New quinolone-based thiosemicarbazones showing activity against plasmodium falciparum and mycobacterium tuberculosis

Article abstract of DOI:10.3390/molecules24091740

Co-infection of malaria and tuberculosis, although not thoroughly investigated, has been noted. With the increasing prevalence of tuberculosis in the African region, wherein malaria is endemic, it is intuitive to suggest that the probability of co-infection with these diseases is likely to increase. To avoid the issue of drug-drug interactions when managing co-infections, it is imperative to investigate new molecules with dual activities against the causal agents of these diseases. To this effect, a small library of quinolone-thiosemicarbazones was synthesised and evaluated in vitro against Plasmodium falciparum and Mycobacterium tuberculosis, the causal agents of malaria and tuberculosis, respectively. The compounds were also evaluated against HeLa cells for overt cytotoxicity. Most compounds in this series exhibited activities against both organisms, with compound 10, emerging as the hit; with an MIC₉₀ of 2 μM against H37Rv strain of M. tuberculosis and an IC₅₀ of 1 μM against the 3D7 strain of P. falciparum. This study highlights quinolone-thiosemicarabazones as a class of compounds that can be exploited further in search of novel, safe agents with potent activities against both the causal agents of malaria and tuberculosis.

Full text of DOI:10.3390/molecules24091740

molecules
Article
New Quinolone-Based Thiosemicarbazones Showing
Activity Against Plasmodium falciparum and
Mycobacterium tuberculosis
1
,
2
3
3,4,5
Richard M. Beteck * , Ronnett Seldon , Audrey Jordaan , Digby F. Warner
,
Heinrich C. Hoppe 6
,7
, Dustin Laming 7 and Setshaba D. Khanye
1,7,
*
1
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Rhodes University,
Grahamstown 6140, South Africa
Drug Discovery and Development Centre (H3-D), Department of Chemistry, University of Cape Town,
Rondebosch 7701, South Africa; ronnett.seldon@uct.ac.za
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology,
University of Cape Town, Observatory 7925, South Africa; audrey.jordaan@uct.ac.za (A.J.);
Digby.Warner@uct.ac.za (D.F.W.)
2
3
4
5
6
7
Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Observatory 7925, South Africa
Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town,
Observatory 7925, South Africa
Department of Biochemistry and Microbiology, Faculty of Science, Rhodes University,
Grahamstown 6140, South Africa; h.hoppe@ru.ac.za
Centre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown 6140, South Africa;
dustinlaming89@gmail.com
*
Correspondence: richmbi1@yahoo.com (R.M.B.); s.khanye@ru.ac.za (S.D.K.); Tel.: +27-46-603-8397 (S.D.K.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 9 March 2019; Accepted: 9 April 2019; Published: 4 May 2019
Abstract: Co-infection of malaria and tuberculosis, although not thoroughly investigated, has been
noted. With the increasing prevalence of tuberculosis in the African region, wherein malaria is
endemic, it is intuitive to suggest that the probability of co-infection with these diseases is likely to
increase. To avoid the issue of drug-drug interactions when managing co-infections, it is imperative
to investigate new molecules with dual activities against the causal agents of these diseases. To this
effect, a small library of quinolone-thiosemicarbazones was synthesised and evaluated in vitro against
Plasmodium falciparum and Mycobacterium tuberculosis, the causal agents of malaria and tuberculosis,
respectively. The compounds were also evaluated against HeLa cells for overt cytotoxicity. Most
compounds in this series exhibited activities against both organisms, with compound 10, emerging as
the hit; with an MIC90 of 2 µM against H37Rv strain of M. tuberculosis and an IC50 of 1 µM against
the 3D7 strain of P. falciparum. This study highlights quinolone-thiosemicarabazones as a class of
compounds that can be exploited further in search of novel, safe agents with potent activities against
both the causal agents of malaria and tuberculosis.
Keywords: Co-infections; thiosemicarbazones; quinolones; malaria; Mycobacterium tuberculosis
1
. Introduction
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis, primarily a
pulmonary infection in humans [
1]. This bacterium currently infects about one-third of the world
population [ ]; however, a healthy immune system suppresses growth and multiplication of Mtb to an
2
extent that the bacterium remains inactive and unable to cause pathology [3]. This is the case for 90%
of people harbouring Mtb [4].
Molecules 2019, 24, 1740; doi:10.3390/molecules24091740
www.mdpi.com/journal/molecules

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When the immune system becomes suppressed or weakened as is the case during treatment of
rheumatoid arthritis, HIV infection, and old age, Mtb reactives, and consequently latent tuberculosis
is converted to active pulmonary tuberculosis [
state include diabetes and malnutrition [ ]. People with active pulmonary tuberculosis suffer from
symptoms of the disease [ ] and can infect others [ ]. An estimated 10.0 million people worldwide were
reported to be infected by active pulmonary tuberculosis, and 1.3 million people died as consequence in
017 [ ]. The disease is currently treated using a first line regiment of four drugs (rifampicin, isoniazid,
5]. Other factors promoting conversion to the active
6
7
8
2
9
pyrazinamide, ethambutol), which must be taken daily for at least six months [10]. The current
management and/or treatment of tuberculosis is complicated, and the situation has been worsened
by the emergence and spread of multi-drug resistant and extensively drug resistant forms of Mtb
strains [11]. There is therefore a great need to research new chemical agents with the potential to inhibit
Mtb infection.
