Double-edged Swords: Diaryl pyrazoline thiazolidinediones synchronously targeting cancer epigenetics and angiogenesis

Article abstract of DOI:10.1016/j.bioorg.2021.105350

In the present study, two novel series of compounds incorporating naphthyl and pyridyl linker were synthesized and biological assays revealed 5-((6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy) naphthalene-2-yl)methylene)thiazolidine-2,4-dione (14b) as the most potent dual inhibitors of vascular endothelial growth factors receptor-2 (VEGFR-2) and histone deacetylase 4 (HDAC4). Compounds 13b, 14b, 17f, and 21f were found to stabilize HDAC4; where, pyridyl linker swords were endowed with higher stabilization effects than naphthyl linker. Also, 13b and 14b showed best inhibitory activity on VEGFR-2 as compared to others. Compound 14b was most potent as evident by in-vitro and in-vivo biological assessments. It displayed anti-angiogenic potential by inhibiting endothelial cell proliferation, migration, tube formation and also suppressed new capillary formation in the growing chick chorioallantoic membranes (CAMs). It showed selectivity and potency towards HDAC4 as compared to other HDAC isoforms. Compound 14b (25 mg/kg, i.p.) also indicated exceptional antitumor efficacy on in-vivo animal xenograft model of human colorectal adenocarcinoma (HT-29). The mechanism of action of 14b was also confirmed by western blot.

Full text of DOI:10.1016/j.bioorg.2021.105350

Journal Pre-proofs
Double-edged Swords: Diaryl Pyrazoline Thiazolidinediones Synchronously
Targeting Cancer Epigenetics and Angiogenesis
Neha Upadhyay, Kalpana Tilekar, Sabreena Safuan, Alan P. Kumar, Markus
Schweipert, Franz-Josef Meyer-Almes, Ramaa C S
PII:
S0045-2068(21)00727-6
DOI:
https://doi.org/10.1016/j.bioorg.2021.105350
YBIOO 105350
Reference:
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Bioorganic Chemistry
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Accepted Date:
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13 July 2021
7 September 2021
Please cite this article as: N. Upadhyay, K. Tilekar, S. Safuan, A.P. Kumar, M. Schweipert, F-J. Meyer-Almes, R.
C S, Double-edged Swords: Diaryl Pyrazoline Thiazolidinediones Synchronously Targeting Cancer Epigenetics
and Angiogenesis, Bioorganic Chemistry (2021), doi: https://doi.org/10.1016/j.bioorg.2021.105350
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Double-edged Swords: Diaryl Pyrazoline Thiazolidinediones
Synchronously Targeting Cancer Epigenetics and Angiogenesis
Neha Upadhyay1, Kalpana Tilekar1, Sabreena Safuan2, Alan P. Kumar3, Markus Schweipert4, Franz-Josef
Meyer-Almes*4, Ramaa C S*1
1Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai, India.
2School of Health Sciences, Health Campus Universiti Sains Malaysia 16150 Kubang Kerian Kelantan.
3Cancer Science Institute of Singapore and Department of Pharmacology, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore
4Department of Chemical Engineering and Biotechnology, University of Applied Sciences, Darmstadt,
Germany.
*Corresponding authors. E-mail address: sinharamaa@yahoo.in, franz-josef.meyer-almes@h-da.de
Keywords: Anti-tumor, HDAC, Multi-targeting, Synthesis, VEGFR-2
ABSTRACT
In the present study, two novel series of compounds incorporating naphthyl and pyridyl linker were synthesized
and biological assays revealed 5-((6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-
2-oxoethoxy) naphthalene-2-yl)methylene)thiazolidine-2,4-dione (14b) as the most potent dual inhibitors of
vascular endothelial growth factors receptor-2 (VEGFR-2) and histone deacetylase 4 (HDAC4). Compounds
13b, 14b, 17f, and 21f were found to stabilize HDAC4; where, pyridyl linker swords were endowed with higher
stabilization effects than naphthyl linker. Also, 13b and 14b showed best inhibitory activity on VEGFR-2 as
compared to others. Compound 14b was most potent as evident by in-vitro and in-vivo biological assessments.
It displayed anti-angiogenic potential by inhibiting endothelial cell proliferation, migration, tube formation and
also suppressed new capillary formation in the growing chick chorioallantoic membranes (CAMs). It showed
selectivity and potency towards HDAC4 as compared to other HDAC isoforms. Compound 14b (25 mg/kg, i.p.)
also indicated exceptional antitumor efficacy on in-vivo animal xenograft model of human colorectal
adenocarcinoma (HT-29). The mechanism of action of 14b was also confirmed by western blot.

1. INTRODUCTION
Cancer is the most prevalent disease worldwide which is controlled by both genetic and epigenetic
modifications. To combat cancer, it is essential for a molecule to have target specificity as well as potency.
However, administration of single targeting agent might not always produce desired pharmacological effects
due to target mutation or development of resistance.[1,2] In addition, commencement and progression of
cancer rely on multiple receptor or signaling pathway, therefore, alternative strategies to target cancer is either
use of combination therapy or development of multi-targeting agents.[3,4] Limitations of using combination
therapy are dose-limiting toxicities, drug-drug interactions and cost of two separate agents. A single agent
simultaneously targeting multiple pathways may provide therapeutic benefits in cancer treatment due to
synergistic potential.[5–7] In dual/multi-target approach, the concept of “magic bullets” exists which is “a
single drug with multi-target specificity and high potency”,[8] therefore, dual/multi-targeting approach could
be used extensively for the development of potential agents in cancer treatment.
Angiogenesis plays a fundamental role in many physiological processes such as embryonic development,
wound healing, growth of endometrium during menstrual cycle and ovulation. However, excessive
angiogenesis is often linked with numerous pathologies, including chronic inflammation, arthritis, psoriasis,
proliferative blinding retinopathy and cancer.[9,10] There are varieties of molecules involved in regulation
of angiogenesis, among which, vascular endothelial growth factors (VEGF) and their kinase receptors
remains the most prominent ones. The interaction of VEGF with VEGF receptors (VEGF receptor 2 -
VEGFR-2/KDR/Flk1) starts critical downstream signaling pathways that bring about tumor angiogenesis.
Thus, VEGF/VEGFR pathway signifies a rational target for therapeutic intervention.[11–14] FDA has
approved bevacizumab & ramucizumab antibody that target VEGF and other synthetic agents such as
sorafenib, sunitinib, pazopanib, vandetanib, axitinib and regorafenib.[5,15] However, their use as
monotherapy is limited due to drug resistance, low efficacy, aggressive relapse and metastasis of tumor post
withdrawal of drugs.[16–20] Resistance is the major concern among all, thus, over decades researchers are
directing their focus on potential treatment of resistance.[5]
Histone deacetylase (HDAC) is a key enzyme involved in epigenetic alterations thus, it is well known
therapeutic target in anticancer drug discovery.[21,22] However, monotherapy of HDAC inhibitors fails to
display any significant effectiveness against solid tumors.[23] So far there are various FDA approved non-
selective HDAC inhibitors used for hematologic cancer such as vorinostat, panobinostat and belinostat, but
they are associated with several side effects such as mutagenicity, thrombocytopenia, leukopenia, diarrhoea
and fatigue.[24,25] Hydroxamic acids are under suspicion in being potentially mutagenic besides the intrinsic
propensity for unselectivity.[26] Thus, best alternative strategy to circumvent potential mutagenicity and
unselectivity issues associated with hydroxamate function would be to develop non-hydroxamate ZBGs. A
suitable example would be prodrug romidepsin (FK228) which contain thiol ZBG and has been approved for
cutaneous and peripheral T-cell lymphoma.[27] On the similar lines various HDAC inhibitors with non-
hydroxamate ZBGs have been reported.[28–30]

Interestingly, HDAC inhibitors are also know to inhibit angiogenesis by downregulation of VEGF and
suppression of neovascularization through alteration of other genes directly involved in angiogenesis.[31–34]
Literature reports also state that HDAC inhibitors mediate anti-angiogenesis actions by several mechanisms
viz. suppression of hypoxia inducible factor-1α (HIF-1α), initiating cell cycle arrest, and inducing apoptosis
and autophagy.[35,36] Furthermore, in 2011, cabozantinib was developed using multitarget approach, which
was reported as dual inhibitor of VEGFR-2 and c-Met and is successfully used for treatment of renal cell
carcinoma.[37–39] Various dual targeting agents are evident, wherein, VEGFR-2 is one of the target along
with other potential targets[1,2,40–43] and same remain true in case of HDAC also.[28,44–48] Additionally,
multi-target 2-indolinone derivatives were reported as protein kinase and HDAC inhibitors.[49] In clinical
studies HDAC inhibitors have also been used in combination with anti-angiogenic agents for the treatment of
sarcomas.[50] HDACs are known to involve in VEGF-induced angiogenesis, endothelial cell proliferation
and migration; and also several reports support selection of these two as potential druggable targets.[35,51]
Thus, by using multitarget approach development of agents simultaneously targeting VEGFR-2 and HDAC
would attain anti-tumor effects along with reduced side effects associated to single-target therapy.
Herein, we designed this research project with aim to identify agent/s with inhibition capability of VEGFR-
2 and HDAC. After successful synthesis and characterization of all compounds they were comprehensively
screened for their biological potency. Synthesized compounds were subjected to in-vitro and in-vivo assays
by which most potent compound 14b was identified with simultaneous VEGFR-2 and HDAC inhibition
potential.
2. RESULTS AND DISCUSSION
2.1 Rationale of Designing. A few literatures put forward development of VEGFR-2 and HDAC multi-acting
candidates by structural hybridization strategy.[6,7,42] Most of this study led to dual inhibitors with
micromolar to nanomolar inhibitory concentrations which were in comparative range of the standard drugs.
However, most of the reported dual inhibitors incorporated hydroxamate moiety as the ZBG and according
to literatures, the physicochemical properties associated with hydroxamic acid ZBG attain some major
drawbacks including significant off-target effects, easy metabolism, poor stability, and weak binding to
isozymes of class IIa.[26,52–54] For these reasons alternative ZBGs have been explored.
To design dual targeting VEGFR-2 and HDAC inhibitors, it was essential to design such unique structure
which will constitute pharmacophoric requirements of both targets. Common features of VEGFR-2 inhibitors
are: a hinge-binding group that binds to ATP pocket (by hydrogen bond with the NH backbone of Cys919
residue); a linker that traverses to gatekeeper residue; a hydrogen bonding moiety that interact with DGF
motif (with Glu885, Cys1045 and Asp1046), and a hydrophobic tail that occupies allosteric binding pocket
formed by DFG-out flip.[55–58] On the other hand, pharmacophoric features of HDAC inhibitors are: CAP
portion; a linker, and ZBG[21] (Figure 2). Accordingly, dual targeting agents were designed by considering
the structural necessities of both targets. We have previously reported 5-benzylidene-2,4-
thiazolidinedione[59] and 5-pyridin-4-yl-2-thioxo-[1,3,4]oxadiazol-3-yl[60] analogs as VEGFR-2 inhibitors

