Synthesis and antiproliferative activity of some 3-(pyrid-2-yl)-pyrazolines

Article abstract of DOI:10.1039/c3md00077j

The synthesis and antiproliferative activity of eleven 3-(pyrid-2-yl)- pyrazolines in two cancer cell lines are reported. X-ray crystallography was obtained of the lead compound 8i which was screened in the NCI 60 human tumour cell line and displayed sub-micromolar activity. Cell cycle analysis, in vitro tubulin assay and confocal microscopy are also reported and suggest that the lead compound disrupts microtubule formation.

Full text of DOI:10.1039/c3md00077j

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Synthesis and antiproliferative activity of some 3-(pyrid-
2-yl)-pyrazolines†
Cite this: DOI: 10.1039/c3md00077j
Alexander Ciupa,a Paul A. De Bank,a Mary F. Mahon,b Pauline J. Wooda
a
*
and Lorenzo Caggiano
The synthesis and antiproliferative activity of eleven 3-(pyrid-2-yl)-pyrazolines in two cancer cell lines are
reported. X-ray crystallography was obtained of the lead compound 8i which was screened in the NCI
60 human tumour cell line and displayed sub-micromolar activity. Cell cycle analysis, in vitro tubulin
assay and confocal microscopy are also reported and suggest that the lead compound disrupts
microtubule formation.
Received 6th March 2013
Accepted 23rd April 2013
DOI: 10.1039/c3md00077j
www.rsc.org/medchemcomm
Me) and the corresponding oxidised pyrazole derivative, which
Introduction
are “turn on” uorescent sensors for Cd2+ and Zn2+, respec-
tively.19 Our interests in pyrazolines derived from 2-acetylpyr-
idine 3 originated from possible chelation of ions in these
systems by the pyridine nitrogen, the basic pyrazoline nitrogen
(N2) and co-ordinating groups (R2, Scheme 1). Compounds of
this structure have been reported as ligands for gold and display
antiproliferative activities.20 In addition, the 3-pyridyl motif
would afford compounds with improved pharmacological
properties. N1-Substituted 3-(pyrid-2-yl)-pyrazolines display
various biological activities, including antitubercular,21 anti-
mycobacterial,22 antifungal,23 anti-inammatory24,25 and anti-
proliferative20,24 activities. We therefore wished to investigate
novel compounds of this class and examine their anti-
proliferative activity.
Numerous pyrazoline derivatives possess potent biological
activities,1,2 including antiproliferative,3–6 antimalarial7 and
antibacterial activities.8 The inhibition of various processes
such as xanthine oxidase,9 COX-2,10 monoamine oxidase11–17
and, of particular relevance to the current study, tubulin poly-
merisation18 have also been reported (selected examples are
shown in Fig. 1).
We have previously reported the synthesis and physico-
chemical properties of the simple pyrazoline 8a (R1 ¼ H, R2 ¼
Fig. 1 Examples of biologically active pyrazolines 1 and 2.
aMedicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath, BA2 7AY, UK. E-mail: l.caggiano@bath.ac.uk; Fax: +44 (0)1225
386114; Tel: +44 (0)1225 385709
bX-ray Crystallography Unit, Department of Chemistry, University of Bath, Claverton
Down, Bath, BA2 7AY, UK
† Electronic supplementary information (ESI) is available: Experimental
procedures, characterisation data, 1H and 13C NMR spectra, HPLC traces and
MTS assays for all compounds. In addition, NCI data and cell cycle analysis of
compound 8i together with X-ray crystallography data for pyrazolines 8e and 8i
are provided. CCDC 926530 and 926531. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c3md00077j
Scheme 1 Synthesis of 3-(pyrid-2-yl)-pyrazolines 8 and 9 (see Table 1 for R2
groups and yields).
