Synthesis and cytotoxicity studies of 3,5-diaryl N-acetyl pyrazoline - Isatin hybrids

Article abstract of DOI:10.1007/s00044-014-1001-5

Numerous reports highlighting the cytotoxic effects of 3,5-diaryl N-acetyl-pyrazolines and isatin tempted us to synthesise conjugates of the functionalities via alkyl armed triazole tetheration. The hybrids were synthesized by click chemistry approach and were evaluated against a panel of cell lines i.e. viz HeLa (cervix cancer), CAKI-I (Renal cancer), PC-3 (Prostate cancer) and Mia-paca-2 (pancreatic cancer). The hybrids were classified into right-handed and left-handed conjugates on the basis of the placement of the isatin ring. The length of the alkyl armed triazole linker was varied from 2 to 6. Structure activity relationship has also been presented. A preliminary cytotoxic assay was performed on the series of 3,5-diaryl N-acetyl-pyrazolines and only the potent 3,5-diaryl N-acetylpyrazolines were selected for their inclusion in the hybrid scaffold. Among the cell lines employed, HeLa cell line was the most sensitive towards the exposure of test compounds. Out of all the compounds evaluated, two right-handed conjugates MI-7b and MI-8b and two left-handed conjugates MI-4b, MI-6b displayed significant cytotoxic potential and exhibited an IC₅₀ range from 1.3 to 3.5 μM against HeLa Cell line.

Full text of DOI:10.1007/s00044-014-1001-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1001-5
ORIGINAL RESEARCH
Synthesis and cytotoxicity studies of 3,5-diaryl N-acetyl
pyrazoline—isatin hybrids
•
Manmohan Sharma Sahil Sharma Abhishek Buddhiraja
•
•
•
A. K. Saxena Kunal Nepali P. M. S. Bedi
•
Received: 3 December 2013 / Accepted: 14 March 2014
Ó Springer Science+Business Media New York 2014
Abstract Numerous reports highlighting the cytotoxic
effects of 3,5-diaryl N-acetyl-pyrazolines and isatin temp-
ted us to synthesise conjugates of the functionalities via
alkyl armed triazole tetheration. The hybrids were syn-
thesized by click chemistry approach and were evaluated
against a panel of cell lines i.e. viz HeLa (cervix cancer),
CAKI-I (Renal cancer), PC-3 (Prostate cancer) and Mia-
paca-2 (pancreatic cancer). The hybrids were classified into
right-handed and left-handed conjugates on the basis of the
placement of the isatin ring. The length of the alkyl armed
triazole linker was varied from 2 to 6. Structure activity
relationship has also been presented. A preliminary cyto-
toxic assay was performed on the series of 3,5-diaryl N-
acetyl-pyrazolines and only the potent 3,5-diaryl N-acetyl-
pyrazolines were selected for their inclusion in the hybrid
scaffold. Among the cell lines employed, HeLa cell line
was the most sensitive towards the exposure of test com-
pounds. Out of all the compounds evaluated, two right-
handed conjugates MI-7b and MI-8b and two left-handed
conjugates MI-4b, MI-6b displayed significant cytotoxic
potential and exhibited an IC50 range from 1.3 to 3.5 lM
against HeLa Cell line.
Keywords Molecular hybridization Á Hybrids Á
Isatin Á Cell lines Á Cytotoxic
Introduction
Cancer, the uncontrolled, rapid and pathological prolifer-
ation of abnormal cells, is one of the most formidable
afflictions in the world. (Fadeyi et al., 2008; Rostom,
2006). Over the years, the design of cancer chemotherapy
has become increasingly sophisticated. Yet, there is no
cancer treatment that is 100 % effective against dissemi-
nated cancer. At present cancer therapy interfering with a
single biological molecule or pathway has been success-
fully utilized (Sawyers, 2004; Petrelli and Giordano, 2008;
Overington et al., 2006). Still the problem of drug resis-
tance and a general belief that agents modulating more than
one target could have superior efficacy compared to single
target drugs (Bode and Dong, 2009; Zhan and Liu, 2009)
has initiated the search for molecules modulating multiple
targets. Modulating multiple targets simultaneously can be
achieved either by the combination of multiple drugs with
different mechanisms or by single chemical entity that
could modulate several targets of a multifactorial disease.
(Morphy and Rankovic, 2005; Hopkins, 2008) The
molecular hybridization (MH) is a strategy of rational
design of new ligands or prototypes based on the recog-
nition of pharmacophoric sub-units in the molecular
structure of two or more known bioactive derivatives
which, through the adequate fusion of these sub-units,
provides new hybrid architectures (Viegas-Junior et al.,
2007). With this background and promising results
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1001-5) contains supplementary
material, which is available to authorized users.
