Synthesis of thiazolyl hydrazonothiazolamines and 1,3,4-thiadiazinyl hydrazonothiazolamines as a class of antimalarial agents

Article abstract of DOI:10.1002/ardp.201900079

Novel thiazolyl hydrazonothiazolamines and 1,3,4-thiadiazinyl hydrazonothiazolamines were synthesized by a facile one-pot multicomponent approach by the reaction of 2-amino-4-methyl-5-acetylthiazole, thiosemicarbazide or thiocarbohydrazide and phenacyl br

Full text of DOI:10.1002/ardp.201900079

Received: 26 March 2019
Revised: 14 August 2019
Accepted: 1 September 2019
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DOI: 10.1002/ardp.201900079
F U L L P A P E R
Synthesis of thiazolyl hydrazonothiazolamines and
1,3,4‐thiadiazinyl hydrazonothiazolamines as a class
of antimalarial agents
Kodam Sujatha1
Rajeswar Rao Vedula1
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Naidu Babu Ommi2
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Anwita Mudiraj2
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Phanithi Prakash Babu2
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1Department of Chemistry, National Institute
of Technology, Warangal, Telangana, India
Abstract
2Department of Biotechnology and
Bioinformatics, School of Life Sciences,
University of Hyderabad, Hyderabad,
Telangana, India
Novel thiazolyl hydrazonothiazolamines and 1,3,4‐thiadiazinyl hydrazonothiazola-
mines were synthesized by a facile one‐pot multicomponent approach by the reaction
of 2‐amino‐4‐methyl‐5‐acetylthiazole, thiosemicarbazide or thiocarbohydrazide and
phenacyl bromides or 3‐(2‐bromoacetyl)‐2H‐chromen‐2‐ones in acetic acid with good
to excellent yields. These new compounds were screened in vitro for their
antimalarial activity; among them, four compounds, 4h, 4i, 4k, 4l, showed moderate
activity with half‐maximal inhibitory concentration (IC50) values of 3.2, 2.7, 2.7, and
2.8 and 3.2, 3.2, 3.1, and 3.5 μM against chloroquine‐sensitive and ‐resistant strains of
Plasmodium falciparum, respectively. Compound 4l inhibited the ring stage growth of
P. falciparum 3D7 at an IC90 concentration of 12.5 µM in a stage‐specific assay
method, where the culture is incubated with specific stages of P. falciparum for 12 hr,
and no activity was found against the trophozoite and schizont stages, confirming
that 4l may have potent action against the ring stage of P. falciparum.
Correspondence
Phanithi Prakash Babu, Department of
Biotechnology and Bioinformatics, School of Life
Sciences, University of Hyderabad, Hyderabad,
Telangana 500046, India.
Email: prakash@uohyd.ac.in
Rajeswar Rao Vedula, Department of Chemistry,
National Institute of Technology, Warangal,
Telangana 506004, India.
Email: rajeswarnitw@gmail.com
K E Y W O R D S
2‐amino‐4‐methyl‐5‐acetylthiazole, antimalarial activity, bithiazoles, multicomponent reactions,
thiazolothiadiazines
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1
INTRODUCTION
modern organic synthesis. MCRs are good synthetic approaches for
functionalized heterocyclic compounds without any side products.[3,4]
Malaria is a parasitic disease transmitted to humans by the female
Anopheles mosquito and continues to remain a lethal infectious
disease. According to WHO 2017 report,[1] an estimated 216 million
cases of malaria occurred worldwide in 2016, and India accounts for
6% of it. Plasmodium falciparum, the most virulent species of this
parasite has developed resistance to most available antimalarial
Thiazole scaffolds (Figure 1) are found in many natural products[5]
and possess diverse medicinal and pharmaceutical applications such as
antitubercular,[6,7] anticonvulsant,[8] anticancer,[9] antiviral,[10] antimi-
crobial,[11] antimalarial[12] and anti‐inflammatory activity.[13]
Coumarin is an important pharmacophore having many applica-
tions in the fields of medicinal chemistry as well as pharmaceuticals
like antifungal,[14] anti‐HIV agents,[15] anti‐Alzheimers[16]and also acts
as a luminescent material.[17] When the coumarin ring is attached with
the thiazole ring, it exhibits improved biological activities like being an
anti‐inflammatory and anti‐analgesic agent.[18] On the other hand, thia-
diazines are also versatile biologically important heterocyclic mole-
cules[19] with proven applications as antidepressant,[20] antihyperten-
sive[21] and antiproliferative agents.[22]
drugs. This has been
a constant challenge to malaria control
initiatives necessitating the search for novel and structurally diverse
antimalarial drugs as a viable strategy to combat this issue.
Multicomponent reactions (MCRs) are modern methods for the
synthesis of drug molecules.[2] The advantages of MCRs are
convergent, one pot and sequential assembling of starting materials
to get the final product in a short time. MCRs play a vital role in
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Arch Pharm Chem Life Sci. 2019;e1900079.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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Micrococcinic acid
Antibiotic
Mycobacterium tuberculosis
Gyr B inhibitor
Wang 354
Antiviral agent
Anti-malarial
Thia-netropsin
Antitumor agent
FIGURE 1 Biologically active molecules
Furthermore, thiazoles attached to coumarins, thiadiazine, and
thiosemicarbazones are documented to exhibit an enhanced spec-
trum of biological activities.[23,24] To the best of our knowledge,
coumarinyl hydrazino thiazole and coumarinyl hydrazino thiadiazine
moieties and their antiplasmodial activity are hitherto unreported.
In continuation of our earlier work on nitrogen and sulphur
heterocycles,[25,26] we are now reporting the synthesis of thiazolyl
hydrazonothiazolamines and 1,3,4‐thiadiazinyl hydrazonothiazola-
mines having aryl/3‐coumarinyl moieties via a one‐pot multi-
component method and their antimalarial activity against P.
falciparum in vitro.
thiosemicarbazide, phenacyl bromide, or 3‐(2‐bromoacetyl)‐2H‐
chromen‐2‐ones in good yields.
This is a three‐component condensation reaction. In this reaction,
the 2‐amino‐4‐methyl‐5‐acetylthiazole first reacts with thiosemicar-
bazide to give the corresponding thiosemicarbazone of 2‐amino‐
4‐methyl‐5‐acetylthiazole. The in‐situ formed thiosemicarbazones
react with phenacyl bromides or 3‐(2‐bromoacetyl)‐2H‐chromen‐2‐
ones to give final product 4 by Hantzsch thiazole synthesis (Scheme 1).
In contrast, 2‐amino‐4‐methyl‐5‐acetylthiazole on reaction with
thiocarbohydrazide gave the thiocarbohydrazone of 2‐amino‐4‐
methyl‐5‐acetylthiazole. This undergoes cyclization with α‐halo
ketones such as phenacyl bromides or 3‐(2‐bromoacetyl) coumarins
to yield the target compound 6, as given in Scheme 2. The advantage
of our reaction is that without isolation of intermediate thiosemi-
carbazone of 1 or thiocarbohydrazone of 1, we have synthesized the
final products (4 and 6) in a single step. In addition to this, the
reaction involves concomitant formation of C═N, C–N, and C–S
bonds.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Thiazolyl hydrazonothiazolamines were synthesized by a one‐pot
multicomponent method using 2‐amino‐4‐methyl‐5‐acetylthiazole,
SCHEME 1 Synthesis of thiazolyl hydrazonothiazolamines

