Design, synthesis, in vitro and in vivo biological evaluation of pyranone-piperazine analogs as potent antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2021.113516

The current therapeutic regimen for visceral leishmaniasis is inadequate and unsatisfactory due to toxic side effects, high cost and emergence of drug resistance. Alternative, safe and affordable antileishmanials are, therefore, urgently needed and toward these we synthesized a series of arylpiperazine substituted pyranone derivatives and screened them against both in vitro and in vivo model of visceral leishmaniasis. Among 22 synthesized compounds, 5a and 5g showed better activity against intracellular amastigotes with an IC₅₀ of 11.07 μM and 15.3 μM, respectively. In the in vivo, 5a significantly reduced hepatic and splenic amastigotes burden in Balb/c mice model of visceral leishmaniasis. On a mechanistic node, we observed that 5a induced direct Leishmania killing via mitochondrial dysfunction like cytochrome c release and loss of membrane potential. Taken together, our results suggest that 5a is a promising lead for further development of antileishmanial drugs.

Full text of DOI:10.1016/j.ejmech.2021.113516

European Journal of Medicinal Chemistry 221 (2021) 113516
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, in vitro and in vivo biological evaluation of
pyranone-piperazine analogs as potent antileishmanial agents
,
,
c
,
,
Shachi Mishra a 1, Naveen Parmar b 1, Pragya Chandrakar b c, Chandra Prakash Sharma a,
,
,
,
,
a,
, c, *
Sajiya Parveen c a, Ravi P. Vats c a, Anuradha Seth b c, Atul Goel c **, Susanta Kar b
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow,
226031, India
b Molecular Parasitology & Immunology Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow,
226031, India
c Academy of Scientific and Innovative Research (AcSIR), Sector 19, Kamla Nehru Nagar, Ghaziabad, Uttar Pradesh, 201 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The current therapeutic regimen for visceral leishmaniasis is inadequate and unsatisfactory due to toxic
side effects, high cost and emergence of drug resistance. Alternative, safe and affordable antileishmanials
are, therefore, urgently needed and toward these we synthesized a series of arylpiperazine substituted
pyranone derivatives and screened them against both in vitro and in vivo model of visceral leishmaniasis.
Among 22 synthesized compounds, 5a and 5g showed better activity against intracellular amastigotes
Received 17 July 2020
Received in revised form
23 April 2021
Accepted 24 April 2021
Available online 4 May 2021
with an IC50 of 11.07 mM and 15.3 mM, respectively. In the in vivo, 5a significantly reduced hepatic and
splenic amastigotes burden in Balb/c mice model of visceral leishmaniasis. On a mechanistic node, we
observed that 5a induced direct Leishmania killing via mitochondrial dysfunction like cytochrome c
release and loss of membrane potential. Taken together, our results suggest that 5a is a promising lead
for further development of antileishmanial drugs.
Keywords:
Visceral leishmaniasis (VL)
2-Pyranone
Phenylpiperazine substituted pyranones
Antileishmanial activity
Mitochondrial dysfunction
Apoptosis
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
candidate, chemotherapeutic regimen remains a basis for the
treatment of VL. Pentavalent antimonials (Pentostam and Glu-
Leishmaniasis is a disease caused by obligate intra-macrophage
protozoan parasite that belongs to the genus, Leishmania. Visceral
leishmaniasis (VL) also known as kala azar is mainly caused by
Leishmania donovani in Indian subcontinent and East Africa, and
Leishmania infantum in Europe and Latin America [1]. VL is a lethal
form of leishmaniasis, and is responsible for high mortality and
morbidity rate if left untreated. Since there is no effective vaccines
cantime) were used as a first line treatment for VL for more than
half a century [2], however, rates of resistance have been well
documented and reported to be higher than 60% in parts of the
state of Bihar, in north-east India. In addition, therapeutic options
for VL have been improved by using of oral drug miltefosine, a
variety of liposomal formulations of amphotericin B, aminoglyco-
side antibiotic paromomycin and pentamidine [3]. However,
Abbreviations: CC50, 50% reduction of viability of treated cells with respect to untreated cells; DMSO, dimethyl sulfoxide; FACS, Fluorescence-Activated Cell Sorting; FBS,
fetal bovine serum; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; IC50, half-maximal inhibitory concentration; ip, intraper-
itoneal; IR, infrared spectroscopy; LDU, Leishmania donovani unit; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SI, selectivity index; MMP, mito-
chondrial membrane potential; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; NMR, nuclear magnetic resonance; TLC, thin-
layer chromatography; VL, visceral leishmaniasis.
* Corresponding author. Molecular Parasitology & Immunology Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road,
Lucknow, 226031, India.
** Corresponding author. Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow,
226031, India.
E-mail addresses: atul_goel@cdri.res.in (A. Goel), susantakar@cdri.res.in (S. Kar).
S.M. and N.P. contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113516
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
concerns like teratogenicity, gastrointestinal side effects, high cost,
long elimination half life and parenteral administration for longer
duration are associated with these chemotherapeutic agents [4].
These issues emphasize an urgent need of antileishmanial drug
discovery programme to identify novel, safer and cheaper chemo-
therapeutic regimen against leishmaniasis. L. donovani has the ca-
pacity to successfully survive within hostile environment and
avoiding host immune defense mechanisms by intracellular growth
[5]. Therefore, the introduction of novel synthetic compounds with
improved pharmacological properties and effective killing of
intracellular forms of Leishmania parasite is essential for the
treatment of any form of leishmaniasis. 2-Pyranone belongs to
privileged class of heterocyclic scaffold and due to its pharmaco-
phoric properties, has attracted much attention. Most of the com-
pounds display strong biological activities such as cytotoxic,
antibiotic, antihyperglycemic and antifungal activities [6e9]. Some
pyranones and their isosteres have also been found to exhibit
promising antileishmanial activity (Fig. 1) [10,11]. Further, pipera-
zines are also an important heterocyclic scaffold and several dia-
rylpiperazine analogs possess good antileishmanial activity (Fig. 1)
[12e16]. Therefore, in our endeavor to find new antileishmanials
with better efficacy and less toxicity, we designed, synthesized and
screened a series of arylpiperazine substituted pyranone de-
rivatives against both in vitro and in vivo experimental model of VL.
To further extend the SAR, ring transformation of pyranone core
was done with dihydrothiophen-3(2H)-one and tetrahydro-4H-
thiopyran-4-one.
the conjugate base of 6a,b at the electrophilic C-6 position of
substituted 2H-pyran-2-ones 5a,c,g,i gave the respective ring
transformed products 7a-e in good yields (Scheme 2).
2.1.1. In silico prediction of physicochemical properties of 5a-q and
7a-e
In silico predictions of physicochemical properties, pharmaco-
kinetics and toxicity profiling provide useful insight for drugability
of newly synthesized derivatives. The molecular descriptors such as
TPSA, log P, nRotB, and molecular weight, number of hydrogen
bond donors and acceptors of Lipinski's rule were calculated for all
the synthesized compounds using Molinspiration software [18].
Lipinski's rule of five (RO5) states that most “drug like” molecules
have log P ꢀ 5, molecular weight ꢀ 500, number of hydrogen bond
acceptors <10, and number of hydrogen bond donors ꢀ 5 [19]. The
expansion of these rules evolved the nRot bond and PSA descriptors
in which it is desirable that there are 10 or fewer rotatable bonds
and a polar surface area equal to or less than 140 Å2 for excellent
oral absorption of a drug candidate [20,21]. TPSA is the surface
areas that are occupied by oxygen and nitrogen atoms and by
hydrogen atoms linked to them. Therefore, it is closely related to
the hydrogen bonding potential of a compound. MiLogP (octanol/
water partition coefficient) is calculated by the methodology
developed by Molinspiration as a sum of fragment based contri-
butions and correction factors [18]. In silico study revealed that all
the synthesized compounds (5a-q and 7a-e) have TPSA
values < 140 Å2, nRotB ¼ 2 to 4, hydrogen bond acceptors ¼ 4 to 7,
hydrogen bond donor ¼ 0 to 1, and molecular volume 334 to 432
(Å3) thus, they are foreseen to display good oral absorption. For a
molecule being well absorbed, miLogP value must be in the range of
- 0.4 to þ5. Our results indicated that, pyranone-piperazine series
5a-q (except 5l and 5m) was found to have miLogP values within
the acceptable range and thus they were predicted to have good
oral absorption (Table 1). While in series of 1,3-dihydrobenzo[c]
thiophene-4-carbonitrile (7a-c) and 8-(aryl/heteroaryl)iso-
thiochromane-5-carbonitrile (7d, e), it is noteworthy that all
analyzed compounds have one or two violations of Lipinski's rule
(logP and molecular weight, Table 1) except 7b.