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pyrazoline series 8, the N–Me derivative 8a displayed weak
activity, which improved two-fold with the N–Ph analogue 8b
(entries 2 and 3). The introduction of an N-acetyl group (8c),
which provides additional Lewis basic sites, resulted in partial
loss of activity although it did exhibit some selectivity for the
HT29 cell line (entry 4). The triuoroacetyl analogue 8d, which
has a similar steric requirement as the acetyl but is a much less
effective Lewis base, resulted in complete loss of anti-
proliferative activity in both cell lines (entry 5).
The carbothioamide 1 shown in Fig. 1 displayed potent
antiproliferative activity4 and similar compounds also show
promise.5,6 The related derivative 8e was previously reported to
exhibit activity as a gold complex with HeLa and A549 cancer
cell lines.20 As the free ligand, however, the carbothioamide 8e
gave poor inhibition of cell growth in the HT29 and MDA-MB-
231 cell lines, while the monomethyl analogue 8f exhibited
much improved activities for both cell lines (entries 6 and 7,
Table 1).
X-ray crystallography of the carbothioamide 8e indicated a
putative intramolecular H-bond to the basic pyrazoline nitrogen
(shown in Fig. 2) and an intermolecular H-bond with the pyri-
dine nitrogen (shown in the ESI†). Likewise, the monomethyl
derivative 8f could also possess intramolecular co-ordination to
the remaining N–H. Although these interactions are present in
the solid state, in aqueous solution competing intermolecular
H-bonding to the solvent will undoubtedly occur, affording
different conformations such as that observed in co-ordination
with gold.20 To investigate further we attempted to generate the
dimethyl analogue 8g, as this would remove the interaction
altogether. Attempts to obtain pyrazoline 8g either by methyl-
ation of the carbothioamide 8e or 8f, or by reaction of the N–H
Results and discussion
Continuing our research interests in pyrazolines derived from 2-
acetylpyridine 3, we synthesised various derivatives following
the sequence shown in Scheme 1. Claisen–Schmidt condensa-
tion of 2-acetylpyridine 3 with an aromatic aldehyde gave the
corresponding chalcone 4 (97%) or 5 (85%) in excellent yields.
Treatment with an excess of hydrazine hydrate gave the N–H
pyrazoline derivative 6 or 7 which could be characterised by 1H
NMR. Attempts to purify these compounds by silica gel column
chromatography however led to decomposition, as was previ-
ously observed with similar substrates,22,23,26 possibly due to
autoxidation.27 Instead, the N–H pyrazolines 6 and 7 were
treated directly with the desired electrophile to afford the N-
substituted pyrazolines 8 and 9 which could be puried, in a
one-pot transformation in good yields.
The N–Me and N–Ph substituted pyrazolines 8a,b and 9b
were synthesised by reaction of the chalcone 4 or 5 with the
commercially available substituted hydrazine NH2NHMe or
NH2NHPh, affording the corresponding products 8a (72%),19 8b
(49%) or 9b (34%) directly ((iv), Scheme 1). Low yields with
arylhydrazines have been previously reported,26 and similar
transformations suggest that the reaction proceeds via initial
1,2-addition followed by cyclisation and not 1,4-conjugate
addition and subsequent cyclisation, which could generate
different isomers.19,28
Compounds were examined for antiproliferative activity in vitro
using the MTS assay29 with human colon carcinoma (HT29) and
highly metastatic human breast carcinoma (MDA-MB-231) cell
lines. The results are reported as the concentration required to
inhibit 50% cell proliferation (IC50) and are shown in Table 1.
The unsubstituted chalcone 4 has been reported to display
modest antiproliferative activity in various cancer cell lines.30
Likewise, the 3,4,5-trimethoxy derivative 5 showed good activity
in both the cell lines examined in this study (entry 1, Table 1). As
can be seen in Table 1, the R2 substituent had a great effect on
the antiproliferative activity of the pyrazolines. In the C5-phenyl
pyrazoline
6 with dimethylthiocarbamoyl chloride, were
however unsuccessful as previously observed in similar
substrates.26
The effect of an N-benzoyl substituent (8h) was also investi-
gated and exhibited no observable antiproliferative activity in
the two cell lines examined (entry 8, Table 1). Following our
Table 1 Yields and antiproliferative activities against human colon (HT29) and breast (MDA-MB-231) cancer cell lines
IC50a (mM)
Entry
Comp.
