Design, synthesis, topoisomerase I & II inhibitory activity, antiproliferative activity, and structure-activity relationship study of pyrazoline derivatives: An ATP-competitive human topoisomerase IIα catalytic inhibitor

Article abstract of DOI:10.1016/j.bmc.2016.03.017

A series of pyrazoline derivatives (5) were synthesized in 92-96% yields from chalcones (3) and hydrazides (4). Subsequently, topo-I and IIα-mediated relaxation and antiproliferative activity assays were evaluated for 5. Among the tested compounds, 5h had

Full text of DOI:10.1016/j.bmc.2016.03.017

Accepted Manuscript
Design, synthesis, topoisomerase I & II inhibitory activity, antiproliferative ac-
tivity, and structure-activity relationship study of pyrazoline derivatives: An
ATP-competitive human topoisomerase IIα catalytic inhibitor
Pervez Ahmad, Hyunjung Woo, Kyu-Yeon Jun, Adnan A. Kadi, Hatem A.
Abdel-Aziz, Youngjoo Kwon, A.F.M. Motiur Rahman
PII:
S0968-0896(16)30166-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.017
BMC 12866
Reference:
Bioorganic & Medicinal Chemistry
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7 March 2016
8 March 2016
Please cite this article as: Ahmad, P., Woo, H., Jun, K-Y., Kadi, A.A., Abdel-Aziz, H.A., Kwon, Y., Motiur Rahman,
A.F.M., Design, synthesis, topoisomerase I & II inhibitory activity, antiproliferative activity, and structure-activity
relationship study of pyrazoline derivatives: An ATP-competitive human topoisomerase IIα catalytic inhibitor,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.03.017
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Design, synthesis, topoisomerase I & II inhibitory activity,
antiproliferative activity, and structure-activity relationship study of
pyrazoline derivatives: an ATP-competitive human topoisomerase
IIα catalytic inhibitor
Pervez Ahmad,a,# Hyunjung Woo,b,# Kyu-Yeon Jun,b Adnan A. Kadi,a Hatem A.
Abdel-Aziz,a,c Youngjoo Kwon,b,* A. F. M. Motiur Rahmana,*
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud
b
University, Riyadh 11451, Saudi Arabia; College of Pharmacy, Graduate School of
Pharmaceutical Sciences, Ewha Womans University, Seoul, 120-750, Republic of
Korea. Department of Applied Organic Chemistry Department, National Research
Center, Dokki, Cairo 12622, Egypt.
*Corresponding Authors: Tel: +82232774653, fax: +82232772851, email:
ykwon@ewha.ac.kr (Y. Kwon) Tel: +966114670237, fax: +966114676220, email:
afmrahman@ksu.edu.sa (A. F. M. Motiur Rahman)
#Authors are equal contributor
1

Abstract
A series of pyrazoline derivatives (5) were synthesized in 92-96 % yields from
chalcones (3) and hydrazides (4). Subsequently, topo-I and IIα-mediated relaxation
and antiproliferative activity assays were evaluated for 5. Among the tested
compounds, 5h had a very strong topo-I activity of 97% (camptothecin, 74%) at
concentration of 100μM. Nevertheless, all the compounds 5a-5i showed significant
topo II inhibitory activity in the range of 90-94% (Etoposide, 96%) at the same
concentration. Cytotoxic potential of these compounds was tested in a panel of three
human tumor cell lines, HCT15, BT474 and T47D. All the compounds showed strong
activity against HCT15 cell line with IC50 at the range of 1.9-10.4 μM (Adriamycin,
23.0; Etoposide, 6.9; and Camptothecin, 7.1 μM). Moreover, compounds 5c, 5f and 5i
were observed to have strong antiproliferative activity against BT474 cell lines. Since,
compound 5d showed antiproliferative activity at a very low IC50 thus 5d was then
selected to study on their mode of action with diverse methods of ATP competition
assay, ATPase assay and DNA-topo II cleavable complex assay and the results
revealed that it functioned as a ATP-competitive human topoisomerase IIα catalytic
inhibitor. Further evaluation of endogenous topo-mediated DNA relaxation in cells
has been conducted to find that, 5d inhibited endogenous topo-mediated pBR322
plasmid relaxation is more efficient (78.0 ± 4.7% at 50 µM) than Etoposide (36.0 ±
1.7 % at 50 µM).
Keywords
Pyrazoline derivatives; Antiproliferative activity; Topoisomerase; ATP-Competitive
Inhibitor;
2

Introduction
Pyrazolines have shown a variety of biological properties [1-41] including antitumor
activity [21, 22, 33], anticancer activity on colon cancers cell line [42]. Recently,
pyrazoline-based selective fluorescent probe for detecting reduced glutathione and its
application in living cells and serum has been reported [43]. It should be noted that,
number of drugs are in market containing pyrazole/pyrazoline/pyrazolidine moieties
have been used for various treatments, e.g. Celecoxib [44], Famprofazone [45],
Deracoxib [46], Sulfinpyrazone [47], Apixaban [48], Allopurinol [49], Rimonabant
[50], Ruxolitinib [51], Sulfaphenazole [52], and Phenazone [53]. In addition, some of
the derivatives are under pre-clinical studies, e.g. SLV-330 [54, 55] and E-6087 [56,
57] (Chart 1). However, neither systematic SAR studies nor mechanistic studies have
not been pursues as yet.
Chart 1. Structures of some drugs containing pyrazole / pyrazoline / pyrazol-3-ones /
pyrazoline-3,5-dione moiety
4