2. Results and discussion
2.1. Chemistry
A series of 2H-pyranones appended with arylpiperazine was
synthesized as depicted in Scheme 1. The precursors 2H-pyranones
derivatives 3a-j were prepared from ketene-S,S-acetal (1) and
substituted cyclic or acyclic methylene carbonyl compounds 2a-j
through carbanion-induced ring formation reaction [17]. Further
for the synthesis of our designed piperazinyl-pyranone hybrid
molecule, the methylsulfanyl group of 3a-j was replaced with
arylpiperazine 4a by refluxing in methanol for 1e4 h to furnish 5a-j
in good yields. In order to establish structure-activity relationships,
different electron withdrawing and electron releasing groups were
incorporated onto the arylpiperazine substrates 4b-d to furnish
pyranone-piperazine derivatives 5k-q. Further, Michael addition of
2.1.2. Theoretical toxicity profling of synthesized compounds 5a-q
and 7a-e
In order to evaluate the fragment-based toxicity risks (muta-
genic, tumorigenic and irritant effects) of the synthesized de-
rivatives and also for comparison with standard drug Miltefosine,
Fig. 1. Strategy for designing pyranone and piperazine (molecular hybrid approach) analogs as Anti-leishmanial agents.
2

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Scheme 1. Synthesis of 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-oxo-6-(aryl/heteroaryl)-2H-pyran-3-carbonitrile (5a-q).
3

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Scheme 2. Synthesis of 5-(4-(2-hydroxyphenyl)piperazin-1-yl)-7-(aryl/hetero-aryl)-1,3-dihydrobenzo[c]thiophene-4-carbonitrile (7a-c) and 6-(4-(2-hydroxy-phenyl)piperazin-1-
yl)-8-(aryl/heteroaryl)lisothiochromane-5-carbonitrile (7d,e).
the online web free software OSIRIS property explorer [22] was
used. This software comprehends the database of known drugs and
nondrug-like Fluka compounds to assess the frequency of each
fragment in the single molecule. All results using OSIRIS software
are shown in Table 2. The theoretical toxicity analysis (tumorigenic,
mutagenic profiles) of the synthesized compounds revealed that
most of the compounds showed no risk of being mutagenic and
tumorigenic effect, like the antileishmanial drug Miltefosine, while
two compounds namely 5f and 7c showed some risk of mutagenic
and tumorigenic effect. All the synthesized compounds were pre-
dicted to have some irritant effect (Table 2). Overall, based on the in
silico studies, it was observed that a series of 5a-q showed good
drug-likeness as compared to the compounds 7a-e.
Initially all the synthesized derivatives were evaluated at 25
mM
and 50 M concentration against both life stages of the parasite
m
taking miltefosine as a reference drug. The anti-leishmanial activity
of all the synthesized compounds is represented in Table 3. We
observed that two compounds 5a and 5g showed more than 80%
inhibition of intracellular amastigotes and extracellular promasti-
gotes at both concentrations (Table 3). In order to establish
structureꢁactivity relationships (SARs), different electron donating
and withdrawing substituents at C4 and C6 positions of pyranones
5a-q were incorporated. So the in vitro antileishmanial activity
profile of the compounds 5a-e revealed that the substitution of
electron withdrawing groups (EWG) such as chloro (5b) and bromo
(5c) to the benzene ring led to marked reduction in both anti-
promastigote and anti-amastigote activity. Again, substitution of
electron donating groups such as methyl (5d) or methoxy (5e) at
the same position (C6) also displayed less parasitic inhibition than
the unsubstituted phenyl moiety (5a). Replacing the phenyl ring
with more bulkier substituent like 1-naphthyl moiety (5f) resulted
2.2. In vitro anti-leishmanial activity against extracellular
promastigotes and intracellular amastigotes
To check the anti-leishmanial potential, we screened the syn-
thesized pyranone-piperazine derivatives of 5a-q and 7a-e against
both extracellular and intracellular stages of Leishmania parasites.
in significant inhibition (78.6 and 80.8%) at 50
mM concentration
against both promastigote and amastigote respectively but the
4

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Table 1
Molecular properties calculated by Molinspiration.
Entry
M.W
LogP
HBA
HBD
TPSA(Å2)
nRotB
Lipinski's Violation
Molecular Volume (Å3)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
373.41
407.86
452.31
387.44
403.44
423.47
379.44
458.34
385.42
399.45
391.86
426.30
470.75
421.88
391.86
393.47
466.33
413.55
419.57
425.56
433.60
506.47
407.58
3.66
4.34
4.47
4.11
3.72
4.82
3.44
4.38
3.74
3.76
4.56
5.24
5.37
4.61
4.58
3.72
4.75
5.02
4.18
5.10
5.01
6.04
ꢁ0.21
6
6
6
6
7
6
6
6
6
6
5
5
5
6
5
6
6
4
4
4
4
4
5
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
1
1
1
1
1
0
80.71
80.71
80.71
80.71
89.94
80.71
80.71
80.71
80.71
80.71
60.48
60.48
60.48
69.71
60.48
69.71
69.71
50.50
50.50
50.50
50.50
50.50
58.60
3
3
3
3
4
3
3
3
2
2
3
3
3
4
3
4
4
3
3
2
3
3
20
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
1
0
1
1
2
0
334.22
347.76
352.11
350.78
359.77
378.21
324.93
342.82
340.18
356.98
339.74
353.28
357.63
365.29
339.74
342.46
369.64
374.56
365.27
380.52
382.07
409.25
432.32
5q
7a
7b
7c
7d
7e
Miltefosine
M.W.: molecular weight; LogP: logarithm of n-octanol-water partition coefficient; HBA (nON): number of hydrogen bond acceptors or no of oxygen and nitrogen atom; HBD
(nOHNH): number of hydrogen bond donors or number of NH, OH bond; TPSA: topological polar surface area; nRotB: number of rotatable bonds.
Table 2
Theoretical toxicity evaluation of synthesized compounds 5a-q and 7a-e.
Entry
Mutagenic
Tumorigenic
Irritant
Entry
Mutagenic
Tumorigenic
Irritant
5a
5b
5c
5d
5e
5f
5g
5h
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þþ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(ꢁ)
5l
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þþ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þþ)
(ꢁ)
(ꢁ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(þþ)
(ꢁ)
5m
5n
5o
5p
5q
7a
7b
7c
7d
7e
(ꢁ)
5i
5j
5k
(ꢁ)
(ꢁ)
(ꢁ)
Miltefosine
The scale of toxicity risk ranges from none (ꢁ), low (þ), high (þþ) calculated by using OSIRIS Property Explorer.
activity was lesser than 5a. Interestingly, when phenyl moiety was
replaced with thienyl ring (5g) at C6 position of pyranone frame-
work, the remarkable inhibitory activity was observed (z90%) at
weak to moderate activity, implying that pyranone ring play an
important role is displaying anti-leishmanial activity. The in vitro
activity profile of 5a-q and 7a-e was well in agreement with the in
silico analysis of the compounds.
50
mM concentration against both life stages of the parasite
(Table 3). Further, by blocking the rotational freedom of phenyl ring
at C5eC6 positions of the pyranone ring to form indeno[1,2-b]py-
ran-2(5H)-one (5i) and 5,6-dihydro-2H-benzo[h]chromen-2-one
(5j) compounds resulted in lesser antileishmanial activity as
compared to 5a.
Further in order to examine SAR at C4 position of pyranone ring,
different phenyl piperazine derivatives linked with electron with-
drawing and releasing groups (EWG and ERG) were employed to
prepare compounds (5k-q) and further subjected to in vitro anti-
leishmanial activity evaluation (Table 3). When hydroxy group of
phenylpiperazine in 5a was replaced by electron withdrawing
chloro group (5k), it displayed moderate anti-leishmanial activity
whereas when hydroxy group of phenylpiperazine in 5g was
replaced by an electron donating methoxy group (5p), the anti-
leishmanial activity was significantly decreased possibly due to
inhibition of hydrogen bonding with the receptor.
Overall, on the basis of in silico (physicochemical) and in vitro
studies, it was found that compounds 5a and 5g exhibited anti-
leishmanial efficacy superior to other derivatives. So compounds 5a
and 5g were selected for further in vivo studies.
The efficacy of 5a and 5g on the inhibition of promastigote and
amastigote multiplication was then checked in a time-dependent
fashion by MTT assay and luciferase assay respectively. A time-
dependent killing of promastigotes and amastigotes were
observed treated with 5a and 5g with maximum inhibition
observed at 72 h post-treatment for both the compounds (Fig. 2A
and B). Similarly, dose response analysis revealed dose dependent
inhibition of amastigotes up to 50
>89% by both 5a and 5g which was remained same up to 100
(Fig. 2C) as determined by luciferase assay. The IC50 was found to be
11.07 1.15 M and 15.3 2.1 M for 5a and 5g respectively against
mM with a maximum killing of
mM
m
m
intracellular amastigotes at 72 h post treatment, as compared to
IC50 of reference drug, miltefosine, which was reported to be
The synthesized dihydrothiophen-3(2H)-one (7a-c) and tetra-
hydro-4H-thiopyran-4-one (7d-e) showed either no inhibition or
12.77
0.47 mM. Since compounds 5d, 5e, 5f, and 5h showed
5

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Table 3
visceral leishmaniasis, therefore in this context, we measured the
level of host protective proinflammatory cytokines and anti-
inflammatory cytokines. However, we did not find increased level
In vitro anti-leishmanial activity of 5a-q and 7a-e.