(Figure 2; compound 10 and 11 respectively), with a conclusion that heavy hydrophobic moiety was essential
to occupy large DFG motif. Thus, 5-benzylidene-2,4-thiazolidinediones analog was modified by replacing
terminal aryl group with bulkier diaryl-substituted pyrazoline (ring A and B) and other structural features
were retained. The diaryl-substituted pyrazoline ring was the best choice due to the fact that: they have been
identified as appropriate surface recognition motif in designing of HDAC inhibitors (Figure 1; compound 5–
9)[61–67]; they exhibit excellent antiproliferative potential[68–74]; and more interestingly, pyrazoline
bearing analogs have been reported to show excellent VEGFR-2 inhibition capability (Figure 1; compound 3
and 4)[42,43,75–78] as well as anti-angiogenesis potency (Figure 1; compound 1 and 2).[79–82]
Thiazolidinedione (TZD) is a privileged scaffold which has been explored for its antiproliferative potential
in the field of anticancer drug discovery.[83,84] We have previously reported HDAC inhibitors (Figure 2;
compound 12 and 13)[29,85] where TZD was placed at the zinc binding tail and served as replacement of
classical hydroxamate ZBG, led to a novel non-hydroxamate ZBG. Additionally, our previously reported
VEGFR-2 inhibitors (Figure 2 compound 10) also hold TZD; hence, keeping the TZD scaffold would ensure
retention of VEGFR-2 inhibition along with HDAC inhibitory potential. Thus, while designing the dual
inhibitors, TZD scaffold was projected at the tail portion. In particular, many non-classical HDAC inhibitors
contain cyclic ring as a linker instead of straight chain with potent HDAC inhibitory activity[21,86,87] and
similar was found true with our recently reported selective-HDAC8 inhibitors (Figure 2 compound 13),[29]
which contains naphthalene as cyclic linker. Thus, two different cyclic rings (ring C) viz. naphthalene and
pyridine were incorporated as linker to give completely novel structure and also to compare their structure
activity relationship as VEGFR-2 and HDAC inhibitors.

Figure 1. Different VEGFR-2 and HDAC inhibitors.

Figure 2. Designing strategy of novel VEGFR-2 and HDAC dual inhibitors. HBA – hydrogen bond acceptor;
HBD – hydrogen bond donor; SRC – surface recognition cap; ZBG – zinc binding group.
2.2 Chemistry. All intermediates and final compounds were synthesized by using 3 different schemes
(scheme I, II and III). The detailed procedure of synthesis and other spectral observations have been presented
in the experimental section. The purity of all the final compounds were confirmed by HPLC which was >95%.
Structures of all the intermediates were confirmed by 1H-NMR and FTIR and that of all the final compounds
by different spectroscopies viz.1H-NMR, 13C-NMR, FTIR and Mass.
Scheme I outline the synthesis of intermediates viz. chalcones and, chloroacetylated pyrazolines. First step
was synthesis of different chalcones (3a-3c, 3e, 3g, 3i, 6a-6g, 6j, and 9b-9g), which occurred via renowned
Claisen-Schmidt condensation reaction. Substituted acetophenone (1, 5 and 8) were reacted with different
aromatic aldehydes (2a-2g, 2i, and 2j) in basic medium to obtain respective chalcone derivatives (3a-3c, 3e,
3g, 3i, 6a-6g, 6j, and 9b-9g). Chloroacetylated pyrazoline intermediates (4a-4c, 4e, 4g, 4i, 7a-7g, 7j, and
10b-10g) were synthesized by refluxing different chalcones (3a-3c, 3e, 3g, 3i, 6a-6g, 6j, and 9b-9g) with
hydrazine hydrate and chloroacetyl chloride in chloroform.
Scheme II outlines the synthesis of final compounds (13a-13c, 13e, 13g, 13i, 14a-14c, 14e, 14g, 15e, and
15g) which proceed via two steps: 1) Knoevenagel condensation of 2,4-thiazolidinedione (11) with 6-
hydroxy-2-naphthaldehyde, in the presence of piperidine benzoate in toluene to obtain 5-((6-
hydroxynaphthalen-2-yl)methylene)thiazolidine-2,4-dione (12); 2) Reaction of 12 with the respective
chloroacetylated pyrazolines (4a-4c, 4e, 4g, 4i, 7a-7g, 7j, and 10b-10g) in the presence of K2CO3 in DMF to
obtain the respective final compounds (13a-13c, 13e, 13g, 13i, 14a-14c, 14e, 14g, 15e, and 15g).
Scheme III represents the synthesis of final compounds 17a-17b, 17d-17f, 17h, 17i, 19a-19f, and 21b-21f.
In this route of synthesis, appropriate changes were made at different steps, as by following the same synthetic
routes (Scheme I and II) product was not obtained. Hence, the scheme was reversed by first coupling 6-
hydroxynicotinaldehyde with various chloroacetylated intermediates (4a-4b, 4d-4f, 4h, 4i, 7a-7f, and 10b-
10f) in the presence of K2CO3 in DMF to obtain respective aldehyde containing intermediates (16a-16b, 16d-
16f, 16h, 16i, 18a-18f, and 20b-20f). Further, Knoevenagel condensation of 2,4-thiazolidinedione (11) in 2-
methoxyethanol and catalytic amount of piperidine along with the aldehyde intermediates (16a-16b, 16d-16f,
16h, 16i, 18a-18f, and 20b-20f) was carried out to give the corresponding target compounds (17a-17b, 17d-
17f, 17h, 17i, 19a-19f and 21b-21f).

Scheme I. Synthesis of intermediates (3a-3i, 6a-6g, 6j, 9b-9g, 4a-4i, 7a-7g, 7j, and 10b-10g). Reagents and
conditions: (a) Aq. NaOH, EtOH, RT, 5-6 h, 80-93%; (b) NH2NH2.H2O, CHCl3, 80 °C, 12 h, then K2CO3,
ClCH2COCl, RT, 12 h, 50-64%.
b
a
N
N
O
2a-2i
O
Cl
Substituted
Ar
1
Substituted Ar
3a-3i
O
4a-4i
F
F
F
a
b
N
N
O
2a-2g, 2j
Cl
O
Substituted
Substituted Ar
O
Ar
5
6a-6g, 6j
7a-7g, 7j
F
F
F
a
F
b
F
N
N
O
F
2b-2g
O
Cl
Substituted Ar
9b-9g
Substituted
O
Ar
8
10b-10g
2a- benzaldehyde
2b- o-chlorobenzaldehyde
2c- 2-furaldehyde
2f- 2,4-difluorobenzaldehyde
2g- p-methylbenzaldehyde
2h- 3-nitrobenzaldehyde
2d- 4-trifluoromethylbenzaldehyde
2e- p-fluorobenzaldehyde
2i- thiophene-2-carboxaldehyde
2j- 3,4-dichlorobenzaldehyde

Scheme II. Synthesis of final compounds (13a-13c, 13e, 13g, 13i, 14a-14c, 14e, 14g, 15e, and 15g). Reagents
and conditions: (c) 6-hydroxy-2-naphthaldehyde, piperidine benzoate, toluene, reflux, 4 h, 86%; (d) K2CO3,
DMF, RT, 24 h, 52-70%.
O
NH
O
NH
c
S
S
HO
O
O
11
O
12
d
O
NH
N
N
S
4a-4c,
4e, 4g, 4i
HO
NH
O
O
12
S
Substituted
O
Ar
O
13a-13c, 13e, 13g, 13i
F
O
O
N
N
d
NH
O
S
NH
HO
O
S
7a-7c, 7e, 7g
Substituted
Ar
O
O
12
14a-14c, 14e, 14g
F
O
F
O
d
NH
O
N
N
S
HO
10e, 10g
NH
O
S
12
Substituted
Ar
O
O
15e,15g

Scheme III. Synthesis of final compounds (17a-17b, 17d-17f, 17h, 17i, 19a-19f, and 21b-21f). Reagents and
conditions: (e) K2CO3, DMF, RT, 48 h, 52-80%; (f) 2,4-thiazolidinedione, 2-methoxyethanol, piperidine,
reflux, 3 h, 54-88%.
CHO
O
N
N
N
CHO
N
N
N
N
f
e
HO
NH
O
O
S
Substituted
Substituted
O
Ar
O
Ar
O
4a-4b, 4d-4f, 4h, 4i
16a-16b, 16d-16f, 16h, 16i
F
17a-17b, 17d-17f, 17h, 17i
F
CHO
N
O
f
e
HO
N
N
N
N
CHO
N
N
NH
O
S
O
Substituted
Substituted
Ar
7a-7f
O
O
Ar
O
18a-18f
19a-19f
F
F
F
F
O
CHO
N
N
N
N
CHO
N
N
f
e
N
NH
HO
O
O
S
Substituted
Ar
Substituted
O
O
Ar
O
10b-10f
20b-20f
21b-21f
2.3 In-vitro HDAC Enzyme Inhibition Assay. Preliminary Screening on Class I and Class II HDAC
Isoform. To determine the effects of pyrazoline-based thiazolidinediones (Table 1; compounds 17a-15g) on
the activity of HDACs, all thirty-one compounds were preliminary screened at 50 µM concentration on two
HDAC isoforms viz. HDAC4 (representative of Class IIa) and HDAC8 (representative of Class I). Test
compounds showed excellent inhibitory effects on HDAC4 and significantly lower activity on HDAC8.
Based on the results obtained in primary screening, compounds with residual enzyme activity < 50% were
taken further to establish a dose-response curve. Many compounds showed considerable selectivity for
HDAC4 over HDAC8 with greatest selectivity factors of >100 [IC50(HDAC8)/IC50(HDAC4)] for compounds
13b, 14b and 15g. In contrast, compound 13c and 14c, both of them containing a furyl group attached to the
pyrazoline ring, showed comparable activities against HDAC4 and HDAC8 (supplementary data). All
pyrazoline-based thiazolidinediones displayed significant inhibitory activity on HDAC4 (in low to sub-
micromolar range). The best activities against HDAC4 were comparable to “Cpd 6” (Table 1).[88] There was
visible difference in the HDAC4 activity of compounds containing –pyridyl and –naphthyl linker.
Compounds with –pyridyl linker (17a-17b, 17d-17f, 17h, 17i, 19a-19f, and 21b-21f) offered considerable
inhibitory potency, IC50 below 10 µM, except for 17a. Interestingly, all compounds incorporating the naphthyl
linker (13a-13c, 13e, 13g, 13i, 14a-14c, 14e, 14g, 15e, and 15g) demonstrated enhanced potency, IC50 below
1 µM, which proved our hypothesis of selecting pyridine and naphthalene ring as linker. For representative