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SCHEME 2 Synthesis of 1,3,4‐thiadiazinyl hydrazonothiazolamines
The structures of all the newly synthesized compounds were
confirmed by their spectral data and are summarized in the
Supporting Information. The infrared (IR) spectrum of compound 4f
showed peaks at 3,436 and 1,622 cm−1 due to amino and –C═N–
groups, respectively. The 1H NMR spectrum of the compound 4f
showed a characteristic singlet peak at δ 7.12 ppm corresponding to
the newly formed thiazole CH proton and the remaining protons
were observed in the usual expected region. 13C NMR of compound
4f gave a characteristic peak at 103.4, which further confirms the
newly formed thiazole carbon. The compound 4f exhibited the
molecular ion peak at m/z 344 (M+1)+.
compounds showed IC50 at 3.12 µM concentration against P.
falciparum 3D7 culture (SI data). In general, the thiazolyl hydrazo-
nothiazolamine series having halogenated and electron‐rich coumarin
substituents showed good activity compared with simple phenyl
substituted thiazolyl hydrazonothiazolamines. Presence of electron‐
withdrawing –NO2 group on the benzene ring in 4e compound
caused a complete loss of antimalarial activity in the 3D7 strain
compared with analogs having halogen atoms and electron‐donating
groups. In contrast, the simple phenyl or coumarin substituted 1,3,4‐
thiadiazinyl hydrazonothiazolamine series showed no activity. Only
three compounds from this series (6b, 6c, and 6e) showed activity
around 6.15–6.25 µM against the CQ‐sensitive strain of P. falciparum.
Hence, in both series, the active compounds were 4h, 4i, 4k, and 4l.
The IC50 of chloroquine against 3D7 is 26 2.5 nM and that of Dd2 is
184 10.6 nM; whereas the antimalarial activity of these four
compounds cannot be compared to that of chloroquine, the
functional group modifications at thiazole or coumarin moiety have
tremendous prospects in further development and this work is in
progress in this laboratory.
The IR spectrum of compound 6a showed prominent peaks at
3,400 and 1,605 cm−1 for amino and –C═N– groups, respectively.
The 1H NMR spectrum of the compound 6a showed a characteristic
singlet peak at
δ 3.94 ppm, corresponding to newly formed
thiadiazine CH2 protons, and the remaining protons were observed
in the usual expected region. 13C NMR of compound 6a gave a
characteristic peak at 22.2, corresponding to newly formed thiadia-
zine CH2 carbon. Compound 6a exhibited a molecular ion peak at m/z
343 (M−1)+.
To check the toxicity of the compounds against normal
cell cytotoxicity, tests were carried out against mice macrophage
J774.2 cells using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetra-
zolium bromide (MTT) colorimetric method. Compounds 4i and 4k
had a cytotoxic concentration 50 (CC50) of 500 µM, whereas 4h and
4l showed a CC50 of 250 µM, respectively (Table 2).
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2.2
Biological evaluation
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2.2.1
In vitro antimalarial activity and cytotoxicity
of compounds
The antimalarial property of all derivatives were evaluated in vitro
against the chloroquine (CQ)‐resistant (Dd2) and CQ‐sensitive (3D7)
strains of P. falciparum parasite. Thirteen compounds exhibited half‐
maximal inhibitory concentration (IC50) values below 6.30 μΜ against
both sensitive and resistant 3D7 and Dd2 strains. All IC50 values
against CQ‐sensitive and ‐resistant strains are tabulated in Table 1.
Compound 6g showed an IC50 value of 12.5 0.12 μΜ and the
remaining nine compounds showed no activity (>24 μΜ) in the 3D7
strain. Four compounds 4h, 4i, 4k, 4l showed reasonable activity with
an IC50 value of 3.2, 2.7, 2.7, and 2.8 µM against the CQ‐sensitive
strain of P. falciparum. These compounds also showed inhibition of
the CQ‐resistant strain with an IC50 value of 3.25, 3.25, 3.13, and
3.5 µM (Table 1). Furthermore, lactate dehydrogenase (LDH) assay
was conducted to measure the cytotoxicity of 4h, 4i, 4k, and 4l
against synchronized P. falciparum 3D7 culture in vitro. These four
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2.2.2
Inhibition of β‐hematin crystallization
Hemozoin formation is unique to Plasmodium. P. falciparum is an
intraerythrocytic parasite that grows and multiplies by converting
the toxic by‐product heme (ferriprotoporphyrin IX) released from
hemoglobin degradation into nontoxic hemozoin. This occurs in the
digestive food vacuole of the parasite, and inhibition of hemozoin
formation proves fatal for the parasite, and thus forms an attractive
antimalarial drug target. The inhibition of β‐hematin formation was
evaluated for active compounds. Results showed that compound 4h
inhibited β‐hematin formation with an IC50 value of 62.5 µM similar
to that of chloroquine, whereas the other compounds did not show
any activity against β‐hematin crystallization like the negative control
of pyrimethamine (Figure 2).