On the other hand, malaria is currently the third leading cause of deaths from a single infective
agent. It caused ill health in 216 million people, and reportedly led to the death of 445,000 people in
2
9
016 [12]. The disease is the biggest burden to Sub-Sahara Africa, wherein 88% of malarial cases and
0% of the reported deaths occurred [13]. Plasmodium falciparum is one of the five Plasmodium species
responsible for malaria infection in humans [14]. It is the most virulent of all species [15] and accounts
for 90 – 99% of the global malaria cases [16]. Although the death toll due to malaria has witnessed
a decline over the past fifteen years [17], malaria is still a great global health challenge, with at least
40% of the world‘s population currently living in places endemic to this disease [18] and 90 countries
still having ongoing malaria transmission [16]. The current scenario of malaria is worrisome, and
is characterised by the emergence and spread of parasites resistant to all current treatment options,
including artemisinins, the current treatment gold standard [19,20]. Artemisinin combination therapy
(ACTs) is the recommended treatment for uncomplicated malaria [21], while quinine or artesunate
administered parenterally is recommended for the treatment of complicated malaria [22].
A great common trait between malaria and tuberculosis is the development of resistance. A careful
look into the history of malarial chemotherapy and treatment of tuberculosis suggests that the
pathogens responsible for these diseases have an inherent ability to develop resistance over any drug
used against them, irrespective of whether the drug(s) is used in mono- or combination therapy [23,24].
It is noteworthy to emphasise that these two diseases share common endemicity and the issue of
co-infection with both diseases has been reported [25]. With increasing prevalence of TB in the African
region (the area with the highest malaria prevalence), there is likely to be a high prevalence of TB and
malaria co-infection. Furthermore, it has been noted that malaria causes a deterioration in TB treatment
outcome [25] and commonly deployed anti-malarials such as chloroquine and quinine are counter
indicative with fluoroquinolones, second line anti-TB agents [26]. Taking these in consideration,
it should be intuitive to search for new molecules with the potential to act as anti-malarial and
anti-TB agents.
Thioacetazone (1, Figure 1), a thiosemicarbazone-containing compound, is a potent anti-TB drug.
Inherent toxicities issues associated with the use of this drug especially in situations of HIV infection,
a common TB co-infection, has jeopardised further clinical deployment of this drug [27]. Other
thiosemicarbazone containing compounds (2, Figure 1) have been reported to exhibit anti-malarial
activity [28]. Fluoroquinolones (3, Figure 1), a sub-class of quinolones, are recommended second line
therapy for TB [29]. More importantly, fluoroquinolones have also been reported for their anti-malarial
potential (
4, Figure 1) [30]. In recent work, we reported quinolones incorporating a hydrazide-hydrazone
moiety ( , Figure 1), which exhibited potent activity against Mtb [31]. Considering these literatures
5
precedents, we reasoned that design and development of novel compounds incorporating both
thiosemicarbazone and quinolone frameworks might be worth exploiting as potential compounds
to target both malaria and TB infections. Herein, we report the synthesis of heterocyclic compounds
with thiosemicarbazone and quinolone motifs at their core structure. In vitro biological evaluations
showcase these compounds as potent inhibitors of both P. falciparum and M. tuberculosis.

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Figure 1. Thiosemicarbazones and quinolones exhibiting antimalarial and anti-TB activity.
. Results and Discussion
2
2
.1. Chemistry
Our target compounds were obtained following the synthetic routes presented in Scheme 1
below. Briefly, the reduction of p-nitroacetophenone using reduced iron powder and ammonium
chloride in refluxing ethanol afforded p-aminoacetophenone . Compound was condensed with
diethyl ethoxy methylenemalonate, and following a classical Gould-Jacobs cyclization procedure for
quinolone synthesis [32], later cyclised in boiling diphenylether to obtain intermediate in 50–70%
yields. Compound , just like most quinolones reported in literature, is practically not soluble in any
6
7
7
8
8
solvent [32]; this partly could be attributed to pi-pi stacking promoted by the highly planar nature
of this fragment in particular, and the quinolone nucleus in general. It was therefore essential to
modify this fragment in such a way as to disrupt planarity, and/or introduce hydrophilic moieties
before appending a thiosemicarbazone unit, this was achieved through N-alkylation of the secondary
amine and aminolysis of the ester functionalities in
8. N-alkylation of the secondary amine was
achieved following treatment with alkyl/aryl halides under refluxing DMF using K CO as a base to
2
3
afford intermediate
hydrophilic amines in the presence of DBU under refluxing conditions to obtain 10 as amide derivative
in 40% yield. Treatment of , or 10 with equimolar amounts of thiosemicarbazide and a few drops of
acetic acid gave target compounds 11–21 in 60–80% yields. The structures of target compounds were
9 in 50–70% yields. Compound 9 was subjected to aminolysis by treatment with
9
1
13
confirmed using H and C NMR, high resolution mass spectrometry analysis also confirmed the
pseudomolecular ion peaks consistent with structures of achieved compounds. (See Supplementary
1
Materials). The signal appearing at ca 10.11 ppm on the H NMR spectra of compound 11
–19 is
1
indicative of the presence of an amide proton (-CONH-), this signal is absent in the H NMR spectra
of compound 20 and 21, both of which lack the corresponding amide. The peak at ca 10.37 ppm on
1
the H NMR spectra of all target compounds is attributable to the hydrazinic proton (-C(=S)NH-N=).