most active compounds 13b and 14b, the selectivity profile was determined over a broad panel of zinc-
dependent HDAC isoenzymes (Table 2) demonstrating very similar isoenzyme selectivity compared to
reference compound “Cpd6”.[88]
Overall HDAC4 and HDAC8 enzyme inhibition assay results suggest that, novel series of pyrazoline-based
TZDs are HDAC4-selective inhibitors. Additionally, it was noteworthy that, in compound 13 (Figure 2), the
“ortho-position” of the TZD warhead with respect to oxo-linker chain headed to “HDAC8-isoenzyme
selectivity” (HDAC8 – 2.7 μM and HDAC4 – 15 μM),[89] whereas, “para-positioning” shifted the isoenzyme
selectivity towards HDAC4.[28] Similar was found true with the proposed pyrazoline-TZDs, wherein, the
setting of TZD warhead was at “para-position” to the oxo-linker indicating HDAC4-isoenzyme selectivity
(Table 1 and Table 2).
Initially to find the effects of various linkers (bicyclic/heterocyclic/aromatic), the arylidene type Knoevenagel
derivatives of TZD containing these rings were synthesized and evaluated for their effects on HDAC4 and
HDAC8 isoforms, wherein, Knoevenagel derivatives containing naphthyl and pyridyl ring exhibited
relatively weak HDAC inhibitory activity (data not shown here). Comparison of the inhibitory potential of
these Knoevenagel derivatives with that of the final compounds demonstrated that attachment to diaryl-
pyrazoline ring enhanced the HDAC4 inhibitory capacity several folds as compared to Knoevenagel
derivatives.
Precisely, HDACs contribute significantly in the development and growth of tumorigenesis. The, hypoxia-
derived transcription factor HIF-1α regulates the gene expression involved in different cellular signaling
pathways of angiogenesis (via expression of VEGF). In multiple malignancies, VEGF-overexpression
mediated by stabilization of HIF-1α has been identified.[35,90] The posttranslational modifications such as
acetylation driven by HATs and HDACs are critical for HIF-1α signaling. More interestingly, class II HDACs
(such as HDAC4) are vital for the stability of HIF-1α protein and its transcriptional activity; and HDAC4 has
been shown to regulate the acetylation levels of HIF-1α.[12] In a nutshell, HDAC4 is directly or indirectly
linked to the management of angiogenesis, selective-HDAC4 inhibitors are therefore supposed to be
therapeutically effective.
The results demonstrated HDAC4 selectivity of these compounds and to explore their anti-angiogenesis
potential, compounds with best inhibitory activity were comprehensively evaluated through various in-vitro
and in-vivo follow-up assays.
Table 1. Preliminary screening and IC50 determination of pyrazoline-based thiazolidinediones on HDAC4
and HDAC8. Residual activities are shown as means ± standard deviation, n=3.
Sr.
Code
Residual HDAC4
Residual HDAC8
HDAC4 IC50
(µM)
HDAC8
No.
activity (%) at 50 µM
activity (%) at 50 µM
IC50 (µM)
1
13a
16 ± 1
100 ± 5
3.8 ± 0.1
>50

2
13b
13c
13e
13g
13i
12 ± 1
14 ± 1
59 ± 3
1.3 ± 0.3
70 ± 2
68 ± 2
6.7 ± 1.5
27 ± 1
75 ± 4
1.0 ± 0.5
84 ± 7
51 ± 4
63 ± 3
40 ± 1
100 ± 2
85 ± 7
100 ± 8
100 ± 5
100 ± 5
97 ± 5
66 ± 3
100 ± 8
32 ± 3
74 ± 5
70 ± 5
93 ± 3
93 ± 4
0.34 ± 0.02
1.1 ± 0.2
0.7 ± 0.1
0.7 ± 0.1
0.35 ± 0.02
0.8 ± 0.2
0.36 ± 0.2
1.7 ± 0.3
0.8 ± 0.4
0.8 ± 0.4
0.6 ± 0.1
0.4 ± 0.1
18 ± 4
31 ± 3
1.6 ± 0.4
>50
3
4
10 ± 1
5
12 ± 1
>50
6
6.7 ± 0.6
5 ± 0.8
15 ± 1
14 ± 10
>50
7
14a
14b
14c
14e
14g
15e
15g
17a
17b
17d
17e
17f
17h
17i
8
>50
9
10 ± 1
4.7 ± 0.5
>50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
9.4 ± 1.7
8.0 ± 1.3
11 ± 1
>50
>50
5.6 ± 0.3
22 ± 1
>50
>50
21 ± 1
3.3 ± 1
>50
19 ± 1
2.7 ± 0.2
2.5 ± 1.5
1.9 ± 0.4
5.2 ± 0.5
5.9 ± 0.5
2.1 ± 0.4
2.2 ± 0.3
>50
>50
11 ± 2
>50
14 ± 2
>50
10 ± 1
>50
6.4 ± 0.9
10 ± 1
>50
19a
19b
19c
19d
19e
19f
>50
7.2 ± 0.7
25 ± 1
>50
>50
3.5 ± 0.7
11 ± 1
2.3 ± 0.2
2.1 ± 0.6
2.4 ± 0.2
>50
>50
5.1 ± 0.6
>50

27
28
29
30
31
32
33
34
21b
21c
15 ± 1
13 ± 1
8.2 ± 1.3
4.3 ± 0.4
4.9 ± 0.3
95 ± 1
-
98 ± 9
96 ± 2
98 ± 7
81 ± 4
61 ± 5
93 ± 3
-
4.3 ± 0.3
10 ± 2
1.9 ± 0.2
3.1 ± 0.2
0.9 ± 0.6
>50
30.4
>50
>50
>50
>50
>50
>50
0.36
21d
21e
21f
S1[91]
Cpd 6[88]
Cpd 31[92]
0.22
-
-
0.02
2.4 HDAC-Profiling on a Panel of HDACs (HDAC1-HDAC8). In the primary screening on the two HDAC
isoforms (HDAC4 and HDAC8), compounds 13b and 14b with naphthyl linker, and compounds 17f and 21f
with pyridyl linker were found with greatest inhibitory potential; thus, these four compounds were evaluated
on a panel of HDAC (HDAC1-HDAC8) to confirm their selectivity towards HDAC4 (Table 2). In particular,
these compounds were found to be potent and highly selective towards HDAC4 as compared to other isoforms
(Figure 3).
Table 2 – Selectivity profile of pyrazoline-based thiazolidinediones on HDAC isoenzymes.
IC50 (µM)
Code
HDAC1
HDAC2
HDAC3
HDAC4
HDAC6
HDAC7
HDAC8
13b
24 ± 2
37 ± 2
22 ± 1
0.34 ±
0.02
20 ± 1
>50
31 ± 3
14b
17f
18 ± 1
34 ± 3
>50
45 ± 2
*ND
*ND
>50
16 ± 1
34 ± 5
29 ± 3
12
0.36 ± 0.2
1.9 ± 0.4
0.9 ± 0.6
0.22
15 ± 1
>50
>50
17
6.3 ± 0.3
>50
>50
>50
>50
>50
21f
>50
Cpd 6[88]
>50
>50
*ND – Not determined.

Figure 3. Dose response curve of compounds 13b, 14b, 17f, and 21f on different HDAC isoforms.
2.5 Thermal Shift Assay on HDAC4. Thermal shift assay is a modern method used to assess the binding of
ligands to a target protein. This method is used to demonstrate in-vitro target engagement (TE) of ligands.
Binding of a ligand to a target protein shifts the melting temperature of the protein producing a thermal shift.
Test compounds with HDAC4 inhibitory potential (13b, 14b, 17f, and 21f) were subjected to this assay with
HDAC4 isoform to examine the thermal stabilization by the ligands. The melting temperature of unbound
HDAC4 was 57.3 °C, and those of the protein in the presence of the test compounds were 57.5 °C, 57.9 °C,
60.4 °C, and 58.8 °C, for 13b, 14b, 17f, and 21f, respectively (Figure 4). In particular, all of the inhibitors
were able to stabilize HDAC4, thereby confirming the interaction between HDAC4 and these compounds.
Inhibitors 17f and 21f with pyridyl linker showed significantly higher stabilizing effects than 13b and 14b
with naphthyl linker.

Figure 4. Thermal stabilization of HDAC4 upon binding of test compounds 13b, 14b, 17f, and 21f. A)
Residual native HDAC4 in terms of band intensity in protein gel as a function of temperature in the presence
of indicated compounds, B) Thermal stabilization of HDAC4 given as difference between the melting point
of HDAC4 in the presence of indicated compound minus the melting point of unbound enzyme.
2.6 HUVECs Cytotoxicity Assay. Human Umbilical Vein Endothelial Cells (HUVECs) are well known
representative of endothelial cells to determine cytotoxicity potential of novel antiangiogenic compounds in-
vitro.[93,94] Sixteen compounds (Table 3) with highest HDAC4 inhibitory potential were selected to
determine their effects on HUVECs proliferation and MTT assay was performed. Staurosporine (STS) was
used as positive control. Different concentrations (10, 1, 0.1, 0.01, and 0.001 µM) of test compounds and STS
were used to determine their inhibitory effects. It was noted that 13a, 13b, 13e, 14b, 14c and 19c exhibited
good inhibitory effects on HUVEC proliferation with IC50 <10 µM (Table 3); while, 13c, 13g, 13i, 15g, 17e,
19d, 19e, 19f, 21b, and 21c showed poor inhibition >10 µM. However, 13a, 13b, 14b, and 14c showed better
inhibitory activity on HUVECs proliferation as compared to other compounds.
Table 3. Cytotoxicity of Compounds 13a-13c, 13e, 13g, 13i, 14b-14c, 15g, 17e, 19c-19f, 21b, and 21c on
HUVECs.
Sr. No. Code IC50 (µM)a Sr. No. Code IC50 (µM)a
1
2
3
4
5
6
13a
13b
13c
13e
13g
13i
0.6
1
10
11
12
13
14
15
17e
19c
19d
19e
19f
21b
>10
3
>10
6
>10
>10
>10
>10
>10
>10

