First identification of isatin-β-thiosemicarbazones as novel inhibitors of New Delhi metallo-β-lactamase-1: Chemical synthesis, biological evaluation and molecular simulation

Article abstract of DOI:10.1016/j.cclet.2017.09.035

New Delhi metallo-β-lactmase-1 (NDM-1) catalyzes the hydrolysis of β-lactam antibiotics and cleaves the β-lactam ring of the molecule, conferring bacterial resistance against these medicines. In an effort to discover novel agents to treat this superbug, a

Full text of DOI:10.1016/j.cclet.2017.09.035

Accepted Manuscript
Title: First identification of isatin-β-thiosemicarbazones as
novel inhibitors of New Delhi metallo-β-lactamase-1:
Chemical synthesis, biological evaluation and molecular
simulation
Authors: Guo-Qing Song, Wei-Min Wang, Zai-Shun Li, Ying
Wang, Jian-Guo Wang
PII:
S1001-8417(17)30377-7
DOI:
Reference:
http://dx.doi.org/10.1016/j.cclet.2017.09.035
CCLET 4241
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
30-6-2017
14-8-2017
13-9-2017
Please cite this article as: Guo-Qing Song, Wei-Min Wang, Zai-Shun Li, Ying
Wang, Jian-Guo Wang, First identification of isatin-␤-thiosemicarbazones
as novel inhibitors of New Delhi metallo-␤-lactamase-1: Chemical
synthesis, biological evaluation and molecular simulation, Chinese Chemical
Lettershttp://dx.doi.org/10.1016/j.cclet.2017.09.035
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Communication
First identification of isatin-β-thiosemicarbazones as novel inhibitors of New Delhi
metallo-β-lactamase-1: Chemical synthesis, biological evaluation and molecular
simulation
Guo-Qing Songa,1, Wei-Min Wanga,c,1, Zai-Shun Lia, Ying Wangb,*, Jian-Guo Wanga,*
a State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, National Pesticide Engineering Research Center, College of Chemistry, Nankai
University, Tianjin 300071, China
b Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China
c Patent examination cooperation Jiangsu center of the patent office, SIPO, Suzhou 215163, China
*corresponding authors.
E-mail addresses: nkwjg@nankai.edu.cn (J.-G. Wang), ying.wang@htmdc.org (Y. Wang)
1 These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
New Delhi metallo-β-lactmase-1 (NDM-1) catalyzes the hydrolysis of β-lactam
antibiotics and cleaves the β-lactam ring of the molecule, conferring bacterial
resistance against these medicines. In an effort to discover novel agents to treat this
superbug, an old drug methisazone was found to be a weak NDM-1 inhibitor, with an
IC50 of 297.6 µmol/L. Based on this result, a series of isatin-β-thiosemicarbazones
(IBTs) were synthesized and biologically evaluated as novel NDM-1 inhibitors. Nine of
the IBT compounds showed IC50 values of <10 µmol/L, the best of which was 2.72
µmol/L. Comparative field analysis (CoMFA) contour maps were generated to depict
the structural features and molecular docking was performed to understand the
possible binding mode of these inhibitors. The present research hereby has provided
valuable information for further discovery of NDM-1 inhibitors.
Received 30 June 2017
Received in revised form 14 August 2017
Accepted 6 September 2017
Available online
Keywords: New Delhi metallo-β-lactmase-1
Isatin-β-thiosemicarbazones
In vitro enzyme inhibition
Molecular docking
The invention of antibiotics was regarded as one of the greatest medicinal improvements in the 20th century,
because thousands of patients had been rescued from fatal bacterial infection. However, with the abuse of
antibiotics, resistance against these drugs to bacteria has become a global problem gradually. In 2008, Rice termd
“the ESKAPE bugs” to describe Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter
baumanni, Pseudomonas aeruginosa and Enterobacter as the most common resistant bacteria species in hospitals
[1]. In 2010, a carbapenemase named New Delhi metallo-beta-lactamase-1 (NDM-1), was reported to cause Gram-
negative bacterial resistance to almost all available β-lactam antibiotics and the rapid spread of the plasmid-
encoded NDM-1 gene in some countries subsequently indicated that “superbug” era was arriving [2,3].
NDM-1 belongs to the Class B metallo-β-lactamase (MBL) superfamily, which contains enzymes that hydrolyze β-
lactams and cleaves the β-lactam ring of the compound, leading to antibiotic-resistant infections [4]. Crystal
structures of NDM-1 had been elucidated by different research groups, either the free enzyme alone or that in a
bound form [4-8].Two catalytic zinc ions have been found in the active site of the enzyme, which play essential
roles for the biochemical function of this metallolactamase. The NDM-1 crystal structures were bound either with
the hydrolyzed state of an antibiotic drug, or an inhibitor such as L-captopril. These achievements have not only
provided structural insights into β-lactam recognition and inhibition mechanism, but also offered opportunities to
design novel inhibitors targeting NDM-1 in a rational way [7,8].
Very few medicinal antibiotics can inhibit resistant bacteria with NDM-1 gene effectively, including colistin and
tigecycline. From 2000 to 2013 only 22 new antibiotics were approved, but none of them was indicated for Gram-
negative bacteria [9]. It is therefore a strong demand to develop novel inhibitors tackle such a bacterial resistance.
Rao et al. reported that captopril is an NDM-1 inhibitor with IC50 of 7.9 µmol/L [4], based on which some captopril
analogues were synthesized and evaluated [10]. Proschak et al. accessed some approved drugs containing thiols

