Synthesis, characterization and anti-tumor activity of novel thymoquinone analogs against pancreatic cancer

Article abstract of DOI:10.1016/j.bmcl.2013.03.003

Thymoquinone (TQ), isolated from the seeds of Nigella sativa, show moderate efficacy against pancreatic cancer. In the present work we report synthesis and characterization of novel TQ analogs appended with gallate and fluorogallate pharmacophores and eva

Full text of DOI:10.1016/j.bmcl.2013.03.003

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3101–3104
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization and anti-tumor activity of novel
thymoquinone analogs against pancreatic cancer
Mujahid Yusufi a, , Sanjeev Banerjee b, , Momin Mohammad c, Sandhya Khatal d,
K. Venkateswara Swamy d, Ejazuddin M. Khan a, Amro Aboukameel b, Fazlul H. Sarkar b,c,
,
⇑
Subhash Padhye a,b,
⇑
a ISTRA, Department of Chemistry, Abeda Inamdar College, University of Pune, Azam Campus, Pune 411001, India
b Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA
c Department of Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA
d Department of Bioinformatics and Computer Science, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune 411044, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thymoquinone (TQ), isolated from the seeds of Nigella sativa, show moderate efficacy against pancreatic
cancer. In the present work we report synthesis and characterization of novel TQ analogs appended with
gallate and fluorogallate pharmacophores and evaluation of their effects against pancreatic cancer cell
lines for cell viability and induction of apoptosis. The efficacy of the analogs alone or in combination with
Gemcitabine was assessed in vitro. LC–MS spectra of ATQTHB and ATQTFB showed major peaks corre-
sponding to expected M+1 fragment at 316.34 and 322.34 respectively. Molecular docking studies
revealed good fit for these analogs in the COX-2 protein cavity with better binding energies compared
to parent TQ compound. Present TQ analogs exhibit superior anti-proliferative activity, excellent
chemo-sensitizing activity against pancreatic cancer in vitro and in combination with Gemcitabine.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 9 January 2013
Revised 25 February 2013
Accepted 1 March 2013
Available online 14 March 2013
Keywords:
Thymoquinone analogs
Molecular Docking
Pancreatic cancer
Thymoquinone
Chemosensitisation
Gemcitabine
Pancreatic cancer is the fourth most common cancer with a five
year survival rate of less than 3% amongst afflicted patients. In Uni-
ted States, approximately 43,920 new cases of pancreatic cancer
are expected to be diagnosed in 2012, with 37,390 deaths antici-
pated.1 The high rate of mortality associated with this disease is
in part due to indolent nature of tumor growth presenting metas-
tasis at time of diagnosis as well as, intrinsic and drug induced
resistance to commonly used chemotherapies. As a consequence,
disease-free survival time even after complete resection of the tu-
mor and adjuvant treatments is less favourable. Since, currently
available cytotoxic chemotherapeutic agents and other modalities
of treatment have been found to be inadequate there is a dire need
to develop novel translational drugs from clinical perspective for
the treatment of pancreatic cancer.
reported in literature for use in cancer therapy.3,5,6 However, de-
spite limited attempts originating from various laboratories, none
of the analogs have proven efficacious as monotherapy or as ther-
apeutic adjunct against pancreatic cancer.
In our previous endeavor, we have synthesized di-substituted
benzoquinones bearing structural similarity with TQ which have
shown promising results against pancreatic cancer cell lines.7 In
the same study we also reported that TQ exhibits growth inhibition
and chemo-sensitization to standard chemo drugs like Gemcita-
bine and Oxaliplatin against chemo-resistant pancreatic cancer cell
line-MiaPaCa-2 (7). Recently, Effenberger et al. have reported ter-
pene conjugates of TQ which have been found to be more potent
than TQ against human HL-60 leukemia, multidrug-resistant KB-
V1/Vbl cervix, 518A2 melanoma and MCF-7/Topo breast cancer
cells.8 The 4-acylhydrazones and 6-alkyl analogs of TQ have also
been found to be inhibitory against MCF-7/Topo breast cancer cells
and HL-60 leukemia cells.9
In order to broaden the scope and propensities of these activi-
ties it is necessary to prepare different analogs of TQ through mod-
ification of its carbonyl functionalities or through nucleophilic
additions of desirable groups and linkages. However, such modifi-
cations have not provided significant therapeutic advantage. Tak-
ing clue from our previous efforts where we showed that the
Thymoquinone (TQ, 2-methyl, 5-isopropyl 1,4-benzoquinone)
isolated from the seeds of Nigella sativa has been shown to exhibit
anti tumor activity against breast, lung, prostate, liver, colon and
pancreatic cancer.2–4 We recently summarized the biological basis
and therapeutic activities of TQ and reviewed existing analogs
⇑
Corresponding authors. Tel.: +91 8390025533 (S.P.).
E-mail address: sbpadhye@hotmail.com (S. Padhye).
These authors contributed equally.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.003