M. Sharma Á S. Sharma Á K. Nepali (&) Á P. M. S. Bedi
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar 143005, Punjab, India
e-mail: kunal_8855@rediffmail.com
A. Buddhiraja
Faculty of Pharmaceutical Sciences, Shoolini University, Solan,
Himachal Pradesh, India
A. K. Saxena
Indian Insitute of Integrative Medicine, Jammu, India
123

Med Chem Res
3,5 -DIARYL N-ACETYL
PYRAZOLINE (DNAP)
ISATIN
O
N
N
OCH3
O
O
N
R
Linker
N
O
N
O
R
OH
DNAP-ISATIN HYBRIDS
3,5 -DIARYL N-ACETYL
PYRAZOLINE (DNAP)
O
3,5 -DIARYL N-ACETYL
PYRAZOLINE (DNAP)
ISATIN
Molecular
Hybridization
O
N
H
O
ISATIN
O
ISATIN
N
N
R
N
N
O
R
N
Linker
O
O
HO
3,5 -DIARYL N-ACETYL
PYRAZOLINE (DNAP)
DNAP-ISATIN HYBRIDS
Fig. 1 1 DNAP-Isatin hybrids
achieved with hybrids structure-based design approach,
hybrids of 3,5-diaryl-4,5-dihydro-pyrazolines and isatin
were designed in view of the promising anticancer poten-
tial displayed by these functionalities.
benzothiazole analogues (Solomon et al., 2009), isatin-4-
pirazinylquinoline hybrids (Solomon et al., 2010) and
isatin-thiazolidinone hybrid (Ramshid et al., 2010) clearly
indicates the success of isatin-based molecular hybrids and
justifies its inclusion in the conjugates designed by our
research group.
3,5-Diaryl-4,5-dihydro-pyrazolines have been well
explored to be endowed with anticancer potential and
inhibitory effects on various enzymes. Zhou et al., (2013)
recently reported 1-(5-(3,4-dihydroxyphenyl)-3-(4-hydroxy-
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone as potent
tyrosinase inhibitor with IC50 value of 0.301 lM. Chloro-
quinolinyl-based pyrazoline derivatives (Insuasty et al.,
2013) displayed remarkable antitumor activity against can-
cer cell lines, with low GI50 values ranging from 0.13 to
0.99 lM. Manna et al., (2005) reported some appropriate
pyrazolines capable of inhibiting P-glycoprotein-mediated
multidrug resistance by direct binding to a purified protein
domain containing an ATP-binding site and a modulator
interacting region. Ethiraj et al., (2012) also reported pyr-
azolines derived from methoxy-substituted naphthyl chal-
cones with potent cytotoxicity against HCT15, A549 and
HeLa, respectively. Thus, the numbers of reports on the
anticancer profile of N-acetyl pyrazolines along with their
synthetic accessibility and ease of synthesis led to their
inclusion within the designed hybrids chemical architecture.
Number of recent reports on cell killing potential of
Uracil-isatin conjugates (Kumar et al., 2012), triazole
tethered isatin conjugates (Singh et al., 2012), isatin–
With this background and in continuation of our ongo-
ing research on 3,5-diaryl-4,5-dihydro-pyrazolines based
conjugates (Singh et al., 2013), hybrids of the two func-
tionalities (Fig. 1) were synthesized and evaluated for
cytotoxic activity against four human cancer cell lines viz
HeLa (cervix cancer), CAKI-I (Renal cancer), PC-3
(Prostate cancer) and Miapaca-2 (pancreatic cancer). The
selection of triazole for tethering 3,5-diaryl N-acetyl pyr-
azoline (DNAP) and isatin was attributed to its moderate
dipole character, hydrogen bonding capability, rigidity and
stability under in-vivo conditions. (Kamal et al., 2008;
Wang et al., 2010; He et al., 2010; Kolb and Sharpless,
2003).
Results and discussion
In our efforts to discover potent DNAP-ISATIN anticancer
hybrids, we explored a series of DNAPs as cytotoxic
agents for their inclusion in the hybrids chemical archi-
tecture. The DNAPs were synthesized as per Scheme 1.