and identified some good NDM-1 inhibitors [6]. Aspergillomarasmine A (AMA) was shown to be a rapid and potent
NDM-1 inhibitor, the IC50 of which is 4.0 µmol/L [11]. Ebselen was reported to be a potent covalent inhibitor of
NDM-1, which binds to Cys211 to inactivate the enzyme [12]. Cheng et al. discovered some active compounds via
multistep virtual screening and the most potent inhibitor VNI-41 has an IC50 of 29.6 µmol/L [13]. Yang et al.
demonstrated that triazolylthioacetamide is a promising scaffold for the development of NDM-1inhibitors, and the
best IC50 value is 0.15 µmol/L for compound 4a in their study [14].
Isatin is an endogenous natural product and its derivatives possess a wide range of biological activities, such as
antibacterial, antitubercular, antimalarial, antifungal and antiviral activities [15-20]. Some drugs contain isatin as an
active scaffold, for instance, indirubin and methisazone, which have been used to treat chronic myelogenous
leukemia and smallpox infection, respectively [21,22]. It is a common knowledge that the most fruitful basis for the
discovery of a new drug is to start with an old drug [6,23]. In the course of screening new chemical agents for
resistant bacteria, we had evaluated the inhibitory activity of some medicines containing isatin structure against
purified NDM-1. Methisazone was found to display some activity in the screening process, although the IC50 value
for NDM-1 of this old drug is only 297.6 µmol/L. Inspired by this result, we then synthesized some isatin-β-
thiosemicarbazone (IBT) derivatives designed from methisazone and tested their in vitro inhibitory activities.
Enhanced biological potencies were observed for the IBT compounds and many IC50 values against NDM-1 are at
micromolar level. Thus we have identified for the first time that a series of IBT compounds are new NDM-1
inhibitors, which is meaningful for further drug discovery. Fig. 1 illustrated the chemical structures of some
reported NDM-1 inhibitors, isatin, indirubin and methisazone.
The synthetic method for the IBT compounds was similar to our previous procedure, in which the target
compounds were chemically prepared by a straightforward condensation of isatin and thiosemicarbazide [24]. The
yields of the reaction were generally satisfactory, in the range of 65%~80%. For examples, the yield of compound 6
is 58% while that of compound 7 is 81%. The synthetic scheme, chemical structures of compounds 1-14 and their in
vitro inhibitory activities against NDM-1 are shown in Table 1. The inhibitory potencies of the IBT derivatives are
sorted in ascending order. Compounds 3, 4, 6, 7 and 13 are new compounds that have not been characterized
before, whereas the other compounds had been reported and detailed elsewhere [24-26]. Detailed experimental
procedures, analytical data for all previously unreported compounds, 1H NMR, 13C NMR and HRMS figures for
selected compounds, relevant biological data and inhibition curves for all NDM-1 inhibitors are in Supporting
information.
From the enzymatic inhibitory data, it can be seen that methisazone (compound 1) exhibits a weak IC50 of 297.60
µmol/L. Compound 2 has a close structure to methisazone and the IC50 data improves to 225.9 µmol/L. When a
phenyl ring is attached to the thiourea part of the compound, there is a significant improvement of the biological
activity. Compounds 3-14 display desirable NDM-1 inhibition data, among which compound 14 is the strongest
one, with an IC50 value of 2.72 µmol/L. Compound 3 has an IC50 data of 27.69 µmol/L, compound 4 has an IC50 data
of 14.82 µmol/L and the IC50 data for compound 5 is 10.72 µmol/L, respectively. The IC50 data of the other nine
compounds (6-14) were <10 µmol/L. Fig. 2 shows the inhibition curve of compound 14.
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Fig. 2. Inhibition curve of compound 14 against NDM-1.
The results are interesting because this is the first identification of IBT structures as NDM-1 inhibitors, indicating
their potential use to treat bacterial infections associated with New Delhi metallo-β-lactamase-1, ideally when used
in combination with clinical antibiotics. Indeed, some IBT compounds had been recognized as strong inhibitors of
Methicillin-resistant Staphylococcus aureus (MRSA) in our previous work, many of which were much better than
the control drug vancomycin [24]. Bearing this in mind, soon after we discovered that IBTs are NDM-1 inhibitors, all
the fifty-one IBTs in that paper were subjected to NDM-1 assay, however only a few compounds (5, 8-12, 14) were
active against NDM-1. Not every IBT derivative displayed both MRSA inhibition and NDM-1 inhibition, therefore
there should be no direct relationship between the two different biological activities. The bacterial strains for
MRSA are Gram-positive, while NDM-1 only exists in Gram-negative bacteria strains.
Compared with the reported NDM-1 inhibitors, the IBT compounds are distinct in their chemical structures. The
IC50 data of most known NDM-1 inhibitors are at micromolar level, similar to the potency of the IBT inhibitors in
this study. This means that the IBT compounds studied here are a new family of NDM-1 inhibitors. Methisazone