R1
R2
Yieldb (%)
HT29
MDA-MB-231
1
2
3
4
5
6
7
8
5
8a
8b
8c
8d
8e
8f
8h
8i
9b
9c
9i
3,4,5-OMe
H
H
H
H
H
H
H
—
Me
Ph
Ac
85
72
49
72
65
67
89
76
75
34
80
84
9.6 ꢀ 0.9
9.7 ꢀ 4.3
115.8 ꢀ 11.6
53.8 ꢀ 3.94
161.3 ꢀ 21.9
>500
135 ꢀ 31.2
60.2 ꢀ 19.6
294.1 ꢀ 71.1
>500
CF3C¼O
NH2C¼S
350.2 ꢀ 42.0
25.6 ꢀ 4.0
>500
2.53 ꢀ 0.39
26.2 ꢀ 3.4
>500
140.5 ꢀ 42.0
20.7 ꢀ 0.79
>500*
0.69 ꢀ 0.10
4.36 ꢀ 0.85
52.5 ꢀ 6.5
11.4 ꢀ 0.75
0.008 ꢀ 0.001
NHMeC¼S
Bz
9
H
3,4,5-Trimethoxy-benzoyl
Ph
Ac
10
11
12
13
3,4,5-OMe
3,4,5-OMe
3,4,5-OMe
3,4,5-Trimethoxy-benzoyl
22.5 ꢀ 2.50
Colchicinec
0.008 ꢀ 0.001
a
IC50 is the concentration that inhibits 50% cell proliferation. Values are the mean from three independent experiments, except * (one), with
quadruplicate readings in each. b Yield over two steps from the chalcone (steps (ii) and (iii), Scheme 1). c Colchicine was used as a positive control.
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Table 2 NCI 60 cell line screen of pyrazoline 8i (NSC 761258)
Panel
Cell line
GI50a (mM)
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
HOP-62
2.08
0.747
4.46
66.3
0.415
0.952
>100
1.82
Non-small cell
Lung
HOP-92
>100
1.33
3.45
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
>100
0.505
0.688
0.585
2.32
0.586
0.848
0.431
0.711
0.567
33.4
Colon
Fig. 2 X-ray crystal structures of pyrazolines 8e and 8i with ellipsoids repre-
sented at 30% probability.‡
CNS
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
1.06
1.89
previous studies using 3,4,5-trimethoxyphenyl substituents,31
which have been shown to play crucial roles in activity,32–36 we
investigated this substitution pattern in the N-benzoyl group.
Remarkably, the corresponding 3,4,5-trimethoxybenzoyl deriv-
ative 8i displayed potent activity and selectivity for the highly
metastatic human breast carcinoma (MDA-MB-231, entry 9).
The X-ray crystal structure of pyrazoline 8i was also obtained
and is shown in Fig. 2. As with pyrazoline 8e, it shows that in the
solid state the amide adopted the E-conformation.
Continuing our interest in the 3,4,5-trimethoxyphenyl motif,
we investigated the effect of this substitution pattern in three
examples in the pyrazoline series 9. In all three examples, the
compounds exhibited increased potency for the MDA-MB-231
cell line, as previously observed with derivative 8i which also
possesses the 3,4,5-trimethoxyaryl motif. The N–Ph derivative
9b was more active in both cell lines than the corresponding
C5-phenyl analogue 8b (entries 10 and 3, Table 1). Surprisingly,
the N–Ac derivative 9c showed no activity in HT29 (>500 mM), yet
exhibited improved activity and selectivity for MDA-MB-231
(52.5 mM) compared to pyrazoline 8c (entries 11 and 4).