123

Med Chem Res
O
O
O
O
C
OCH3
OH
CH3
OCH3
OH
N
N
CH3
a
H
b
+
OCH3
OH
R
R
R
O
N
O
O
C
O
CH3
CH3
H
N
a
b
R
+
R
HO
HO
R
HO
Scheme 1 Reagents and conditions: a CH3COOH, H2SO4, stirring, 48 h; b NH2NH2.H2O, CH3COOH, 2 h, reflux
The DNAPs were evaluated for their anticancer activity
against four human cancer cell lines viz HeLa (cervix
cancer), CAKI-I (Renal cancer), PC-3 (Prostate cancer) and
Miapaca-2 (pancreatic cancer) using sulforhodamine B
assay. Among the series, only the potent pyrazolines were
selected for the synthesis of the target hybrids.
range of 5.3–11 lM). Among the cell lines employed,
HeLa cell line was the most sensitive towards the exposure
of test compounds. Out of all the compounds evaluated,
two right-handed conjugates MI-7b and MI-8b and two
left-handed conjugates MI-4b, MI-6b displayed significant
cytotoxicity and exhibited an IC50 value ranging from 1.3
to 3.5 lM against HeLa cell line. The compounds having
IC50 value more than 100 lM were considered inactive.
The most active hybrid i.e. MI-6b possessed an IC50 value
of 1.3 lM and displayed striking selectivity i.e. around 10
folds towards HeLa cell lines. MI-4b was the second most
potent hybrids of the series against HeLa cell lines with an
IC50 values of 1.7 lM. While most of the hybrids were
inactive against other cell lines, the hybrids with a propyl
chain were found to be endowed with moderate cytotox-
icity against other cell lines. MI-4b inhibited CAKI-I, PC-
3, Miapaca-2 cell lines with an IC50 value of 18, 37 and
67 lM. MI-6b inhibited CAKI-I, PC-3, Miapaca-2 cell
lines with an IC50 value of 14, 31 and 35 lM. MI-8b
inhibited CAKI-I, PC-3, Miapaca-2 cell lines with an IC50
value of 18, 27 and 38 lM (Table 2).
Table 1 clearly indicates that the N-acetyl pyrazolines
displayed significant cytotoxicity against PC-3 and HeLa
cell lines. Among the series, M-8 exhibited remarkable
cytotoxicity against HeLa cell lines with an IC50 value of
5.1 lM, and M-6 was endowed with significant potential
against PC-3 cell lines with an IC50 value of 8 lM. Overall,
five pyrazolines M-3, 4, 6, 7 and 8 were selected for the
inclusion in the hybrids chemical architecture. The pyraz-
olines lacking the methoxy groups and bearing halo groups
and nitro groups displayed poor cytotoxic profile. Keeping
in view, the results of the cytotoxic studies of pyrazolines,
we hypothesized that incorporating the structural features
of M-3, 4, 6, 7 and 8 would result in potent cytotoxic
hybrids.
The potent pyrazolines were propargylated for conden-
sation with isatin azides (Scheme 2).
A closure look into the structure activity relationship for
the inhibitory effects against HeLa cell lines indicated that
the length of the linker connecting the two functionalities
remarkably influences the cytotoxic activity. The cytotoxic
potential of the hybrids significantly improved on
increasing the length of the linker from n = 2 to n = 3 and
it was drastically decreased on increasing the chain length
i.e. n [ 3. Thus, the optimum length of the linker is n = 3
as hybrids with propyl chain (MI-4b, MI-6b, MI-7b, MI-
8b) exhibited potent cytotoxicity in addition to selectivity
towards the HeLa cell lines. All the hybrids with linker
length n [ 3 were almost inactive with most of them dis-
playing an IC50 value of [100 lM (mentioned as NA).
Apart from the length of linker, the substituents enhancing
the electron charge density at the periphery, i.e. placement
of methoxy phenyl rings and naphthyl ring, were found to
The target hybrids (MI) were synthesized by utilizing
regioselective azide-alkyne cycloaddition reaction of the
acetylenic DNAP and the isatin azides. Linker length was
varied from 2 to 6. The hybrids were classified as right-
handed and left-handed conjugates on the basis of the
placement of isatin ring (Scheme 3).
The synthesized hybrids (MI) were evaluated for their
anticancer activity against four human cancer cell lines viz
HeLa (cervix cancer), CAKI-I (Renal cancer), PC-3
(Prostate cancer) and Miapaca-2 (pancreatic cancer). The
results of the cytotoxicity studies are presented in Table 1.
