Synthesis and anticancer activity of novel curcumin-quinolone hybrids

Article abstract of DOI:10.1016/j.bmcl.2015.06.068

A number of new curcumin-quinolone hybrids were synthesised from differently substituted 3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most potent compound 14, was analysed for its mode of action using various cell biology experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle progression in S and G2/M phase.

Full text of DOI:10.1016/j.bmcl.2015.06.068

Accepted Manuscript
Synthesis and Anticancer activity of Novel Curcumin-Quinolone Hybrids
Saiharish Raghavan, Prasath Manogaran, Krishna Kumari Gadepalli
Narasimha, Balasubramaninan Kalpattu Kuppusami, Palanivelu Mariyappan,
Anjana Gopalakrishnan, Ganesh Venkatraman
PII:
S0960-894X(15)00661-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.068
BMCL 22858
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2015
4 June 2015
19 June 2015
Please cite this article as: Raghavan, S., Manogaran, P., Gadepalli Narasimha, K.K., Kalpattu Kuppusami, B.,
Mariyappan, P., Gopalakrishnan, A., Venkatraman, G., Synthesis and Anticancer activity of Novel Curcumin-
Quinolone Hybrids, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.06.068
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Synthesis and Anticancer activity of Novel Curcumin-Quinolone Hybrids
Saiharish Raghavana, Prasath Manogaranb, Krishna Kumari Gadepalli Narasimhaa, Balasubramaninan Kalpattu
Kuppusamia,c, Palanivelu Mariyappana, Anjana Gopalakrishnanb and Ganesh Venkatramanb
aDepartment of Medicinal Chemistry, Sri Ramachandra University, Chennai – 600 116, India
bDepartment of Human Genetics, Sri Ramachandra University, Chennai – 600 116, India
cPresent Address: Department of Biotechnology, IIT-Madras, Chennai – 600 036, India
Keywords
Curcumin–Quinolone hybrids
MTT Assay
Annexin-V
Cell cycle analysis
Abstract
A number of new curcumin-quinolone hybrids were synthesised from differently substituted
3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel
of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most
potent compound 14, was analysed for its mode of action using various cell biology
experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology
under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE
assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle
analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle
progression in S and G2/M phase.
Curcumin (diferuloylmethane or (1E,6E)-1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione) 18 is currently acclaimed to be one of the most widely researched
naturally occurring chemopreventive agent and it has been subjected to several in vitro, in
vivo and clinical trial studies1, but it suffers from limitations2. In order to circumvent these
problems and to promote pharmacological properties, several modifications in aromatic ring
and linker chain of curcumin have been attempted and various analogues have been
prepared3. But synthesis of curcumin hybrids such as curcumin-diaminothiazole hybrid4 and
curcumin-thalidomide hybrid5 and evaluation of their anti cancer potential has opened up new
avenues for research6. 3-substituted-2-quinolone moiety is an important structural feature
present in numerous compounds with promising anti-cancer activities as they are found to be
tyrosine kinase inhibitors7. Cytotoxic activity of quinolone derivatives as non nucleosidic
MGMT inhibitors have been reported previously8. 4-quinolones were synthesised and
evaluated as potential antitumor topoisomerase I inhibitors9. 2-quinolone based chalcones
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

have been proven to display anti-tumour activity10. 2-phenyl-4-quinolones were reported to
exhibit inhibitory activity on several cell lines11. In line of these reports, in this study we have
synthesized new curcumin-quinolone hybrids from differently substituted 3-formyl-2-
quinolones and vanillin which retain important pharmacophores (α,β-unsaturated ketone
moiety and 2-quinolone moiety) to improve potency and selectivity. The proposed hybrids
may exhibit the cytotoxicity of curcumin as well as tumour antibiotic effect of 3-formyl-2-
quinolones.
The synthesis of these curcumin-quinolone hybrids 9-16 and diquinolone 17 are outlined in
Schemes 1 and 2 respectively. 3-formyl-2-quinolones 1-6 were prepared from corresponding
arylacetamides using Vilsmeier-Haack cyclization as per standard procedure12. These
quinolones were condensed with feruloyl acetone difluoroboronite complex (prepared from
feruloyl acetone13and BF3 etherate) to form difluoroboronite complexes of curcumin-
quinolone hybrids which were isolated and characterised by NMR studies. These
difluoroboronite complexes required prolonged refluxing in aqueous methanol for
decomplexation14. In order to reduce the reaction time, the reaction was tried in various other
solvents and different experimental conditions. But under microwave irradiation conditions in
aqueous methanol, not only the reaction was faster (3-5 minutes) but the yield was also
better. For improving the solubility and to facilitate the testing, nitrogen of 3-formyl-2-
quinolones was substituted with allyl, propargyl and benzyl groups. Quinolone hybrid 12
with propargyl substituent at nitrogen had better solubility and improved toxicity on all four
cell lines when compared to allyl and benzyl substituted quinolone hybrids 10 and 11. Hence
different hybrids were made with N-propargyl quinolone structural unit (13-16). All these
new compounds were characterised by NMR and HR-MS techniques.
Scheme 1. Synthesis of quinolone hybrids (9-16): a. (CH3CO)2O/ CH3COOH / 30 min / 50-600C; b. POCl3 /
DMF / 80-900C / 4-16 hrs; c. 4N HCl reflux / 6-8 hrs; d. Allyl bromide or propargyl bromide or benzyl bromide
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