7
8
9
14b
14c
15g
2
16
17
21c
>10
0.5
0.7
>10
STSb
aAssays were performed in duplicate (n=2); bSTS represents staurosporine.
2.7 VEGFR-2 Inhibition Assay. Compounds with best inhibitory activity on HUVECs were selected to
determine their effects on VEGFR-2 phosphorylation. Phosphorylated VEGFR-2 activates many signaling
pathways which leads to endothelial cell proliferation, migration and differentiation.[95,96] In-vitro cell-
based ELISA method was used to determine the effects of test compounds as well as positive control STS on
pVEGFR-2 by primary screening at 10 µM concentration. These test compounds with visible structural
differences showed poor to good % VEGFR-2 inhibition. Moreover, compounds 13b and 14b showed >50%
VEGFR-2 inhibition and both structurally comprised of naphthalene at ring C. On the contrary, 19c with
pyridine at ring C showed no significant activity (Table 4). Thus, we assume that compounds with
naphthalene as central aryl moiety had more potential for pVEGFR-2 inhibition as compared to pyridine. For
more detailed comparison, we compared the IC50 of 13b and 14b with our previously reported VEGFR-2
inhibit 3i and sorafenib and found that our current series of compounds showed less potency towards VEGFR-
2, even though overall anti-angiogenic effects of 14b were much better than compound 3i.[59] The possible
reason could be that the other effects such as inhibition of migration (supplementary data) or tube formation
(apart from VEGFR-2 inhibition) are more pronounced leading to overall improved anti-angiogenic effects.
Thus, incorporation of the diaryl-pyrazoline moiety led to dual inhibition of HDAC and VEGFR-2, and more
structural modification are needed to further improve the VEGFR-2 inhibitory potential of this series of
compounds.
Table 4. VEGFR-2 Inhibitory Activity of Compounds 13a, 13b, 13e, 14b, 14c, and 19c.
Sr. No.
Code
13a
% Inhibition at 10 µMa IC50 (µM)a
1
2
3
4
5
6
7
8
23.45
52.35
48.17
64.12
35.65
18.52
87.85
NDb
NDb
5
13b
13e
NDb
5
14b
14c
NDb
NDb
0.5
0.5
19c
STSc
3i[59]

9
Sorafenib[59]
NDb
0.1
aAssays were performed in duplicate (n=2); bNot determined; cSTS represents staurosporine.
2.8 Capillary Tube Formation Assay. The process of capillary-like tube formation of endothelial cells is
considered as representative of later stages of angiogenesis. Thus, in-vitro tube formation assay could be
performed for the test compounds to govern their anti-angiogenesis potency.[93,94,97,100] Compounds 13b
and 14b displayed substantial inhibitory activity against HUVECs, pVEGFR-2 and endothelial cell migration
(supplementary data); so, these two were further evaluated to determine their effects on HUVECs tube
formation. STS-treated HUVECs, after 24 h, reduced the number of capillaries to a great extent as compared
to control which formed hollow capillary-like networks (Figure 5). However, 14b exhibited significant
decrease in tube formation as compare to 13b and control [Figure 5 and supplementary data (p=0.1)]. Thus,
results indicated that, 14b exhibited anti-angiogenesis activity by reducing HUVECs tube formation.
Figure 5. Graphical representation of intersection counts with different treatments on HUVECs after 48 h of
untreated, staurosporine (10 µM), 13b (10 µM), and 14b (10 µM) respectively. Error bar represents SEM,
n=3, ***p≤0.0001, **p≤0.001 (student’s unpaired t-test).
2.9 MTT Cytotoxicity Assay. In-vitro HDAC inhibitory and anti-angiogenesis assays revealed two best
compounds, 13b and 14b. These two were further tested for their cytotoxicity potential against 4 cell lines
viz. MCF-7 (human breast cancer), K562 (leukaemia), A549 (human lung cancer) and HT-29 (human
colorectal adenocarcinoma) along with two positive controls paclitaxel and cis-platin. Results showed that
test compounds displayed moderate to good cytotoxicity in mid-micromolar range on the four cell lines (Table
5). Paclitaxel was more potent than test compounds whereas cis-platin showed variation in activity.
Compound 13b presented better inhibitory activity than cis-platin on K562, A549 and HT-29 cell lines, while
comparatively poor inhibition on MCF-7, while, 14b showed significantly better cytotoxicity against K562
and HT-29; specifically, HT-29 appears to be most affected. Thus, cytotoxicity results indicated that 13b and
14b had encouraging cytotoxicity on these cell lines.
Table 5. Cytotoxicity of Compounds 13b and 14b.

Code
IC50 (μM)a
K562 A549
MCF-7
HT-29
13b
14b
24.40±2.42 5.83±1.1 9.63±1.06 8.66±1.30
16.92±3.0 8.92±1.25 17.99±1.51 8.20±0.42
Paclitaxel
0.35
0.29
0.32
0.28
Cis-platin 10.57±1.1 58.4±1.4 16.68±1.74 10.6±1.2
aAssays were performed in replicates (n=6). ND – Not determined.
2.10 In-vivo Chick Chorioallontoic Membrane (CAM) Assay. The CAM of chick offers a reliable system
to study the anti-angiogenesis effects of compounds in-vivo. Chick CAMs are easy to access outside the
embryo and it offers a simple approach to study complex biological systems.[100] Compounds 13b and 14b
along with positive control STS, were subjected to CAM assay to govern their in-vivo anti-angiogenic
potency. After 12 days of implanting sponges loaded with VEGFs, STS, and test samples into the CAMs,
average number of blood vessels was determined. STS substantially decreased the branching of capillaries
(Figure 6) as compared to untreated. Importantly, 14b-treated CAMs showed a significant reduction in the
branching of blood capillaries as compared to untreated and 13b-treated, further supporting its anti-
angiogenesis mechanism.
Figure 6. Graphical representation of CAM assay. Error bars represent SEM, n=5, ****p≤0.00001,
***p≤0.0001, **p≤0.01 (student’s unpaired t-test). STS represents staurosporine. UT represents untreated.
2.11 Western Blotting. With aim to determine effects of 14b on expression levels of VEGFR-2, western
blot was performed on HUVEC cells. STS (10 µM) was used as positive control and TBP as loading control.
In proteins, phosphorylation is an essential post-translational modification. Phosphorylated VEGFR-2
(tyrosine residues) transduces the signals to several downstream ligands in order to regulate the angiogenesis
events such as endothelial cell proliferation, migration and survival.[101] Upon western blot, phosphorylated

VEGFR-2 expression level was significantly decreased in the presence of 14b (10 µM) as compared to STS
at the same concentration (Figure 7).
Tumoral hypoxia is a major factor that regulates tumor angiogenesis which is driven by a key mediator
hypoxia inducible factor (HIF)-1α. It is known to be the chief controller of hypoxia-induced
angiogenesis.[102,103] Thus, to analyse whether HIF-1α was responsible to elicit anti-angiogenesis response,
western blot was performed to evaluate the change in the expression levels of HIF-1α induced by 14b.
Compound 14b caused slight decrease in expression levels of HIF-1α as compared to the positive control,
which further provided support for anti-angiogenesis potency of 14b by VEGFR-2 inhibition.
In particular, caspase 3 are key regulators of apoptosis which are produced in inactive-proenzyme form, and
they are cleaved further to form smaller subunits which combine together to form active-caspase 3 enzyme.
This active-caspase 3 promotes various degradation pathways and DNA fragmentation during
apoptosis.[104–106] On western blot of 14b, the cleaved caspase 3 expression level was enhanced
significantly suggesting its potential to increase the production of cleaved caspase 3.
Figure 7. Western blotting of VEGFR-2 expression levels on HUVEC treated with staurosporine and
compound 14b at 10 µM for 5 h. TBP was used as loading control. Expression levels of hypoxia inducible
factor (HIF-1α), phosphorylated VEGFR-2 (pVEGFR-2), and cleaved caspase 3. STS represents
staurosporine. UT represents untreated.
2.12 Antitumor Activity Studies on Xenograft Models. To access the in-vivo antitumor efficacy of 14b,
HT-29 (human colorectal adenocarcinoma) xenograft model was established. For this assay, 18 SCID mice
were used and tumors were generated to a specific size. Then after the tumor bearing mice were separated
into three groups each containing six mice, wherein, the untreated or control group received saline; the
positive control group were injected with 20 mg/kg of doxorubicin and the test group were introduced with
25 mg/kg of 14b intraperitoneally on 1-5, 8-12, 15-18 day. The tumor volume of each mouse was measured

every two days and documented. At the end of experiment, mice were sacrificed, and tumors were incised,
immediately imaged and volume was measured (supplementary data).
In vehicle-treated group, the tumors developed in exponential manner (Figure 8), whereas, compound 14b
significantly reduced the tumor volumes as presented by tumor size (Figure 8A) as well as tumor growth
curve (Figure 8B). Strikingly, 14b-treated xenograft model produced tumor volume reduction ranging from
30 to 64% and 3 to 44% in comparison to vehicle-treated and doxorubicin-treated respectively, after 18 days
of administration in animal. Moreover, 14b reduced the tumor growth ranging from 30 to 51% as compared
to vehicle-treated, additionally, 66% of 14b-treated animals exhibited greater reduction in tumor growth as
compared to doxorubicin whereas, 34% animals showed 15 to 19% reduction.
Thus, it is worth mentioning that the tumor size and volume of 14b was comparable to doxorubicin, therefore,
it will be reasonable to tick on the in-vivo antitumor efficacy of pyrazoline-TZD analog 14b. Furthermore, in
the course of experiment, 14b-treated mice group showed no significant evidence of toxicity or weight loss.
Figure 8. In-vivo antitumor efficacy of 14b on human colorectal adenocarcinoma (HT-29) xenograft model.
(A) Excised tumors of doxorubicin-treated (Dose – 20 mg/kg i.p.), 14b-treated (Dose – 25 mg/kg i.p.) and
vehicle-treated (saline i.p.). (B) Growth curve of tumor volume (mm3) vs. time (days) of vehicle-treated
(represented as mean ± SEM, blue line, n=6), 14b-treated (represented as mean ± SEM, green line, n=6) and
doxorubicin-treated (represented as mean ± SEM, red line, n=6). UT represents untreated. Dox represents
doxorubicin. i.p. represents intraperitoneally. Error bars represent mean ± SEM n=6; ***, p < 0.001.
2.13 Molecular Docking Study. A comprehensive docking study was performed to rationalize the structure
activity data and reveal the molecular determinants of protein-ligand binding. The docking procedure was
validated by redocking of the representative ligands in the crystal structure of VEGFR-2 (PDB-ID: 1YWN)
and HDAC4 (PDB-ID: 2VQJ and 4CBY).[88] The GBVI/WSA dG docking score of the redocked ligand was
-13.6 on HDAC4 (PDB-ID: 4CBY), -13.9 on HDAC4 (PDB-ID 2VQJ) and -11.2 on VEGFR-2
(supplementary data). The ligands of PDB-ID’s 1YWN and 4CBY redocked into the protein-receptor yielded
an almost perfect overlap between docked and x-ray pose with RMSD-values of less than 0.4 Å
(supplementary data). The thiophene linker and trifluoromethyl warhead of the redocked ligand in HDAC4
(PDB ID: 2VQJ) showed a good RMSD value of 0.4 Å with respect to the crystal structure. Since the aromatic
head group of the trifluoromethylketone ligand protrudes into free solution, this part of the molecules was