Compound code Antipromastigote activity
Antiamastigote activity
25 50
of host defensive IFN-g (Fig. 3C) and IL-12 (Fig. 3D), in culture su-
25
m
M
50
m
M
m
M
mM
pernatant isolated from splenocytes of 5a treated mice infected
with L. donovani. Similarly, expression of disease progressive IL-10
(Fig. 3E) and TGF-b (Fig. 3F) were also not downregulated in sple-
nocytes of 5a treated mice infected with L. donovani. On the con-
trary, standard anti-leishmanial drug Miltefosine markedly
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
82.7 9.4
NI
NI
54.7 6.2
78.3 8.4
77.3 5.7
81.3 9.5
52.1 4.3
14.2 2.4
35.9 3.9
85.2 8.2
25.01 3.1
NI
72.8 6.9
86.3 7.4
78.6 6.4
90.2 7.2
88.1 7.2
82.2 6.3
81.3 7.4
84.2 6.3
NI
95.7 3.2
NI
14.49 1.5
77.3 4.9
51.8 4.9
71.4 6.3
84.7 5.6
74.8 5.2
37.8 2.6
37.8 4.8
35.07 2.9
83.2 5.2
84.4 5.3
80.8 5.4
89.3 6.2
85.07 6.8
60.03 4.8
64.8 4.1
increase host defensive IFN-
significant attenuation of IL-10 and TGF-
g
and IL-12 (Fig. 3C and D) along with
(Fig. 3E and F). These
b
observations allowed us to speculate for the direct killing of
L. donovani parasite by compound 5a as it did not possess any
immunomodulatory properties.
46.88 3.05 72.91 1.49 49.24 3.31 73.65 1.9
55.21 3.15 58.30 1.09 73.10 3.13 71.02 1.2
5 m
5n
5ꢂ
5p
5q
7a
7b
7c
7d
7e
57.92 3.19 66.46 3.28 60.5 3.57
60.32 0.89 69.21 1.64 50.96 3.4
44.01 2.94 57.31 2.89 47.44 3.87 72.13 3.74
48.11 2.27 62.24 3.77 52.18 5.47 71.19 2.7
56.63 0.75 61.17 1.99 43.31 4.53 58.34 2.76
68.51 2.71
62.45 1.56
2.4. 5a induces mitochondrial apoptosis in extracellular
promastigotes
To examine whether L. donovani promastigotes undergo
apoptosis which is a well known mode of direct killing of parasite,
untreated and 5a treated promastigotes were stained with annexin
V and propidium iodide. As shown in Fig. 4A, flow cytometric
analysis revealed that 5a exposure for different time periods at
82.1 6.3
NI
NI
NI
87.8 6.9
4.8 2.6
NI
NI
NI
60.1 4.9
45.5 3.9
65.02 5.3
71.02 6.3
91.2 0.7
79.3 5.2
57.3 4.7
72.6 3.9
83.3 5.7
95.3 0.6
NI
NI
33.3 4.1
99.7 0.2
Miltefosine
99.4 0.4
maximum inhibitory concentration (50 mM) resulted in increased
Percent inhibition is expressed as mean standard deviation of at least three in-
dependent experiments done in replicate for each compound. NIeNo Inhibition.
populations of apoptotic cells (16.6% at 24 h, 41.4% at 48 h and 54.5%
at 72 h post treatment) as compared to vehicle treated promasti-
gotes (Fig. 4A). Depolarization of mitochondrial membrane poten-
tial (MMP) is one of the characteristic features of mitochondrial
dysfunction mediated apoptosis. Therefore, to investigate whether
significant antileishmanial activity (>80% inhibition) against
intracellular amastigote form at 50
effect in a dose-dependent manner (3.125e50
IC50 of all four compounds against intracellular amastigotes fol-
lowed a range between 13 and 24 M, which was higher than 5a
(Fig. 2E). However, in terms of maximum inhibition of intracellular
parasite growth, compounds 5a and 5g showed most superior ef-
ficacy (~90% killing of amastigotes), hence we proceeded with these
two compounds for further safety index determination.
The cytotoxicity assay was performed next to differentiate be-
tween actual inhibitory activity against amastigotes or cytotoxicity
against macrophages and we found that both these compounds
were non toxic for macrophages even at higher concentration with
mM, we proceeded to check their
5a treatment (both IC50 and 50 mM concentration) in promastigotes
mM). The observed
is associated with mitochondrial dysfunction, we analyzed MMP
changes in treated promastigotes by staining with JC-1 dye which
at a lower potential forms monomers and emits green fluorescence
in the cytoplasm. Flow cytometric analysis also revealed a time
dependent loss in MMP (as evident by increase in green fluores-
cence) in 5a treated promastigotes which was maximum at 72 h
post treatment (Fig. 4B). The loss in MMP was drastically reduced
(22.9%) when the parasite was treated with IC50 of 5a (Fig. 4C). The
maximum fluorescence signal shifted from red to green was
observed in CCCP-treated parasites (a mitochondrial uncoupler,
used as a positive control), which suggested JC-1 sensitivity toward
membrane potential in leishmanial cells. Cytochrome c release
from the dysfunctional mitochondria to cytosol is an important
event in apoptosis [23] and therefore we next examined whether
the translocation of cytochrome c from mitochondria to cytosol is
initiated by 5a treatment in promastigotes or not. We observed
very little abundance of cytochrome c in the cytosolic fractions of
control promastigotes thereby suggesting intact mitochondria.
However, cytosolic abundance of cytochrome c in 5a treated pro-
m
CC50 of 350.8 mM and 310.9 mM for 5a and 5g, respectively (Fig. 2D).
Altogether, these results prompted us to evaluate the anti-
leishmanial effect of both these derivatives in in vivo model of VL
of L. donovani.
2.3. Evaluation of anti-leishmanial potential against Balb/c mice
model of VL
The in vitro active compounds with a good safety profile (5a and
5g) were further selected for in vivo screening which was con-
ducted in Balb/c mice (acute model) infected with L. donovani. For
these, 15 days infected Balb/c mice were administered intraperi-
toneally with 5a and 5g for five consecutive days at a dose of 50 mg/
kg. Animals were sacrificed 1 wk post treatment to assess organ
parasite burden. Compound 5a showed better antileishmanial ef-
ficacy than 5g as we observed a significant reduction of intracel-
lular amastigotes in liver and spleen tissue of Balb/c mice after 1 wk
post treatment. The reduction in amastigotes multiplication in the
liver and spleen of L. donovani -infected Balb/c mice treated with 5a
were found to be 67.3%, and 56.9%, respectively (Fig. 3A and B).
Miltefosine was used as standard reference drug showed >97%
inhibition of organ parasite burden in infected Balb/c mice, when
given at a dose of 25 mg/kg/day for 5 days (data not shown). Since
cytokines play an important role in the progression or resolution of
mastigotes gradually increased, starting at 12 h and being
maximum between 48 h and 72 h, along with its gradual disap-
pearance from mitochondria, as observed by immunoblotting
(Fig. 4D). To further confirm the induction of apoptosis in 5a treated
promastigotes, we detected DNA fragmentation as a marker of
apoptosis by terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) staining. As shown in Fig. 4D, the promastigotes
treated with 5a showed significant TUNEL positivity as evident by
bright green fluorescence in the nucleus because of DNA frag-
mentation, which was not detected in non-treated and vehicle
(0.1% DMSO) treated promastigotes (Fig. 4E).
3. Conclusion
A novel series of arylpiperazine containing pyranones and
benzene cored arylpiperazine was prepared. All synthesized
6

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Fig. 2. In vitro anti-leishmanial efficacy of synthesized derivatives: (A) Percentage inhibition of 5a- and 5g-treated promastigotes by MTT assay as described in Experimental
Section. (B) Percentage inhibition of 5a- and 5g-treated amastigotes by giemsa staining in J774 macrophages (C) Percentage inhibition of 5a- and 5g-treated amastigotes in J774
macrophages by luciferase assay as described in Experimental Section.(D) Determination of cellular cytotoxicity of 5a- and 5g-treated macrophages using MTT assay. (E) Deter-
mination of IC50 of active compounds against intracellular amastigotes as described in Experimental Section.
compounds were characterised by spectroscopic techniques (1H
NMR, 13C NMR, ESMS, HRMS and IR) and were screened for anti-
leishmanial activity. Among them, 5a and 5g showed better activ-
study could constitute an opportunity for new derivatives for the
treatment of fatal disease caused by Leishmania parasite.
ity against intracellular amastigotes with an IC50 of 11.07
mM and
4. Experimental Section
15.3 M, respectively. These two compounds further considered for
m
anti-leishmanial activity in in vivo model for experimental visceral
leishmaniasis. Additionally, we observed that compound 5a
showed more promising antileishmanial efficacy than 5g in Balb/c
model of VL. On a mechanistic view point, 5a induced direct
Leishmania killing by mitochondrial dysfunction like cytochrome c
release and loss of membrane potential. Our results suggest that 5a
is a promising lead for further development of antileishmanial
drugs. As it is important to continuously develop alternate solu-
tions to decrease mortality rate caused by Leishmania parasite, our
4.1. In silico study
The in silico analysis of the synthesized compounds was done by
using free access Molinspiration [18] (http://www.molinspiration.
com). For the theoretical study of toxicity (mutagenic, tumori-
genic, and irritant effects) profiles of synthesized compounds 5a-q
and 7a-e, the online web free OSIRIS Property Explorer [22]
(https://www.organic-chemistry.org/prog/peo/) computer pro-
gram was used.