The thioamide protons (-C(=S)NH ) could be observed at two different chemical shifts, ca 8.3 ppm
2
and 8.00 ppm. These differences in chemical shift to protons attached to the same atom have been
attributed to the lack of free rotation brought about by the formation of carbon-nitrogen double bond
character around C-N bond of the thioamide [33].

Molecules 2019, 24, 1740
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Scheme 1. Synthesis of target compounds 11
NH Cl; (ii) Acetonitrile, diethyl ethoxymethylenemalonate, reflux 12 h; (iii) Diphenyl ether, 250 C,
–
21a. a Reagents and conditions: (i) Reduced Fe powder,
◦
4
5
min; (iv) K CO , DMF, alkyl/arylhalide (1.2-5 eqv), 7 h; (v) Amine (5 eqv), DBU (1.2 eqv), CHCl ,
2 3 3
reflux 12 h; thiosemicarzide, AcOH (cat), EtOH, reflux overnight.
2
.2. Pharmacology
All target compounds were screened for in vitro inhibitory activity against the 3D7 strain of
P. falciparum and the M. tuberculosis H37Rv strain. Target compounds were also evaluated in vitro
against human cervix adernocarcinoma (HeLa) cell for overt cytotoxicity. Emetine, a cytotoxic agent,
was used as a reference in the latter assay. At 20 µM, none of the compounds suppressed HeLa cell
viability below 80%, suggesting that they pose little cytotoxicity risk at this concentration. Target
compounds were evaluated for their potential to inhibit the growth of M. tuberculosis in vitro using
middlebrook 7H9 media supplemented with casitone, glucose and tyloxpol [34]. The H37Rv strain
of Mtb was deployed in this study, and rifampicin was used as a reference. The anti-Mtb activity of
compounds in this study is reported as the minimum inhibitory concentration required to inhibit 90%
(
MIC ) of the bacteria population and the data is summarised in Table 1 below. Except compound 11,
90
all compounds were active against Mtb; exhibiting activity in the range of 2–100 µM.
The anti-Mtb potential of this series appeared to be strongly influenced by the nature of substituents
at position -1 and -3 of the quinolone ring. For-example, comparing compound 20 (MIC ; 102.5 M)
M) indicates that an amide at position -3 promotes activity over an
ester group. This observation is also evident when comparing compound 21 (MIC ; 31.6 M) with
M). Structure-activity relationship analysis further suggests that activity
µ
90
with compound 14 (MIC ; 10.3
µ
90
µ
90
compound 16 (MIC ; 4.8
µ
9
0

Molecules 2019, 24, 1740
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increases with increasing hydrophilicity of the amide moiety at position -3. This is observed when
comparing the structure and activity of, for example, compound 17 (MIC ; 44.2 M) with compound
M) versus compound 12 (MIC ; 73.4 M). Moreover,
M) with compound 12 (MIC90;
3.4 µM) highlights that the presence of benzyl unit at position -1 seems to be favoured over an alkyl
µ
90
1
3
(MIC ; 2.7
µ
M), compound 11 (MIC ; >125
µ
µ
9
0
90
90
comparing the structure and activity of compound 13 (MIC ; 2.7
µ
90
7
chain. There, is however, lack of a clear correlation of how the substitution pattern on the benzyl unit
influences activity.
Table 1. In vitro antiplasmodial and antimycobacterial activities of quinolone-based thiosemicarbazones.
MIC90 (µM)
IC50 (µM)
Pf 3D7
Compound
ClogP a
H37Rv
1
1
1
1
1
1
1
1
1
2
2
1
2
3
4
5
6
7
8
9
0
1
>125
73.4
2.7
10.3
2.0
3.7
na
−0.02
−1.17
0.18
0.95
0.75
1.38
1.07
1.84
2.27
2.55
3.52
-
4.1
4.7
1.2
3.1
24.6
9.9
na
na
na
0.012
-
4.8
44.2
15.0
10.2
102.5
31.6
-
CQ
RF
0.062
-
a
Calculated using ACD Chemsketch freeware version 12.0, na = not active, RF = Rifampicin, CQ = Chloroquine.
To investigate anti-plasmodial activity, target compounds were screened in vitro against the
chloroquine sensitive strain (3D7) of P. falciparum in a pLDH assay using chloroquine as the reference.