/ DMF / K2CO3 / RT / 3-4 hrs; e. Feruloyl acetone difluoroboronite complex / n-BuNH2 / EA / RT / overnight; f.
Aq. methanol (MeOH : H2O 90:10) / Microwave / 3-5 min / 100-1500C
F
F
B
O
O
O
OH
a.
b.
N
O
N
H
O
O
N
H
N
H
O
O
N
H
H
Scheme 2. Synthesis of diquinolone 17. a. Acetylacetone difluoroboronite complex (prepared from
acetylacetone and BF3 etherate) / n-BuNH2 / EA / RT / overnight; b. Aq. methanol (MeOH : H2O 90:10) /
Microwave / 3-5 min / 100-1500C
3-formyl -2-quinolones (Figure 1) and curcumin-quinolone hybrids (9-16 in Figure 2) were
screened for cytotoxicity using MTT assay on a panel of representative cell lines and the
results are tabulated in Tables 1&2 respectively.
Figure 1. 3-formyl-2-quinolones
Table 1. List of parent quinolones synthesised and their IC50 values
Entry Compound No.
No.
R
RI
IC50 Values (µM)
A549
>100
>100
>100
>100
>100
>100
>100
>100
MCF-7
>100
>100
SKOV3
>100
>100
>100
>100
>100
>100
>100
>100
H460
>100
>100
>100
>100
>100
>100
>100
>100
1.
2.
3.
4.
5.
6.
7.
8.
1
2
3
4
5
6
7
8
-H
-H
-H
-CH2-CH=CH2
-CH2-C6H5
-H
-H
31.9 1.19
>100
>100
-CH2-C C-H
-CH2-C C-H
-CH2-C C-H
-CH2-C C-H
-CH2-C C-H
-Cl
-F
>100
>100
>100
-OCH3
-CH3
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

O
OH
HO
OH
O
O
Curcumin
18
O
OH
R
N
O
OH
RI
O
Curcumin-Quinolone hybrid
9-16
O
OH
N
H
O
O
N
H
Diquinolone
17
18
Figure 2. Curcumin ( ), Curcumin-Quinolone hybrids (
9-16 17
) and Diquinolone ( )
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

Table 2. List of curcumin-quinolone hybrids (9-16), diquinolone (17) and curcumin (18) synthesized and their
IC50 values
Entry
No.
Compound
No.
R
RI
IC50 Values (µM)
MCF-7 SKOV3
A549
H460
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
9
-H
-H
-H
*
*
*
*
10
11
12
13
14
15
16
17
18
-CH2-CH=CH2
-CH2-C6H5
-CH2-C C-H
-CH2-C C-H
-CH2-C C-H
-CH2-C C-H
-CH2-C C-H
-
31.3 2.18
*
23.5 2.69
*
26.0 2.60
*
**
-H
*
-H
21.3 1.51
20.62 1.18
23.9 2.5
40.45 2.9
**
15 1.56
>100
19 2.38
14.04 2.97
12.8 0.21
>100
25.4 1.5
38.14 0.81
21.75 0.55
44.3 1.45
**
-OCH3
-CH3
-F
36.2 1.99
25.0 2.3
**
-Cl
-
**
69.1 1.62
60.2 2.6
31.5 1.2
32.2 1.90
34.0 2.3
43.63 0.63
63.3 1.25
82.5 0.5
-
-
*Not active upto 10 µM and beyond that it is not soluble
** Not active upto 30 µM and beyond that it is not soluble
These results indicate dose dependant decrease in cell viability and the hybrids are more
potent than 3-formyl-2-quinolones and curcumin. The IC50 of each compound was calculated
using semi log curve fitting with regression analysis. Among the curcumin-quinolone
hybrids, compound 14 was found to show maximum activity against SKOV3 cells with the
least observed IC50 value of 12.8 µM and hence taken up for further biological experiments.
At IC50 concentration, the compound was not toxic to normal fibroblast cell line NIH3T3
with cell viability of 74.5%.
To observe the effect on cell morphology, SKOV3 cells were treated with 12.8 µM (IC50
value) of 14 for 24 hours. Phase contrast microscopy images of treated cells are shown in
Figure 3. Cell shrinkage and development of bubble like blebs on the membrane were
observed. Cells with ruptured cell membranes were also found.
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