intrinsically flexible and thus not considered for the calculation of RMSD. Consequently, we assumed that
all parameters were well configured for docking of pyrazoline TZD-analogs.
Compounds with identical absolute steric configuration at the pyrazole ring as (S)-14b, showed better scores
on VEGFR-2 than their steric counterparts (supplementary data). Interestingly, all analogs with (S)-14b
configuration showed similar binding poses, but the corresponding stereo isomers consistently adopted an
inverse binding pose (supplementary data). The reason behind this finding was that the o-chlorophenyl group
of (S)-14b and corresponding stereo isomers shows perfect fit into a hydrophobic sub pocket flanked by
L1017, I1042, V896, V897, I890, I886 and L887, while the same group of (R)-14b would clash with this sub
pocket. (S)-14b as a whole snuggled perfectly in the prolonged binding channel of VEGFR-2. The TZD war
head was found to tightly fixed by two hydrogen bonds with L838 and A1048 (Figure 9). The carboxamide-
oxygen accepts H-bond with the backbone of Asp1044 which may add to affinity.
Two representatives of TZD analogs with naphthyl-linker and the most potent TZD with pyridyl-linker were
docked into the crystal structure of the open (PDB-ID: 2VQJ) and closed (PDB-ID: 4CBY) conformation of
HDAC4, HDAC4o and HDAC4c, respectively. The docking scores for the most potent compounds (S)-14b
and (S)-13i with the same absolute configuration at the pyrazoline ring were better for HDAC4o than for
HDAC4c suggesting preferred binding to the open conformation. It is noteworthy, that the catalytic zinc ion
in the complex between TZD-ligands and both, HDAC4o and HDAC4c, shows tetragonal bipyramidal
coordination with similar distances between the electronegative heavy atoms and the central zinc ion (Figure
S12, supplementary data).
Interestingly, for all TZD-analogs the enantiomer with the same absolute configuration at the pyrazoline ring
as (S)-14b shows significantly higher docking scores for binding to HDAC4o, than the corresponding
enantiomer (supplementary data). In agreement with the activity data, 13i and 14b with naphthyl-linker
showed higher scores compared with 21d (pyridyl-linker). If the linker was removed and the TZD-warhead
was replaced, as seen with compound S1, the activity drops dramatically below an IC50-value of 50 µM (Table
1). This correlates well with a lower docking score, when compared with the score of active compounds 13i,
14b and 21d (supplementary data). Looking at the binding pose of (S)-14b at the binding groove adjacent to
the active site pocket of HDAC4 reveals that the TZD-warhead plays a pivotal role in the molecular
recognition of the pyazoline TZD-analogs by HDAC4o (Figure 10). The TZD-group binds to the catalytic
zinc ion and forms a hydrogen bond to H159 as well as a Pi-Pi interaction with the sidechain of H198. The
naphthyl-linker fits perfectly into the binding groove of HDAC4o, which was flanked by hydrophobic amino
acids P155, P156, F168, L334 and I38. The linker is optimally oriented to form further Pi-stacking with F871.
The naphthyl-linker shows also a two-fold cation-Pi-interaction with R37. In addition, two hydrogen bonds
with R37 and R154 contribute to the stabilization of the protein-ligand complex. One ring of the branched
pyrazoline capping group occupies a deeper sub-pocket forming Pi-Pi-stacking with H332 and Pi-alkyl
interactions with L334 (Figure 10). This was consistent with similar activities of TZD-analogs with the same
linker and different smaller substituents at the aromatic rings of the capping group. Altogether, docking results
were in agreement with the dual activity of the pyrazoline TZD-analogs against HDAC4 and VEGFR-2.

Figure 9. (S)-14b docked into the binding pocket of VEGFR-2 (PDB-ID: 1YWN). A) Binding pose with H-
bonds to L838, A1048, D1044 (dotted green lines), Pi-Pi-interactions (dotted magenta line) and multiple Pi-
alkyl and alkyl-interactions (dotted pink lines). B) 2D diagram of protein-ligand interactions.
Figure 10. A) 3D-docking pose of (S)-14b within HDAC4o (PDB-ID: 2VQJ). The ligand forms multiple
hydrogen bonds (dotted green lines), Pi-Pi-interactions (dotted magenta lines), Pi-alkyl-interactions (dotted
pink lines), Pi-cation interactions (dotted light brown lines) and zinc complexation (dotted gray line) in the
binding grove adjacent to the catalytic site pocket. B) 2D diagram of protein-ligand interactions.

3. CONCLUSIONS
In this paper, we have rationally designed and developed novel pyrazoline TZD-analogs simultaneously
targeting VEGFR-2 and HDAC, wherein, two different series exploring naphthyl and pyridyl linker were
constructed. The naphthyl linker demonstrated superior HDAC inhibition with selectivity towards HDAC4
as well as impressive VEGFR-2 inhibition.
Further in depth in-vitro assays were performed of the two most active compounds incorporating naphthyl
linker, 13b and 14b. These structurally different analogs, 13b and 14b have exhibited similar inhibitory
effects on VEGFR-2 (5 µM), whereas they stated pronounced inhibition of HDAC4 with IC50 of 0.34 µM and
0.36 µM respectively. Compounds with naphthyl linker (13b, 14b) and pyridyl linker (17f, 21f) were able to
stabilize HDAC4 as evident by shift in the melting temperatures confirming its interaction with HDAC4
isoform. Additionally, the anti-angiogenic potential of 13b and 14b was evident by different assays such as
HUVEC proliferation, migration (supplementary data), and tube formation. Compound 13b (1 µM) was
found to have better inhibitory concentration on HUVECs proliferation than that of 14b (2 µM), whereas they
both had similar effects on cell mobility of HUVECs. In capillary tube formation, compound 14b strikingly
reduced the number of capillaries as compared to 13b. The in-vivo assay displayed higher potency of 14b to
attenuate the neovascularization in the growing CAMs in comparison to 13b. These two non-hydroxamate
analogs were examined for their effects on cancer cell viability and they significantly reduced it.
In addition, western blot analysis revealed that 14b considerably decreased phosphorylated VEGFR-2 and
HDAC4 expression levels while increased the same of cleaved caspase 3, a key regulator of apoptosis.
Additionally, it must be emphasized that 14b (25 mg/kg, i.p.) showed uncompromised in-vivo efficacy (tumor
growth and volume reduction) on HT-29 tumor xenograft model comparable to doxorubicin (20 mg/kg, i.p.).
Since, the present results highlight the effects of subtle change of linker on the inhibitory potency and
selectivity against these targets, intense efforts are being directed towards optimization of 14b by linker
modification.

4. EXPERIMENTAL
4.1 Chemistry
The final compounds were synthesized using 3 different schemes (scheme I, II and III). In scheme I, synthesis
of intermediates is mentioned, while scheme II and scheme III comprised of synthesis of final compounds.
All the required reagents, solvents and chemicals were procured from commercial sources viz. Sigma Aldrich,
S.D. Fine Chem. Ltd., Himedia and were utilized without any further purification. The reactions were
monitored at each step by Thin Layer Chromatography (TLC) using Merck precoated silica gel 60 F-254
plates under short wave UV-light (254 nm) to identify the UV absorbing spots for completion of reaction and
also to trace presence of any impurities. All intermediates were purified by recrystallization using suitable
solvents such as chloroform, methanol, ethanol etc. All the final molecules were purified by column
chromatography technique on silica gel 60 (60 to 120 mesh) using combination of different solvents. Melting
point of all the intermediates were obtained by using VEEGO, MODEL: VMP-DS Melting Point apparatus
and that of the final compounds were obtained using a DSC1 STAR system differential scanning calorimeter
from Mettler Toledo. Purity of all final compounds was determined using an Agilent 1200 high-performance
liquid chromatography (HPLC) system; software- EZ chrome Elite. The chromatographic column used was
HemochromIntsil A31 C18 5U 150 mm × 4.6 mm Sn-B180127, detection at 300 nm. UV−visible detector
was used with the flow rate of 1 mL/min. Oven temperature maintained was 30 °C; gradient elution with a
run time of 10 min using Methanol: Formic Acid (1%) (Formic acid: in 1000 mL double distilled water 1 mL
formic acid was added) in 80:20/90:10 ratio.
The structures of all the compounds were confirmed by different spectroscopic techniques such as: FTIR, 1H-
NMR, 13C-NMR, Mass. All the intermediates were structurally characterized by FTIR and 1H-NMR and final
1
compounds by FTIR, H-NMR, 13C-NMR and Mass spectrometry. IR was recorded using JASCO FT/IR-
1
4100 typeA spectrometer using manual sampling method. H-NMR spectra were recorded using Bruker
Avance 400 MHz spectrometer using different solvents. All shifts of 1H-NMR are reported in δ (ppm) units
relative to the signals for the solvent DMSO (δ- 2.50 ppm). All coupling constants (J values) are reported in
hertz (Hz). NMR abbreviations are as: bs, broad singlet; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
and dd, doublet of doublets. 13C-NMR was recorded on Bruker Avance Spectrometer at 100 MHz using
solvent DMSO-d6. Mass spectrum was documented on LC-MS Agilent Technologies 1260 Infinity
instrument.
Different chalcones were synthesized by procedure previously reported with minor modifications [91,107].
To ethanolic solution of NaOH (10%, 20 mL), substituted acetophenones (0.04 mol) and different aldehydes
(0.04 mol) was added dropwise under ice bath and stirred for 5 h at RT. Reaction mixture was kept in
refrigerator overnight, filtered and washed with cold water. Crude obtained was recrystallized using ethanol
to obtain appropriate chalcones (3a-3i, 6a-6g, 6j, and 9b-9g) (Scheme I). Following are the unpublished
chalcone intermediates, others have been reported elsewhere.[91,107]