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M. Yusufi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3101–3104
gallate pharmacophore and its fluorinated analog when appended
to chalcones and curcumin enhance the anticancer effect of the
parent compounds by several folds, we were motivated to combine
these pharmacophores with TQ.10,11 Conceptually, these may be-
have as novel and potent agents against pancreatic cancer because
of the retention and accompanying pleiotropic effects exhibited by
TQ. In the present work we have employed a different strategy by
synthesizing 3-aminothymoquinone (ATQ) as the initial building
block and then synthesizing its Schiff base derivatives ATQTHB
and ATQTFB with 2,3,4-trihydroxybenzaldehyde and 2,3,4-triflu-
orobenzaldehyde respectively.
The starting building compound, viz. 3-amino Thymoquinone
(ATQ) was synthesized with the procedure described by Moore
and co-workers with slight modifications.12 A mixture of TQ
(1 mmol, 0.164 g) and sodium azide (1.3 mmol, 0.084 g) in ethanol
was refluxed for 3 h in the presence of 3 ml of glacial acetic acid
(Fig 1). The compounds ATQTHB and ATQTFB were synthesized
by condensation of equimolar quantities of ATQ (0.179 mg,
1 mmol) and 2,3,4-tri-hydroxy and 2,3,4-trifluoro benzaldehyde
respectively in absolute alcohol in presence of 0.5 ml of concen-
trated hydrochloric acid as catalyst with continuous stirring at
75 °C for 4 h (Fig. 1). The reaction mixture was poured on crushed
ice and the resulting precipitate was filtered, washed with water
and purified by Column chromatography. The details of synthesis
and spectroscopic characterization are provided in Supplementary
material. The LC-MS spectra of ATQTHB and ATQTFB (Fig. 2A–D)
showed major peaks corresponding to expected M+1 fragment at
316.34 (with retention time of 2.08 min) and 322.34 (with reten-
tion time of 2.77 min) respectively. The IR spectra of ATQ showed
an intense carbonyl band at 1647 cmÀ1 and two medium intensity
peaks at 3254 and 3310 cmÀ1 due to N–H stretches of the amino
group. ATQTHB exhibited quinone carbonyl stretch in the range
1637–1639 cmÀ1 while the azomethine band was observed in the
region 1595–1599 cmÀ1. The broad band at 3375–3495 cmÀ1 was
assigned to hydroxyl groups on the side chain. ATQTFB compound
showed the carbonyl band at 1637 cmÀ1 while the azomethine
band was observed in the range 1556–1564 cmÀ1. A distinct band
observed at 1058 cmÀ1 was due to C–F stretching vibration.16 The
1H NMR spectrum of parent ATQ compound showed proton of the
amino group at 6.42 ppm which was found to be absent in the con-
densed Schiff base products (ATQTHB and ATQTFB). Additionally
appearance of the azomethine hydrogen in the region 9.0–
9.18 ppm confirmed the Schiff Base formation. The aromatic
hydrogen atoms were located in the range of 6.9–8.0 ppm. In case
of ATQTFB downfield values of aromatic hydrogen atoms were due
to the presence of fluorine and aromatic signals appearing as mul-
tiplets were due to ortho- and meta-coupling of neighbouring
hydrogen or fluorine atoms with coupling constants in the range
of 7, 2 and 14, 11 Hz respectively. On the other hand the protons
of hydroxyl groups in ATQTHB appeared as three singlet in the
range of 10.20–11.42 ppm. The 13C NMR of ATQ exhibited signals
from the aliphatic carbons in the range of 8.59–25.84 ppm, olefinic
carbon atoms at 106.6–148.9 ppm and quinone carbonyl carbons
at 183.6 and 184.7 ppm respectively. In both these compounds
the aliphatic carbon signals were found in the range of 9.9–
10.1 ppm, while the olefinic and aromatic carbons atoms appeared
in the range of 102–149 ppm. The azomethine and carbonyl car-
bons were observed in the range of 150–162.7 ppm.17
The COX pathway is known to elicit growth related signals and
regulate cell proliferation contributing to development of pancre-
atic cancer.18–23 The majority of human pancreatic carcinomas
show over-expression of COX-2, while their benign counterpart
lack this expression24–26 suggesting that these tumors can be tar-
geted selectively through COX pathway. The COX-2 inhibitor Cele-
coxib has been shown to reduce pancreatic cancer growth in
animal models and humans.27–30 Arafat and co-workers31 have
shown that TQ induced apoptosis and inhibited proliferation in
pancreatic ductal adenocarcinoma cells as well as synthesis of
MCP-1, TNF-a, interleukin (IL)-1beta and COX-2 in dose- and
time-dependent manner. TQ has also been shown to exert an
O
O
H
O
O
R1
R1 Conc. HCl
CH3COOH
R2
R3
+
+
NaN3
N
Reflux, 3 Hrs.
Reflux, 4 Hrs.
R2
NH2
O
O
ATQ
R3
O
TQ
ATQTHB R1=R2=R3 = OH
ATQTFB R1=R2=R3 = F
Figure 1. Schematic representation of synthesis of 3-amino-TQ and its Schiff Base analogs.
Figure 2. (A and B) LC–MS spectra of ATQTHB. (C and D) LC–MS spectra of ATQTFB.