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TABLE 1 Compound structures (4a–l, 6a–k) and half‐maximal inhibitory concentration (IC50) values with 3D7 strain and Dd2 strain
Compound code
Compound structure
IC50 (µM)
IC50 (µM)
3D7 strain
Dd2 strain
4a
6.37 0.87
6.783 0.21
4b
5.375 0.37
6.23 0.15
4c
5.7917 0.39
5.85 0.83
4d
5.375 0.37
6.25 0.41
4e
>50
nd
4f
4.166 0.83
5.23 0.35
4g
4.166 0.83
5.12 0.20
(Continues)

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TABLE 1 (Continued)
Compound code
Compound structure
IC50 (µM)
IC50 (µM)
3D7 strain
Dd2 strain
4h
3.208 0.43
3.25 0.88
4i
2.706 0.20
3.251 0.42
4j
5.375 0.37
6.12 0.20
3.138 0.42
3.521 0.31
4k
2.708 0.20
4l
2.85 0.20
6a
25
nd
(Continues)

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TABLE 1 (Continued)
Compound code
Compound structure
IC50 (µM)
IC50 (µM)
3D7 strain
Dd2 strain
6b
6.15 0.28
7.32 0.43
6c
6.15 0.10
6.534 0.22
6d
26 0.44
nd
6e
6.25 0.78
nd
6f
25.8 0.26
nd
6g
12.5 0.12
nd
(Continues)

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TABLE 1 (Continued)
Compound code
Compound structure
IC50 (µM)
IC50 (µM)
3D7 strain
Dd2 strain
6h
26.2 0.40
nd
6i
24 0.36
nd
6j
25.2 0.24
nd
6k
24 0.13
nd
Note: IC50 of chloroquine against 3D7 is 26 2.5 nM and Dd2 is 184 10.6 nM.
Abbreviation: nd, not determined.
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2.2.3
Effect of compound 4l on the growth of P.
(UR + 4l) when compared to control. The survived parasites of ring
stage treated and unremoved cultures were further grown and
infected fresh red blood cell (RBC) forming rings at 48 hr after
removal of the compound (Figure 3b), but we found they could not
grow further, and the morphology remained at the ring stage even
after 60 and 72 hr when compared with the control. The control
attained the trophozoite stage at a similar cycle time. Trophozoite
(T + 4l) and schizont (S + 4l) stage treated parasites have more
parasitemia than the ring stage treated culture and their growth
morphology is similar to control at 60 and 72 hr. The compounds 4h
and 4k have shown ring stage growth inhibition of the parasite in
unremoved culture (S.I) and because of their highest IC90
falciparum 3D7 culture
To check the growth inhibition activity of compound 4l against P.
falciparum, the parasite culture was treated with an IC90 concentration
of 12.5 µM at their corresponding stages, that is, ring (R), trophozoite
(T), and schizont (S) stages for 12 hr. The compound was removed after
12 hr by several washes and supplemented with complete Roswell
Park Memorial Institute (RPMI) media for further growth of the
parasite. The parasitemia was estimated from Giemsa stained smears.
Figure 3a shows that there is no increase of parasitemia at 48 hr in the
ring stage treated culture (R + 4l) similar to unremoved culture

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4
EXPERIMENTAL
Chemistry
TABLE 2 Antimalarial activities of active compounds against
blood‐stage parasites and in vitro cytotoxicity
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IC50 (µM)a
4.1
CC50 (µM)b
Compound
pf3D7
pfDd2
J774.2
250
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4.1.1
General
4h
4i
3.208 0.43
2.706 0.20
2.708 0.20
2.85 0.20
3.25 0.88
3.251 0.42
3.138 0.42
3.521 0.31
3‐(2‐Bromoacetyl) coumarins were prepared by bromination of
different 3‐acetyl coumarins in dry chloroform. The remaining
chemicals used in the present work were purchased from
commercial sources and used without any purification. Melting
points were determined in open capillaries with a Stuart melting
point apparatus (Mumbai, India) and were uncorrected. IR
spectra were recorded on a PerkinElmer Spectrum 100 s (Perkin
Elmer corporate office Waltham, MA). 1H NMR spectra were
recorded on a Bruker WM‐400 spectrometer in δ ppm using TMS
as the standard (Switzerland), ESI‐MS spectra were recorded on
a Jeol JMSD‐300 spectrometer (Tokyo, Japan). Elemental ana-
lyses were performed on a Carlo Erba EA 1108 automatic
elemental analyzer (France), compound purity was checked
with TLC plates (E. Merck, Mumbai, India).
500
4k
4l
500
250
Abbreviations: CC50, cytotoxic concentration 50; IC50, half‐maximal
inhibitory concentration.
aBlood‐stage antiplasmodial activity was determined against the CQ‐
sensitive strain 3D7 and CQ‐resistant strain Dd2 of P. falciparum.
bCytotoxicity was determined against a mouse macrophage (J774.2) cell
line; data are expressed as the CC50, which is the concentration required
to reduce cell viability by 50%.
concentration they did not show growth inhibition in transiently
treated cultures at the specific stage of the parasite with 12.5 µM
concentration. These results show that the compound 4l is more active
in inhibiting the growth of the ring stage of the P. falciparum 3D7.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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3
CONCLUSIONS
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4.1.2
General procedure for the synthesis of
compounds 4
In conclusion, we have developed novel thiazolyl hydrazonothia-
zolamines and 1,3,4‐thiadiazinyl hydrazonothiazolamine deriva-
tives via a one pot multicomponent approach and evaluated their
antimalarial activity. Four compounds showed good antimalarial
potency with low cytotoxicity as observed in macrophage cell
lines. The method had the advantages of mild reaction conditions,
easy workup, no column chromatographic purification and good
to better yields.
An equimolar amount of 2‐amino‐4‐methyl‐5‐acetylthiazole (1 mmol),
thiosemicarbazide (1 mmol), and phenacyl bromide (1 mmol) or 3‐(2‐
bromoacetyl)‐2H‐chromen‐2‐one (1 mmol) was taken in
a round
bottom flask and refluxed in acetic acid (2 ml) at 70°C for about 2 hr.
The progress of the reaction was monitored through TLC using ethyl
acetate and n‐hexane (40%). After completion of the reaction, the solid
separated was filtered, dried and recrystalized from methanol to give 4.
(E)‐4‐Methyl‐5‐(1‐(2‐(4‐phenylthiazol‐2‐yl)hydrazono)ethyl)thiazol‐
2‐amine (4a)
Yield 90%. White solid, mp: 195–196°C; IR (KBr, νmax, cm−1): 3,448
(NH2 stretching), 1,619 (–C═N– stretching), 1,578 (C═C); 1H NMR
(400 MHz, DMSO‐d6, ppm): δ 2.33 (s, 3H, CH3), 2.41 (s, 3H, CH3),
7.30–7.33 (m, 2H), 7.42 (t, J = 8.0 Hz, 2H), 7.85 (d, J = 8.0 Hz, 2H),
9.39 (s, 2H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.1, 15.8, 104.4,
119.1, 125.9, 128.1, 129.1, 133.8, 134.5, 141.9, 149.8, 167.8, 169.5;
MS (ESI) m/z: 330 (M+H)+; Anal. calcd. for C15H15N5S2: C, 54.69; H,
4.59; N, 21.26; S, 19.47. Found: C, 54.62; H, 4.63; N, 21.21; S, 19.42.
(E)‐5‐(1‐(2‐(4‐(4‐Fluorophenyl)thiazol‐2‐yl)hydrazono)ethyl)‐4‐
methylthiazol‐2‐amine (4b)
Yield 81%. White solid, mp: 211–213°C; IR (KBr, νmax, cm−1): 3,442
(NH2 stretching), 1,621 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.33 (s, 3H, CH3), 2.41 (s, 3H, CH3), 7.25 (t,
J = 8.8 Hz, 2H), 7.32 (s, 1H), 7.41 (t, J = 8.8 Hz, 1H), 7.87–7.90 (m, 2H),
7.95–7.99 (m, 1H), 13.36 (s, 1H); 13C NMR (100 MHz, DMSO‐d6,
ppm): δ 14.1, 15.8, 115.6, 115.8, 116.0, 116.4, 116.7, 128.0, 130.0,
130.1, 150.8, 164.5, 167.7; MS (ESI) m/z: 348 (M+H)+; Anal. calcd. for
FIGURE 2 β‐Hematin crystallization assay. NP‐40 polymerizes
free synthetic hemin into β‐hematin, which was dissolved in 0.36 M
NaOH, 2% (wt/vol) sodium dodecyl sulfate and has absorbance at
400 nm. Increasing the concentration of 4h inhibits the
polymerization reactions, which have shown less absorbance and are
plotted against the respective concentration