3-(3-nitrophenyl)-1-phenylprop-2-en-1-one (3h). Yellow crystalline solid. 81% yield (6.7 g). M.P. 127-
129°C. FTIR (cm-1) 3068, 1660, 1577, 1523. 1H NMR (400 MHz, CDCl3) δ ppm 7.44 – 7.56 (m, 2H), 7.60
– 7.64 (m, 2H), 7.67 (s, 1H), 7.82 (s, 1H), 7.91 – 7.93 (m, 1H), 8.03 – 8.06 (m, 2H), 8.25 – 8.27 (m, 1H), 8.51
– 8.52 (m, 1H).
3-(2-chlorophenyl)-1-(2,4-difluorophenyl)prop-2-en-1-one (9b). Yellow crystals. 87% yield (9.8 g). M.P.
110-111°C. FTIR (cm-1) 3074, 1685, 1599, 1261, 1232, 752. 1H NMR (400 MHz, CDCl3) δ ppm 6.88–6.93
(m, 1H), 6.97–7.02 (m, 1H), 7.26–7.38 (m, 3H), 7.42–7.44 (m, 1H), 7.71–7.74 (m, 1H), 7.87–7.93 (m, 1H),
8.16 (d, J= 15.9 Hz, 1H).
1-(2,4-difluorophenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (9d). Yellow crystals. 90% yield
(8.5 g). M.P. 108-109°C. FTIR (cm-1) 3124, 3076, 1664, 1543, 1519, 1288, 1265, 1230. 1H NMR (400 MHz,
CDCl3) δ ppm 6.89 – 6.95 (m, 2H), 6.99 – 7.03 (m, 1H), 7.43 – 7.47 (m, 1H), 7.65 – 7.73 (m, 4H), 7.89 –
7.95 (m, 1H).
1-(2,4-difluorophenyl)-3-(4-fluorophenyl)prop-2-en-1-one (9e). Yellow crystals. 90% yield (8.5 g). M.P.
1
64-65°C. FTIR (cm-1) 3101, 3076, 1660, 1587, 1506, 1263, 1222. H NMR (400 MHz, CDCl3) δ ppm 6.89 –
6.93 (m, 1H), 6.94 – 7.02 (m, 4H), 7.44 – 7.45 (m, 2H), 7.61 – 7.66 (m, 1H), 7.88 – 7.94 (m, 1H).
1,3-bis(2,4-difluorophenyl)prop-2-en-1-one (9f). Yellow crystals. 89% yield (8 g). M.P. 63-65°C. FTIR
(cm-1) 3086, 1664, 1595, 1265, 1234. 1H NMR (400 MHz, CDCl3) δ ppm 6.69 – 6.81 (m, 1H), 6.86 – 7.05
(m, 4H), 7.40 – 7.45 (m, 1H), 7.60 – 7.66 (m, 1H), 7.87 – 7.93 (m, 1H).
Various pyrazoline derivatives were synthesized by previously reported procedure with suitable
modifications [91,107]. To solution of different chalcones (3a-3i, 6a-6g, 6j, and 9b-9g) (0.02 mol) in
chloroform, hydrazine hydrate (0.04 mol) was added and refluxed for 12 h. The reaction mixture was cooled
under ice-bath and K2CO3 (0.05 mol) was added to this mixture and stirred for 15 min. Chloroacetyl chloride
(0.03 mol) was added dropwise with stirring under ice-bath and further stirred at RT. After 12 h, reaction was
stopped and chloroform layer was washed several times with water to remove excess of K2CO3 and
evaporated to obtain solid. Crude was purified by extracting with diethylether to get appropriate
chloroacetylated pyrazolines (4a-4i, 7a-7g, 7j, and 10b-10g) (Scheme I). Following are the unpublished
chloroacetylated intermediates, others have been reported elsewhere.[91,107]
2-chloro-1-(5-(3-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone (4h). White colour solid.
1
59% yield (5.6 g). M.P. 146-148°C. FTIR (cm-1) 1670, 1600, 1572, 1588, 1523, 1500, 688. H NMR (400
MHz, CDCl3) δ ppm 3.23 (d, J= 18.0 Hz, 1H), 3.85-3.92 (m, 1H), 4.54-4.64 (m, 2H), 5.68 (d, J= 12.0 Hz,
1H), 7.43-7.55 (m, 4H), 7.60-7.62 (m, 1H), 7.74-7.77 (m, 2H), 8.11-8.16 (m, 1H), 8.76 (s, 1H).
2-chloro-1-(5-(2-chlorophenyl)-3-(2,4-difluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethenone
(10b).
White colour solid. 70% yield (4.7 g). M.P. 114-115°C. FTIR (cm-1) 1668, 1600, 1504, 1263, 1211, 858, 752.
1H NMR (400 MHz, CDCl3) δ ppm 3.16–3.23 (m, 1H), 3.90–3.99 (m, 1H), 4.52 (d, J= 13.28 Hz, 1H), 4.65
(d, J= 13.28 Hz, 1H), 5.90 (d, J= 11.9 Hz, 1H), 6.83–6.89 (m, 1H), 6.94–6.99 (m, 1H), 7.08–7.10 (m, 1H),
7.20–7.26 (m, 2H), 7.39–7.41 (m, 1H), 7.94–8.00 (m, 1H).

2-chloro-1-(3-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)
ethanone (10d). Cream colour solid. 57% yield (4.8 g); M.P. 123-124°C. FTIR (cm-1) 1676, 1600, 1512,
1274, 1246, 839. 1H NMR (400 MHz, CDCl3) δ ppm 3.23 – 3.25 (m, 1H), 3.86 – 3.95 (m, 1H), 4.48 – 4.95
(m, 2H), 4.60 (d, J= 11.8 Hz, 1H), 6.85 – 6.91 (m, 1H), 6.96 – 7.01 (m, 1H), 7.36 (d, J= 8.4 Hz, 2H), 7.60 (d,
J= 8.0 Hz, 2H), 7.97 – 8.03 (m, 1H).
2-chloro-1-(3-(2,4-difluorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone
(10e).
Pale yellow solid. 60% yield (4 g). M.P. 97-98°C. FTIR (cm-1) 1672, 1602, 1504, 1417, 1259, 1219, 815. 1H
NMR (400 MHz, CDCl3) δ ppm 3.17 (d, J= 17.6 Hz, 1H), 3.74 – 3.82 (m, 1H), 4.50 – 4.61 (m, 2H), 5.73 (d,
J= 11.8 Hz, 1H), 6.68 – 6.79 (m, 2H), 7.10 – 7.20 (m, 3H), 7.71 – 7.76 (m, 2H).
1-(3,5-bis(2,4-difluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-chloroethanone (10f). Off white colour
solid. 63% yield (5.3 g). M.P. 112-114°C. FTIR (cm-1) 1678, 1602, 1500, 1444, 1259, 1219, 844. 1H NMR
(400 MHz, CDCl3) δ ppm 3.25 – 3.21 (m, 1H), 3.82 – 3.91 (m, 1H), 4.48 – 4.59 (m, 2H), 5.70 (d, J= 12.0 Hz,
1H), 6.79 – 6.90 (m, 3H), 6.95 – 7.00 (m, 1H), 7.15 – 7.20 (m, 1H), 7.95 – 8.01 (m, 1H).
5-((6-hydroxynaphthalen-2-yl)methylene)thiazolidine-2,4-dione (12). This intermediate was synthesized
by previously reported procedure[29]. In brief, toluene (10 mL), 2,4-thiazolidinedione (11) (4.68 g, 0.04
mmol) and 6-hydroxy-2-naphthaldehyde (6.88 g, 0.04 mmol) was taken together in RBF. To this mixture,
catalytic amount of piperidinium benzoate was dispersed and refluxed in Dean Stark apparatus. The reaction
was continuously monitored by TLC, after 4 h reaction was stopped and mixture was cooled to RT. Solid
precipitated was collected by filtering under vacuum and washed with water to obtained intermediate 12
(Scheme II). Yellow shiny crystals. 86% yield (10 g). M.P. charred at 300 °C. FTIR (cm-1) 3390, 3119, 1672,
1662, 1587. 1H-NMR (400 MHz, DMSO-d6) δ ppm 7.16-7.19 (m, 2H), 7.55 (dd, J = 1.8, 8.68 Hz, 1H), 7.80
(d, J = 8.68 Hz, 1H), 7.88-7.90 (m, 2H), 8.04 (d, J = 0.96 Hz, 1H), 10.21 (s, 1H), 12.59 (bs, 1H).
To the solution of K2CO3 (0.007 mol) in dimethylformamide (10 mL), 6-hydroxynicotinaldehyde (0.008 mol)
was added with stirring. In this mixture, appropriate chloroacetylated intermediate (0.004 mol) (4a-4b, 4d-4f,
4h, 4i, 7a-7f, and 10b-10f) was added was added and stirred for 48 h at RT. Reaction was immobilized by
adding water (40 mL), precipitated residue was collected by filtering under vacuum and washed several times
with water. Residue collected was purified by recrystallization technique with methanol (25 mL) to give
respective intermediates (16a-16b, 16d-16f, 16h, 16i, 18a-18f, and 20b-20f) (Scheme III).
6-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde (16a). Grey colour solid.
1
55% yield (1 g). M.P. charred at 130 °C. FTIR (cm-1) 2868, 1656, 1606, 1543, 1502, 1265. H-NMR (400
MHz, DMSO-d6) δ ppm 3.23 (dd, J = 4.8, 18.4 Hz, 1H), 3.92-3.99 (m, 1H), 5.27 (d, J = 16.4 Hz, 1H), 5.36
(d, J = 16.4 Hz, 1H), 5.59 (dd, J = 4.8, 11.6 Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H), 7.24-7.32 (m, 3H), 7.32 – 7.35
(m, 2H), 7.47-7.51 (m, 3H), 7.80 (dd, J = 2.4, 9.6 Hz, 1H), 7.84-7.86 (m, 2H), 8.55 (d, J = 2.4 Hz, 1H), 9.58
(s, 1H).
6-(2-(5-(2-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde (16b).
Grey colour solid. 59% yield (0.9 g). M.P. charred at 250 °C. FTIR (cm-1) 2862, 1654, 1602, 1541, 1502,
1267, 846. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.11 (dd, J = 5.16, 18.24 Hz, 1H), 4.02-4.09 (m, 1H), 5.27