0.277
0.868
1.13
>100
0.801
0.273
0.699
1.51
0.432
>100
0.541
7.13
0.719
5.95
Melanoma
Ovarian
Renal
4.01
3.28
0.519
0.762
1.54
0.943
4.29
0.493
1.03
3.53
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
>100
5.01
>100
4.26
0.324
0.989
9.18
5.13
>100
0.417
‡ Both pyrazolines 8e and 8i were racemic, but single compounds are shown to
represent the structures obtained by X-ray crystallography. Crystal data for
pyrazoline 8e, CCDC 926530. Formula: C15H14N4S1. M ¼ 282.36, monoclinic.
Prostate
Breast
˚
˚
˚
Unit cell parameters: a ¼ 9.7950(2) A, b ¼ 14.7280(3) A, c ¼ 10.0360(2) A. a ¼
MCF7
3
ꢁ
ꢁ
ꢁ
˚
90 , b ¼ 107.768(1) , g ¼ 90 , V ¼ 1378.74(5) A , T ¼ 150(2) K, space group
P21/n, Z ¼ 4, 24456 reections collected, 3152 independent reections [R(int) ¼
0.0669]. Final R indices [I > 2s(I)] R1 ¼ 0.0392 and wR2 ¼ 0.0883. R indices (all
data) R1 ¼ 0.0608 and wR2 ¼ 0.0979. Crystal data for pyrazoline 8i, CCDC
926531. Formula: C24H23N3O4. M ¼ 417.45, orthorhombic. Unit cell parameters:
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
ꢁ
ꢁ
ꢁ
˚
˚
˚
a ¼ 6.9770(1) A, b ¼ 22.0950(2) A, c ¼ 26.6010(3) A. a ¼ 90 , b ¼ 90 , g ¼ 90 ,
a
3
GI50 is concentration required to inhibit growth by 50% as dened by
the National Cancer Institute (NCI).
˚
V ¼ 4100.73(8) A , T ¼ 150(2) K, space group Pbca, Z ¼ 8, 56599 reections
collected, 4679 independent reections [R(int) ¼ 0.0645]. Final R indices [I >
2s(I)] R1 ¼ 0.0422 and wR2 ¼ 0.0882. R indices (all data) R1 ¼ 0.0649 and wR2
¼
0.0991.
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Fig. 3 Cell cycle analysis. (A) HT29 cells only, (B) HT29 + colchicine (100 nM), (C)
HT29 + 8i (1 mM), and (D) HT29 + 8i (5 mM).
Fig. 5 Confocal microscopy showing tubulin, Dm1a (green) and DNA, DAPI
(blue). (A) HT29 cells only, (B) HT29 + colchicine (100 nM) and (C) HT29 + 8i
(5 mM).
led to cell cycle arrest in the G2/M phase, shown in Fig. 3B. In
the presence of pyrazoline 8i at 1 mM (Fig. 3C) and 5 mM
(Fig. 3D), 50% and 95% respectively of the cells also arrested in
G2/M phase, suggesting that pyrazoline 8i affects microtubule
polymerisation.
To examine this interaction an in vitro tubulin polymerisation
assay was performed in the presence of pyrazoline 8i (20 mM) or
paclitaxel (5 mM), which is known to polymerise microtubule
formation. As can be seen in Fig. 4, the presence of paclitaxel led
to an increased optical density (OD) compared to the control
experiment, due to increased microtubule formation. The pres-
ence of pyrazoline 8i however gave a lower OD reading than the
control, implying that pyrazoline 8i inhibits microtubule poly-
merisation and is consistent with the cell cycle analysis.