has been used as an old antiviral drug for treating smallpox infection for a half century [22], it is likely that suitable
modifications on the structure of methisazone might bring a new drug with clinical safety.
To further understand the structure-activity relationship of these inhibitors, a comparative field analysis
(CoMFA) model was constructed subsequently. The leave-one-out q2 is 0.644 at the optimum components of 2,
and the non-cross-validated r2 is 0.917, with a standard error of estimate of 0.204. The steric and electrostatic
contributions are 59.4% and 40.6%, respectively. Considering the fact that only fourteen IBT compounds are in the
database, the statistical result is fairly satisfactory. Compound 14 has been selected as a template to depict the
structural features of the NDM-1 inhibitors. For the steric contour map (Fig. 3a), a bulky group will be favorable for
better NDM-1 inhibition in the green contour region and such a group is likely to decrease the biological activity in
the yellow contour space. The green map is hidden behind the yellow contour region in the phenyl part (in the
right side of the figure). For the electrostatic contour map (Fig. 3b), a positively charged group will increase the
activity of the compound in the blue contour region, meanwhile, a negatively charged group is favorable to
enhance the activity in the red region. It seems that the electrostatic contour map is better than the steric contour
map for further molecular design from this CoMFA model. Details for molecular simulation and experimental
biological activity versus predicted biological activity from CoMFA model are in Supporting information.
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Fig. 3. Three-dimensional contour maps from the CoMFA model of the NDM-1 inhibitors.
The possible binding mode of the NDM-1 inhibitors of IBT family was then generated via molecular docking
simulation. In this procedure 4EXS from protein databank was used because this pdb file contains a reported NDM-
1 inhibitor L-captopril [8]. Fig. 4 illustrates the interaction of compound 14 and the neighboring residues of NDM-1
drawn by LIGPLOT [27]. It could be seen that many residues (Thr34, Val73, Trp93, Gly219, Asn220 and Gly222) form
hydrophobic interactions with the inhibitor, while two residues (Gly69 and His189) have hydrogen bonding
contacts with the inhibitor. Two zinc ions in the structure form several coordination bonds with the sulfur atom in
the IBT molecule, suggesting their essential roles in the inhibition process. This is in agreement with the binding
modes of some reported NDM-1 inhibitors or hydrolyzed antibiotics, in which the zinc ions also serve as chelating
anchors for the biochemical function of this enzyme.
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Fig. 4. 2D representation of 14 bound with NDM-1 from molecular docking.
In summary, by screening the activity groups on the isatin fragments, isatin-β-thiosemicarbazone scaffold has
been studied as novel inhibitors of New Delhi metallo-β-lactamase-1, the best IC50 value was found as low as 2.72
µmol/L. This new class of compounds is different from the published NDM-1 inhibitors, which is interesting for
further research. The quantitative structure-activity relationship analysis and the proposed binding mode
suggested by molecular docking have provided important clues on further structural modification. In face of the
superbug attack, more and more research should be carried out in this area. The present study has hence shed
light on the design and discovery of novel NDM-1 inhibitors based on the IBT scaffold.
Acknowledgments
This work was supported by the “111” Project of Ministry of Education of China (No. B06005), the National
Natural Science Foundation of China (No. 21672114) and the National Basic Research Program of China (No.
2013CB734004).
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Fig. 1. Chemical structures of reported NDM-1 inhibitors and some drugs containing isatin scaffold.
Fig. 2. Inhibition curve of compound 14 against NDM-1.
Fig. 3. Three-dimensional contour maps from the CoMFA model of the NDM-1 inhibitors.
Fig. 4. 2D representation of 14 bound with NDM-1 from molecular docking.

Table 1
IBT NDM-1 inhibitors and their biological activities.
R2
Compd
R1
Yield (%)a
IC50 (µmol/L)
297.60±77.54
1
2
methisazone
225.90±77.49
3
4
5
6
7
CH3
OCH3
H
COOH
Br
Br
Cl
Cl
Cl
Cl
H
H
H
H
H
70
67
27.69±4.63
14.82±6.52
10.72±1.38
7.75±1.13
6.29±0.94
6.03±1.25
4.91±1.66
4.65±0.80
3.51±0.51
3.35±0.84
3.30±0.57
2.72±0.93
58
81
8
9
o-F
o-F
o-OCH3
p-F
p-Br
H
m-CH3
10
11
12
13
14
I
Br
65
a Only yields of the new compounds were given.