(d, J = 16.6 Hz, 1H), 5.45 (d, J=16.6 Hz, 1H), 5.80 (dd, J = 5.0, 11.8 Hz, 1H), 6.42-6.54 (m, 1H), 7.22-7.33
(m, 4H), 7.48-7.50 (m, 3H), 7.80–7.95 (m, 3H), 8.57 (d, J = 2.2 Hz, 1H), 9.60 (s, 1H).
6-(2-oxo-2-(3-phenyl-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethoxy)
nicotinaldehyde (16d). Grey colour solid. 58% yield (1.1 g). M.P. charred at 270 °C. FTIR (cm-1) 2848,
1651, 1600, 1539, 1500, 1325, 1267. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.27 (d, J = 4.8 Hz, 1H), 3.95-
4.03 (m, 1H), 5.27 (d, J = 16.4 Hz, 1H), 5.37 (d, J = 16.4 Hz, 1H), 5.70 (dd, J = 5.2, 12.0 Hz, 1H), 6.49-6.54
(m, 2H), 7.48-7.52 (m, 3H), 7.70 (d, J = 8.0 Hz, 2H), 7.79-7.84 (m, 4H), 8.54-8.6 (m, 1H), 9.58 (s, 1H).
6-(2-(5-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde (16e).
Grey colour solid. 60% yield (0.95 g). M.P. 130-132 °C. FTIR (cm-1) 3064, 1658, 1600, 1545, 1512, 1383,
1269. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.24 (dd, J = 4.8, 18.4 Hz, 1H), 3.91-3.96 (m, 1H), 5.24 (d, J =
16.4 Hz, 1H), 5.34 (d, J = 16.4 Hz, 1H), 5.60-5.61 (m, 1H), 6.50 (d, J = 9.2 Hz, 1H), 7.15 (t, J = 8.8 Hz, 2H),
7.28-7.32 (m, 2H), 7.50-7.52 (m, 3H), 7.79-7.86 (m, 3H), 8.54 (d, J = 2.4 Hz, 1H), 9.58 (s, 1H).
6-(2-(5-(2,4-difluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde
(16f). Grey colour solid. 59% yield (1.2 g). M.P. 137-139 °C. FTIR (cm-1) 3049, 1656, 1649, 1546, 1502,
1352, 1269. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.19 (dd, J = 5.2, 18.4 Hz, 1H), 4.01-4.08 (m, 1H), 5.26
(d, J = 16.4 Hz, 1H), 5.43 (d, J = 16.4 Hz, 1H), 5.80 (dd, J = 5.2, 12.0 Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H), 7.21-
7.24 (m, 1H), 7.29-7.36 (m, 4H), 7.47-7.50 (m, 1H), 7.80 (dd, J = 2.4, 9.6 Hz, 1H), 7.89-7.92 (m, 2H), 8.56
(d, J = 2.4 Hz, 1H), 9.59 (s, 1H).
6-(2-(5-(3-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde
(16h).
Brown colour powder. 56% yield (1.1 g). M.P. charred at 240 °C. FTIR (cm-1) 3068, 1654, 1602, 1527, 1502,
1348, 1269. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.36 (dd, J = 5.2, 18.4 Hz, 1H), 3.97-4.04 (m, 1H), 5.26
(d, J = 16.4 Hz, 1H), 5.37 (d, J = 16.4 Hz, 1H), 5.78 (dd, J = 5.2, 12.0 Hz, 1H), 6.50 (d, J = 9.6 Hz, 1H), 7.51-
7.52 (m, 3H), 7.65 (t, J = 8.4 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.78-7.81 (m, 1H), 7.82-7.87 (m, 2H), 8.13-
8.14 (m, 2H), 8.54 (d, J = 2.4 Hz, 1H), 9.59 (s, 1H).
6-(2-oxo-2-(3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethoxy)nicotinaldehyde
(16i).
Grey colour powder. 52% yield (0.8 g). M.P. charred at 210 °C. FTIR (cm-1) 3061, 1651, 1604, 1547, 1500,
1386, 1267. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.44 (dd, J = 4.4, 18.4 Hz, 1H), 3.90-3.98 (m, 1H), 5.21
– 5.30 (m, 2H), 5.90 (dd, J = 4.4, 11.6 Hz, 1H), 6.49-6.51 (m, 1H), 6.95-6.96 (m, 1H), 7.06-7.07 (m, 2H),
7.42-7.44 (m, 2H), 7.50-7.53 (m, 2H), 7.79-7.89 (m, 2H), 8.54-8.59 (m, 1H), 9.59 (s, 1H).
6-(2-(3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde (18a).
Grey colour solid. 52% yield (1.3 g). M.P. 139-141 °C. FTIR (cm-1) 3028, 1658, 1600, 1543, 1504, 1398,
1327, 1263. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.17 (dd, J = 5.2, 18.4 Hz, 1H), 4.02-4.12 (m, 1H), 5.26
(d, J = 16.4 Hz, 1H), 5.43 (d, J = 16.4 Hz, 1H), 5.80 (dd, J = 5.2, 12.0 Hz, 1H), 7.21-7.23 (m, 1H), 7.29-7.33
(m, 2H), 7.46-7.52 (m, 4H), 7.79-7.87 (m, 4H), 8.54-8.58 (m, 1H), 9.59 (s, 1H).
6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde (18b). Grey colour solid. 53% yield (1 g). M.P. charred at 150 °C. FTIR (cm-1) 3076, 1658,
1600, 1545, 1514, 1269, 1200, 752. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.11 (dd, J = 5.2, 17.92 Hz, 1H),
3.85-3.92 (m, 1H), 5.19 (d, J = 16.6 Hz, 1H), 5.23 (d, J = 16.6 Hz, 1H), 5.92 (dd, J = 5.16, 11.8 Hz, 1H), 6.59

(d, J = 9.52 Hz, 1H), 7.16 (t, J = 8.6 Hz, 2H), 7.19-7.27 (m, 3H), 7.37 (t, J = 6.4 Hz, 1H), 7.74–7.81 (m, 3H),
7.91 (d, J = 2.16 Hz, 1H), 9.56 (s, 1H).
6-(2-(3-(4-fluorophenyl)-5-(furan-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde
(18c). Grey colour solid. 49% yield (0.9 g). M.P. charred at 173 °C. FTIR (cm-1) 3282, 1664, 1606, 1546,
1510, 1274, 1220. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.48 (dd, J = 5.08, 17.68 Hz, 1H), 3.59-3.66 (m,
1H), 5.03 (d, J = 16.72 Hz, 1H), 5.39 (d, J = 16.72 Hz, 1H), 5.67 (dd, J = 5.04, 11.56 Hz, 1H), 6.30-6.37 (m,
2H), 6.57 (d, J = 9.48 Hz, 1H), 7.14 (t, J = 8.6 Hz, 2H), 7.28-7.30 (m, 1H), 7.76–7.80 (m, 3H), 7.90 (d, J =
2.2 Hz, 1H), 9.55 (s, 1H).
6-(2-(3-(4-fluorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde (18d). Grey colour solid. 56% yield (1.1 g). M.P. charred at 215 °C. FTIR (cm-1) 3205,
1656, 1602, 1545, 1514, 1323, 1224, 1269. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.30 (dd, J = 5.2, 18.4 Hz,
1H), 3.95-4.02 (m, 1H), 5.27 (d, J = 16.4 Hz, 1H), 5.36 (d, J = 16.4 Hz, 1H), 5.70 (dd, J = 5.2, 12.0 Hz, 1H),
6.51 (d, J = 9.6 Hz, 1H), 7.33-7.37 (m, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.78-7.82 (m,
1H), 7.89-7.92 (m, 2H), 8.54 (d, J = 2.4 Hz, 1H), 9.58 (s, 1H).
6-(2-(3,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde (18e). Grey
colour solid. 51% yield (0.85 g). M.P. 135-137 °C. FTIR (cm-1) 3064, 1656, 1602, 1546, 1510, 1384, 1220,
1269. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.28 (dd, J = 4.8, 18.4 Hz, 1H), 3.90-3.97 (m, 1H), 5.24 (d, J =
16.4 Hz, 1H), 5.33 (d, J = 16.4 Hz, 1H), 5.60 (dd, J = 4.8, 11.6 Hz, 1H), 6.50 (d, J = 9.6 Hz, 1H), 7.15 (t, J =
8.8 Hz, 2H), 7.28-7.37 (m, 4H), 7.80 (dd, J = 2.4, 9.6 Hz, 1H), 7.89-7.93 (m, 2H), 8.53 (d, J = 2.4 Hz, 1H),
9.58 (s, 1H).
6-(2-(5-(2,4-difluorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde (18f). Grey colour solid. 54% yield (0.9 g). M.P. 150-152 °C. FTIR (cm-1) 3064, 1656, 1602,
1
1543, 1502, 1386, 1330, 1309, 1267. H-NMR (400 MHz, DMSO-d6) δ ppm 3.13-3.18 (m, 1H), 4.07-4.14
(m, 1H), 5.24 (d, J = 16.8 Hz, 1H), 5.41 (d, J = 16.4 Hz, 1H), 5.77 (dd, J = 5.2, 12.0 Hz, 1H), 6.50-6.53 (m,
1H), 7.22-7.27 (m, 2H), 7.29-7.34 (m, 2H), 7.39-7.45 (m, 1H), 7.47-7.50 (m, 1H), 7.79-7.82 (m, 1H), 7.97-
8.04 (m, 1H), 8.54-8.56 (m, 1H), 9.58-9.59 (m, 1H).
6-(2-(5-(2-chlorophenyl)-3-(2,4-difluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde (20b). Grey solid. 80% yield (2 g). M.P. 200-202 °C. FTIR (cm-1) 3190, 1662, 1604, 1545,
1
1444, 1307, 1226, 1269, 756. H-NMR (400 MHz, CDCl3) δ ppm 3.12–3.19 (m, 1H), 4.07–4.14 (m, 1H),
5.24 (d, J = 16.52 Hz, 1H), 5.41 (d, J = 16.52 Hz, 1H), 5.78 (dd, J = 5.2, 12.0 Hz, 1H), 6.49-6.53 (m, 1H),
7.22–7.30 (m, 2H), 7.30–7.34 (m, 2H), 7.40–7.50 (m, 2H), 7.78–7.82 (m, 1H), 7.98–8.04 (m, 1H), 8.54–8.57
(m, 1H), 9.59 (s, 1H).
6-(2-(3-(2,4-difluorophenyl)-5-(furan-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde
(20c). Grey colour solid. 69% yield (0.94 g). M.P. 167-169 °C. FTIR (cm-1) 3091, 1658, 1600, 1543, 1504,
1
1398, 1379, 1263. H-NMR (400 MHz, CDCl3) δ ppm 3.55–3.62 (m, 1H), 3.68–3.76 (m, 1H), 5.03 (d, J =
16.76 Hz, 1H), 5.38 (d, J = 16.72 Hz, 1H), 5.66 (dd, J = 5.16, 11.6 Hz, 1H), 6.30-6.31 (m, 1H), 6.37 (d, J =
3.2 Hz, 1H), 6.59 (d, J = 9.48 Hz, 1H), 6.88–6.94 (m, 1H), 6.97–7.01 (m, 1H), 7.28-7.32 (m, 1H), 7.80 (dd, J
= 2.4, 9.48 Hz, 1H), 7.90 (d, J = 2.32 Hz, 1H), 7.97–8.03 (m, 1H), 9.6 (s, 1H).