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FIGURE 3 Effect of short term treatment of compound 4l at various plasmodial stages. (a) Growth of parasitemia after treatment with
compound 4l at various Plasmodium falciparum 3D7 growth stages; 12 hr postinfection cultures were treated with compound 4l at various stages
corresponding to ring (R + 4l), trophozoite (T + 4l) and schizont (S + 4l) stages. After that, the compound was removed and the parasitemia was
monitored for up to 72 hr postinfection. Results are represented as the mean of three individual experiments standard error. (b) Giemsa
stained images represent the morphology of the parasite at the corresponding stages. Representation used is R + 4l (closed circles), T + 4l (open
circles), S + 4l (open square), unremoved 4l (open diamond), control (closed diamond)
C15H14FN5S2: C, 51.85; H, 4.06; N, 20.16; S, 18.46. Found: C, 51.81;
H, 4.12; N, 20.20; S, 18.41.
DMSO‐d6, ppm): δ 2.31 (s, 3H, CH3), 2.41 (s, 3H, CH3), 7.40 (s, 1H),
7.50–7.53 (m, 1H), 7.70 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 9.37 (s, 2H),
11.56 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.3, 15.8,
110.1, 118.8, 127.9, 130.2, 132.0, 132.4, 132.6, 133.0, 134.6, 141.9,
146.05, 167.7, 168.7; MS (ESI) m/z: 416 (M+NH4)+; Anal. calcd. for
(E)‐5‐(1‐(2‐(4‐(2,4‐Dichlorophenyl)thiazol‐2‐yl)hydrazono)ethyl)‐4‐
methylthiazol‐2‐amine (4c)
Yield 87%. White solid, mp: 170–172°C; IR (KBr, νmax, cm−1): 3,448
(NH2 stretching), 1,624 (–C═N– stretching); 1H NMR (400 MHz,
C15H13Cl2N5S2: C, 45.23; H, 3.29; N, 17.58; S, 16.10. Found: C, 45.27;
H, 3.23; N, 17.52; S, 16.17.