0.1 mM
Cisplatin
Vehicle Control
12.8 µM Compound 14
Figure 3. Morphology of SKOV3 cells treated with vehicle control, compound 14 at IC50 and 0.1mM Cisplatin
treated cells as positive control
As the morphological changes observed were associated with apoptosis, Annexin V/PE
staining was carried out to confirm apoptosis as the mode of cell death. SKOV3 cells treated
with compound 14 were stained with Annexin V PE/7 AAD and quantified by flow
cytometry. Figure 4A shows the distribution of live, early & late apoptotic and dead cells on
treatment with 12.8µM compound 14 and compared with the vehicle control. It was therein
observed that 12.8µM of compound 14 significantly induced apoptosis in SKOV3 cells in 24
hours. Apoptosis analysis revealed that the percentage of late apoptotic cells was found to be
62.5% in compound 14 treated cells as compared to 9.0% in vehicle control. While the
percentage of early apoptotic cells reduced from 6.8% to 2.5%, the percentage of late
apoptotic cells was found to increase from 9.0% to 62.5%. In total, there were 72.3% of dead
cells in compound 14 treated cells while it was only 16.5% in vehicle control. Thus, we could
observe that compound 14 strongly induced apoptosis at its IC50 concentration on SKOV3
cells.
A comparison of the distribution of cells in various stages of apoptosis when treated with
12.8µM of compound 14 and vehicle control is shown in Figure 4B.
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

Figure 4. Compound 14 treated SKOV3 cells in various stages of apoptosis (A and B)
To examine how compound 14 triggers apoptosis, generation of ROS and the effects of
compound 14 on cell cycle machinery were investigated.
Studies have shown that generation of ROS after treatment with cytotoxic agents trigger
apoptosis15. The cytotoxicity of curcumin has also been attributed to generation of ROS,
where in approximately 1.5 fold increase in ROS generation was reported in Rh30 cells
within 1-2 hours of treatment with 10µM of curcumin16. In the present study, we analyzed
generation of ROS through flow cytometry assay using the dye DCFDA (dichloro fluorescein
diacetate). When cells were treated at IC50 concentration, there was an increase in ROS
production. As seen in Figure 5, 61.5% ROS generation was observed in cells treated with
12.8µM of compound 14 after 3 hours when compared to 20.9% in vehicle control
(approximately 3 fold). This confirms the generation of ROS as a mechanism of action for
this compound.
Figure 5. Percentage of ROS generated in SKOV3 cells treated with vehicle control (A) and IC50 concentration
of compound 14 (B)
Another mode by which apoptosis can be induced is by causing cell cycle arrest17. To
determine the effect of 14 on SKOV3 cell cycle, cell cycle analysis was performed by hypo
propidium iodide method. On treatment with 12.8µM of 14, there was an increase in the
number of cells in the S phase (19.49%) and G2/M phase (25.0%) of the cell cycle as well as
a significant reduction in number of cells in the G0/G1 phase (54.43%) as compared to
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

vehicle control which showed 11.4% of cells in S phase and 15.43% of cells in G2/M phase
and 72.52% of cells in G0/G1 phase of the cell cycle (Figure 6A). This indicates that
compound 14 induces cell cycle arrest in the S phase and G2/M phase rather than G0/G1
phase. These findings are in concordance with reported data wherein curcumin was found to
arrest cells in the G2/M phase from 12% in control to 33% at 50µM concentration in HT-29
cells and from 17% in control to 32% at 50µM concentration in HCT-15 cells18 as well as
from 9% in control to 28% at 10µM concentration in MCF-7 cells19 in 24 hours. A
comparison of the distribution of cells in various stages is shown in Figure 6B. All
experimental results were found to be statistically significant by Students t test with p<0.05.
Figure 6. A) Distribution of SKOV3 cells in various stages of the cell cycle on treatment with vehicle control
and IC50 concentration of compound 14. Region M2 represents G0/G1 phase, region M3 represents the S phase
and region M4 represents G2/M phase. B) Graphical representation of cell cycle analysis on SKOV3 cells
treated with compound 14. *VC= Vehicle control
In summary, compound 14 was found to be the most potent amongst the curcumin-quinolone
hybrids tested, inducing morphological changes like cell shrinkage and formation of blebs in
SKOV3 cells. ROS generation, S and G2/M phase arrest were found to be the mechanisms of
action of compound 14 on SKOV3 cells triggering apoptotic cell death. In the present study,
diquinolone 17 with seven carbon linker showed better cytotoxicity than diquinolone with
five carbon linker reported in the literature10 which can be attributed to the presence of β-
diketone moiety. The hybrids possessing one vanillin and one quinolone moiety linked by a
seven carbon chain showed better activity than both curcumin and diquinolone supporting the
concept of synthesising hybrids with active pharmacophores.
Acknowledgements
The authors thank SERB, Department of Science and Technology, Government of India for
funding the project (SR/S1/OC-64/2011), CRF, Sri Ramachandra University, Chennai for
NMR analysis, SAIF-IITM for HRMS analysis and Anju Chadha, Professor, Department of
Biotechnology, IIT-M for microwave facility.
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