6-(2-(3-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde (20d). Grey colour solid. 63% yield (1.5 g). M.P. charred at 260 °C. FTIR (cm-1) 3064,
1658, 1602, 1543, 1514, 1384, 1352, 1327, 1269. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.23-3.30 (m, 1H),
4.00-4.08 (m, 1H), 5.24 (d, J = 16.4 Hz, 1H), 5.34 (d, J = 16.4 Hz, 1H), 5.67 (dd, J = 5.2, 12 Hz, 1H), 6.50
(d, J = 9.6 Hz, 1H), 7.23-7.28 (m, 1H), 7.40-7.46 (m, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H),
7.78-7.82 (m, 1H), 7.98-8.04 (m, 1H), 8.53-8.54 (m, 1H), 9.57-9.59 (m, 1H).
6-(2-(3-(2,4-difluorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
nicotinaldehyde (20e). Grey colour solid. 61% yield (1.4 g). M.P. charred at 250 °C. FTIR (cm-1) 3047, 1654,
1602, 1541, 1504, 1386, 1354, 1330, 1267. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.20-3.32 (m, 1H), 3.96-
4.04 (m, 1H), 5.22 (d, J = 16.4 Hz, 1H), 5.31 (d, J = 16.4 Hz, 1H), 5.57 (dd, J = 5.2, 12.0 Hz, 1H), 6.47-6.51
(m, 1H), 7.13-7.17 (m, 2H), 7.23-7.33 (m, 2H), 7.40-7.43 (m, 1H), 7.75-7.81 (m, 2H), 7.98-8.04 (m, 1H),
8.50-8.55 (m, 1H), 9.56-9.59 (m, 1H).
6-(2-(3,5-bis(2,4-difluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)nicotinaldehyde
(20f).
Grey colour solid. 67% yield (1.6 g). M.P. 179-181 °C. FTIR (cm-1) 3066, 1658, 1600, 1541, 1504, 1398,
1379, 1350, 1327, 1263. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.26-3.30 (m, 1H), 3.98-4.05 (m, 1H), 5.24
(d, J = 16.8 Hz, 1H), 5.29 (d, J = 16.8 Hz, 1H), 5.66 (dd, J = 5.6, 12.0 Hz, 1H), 6.50 (d, J = 9.6 Hz, 1H), 7.05-
7.07 (m, 1H), 7.22-7.28 (m, 2H), 7.32-7.38 (m, 1H), 7.41-7.47 (m, 1H), 7.80 (dd, J = 2.4, 9.6 Hz, 1H), 7.97-
8.03 (m, 1H), 8.53-8.54 (m, 1H), 9.58 (s, 1H).
The final product was synthesized by similar route mentioned previously[29,84]. 5-((6-hydroxynaphthalen-
2-yl)methylene)thiazolidine-2,4-dione (12) (1 mol) and potassium carbonate (1.5 mol) was added in dimethyl
formamide (10 mL) and stirred for 5 min at RT. To this solution chloroacetylated pyrazolines (4a-4c, 4e, 4g,
4i, 7a-7c, 7e, 7g, 10e, and 10g) (1.5 mol) were added with stirring. The reaction mixture was allowed to stir
for 24 h at RT, and water was added (50 mL) to stop the reaction. Precipitated solid was collected by filtering
and washed thoroughly with water. Crude obtained were purified by column chromatography (hexane:
ethylacetate, 1:1 to 0.5:1.5) get respective final compounds (13a-13c, 13e, 13g, 13i, 14a-14c, 14e, 14g, 15e,
and 15g) (Scheme II).
5-((6-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)naphthalen-2-yl)methylene)
thiazolidine-2,4-dione (13a). Yellow colour powder. 70% yield (0.7 g). M.P. 260.3 °C. FTIR (cm-1) 3389,
1739, 1680, 1622, 1579, 1494, 1475, 1427, 1267, 817. 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.20 (dd, J =
4.8, 18.4 Hz, 1H), 3.89-3.97 (m, 1H), 5.31 (d, J = 16.4 Hz, 1H), 5.47 (d, J = 16.0 Hz, 1H), 5.62 (dd, J = 4.4,
11.6 Hz, 1H), 7.23-7.24 (m, 1H), 7.27-7.29 (m, 2H), 7.29-7.32 (m, 2H), 7.34-7.36 (m, 2H), 7.50-7.51 (m,
3H), 7.63 (d, J = 8.4 Hz, 1H), 7.87-7.91 (m, 4H), 7.95 (d, J = 8.8 Hz, 1H), 8.10 (s, 1H), 12.60 (bs, 1H). 13C-
NMR (100 MHz, DMSO-d6) δ 41.86, 59.92, 65.58, 107.19, 119.70, 122.31, 125.47, 126.51, 126.93, 127.35,
127.77, 128.25, 128.31, 128.68, 128.79, 130.39, 130.64, 130.74, 130.84, 132.10, 134.72, 141.83, 155.48,
157.67, 164.50, 167.32, 167.93. Theoretical mass: 533.60, LC-MS (m/z, I %): 532.1 [(M-H)+, 100%]. HPLC
Purity: % Area 95.82, Retention Time 5.65 mins.
5-((6-(2-(5-(2-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)naphthalen-2-yl)
methylene)thiazolidine-2,4-dione (13b). Pale yellow colour powder. 69% yield (0.72 g). M.P. 281.5 °C.

FTIR (cm-1) 3379, 1737, 1676, 1626, 1587, 1498, 1475, 1440, 1257, 837, 750. 1H-NMR (400 MHz, DMSO-
d6) δ ppm 3.15 (dd, J = 5.2, 18.0 Hz, 1H), 3.97-4.05 (m, 1H), 5.33 (d, J = 16.4 Hz, 1H), 5.54 (d, J = 16.4 Hz,
1H), 5.83 (dd, J = 4.8, 12.0 Hz, 1H), 7.29-7.31 (m, 4H), 7.37 (s, 1H), 7.49-7.51 (m, 4H), 7.64 (d, J = 8.8 Hz,
1H), 7.86-7.88 (m, 2H), 7.89-7.91 (m, 1H), 7.94-7.95 (m, 1H), 7.95-7.97 (m, 1H), 8.10 (s, 1H), 12.59 (bs,
1H). 13C-NMR (100 MHz, DMSO-d6) δ 40.64, 57.72, 65.52, 107.21, 119.70, 122.29, 126.52, 126.95, 127.64,
127.76, 128.26, 128.31, 128.79, 129.15, 129.67, 130.40, 130.58, 130.71, 130.78, 130.86, 132.12, 134.74,
138.40, 146.50, 155.61, 157.65, 164.63, 167.93. Theoretical mass: 568.04, LC-MS (m/z, I %): 566.0 [(M-
2H)+, 100%]. HPLC Purity: % Area 97.42, Retention Time 4.12 mins.
5-((6-(2-(5-(furan-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)naphthalen-2-yl)
methylene)thiazolidine-2,4-dione (13c). Yellow colour powder. 66% yield (0.75 g). M.P. 269.7 °C. FTIR
(cm-1) 3302, 1737, 1669, 1643, 1595, 1572, 1535, 1500, 1462, 1444, 1269, 815. 1H-NMR (400 MHz, DMSO-
d6) δ ppm 3.45 (dd, J = 3.2, 8.4 Hz, 1H), 3.76-3.84 (m, 1H), 5.27 (d, J = 16.0 Hz, 1H), 5.35 (d, J = 16.0 Hz,
1H), 5.72 (dd, J = 4.8, 12.0 Hz, 1H), 6.39 (s, 2H), 7.28-7.30 (m, 2H), 7.51-7.52 (m, 3H), 7.58-7.60 (m, 1H),
7.60-7.62 (m, 1H), 7.86-7.89 (m, 4H), 7.96 (d, J = 8.8 Hz, 1H), 8.10 (s, 1H), 12.60 (bs, 1H). 13C-NMR (100
MHz, DMSO-d6) δ 38.12, 53.55, 65.59, 107.24, 107.28, 110.51, 119.64, 122.37, 126.50, 126.89, 127.76,
128.26, 128.33, 128.82, 130.39, 130.60, 130.70, 130.86, 132.06, 134.71, 142.50, 152.18, 155.66, 164.62,
167.38, 167.97. Theoretical mass: 523.56, LC-MS (m/z, I %): 522.0 [(M-H)+, 100%]. HPLC Purity: % Area
96.34, Retention Time 5.01 mins.
5-((6-(2-(5-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)naphthalen-2-yl)
methylene)thiazolidine-2,4-dione (13e). Yellow colour powder. 68% yield (0.7 g). M.P. 283.3 °C. FTIR
(cm-1) 3371, 1722, 1672, 1627, 1589, 1477, 1438, 1274, 1193, 862. 1H-NMR (400 MHz, DMSO-d6) δ ppm
3.20 (dd, J = 4.8, 18.4 Hz, 1H), 3.87-3.95 (m, 1H), 5.30 (d, J = 16.0 Hz, 1H), 5.46 (d, J = 16.4 Hz, 1H), 5.62
(dd, J = 4.8, 11.6 Hz, 1H), 7.14 (t, J = 8.8 Hz, 2H), 7.27-7.29 (m, 2H), 7.30-7.36 (m, 2H), 7.51 (s, 3H), 7.62
(d, J = 8.4 Hz, 1H), 7.88-7.90 (m, 4H), 7.94 (d, J = 8.8Hz, 1H), 8.08 (s, 1H), 12.59 (bs, 1H). 13C-NMR (100
MHz, DMSO-d6) δ 41.72, 59.28, 65.57, 107.18, 115.27, 115.48, 119.66, 122.32, 126.49, 126.94, 127.67,
127.76, 128.24, 128.32, 128.78, 130.38, 130.66, 130.69, 130.84, 132.09, 134.71, 138.01, 155.47, 157.66,
160.12, 162.54, 164.55, 167.32, 167.94. Theoretical mass: 551.59, LC-MS (m/z, I %): 550.1 [(M-H)+, 100%].
HPLC Purity: % Area 95.49, Retention Time 5.09 mins.
5-((6-(2-oxo-2-(3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethoxy)naphthalen-2-yl)methylene)
thiazolidine-2,4-dione (13g). Pale yellow colour powder. 65% yield (0.66 g). M.P. 261.8 °C. FTIR (cm-1)
3381, 3111, 1728, 1672, 1627, 1587, 1475, 1440, 1273, 862. 1H-NMR (400 MHz, DMSO-d6) δ ppm 2.24 (s,
3H), 3.17 (dd, J = 4.68, 18.2 Hz, 1H), 3.85-3.92 (m, 1H), 5.30 (d, J = 16.04 Hz, 1H), 5.45 (d, J = 16.04 Hz,
1H), 5.57 (dd, J = 4.6, 11.64 Hz, 1H), 7.12-7.18 (m, 4H), 7.26-7.29 (m, 2H), 7.47-7.51 (m, 3H), 7.61 (dd, J =
1.64, 8.68 Hz, 1H), 7.85-7.88 (m, 4H), 7.94 (d, J = 8.88 Hz, 1H), 8.08 (s, 1H), 12.61 (bs, 1H). 13C-NMR (100
MHz, DMSO-d6) δ 20.57, 27.98, 42.01, 59.98, 115.78, 116.00, 120.83, 124.50, 125.55, 129.18, 129.35,
134.31, 134.91, 138.60, 140.10, 155.06, 161.79, 162.76, 177.13. Theoretical mass: 547.62, LC-MS (m/z, I
%): 546.2 [(M-H)+, 100%]. HPLC Purity: % Area 97.15, Retention Time 4.17 mins.