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(E)‐5‐(1‐(2‐(4‐(4‐Bromophenyl)thiazol‐2‐yl)hydrazono)ethyl)‐4‐
methylthiazol‐2‐amine (4d)
11.59 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.5, 15.8,
111.6, 116.3, 118.9, 119.5, 120.8, 125.2, 129.2, 132.2, 135.0, 138.5,
152.7, 159.1, 167.7, 168.9; MS (ESI) m/z: 398 (M+1)+; Anal. calcd. for
C18H15N5O2S2: C, 54.39; H, 3.80; N, 17.62; S, 16.13. Found: C, 54.33;
H, 3.84; N, 17.67; S, 16.17.
Yield 92%. White solid, mp: 185–187°C; IR (KBr, νmax, cm−1): 3,427
(NH2 stretching), 1,624 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3), 2.40 (s, 3H, CH3), 7.41 (s, 1H),
7.61 (d, J = 11.6 Hz, 2H), 7.78–7.83 (m, 3H), 9.29 (s, 2H); 13C NMR
(100 MHz, DMSO‐d6, ppm): δ 13.8, 15.3, 104.9, 120.6, 127.5, 128.9,
131.5, 132.0, 133.5, 133.9, 141.4, 164.0, 167.3, 169.1, 171.9; MS
(ESI) m/z: 410 (M+2)+; Anal. calcd. for C15H14BrN5S2: C, 44.12; H,
3.46; N, 17.15; S, 15.71. Found: C, 44.16; H, 3.41; N, 17.11; S, 15.76.
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
thiazol‐4‐yl)‐6‐bromo‐2H‐chromen‐2‐one (4i)
Yield 91%. White solid, mp: 209–211°C; IR (KBr, νmax, cm−1): 3,400
(NH2 stretching), 1,718 (lactone C═O stretching), 1,629 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3),
2.40 (s, 3H, CH3), 7.42 (d, J = 8.8 Hz, 1H), 7.75–7.77 (m, 1H), 7.83 (s,
1H), 8.07 (s, 1H), 8.44 (s, 1H), 9.20 (s, 2H), 11.62 (s, 1H); 13C NMR
(100 MHz, DMSO‐d6, ppm): δ 14.4, 15.9, 112.6, 116.9, 118.5, 121.5,
130.9, 131.2, 134.4, 136.9, 137.4, 144.2, 151.7, 158.6, 167.7, 169.0,
169.8, 172.5; MS (ESI) m/z: 476 (M)+; Anal. calcd. for
C18H14BrN5O2S2: C, 45.38; H, 2.96; N, 14.70; S, 13.46. Found: C,
45.41; H, 3.00; N, 14.73; S, 13.41.
(E)‐4‐Methyl‐5‐(1‐(2‐(4‐(4‐nitrophenyl)thiazol‐2‐yl)hydrazono)ethyl)‐
thiazol‐2‐amine (4e)
Yield 90%. White solid, mp: 207–209°C; IR (KBr, νmax, cm−1): 3,430
(NH2 stretching), 1,638 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.29 (s, 3H, CH3), 2.35 (s, 3H, CH3), 7.70 (s, 1H),
8.07–8.15 (m, 4H), 8.28 (d, J = 8.8 Hz, 2H), 11.37 (s, 1H); 13C NMR
(100 MHz, DMSO‐d6, ppm): δ 16.2, 16.4, 109.3, 118.8, 124.5, 126.7,
141.1, 143.2, 146.6, 155.2, 156.7, 167.4, 170.1; MS (ESI) m/z: 375
(M+1)+; Anal. calcd. for C15H14N6O2S2: C, 48.11; H, 3.77; N, 22.44; S,
17.13. Found: C, 48.16; H, 3.71; N, 22.40; S, 17.10.
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
thiazol‐4‐yl)‐6‐chloro‐2H‐chromen‐2‐one (4j)
Yield 90%. White solid, mp: 203–205°C; IR (KBr, νmax, cm−1): 3,397
(NH2 stretching), 1,723 (lactone C═O stretching), 1,623 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3),
2.39 (s, 3H, CH3), 7.49 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 9.2 Hz, 1H), 7.83
(s, 1H), 7.96 (s, 1H), 8.46 (s, 1H), 8.96 (s, 2H), 11.57 (s, 1H); 13C NMR
(100 MHz, DMSO‐d6, ppm): δ 14.6, 16.0, 102.0, 105.9, 109.0, 112.5,
112.7, 118.2, 119.0, 120.9, 128.0, 131.6, 135.3, 137.3, 144.4, 151.3,
159.9, 169.1; MS (ESI) m/z: 432 (M+1)+; Anal. calcd. for
C18H14ClN5O2S2: C, 50.05; H, 3.27; N, 16.21; S, 14.85. Found: C,
