and 2.2 Å, respectively.20 The terminal aro-
matic ring in 3g interacts with a hydrophobic patch in the surface
ments in potency (from 1: Ki ꢀ1 mM to 4i: Ki ꢀ70
tained. In contrast, optimisation of acylated thiosemicarbazides 3
has led to several compounds with Ki values as low as 11 M, com-
parable with the potency of -captopril (12.5 M). All of the newly
lM) were ob-
l
L
l

386
Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 380–386
the recipient of a scholarship from the Pakistan Higher Education
Commission (International Research Support Initiative Program).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.116.
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Acknowledgements
This work was supported by a National Health and Medical Re-
search Council of Australia Project Grant (631443). Mr. Faridoon is