Though numbers of pyrazoline derivatives have been reported with various biological
activities, and a numbers of drugs are currently available in market with
pyrazole/pyrazoline/pyrazolidine moieties, their intriguing structures and various
biological activities attracting the attention of researcher to synthesize novel
pyrazoline derivatives for the development of potential anticancer therapeutics. Since,
DNA is one of the most important pharmacological targets of many drugs. DNA
topoisomerases are enzymes that transiently break one or two strands of DNA, which
allow to solve various DNA topological problems generated during vital cellular
processes [58-67]. Because of the crucial role of topoisomerases for the maintenance
and replication of DNA during proliferation, topoisomerase inhibitors are among the
most potent anticancer agents to date. Our research group has been focusing on
development of human topo IIα catalytic inhibitors [58-62] especially using chalcone
moiety [63-66], to enhance topo IIα catalytic inhibitory activity. As part of our broad
interest finding novel topo inhibitors, we have designed and synthesized a series of
novel pyrazoline derivatives, derived from various chalcones and substituted 2-
carbohydrazide, evaluated their cytotoxic potential against various human cancer cell
lines, topo I & II inhibitory activities by assessing the relaxation of supercoiled pBR
322 plasmid DNA, evaluated mode of action of the most active compound by
performing DNA-topo cleavage complex, topo IIα-mediated ATP competition assay
and ATPase assay, studied molecular docking, and evaluated the effect of compound
on cell by endogenous topo inhibition assay for the development of new anticancer
therapeutics.
5

Results and discussion
Chemistry
Claisen-Schmidt reaction of commercially available inexpensive acetophenone
derivatives (1a-1d) and aromatic aldehydes (2a-2d) in presence of catalytic amount of
sodium hydroxide (10%) in ethanol obtained corresponding chalcones (3a-3e) in
quantitative yields (>95%) (Scheme 1) [68]. Structures of the compounds 3a-3e were
resolved by systematic analysis of nuclear magnetic resonance (NMR) and mass
spectroscopic (MS) data and were compared with the reported values.
Scheme 1. Synthesis of chalcones (3a-e)
On the other hand, 5-nitrofuran-2-carbohydrazide (4b) was synthesized using ethyl 5-
nitrofuran-2-carboxylate (4e) with hydrazine hydrate in absolute ethanol at 0C for
1h, and benzo[d]thiazole-2-carbohydrazide (4c) was synthesized using ethyl
benzo[d]thiazole-2-carboxylate (4f) with hydrazine hydrate in refluxing ethanol for
6

overnight (Scheme 2). Furan-2-carbohydrazide (4a) and isonicotinohydrazide (4d)
were obtained from commercial source.
O
H2N NH2
O2N
O2N
CO2Et
CO2Et
O
O
4e
0 oC, 1h
abs. EtOH
HN
O
NH2
4b
4c
N
S
H2N NH2
N
S
reflux, 8h
abs. EtOH
HN NH2
4f
N
O
O
O
HN
HN
NH2
4d
4a
NH2
Scheme 2. Synthesized and commercial hydrazides (4a-4d)
Synthesis of pyrazoline derivatives 5a-5i was straightforward as shown in Scheme 3.
Intermediate chalcones (3a-3e) and hydrazide (4a-4d) were reacted to obtain
pyrazoline derivatives 5a-5i in presence of catalytic amount of glacial acidic acid in
ethanol [69]. Purification of the compounds were accomplished by flash column
chromatography on silica gel eluting with ethyl acetate and n-hexane (1:1) obtained
analytical pure 5a-5i (92-96 % yields).
7

Scheme 3. Synthesis of pyrazoline derivatives (5a-5i)
The structures of the compounds were characterized using spectroscopic data as
follows: The IR spectra on KBr gives absorption bands in the regions 1612-1510 cm-1
corresponding to C=N stretching and 1704-1626 cm-1 corresponding to amide
carbonyl (N-C=O) stretching. Characteristic methylene protons (4-Hα and 4-H) for
the compounds (5a-5f) were shown at 3.52-4.04 ppm for 4-Hα and at 3.13-3.50 ppm
for 4-H as double doublet with the coupling constant ‘J’ approximately 18.0 & 11.5
Hz for 4-Hα and 18.0 & 4.5 Hz for 4-H, respectively. Another characteristic vicinal
methine proton 5-H was appeared at 5.68-5.92 ppm as double doublet with the
coupling constant 11.5 and 4.5 Hz. Synthesized pyrazoline derivatives also
characterized by mass spectrometer, all the derivatives gave protonated [M+H]+
8

and/or sodium adduct [M+Na]+ of molecular mass in positive mode or deprotonated
[M-H]- molecular mass in negative mode. In addition, the structures of the pyrazoline
derivatives also confirmed by elemental analysis data found for carbon, hydrogen and
nitrogen (CHN) percentage (details are given in experimental section).
Recombinant topoisomerase I and II inhibitory activities of compounds 5a-5i.
The conversion of supercoiled plasmid DNA to relaxed DNA by recombinant topo I
and II was examined in the presence of each 5a-5i for measuring their topo I and II
inhibitory activities. Well-known topo I and II inhibitors, camptothecin and Etoposide
were used as positive controls. The reaction products of topo I and II relaxation assays
were analysed by electrophoretic mobility and developed in ethidium bromide in the
presence of UV light. The inhibitory activities were evaluated at 100 μM for all
compounds. Only those compounds with moderate to significant activity were tested
further at 20 μM concentration.
100 M
(A)
D T C 5a 5b 5c 5d 5e 5f 5g 5h 5i
Relaxed DNA
Supercoiled DNA
20 M
(B)
D T C 5h
Relaxed DNA
Supercoiled DNA
Figure 1. Recombinant topo I inhibitory activities of compounds 5a-5i at 100 M (A)
and 20 M (B). Lane D: pBR322 DNA only; Lane T: pBR322 DNA + Topo I; Lane
C: pBR322 DNA + Topo I + Camptothecin; Lanes 5a-5i: pBR322 DNA + Topo I +
compounds 5a-5i.
9