It was observed that incorporation of DNAP in the hybrid
scaffold resulted in diminished cytotoxic effect against PC-
3 cell lines, whereas the DNAP (M-3, 4, 6, 7, 8) displayed
significant cytotoxicity against PC-3 cell line (IC50 value
123

Med Chem Res
Table 1 IC50 values of 3,5-diaryl N-acetyl pyrazolines
O
N
N
R1
R2
R5
R3
R4
Code
R1
R2
R3
R4
R5
IC50 (lM)
MiaPaca-2
HeLa
CAKI-I
PC-3
M-l
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
H
OH
65
74
21
10
97
7
NA
NA
76
13
21
11
9
83
M-2
M-3
M-4
M-5
M-6
M-7
Cl
OH
NA
57
OCH3
OH
NO2
OH
OH
OCH3
OH
53
42
OCH3
OCH3
NA
48
31
8
NA
31
OCH3
8
51
10
46
O
N N
OCH3
OH
M-8
M-9
M-10
OCH3
H
OCH3
OH
OCH3
OCH3
H
OH
H
5.1
18.9
81
21
5.3
17
32
H
H
38.7
NA
51
H
Br
OCH3
OH
29.3
NA
Bold values indicate potent compounds
Scheme 2 Reagents and
conditions: a Propargyl
bromide, K2CO3, DMF, stirring,
24 h
O
O
CH3
N
N
CH3
N
N
OCH3
O
a
OCH3
OH
R
R
O
O
CH3
CH3
N
N
N
N
a
R
R
O
HO
be beneficial in potentiating the cytotoxic activity. Among
the right-handed conjugates, MI-8b possessing a trimeth-
oxy phenyl ring and MI-7b possessing a naphthyl ring in
place of trimethoxy phenyl ring were the two active
hybrids. This further indicated that the naphthyl ring can be
a surrogate for the trimethoxy phenyl ring in the hybrid
scaffold; however, MI-8b was two folds more active than
MI-7b. Among the left-hand conjugates, MI-4b and MI-6b
were the potent hybrids. Both of them possessed methoxy
substituent/s at the periphery of the hybrid structure.
123

Med Chem Res
O
CH3
O
N
N
R
O
CH3
OCH3
O
N
N
O
N
i
OCH3
O
O
O
N
( )n
N
N
N
R
N3
Right handed conjugates
O
O
O
O
CH3
N
CH3
N
N
N
( )n
N
N
N
O
N
O
i
X
O
R
O
N
Left handed conjugates
DNAP-Isatin hybrids
N3
a = 2 (n), b = 3 (n), c = 4 (n), d = 5 (n), e = 6 (n)[alkyl chain length}
Scheme 3 (i) CuSO4Á5H2O, Sodium Ascorbate, DMF, stirring, 2 h
Doubly substituting the phenyl ring with methoxy groups
was found to be beneficial as MI-6b was found to be more
active than MI-4b. As far as the competition between the
right-hand and left-hand conjugates is concerned, the left-
hand conjugates were found to be more toxic to the HeLa
cells as compared to right-hand conjugates. Overall, the
MI-4b and MI-6b are good hits among the hybrids as they
are endowed with promising results and will be further
investigated in detail.
functionality of the hybrids were observed as important
structural prerequisites for the activity. Overall, the MI-4b
and MI-6b are good hits among the hybrids as they are
endowed with significant cytotoxicity. The potential of
these hybrids to selectively kill the HeLa cells makes them
promising candidates for detailed investigation. In future
both of them will be utilized as leads for the synthesis of
derivatives with diverse permutation and combination.
Experimental
Conclusion
The reagents were purchased from Sigma Aldrich, Merck,
CDH, Loba chem., Spectro chem., India and used without
further purification. All yields refer to isolated products
after purification. Products were characterized by spectral
Thus, in the present study, DNAP-Isatin bifunctional
hybrids were synthesized by a cycloaddition reaction via
click chemistry. The synthesized hybrids were evaluated
for their anticancer activity against four human cancer cell
lines viz HeLa (cervix cancer), CAKI-I (Renal cancer), PC-
3 (Prostate cancer) and Miapaca-2 (pancreatic cancer)
using sulforhodamine B assay. The most active hybrid i.e.
MI-6b possessed an IC50 value of 1.3 lM (HeLa Cell line)
and displayed striking selectivity i.e. around 10 folds
towards HeLa cell lines. MI-4b was the second most potent
hybrid of the series against HeLa cell lines with an IC50
values of 1.7 lM. Structure activity relationship revealed
that both electronic effects as well as length of the linker
remarkably influenced the cytotoxic activity of the hybrids.
The chain length i.e. n = 3 and substitution of methoxy
groups on the phenyl rings of N-acetyl pyrazoline
1
techniques; H NMR and 13C NMR spectra were recorded
on Bruker Advance II 400 NMR Spectrometer and JEOL
AL 300 NMR Spectrometer. The spectra were measured in
DMSO-d6 relative to TMS (0.00 ppm). Melting points
were determined in open capillaries and were uncorrected.