7

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Fig. 3. Effect of 5a and 5g on L. donovani infected Balb/c mice. (A) liver and (B) spleen parasite burden were assessed in 5a- and 5g-treated Balb/c mice using stamps esmear method
as described in Experimental Section. Miltefosine was used as a standard anti-leishmanial reference drug. The level of IFN- (C), IL-12 (D), IL-10 (E) and TGF- (F) were determined
g
b
in the supernatant harvested from splenocytes of different group of Balb/c mice as described in Experimental Section. Significance: infected vs infected þ 5a and infected þ vehicle
vs infected þ 5a ns, non significant, *p < 0.05, **p < 0.01 and ***p < 0.001.
4.2. Synthesis of compounds
2,5-dihydroindeno[1,2-b]pyran-3-carbonitrile and 2-(piperazin-1-
yl)phenol (4, 1.2 mmol) was taken and for 5j a mixture of 4-
(methylthio)-2-oxo-5,6-dihydro-2H-benzo[h]chromene-3-
carbonitrile and 2-(piperazin-1-yl)phenol (4, 1.2 mmol) was taken
following the above method. Same procedure was followed for
other derivatives.
4.2.1. General method for preparation of 2-(piperazin-1-yl)phenol
substituted 2H-Pyranones 5a-q
A
mixture of 6-aryl-3-cyano-4-methylsulfanyl-2H-pyran-2-
ones (3a-h, 1 mmol) and 2-(piperazin-1-yl)phenol (4, 1.2 mmol)
was refluxed in methanol for 1e4 h. After completion, the reaction
was cooled to room temperature and was filtered off and washed
with methanol. Crude product was purified by silica gel column
chromatography using 2% methanol in chloroform as eluent.
Further for the synthesis of 5i, a mixture of 4-(methylthio)-2-oxo-
4.2.2. General method for preparation of 7a-e
A mixture of 6-(aryl)-4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-
oxo-2H-pyran-3-carbonitrile (5a,c,g,i,
1
mmol,
1
equiv.),
substituted acetophenones (6a and 6b, 1.2 mmol, 1.2 equiv.), and
8

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
Fig. 4. 5a induces apoptosis cell death in L. donovani promastigotes via mitochondrial depolarization. (A) Flow cytometry analysis of Log phase L. donovani promastigotes
treated with 50
M of 5a. Representative quadrant showing viable parasite (lower left as annexin V-PI-), early apoptotic (lower right as annexin VþPIꢁ), late apoptotic (upper right as
annexin VþPIþ) and necrotic (upper left as annexin V-PIþ). (B & C) Promastigotes were exposed to 50
M of 5a for 24e72 h or IC50 of 5A for 72 h and then stained with JC-1(2 M).
Loss in mitochondrial membrane potential was determined using flow cytometry. CCCP was used as a positive control. (D) Western blot for cytochrome c in mitochondria and
cytosol fractions of L. donovani promastigotes treated with 50 M 5a for 12e72 h. Actin was used as a loading control for cytosol fractions and COX-4 was used as a control for
mitochondrial fractions. (E) Log phase promastigotes were treated with 50 M 5a for 72 h. After fixation and permeabilization, non-treated and treated promastigotes were stained
m
m
m
m
m
with labeling solution of Tdt enzyme and BrdUTP and analyzed by confocal microscopy.
KOH (2 mmol, 2 equiv.) in dry DMF (10 mL) was stirred at 25 ꢂC for
1 h. The progress of the reaction was monitored by TLC and on
completion the reaction mixture was poured onto crushed ice with
vigorous stirring and finally neutralized with 10% HCl. The precip-
itate obtained was filtered and purified on a silica gel column using
5% ethyl acetate in hexane as the eluent.
139.1, 150.7, 159.8, 161.0, 162.3 ppm; MS (ESI) 374 [M þ H]þ; HRMS
calculated for C22H20N3O3 [M þ H]þ 374.1505, found: 374.1494.
4.2.4. 6-(4-chlorophenyl)-4-(4-(2-hydroxyphenyl)piperazin-1-yl)-
2-oxo-2H-pyran-3-carbonitrile (5b)
Light yellow; Yield 68%; Rf ¼ 0.38 (Chloroform/Methanol, 10:1,
v/v); mp(Chloroform/Methanol): 283e285 ꢂC; IR (KBr)
nmax ¼ 1682 cmꢁ1 (C]O str.), 2210 cmꢁ1 (CN str.), 3351 (OH str.); 1H
4.2.3. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-oxo-6-phenyl-2H-
pyran-3-carbonitrile (5a)
NMR (400 MHz, DMSO‑d6):
d
¼ 3.11 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 4.08
Off-white solid, Yield 72%; Rf ¼ 0.40 (Chloroform/Methanol,
10:1, v/v); mp(Chloroform/Methanol): 267e269 ꢂC; IR (KBr)
nmax ¼ 1702 cmꢁ1 (C]O str.), 2210 cmꢁ1 (CN str.), 3357 (OH str.); 1H
(t, J ¼ 4.2 Hz, 4H, 2NCH2), 6.73e6.92 (m, 4H, AreH), 7.18 (s, 1H,
C5eH), 7.63 (d, J ¼ 8.8 Hz, 2H, AreH), 8.02 (d, J ¼ 8.8 Hz, 2H, AreH),
9.10 (s, 1H, OH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 49.9,
NMR (400 MHz, DMSO‑d6):
d
¼ 3.11 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 4.08
50.8, 71.3, 96.6, 116.2, 118.1, 119.6, 120.0, 124.1, 128.6, 129.6, 129.9,
137.0, 139.2, 150.7, 158.6, 160.9, 162.2 ppm; MS (ESI) 408 [M þ H]þ,
410 [M þ 2 þ H]þ; HRMS calculated for C22H19ClN3O3 [M þ H]þ
408.1115, found: 408.1103 and [M þ 2 þ H]þ 410.1085, found:
410.1074.
(t, J ¼ 4.2 Hz, 4H, 2NCH2), 6.73e6.92 (m, 4H, AreH), 7.15 (s, 1H,
C5eH), 7.53e7.59 (m, 3H, AreH), 7.98e8.00 (m, 2H, AreH), 9.11 (s,
1H, OH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 49.8, 50.8, 71.2,
79.6, 96.2, 116.2, 118.1, 119.6, 119.9, 124.1, 126.8, 129.5, 131.0, 132.2,
9

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
4.2.5. 6-(4-bromophenyl)-4-(4-(2-hydroxyphenyl)piperazin-1-yl)-
2-oxo-2H-pyran-3-carbonitrile (5c)
Light yellow; Yield 66%; Rf ¼ 0.40 (Chloroform/Methanol, 10:1,
v/v); mp(Chloroform/Methanol): 281e283 ꢂC; IR (KBr)
nmax ¼ 1684 cmꢁ1 (C]O str.), 2210 cmꢁ1 (CN str.), 3350 (OH str.); 1H
120.0, 124.1, 129.5, 130.2, 132.4, 134.4, 139.2, 150.7, 155.9, 160.9,
161.8 ppm; MS (ESI) 380 [M
C
þ
H]þ; HRMS calculated for
20H18N3O3S [M þ H]þ 380.1069, found: 380.1057.
4.2.10. 6-(5-Bromothiophen-2-yl)-4-(4-(2-hydroxyphenyl)
piperazin-1-yl)-2-oxo-2H-pyran-3-carbonitrile (5h)
NMR (400 MHz, DMSO‑d6):
d
¼ 3.11 (s, 4H, 2NCH2), 4.08 (s, 4H,
2NCH2), 6.74e6.91 (m, 4H, AreH), 7.20 (s, 1H, C5eH), 7.77 (d,
J ¼ 8.7 Hz, 2H, AreH), 7.95 (d, J ¼ 8.7 Hz, 2H, AreH), 9.11 (s, 1H, OH)
Yellow solid; Yield 50%; Rf ¼ 0.35 (Chloroform/Methanol,10:1, v/
v);
mp(Chloroform/Methanol):
274e276
ꢂC;
IR
(KBr)
ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 49.9, 50.9, 71.4, 96.7,
nmax ¼ 1716 cmꢁ1 (C]O str.), 2201 cmꢁ1 (CN str.), 3338 (OH str.); 1H
116.3, 118.2, 119.7, 120.1, 124.2, 126.1, 128.8,130.3,132.6,139.2, 150.8,
158.8, 151.0, 162.3 ppm; MS (ESI) 452 [M þ H]þ, 454 [M þ 2 þ H]þ;
HRMS calculated for C22H19BrN3O3 [M þ H]þ 452.0610, found:
452.0604, [M þ 2 þ H]þ 454.0589, found: 454.0584.
NMR (400 MHz, DMSO‑d6):
d
¼ 3.10 (s, 4H, 2NCH2), 4.04 (s, 4H,
2NCH2), 6.73e6.91 (m, 4H, AreH), 7.09 (s, 1H, C5eH), 7.44 (d,
J ¼ 4.1 Hz, 1H, AreH), 7.89 (d, J ¼ 4.1 Hz, 1H, AreH), 9.10 (s, 1H, OH)
ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 49.9, 50.7, 70.8, 94.7,
116.2, 118.1, 119.6, 120.0, 124.1, 130.7, 132.9, 135.8, 139.1, 145.8, 150.7,
154.5, 160.7, 161.6 ppm; MS (ESI) 458 [M þ H]þ, 460 [M þ 2 þ H]þ;
HRMS calculated for C20H17BrN3O3S [M þ H]þ 458.0174, found:
458.0167and [M þ 2 þ H]þ 460.0154, found: 460.0139.