Activity of these compounds together with the reference compound is reported as IC , concentration
50
required to inhibit 50% parasite growth and is incorporated in Table 1. Most compounds in this series
exhibited activity in the low micromolar range (IC50 less than 5 M). The anti-plasmodial activity of
this series follows the same pattern noted for anti-Mtb activity. For example, comparing compound
(not active against 3D7) and (IC ; 4 M), both of which differ only on the nature of substituent
µ
1
2
8
µ
50
at position -1, suggest that a benzyl unit at this position leads to favourable anti-plasmodial activity.
Compound 20, bearing an ethyl ester at position -3, is devoid of activity, while compound 14, its amide
derivative, exhibited an IC50 value of 4
µM. This observation points to the fact that an amide at this
position greatly enhances activity over an ester moiety.
3
. Materials and Methods
3
.1. General Information
Chemicals and solvents deployed in this study were purchased from various chemical vendors:
Sigma-Aldrich (Pty) Ltd. (Johannesburg, South Africa), Merck (Pty) Ltd. (Johannesburg, South Africa)
and were used as purchased. The progress of the reactions was monitored by thin layer chromatography
(
TLC) using Merck 60F254 silica gel plates (Merck, Johannesburg, South Africa) supported on aluminum
and the plates were visualized under ultraviolet (UV 254 and 366 nm) light and in iodine flasks. Where
necessary, the crude compounds were purified by means of a silica gel column chromatography using
1
Merck Kieselgel 60 Å: 70–230 (0.068–0.2 mm) silica gel mesh (Merck, Johannesburg, South Africa). H
and 13C NMR spectra were recorded on Bruker Biospin 300 MHz spectrometer, and the chemical shifts
(δ) are given in values referenced to deuterated DMSO-d and are reported in parts per million (ppm).
6
1
13
Proton chemical shifts for deuterated DMSO appear at 2.5 ppm for H and 39.5 ppm for C-NMR.

Molecules 2019, 24, 1740
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Proton coupling patterns are abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), and
m (multiplet). Coupling constant (J) are reported in Hz. The high-resolution mass spectrometric data
(
HRS-MS) of final compounds were recorded on Waters Synapt G2 Mass Spectrometer (Stellenbosch
University, South Africa) using electrospray ionization (ESI) in the positive ionization mode. Melting
points were obtained using a Reichert hot stage microscope and are reported as obtained. The starting
compounds 7–10 were synthesized from the commercial accessible p-nitroacetophenone 6 as previously
described in the literature [34]. Purity was determined by HPLC (Agilent, Santa Clara, CA, USA), and
all compounds were confirmed to have purity >95% using a similar method previously described [35].
3
.2. General Synthetic Procedure for the Quinolone-Thiosemicarbazone Derivatives, 11–21
In a round bottom flask containing 400 mg of compound or 10 dissolved in 95% ethanol (20 mL)
9
was added 1.2 equivalence of thiosemicarbazide followed by a few drops of glacial acetic acid. The
reaction mixture was stirred under reflux overnight, left to cool to room temperature. The formed
precipitates were filtered, washed with 2
compounds, 11 – 21, in 60–80% yields.
×
10 mL ice cold ethanol and air dried to obtain target
(
3
E)-N-(3-(1H-imidazol-1-yl)propyl)-6-(1-(2-carbamothioylhydrazono)ethyl)-1-ethyl-4-oxo-1,4-dihydroquinolone-
-carboxamide, 11. Brown powder. Yield = 80% (0.35 g). m.p.: 187–189 C. H-NMR (300 MHz,
◦
1
DMSO-d6)
δ
H
(ppm): 10.37 (s, 1H, CSNHN), 10.04 (t, J = 5.8 Hz, 1H, CONH), 8.86 (s, 1H, Ar-H), 8.69 –
8
7
2
.47 (m, 3H, Ar-H), 8.36 (s, 1H, NH), 8.03 (s, 1H, NH), 7.83 (d, J = 9.2 Hz, 1H, Ar-H), 7.68 (s, 1H, Ar-H),
.22 (s, 1H, Ar-H), 4.53 (q, J = 6.9 Hz, 2H, -CH2CH ), 4.03 (t, J = 6.9 Hz, 2H, -CH2-), 3.60–3.29 (m, 6H,
3
×
-CH overlap with dH O), 2.40 (s, 3H, CH ), 1.39 (t, J = 6.9 Hz, 3H, -CH2CH3). 13C NMR (75 MHz,
2, 2 3
DMSO-d )
δ (ppm): 179.9, 173.1, 165.4, 148.1, 146.4, 140.0, 137.4, 134.6, 131.4, 128.3, 126.9, 124.7, 120.1,
C
6
1
4
17.9, 111.9, 56.3, 48.2, 44.6, 35.5, 15.2, 14.2. m/z (ESI-MS) found 440.1860, calcd for C H N O S
21 26 7 2
40.1869 [M + H] . HPLC purity > 96%, rt = 8.2 min.