Procedure for the synthesis of 1,3-Diarylpropenones:
Concentrated sulphuric acid (2.5 ml) was added slowly to
the stirring solution of various substituted benzaldehydes
(1 mmol) and acetophenones (1 mmol) in glacial acetic
acid in a 500 ml round-bottom flask. The stirring was
continued for 48 h keeping the temperature of reaction
123

Med Chem Res
Procedure for the synthesis of O-propargylated DNAP
Table 2 In-vitro cytotoxic activity of hybrids against human cancer
cell lines
To the stirred suspension of K2CO3 (1.5 mmol) in dry
DMF, the pyrazole (1 mmol) was added. To the reaction
mixture, propargyl bromide (1.5 mmol) was added. The
reaction was kept on stirring at 0 °C under anhydrous
condition for 24 h. After completion of reaction, as evident
by TLC, the reaction mixture was poured onto crushed ice.
Precipitates were then filtered out. Propargylated pyrazole
was obtained in good yield.
Sample
Cell line
HeLa
n
CAKI-I
PC-3
MiaPaca-2
IC50
IC50
(lM)
IC50
(lM)
IC50
(lM)
(lM)
MI-3a
MI-4a
MI-6a
MI-7a
MI-8a
MI-3b
MI-4b
MI-6b
MI-7b
MI-8b
MI-3c
MI-4c
MI-6c
MI-7c
MI-8c
MI-3d
MI-4d
MI-6d
MI-7d
MI-8d
MI-3e
MI-4e
MI-6e
MI-7e
MI-8e
Adriamycin
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
48
NA
32
83
NA
NA
51.2
88
16.2
13.31
NA
18.21
31
47
38.4
NA
29
45.6
69.5
42.2
75
59.5
NA
67
Procedure for the synthesis of Isatin azide
NA
18
1.7
1.3
3.5
2.4
NA
39.7
36.2
NA
43
37
A mixture of 1-(bromo alkyl) indoline (1 mmol) in DMF
and sodium azide (1 mmol) was stirred for 2 h at room
temperature. After completion of reaction, as monitored by
TLC, reaction mixture was extracted with ethyl acetate.
Ethyl acetate layer was evaporated to get the desired
product in good yield.
14
31
35
21
NA
27
NA
38
18
30
NA
NA
NA
NA
32
NA
NA
NA
96
NA
NA
73
NA
NA
NA
NA
NA
NA
87
NA
NA
94
Procedure for the synthesis of DNAP-Isatin hybrids
90
90
NA
75
NA
82
To the stirred solution, O-propargylated DNAP (1 mmol)
and Isatin azide (1.5 mmol) in DMF, copper sulphate
(0.055 mmol) and sodium ascorbate (0.143 mmol) were
added in succession, at room temperature. On completion
of reaction, as monitored by TLC, the reaction mixture was
poured onto crushed ice. Precipitates were then filtered out.
Desired product was obtained in good yield.
NA
78
91
NA
NA
78
95
NA
NA
85
NA
94
NA
88
NA
78
NA
NA
93
NA
77
NA
NA
0.15
NA
76
The physical data of characteristic compounds are pro-
vided below.
NA
0.13
0.21
0.17
Bold values indicate potent compounds
1-(5-(4-((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-(4-methoxy-
phenyl)pyrazol-1-yl)ethanone (MI-3a) Yield: 71 %. mp
86–88 °C; 1H NMR (CDCl3, 300 MHz, d, TMS = 0): 7.68
(2H, d, J = 7.8. Hz), 6.93 (2H, d, J = 7.8 Hz), 6.51–6.90
(4H,m) 6.95–7.64 (4H, m), 5.52 (1H, bs), 5.17 (2H, s), 4.67
(2H, bs), 4.23 (2H, bs), 3.85 (3H, s), 3.81(3H, s), 3.69 (1H,
bs), 3.11 (1H, bs), 2.40 (3H, s). 13C NMR (CDCl3,
75 MHz, d, TMS = 0): 184.2, 168.4, 158.5, 153.4, 150.5,
148.6, 147.5, 147.5,146.4,146.4, 138.4, 137.9, 135.4, 128.
4, 125.4, 124.5, 123.5, 118.4, 114.5, 110.5, 61.6, 56.2, 46.6,
42.8, 40.7, 21.56. MS: m/z = 594.1524. Anal. Calcd. for
C32H30N6O6 : C, 64.64; H, 5.09; N, 14.13; O, 16.14, Found
C, 64.78; H, 5.01; N, 14.07.
mixture below 5 °C. The completion of the reaction was
monitored by TLC. After the completion, the reaction
mixture was poured over crushed ice and extracted thrice
with ethylacetate. The organic layer was pooled and con-
centrated under reduced pressure. The concentrated residue
was adsorbed on silica gel (60–120 mesh), and the desired
product was isolated by column chromatography employ-
ing increasing % age of ethylacetate in hexane.