50.12; H, 3.24; N, 16.16; S, 14.88.
(E)‐4‐Methyl‐5‐(1‐(2‐(4‐(p‐tolyl)thiazol‐2‐yl)hydrazono)ethyl)thiazol‐
2‐amine (4f)
Yield 85%. White solid, mp: 213–215°C; IR (KBr, νmax, cm−1): 3,436
(NH2 stretching), 1,622 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.78 (s, 3H,
CH3), 6.97 (d, J = 8.8 Hz, 2H), 7.12 (s, 1H), 7.77 (d, J = 8.8 Hz, 2H), 9.38
(s, 2H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.1, 15.8, 21.2,
103.4, 125.9, 129.6, 130.0, 131.8, 133.7, 142.1, 151.7, 167.7, 169.2,
169.8; MS (ESI) m/z: 344 (M+1)+; Anal. calcd. for C16H17N5S2: C,
55.95; H, 4.99; N, 20.39; S, 18.67. Found: C, 55.91; H, 4.92; N, 20.31;
S, 18.64.
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
thiazol‐4‐yl)‐6,8‐dibromo‐2H‐chromen‐2‐one (4k)
(E)‐5‐(1‐(2‐(4‐(4‐Methoxyphenyl)thiazol‐2‐yl)hydrazono)ethyl)‐4‐
methylthiazol‐2‐amine (4g)
Yield 85%. White solid, mp: 238–240°C; IR (KBr, νmax, cm−1): 3,392
(NH2 stretching), 1,723 (lactone C═O stretching), 1,630 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.31 (s, 3H, CH3),
2.39 (s, 3H, CH3), 7.83 (s, 1H), 8.08–8.11 (m, 2H), 8.38 (s, 1H), 9.07 (s,
2H), 11.58 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.6, 15.8,
110.2, 113.2, 116.9, 119.0, 122.4, 130.5, 135.3, 136.1, 136.3, 141.8,
143.9, 148.4, 157.9, 167.7, 168.9, 172.3; MS (ESI) m/z: 555 (M)+;
Anal. calcd. for C18H13Br2N5O2S2: C, 38.93; H, 2.36; Br, 28.78; N,
12.61; S, 11.55. Found: C, 38.97; H, 2.31; N, 12.66; S, 11.50.
Yield 82%. White solid, mp: 188–190°C; IR (KBr, νmax, cm−1): 3,420
(NH2 stretching), 1,615 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.78 (s, 3H,
OCH3), 3.89 (s, 1H, NH), 6.97 (d, J = 8.8 Hz, 2H), 7.12 (s, 1H), 7.77 (d,
J = 8.8 Hz, 2H), 9.38 (s, 2H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ
14.1, 15.7, 55.6, 102.1, 114.4, 115.1, 119.1, 127.2, 130.5, 132.4,
133.2, 133.7, 141.9, 159.3, 167.8, 169.5; MS (ESI) m/z: 360
(M+1)+; Anal. calcd. for C16H17N5OS2: C, 53.46; H, 4.77; N, 19.48;
S, 17.84. Found: C, 53.49; H, 4.71; N, 19.42; S, 17.88.
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
thiazol‐4‐yl)‐8‐methoxy‐2H‐chromen‐2‐one (4l)
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
thiazol‐4‐yl)‐2H‐chromen‐2‐one (4h)
Yield 86%. White solid, mp: 216–218°C; IR (KBr, νmax, cm−1): 3,407
(NH2 stretching), 1,710 (lactone C═O stretching), 1,624 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3),
2.39 (s, 3H, CH3), 3.93 (s, 3H, OCH3), 7.33 (t, J = 7.2 Hz, 3H), 7.80 (s,
1H), 8.15 (s, 1H), 8.51 (s, 1H), 9.14 (s, 2H); MS (ESI) m/z: 426 (M−1)+;
Anal. calcd. for C19H17N5O3S2: C, 53.38; H, 4.01; N, 16.38; S, 15.00.
Found: C, 55.32, H, 4.12; N, 16.32; S, 14.96.
Yield 93%. White solid, mp: 255–257°C; IR (KBr, νmax, cm−1): 3,401
(NH2 stretching), 1,706 (lactone C═O stretching), 1,577 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3),
2.40 (s, 3H, CH3), 7.40 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.64
(t, J = 7.2 Hz, 1H), 7.81 (t, J = 7.6 Hz, 2H), 8.54 (s, 1H), 9.17 (s, 2H),