As shown in Figure 1, most of the compounds did not exhibit topo I inhibitory
activity at 100 μM concentration while compound 5h showed very strong topo I
inhibitory activity of 97% (as compared to 74% of camptothecin) at 100 μM
concentration and weak topo I inhibitory activity of 4% (as compared to 25% of
camptothecin) at 20 μM concentration.
In case of topo II inhibitory evaluation, we found all the compounds 5a-5i showed
significant topo II inhibitory activity in the range of 90 - 94% (as compared to 96 %
of Etoposide) at 100 μM concentration but inactive or very weak at 20 μM
concentration (only compounds 5b and 5h inhibited topo II by 3% and 1% as
compared to 41% of Etoposide).
100 M
(A)
D T E 5a 5b 5c 5d 5e 5f 5g 5h 5i
Relaxed DNA
Supercoiled DNA
20 M
(B)
D T E 5a 5b 5c 5d 5e 5f 5g 5h 5i
Relaxed DNA
Supercoiled DNA
Figure 2. Recombinant topo II inhibitory activities of compounds 5a-5i at 100 M (A)
and 20 M (B). Lane D: pBR322 DNA only; Lane T: pBR322 DNA + Topo II; Lane
E: pBR322 DNA + Topo IIα + Etoposide; Lanes 5a-5i: pBR322 DNA + Topo II +
compounds 5a-5i.
Topo I and topo II inhibitory activities of compounds 5a-5i at both 100 and 20 μM
concentrations are summarized in Table 1. It should be noted that, compound 5h
showed considerable dual topo I and II inhibitory activities.
10

Table 1. Recombinant topo I and II inhibitory activities of compounds 5a-5i
Compounds
Topo I (% inhibition)
Topo II (% inhibition)
Concentration
100 μM
73.6
-
20 μM
100 μM
-
20 μM
-
24.6
Camptothecin
-
-
95.9
91.6
92.6
91.2
89.7
92.5
92.8
94.1
93.1
76.0
41.1
0.0
2.9
0.0
0.0
0.0
0.0
0.0
1.3
0.0
Etoposide
0.0
5a
5b
5c
5d
5e
5f
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
5g
5h
5i
96.8
0.0
4.0
-
Antiproliferative activity assays of compounds 5a-5i.
For the evaluation of antiproliferative activity of the compounds 5a-5i, three different
human cancer cell lines, HCT15 (human colorectal adenocarcinoma cell line), BT474
(human ductal carcinoma cell line) and T47D (Human ductal breast epithelial tumor
cell line) were used. Antiproliferative activity evaluation was performed for 5a-5i
since all had significant topo II inhibitory activity. Antiproliferative activity
11

evaluation results against human cancer cell lines are listed in Table 2 as values of
IC50. Most of the compounds showed strong antiproliferative activity against HCT15
cells with the IC50 values of 1.9-10.4 μM, whereas, controls gave IC50 values of 23.0,
6.9 and 7.1 μM for Adriamycin, Etoposide and Camptothecin, respectively. Among
the compounds, 5b and 5h did not show antiproliferative activity (IC50 > 50). On the
other hand, strong antiproliferative activity against BT474 cells with IC50 values of
6.3-7.2 μM was observed for compounds 5c, 5d, 5f and 5i, whereas controls gave IC50
values of 3.2, 6.6 and 7.0 μM for adriamycin, Etoposide and Camptothecin,
respectively. Moderate to strong antiproliferative activity was observed for the
compounds 5a, 5d, 5g and 5i against human ductal breast epithelial tumor cell line
(T47D).
12

Table 2. Antiproliferative activity assays of compounds 5a-5i
IC ( M)

50
Compound
HCT15
23.0 ± 7.6
6.9 ± 1.1
7.1 ± 2.3
3.9 ± 0.3
> 50
BT474
3.2 ± 0.1
6.6 ± 0.1
7.0 ± 1.2
> 50
T47D
2.9 ± 0.1
6.4 ± 0.3
4.1 ± 0.1
27.3 ± 2.2
> 50
Adriamycin
Etoposide
Camptothecin
5a
5b
5c
5d
5e
5f
> 50
1.9 ± 0.8
4.1 ± 0.01
10.4 ± 1.1
2.6 ± 1.2
4.3 ± 0.1
> 50
6.3 ± 0.1
> 50
7.2 0.03
6.6 ± 0.8
> 50

> 50
7.2 ± 1.1
> 50
> 50
7.5 ± 1.1
> 50
5g
5h
5i
> 50
3.1 ± 0.2
6.5 ± 0.1
7.9 ± 0.8
Structure Activity Relationships (SARs) Study.
The structural activity relationships (SARs) was analyzed based on the
antiproliferative activity results of compounds 5a-5i against HCT15, BT474 and
T47D cells lead to the following assumptions. As depicted in Table 3, compound 5a
showed 3.9 µM of IC50 against HCT15 cells while Adriamycin, Etoposide and
Camptothecin showed 23.0, 6.9 and 7.1 μM, respectively. On the other hand,
compound 5a showed weak or very weak antiproliferative activity against T47D cells
(IC50 = 27 μM) and BT474 cells (IC50 > 50 µM). Introducing substituents in phenyl
ring-A with 2’-OCH3, 4-NO2 group instead of 4-OCH3 in phenyl ring-B and 5-NO2 in
13