+
(
-
E)-6-(1-(2-carbamothioylhydrazono)ethyl)-1-ethyl-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1,4-dihydroquinolone
◦
3-carboxamide, 12. Yellow powder. Yield = 60% (0.2 g). m.p.: 183–185 C. H-NMR (300 MHz, DMSO-d )
6
1
δ
H
(ppm): 10.35 (s, 1H, CSNHN), 10.12 (t, J = 4.9 Hz, 1H, CONH), 8.86 (s, 1H, Ar-H), 8.69 – 8.47 (m, 2H,
Ar-H), 8.36 (s, 1H, NH), 8.03 (s, 1H, NH), 7.83 (d, J = 8.9 Hz, 1H, Ar-H), 5.31 (s, 1H, OH), 4.53 (q, J = 6.8
Hz, 2H, -CH2CH ), 3.74 – 3.54 (m, 5H, -NH-, 2 -CH -), 3.21–2.95 (m, 4H, 2 -CH -), 2.39 (s, 3H, CH ),
×
×
3
2
2
3
13
1
.43 (t, J = 6.8 Hz, 3H, -CH2CH3). C-NMR (75 MHz, DMSO-d ) δ (ppm): 179.4, 175.4, 165.9, 148.7,
6 C
1
4
46.8, 140.0, 135.1, 131.9, 127.9, 124.7, 117.9, 111.6, 56.8, 49.5, 49.1, 46.9, 35.9, 15.1, 14.2. m/z (ESI-MS) found
+
19.1971, calcd for C H N O S 419.1966 [M + H] . HPLC purity > 96%, rt = 6.4 min.
19
27
6
3
(
3
δ
E)-1-benzyl-6-(1-(2-carbamothioylhydrazono)ethyl)-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1,4-dihydroquinolone-
◦
-carboxamide, 13. White powder. Yield = 70% (0.34 g). m.p.: 197–199 C. H-NMR (300 MHz, DMSO-d )
6
1
(ppm): 10.35 (s, 1H, CSNHN), 10.10 (t, J = 5.6 Hz, 1H, CONH), 9.08 (s, 1H, Ar-H), 8.64 – 8.44 (m, 2H,
H
Ar-H), 8.32 (s, 1H, NH), 8.02 (s, 1H, NH), 7.67 (d, J = 9.2 Hz, 1H, Ar-H), 7.47 – 7.10 (m, 5H, Ar-H), 5.83 (s,
H, -CH2Ar), 5.30 (s, 1H, OH), 3.94 – 3.51 (m, 5H, -NH-, 2 -CH -), 3.16 (t, J = 5.6 Hz, 2H, -CH2-), 3.05 (t,
J = 6.7 Hz, 2H, -CH2-), 2.36 (s, 3H, -CH3). 13C-NMR (75 MHz, DMSO-d )
2
×
2
δ
C
(ppm): 179.9, 175.8, 165.9,
6
1
4
49.1, 146.4, 139.9, 136.8, 135.5, 131.4, 129.6, 129.2, 128.3, 127.9, 127.4, 126.9, 124.22, 119.2, 111.9, 56.8, 56.3,
+
9.9, 46.8, 35.9, 14.2. m/z (ESI-MS) found 481.1930, calcd for C H N O S 481.1925 [M+H] . HPLC purity
24
29
6
3
>
96%, rt = 8.5 min.
(
E)-1-(4-bromobenzyl)-6-(1-(2-carbamothioylhydrazono)ethyl)-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1,4-
◦
1
dihydroquinolone-3-carboxamide, 14. White powder. Yield = 80% (0.34 g). m.p.: 200–202 C. H-NMR
300 MHz, DMSO-d6) (ppm): 10.36 (s, 1H, CSNHN), 10.11 (t, J = 5.8 Hz, 1H, CONH), 9.08 (s, 1H,
(
δ
H
Ar-H), 8.61 – 8.44 (m, 2H, Ar-H), 8.33 (s, 1H, NH), 8.01 (s, 1H, NH), 7.64 (d, J = 9.8 Hz, 1H, Ar-H), 7.55
d, J = 8.4 Hz, 2H, Ar-H), 7.19 (d, J = 8.4 Hz, 2H, Ar-H), 5.82 (s, 2H, -CH2Ar), 5.27 (s, 1H, OH), 3.77 –
.63 (m, 5H, -NH-, 2 -CH -), 3.16 (t, J = 6.1 Hz, 2H, -CH -), 3.09 – 3.00 (m, 2H, -CH -), 2.36 (s, 3H,
CH3). 13C-NMR (75 MHz, DMSO-d )
(
3
-
×
2
2
2
δ (ppm): 179.5, 175.4, 165.4, 146.4, 144.1, 140.0, 136.4, 135.1,
H
6

Molecules 2019, 24, 1740
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1
5
34.6, 132.4, 129.6, 127.9, 124.2, 121.9, 118.3, 111.9, 56.3, 55.4, 50.0, 47.2, 35.9, 14.2. m/z (ESI-MS) found
+
59.1123, calcd for C H BrN O S 559.1127 [M + H] . HPLC purity > 96%, rt = 9.4 min.