SUJATHA ET AL.
11 of 13
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CH2 of thiadiazine), 7.48–7.51 (m, 3H), 7.75 (s, 1H), 11.43 (s, 1H); 13
C
|
4.1.3
General procedure for the synthesis of
compounds 6
NMR (100 MHz, DMSO‐d6, ppm): δ 16.4, 17.5, 25.4, 120.2, 128.1,
129.8, 132.5, 132.8, 135.3, 146.3, 149.6, 153.6, 159.9, 162.8, 168.2;
MS (ESI) m/z: 413 (M+1)+; Anal. calcd. for C15H14Cl2N6S2: C, 43.59;
H, 3.41; N, 20.33; S, 15.51. Found: C, 43.55; H, 3.47; N, 20.37; S,
15.56.
Equimolar amounts of 2‐amino‐4‐methyl‐5‐acetylthiazole (1 mmol),
thiocarbohydrazide (1 mmol) and phenacyl bromide (1 mmol) or 3‐(2‐
bromoacetyl)‐2H‐chromen‐2‐one (1 mmol) were taken in a round
bottom flask and refluxed in acetic acid (2 ml) at 70°C for about 2 hr.
The progress of the reaction was monitored through TLC using ethyl
acetate and n‐hexane (40%). After completion of the reaction, the
solid separated was filtered, dried and recrystalized from methanol
to give 6.
(E)‐5‐(1‐(2‐(5‐(4‐Bromophenyl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hydrazono)‐
ethyl)‐4‐methylthiazol‐2‐amine (6e)
Yield 85%. White solid, mp: 193–195°C; IR (KBr, νmax, cm−1): 3,400
(NH2 stretching), 1,624 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.34 (s, 3H, CH3), 2.37 (s, 3H, CH3), 3.92 (s, 2H,
CH2 of thiadiazine), 7.65 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H),
8.03 (s, 2H), 11.46 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ
16.2, 17.0, 21.9, 120.3, 123.3, 128.2, 132.0, 134.7, 143.5, 145.6,
153.1, 159.8, 167.9; Anal. calcd. for C15H15BrN6S2: C, 42.56; H, 3.57;
N, 19.85; S, 15.15. Found: C, 42.62; H, 3.52; N, 19.81; S, 15.19.
(E)‐4‐Methyl‐5‐(1‐(2‐(5‐phenyl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hydrazono)‐
ethyl)thiazol‐2‐amine (6a)
Yield 89%. White solid, mp: 230–232°C; IR (KBr, νmax, cm−1): 3,400
(NH2 stretching), 1,605 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.35 (s, 3H, CH3), 2.39 (s, 3H, CH3), 3.94 (s, 2H,
CH2 of thiadiazine), 7.45–7.46 (m, 3H), 7.79–7.81 (m, 2H), 8.53 (s,
2H), 11.47 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 15.9, 18.7,
22.2, 120.4, 126.3, 129.1, 130.0, 135.3, 147.0, 152.3, 160.9, 168.0,
170.9; MS (ESI) m/z: 343 (M−1)+; Anal. calcd. for C15H16N6S2: C,
52.30; H, 4.68; N, 24.40; S, 18.62. Found: C, 52.34; H, 4.61; N, 24.45;
S, 18.65.
(E)‐5‐(1‐(2‐(5‐(4‐Methoxyphenyl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hydrazo-
no)ethyl)‐4‐methylthiazol‐2‐amine (6f)
Yield 85%. White solid, mp: 240–242°C; IR (KBr, νmax, cm−1): 3,380
(NH2 stretching), 1,604 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.36 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.80 (s, 3H,
OCH3), 3.92 (s, 2H, CH2 of thiadiazine), 7.01 (d, J = 9.2 Hz, 2H), 7.76
(d, J = 9.2 Hz, 2H), 9.07 (s, 2H), 11.42 (s, 1H); 13C NMR (100 MHz,
DMSO‐d6, ppm): δ 14.3, 15.1, 21.8, 55.3, 114.0, 120.0, 127.1, 127.4,
135.8, 146.8, 150.8, 160.4, 161.6, 167.6; MS (ESI) m/z: 375 (M+1)+;
Anal. calcd. for C16H18N6OS2: C, 51.32; H, 4.84; N, 22.44; S, 17.12.
Found: C, 51.35; H, 4.80; N, 22.47; S, 17.16.
(E)‐4‐Methyl‐5‐(1‐(2‐(5‐(4‐nitrophenyl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hy-
drazono)ethyl)thiazol‐2‐amine (6b)
Yield 92%. White solid, mp: 194–196°C; IR (KBr, νmax, cm−1): 3,400
(NH2 stretching), 1,663 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.32 (s, 3H, CH3), 2.37 (s, 3H, CH3), 4.04 (s, 2H,
CH2 of thiadiazine), 8.04 (d, J = 9.2 Hz, 2H), 8.30 (d, J = 9.2 Hz, 2H),
8.66 (s, 2H), 11.84 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ
15.9, 18.6, 29.9, 120.2, 121.7, 124.3, 127.2, 141.5, 143.9, 147.9,
153.1, 157.8, 159.5, 168.1, 170.9; MS (ESI) m/z: 390
(M+1)+; Anal. calcd. for C15H15N7O2S2: C, 46.26; H, 3.88; N, 25.18;
S, 16.47. Found: C, 46.22; H, 3.82; N, 25.14; S, 16.52.
(E)‐5‐(1‐(2‐(5‐([1,11‐Biphenyl]‐4‐yl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hydra-
zono)ethyl)‐4‐methylthiazol‐2‐amine (6g)
Yield 86%. White solid, mp: 236–238°C; IR (KBr, νmax, cm−1): 3,434
(NH2 stretching), 1,627 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.34 (s, 3H, CH3), 2.43 (s, 3H, CH3), 4.01 (s, 2H,
CH2 of thiadiazine), 7.41 (d, J = 6.8 Hz, 2H), 7.49 (t, J = 7.2 Hz, 2H),
7.73–7.79 (m, 4H), 7.91 (d, J = 8.0 Hz, 1H), 9.30 (s, 2H), 11.65 (s, 1H);
13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.3, 16.7, 29.7, 127.1, 128.3,
129.5, 139.6, 168.2, 169.9, 189.5; MS (ESI) m/z: 421
(M+1)+; Anal. calcd. for C21H20N6S2: C, 59.97; H, 4.79; N, 19.98; S,
15.25. Found: C, 60.00; H, 4.13; N, 19.94; S, 15.20.
(E)‐5‐(1‐(2‐(5‐(4‐Fluorophenyl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hydrazono)‐
ethyl)‐4‐methylthiazol‐2‐amine (6c)
Yield 90%. White solid, mp: 204–206°C; IR (KBr, νmax, cm−1): 3,437
(NH2 stretching), 1,626 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.37 (s, 3H, CH3), 2.42 (s, 3H, CH3), 3.96 (s, 2H,
CH2 of thiadiazine), 7.31 (t, J = 8.8 Hz, 2H), 7.84–7.87 (m, 2H), 9.17 (s,
2H), 11.58 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 14.6, 15.6,
22.3, 116.0, 116.2, 120.4, 128.6, 128.7, 131.8, 135.8, 146.2, 151.6,
161.7, 164.6, 168.1; MS (ESI) m/z: 363 (M+1)+; Anal. calcd. for
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
6H‐1,3,4‐thiadiazin‐5‐yl)‐2H‐chromen‐2‐one (6h)
Yield 90%. White solid, mp: 214–216°C; IR (KBr, νmax, cm−1): 3,392
(NH2 stretching), 1,710 (lactone C═O stretching), 1,605 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm):δ; 2.37 (s, 3H, CH3),
2.40 (s, 3H, CH3), 2.43 (s, NH), 3.86 (s, 2H, CH2 of thiadiazine),
7.30–7.40 (m, 3H, ArH), 7.65–7.75 (m, 3H, 1 ArH and NH2), 8.45 (s,
1H, coumarin 4th proton); 13C NMR (100 MHz, DMSO‐d6, ppm): δ
16.6, 17.7, 23.5, 116.1, 118.9, 119.9, 123.7, 125.0, 129.4, 132.7,
141.3, 144.3, 146.7, 153.5, 153.6, 159.2, 159.4, 167.6; MS (ESI) m/z:
C
15H15FN6S2: C, 49.71; H, 4.17; N, 23.19; S, 17.69. Found: C, 49.67;
H, 4.21; N, 23.15; S, 17.73.
(E)‐5‐(1‐(2‐(5‐(2,4‐Dichlorophenyl)‐6H‐1,3,4‐thiadiazin‐2‐yl)hydra-
zono)ethyl)‐4‐methylthiazol‐2‐amine (6d)
Yield 87%. White solid, mp: 211–213°C; IR (KBr, νmax, cm−1): 3,431
(NH2 stretching), 1,627 (–C═N– stretching); 1H NMR (400 MHz,
DMSO‐d6, ppm): δ 2.33 (s, 3H, CH3), 2.36 (s, 3H, CH3), 3.79 (s, 2H,