furan ring in compound 5b almost did not show antiproliferative activity at 50 µM
treatment with all tested cells (IC50 > 50 µM). While replacing furan ring of
compound 5a by benzo[d]thiazole ring to be compound 5c, antiproliferative activity
increased greatly with IC50 values of 1.9 μM against HCT15 cells and 6.3 μM against
BT474 cells, where IC50 values of adriamycin, Etoposide and camptothecin were 3.2,
6.6 and 7.0 μM, respectively. Keeping benzo[d]thiazole ring as 5c and introducing
substituent on ring-A with 4’-Cl in compound 5d improved antiproliferative activity
(IC50 = 4.1 µM against HCT15, 7.2 µM against BT474 and 6.6 µM against T47D
cells) compared to compound 5a. However, replacing furan ring of compound 5a or
benzo[d]thiazole ring of compound 5c by 4-pyridyl ring to be compound 5e showed
less antiproliferative activity (IC50 = 10 µM) against HCT15 cells, and almost no
antiproliferative activity with other two cell lines. Interestingly, replacing furan ring
of compound 5b with 4-pyridyl ring to be compound 5f showed much stronger
antiproliferative activity (IC50 = 2.6 µM) than all control compounds against HCT15
cells and similar antiproliferative activity (IC50 = 7.2 µM) to Etoposide and
camptothecin and weaker than Adriamycin against BT474 cells. The subtle
modification on the skeleton of compounds 5g-5i also showed dramatic change in
antiproliferative activity. Introducing methoxy group (-OCH3) in 4’-position of phenyl
ring-B of compound 5a and replacing ring-B by ring-C to be compound 5g
ameliorated antiproliferative activity (IC50 = 4.3 µM) against HCT15 cells and showed
moderate antiproliferative activity (IC50 = 7.5 µM) against T47D cells. Unfortunately,
replacement of 4’-OCH3 of ring-A of 5g with 4’-Cl to be compound 5h deteriorated
antiproliferative activity (IC50 > 50µM). Interestingly, replacing furan ring of
compound 5h by 4-pyridyl ring to be compound 5i showed excellent antiproliferative
14

activity (IC50 = 3.1 µM) against HCT15 cells and moderate antiproliferative activity
(IC50 = 6.5 µM and 7.9 µM) against BT474 and T47D cells, respectively.
15

Table 3. Summarized SARs study results of pyrazoline derivatives 5a-5i
Antiproliferative activity (IC50 in
Entry
R
M)
3.9 ± 0.3 against HCT15 cells
5a
R1=H; R2=4-OCH3; R3=
R1=2’-OCH3;
R2=4-NO2;
R3=
Not active
5b
5c
1.9 ± 0.8 against HCT15 cells
6.3 ± 0.1 against BT474 cells
4.1 ± 0.01 against HCT15 cells
7.2 ± 0.03 against BT474 cells
6.6 ± 0.8 against T47D cells
R1=H; R2=4-OCH3; R3=
5d
R1=4’-Cl; R2=4-OCH3; R3=
10.3 ± 1.1 against HCT15 cells
5e
5f
R1=H; R2=4-OCH3; R3=
2.2 ± 1.2 against HCT15 cells
7.2 ± 1.1 against BT474 cells
R1=2’-OCH3; R2=4-NO2; R3=
4.3 ± 0.1 against HCT15 cells
7.5 ± 1.1 against T47D cells
5g
5h
5i
R1=4’-OCH3; R3=
Not active
R1=4’-Cl; R3=
R1=4’-Cl; R3=
3.1 ± 0.2 against HCT15 cells
6.5 ± 0.1 against BT474 cells
16

7.9 ± 0.8 against T47D cells
All the compounds 5a-5i showed moderate to significant antiproliferative activity
even much more potential than control drugs in some cases. Interestingly, most of the
compounds 5a-i did not show topo I inhibitory activity at all, except for compound
5h, which has 4’-Cl substituent attached with 4’-position of ring-A in the skeleton of
compounds 5g-5i. It inhibited topo I by 97% at 100 μM concentration and 4% at 20
μM concentration, whereas camptothecin inhibited 74%, and 24% at 100 μM and 20
μM, respectively. On the other hand, all compounds 5a-5i inhibited topo II activity.
Substituents did not affect very much to increase or decrease the topo II inhibitory
activity. The topo II inhibitory activity of compounds 5a-5i was comparable with that
of Etoposide at 100 μM concentration.
Molecular docking study of compounds 5a-5i.
As discussed above, compounds 5a-5i inhibited topo II specifically than topo I except
for compound 5h. Topoisomerases resolve DNA topological problems encountered
during DNA replication, transcription, recombination, repair and chromatin assembly
through breaking and relegating single strand for topo I and double strand of DNA
for topo II, respectively [70]. Beside the difference of breaking single or double
strands of DNA, topo II is different from topo I in that an ATP binds to the ATPase
domain [71]. Therefore, we first checked whether compounds 5a-5i can bind to the
ATPase domain with molecular docking study using coordinates (PDB entry 1ZXM)
previously reported for the co-crystal structure of the adenosine 5′-(β,γ-imido)
triphosphate tetralithium salt hydrate (AMP-PNP) bound to the ATP-binding domain
of human topo II [61, 72]. AMP-PNP is a structural analogue of ATP that has
comparable binding affinity for the ATP-binding pocket of topo II but is non-
17

hydrolyzable which enables to co-crystallize with topo II. AMP-PNP was used as a
reference ligand to identify the active site and to validate our docking process by re-
docking. Compounds 5a-5i showed favourable binding to the ATP-binding domain in
order of 5b>5f>5d. All compounds have a chiral center on the pyrazoline ring. The R
and S isomers of all compounds were prepared and molecular docking study were
done for all the isomers. Compound 5f bind to the ATP binding pocket better than
compound 5d. However, the docking result of the two stereoisomers of compound 5f
did not align well (Figure 3A). On the other hand, the two stereoisomers of 5d flipped
over in order to occupy the same binding site (Figure 3). As shown in detailed view of
compound 5d bound to the ATP-binding domain of human topo II (Figure 3),
compound 5d fits well into the ATP-binding cleft drawn in space-filled mode (Figure
3A) and overlays with AMP-PNP (Figure 3B). Compound 5d has hydrogen-bonds
with residues of Arg162 and Gly164 and hydrophobic interactions with Asn91, Asn95,
Asn120, Ile141, Phe142, Thr147, Ser148, Ser149, Arg162, Asn163, Gly164, Tyr165,
Gly166, Ala167, Gln376, and Lys378 of the topo II ATP-binding pocket. In
consideration of the preference from the results of the docking study and
antiproliferative activity, compound 5d was chosen as a representative compound to
determine whether it competes with ATP and inhibits ATP hydrolysis, leading to
inhibition of topo II catalytic activity.
18