Confocal microscopy was performed on HT29 cells to visualise
the effects of pyrazoline 8i in microtubule formation, using Dm1a
tubulin antibodies (Fig. 5). The effects of 100 nM colchicine,
which inhibits microtubule polymerisation, can be seen in
Fig. 5B, which contains much less tubulin compared to the
control experiment (Fig. 5A). Likewise, compared to the control
there is less tubulin present in cells treated with 5 mM pyrazoline
8i (Fig. 5C), which again supports the data obtained from the cell
cycle analysis and in vitro tubulin polymerisation assay that pyr-
azoline 8i disrupts microtubule formation. The results are
consistent with previous reports of similar compounds which
were also shown to interfere with microtubule assembly.18
Fig. 4 In vitro tubulin polymerisation assay. Paclitaxel (5 mM) and pyrazoline 8i
(20 mM).
Incorporating the 3,4,5-trimethoxy-substitution in both the C5-
aryl and N1-benzoyl positions of the pyrazoline ring (9i) did not
further improve activity compared to compound 8i (entries 12
and 9). On the basis of these preliminary investigations,
compound 8i was selected for further evaluation by the National
Cancer Institute (NCI) and the results are shown in Table 2.
Pyrazoline 8i (NSC 761258) displayed promising GI50 values
across the NCI 60 human tumour cell line panel and exhibited
sub-micromolar activity in a range of cancer cell lines, including
the multidrug resistant ovarian cell line NCI/ADR-RES (0.519 mM).
Also of interest is that one of the most susceptible ovarian cancer
cell lines, OVCAR-3, is particularly sensitive to tubulin disruptors,37
suggesting that pyrazoline 8i may be inhibiting cell proliferation
through the disruption of tubulin. Although COMPARE analysis of
the NCI 60 cancer cell line data did not afford strong correlations
with compounds of known biological activity, drugs which inhibit
the polymerization of tubulin did feature (see ESI†).
To investigate potential interaction with tubulin dynamics,
cell cycle analysis was performed with HT29 cells with pyrazo-
line 8i and colchicine, a known inhibitor of microtubule poly-
Conclusions
merisation. Cell cycle analysis was performed three times and In conclusion, we report the two-step synthesis of some 3-(pyrid-
the results shown in Fig. 3 are representative of the complete 2-yl)-pyrazolines and their antiproliferative activities. The lead
study (shown in the ESI†). The presence of 100 nM of colchicine pyrazoline 8i was selected from this preliminary study and
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¨
˘
¨
further investigated against the NCI 60 cancer cell line (NSC 12 N. Gokhan-Kelekçi, S. Yabanoglu, E. Kupeli, U. Salgin,
¨
¨
761258) and exhibits sub-micromolar activity in a number of
cell lines. The mode of action was explored by cell cycle analysis,
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in vitro microtubule assembly assay and confocal microscopy 13 A. Sahoo, S. Yabanoglu, B. N. Sinha, G. Ucar, A. Basu and
with a-tubulin antibody Dm1a which suggest that the novel
compound 8i inhibits microtubule polymerisation.
V. Jayaprakash, Bioorg. Med. Chem. Lett., 2010, 20, 132–
136.
The promising antiproliferative activity of 3-(pyrid-2-yl)-pyr- 14 F. Chimenti, A. Bolasco, F. Manna, D. Secci, P. Chimenti,
`
azoline 8i and the ease of its synthesis provide the potential for
further development, which is currently under investigation
and will be reported in due course.
O. Befani, P. Turini, V. Giovannini, B. Mondovı, R. Cirilli
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15 F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti,
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Acknowledgements
16 F. Chimenti, A. Bolasco, F. Manna, D. Secci, P. Chimenti,
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We wish to thank Dr Timothy J. Woodman, Dr Anneke Lubben
and Dr Adrian Rogers (University of Bath) for their assistance
with the NMR, mass spectra and confocal spectroscopy,
respectively. We also wish to thank Dr Andrew Chalmers for
the antibodies used in the confocal experiments and to Cancer
Research at Bath (CR@B) for funding the confocal imaging.
We are extremely grateful to the National Cancer Institute
(NCI) for conducting the in vitro testing and the University of
Bath for providing a studentship for AC. We also wish to
acknowledge RCUK and the University of Bath for the fellow-
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