Synthesis and antituberculosis activity of new acylthiosemicarbazides designed by structural modification

Article abstract of DOI:10.1002/ddr.21626

Acylthiosemicarbazides 8a–n were designed by structural modification of lead Compound 7. The syntheses of 8a–n involve a five-step procedure starting from carboxylic acids. Compounds 8a–n were tested against three Mycobacterium tuberculosis strains to measure their inhibitory antituberculosis activities. These activities could be explained according to the presence or absence of the chlorine substituent in the aromatic ring of the amide joined to the thiosemicarbazide core. Thiosemicarbazide derivative 8n is a candidate for the development of novel antitubercular agents. Ongoing studies are focused on exploring the mechanism by which these compounds inhibit M. tuberculosis cell growth.

Full text of DOI:10.1002/ddr.21626

Received: 3 July 2019
Revised: 8 October 2019
Accepted: 9 November 2019
DOI: 10.1002/ddr.21626
R E S E A R C H A R T I C L E
Synthesis and antituberculosis activity of new
acylthiosemicarbazides designed by structural modification
Roberto Martínez1
| Clara I. Espitia-Pinzón2 | Mayra Silva Miranda3
|
Rosa María Chávez-Santos1 | Gustavo Pretelin-Castillo1 | Aldahir Ramos-Orea1
Angela M. Hernández-Báez | Sandra Cotlame-Pérez1 | Rogelio Pedraza-Rodríguez1
ꢀ
|
1
1Universidad Nacional Autónoma de México,
Abstract
Instituto de Química, Ciudad Universitaria,
Ciudad de México, México
Acylthiosemicarbazides 8a–n were designed by structural modification of lead
2Instituto de Investigaciones Biomédicas,
Compound 7. The syntheses of 8a–n involve a five-step procedure starting from car-
Universidad Nacional Autónoma de México,
Ciudad Universitaria, Ciudad de México,
México
boxylic acids. Compounds 8a–n were tested against three Mycobacterium tuberculosis
strains to measure their inhibitory antituberculosis activities. These activities could
be explained according to the presence or absence of the chlorine substituent in the
aromatic ring of the amide joined to the thiosemicarbazide core. Thiosemicarbazide
derivative 8n is a candidate for the development of novel antitubercular agents.
Ongoing studies are focused on exploring the mechanism by which these compounds
inhibit M. tuberculosis cell growth.
3Catedrática CONACYT adscrita al Instituto de
Investigaciones Biomédicas, Universidad
Nacional Autónoma de México, Ciudad
Universitaria, Ciudad de México, México
Correspondence
Roberto Martínez, Universidad Nacional
Autónoma de México, Instituto de Química,
Ciudad Universitaria, Ciudad de México,
04510 México.
K E Y W O R D S
Email: robmar@unam.mx
acylthiosemicarbazides, antituberculosis agents, design, isosteric modification, synthesis
Funding information
DGAPA and UNAM, Grant/Award Numbers:
NUATEI-IIB-UNAM, PAPIIT IN208015
1
|
INTRODUCTION
designing better drugs. On the other hand, a thiosemicarbazide core is
present in many derivatives with diverse biological activities (Figure 1).
Tuberculosis is an infectious disease caused by the bacillus Mycobacterium
tuberculosis. According to data published by the World Health Organiza-
tion (WHO), tuberculosis is among the top 10 causes of death worldwide
(World Health Organization, 2018). The increase in the number of tuber-
culosis cases, the presence of M. tuberculosis strains resistant to commonly
used drugs, the long duration of treatment, and the adverse effects of
available medications used have led medicinal chemistry researchers to
search for new compounds that are more effective against this disease.
From this perspective, the synthesis of chemical compounds with a combi-
nation of characteristics such as high selectivity, few side effects, and the
desired pharmacological response at a low dose is important.
For example, 1-(4-chlorophenylacyl)-4-arylthio semicarbazide deriva-
tives 1 have been reported as urease inhibitors (Ali et al., 2018), novel
indole-based thiosemicarbazides 2 act as antiviral agents (Cihan-
ꢁ
Üstündag, Gürsoy, Naesens, Ulusoy-Güzeldemirci, & Çapan, 2016),
diacyl thiosemicarbazides 3 are potent Gram-positive antibacterial
agents (Paneth et al., 2016), butylated hydroxytoluene derivatives
4 function as potential antioxidants (Ariffin, Rahman, Yehye, Alhadi, &
Kadir, 2014), 5 have antimicrobial and antitubercular activities (Rane
et al., 2014), and 19-carboxyl-modified novel isosteviol derivatives
6 exhibit antineoplastic activity (Liu et al., 2017).