4.2.6. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-oxo-6-(p-tolyl)-
2H-pyran-3-carbonitrile (5d)
Light yellow; Yield 62%; Rf ¼ 0.37 (Chloroform/Methanol, 10:1,
v/v); mp(Chloroform/Methanol): 288e290 ꢂC; IR (KBr)
nmax ¼ 1682 cmꢁ1 (C]O str.), 2203 cmꢁ1 (CN str.), 3381 (OH str.); 1H
4.2.11. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-oxo-2,5-
dihydroindeno[1,2-b]pyran-3-carbonitrile (5i)
NMR (400 MHz, DMSO‑d6):
d
¼ 2.39 (s, 3H, CH3), 3.11 (s, 4H, 2NCH2),
4.08 (s, 4H, 2NCH2), 6.74e6.92 (m, 4H, AreH), 7.10 (s, 1H, C5eH),
7.37 (d, J ¼ 8.1 Hz, 2H, AreH), 7.90 (d, J ¼ 8.2 Hz, 2H, AreH), 9.11 (s,
Brown solid; Yield 47%; Rf ¼ 0.40 (Chloroform/Methanol, 10:1, v/
v);
mp(Chloroform/Methanol):
258e260
ꢂC;
IR
(KBr)
1H, OH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 21.5, 49.7, 50.7,
nmax ¼ 1732 cmꢁ1 (C]O str.), 2202 cmꢁ1 (CN str.), 3353 (OH str.); 1H
71.0, 95.3, 116.2118.2, 119.6, 124.0, 126.7, 128.2, 130.0, 139.1, 142.5,
150.7, 160.0, 161.1, 162.3 ppm; MS (ESI) 388 [M þ H]þ; HRMS
calculated for C23H22N3O3 [M þ H]þ 388.1661, found: 388.1651.
NMR (400 MHz, DMSO‑d6):
d
¼ 3.17 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 4.02
(t, J ¼ 4.3 Hz, 4H, 2NCH2), 4.14 (s, 2H, Ar-CH2), 6.74e6.94 (m, 4H,
AreH), 7.49e7.58 (m, 2H, AreH), 7.66e7.71 (m, 2H, AreH), 9.13 (s,
1H, OH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 36.3, 51.2, 72.9,
4.2.7. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-6-(4-
methoxyphenyl)-2-oxo-2H-pyran-3-carbonitrile (5e)
Light yellow; Yield 61%; Rf ¼ 0.32 (Chloroform/Methanol, 10:1,
v/v); mp(Chloroform/Methanol): 250e252 ꢂC; IR (KBr)
nmax ¼ 1687 cmꢁ1 (C]O str.), 2203 cmꢁ1 (CN str.), 3377 (OH str.); 1H
112.1, 116.4, 118.7, 119.8, 120.2, 120.7, 124.4, 125.6, 128.4, 131.0, 133.8,
139.3, 144.0, 150.8, 162.3, 162.8 ppm; MS (ESI) 386 [M þ H]þ; HRMS
calculated for C23H20N3O3 [M þ H]þ 386.1505, found: 386.1498.
4.2.12. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-oxo-5,6-dihydro-
2H-benzo[h]chromene-3-carbonitrile (5j)
Dark Yellow solid; Yield 44%; Rf ¼ 0.40 (Chloroform/Methanol,
10:1, v/v); mp(Chloroform/Methanol): 260e262 ꢂC; IR (KBr)
nmax ¼ 1730 cmꢁ1 (C]O str.), 2210 cmꢁ1 (CN str.), 3357 (OH str.); 1H
NMR (400 MHz, DMSO‑d6):
d
¼ 3.11 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 3.84
(s, 3H, OMe), 4.06 (t, J ¼ 4.2 Hz, 4H, 2NCH2), 6.73e6.91 (m, 4H,
AreH), 7.02 (s, 1H, C5eH), 7.09 (d, J ¼ 8.9 Hz, 2H, AreH), 7.96 (d,
J ¼ 8.9 Hz, 2H, AreH), 9.10 (s, 1H, OH) ppm; 13C NMR (100.6 MHz,
DMSO‑d6):
d
¼ 49.8, 50.8, 56.0, 70.6, 94.4, 115.0, 116.2, 118.3, 119.6,
NMR (400 MHz, DMSO‑d6):
d
¼ 2.73e2.77 (m, 2H, CH2), 2.85e2.88
120.0, 123.2, 124.1, 128.7, 139.2, 150.7, 160.0, 161.3, 162.5, 162.6 ppm;
MS (ESI) 404 [M þ H]þ; HRMS calculated for C23H22N3O4 [M þ H]þ
404.1610, found: 404.1606.
(m, 2H, CH2), 3.12 (s, 4H, 2NCH2), 3.73 (s, 4H, 2NCH2), 6.74e6.94 (m,
4H, AreH), 7.35e7.48 (m, 3H, AreH), 7.68 (d, J ¼ 6.84, 1H, AreH),
9.11 (s, 1H, OH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 23.5,
27.7, 51.4, 52.1, 79.7, 110.3, 116.5, 118.0, 119.9, 120.4, 124.3, 124.4,
128.1, 128.3, 128.5, 132.3, 139.1, 139.8, 150.9, 156.8, 161.8, 167.2 ppm;
MS (ESI) 400 [M þ H]þ; HRMS calculated for C24H22N3O3 [M þ H]þ
400.1661, found: 415.1658.
4.2.8. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-6-(naphthalen-1-yl)-
2-oxo-2H-pyran-3-carbonitrile (5f)
Off white solid; Yield 58%; Rf ¼ 0.36 (Chloroform/Methanol,10:1,
v/v); mp(Chloroform/Methanol): 247e248 ꢂC; IR (KBr)
nmax ¼ 1702 cmꢁ1 (C]O str.), 2211 cmꢁ1 (CN str.), 3369 (OH str.); 1H
4.2.13. 4-(4-(2-chlorophenyl)piperazin-1-yl)-2-oxo-6-phenyl-2H-
pyran-3-carbonitrile (5k)
NMR (400 MHz, DMSO‑d6):
d
¼ 3.11 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 4.07
(t, J ¼ 4.4 Hz, 4H, 2NCH2), 6.75e6.91 (m, 4H, AreH), 6.99 (s, 1H,
C5eH), 7.62e7.66 (m, 3H, AreH), 7.80e7.82 (m, 1H, AreH),
8.04e8.16 (m, 3H, AreH), 9.10 (s, 1H, OH) ppm; 13C NMR
Yellow solid; Yield 62%; Rf ¼ 0.35 (Chloroform/Methanol,10:1, v/
v);
mp(Chloroform/Methanol):
216-214
ꢂC;
IR
(KBr)
nmax ¼ 1700 cmꢁ1 (C]O str.), 2207 cmꢁ1 (CN str.); 1H NMR
(100.6 MHz, DMSO‑d6):
d
¼ 49.8, 50.7, 71.3, 101.1, 116.2, 118.2, 119.6,
(400 MHz, DMSO‑d6):
d
¼ 3.18 (t, J ¼ 4.8 Hz, 4H, 2NCH2), 4.08 (t,
124.1, 125.3, 125.7, 127.1, 128.0, 128.9,129.1, 130.0, 130.2, 131.9, 133.6,
139.1, 150.7, 160.9, 161.9, 162.7 ppm; MS (ESI) 424 [M þ H]þ; HRMS
calculated for C26H22N3O3 [M þ H]þ 424.1661, found: 424.1654.
J ¼ 4.8 Hz, 4H, 2NCH2), 7.08e7.13 (m, 1H, AreH), 7.17e7.21 (m, 2H,
AreH), 7.31e7.36 (m, 1H, AreH), 7.46 (dd, J1 ¼ 7.9, J2 ¼ 1.5 Hz, 1H,
AreH), 7.56e7.59 (m, 3H, AreH), 7.99e8.01 (m, 2H, AreH) ppm; 13
C
NMR (100.6 MHz, DMSO‑d6):
d
¼ 49.8, 51.3, 71.6, 96.3, 118.1, 121.7,
4.2.9. 4-(4-(2-hydroxyphenyl)piperazin-1-yl)-2-oxo-6-(thiophen-
2-yl)-2H-pyran-3-carbonitrile (5g)
125.0, 126.8, 128.1, 128.7, 129.5, 130.9, 131.0, 132.3, 148.4, 159.9,
161.3, 162.3 ppm; MS (ESI) 392 [M þ H]þ; HRMS calculated for
Brown solid; Yield 55%; Rf ¼ 0.31 (Chloroform/Methanol, 10:1, v/
C
22H18ClN3O2 [M þ H]þ 392.1166, found: 392.1158.