24
28
6
3
(
1
E)-6-(1-(2-carbamothioylhydrazono)ethyl)-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1-(4-(trifluoromethyl)benzyl)-
,4-dihydroquinolone-3-carboxamide, 15. White powder. Yield = 80% (0.35 g). m.p.: 199–201 C. H-NMR
◦
1
(300 MHz, DMSO-d6)
δ
H
(ppm): 10.37 (s, 1H, CSNHN), 10.11 (s, 1H, CONH), 9.12 (s, 1H, Ar-H), 8.56
(s, 1H, Ar-H), 8.32 (s, 1H, CSNH), 8.01 (s, 1H, CSNH), 7.87 – 7.27 (m, 6H, Ar-H), 5.97 (s, 2H, -CH2Ar),
5
.26 (s, 1H, OH), 3.79 – 3.50 (m, 4H, 2 -CH ), 3.10 – 2.87 (m, 5H, -NH-, 2 -CH ), 2.36 (s, 3H, -CH ).
×
×
2
2
3
1
3
C-NMR (75 MHz, DMSO-d6)
δ (ppm): 179.9, 175.8, 165.9, 149.6, 146.4, 141.4, 140.5, 135.5, 135.0, 131.7,
C
1
27.6, 127.5, 126.3, 124.7, 122.9, 118.3, 112.5, 56.8, 55.4, 49.5, 47.3, 35.9, 14.2. m/z (ESI-MS) found 549.1790,
+
calcd for C H F N O S 549.0896 [M + H] . HPLC purity > 96%, rt = 9.4 min.
25
28
3
6
3
(
E)-6-(1-(2-carbamothioylhydrazono)ethyl)-1-(2,4-dichlorobenzyl)-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-
,4-dihydroquinolone-3-carboxamide, 16. Off white powder. Yield = 80% (0.32 g). m.p.: 205–207 C.
◦
1
1
H-NMR (300 MHz, DMSO-d6)
δ (ppm): 10.39 (s, 1H, CSNHN), 10.10 (t, J = 5.0 Hz, 1H, CONH), 9.12
H
(
s, 1H, Ar-H), 8.69 – 8.47 (m, 2H, Ar-H), 8.36 (s, 1H, CSNH), 8.05 (s, 1H, CSNH), 7.78 – 7.50 (m, 3H,
Ar-H), 7.15 (dd, J = 7.8, 5.8 Hz, 1H, Ar-H), 5.85 (s, 2H, -CH2Ar), 5.32 (t, J = 4.7 Hz, 1H, OH), 3.92 – 3.54
m, 5H, -NH-, 2 -CH -), 3.16 (t, J = 5.9 Hz, 2H, -CH -), 3.05 (t, J = 5.0 Hz, 2H, -CH -), 2.36 (s, 3H, -CH ).
(
×
2
2
2
3
1
3
C-NMR (75 MHz, DMSO-d )
δ (ppm): 179.4, 176.3, 165.4, 149.9, 146.4, 145.9, 139.6, 137.7, 135.5,
C
6
1
32.4, 131.9, 131.4, 131.0, 129.6, 127.4, 124.7, 118.8, 111.9, 57.2, 55.4, 49.5, 46.8, 35.9, 14.2. m/z (ESI-MS)
+
found 549.1126, calcd for C H Cl N O S 549.1119 [M + H] . HPLC purity > 96%, rt = 9.1 min.
24
27
2
6
3
(
1
E)-1-benzyl-6-(1-(2-carbamothioylhydrazono)ethyl)-N-(2-methoxyethyl)-4-oxo-1,4-dihydroquinolone-3-carboxamide,
◦
7. Off white powder. Yield = 73% (0.34 g). m.p.: 199–200 C. H-NMR (300 MHz, DMSO-d ) δ (ppm):
6 H
1
1
1
3
(
0.43 (s, 1H, CSNHN), 10.17 (t, J = 5.0 Hz, 1H, CONH), 9.14 (s, 1H, Ar-H), 8.70 – 8.50 (m, 2H, Ar-H), 8.40 (s,
H, NH), 8.11 (s, 1H, NH), 7.73 (d, J = 8.7 Hz, 1H, Ar-H), 7.48 – 7.16 (m, 5H, Ar-H), 5.88 (s, 2H, -CH2Ar),
.66 – 3.52 (m, 4H, 2
-CH -), 3.37 (s, 3H, OCH3), 2.42 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6)
ppm): 179.9, 175.8, 164.4, 146.8, 140.5, 136.8, 134.6, 131.9, 130.1, 129.6, 129.2, 128.3, 127.9, 127.4, 126.9, 125.1,
18.3, 111.6, 71.7, 58.5, 57.2, 38.7, 14.2. m/z (ESI-MS) found 452.1660, calcd for C H N O S 452.0981
×
δ
C
2
1
23
26
5
3
+
[M + H] . HPLC purity > 96%, rt = 9 min.