Procedure for the synthesis of 3, 5 diaryl N-Acetyl
pyrazoline (DNAP)
1-(3-(4-((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-4,5-dihydro-5-(4-methoxyphenyl)pyr-
azol-1-yl)ethanone (MI-4a) Yield: 71 %. mp 87–92 °C;
1H NMR (DMSO-d6, 300 MHz, d, TMS = 0): 8.29 (1H,
s), 7.68 (2H, d, J = 8.2 Hz), 7.48 (2H, m), 7.03–7.09 (6H,
m), 6.83 (2H, d, J = 6.6 Hz), 5.48 (1H, bs), 5.11 (2H, s), 4.
A mixture of diarylpropenone (1 mmol) in acetic acid and
hydrazine hydrate (1 mmol) was refluxed for 2 h. The
mixture was then poured onto crushed ice to get crude
pyrazole, which was purified by crystallization from
methanol to afford the desired product.
123

Med Chem Res
71 (2H, bs), 4.19 (2H, bs), 3.76 (3H, s), 3.68 (1H, dd, bs),
3.11 (1H, bs), 2.31 (3H, s). 13C NMR (DMSO-d6, 75 MHz,
d, TMS = 0): 184.1, 168.2, 158.3, 153.4, 150.2, 148.4,
147.3, 147.4, 146.4, 146.3, 138.4, 137.7, 135.6, 128.2, 125.
1, 124.3, 123.2, 118.2, 114.1, 110.3, 99.9, 61.6, 56.2, 46.6,
42.8, 40.7, 21.5. MS: m/z = 565.1810 (M? ?. Anal. Calcd.
for C31H28N6O5 : C, 65.95; H, 5.00; N, 14.89; O, 14.17,
Found C, 65.73; H, 5.19; N, 14.73.
2, 125.3, 124.1, 123.1, 118.1, 114.3, 110.1, 61.2, 56.2, 54.4,
46.9, 42.2, 40.2, 21.2. Anal. Calcd. for C34H34N6O8 : C, 62.
38; H, 5.23; N, 12.84; O, 19.55, Found C, 62.73; H, 5.09;
N, 12.64.
The characterization data of rest of the compounds has
been provided as supplementary file.
Pharmacological evaluation
1-(3-(4-((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-4,5-dihydro-5-(3,4-dimethoxyphenyl)pyr-
azol-1-yl)ethanone (MI-6a) Yield: 75 %. mp 86–90 °C 1H
NMR (DMSO-d6, 300 MHz, d, TMS = 0): 8.07 (1H, s), 7.
68 (2H, d, J = 7.8 Hz), 7.48 (1H, m), 7.00–7.05 (4H, m),
6.68–6.85 (4H, m), 5.48 (1H, dd, J = 4.2 and 11.4 Hz), 5.
12 (2H, s), 4.71 (2H, bs), 4.13 (2H, bs), 3.77 (3H, s), 3.76
(3H, s), 3.70 (1H, dd, J = 11.4 and 17.7 Hz), 3.10 (1H, dd,
J = 4.2 and 17.7 Hz), 2.32 (3H, s). 13C NMR (DMSO-d6,
75 MHz, d, TMS = 0): 188.0, 172.3, 164.7, 163.2, 158.9,
155.3, 154.0, 153.1, 143.2, 140.1, 133.3, 129.6, 129.1, 128.
4, 122.4, 122.3, 120.0, 117.7, 115.4, 114.8, 66.3, 64.2, 60.7,
60.6, 52.2, 47.7, 44.4, 42.1, 26.9. MS: m/z = 594.1524.
Anal. Calcd. for C32H30N6O6 : C, 64.64; H, 5.09; N, 14.13;
O, 16.14, Found C, 64.93; H, 5.01; N, 13.99.