v);
mp(Chloroform/Methanol):
248e250
ꢂC;
IR
(KBr)
nmax ¼ 1706 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.), 3399 (OH str.); 1H
4.2.14. 6-(4-chlorophenyl)-4-(4-(2-chlorophenyl)piperazin-1-yl)-
2-oxo-2H-pyran-3-carbonitrile (5l)
NMR (400 MHz, DMSO‑d6):
d
¼ 3.10 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 4.05
(t, J ¼ 4.2 Hz, 4H, 2NCH2), 6.73e6.92 (m, 4H, AreH), 7.07 (s, 1H,
C5eH), 7.27e7.29 (m, 1H, AreH), 7.93e7.94 (m, 1H, AreH),
8.04e8.05 (m, 1H, AreH), 9.11 (s, 1H, OH) ppm; 13C NMR
White solid; Yield 55%; Rf ¼ 0.40 (Chloroform/Methanol, 10:1, v/
v);
mp(Chloroform/Methanol):
234e236
ꢂC;
IR
(KBr)
nmax ¼ 1699 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.); 1H NMR
(100.6 MHz, DMSO‑d6):
d
¼ 49.8, 50.7, 70.6, 94.4, 116.2, 118.2, 119.6,
(400 MHz, DMSO‑d6):
d
¼ 3.18 (t, J ¼ 4.9 Hz, 4H, 2NCH2), 4.10 (t,
10

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
J ¼ 4.9 Hz, 4H, 2NCH2), 7.10 (td, J1 ¼ 7.6, J2 ¼ 1.5 Hz, 1H, AreH),
7.18e7.21 (m, 2H, AreH), 7.31e7.36 (m, 1H, AreH), 7.46 (dd, J1 ¼7.9,
J2 ¼ 1.5 Hz, 1H, AreH), 7.63e7.65 (m, 2H, AreH), 8.02e8.04 (m, 2H,
4.2.19. 6-(4-bromophenyl)-4-(4-(2-methoxyphenyl)piperazin-1-
yl)-2-oxo-2H-pyran-3-carbonitrile (5q)
Light yellow solid; Yield 64%; Rf ¼ 0.37 (Chloroform/Methanol,
10:1, v/v); mp(Chloroform/Methanol): 246e247 ꢂC; IR (KBr)
nmax ¼ 1697 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.); 1H NMR
AreH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
d
¼ 49.8, 51.3, 71.7,
96.7, 118.0, 121.7, 125.0, 128.2, 128.6, 129.6, 129.8, 130.9, 137.1, 148.4,
158.7, 161.2, 162.0 ppm; MS (ESI) 426 [M þ H]þ; HRMS calculated
for C22H17Cl2N3O2 [M þ H]þ 426.0776, found: 426.0772.
(400 MHz, CDCl3):
d
¼ 3.27 (t, J ¼ 4.9 Hz, 4H, 2NCH2), 3.90 (s, 3H,
OMe), 4.07 (t, J ¼ 5.0 Hz, 4H, 2NCH2), 6.50 (s, 1H, C5eH), 6.98e6.88
(m, 3H, AreH), 7.10e7.04 (m, 1H, AreH), 7.66e7.59 (m, 2H, AreH),
7.72e7.66 (m, 2H, AreH) ppm; 13C NMR (100.6 MHz, CDCl3):
4.2.15. 6-(4-bromophenyl)-4-(4-(2-chlorophenyl)piperazin-1-yl)-
2-oxo-2H-pyran-3-carbonitrile (5m)
d
¼ 49.7, 50.6, 55.4, 73.3, 94.6, 111.5, 117.0, 118.6, 121.2, 124.1, 126.8,
White solid; Yield 50%; Rf ¼ 0.25 (Chloroform/Methanol, 10:1, v/
127.6, 129.5, 132.4, 139.5, 152.2, 160.2, 160.7, 161.8 ppm; MS (ESI)
466 [M þ H]þ; HRMS calculated for C23H21BrN3O3 [M þ H]þ
466.0766, found: 466.0757 [M þ H]þ and 468.0736 [M þ 2þ H].
v);
mp(Chloroform/Methanol):
240e242
ꢂC;
IR
(KBr)
nmax ¼ 1684 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.); 1H NMR
(400 MHz, DMSO‑d6):
d
¼ 3.18 (t, J ¼ 4.8 Hz, 4H, 2NCH2), 4.10 (t,
J ¼ 4.8 Hz, 4H, 2NCH2), 7.08e7.12 (m, 2H, AreH), 7.18e7.22 (m, 1H,
AreH), 7.32e7.35 (m, 1H, AreH), 7.45e7.47 (m, 1H, AreH),
7.76e7.79 (m, 2H, AreH), 7.94e7.97 (m, 2H, AreH) ppm; 13C NMR
4.2.20. 5-(4-(2-hydroxyphenyl)piperazin-1-yl)-7-phenyl-2,3-
dihydrobenzo[b]thiophene-4-carbonitrile (7a)
Greenish yellow solid; Yield 55%; Rf ¼ 0.45 (Ethylacetate/Hex-
(100.6 MHz, DMSO‑d6):
d
¼ 49.8, 51.3, 71.7, 96.7, 118.0, 121.7, 125.0,
ane, 1:20, v/v); mp(Ethylacetate/Hexane): 175e177 ꢂC; IR (KBr)
126.0, 128.1, 128.7, 128.8, 130.1, 130.9, 132.5, 148.4, 158.7, 161.2,
162.0 ppm; MS (ESI) 470 [M þ H]þ, 472 [M þ 2 þ H]þ; HRMS
calculated for C22H17BrClN3O2 [M þ H]þ 470.0271, found: 470.0221
and [M þ 2 þ H]þ 472.0271, found:472.0194.
nmax ¼ 2210 cmꢁ1 (CN str.); 1H NMR (400 MHz, CDCl3):
d
¼ 3.10 (t,
J ¼ 4.4 Hz, 4H, 2NCH2), 3.33e3.39 (m, 6H, 2NCH2, SCH 2), 3.54e3.58
(m, 2H, SCH 2), 6.87e6.91 (m, 2H, AreH), 6.95e6.97 (m, 1H, AreH),
7.08e7.12 (m, 1H, AreH), 7.24e7.27 (m, 2H, AreH), 7.39e7.53 (m,
5H, AreH) ppm; 13C NMR (100.6 MHz, CDCl3):
d
¼ 32.4, 36.9, 52.7,
4.2.16. 4-(4-(2-chlorophenyl)piperazin-1-yl)-6-(4-
52.9, 103.0, 114.1, 117.0, 118.5, 120.2, 121.7, 126.7, 127.7, 128.5, 128.7,
135.0, 138.5, 139.8, 140.6, 146.5, 151.4, 152.9 ppm; MS (ESI) 414 [M þ
H]þ; HRMS calculated for C25H24N3OS [M þ H]þ 414.1640, found:
414.1623.
methoxyphenyl)-2-oxo-2H-pyran-3-carbonitrile (5n)
White solid; Yield 50%; Rf ¼ 0.37 (Chloroform/Methanol, 10:1, v/
v);
mp(Chloroform/Methanol):
200e202
ꢂC;
IR
(KBr)
nmax ¼ 1698 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.), 3410 (OH str.); 1H
NMR (400 MHz, DMSO‑d6):
d
¼ 3.17 (t, J ¼ 4.8 Hz, 4H, 2NCH2), 3.86
4.2.21. 5-(4-(2-hydroxyphenyl)piperazin-1-yl)-7-(thiophen-2-yl)-
2,3-dihydrobenzo[b] thiophene-4-carbonitrile (7b)
(s, 3H, OMe), 4.08 (t, J ¼ 4.8 Hz, 4H, 2NCH2), 7.04 (s, 1H, C5eH),
7.08e7.12 (m, 3H, AreH), 7.20 (dd, J1 ¼ 8.1, J2 ¼ 1.6 Hz, 1H, AreH),
7.31e7.35 (m, 1H, AreH), 7.46 (dd, J1 ¼ 7.9, J2 ¼ 1.5 Hz, 1H, AreH),
7.96e7.98 (m, 2H, AreH) ppm; 13C NMR (100.6 MHz, DMSO‑d6):
Pale yellow solid; Yield 50%; Rf ¼ 0.40 (Ethylacetate/Hexane,
1:20, v/v); mp(Ethylacetate/Hexane): 193e195 ꢂC; IR (KBr)
nmax ¼ 2218 cmꢁ1 (CN str.); 1H NMR (400 MHz, CDCl3):
d
¼ 3.10 (t,
d
¼ 49.7, 51.3, 56.1, 70.9, 94.5, 114.9, 118.2, 121.7, 123.1, 125.0, 128.1,
J ¼ 4.4 Hz, 4H, 2NCH2), 3.34 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 3.42e3.46 (m,
2H, SCH 2), 3.53e3.57 (m, 2H, SCH 2), 6.87e6.92 (m, 1H, AreH),
6.96e6.98 (m, 1H, AreH), 7.07e7.11 (m, 2H, AreH), 7.12e7.16 (m,
1H, AreH), 7.24e7.27 (m, 2H, AreH), 7.41e7.43 (m, 1H, AreH),
7.47e7.48 (m, 1H, AreH) ppm; 13C NMR (100.6 MHz, CDCl3):
128.7, 128.8, 130.9, 148.4, 160.1, 161.6, 162.3, 162 ppm; MS (ESI) 422
[M þ H]þ; HRMS calculated for C23H20ClN3O3 [M þ H]þ 422.1271,
found: 422.1269.