(
3
E)-1-(4-bromobenzyl)-6-(1-(2-carbamothioylhydrazono)ethyl)-N-(2-methoxyethyl)-4-oxo-1,4-dihydroquinolone-
◦
1
-carboxamide, 18. Brown powder. Yield = 60% (0.25 g). m.p.: 204–206 C. H-NMR (300 MHz, DMSO-d )
6
δ
H
(ppm): 10.34 (s, 1H, CSNHN), 10.07 (t, J = 5.1 Hz, 1H, CONH), 9.09 (s, 1H, Ar-H), 8.64 – 8.47 (m, 2H,
Ar-H), 8.31 (s, 1H, NH), 8.02 (s, 1H, NH), 7.64 – 7.44 (m, 3H, Ar-H), 7.20 (d, J = 8.9 Hz, 2H, Ar-H), 5.80 (s,
H, -CH2Ar), 3.60 – 3.46 (m, 4H, 2
-CH -), 3.26 (s, 3H, OCH3), 2.36 (s, 3H, CH3). 13C-NMR (75 MHz,
DMSO-d6) (ppm): 180.4, 175.8, 164.5, 149.6, 146.4, 139.6, 136.5, 135.1, 132.4, 131.5, 129.2, 127.4, 124.7,
2
×
2
δ
C
1
5
21.5, 118.8, 111.9, 71.3, 58.5, 55.9, 38.7, 13.8. m/z (ESI-MS) found 530.0847, calcd for C H BrN O S
23 25 5 3
30.0861 [M + H] . HPLC purity > 96%, rt = 10.5 min.
+
(
E)-6-(1-(2-carbamothioylhydrazono)ethyl)-1-(2,4-dichlorobenzyl)-N-(2-methoxyethyl)-4-oxo-1,4-dihydroquinolone
◦
1
-3-carboxamide, 19. Brown powder. Yield = 65% (0.27 g). m.p.: 209–211 C. H-NMR (300 MHz, DMSO-d )
6
δ
H
(ppm): 10.34 (s, 1H, CSNHN), 10.05 (s, 1H, CONH), 9.07 (s, 1H, Ar-H), 8.64 – 8.47 (m, 2H, Ar-H), 8.35 (s,
1
6
H, NH), 8.03 (s, 1H, NH), 7.73 – 7.44 (m, 3H, Ar-H), 7.18 (s, 1H, Ar-H), 5.82 (s, 2H, -CH Ar), 3.66 – 3.46 (m,
2
H, 2
×
-CH -, overlap with H O), 3.31 (s, 3H, -OCH3), 2.37 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6)
δ
2 2
179.5, 176.7, 164.1, 146.8, 144.6, 139.9, 138.3, 135.1, 134.6, 132.8, 131.9, 130.5, 129.6, 127.9, 127.4, 124.7, 118.8,
+
112.5, 71.3, 62.6, 58.5, 39.1, 14.7. m/z (ESI-MS): found 520.0857, calcd for C H Cl N O S 520.0861 [M + H] .
23 24 2 5 3
HPLC purity > 96%, rt = 9.4 min.
Methyl-(E)-1-(4-bromobenzyl)-6-(1-(2-carbamothioylhydrazono)ethyl)-4-oxo-1,4-dihydroquinolone-3-carboxylate,
◦
20. Off white powder. Yield = 60% (0.25 g). m.p.: 203–205 C. H-NMR (300 MHz, DMSO-d ) δ (ppm):
6 H
1
10.31 (s, 1H, CSNHN), 8.94 (s, 1H, Ar-H), 8.52 – 8.38 (m, 3H, Ar-H), 8.30 (s, 1H, NH), 7.99 (s, 1H, NH), 7.55
(
3
d, J = 8.7 Hz, 2H, Ar-H), 7.23 (d, J = 8.7 Hz, 2H, Ar-H), 5.69 (s, 2H, -CH2Ar), 3.78 (s, 3H, -OCH3), 2.34 (s,
13
H, -CH3). C-NMR (75 MHz, DMSO-d6) δ (ppm): 179.5, 173.5, 164.9, 150.5, 146.4, 139.2, 135.9, 135.1,
C

Molecules 2019, 24, 1740
8 of 11
1
32.3, 131.05, 129.6, 128.3, 124.7, 121.4, 118.8, 111.0, 55.0, 51.8, 14.7. m/z (ESI-MS) found 487.0437, calcd for
+
C H BrN O S 487.0439 [M + H] . HPLC purity > 96%, rt = 8.5 min.
21
20
4
3
Ethyl-(E)-6-(1-(2-carbamothioylhydrazono)ethyl)-1-(2,4-dichlorobenzyl)-4-oxo-1,4-dihydroquinolone-3-carboxylate,
◦
21. Brown powder. Yield = 60% (0.23 g). m.p.: 211–213 C. H-NMR (300 MHz, DMSO-d ) δ (ppm):
6 H
1
1
–
2
0.35 (s, 1H, CSNHN), 8.92 (s, 1H, Ar-H), 8.58 – 8.41 (m, 2H, Ar-H), 8.33 (s, 1H, NH), 8.03 (s, 1H, NH), 7.75
7.38 (m, 3H, Ar-H), 7.17 (d, J = 9.0 Hz, 1H, Ar-H), 5.72 (s, 2H, -CH2Ar), 4.24 (q, J = 6.7 Hz, 2H, -OCH2-),
13
.34 (s, 3H, CH ), 1.30 (t, J = 6.7 Hz, 3H, -CH ). C-NMR (75 MHz, DMSO-d6)
δ (ppm): 179.5, 173.2,
C
3
3
1
5
64.9, 150.5, 146.4, 140.5, 136.4, 134.6, 133.7, 132.8, 131.9, 129.6, 128.7, 127.9, 126.9, 124.2, 118.4, 111.6, 60.4,
+
4.9, 15.5, 14.2. m/z (ESI-MS) found 491.0610, calcd for C H Cl N O S 491.0615 [M + H] . HPLC purity
22 21 2 4 3
>
96%, rt = 9.4 min.