Figure 3. Compound 5d potentially binds to the ATP binding pocket in the ATPase
domain of human topo IIα. (A) Magnified view of compound 5d (R-5d, magenta; S-
5d, cyan) binding to the active site of ATPase domain of topo IIα. The transparent
channel surface of the ATP binding pocket is represented and coloured by
electrostatic potential (red, most positive; purple, most negative). (B) Overlay view of
compound 5d and AMP-PNP (compound 5d, magenta; AMP-PNP, gray). (C)
Magnified view of the topo IIα ATP binding pocket. The residues of the topo IIα
ATP-binding domain that have van der Waals contact with compound R-5d are
shown as sticks. The stick representation of the complex is colored by the atom type
(R-5d, magenta).
19

20

Compound 5d is ATP-competitive human topo II catalytic inhibitor.
To confirm compound 5d binds to the ATP binding pocket of human topo II, we
performed ATPase assay which evaluates the degree of ATP hydrolysis of topo II.
Compound 5d inhibited the ATP hydrolysis of topo II at a dose-dependent manner
with similar extent to Etoposide, a well-known competitive topo II catalytic inhibitor
(Figure 4A) [73]. The ATP competition assay with topo II was performed as the same
procedure as the topo II relaxation assay, using different concentration of ATP of 1
and 2 mM. Compound 5d inhibited the topo II activity in ATP concentration-
dependent manner (Figure 4B and 4C), while Etoposide did not change the activity as
the ATP concentration increased which is depicted by bar graphs in Figure 4C. It
reflected compound 5d functioned as an ATP-competitive manner but Etoposide did
not. We further performed the cleavage complex assay to investigate compound 5d as
a topo II catalytic inhibitor. Topo II poisons act by stabilizing transiently-formed topo
II enzyme-DNA cleavage complex leading to the formation of linear DNA [74]. In
cleavage complex assay, initially the relaxation of the pBR322 plasmid was allowed
by the addition of topo IIα and then compound 5d was treated. In contrast to
Etoposide, a well-known topo II poison, linear form of DNA did not form in the case
of compound 5d up to 300 µM treatment, which inferred no stabilization of enzyme-
DNA cleavage complex, (Figure 4D). Taken together, compound 5d acted as an ATP-
competitive catalytic inhibitor rather than a topo II poison.
21

Figure 4. The evaluation of compound 5d functioning as an ATP-competitive topo II
catalytic inhibitor. (A) ATPase assay. Topo II-mediated ATPase assay was performed
with ATP dehydrogenase reaction. The free phosphate derived by hydrolysis reaction
of recombinant human topo IIα was detected by malachite green phosphate assay
reagent with incubation for 30 min at room temperature. The extent of hydrolysis
reaction was determined by measuring absorbance of green complex formed between
malachite green, molybdate and free phosphate at a wavelength of 620 nm using
ELISA micro-plate reader. (B) ATP competition assay. Except for the concentration
of ATP, ATP competition assay was performed as following the same process as the
DNA topo II-mediated DNA relaxation assay. The final concentrations of ATP were 1
mM and 2 mM. Etoposide was used as a positive control. (C) The quantitative results
in (B) were depicted. (D) Cleavage complex assay. 100 ng/µL of supercoiled DNA
pBR322 was incubated with 3 unit of recombinant human DNA topo IIα for 5 min
followed by the addition of compound 5d. The linear form of DNA was arrow-
labelled after reaction mixture was electrophoresed in 1% agarose gel in TAE buffer
22

containing 0.5 µg /mL ethidium bromide followed by visualized gel using an Alpha
Tech Imager.
Compound 5d inhibited endogenous topos with better potency than Etoposide.
To confirm the effect of compound 5d on endogenous topo-mediated DNA relaxation
in cells, we prepared nuclear lysates of HCT15 cells treated with compound 5d and
Etoposide, respectively, for 24 hr. Untreated nuclear extract was used as a negative
control to confirm whether endogenous topo relaxes supercoiled pBR322 plasmid by
incubating for 30 min at 37 C as shown in the second lane of Figure 5. Compound 5d
inhibited the endogenous topo-mediated pBR322 plasmid relaxation more efficiently
than Etoposide; % inhibition of DNA relaxation of compound 5d is 78.0 ± 4.7% at 50
µM; that of Etoposide is 36.0 ± 1.7% at the same concentration (Figure 5).
pBR322 (100 ng)
+
-
+
+
+
-
+
-
+
-
Untreated nuclear lysate
50
-
-
-
-
-
-
-
etoposide (µM)
Comp 5d (µM)
Relaxed
Nuclear lysate-
treated with
50
100
Supercoiled
% Inhibition
Figure 5. The effect of compound 5d on endogenous topo-mediated DNA relaxation.
Nuclear lysates of HCT15 cells, prepared after treatment with Etoposide and
compound 5d for 24 hr, were incubated for 30 min with 100 ng of pBR322 plasmid at
37 °C.
23