Figure 1 shows examples of acylthiosemicarbazides that exhibit
biological activity.
Analyses of the drugs launched in the last decade have revealed
that most were derived through modification of known drugs or lead
structures (Flick et al., 2016; Teague, 2011). On the one hand, these
studies support the idea that structural modification is an effective
approach to understanding the mechanism of drug action and to
We are currently engaged in a program aimed at synthesizing origi-
nal heterocyclic compounds that inhibit the growth of M. tuberculosis.
Recently, we discovered that N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-
5-nitrofuran-2-carboxamide (7), which was selected on the basis of a
Drug Dev Res. 2019;1–6.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
MARTÍNEZ ET AL.
FIGURE 1 Examples of acylthiosemicarbazides that exhibit biological activity
structural analysis of a major public database of compounds reported to
Our next approach was to replace the chlorophenyl ring (A)
exhibit antituberculosis activity (Martínez et al., 2019), inhibited the
growth of M. tuberculosis strain H37Rv bacteria, with a minimal inhibitory
concentration (MIC) of 7.8 μg/ml and a selectivity index (SI) of 13.6
(Figure 2). This molecule comprises three units: a phenyl ring (A), a
1,3,4-oxadiazole ring (B), and a five-membered heterocyclic ring (C). The
mechanism of the antiproliferative activity of 7 is still unknown; there-
fore, further exploration of the phenyloxadiazolfurancarboxamide
pharmacophore is warranted.
of 8a with a chlorothiophene (Compound 8b), a chloropyrrole ring
(Compound 8c), or a chlorofuran ring (Compound 8d) via an isosteric
modification (Papadatos & Brown, 2013). We thereafter also replaced
the nitrofuran ring (C) with a nitropyrrole ring, a nitrothiophene ring,
or a nitrophenyl group, which resulted in Compound 8e, 8f, or 8 g,
respectively. In addition, we explored the role of the chloro substitu-
ent attached to the phenyl rings of a thiosemicarbazide moiety
(Compounds 8 h–n) in the inhibition of cell growth of M. tuberculosis
strain H37Rv bacteria.
As a first approach, we opened the 1,3,4-oxadiazole ring (B) of
7 and determined the antituberculosis activity of the product, 8a.
However, 8a showed an MIC > 500 μg/ml. Since a lead compound is
a new chemical entity, that could potentially be developed into a new
drug, by optimizing its beneficial effects and minimizing its side
effects, this result led us to speculate that an exploration of structural
modifications of Compound 8a (Beswick & Naylor, 2012) and an eval-
uation of the antituberculosis activity of the resultant compounds
could lead to compounds with more significant activity than 8a. As
shown in Figure 2, proposed compounds were synthesized to evalu-
ate the effect of the structural modification that lead to two lead
Compounds 7 and 8a.
2
|
MATERIALS AND METHODS
| Chemistry
2.1
The reagents 11a–n, 12a, 12c, and 12e–n were commercial
raw material and were used as purchased from Aldrich. Compounds
8a–n, 10a–n, 12b, and 12d have been prepared following reported
procedures The experimental details are given in the Supporting
Information.

MARTÍNEZ ET AL.
3
2.1.1
|
In vitro antimycobacterial assay
Stock solutions of all compounds were prepared in 100% dimethyl
sulfoxide (DMSO) at a concentration of 10 mg/ml. For the resazurin
microtiter assay (REMA), compounds were diluted in Middlebrook
7H9 broth medium (TH9) without tyloxapol. For reference drugs,
stock solutions with a concentration of 64 μg/ml were prepared and
filtrated using
a membrane with a pore diameter of 0.22 μm
(Millipore; Darmstadt, Germany). All working solutions were kept
refrigerated at −20ꢀC until they were evaluated.
2.1.2
|
Cell culture
The cytotoxicity assays were carried out used Vero cell lines (kidney
of African green monkey) from ATCC. These cells were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine serum and
nonessential amino acids.