3
.3. In Vitro Anti-Plasmodial Assay
The 3D7 strain of Plasmodium falciparum was routinely cultured in medium consisting of RPMI1640
containing 25 mM Hepes (Lonza), supplemented with 0.5% (w/v) Albumax II (ThermoScientific),
2
2 mM glucose, 0.65 mM hypoxanthine, 0.05 mg/mL gentamicin and 2–4% (v/v) human erythrocytes.
◦
Cultures were maintained at 37 C under an atmosphere of 5% CO , 5% O , 90% N . To assess
2
2
2
antiplasmodial activity, three-fold serial dilutions of test compounds in culture medium were added to
parasite cultures (adjusted to 2% parasitaemia, 1% haematocrit) in 96-well plates and incubated for
48 h. Duplicate wells per compound concentration were used. Parasite lactate dehydrogenase (pLDH)
enzyme activity in the individual wells was determined as previously described [35,36].
3
.4. In Vitro Cytotoxicity Assay
As previously described [37], HeLa cells (Cellonex) seeded in 96-well plates were incubated with
2
0 µM test compounds for 24 h and cell viability assessed using a resazurin fluorescence assay.
3
.5. In Vitro Antimycobacterial Assay
The minimum inhibitory concentration (MIC) was determined using the standard broth
microdilution method, where a 10 mL culture of Mycobacterium tuberculosis pMSp12::GFP [38], was
grown to an optical density (OD600) of 0.6–0.7. The medium used was Middlebrook 7H9 supplemented
with 0.03% casitone, 0.4% glucose, and 0.05% tyloxapol [39]. Cultures grown in the medium were
diluted 1:500, prior to inoculation in the MIC assay. The compounds to be tested were reconstituted to a
concentration of 10 mM in DMSO. Two-fold serial dilutions of the test compound were prepared across
a 96-well micro titre plate, after which, 50
µL of the diluted Mtb cultures was added to each well in the
serial dilution. Assay controls used were a minimum growth control (Rifampicin at 2
×
MIC), and a
maximum growth control (5% DMSO). The micro titre plates were sealed in a secondary container and
◦
incubated at 37 C with 5% CO and humidification. Relative fluorescence (excitation 485 nM; emission
2
520 nM) was measured using a plate reader (FLUOstar OPTIMA, BMG LABTECH), at day 7 and day
1
4. The raw fluorescence data were archived and analysed using the CDD Vault from Collaborative
Drug Discovery, in which data were normalised to the minimum and maximum inhibition controls to
generate a dose response curve (% inhibition), using the Levenberg-Marquardt damped least squares
method, from which the MIC90 was calculated (Burlingame, CA www.collaborativedrug.com). The
lowest concentration of drug that inhibited growth of more than 90% of the bacterial population was
considered the MIC .
90
4
. Conclusions
In this study, quinolone-thiosemicarbazones were obtained in high yields through simple and cost
effective classical synthetic transformations without the use of specialised equipment and expensive
catalysts. The compounds reported herein showed no growth inhibition of HeLa cells at a concentration
of 20 µM suggesting the general lack of cytotoxicity by this series. More importantly, this series

Molecules 2019, 24, 1740
9 of 11
exhibited good activities against P. falciparum, and M. tuberculosis, the pathogens responsible for malaria,
and tuberculosis, respectively. Overall, the presented data suggest that quinolone-thiosemicarbazones
are a compound class worth further exploitation in search of cheap molecules with dual activities
against the aforementioned diseases-causing microorganisms.
Supplementary Materials: The following are available online ( H and 13C NMR spectra, and HRMS spectra of
1
compounds 11–21).
Author Contributions: R.M.B. and S.D.K. conceptualized target compounds. R.M.B. executed the synthesis and
compiled the paper. R.S., A.J. and D.F.W. coordinated and performed the anti-mycobacterial assays. H.C.H. and
D.L. coordinated and performed the anti-plasmodial and cytotoxicity assays. S.D.K coordinated the investigation.
Funding: The authors acknowledge the financial support by the National Research Foundation (SDK, Grant
umber 107270), Rhodes University for Postdoctoral funding (RMB) and Rhodes University Sandisa Imbewu (SDK
and HCH) towards this research. The antiplasmodial bioassay component of the project was funded by the South
African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness
and Support Package. All TB screening work was conducted in the MMRU (UCT) with the support of SAMRC
through the Strategic Health Innovation Partnerships (SHIP) initiative (DFW). Our great appreciation to Christian
Nkanga, Department of Chemistry, Rhodes University for HPLC purity data as well as Stellenbosch University
Central for Analytical Facility (CAF) for mass spectrometric analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).