Conclusion
We have designed and synthesized a series of novel pyrazoline derivatives (5a-5i) in
two steps via chalcones (3a-3e) and 2-carbohydrazide (4a-4d). These compounds
were evaluated for their antiproliferative activity and topo I and II inhibitory
activities. Among them 5a, 5c-d, 5f-g and 5i displayed excellent antiproliferative
activity against HCT15 cell lines; 5c, 5d, 5f and 5i against BT474 cell lines; and 5d,
5g and 5i against T47D cell lines. All the pyrazoline derivatives (5a-5i) inhibited topo
II activity with specificity over topo I, except compound 5h (96.8% at 100 μM and
4.0% at 20 μM concentration). We further determined the mode of action of a
representative compound 5d as an ATP-competitive topo II catalytic inhibitor.
Compound 5d inhibited topo II catalytic activity through binding to the ATPase
domain of topo II in ATP-competitive manner without generating the linear DNA
which was confirmed by docking study, ATP competition, ATPase, and cleavage
complex assays using recombinant human topo II. Compound 5d inhibited
endogenous topo (78.0% at 50 µM) with better potency than Etoposide (36.0% at 50
µM). Modification of the structures of the compound and the animal study are in
progress for further development of novel anticancer agents and the results will be
explored in due courses.
Experimental
General Chemistry
Chemicals and solvents were reagent grades and were used without further
purifications. Compounds 4a, 4d, 4e and 4f were obtained from commercial sources.
Thin layer column chromatography (TLC) analysis was carried out on glass-backed
TLC silica plates (silica gel 60 F254, 0.25 mm) impregnated with fluorescence indicator
24

(Merck art. 1.05554). Column chromatography was carried out on 60-120 mesh Merck
silica gel. Melting points were determined on a Fisher ScientificTM digital melting
point apparatus (model number IA9100) and are uncorrected. Infrared spectra (IR)
were recorded using KBr pellets for solids and neat for liquids on a PerkinElmer
Spectrum BX FTIR spectrometer. NMR spectra were obtained using 500 MHz Avance
Bruker Ultra ShieldTM and an Agilent technologies premium shielded 400 MHz
spectrometer and are reported as parts per million (ppm) from the internal standard of
1
13
tetramethylsilane. Chemical shift for H- and C-NMR (δ) are reported in ppm. The
working frequency for 1H- and 13C- were 500/400 MHz and 125/100 MHz,
1
respectively. CDCl3, and DMSO-d6 were used as internal standard in H- as 7.24 and
2.49 ppm, while in 13C- as 77 and 39.5 ppm, respectively. The coupling constants J are
reported in Hertz (Hz). Peaks were described as broad singnals (br), singlet (s),
doublets (d), doublets of doublets (dd), doublets of doublets of doublets (ddd), triplets
(t) and multiplets (m), doublets of triplets (dt), triplets of doublets (td). Mass spectra
were carried out using an Agilent High performance liquid chromatography (HPLC)
1200 connected with Agilent 6320 Ion Trap mass spectrometer fitted with ESI ion
source in positive and/or negative mode. Elemental Analysis were perform using a
PerkinElmer 2400 CHN analyzer.
General procedure for the preparation of Chalcone (3a-3e).
An equimolar mixture of (substituted)-acetophenone and (substituted)-benzaldehyde
in ethanol in presence of alcoholic sodium hydroxide (10%) was stirred for 2-3 hours.
The whole mass of the mixture was poured into ice cold water, solid precipitate was
filtered, dried in open air, obtained quantitative yields of the corresponding chalcones
(3a-3d). Crystallization from ethanol afforded the pure chalcones, and the spectral
25

data were identical to those of literature values and for unreported compounds are
given below.
(E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (3a). White solid; (CAS no. 959-
33-1); (98 %); mp. = 61C [lit. [75] mp. = 60-63C]; 1HNMR (CDCl3, 400 MHz): 
8.01 (dt, J = 6.8, 1.2 Hz, 2H), 7.79 (d, J = 15.6 Hz, 1H), 7.60 (dt, J = 8.4, 1.6 Hz, 2H),
7.56 (dt, J = 8.8 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.49 (t, J = 8.8 Hz, 1H), 7.42 (d, J
= 15.6 Hz, 1H), 6.97 (dt, J = 8.8, 2.0 Hz, 1H), 3.85 (s, 3H, -OCH3); 13CNMR
(CDCl3, 100 MHz):  190.59, 161.69, 144.70, 138.52, 132.54, 130.23, 128.56,
128.41, 127.63, 119.80, 114.42 and 55.40 ppm. ESI mass (m/z) 238.9 [M+H]+, 260.9
[M+Na]+ (calculated exact mass 238.1).
(E)-1-(2-methoxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one (3b). Yellow solid; (96
%); mp. = 71-72C; 1H NMR (CDCl3, 500 MHz):  8.17 (d, J = 8.5 Hz 2H), 7.65 (d, J
= 8.5 Hz 2H), 7.61-7.56 (overlapped, 2H), 7.48-7.42 (overlapped, 2H), 6.98 (d, J =
13
7.5 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 3.86 (s, 3H, -OCH3) ppm; C NMR (CDCl3,
125 MHz):  190.64, 157.45, 147.31, 140.52, 138.18, 132.71, 129.70, 129.63, 127.80,
127.52, 123.11, 119.95, 110.70 and 54.79 ppm. ESI mass (m/z) 284.0 [M+H]+, 305.9
[M+Na]+ (calculated exact mass 283.1).
(E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3c). White solid; (98
%); mp. = 117-118C [lit. [76] mp. = 117C]; 1HNMR (CDCl3, 400 MHz):  7.95 (dt,
J = 8.8, 2.0 Hz, 2H), 7.79 (d, J = 15.6 Hz, 1H), 7.60 (dt, J = 8.8, 1.6 Hz, 2H), 7.46 (dt,
J = 8.8, 2.0 Hz, 2H), 7.36 (d, J = 16.0 Hz, 1H), 6.94 (dt, J = 8.8, 2.0 Hz, 2H), 3.85 (s,
3H, -OCH3); 13CNMR (CDCl3, 100 MHz):  189.18, 161.85, 145.20, 138.93, 136.81,
26