Hala (Cervix cancer), CAKI-I (Renal cancer), PC-3
(Prostate cancer) and MiaPaca-2 (Pancreatic Cancer) cell
lines were procured from the National Cancer Institute,
Frederick, USA. The cells were grown in RPMI-1640 med-
ium supplemented with 10 % heat inactivated foetal bovine
serum, penicillin (100 U/ml), streptomycin (100 lg/ml), L-
glutamine(0.3 mg/ml), pyruvic acid, 0.37 % NaHCO3 and
50 lM of 2-mercaptoethanol at 37 °C in an atmosphere of
95 % air and 5 % CO2 with 90 % humidity. Cells were
cultured in culture medium in a 75 cm2 tissue culture flask
(BD Falcon). Cell density was always kept under 1 9 106/ml
to avoid mutations of the cells. Passaging of the cells was
done four times a week. Experiments were performed with
cells cultivated not longer than twenty passages. Further
passaging of immortalized cell lines is possible but the risk of
genotypic changes increases. Cultivation and experimental
incubation were performed in a cell incubator at 37 °C, 90 %
air humidity and 5 % CO2. Cells grown in logarithmic phase
were treated with synthesized compounds dissolved in
DMSO, while the untreated control cultures received only
the vehicle (DMSO, \0.5 % v/v). The growth inhibitory
effect of different samples towards the Hala, CAKI-I, PC-3
and MiaPaca-2 cells was measured by means of MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bro-
mide) assay. The cleavage and conversion of the soluble
yellowish MTT to the insoluble purple formazan by active
mitochondrial dehydrogenase of living cells have been used
to develop an assay system for measurement of cell prolif-
eration (Kumar et al., 2013).
1-(5-(4-((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)3-methoxyphenyl)-4,5-dihydro-3-(naphthalen-
4-yl)pyrazol-1-yl)ethanone (MI-7a) Yield: 78 %. mp
83–86 °C; 1H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.08
(1H, d, J = 8.7 Hz), 7.89–7.94 (5H, m), 7.51–7.61 (4H,
m), 6.70 (4H, m), 6.50 (1H, d, J = 8.1 Hz), 5.57 (1H, dd,
J = 4.2 and 11.4 Hz), 5.16 (2H, s), 4.66 (2H, bs), 4.21 (2H,
bs), 3.90 (3H, s), 3.88 (1H, dd, J = 11.4 and 17.7 Hz), 3.10
(1H, dd, J = 4.2 and 17.7 Hz), 2.41 (3H, s). MS m/z: 616
(M?).13C NMR (CDCl3, 75 MHz, d, TMS = 0): 182.3,
168.4, 158.5, 154.0, 149.9, 149.7, 146.8, 138.7, 135.7, 134.
1, 133.0, 128.9, 128.5, 128.4, 127.8, 127.3, 127.3, 126.8,
125.6, 124.2, 123. 2, 117.6, 117.3, 114.3, 109.7, 109.4, 99.
9, 59.8, 55.9, 47.9, 42.3, 40.6, 22.0. MS: m/z = 615.2354
(M? ? 1) Anal. Calcd. for C35H30N6O5 C, 68.39; H, 4.92;
N, 13.67; O, 13.02, Found C, 68.69; H, 4.74; N, 13.87.
Conflict of interest The authors declare no conflicts of interest.
1-(5-(4-((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-(3,4,5-tri-
References
methoxyphenyl)pyrazol-1-yl)ethanone
(MI-8a) Yield:
1
Bode AM, Dong ZG (2009) Cancer prevention research—then and
now. Nat Rev Cancer 9:508
Ethiraj KR, Nithya P, Krishnakumar V, Mathew AJ, Khan F (2012)
Synthesis and cytotoxicity study of pyrazoline derivatives of
methoxy substituted naphthyl chalcones. Res Chem Intermed
39:1833–1841
Fadeyi OO, Adamson ST, Myles EL, Okoro CO (2008) Novel
fluorinated acridone derivatives. Part 1: synthesis and evaluation
as potential anticancer agents. Bioorg Med Chem Lett 18:
4172–4186
74 %. mp 90–92 °C, H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 7.90 (1H, s), 7.10–7.47 (4H, m), 6.99 (2H, s),
6.66–6.76 (3H,m), 5.53(1H,dd, J = 4.2 and 11.6 Hz), 5.02
(2H, s), 4.72 (2H, bs), 4.20 (2H, bs), 3.89 (6H, s,), 3.80
(6H, s), 3.69 (1H, dd, J = 11.4 and 17.7 Hz), 3.10 (1H, dd,
J = 4.2 and 17.7 Hz), 2.38 (3H, s). 13C NMR (DMSO-d6,
75 MHz, d, TMS = 0): 183.2, 169.1, 158.2, 153.3, 150.1,
148.4, 147.4, 147.2, 146.4,146.1, 138.3, 137.7, 135.2, 128.