2.1.3
|
Cytotoxicity assay
To assess the cytotoxicity of each tested compound, 10,000 Vero
cells were placed in a 96 well-plate and incubated for 24 hr in
100 μl of RPMI medium. After incubation, the plate was washed
and fresh medium with the compound at a different concentra-
tion was added. Each tested compound was incubated for
48 hr at 37ꢀC in a 5% CO2 atmosphere. Then, 10 μl of 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) sol-
ution (5 mg/ml in sterile Phosphate-Buffered Saline (PBS)) was
added to each well and incubation was allowed to continue for
another 4 hr. The medium was removed, and 100 μl of DMSO
was used to solubilize the formazan. Absorbance was measured
at a wavelength of 570 nm, and cytotoxicity was calculated as %
Toxicity = (1 − [ABS problem/ABS control]) × 100. Controls were
cells without treatment (Mossman, 1983).
FIGURE 2 Proposed compounds to be synthesized to evaluate
the effect of the structural modification of lead Compound 7 and
Compound 8a
2.1.4
|
Mtb inocula preparation
M. tuberculosis H37Rv, Ra, and 209 strains were cultivated in a
7H9–glycerol–10% Albumin Dextrose Catalasa (ADC)–0.01%
tyloxapol medium at 37ꢀC until an Optical Density (O.D.) of 0.4 at
a wavelength of 600 nm was reached. The working bacteria solu-
tion was prepared by 1:25 dilution in 7H9–ADC 10%.
during the long-duration (8 day) incubation. Rifampin (RIF) was
used as a reference drug (sixteen −0.001 μg/ml serial twofold dilu-
tions) in each plate, and controls of DMSO, DMSO + Mtb, medium,
media + Mtb, and only compound to validate the plate were also
included. Compounds were evaluated at various concentrations
from 0.98 to 250 μg/ml and in triplicate in independent assays.
Plates were incubated for 6 days; then, 30 μl of 0.01% resazurin
(wt/vol) (Sigma-Aldrich) was added to each well and the plates were
incubated for an additional 2 days. Visual inspection was used to
determine the colors of each well; blue was interpreted as no
growth and pink as growth. The MIC values were defined from the
first concentration at which blue color was observed.
2.1.5
|
Antimicrobial susceptibility test using
the REMA
The assay used here was previously described (Collins & Franzblau,
1997). In brief, the outer wells of a 96-well plate were each filled
with 200 μl of sterile PBS to prevent dehydration from occurring

4
MARTÍNEZ ET AL.
2.1.6
|
Selectivity index
FICI = (MIC A + B/MIC A) + (MIC B + A/MIC B). MIC A + B represents
the MIC of Drug A associated with Drug B. MIC B + A represents the
MIC of Drug B associated with Drug A. MIC A and MIC B represent
the MIC of Drugs A and B tested separately, respectively. FICI was
considered “synergic” (FICI ≤ 0.5), “antagonism” (FICI > 4.0), and “no
interaction” (FICI > 0.5–4.0).
SI was obtained using the formula SI = IC50 in Vero cells/MIC100
determined using REMA.
Minimal bactericidal concentration (MBC) was assessed for some of
the compounds as described by Molina et al., 2006. Briefly, 5 μl of the
undeveloped duplicates of bacteria suspensions from REMA were
transferred to a new microplate that contained 195 μl of fresh culture
medium per well. Then microplates were incubated as described for
REMA. The MBC values corresponded to the minimum compound
concentration that did not cause a color shift in cultures reincubated
in fresh medium.
3
|
RESULTS
| Chemistry
3.1
Compounds 7 and 8a–d were prepared via the coupling reaction
between isothiocyanates 9a–n, without purification, and hydrazides
10a–d. Isocyanates 9a–n were synthesized via a two-step process (Li,
Wang, & Xue, 2011) that included halogenation of the corresponding
acids 11a–n with oxalyl chloride ([COCl]2) and posterior treatment of
the resulting acid chlorides with potassium thiocyanate (KSCN). In
parallel, key intermediates 10a–n were obtained via a two-step
2.1.7
|
Combinatory activity of 8m and RIF
Combinatory activity between RIF and 8m was analyzed by resazurin
drugs combination microtiter assay. The fractional inhibitory concen-
tration index (FICI) was used to evaluate the combinatory effects:
SCHEME 1 Synthesis route
to Compounds 8a–n

MARTÍNEZ ET AL.