4.2.17. 4-(4-(3-chlorophenyl)piperazin-1-yl)-2-oxo-6-phenyl-2H-
pyran-3-carbonitrile (5o)
d
¼ 32.7, 36.8, 52.6, 52.7, 102.7, 114.1, 116.9, 117.3, 120.2, 121.7, 126.7,
127.0, 128.0, 133.2, 133.8, 138.5, 141.5, 147.2, 151.4, 152.6 ppm; MS
(ESI) 420 [M þ H]þ; HRMS calculated for C23H22N3OS2 [M þ H]þ
420.1204, found: 420.1194.
White solid; Yield 50%; Rf ¼ 0.37 (Chloroform/Methanol, 10:1, v/
v);
mp(Chloroform/Methanol):
200e202
ꢂC;
IR
(KBr)
nmax ¼ 1698 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.), 3410 (OH str.); 1H
NMR (400 MHz, CDCl3):
d
¼ 3.49e3.44 (m, 4H, 2NCH2), 4.12e4.06
4.2.22. 5-(4-(2-hydroxyphenyl)piperazin-1-yl)-3,6-dihydro-2H-
fluoreno[4,3-b]thiophene-4-carbonitrile (7c)
(m, 4H, 2NCH2), 6.51 (s, 1H, C5eH), 6.77e6.82 (m, 1H, AreH),
6.94e6.88 (m, 2H, AreH), 7.20e7.26 (m, 1H, AreH), 7.57e7.48 (m,
3H, AreH), 7.88e7.81 (m, 2H, AreH) ppm; 13C NMR (100.6 MHz,
Pale yellow solid; Yield 45%; Rf ¼ 0.39 (Ethylacetate/Hexane,
1:20, v/v); mp(Ethylacetate/Hexane): 182e184 ꢂC; IR (KBr)
CDCl3):
d
¼ 48.3, 48.8, 94.2, 113.7, 115.7, 117.1, 120.3, 126.3, 129.1,
nmax ¼ 2215 cmꢁ1 (CN str.); 1H NMR (400 MHz, CDCl3):
d
¼ 3.10 (t,
130.3, 130.5, 132.1, 135.2, 150.9, 161.1, 161.6, 161.9 ppm; MS (ESI)
392.05 [M þ H]þ; HRMS calculated for C22H19ClN3O2 [M þ H]þ
422.1271, found: 422.1269.
J ¼ 4.7 Hz, 4H, 2NCH2), 3.49 (t, J ¼ 4.7 Hz, 4H, 2NCH2), 3.53e3.62 (m,
4H, Ar-CH2-Ar, SCH 2), 4.07 (s, 2H, SCH 2), 6.88e6.93 (m, 1H, AreH),
6.97e7.00 (m, 1H, AreH), 7.09e7.13 (m, 1H, AreH), 7.28e7.30 (m,
1H, AreH), 7.38e7.48 (m, 2H, AreH), 7.59 (d, J ¼ 7.3 Hz, 1H, AreH),
7.90 (d, J ¼ 7.6 Hz, 1H, AreH) ppm; 13C NMR (100.6 MHz, CDCl3):
4.2.18. 4-(4-(2-methoxyphenyl)piperazin-1-yl)-2-oxo-6-(thiophen-
2-yl)-2H-pyran-3-carbonitrile (5p)
Off White solid; Yield 68%; Rf ¼ 0.35 (Chloroform/Methanol,
10:1, v/v); mp(Chloroform/Methanol): 242e243 ꢂC; IR (KBr)
nmax ¼ 1700 cmꢁ1 (C]O str.), 2205 cmꢁ1 (CN str.); 1H NMR
d
¼ 33.8, 35.4, 36.7, 52.2, 53.4, 104.9, 114.1, 117.8, 120.1, 121.7, 123.0,
124.6, 126.6, 127.2, 128.0, 131.4, 137.2, 138.9, 139.5, 140.5, 143.7,
145.9,147.4,151.4 ppm; MS (ESI) 426 [M þ H]þ; HRMS calculated for
C
26H24N3OS [M þ H]þ 426.1640, found: 426.1629.
(400 MHz, CDCl3):
d
¼ 3.3e3.2 (m, 4H, 2NCH2), 3.90 (s, 3H, OMe),
4.05 (t, J ¼ 5.0 Hz, 4H, 2NCH2), 6.34 (s, 1H, C5eH), 6.98e6.88 (m, 3H,
AreH), 7.09e7.04 (m, 1H, AreH), 7.16e7.12 (m, 1H, AreH), 7.55 (dd,
J ¼ 5.0, 1.2 Hz, 1H, AreH), 7.69 (dd, J ¼ 3.8, 1.2 Hz, 1H, AreH) ppm;
4.2.23. 6-(4-(2-hydroxyphenyl)piperazin-1-yl)-8-(thiophen-2-yl)
isothiochromane-5-carbonitrile (7d)
Pale yellow solid; Yield 48%; Rf ¼ 0.40 (Ethylacetate/Hexane,
13C NMR (100.6 MHz, CDCl3):
d
¼ 49.7, 50.6, 55.4, 72.6, 93.1, 111.4,
1:20, v/v); mp(Ethylacetate/Hexane): 160e162 ꢂC; IR (KBr)
117.3, 118.5, 121.1, 124.0, 128.7, 129.5, 130.6, 134.1, 139.6, 152.2, 156.7,
nmax ¼ 2215 cmꢁ1 (CN str.); 1H NMR (400 MHz, CDCl3):
d
¼ 2.96 (t,
160.9, 161.6 ppm; MS (ESI) 394 [M þ H]þ; HRMS calculated for
J ¼ 6.3 Hz, 2H, Ar-CH2), 3.10 (t, J ¼ 4.6 Hz, 4H, 2NCH2), 3.29 (t,
C
21H20N3O3S [M þ H]þ 394.1225, found: 394.1216.
J ¼ 6.4 Hz, 2H, SCH2eCH2), 3.37 (t, J ¼ 4.6 Hz, 4H, 2NCH2), 3.76 (s,
11

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
2H, SCH2-Ar), 6.87e6.91 (m, 1H, AreH), 6.95e6.97 (m, 1H, AreH),
7.00 (s, 1H), 7.05e7.06 (m, 1H, AreH), 7.08e7.14 (m, 2H, AreH),
7.24e7.26 (m, 2H, AreH), 7.43e7.44 (m, 1H, AreH) ppm; 13C NMR
well chamber slide. Subsequently, non internalized parasites were
removed and macrophages with intracellular amastigotes were
treated with serial dilution (50 mMe3.125 mM) of the compounds
(100.6 MHz, CDCl3):
d
¼ 25.5, 26.6, 29.7, 52.5, 52.6, 106.7, 114.1,
for 72 h. After completion of incubation, cells were observed under
microscope and their condition was recorded in a scoring sheet.
Furthermore, medium was aspirated from all the wells and equal
volume of PBS and steady glo reagent (15 mg/mL) was added to all
the wells. To assess the luciferase activity, reading was carried out
in a luminometer (Berthold). The IC50 values of active compounds
(5a, 5d, 5e, 5f, 5g and 5h) were calculated by non-linear regression
analysis of dose-response curve using the four-parameter Hill
equations. Miltefosine was used as a reference drug for assay.
116.8,119.2,120.2,121.7,126.7,126.8,127.5,127.8,129.5,138.0,138.5,
140.1, 143.1, 151.3, 154.1 ppm; MS (ESI) 434 [M þ H]þ; HRMS
calculated for C24H24N3OS2 [M þ H]þ 434.1361, found: 434.1348.
4.2.24. 6-(4-(2-hydroxyphenyl)piperazin-1-yl)-8-(thiophen-2-yl)
isothiochromane-5-carbonitrile (7e)
Off white solid; Yield 52%; Rf ¼ 0.42 (Ethylacetate/Hexane, 1:20,
v/v);
mp(Ethylacetate/Hexane):
170e172
ꢂC;
IR
(KBr)
nmax ¼ 2215 cmꢁ1 (CN str.); 1H NMR (400 MHz, CDCl3):
d
¼ 2.95 (t,
J ¼ 6.4 Hz, 2H, Ar-CH2), 3.10 (t, J ¼ 4.3 Hz, 4H, 2NCH2), 3.29 (t,
J ¼ 6.4 Hz, 2H, SCH2eCH2), 3.36 (t, J ¼ 4.4 Hz, 4H, 2NCH2), 3.54 (s,
2H, SCH2-Ar), 6.84 (s, 1H, C5eH), 6.87e6.91 (m, 1H, AreH),
6.95e6.97 (m, 1H, AreH), 7.08e7.12 (m, 1H, AreH), 7.17 (d,
J ¼ 8.4 Hz, 2H, AreH), 7.24e7.26 (m, 2H, AreH), 7.60 (d, J ¼ 8.3 Hz,
4.6. Cell cytotoxicity assay using MTT reagent
Cells were seeded to 96 well plates at a starting density of
1 ꢃ 105 cells/well and cultured overnight. Medium was replaced
and macrophages were incubated with graded concentrations of
2H, AreH) ppm; 13C NMR (100.6 MHz, CDCl3):
d
¼ 25.5, 26.2, 29.5,
compounds (7.81e500
Afterwards, 25 L of MTT (5 mg/mL) was added to cells for 2 h and
incubated at 37 ꢂC and 5% CO2. Medium was aspirated and resulting
formazan was solubilized by adding 150 L of DMSO to each well.
m
M) for another 72 h at 37 ꢂC and 5% CO2.