12 of 13
SUJATHA ET AL.
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413 (M+1)+; Anal. calcd. for C18H16N6O2S2: C, 52.41; H, 3.91; N,
20.37; S, 15.55. Found: C, 52.45; H, 3.95; N, 20.32; S, 15.51.
SYBR Green fluorescence‐based method.[27] Briefly, parasite
culture was synchronized with 5% sorbitol at the ring stage and
further transferred to complete RPMI media till it reached the
mature trophozoites. In 96‐well plates, compounds were incu-
bated with mature trophozoite culture in a proportion of 2%
hematocrit and 1% parasitemia in a total volume of 200 μl for
48 hr in triplicate. After incubation, 100 μl of resuspended culture
was transferred to 96‐well flat‐bottom plates already containing
100 μl of SYBR Green I lysis buffer (2XSYBR Green, 20 mM Tris
base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% wt/vol
Triton X‐100). The plates were incubated for 1 hr at 37°C,
fluorescence associated with SYBR Green I‐intercalated parasitic
DNA was measured at 490 nm excitation and 540 nm emission
using a TECAN Infinite F‐200 spectrophotometer (Tecan Trading
AG, Switzerland) and IC50 of compounds was calculated from
three independent experiments.
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
6H‐1,3,4‐thiadiazin‐5‐yl)‐6‐bromo‐2H‐chromen‐2‐one (6i)
Yield 90%. White solid, mp: 219–221°C; IR (KBr, νmax, cm−1): 3,421
(NH2 stretching), 1,722 (lactone C═O stretching), 1,623 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.37 (s, 3H, CH3),
2.40 (s, 3H, CH3), 3.86 (s, 2H, CH2 of thiadiazine), 7.44 (d, J = 8.8 Hz,
2H), 8.14 (s, 1H), 8.25 (s, 1H), 8.65 (s, 2H), 11.73 (s, 1H); 13C NMR
(100 MHz, DMSO‐d6, ppm): δ 14.7, 15.7, 23.7, 116.9, 11.8, 120.3,
121.2, 125.0, 131.6, 135.3, 140.4, 144.4, 149.6, 152.2, 152.9, 159.2,
161.5, 168.2; MS (ESI) m/z: 490 (M−1)+; Anal. calcd. for
C18H15BrN6O2S2: C, 44.0; H, 3.08; Br, 16.26, N, 17.10; S, 13.05.
Found: C, 39.96; H, 3.12; N, 17.15; S, 12.96.
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
6H‐1,3,4‐thiadiazin‐5‐yl)‐6,8‐dibromo‐2H‐chromen‐2‐one (6j)
Yield 92%. White solid, mp: 226–228°C; IR (KBr, νmax, cm−1): 3,394
(NH2 stretching), 1,730 (lactone C═O stretching), 1,623 (–C═N–
stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.36 (s, 3H, CH3),
2.39 (s, 3H, CH3), 3.85 (s, 2H, CH2 of thiadiazine), 8.05 (s, 1H), 8.16 (s,
1H), 8.28 (s, 1H), 8.72 (s, 2H), 11.75 (s, 1H); 13C NMR (100 MHz,
DMSO‐d6, ppm): δ 15.3, 16.0, 23.6, 110.5, 116.9, 120.4, 122.2, 125.7,
131.3, 135.3, 137.2, 139.8, 141.5, 143.7, 149.8, 153.0, 158.6, 168.1;
MS (ESI) m/z: 571 (M+1)+; Anal. calcd. for C18H14Br2N6O2S2: C,
37.91; H, 2.47; N, 14.74; S, 11.25. Found: C, 37.96; H, 2.42; N, 14.72;
S, 11.29.
|
4.2.2
Cytotoxicity assays (MTT)
Cytotoxicity of compounds was determined by using MTT.[28] The
compounds were seeded at 1 × 104 cells in each well of 96‐well
plates. After 24 hr, the cells were treated with different concentra-
tions of compounds (0.48–500 μM) in twofold serial dilution or
vehicle (0.25% DMSO) for 24 hr. After 24 hr of treatment, 200 µl of
medium containing MTT (0.5 mg/ml) was added to each well of a 96‐
well plate and incubated for 4 hr in a CO2 chamber at 37°C. Reduced
formazan crystals were dissolved in 100 µl of DMSO and absorbance
was measured at 570 nm on a multiplate reader (Tecan infinite‐200).
(E)‐3‐(2‐(2‐(1‐(2‐Amino‐4‐methylthiazol‐5‐yl)ethylidene)hydrazinyl)‐
6H‐1,3,4‐thiadiazin‐5‐yl)‐8‐methoxy‐6‐nitro‐2H‐chromen‐2‐one (6k)
Yield 86%. White solid, mp: 234–236°C; IR (KBr, νmax, cm−1):
3,400 (NH2 stretching), 1,734 (lactone C═O stretching), 1,602
(–C═N– stretching); 1H NMR (400 MHz, DMSO‐d6, ppm): δ 2.32
(s, 3H, CH3), 2.40 (s, 3H, CH3), 3.94 (s, 2H, CH2 of thiadiazine),
4.05 (s, 3H, OCH3), 7.56 (s, 1H), 7.83 (s, 2H), 8.26 (s, 1H), 8.48 (s,
1H), 8.70 (s, 1H); 13C NMR (100 MHz, DMSO‐d6, ppm): δ 16.2,
18.7, 29.8, 56.8, 112.6, 116.7, 119.5, 121.7, 125.7, 139.9, 144.1,
147.3, 148.4, 153.5, 153.9, 158.0, 167.7, 170.9, 188.7; MS (ESI)
m/z: 488 (M+1)+; Anal. calcd. for C19H17N7O5S2: C, 46.81; H,
3.51; N, 20.11; S, 13.15. Found: C, 46.85; H, 3.35; N, 20.15; S,
13.18.
|
4.2.3
assay
NP‐40 mediated β‐hematin crystallization
Compounds were incubated with 100 µM hematin, 1 M acetate
buffer of pH‐4.8 and 30.55 µM NP‐40 in a 96‐well plate at 37°C and
shaken at 55 rpm for 4 hr, the incubation plate was centrifuged at
1,100g for 1 hr at 25°C[29] and after discarding the supernatant,
200 µl of 0.15 M sodium bicarbonate containing 2% sodium dodecyl
sulfate (SDS) was added to each well and centrifugation was
repeated, and the supernatant containing free heme was discarded.
Then, 200 µl 0.36 M sodium hydroxide and 2% SDS was added to
dissolve the synthesized β‐hematin. The absorbance was measured at
400 nm using a multiplate reader (Tecan infinite‐200). β‐Hematin
crystallization inhibition IC50 concentrations of active compounds
were measured by comparing their absorbance values with chlor-
oquine absorbance, a positive control of the β‐hematin crystallization
inhibitor.
|
4.2
Biology
|
4.2.1
Antiplasmodial activity against P. falciparum
3D7 and Dd2 strains
P. falciparum 3D7 and Dd2, chloroquine‐sensitive and ‐resistant
strains, respectively, were maintained at 37°C by the bell jar
candle method in RPMI 1640 medium supplemented with HEPES
25 mM, AlbumAX I 0.5% (wt/vol), hypoxanthine 100 μM, genta-
micin 12.5 μg/ml and sodium bicarbonate 1.77 mM. IC50 of the
compounds against P. falciparum strains was determined using the
|
4.2.4
LDH assay
LDH assay was conducted to measure the cytotoxicity of compounds
4h, 4i, 4k, and 4l against synchronized P. falciparum 3D7 culture in
vitro. The culture was adjusted to 1–1.5% parasitemia and 2%
hematocrit in a 96‐well plate containing serially diluted compounds

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and grown for 48 hr. Later, the percentage of cytotoxicity was
estimated using a Cayman’s LDH cytotoxicity assay kit, where the
LDH released from the P. falciparum due the action compounds
reduces NAD+ to NADH and H+ by oxidation of lactate to pyruvate.
Using NADH and H+, diaphorase reduces a tetrazolium salt (INT) to
colored formazan, which has maximum absorbance at 490–520 nM.
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4.2.5
Stage‐specific growth inhibition assay
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Rajeswar Rao Vedula
http://orcid.org/0000-0002-9762-0868
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