References
1. Ji, JL.; Huang, XF.; Zhu, HL. Anticancer Agents Med Chem. 2012, 12(3), 210
2. Anand, P.; Thomas, SG.; Kunnumakkara, AB.; Sundaram, C.; Harikumar, KB.; Sung,
B.; Tharakan, ST.; Misra, K.; Priyadarsini, IK.; Rajasekharan, KN.; Aggarwal, BB.
Biochem. Pharmacol. 2008, 76(11), 1590
3. Mosley, CA.; Liotta, DC.; Snyder, JP. Adv. Exp. Med. Biol. 2007, 595, 77
4. Juneja, M.; Vanam, U.; Paranthaman, S.; Bharathan, A.; Keerthi, VS.; Reena, JK.;
Rajaram, R.; Rajasekharan, KN.; Karunagaran, D. Eur. J. Med. Chem. 2013, 63, 474
5. Liu, K.; Zhang, D.; Chojnacki, J.; Du, Y.; Fu, H.; Grant, S.; Zhang, S. Org. Biomol.
Chem. 2013, 11(29), 4757
6. Teiten, MH.; Dicato, M.; Diederich, M. Molecules. 2014, 19(12), 20839
7. Renhowe, PA.; Pecchi, S.; Shafer, CM.; Machajewski, TD.; Jazan, EM.; Taylor, C.;
Antonios-McCrea, W.; McBride, CM.; Frazier, K.; Wiesmann, M.; Lapointe, GR.;
Feucht, PH., Warne, RL.; Heise, CC.; Menezes, D.; Aardalen, K.; Ye, H.; He, M.; Le,
V.; Vora, J.; Jansen, JM.; Wernette-Hammond, ME., Harris, AL. J. Med. Chem. 2009,
52(2), 278
8. Ruiz, FM.; Gil-Redondo, R.; Morreale, A.; Ortiz, AR.; Fabrega, C.; Bravo, J. J.
Chem. Inf. Model. 2008, 48(4), 844
9. Shou, KJ.; Li, J.; Jin, Y.; Lv, YW. Chem. Pharm. Bull. 2013, 61(6), 631
10. Abonia, R.; Insuasty, D.; Castillo, J.; Insuasty, B.; Quiroga, J.; Nogueras, M.; Cobo. J.
Eur. J. Med. Chem. 2012, 57, 29
11. Chou, LC.; Tsai, MT.; Hsu, MH.; Wang, SH.; Way, TD.; Huang, CH.; Lin, HY.;
Qian, K.; Dong, Y.; Lee, KH.; Huang, LJ.; Kuo, SC. J. Med. Chem. 2010, 53(22),
8047
12. Srivastava, A.; Singh, RM. Indian. J. Chem., Sec B. 2005, 44B, 1868
13. Wichitnithad, W.; Nimmannit, U.; Wacharasindhu, S.; Rojsitthisak, P. Molecules.
2011, 16(2), 1888
14. Rao, EV.; Sudheer, P. Indian. J. Pharm. Sci. 2011, 73(3), 262
15. Circu, ML.; Aw, TY. Free Radical Biol. Med. 2010, 48(6), 749
16. Han, X.; Xu, B.; Beevers, CS .; Odaka, Y.; Chen, L.; Liu, L.; Luo, Y.; Zhou, H.;
Chen ,W.; Shen, T.; Huang, S. Carcinogenesis. 2012, 33(4), 868
17. DiPaola, RS. Clin. Cancer Res. 2002, 8, 3512
18. Hanif, R.; Qiao, L.; Shiff, SJ.; Rigas, B. J. Lab. Clin. Med. 1997, 130(6), 576
19. Simon, A.; Allais, DP.; Duroux, JL.; Basly, JP.; Durand-Fontanier, S.; Delage, C.
Cancer Lett. 1998, 129(1), 111
Corresponding Authors:a* Tel: 044-9444080916, E-mail address: drkrishna2011@gmail.com (G. N.
Krishna Kumari); b* Tel: 044-24765512, Ext: 237; E-mail address: ganeshv@sriramachandra.edu.in
(Ganesh V)

100
50
0
IC50=12.8µM
10
15
20
Concentration of compound 14…
Cytotoxicity to cancer cells
A549
Induces apoptotic cell death
MCF7
Curcumin- Quinolone hybrid
Increases ROS production
SKOV3
NCI-H460
Arrests cell cycle at S and G2/M phase