52.6,52.7, 106.4, 114.1, 116.9, 118.1, 120.2, 121.7, 122.4, 126.7, 128.8,
130.4, 131.8, 138.3, 138.4, 143.3, 144.2, 151.3, 154.3 ppm; MS (ESI)
m
506 [M
þ
H]þ, 508 [M
þ
2
þ
H]þ; HRMS calculated for
m
C
26H25BrN3OS [M
þ
H]þ 506.0902, found: 506.0889 and
The amount of formazan dye formed was equivalent to the number
of live cells and absorbance was read at 570 nm using a microplate
spectrophotometer (Biotek Powerwave XS2).
[M þ 2 þ H]þ 508.0881, found: 508.0863.
4.3. Cell culture, parasites and infection
4.7. Detection of phosphatidylserine externalization using flow
J774 cells were maintained in RPMI medium supplemented with
fetal bovine serum (Gibco), penicillin (100U/mL) and streptomycin
cytometry
(100
m
g/mL) at 37 ꢂC in a humidified (5% CO2) atmosphere.
Apoptosis was assessed using flow cytometric analysis of control
and 5a treated promastigotes for different time period that were
stained with Alexa fluor 488- Annexin V and PI (Invitrogen). Briefly,
log phase promastigotes (1 ꢃ 106 cells/mL) were seeded into 12
L. donovani axenic promastigotes (MHOM/IN/80/Dd8) transfected
with luciferase gene were maintained in M199 medium supple-
mented with 10% FBS (Hyclone) and G418 antibiotic (20 mg/mL) at
24 ꢂC. For in vitro infection studies, J774 macrophages (4 ꢃ 104 cells/
well) were seeded into 96 well plate and 16 well chamber slide
(Thermo Fisher Scientific) for overnight. Macrophages were infec-
ted with stationary forms of luciferase transfected Dd8 promasti-
gotes at a 1:8 ratio for 24 h. After infection for 24 h and then
treatments with compounds 5a and 5g, cells were fixed and stained
with Giemsa stain.
well plate and were treated with 5a (50 mM) for 24 h, 48 h and 72 h.
After incubation, cells were collected, washed in PBS and resus-
pended in 500 L of 1X annexin binding buffer. Cells were then
m
incubated with annexin V-Alexa fluor 488 and propidium iodide in
the absence of light. After incubation for 15 min, samples were
immediately analyzed via BD FACS calibur flow cytometer. Annexin
V staining was detected as green fluorescence and PI as red
fluorescence.
4.4. Promastigotes assay
4.8. Assessment of mitochondrial membrane potential loss
Log phase L. donovani promastigotes were seeded in 96-well
culture plates with two concentrations (50
m
M and 25
m
M) of all
Fluorophore JC-1 was used to study the loss of mitochondrial
membrane potential which occurs during cell apoptosis. Briefly,
L. donovani promastigotes at 1 ꢃ 106 cells/mL were incubated with
the compounds dissolved in DMSO. As a vehicle control, promas-
tigotes were only treated with 0.1% DMSO. After 72 post treatment
with compounds, cell viability was determined by MTT assay.
50
mM dose of 5a for different time period. Promastigotes were
Briefly, cells were incubated with MTT reagent (20
mL of 5 mg/mL
incubated with CCCP (50 mM final concentration) as a positive
stock in PBS) for 4 h and after incubation cells were transferred to
V-shaped bottom 96 well plate. After centrifugation, medium was
control for induction of depolarization of the mitochondrial
membrane potential. At the end of incubation, cells were harvested
discarded and 150
m
L/well DMSO was added to dissolve the for-
and washed with PBS. To the cell suspension, 10 mL of 200 mM (2 mM
mazan salt. The color intensity of the formazan solution, which
reflects the cell growth condition, was measured at 570 nm
wavelength using a microplate spectrophotometer (Biotek Power-
wave XS2).
final concentration) JC-1 was added and incubated at 37 ꢂC, 5% CO2
for 30 min. Afterwards, the cells were washed, resuspended in PBS
and analyzed by flow cytometry (FACS calibur).
4.9. TUNEL assay for assessment of DNA fragmentation
4.5. Intracellular amastigotes inhibition assay
DNA fragmentation in L. donovani promastigotes treated with 5a
was detected by using APO-Brdu TUNEL Assay Kit (Molecular
probes, USA). Briefly, after incubation with 5a for 72 h, promasti-
gotes were washed with 1X PBS, adhered to poly L-lysine coated
slides, and fixed with 4% paraformaldehyde. Next, promastigotes
were permeabilized with ice-cold 70% ethanol for 30 min on ice.
Briefly, murine J774 macrophage cell (4 ꢃ 104/mL/100
mL/well)
were seeded for 24 h in RPMI supplemented with 10% FBS. Cells
were then washed and infected with stationary form of luciferase
expressing L. donovani promastigotes at a ratio of 1:8 (macro-
phages:parasite) for another 24 h at 37 ꢂC, 5% CO2. After incubation,
infectivity was checked using Giemsa staining in a parallel set of 16
Slides were then incubated in 50 mL of DNA labeling solution (10 ml
12

S. Mishra, N. Parmar, P. Chandrakar et al.
European Journal of Medicinal Chemistry 221 (2021) 113516
of reaction buffer, 0.75
of dH2O) for 60 min at 37 ꢂC. Cells were stained with 100
antibody solution (5 l of Alexa fluor 488 dye labeled anti-BrdU
antibody in 95 l of rinse buffer) for 30 min and followed by
addition of PI/RNaseA staining buffer for another 30 min. Promas-
tigotes were mounted using using Pro-gold anti-fade media
(Thermo Fisher Scientific, USA). The broken DNA ends of apoptotic
promastigotes were analyzed using confocal microscopy (Zeiss LSM
510 META).
m
l of TdT enzyme, 8
m
l of BrdUTP and 31.25
m
l
4.13. Statistical analysis
ml of
m
Data were analyzed using student t-test. The results were
expressed as mean values with standard deviations. Statistical
significance was considered at p < 0.05 and analysis was performed
using Graph Pad Prism version 5.
m
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.10. Ethics Statement
Animal care and experimental procedures were performed in
strict accordance by the Committee for the Purpose of Control and
Supervision of Experiments on Animals (New Delhi, India). The
in vivo experimental protocol was approved by the Institutional
Animal Ethics Committee (registration no. 34/GO/ReBiBt-S/Re-L/
99/CPCSEA) of the Council for Scientific and Industrial
ResearcheCentral Drug Research Institute (Lucknow, India) [IAEC
approval no: IAEC/2016/101 dated 12.5.16]. For experimental
studies, female BALB/c mice (20e25 gm) were housed in climate-
controlled and photo period controlled (12-h light/dark cycles)
animal house and fed with standard rodent pellets and drinking
water ad libitum. Euthanasia of mice was performed by CO2 inha-
lation at the end of the experiments.
Acknowledgment
This study was funded by the Council of Scientific and Industrial
Research (CSIR NWP BSC0114), Department of Biotechnology (BT/
PR32490/MED/29/1457/2019) New Delhi, India and CSIR-CDRI in
house project. AG is thankful to the Department of Atomic Energy
(DAE-SRC) for Outstanding Investigator Project Grant (21/13/2015-
BRNS/35029). SM, NP, CPS and AS are thankful to University Grants
Commission (UGC), New Delhi, Government of India. PC, SP and
RPV are thankful to Council of Scientific and Industrial Research
(CSIR), New Delhi, Government of India for their financial assis-
tance. The transgenic L. donovani promastigotes was a kind gift
from Dr. Neena Goyal, Division of Biochemistry, CSIR-CDRI, Luck-
now, India. Authors thank SAIF-CDRI, Lucknow, India for providing
spectral data, flow cytometry and confocal experiments. This article
has CDRI communication number 10238.
4.11. Infection and treatment in Balb/c mice
Balb/c mice were divided into five groups, with 5 animals/group.
The animals in the group A didn't receive any treatment and termed
as infected group. The infected animals in group B received only 5%
DMSO as vehicle control. The infected animals in group C received
compounds 5a via intraperitoneal route given for 5 consecutive
days at dose of 50 mg/kg. The infected animals in group D received
compounds 5g via intraperitoneal route given for 5 consecutive
days at dose of 50 mg/kg. The infected Balb/c mice in group E
received miltefosine via oral route as reference drug at a dose of
25 mg/kg daily given for 5 consecutive days. To establish Leishmania
infection in animals, mice were intravenously infected with sta-
tionary phase Leishmania donovani promastigotes (2 ꢃ 107). Infec-
tion checking was performed by conducting autopsy of three to
four randomly selected mice after 2 wk of infection. For estimation
of parasite burden, animals were sacrificed at day 7 post-treatment
and tissue samples were obtained from liver and spleen of all
groups of Balb/c mice. Afterwards, impression smears were pre-
pared on glass slides, fixed in methanol, stained with 30% Giemsa
stain for 2 h. Giemsa stained splenic and liver tissue smear was
counted for amastigotes/1000 nucleated cells and was expressed as
LDU (1 LDU ¼ amastigote per nucleated cell ꢃ organ weight in
milligram) [24].
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113516.
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