5
TABLE 1 Anti-M. tuberculosis effects and cytotoxicity levels of
Compounds 8a–n
The removal of the 4-(5-nitrofuranoyl) group from our lead Com-
pound 8a led to increased activity of the synthesized Compounds 8e–
g compared with the activity of 8a (Table 1); however, the inhibitory
activity of Compounds 8e–g was lower than that of Compounds 8b–d
even though Compound 8g exhibited the highest SI (6.08).
REMA-
MIC100
(μg/ml)
MTT IC50,
REMA-MBC
Vero cells
Compound
(μg/ml)
(μg/ml)
SI
8a
>500
7.8
ND
356
0.71
2.60
2.00
3.46
0.39
1.49
6.08
0.784
2.73
12.38
7.42
1.25
16.54
7.22
>16,666
The data for Compounds 8a, 8e, 8f, and 8 g suggest that the anti-
tuberculosis activity can be explained in terms of the distribution of
electronic density across the ring joined to N-4 of the thiosemicar-
bazides (Balaban, Oniciu, & Katritzky, 2004): 3-nitrobenzoyl (8g)
> 4-nitrothiophenoyl (8f) > 4-nitropyrroloyl (8e) > 4-nitrofuranoyl (8d).
Furthermore, we examined the influence of the electron-donating
−OMe group on the antituberculosis activity by synthesizing Com-
pound 8h. Surprisingly, 8h did not inhibit the growth of M. tuberculosis
cells. When the 4-OMe substituent was replaced with a 4-chloro group,
the generated Compound 8i exhibited inhibition levels similar to those
of 8g (R = 3-nitrophenyl) but with a lower SI (SI[8g] = 6.08 vs. SI
[8i] = 2.73). Therefore, we analyzed the role the chloro substituent
plays in the antituberculosis activity by synthesizing compounds with
chloro groups in different positions on their phenyl rings. The incorpo-
ration of a chloro at Position 3 of the D ring of Compound 8i produced
Compound 8j, which exhibits antituberculosis activity equal to that of
8i (MIC = 31.25 μg/ml) but is less toxic (SI[8j] = 12.38 vs. SI[8i] = 2.73).
By contrast, the inhibitory activity of Compound 8k, generated by the
incorporation of a chloro at Position 3 of the E ring of Compound 8j,
was one-half that of 8j, and 8k was also more toxic. These results sug-
gest that derivatization of the E phenyl ring with chloro substituents is
not a suitable approach to maximizing the antituberculosis activity of
thiosemicarbazide compounds.
8b
8c
ND
20.3
15.6
27.0
98.4
186
7.8
15.6
ND
8d
8e
7.8
250
125
31.25
>500
31.25
31.25
62.5
15.6
7.8
ND
8f
125
62.5
ND
8g
190
8h
8i
392
ND
85.3
387
8j
31.25
ND
8k
464
8l
15.6
15.6
ND
78.3
129
8m
8n
Rifampin
7.8
56.3
>1,000
0.06
ND
Note: M. tuberculosis H37Rv ATCC 27294 reference strain: Mtb. MIC:
minimal inhibitory concentration, as determined using the resazurin
microtiter assay (REMA). IC50: half-maximal inhibitory concentration, as
determined by carrying out an MTT assay on Vero cells. MBC: minimal
bactericidal activity. Selectivity index (SI) = IC50/MIC100
.
process that included esterification of acids 12a–n under acidic condi-
tions and hydrazination of the resultant methyl esters (Jorge et al.,
2011) (Scheme 1).
Complete removal of the chloro substituents on the E phenyl ring
resulted in Compound 8l, whose growth inhibition of M. tuberculosis
cells exceeded that of 8k (MIC[8l] = 15.6 vs. MIC[8k] = 62.5); however,
8l was more toxic (SI[8l] = 1.25 vs.SI[8k] = 7.42). Given these results,
we eliminated the 3-chloro substituent from the D phenyl ring of 8l,
resulting in Compound 8m, which displayed an antituberculosis activity
twice that of 8l (MIC[8m] = 7.8 vs. MIC[8l] = 15.6) and was less toxic
(SI[8m] = 16.54 vs. SI[8l] = 1.25). Finally, we eliminated all of the chloro
substituents on Compound 8m, thereby generating 8n, which exhibited
an MIC of 7.8 μg/ml but was more toxic than 8m. Additionally, in the
developing of new antituberculosis drugs, the synergisms between con-
ventional drug treatment and new compound are relevant because this
combination could decrease the toxicity for the long duration of the
treatment (Santos et al., 2018). In the present study, 8m combination
with RIF improves the activity of RIF in M. tuberculosis H37Rv ATCC
27294 susceptible strain with a FICI = 0.149.