130.32, 129.82, 128.85, 127.43, 119.17, 114.70 and 55.42 ppm. ESI mass (m/z) 273.1
[M+H]+, 294.9 [M+Na]+ (calculated exact mass 272.1).
(E)-1-(4-chlorophenyl)-3-(furan-2-yl)prop-2-en-1-one (3d). CAS no. 114570-70-6;
White solid; lit. [77] (95 %); 1HNMR (CDCl3, 400 MHz):  7.95 (dt, J = 8.8, 2.4 Hz,
2H), 7.58 (d, J = 15.2 Hz, 1H), 7.51 (d, J = 1.2 Hz, 1H), 7.44 (dt, J = 8.8, 2.0 Hz, 2H),
7.38 (d, J = 15.2 Hz, 1H), 6.72 (d, J = 2.8 Hz, 1H), 6.50 (dd, J = 3.6, 2.0 Hz, 1H);
13CNMR (CDCl3, 100 MHz):  188.39, 151.52, 145.10, 139.15, 136.44, 131.03,
129.81, 128.89, 118.66, 116.63 and 112.76 ppm. ESI mass (m/z) 232.9 [M+H]+,
254.8 [M+Na]+ (calculated exact mass 233.0).
(E)-1-(4-methoxyphenyl)-3-(furan-2-yl)prop-2-en-1-one (3e). White solid; (98 %);
mp. = 68-69C [lit. [78] mp. = 68C]; 1HNMR (CDCl3, 400 MHz):  8.04 (dt, J = 9.2,
2.0 Hz, 2H), 7.57 (d, J = 15.6 Hz, 1H), 7.51 (dd, J = 2.4, 1.2 Hz, 1H), 7.45 (d, J =
15.2 Hz, 1H), 6.96 (dt, J = 8.8, 2.0 Hz, 2H), 6.68 (d, J = 3.6 Hz, 1H), 6.49 (dd, J =
13
3.6, 2.0 Hz, 1H), 3.87 (s, 3H, -OCH3); CNMR (CDCl3, 100 MHz):  188.05, 163,
40, 151.82, 144.67, 131.08, 130.72, 129.96, 119.21, 115.75, 113.82, 112.58 and 55.47
ppm. ESI mass (m/z) 228.9 [M+H]+, 250.8 [M+Na]+ (calculated exact mass 228.0);
General procedure for the preparation of Hydrazide (4a-4d).
Furan-2-carbohydrazide (4a). (CAS no. 3326-71-4) are commercial available; mp. =
77-79C. 1H NMR (CDCl3, 500 MHz): δ 7.45 (d, J = 1.0 Hz 1H), 7.15 (d, J = 2.5 Hz
1H), 7.50 (dd, J = 3.5, 1.0 Hz, 1H). . 13C NMR (CDCl3, 125 MHz): δ159.47, 146.64,
144.32, 114.76 and 111.99 ppm. ESI mass (m/z) 126.9 [M+H]+ (calculated for 129.1).
27

5-Nitrofuran-2-carbohydrazide (4b). A solution of hydrazine hydrate (11 mmol) and
ethyl 5-nitrofuran-2-carbohydrazide (10 mmol) in absolute ethanol (100 ml.) was
stirred at 0 C for 1 h. the stirring was continued for further 1h at room temperature.
The solid formed was filtered off and washed with ethanol and dried by suction.
Crystallization from methanol afforded 5-nitrofuran-2-carbohydrazide (4b) as pale
1
yellow needles in 73% yield, mp. = 171-172C [lit. [79] mp. = 171-172C]; HNMR
(CDCl3 + DMSO-d6, 400 MHz):  7.22 (d, J = 4.0 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H);
13CNMR (CDCl3 + DMSO-d6, 100 MHz):  156.45, 147.22, 115.82 and 112.19 ppm.
ESI mass (m/z) 171.8 [M+H]+ (calculated exact mass 171.0); CAS no. 5469-78-3.
Benzo[d]thiazole-2-carbohydrazide (4c). A solution of hydrazine hydrate (22 mmol)
and ethyl benzo[d]thiazole-2-carboxylate (10 mmol) in absolute ethanol (50 mL) was
refluxed for 1 h. The solid formed was filtered off and washed with ethanol, dried and
crystallization from ethanol afforded benzo[d]thiazole-2-carbohydrazide (4c) as pale
1
yellow crystals in 90% yield, mp. = 175-176 C [lit. [80] mp. 175-176C]; HNMR
(CDCl3 + DMSO-d6, 400 MHz):  7.99 (ddd, J = 8.0, 1.6, 0.4 Hz, 1H), 7.95 (ddd, J =
8.0, 1.6, 0.4 Hz, 1H), 7.48 (td, J = 7.2, 1.6 Hz, 1H); 7.43 (td, J = 7.2, 1.6 Hz, 1H);
13CNMR (CDCl3 + DMSO-d6, 100 MHz):  163.07, 159.09, 153.11, 136.28, 126.91,
126.76, 124.22 and 122.51 ppm. ESI mass (m/z) 193.8 [M+H]+ (calculated exact mass
193.0); CAS number: 28891-34-1.
1
Isonicotinohydrazide (4d). (CAS no. 54-85-3) are commercial available. HNMR
(CDCl3 + DMSO-d6, 400 MHz):  8.59 (dd, J = 4.4, 1.6 Hz, 2H), 7.67 (dd, J = 4.8,
1.6 Hz, 2H); 13CNMR (CDCl3 + DMSO-d6, 100 MHz):  164.95, 150.18, 140.31 and
121.30 ppm. ESI mass (m/z) 137.9 [M+H]+ (calculated exact mass 137.0).
28