123

Med Chem Res
He R, Chen Y, Chen Y, Ougolkov AV, Zhang JS, Savoy DN,
Billadeau DD, Kozikowski AP (2010) Synthesis and biological
evaluation of triazol-4-ylphenyl-bearing histone deacetylase
inhibitors as anticancer agents. J Med Chem 53:1347
Ramshid PK, Jagadeeshan S, Krishnan A, Mathew M, Nair SA, Pillai
MR (2010) Synthesis and in vitro evaluation of some isatin-
thiazolidinone hybrid analogues as anti-proliferative agents. Med
Chem 6:306
Hopkins AL (2008) Network pharmacology: the next paradigm in
drug discovery. Nat Chem Biol 4:682
Insuasty B, Montoya A, Becerra D, Quiroga J, Abonia R, Robledo S,
Rostom SAF (2006) Synthesis and in vitro antitumour evaluation of
some indeno [1,2-c]pyrazol(in)es substituted with sulfonamide,
sulfonylurea (-thiourea) pharmacophores, and somederived thi-
azole ring systems. Bioorg Med Chem 14:6475
´
Velez ID, Upegui Y, Nogueras M, Cobo J (2013) Synthesis of
novel analogs of 2-pyrazoline obtained from [(7-chloroquinolin-
4-yl)amino] chalcones and hydrazine as potential antitumor and
antimalarial agents. Eur J Med Chem 67:252–262
Sawyers C (2004) Targeted cancer therapy. Nature 432:94
Singh P, Sharma P, Anand A, Bedi PMS, Kaur T, Saxena AK, Kumar
V (2012) Azide-alkyne cycloaddition en route to novel 1H-1,2,3-
triazole tethered isatin conjugates with in vitro cytotoxic
evaluation. Eur J Med Chem 55:455
Singh J, Sharma S, Saxena AK, Nepali K, Bedi PMS (2013)
Synthesis of 1,2,3-triazole tethered bifunctional hybrids by
click chemistry and their cytotoxic studies. Med Chem Res
22:3160–3169
Solomon VR, Hua C, Lee H (2009) Hybrid pharmacophore design
and synthesis of isatin-benzothiazole analogs for their anti-breast
cancer activity. Bioorg Med Chem 17:7585
Solomon VR, Hua C, Lee H (2010) Design and synthesis of anti-
breast cancer agents from 4-piperazinylquinoline: a hybrid
pharmacophore approach. Bioorg Med Chem 18:1563
Viegas-Junior C, Danuell OA, da Silva BV, Barreiro EJ, Fraga CA
(2007) Molecular hybridization: a useful tool in the design of
new drug prototypes. Curr Med Chem 14:1829
Wang M, Xia Y, Fan Y, Rocchi P, Qu F, Iovanna JL, Peng L (2010) A
novel arylethynyltriazole acyclonucleoside inhibits proliferation
of drug-resistant pancreatic cancer cells. Bioorg Med Chem Lett
20:5979
Zhan P, Liu XY (2009) Designed Multiple Ligands: an Emerging
Anti-HIV Drug Discovery Paradigm. Curr Pharm Des 15:1893
Zhou Z, Zhuo J, Yan S, Ma L (2013) Design and synthesis of 3,5-
diaryl-4,5-dihydro-1H-pyrazoles as new tyrosinase inhibitors.
Bioorg Med Chem 21:2156–2162
Kamal A, Shankaraiah N, Devaiah V, Reddy KL, Juvekar A, Sen S,
Kurianb N, Zingdeb S (2008) Synthesis of 1,2,3-triazole-linked
pyrrolobenzodiazepine conjugates employing ‘click’ chemistry:
dNA-binding affinity and anticancer activity. Bioorg Med Chem
Lett 18:1468
Kolb HC, Sharpless KB (2003) The growing impact of click
chemistry on drug discovery. Drug Discov Today 8:1128
Kumar K, Sagar S, Esau L, Kaur M, Kumar V (2012) Synthesis of novel
1H-1,2,3-triazole tethered C-5 substituted uracil-isatin conjugates
and their cytotoxic evaluation. Eur J Med Chem 58:153
Kumar S, Mehndiratta S, Nepali K, Gupta MK, Koul S, Sharma PR, Saxena
AK, Dhar KL (2013) Novel indole-bearing combretastatin analogues
as tubulin polymerization inhibitors. Org Med Chem Lett 3:3
Manna F, Chimenti F, Fioravanti R, Bolasco A, Secci D, Chimenti P,
Ferlini C, Scambia G (2005) Synthesis of some pyrazole
derivatives and preliminary investigation of their affinity binding
to P-glycoprotein. Bioorg Med Chem Lett 15:4632–4635
Morphy R, Rankovic Z (2005) Designed multiple ligands. An
emerging drug discovery paradigm. J Med Chem 48:6523–6543
Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug
targets are there? Nat Rev Drug Discov 5:993
Petrelli A, Giordano S (2008) From single to multi target drugs in
Cancer therapy, when aspecifity becomes an advantage. Curr
Med Chem 15:422
123