3.2
|
In vitro antituberculosis activity
We evaluated the activities of Compounds 8a–n as inhibitors of the
growth of the M. tuberculosis strain H37Rv in 250–0.98 μg/ml serial
twofold dilutions. RIF is usually indicated for the treatment of Mycobac-
terium infections, including TB and the antituberculosis activities of syn-
thesized Compounds 8a–n were compared with the activity of RIF.
4
|
DISCUSSION
We assessed the effects of isosteric replacement of the 1-[4-
chlorophenylbenzoyl] group of Compound 8a, which led to Com-
pounds 8b–d, on the antituberculosis activity (Table 1). Our results
demonstrate that this modification resulted in compounds with anti-
tuberculosis activity superior to that of Compound 8a; however, the
SI of Compounds 8b–d was low (2.60, 2.0, and 3.46, respectively). To
develop compounds with greater antituberculosis activity and
higher SI values than Compounds 8a–d, we also bioisosterically
substituted the 4-(5-nitrofuranoyl) group with 4-(5-nitropyrroloyl) (8e),
4-(5-nitrothiophenoyl) (8f), and 4-(5-nitrobenzoyl) (8g) groups.
5
|
CONCLUSION
The present results indicate that removal of the oxadiazole moiety
from lead Compound 7 results in a Compound (8a) with substantially
lower antituberculosis activity. In addition, isosteric modification of
the 4-chlorophenyl ring (A) of Compound 8a yields compounds with
better antituberculosis activity than 8a but greater toxicity. Likewise,

6
MARTÍNEZ ET AL.
Medicinal Chemistry, 19(16), 5031–5038. https://doi.org/10.1016/j.
bmc.2011.06.034
the bioisosteric modification of the 2-nitrofuran ring (C) gives com-
pounds slightly active but also more toxic. We investigated the role
that the chloro group plays in the antituberculosis activity by synthe-
sizing Compounds 8i–n. As shown in Table 1, the chloro derivative
8m was the most active of the halogenated compounds. These results
suggest that derivatization of the 4-phenyl group (D) with a 4-chloro
substituent results in thiosemicarbazide compounds with the best
antituberculosis performance. Therefore, our results suggest that this
new cytotoxic compound might be useful as a lead compound for
the development of a novel antituberculosis agent. Future studies
will focus on determining the mechanism by which these new
thiosemicarbazides inhibit the growth of M. tuberculosis bacteria.
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ACKNOWLEDGMENTS
Financial support from the DGAPA and UNAM (projects PAPIIT
IN208015 and NUATEI-IIB-UNAM) is gratefully acknowledged. We
also thank R. Patiño, A. Peña, E. Huerta, B. Quiroz, L. Velasco, J. Pérez,
and E. Segura Salinas for technical support.
Mossman, T. (1983). Rapid colorimetric assay for cellular growth and sur-
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CONFLICT OF INTEREST
The authors declare no conflicts of interest.
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Paneth, A., Plech, T., Kapron, B., Hagel, D., Kosikowska, U., Kusmierz, E., …
Paneth, P. (2016). Design, synthesis and biological evaluation of
4-benzoyl-1-dichlorobenzoylthiosemicarbazides as potent Gram-
positive antibacterial agents. Journal of Enzyme Inhibition Medicinal
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1036050
ORCID
Roberto Martínez
https://orcid.org/0000-0001-7342-9332
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
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How to cite this article: Martínez R, Espitia-Pinzón CI, Silva
Miranda M, et al. Synthesis and antituberculosis activity of
new acylthiosemicarbazides designed by structural
modification. Drug Dev Res. 2019;1–6. https://doi.org/10.
1002/ddr.21626
Jorge, S. D., Palace, B. F., Masunari, A., Cechinel, C. A., Ishii, M.,
Mesquita, P. K. F., & Costa, T. L. (2011). Novel benzofuroxan deriva-
tives against multidrug-resistant Staphylococcus aureus strains: Design
using Topliss' decision tree, synthesis and biological assay. Bioorganic &