General procedure for the preparation of Pyrazoline (5a-5i).
A mixture of chalcone (1 equiv.) and hydrazide (1.2 equiv.) was refluxed overnight in
ethanol in the presence of catalytic amount of glacial acetic acid (1-3 drop). Excess
solvent were evaporated and the reaction mixture were pass through silica gel column
chromatography using ethyl acetate and n-hexane as solvent. The spectral data for
unreported compounds follows.
Furan-2-yl(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone
(5a). Light brown solid; (94 %); mp = 243-244C; IR (KBr, cm-1): 3116, 3062, 2953,
1
1648, 1611, 1560, 1512, 1472, 1429, 1246, 1174, 1036 cm-1; H NMR (CDCl3, 500
MHz):  7.23-7.16 (m, 2H), 7.59 (d, J = 2.5 Hz 1H), 7.53 (s, 1H), 7.39-7.37 (m, 3H),
7.14 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 9.0 Hz, 2H), 6.48 (t, J = 1.5 Hz, 1H) 5.68 (dd, J
= 11.5, 4.0 Hz, 1H), 3.68 (s, 3H, -OCH3), 3.66 (overlapped, 1H), 3.13 (dd, J = 18.0,
13
4.0 Hz, 1H), ppm; C NMR (CDCl3, 125 MHz):  159.11, 155.86, 155.40, 146.37,
145.37, 133.67, 131.39, 130.56, 128.87, 127.13, 126.81, 118.94, 114.25, 111.52
60.58, 55.30 and 41.26 ppm; ESI mass (m/z) 347.1 [M+H]+ (calculated exact mass
346.1); Anal. Calculated for C21H18N2O3: C, 72.82; H, 5.24; N, 8.09. Found: C, 72.03;
H, 5.24; N, 7.87.
(3-(2-Methoxyphenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(5-nitrofuran-
2-yl)methanone (5b). Yellow solid; (92 %); mp = 140-141C; IR (KBr, cm-1): 3326,
1
3106, 3060, 2949, 1704, 1596, 1513, 1470, 1257, 1031 cm-1; H NMR (CDCl3, 500
MHz):  7.82 (s, 2H), 7.69 (s, 1H), 7.63 (s, 1H), 7.49 (m, 2H), 7.25 (d, J = 8.5 Hz,
2H), 6.86 (d, J = 8.5 Hz, 2H), 6.58 (s, 1H), 5.80 (dd, J = 11.5, 4.0 Hz, 1H), 3.79 (s,
13
3H, -OCH3), 3.77 (overlapped, 1H), 3.24 (dd, J = 18.0, 4.0 Hz, 1H), ppm; C NMR
30

(CDCl3, 125 MHz):  159.13, 155.86, 155.36, 146.39, 145.36, 133.68, 131.41,
130.55, 128.86, 127.14, 126.80, 118.91, 114.26, 111.50 60.58, 55.30 and 41.25 ppm;
ESI mass (m/z) 434.5 [M-H]- (calculated exact mass 436.0); Anal. Calculated for
C21H16N4O7: C, 57.80; H, 3.70; N, 12.84. Found: C, 57.69; H, 3.77; N, 12.95.
Benzo[d]thiazol-2-yl(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)methanone (5c). Off white solid; (92 %); mp = 199-200C; IR (KBr, cm-1): 3062,
1
2957, 1646, 1610, 1513, 1469, 1417, 1249, 1184, 1036 cm-1; H NMR (DMSO-d6,
500 MHz):  8.29 (d, J = 7.0 Hz, 1H), 8.21 (d, J = 6.0 Hz, 1H), 7.96 (s, 2H), 7.63-
7.56 (m, 5H), 7.25 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.83-5.81 (m, 1H),
4.01 (dd, J = 18, 11.5 Hz, 1H), 3.73 (s, 3H, -OCH3), 3.34 (overlapped, 1H) ppm; 13C
NMR (DMSO-d6, 125 MHz):  158.69, 157.46, 155.05, 151.41, 136.76, 133.19,
131.06, 130.54, 128.99, 127.35, 127.16, 127.15, 126.88, 124.43, 122.53, 114.14,
60.65, 55.10 and 41.57 ppm; ESI mass (m/z) 414.0 [M+H]+, 436.0 [M+Na]+
(calculated exact mass 413.1); Anal. Calculated for C24H19N3O2S: C, 69.71; H, 4.63;
N, 10.16. Found: C, 68.81; H, 4.76; N, 10.21.
Benzo[d]thiazol-2-yl(3-(4-chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)methanone (5d). White solid; (94%); mp = 139-140C; IR (KBr, cm-1):
1
3012, 1918, 1648, 1593, 1515, 1430, 1400, 1333, 1252, 1178, 1031 cm-1; H NMR
(CDCl3, 500 MHz):  8.74 (m, 2H), 7.80 (m, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.39 (d, J
= 8.5 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.75 (dd, J = 11.5,
4.0 Hz, 1H), 3.76 (s, 3H, -OCH3), 3.76 (overlapped, 1H), 3.21 (dd, J = 18.0, 4.5 Hz,
13
1H) ppm; C NMR (CDCl3, 125 MHz):  171.12, 164.25, 159.32, 154.90, 149.70,
141.74, 136.76, 133.19, 130.00, 129.47, 128.09, 127.09. 123.65, 114.43, 60.92, 55.29
31