M. Yusufi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3101–3104
3103
Figure 3. Binding of TQ, ATQTHB and ATQTFB into active site of COX-2 as assessed by molecular docking studies.
TQ, ATQTFB and ATQTHB compounds when docked into the
active site of COX-2 were found to give a good fit confirming
that modification of the parent compound with additional phar-
macophore does not introduce any major steric change in the TQ
moiety except allowing additional hydrogen bonding interactions
(Fig. 3, Table 1). The best binding energy was exhibited by
ATQTHB (À8.1 kcal/mol) followed by ATQTFB (À7.9 kcal/mol)
compared with TQ (À6.50 kcal/mol) respectively. TQ undergoes
hydrogen bonding interactions with three amino acid residues,
viz. SER 530 (3.1 Å), MET 522 (2.7 Å) and VAL 523 (3.2 Å) respec-
tively in the COX cavity via its quinone carbonyl group. Similar
H-bonding interactions are seen for ATQTHB (HIS 388, 2.7 Å)
and ATQTFB (TYR 115, 3.5 Å, SER 119, 3.2 Å, TYR 355, 3.0 Å)
respectively. These interactions lead to stabilization of the com-
pounds in the protein cavity. Based on these considerations,
compound ATQTHB has better stability in the COX-2 protein
cavity than parent TQ molecule and anticipated to show more
enhanced anticancer activity.
Table 1
Docking results and consensus scores of synthesized TQ, ATQTHB and ATQTFB
analogs
Compounds
Binding energy
(Kcal/mol)
No. of
H bonds
Residues
Bond
distance (Å)
TQ
À6.8
3
SER 530
MET 522
VAL 523
HIS 388
TYR 115
SER 119
TYR 355
3.1
2.7
3.2
2.7
3.5
3.2
3.0
ATQ-THB
ATQ-TFB
À8.1
À7.9
1
3
anti-inflammatory effect in a mouse model of ovalbumin-induced
allergic airway inflammation by inhibiting COX-2 protein expres-
sion.32 Hence, we have conducted a molecular docking study of
TQ and its derivatives in the COX-2 protein cavity by using Auto-
13–15
dockVina.
Figure 4. Cytotoxic activity of ATQTHB and ATQTFB against (A) MiaPaCa-2 and (B) BxPC-3 cell lines (⁄p <0.05), (C) histone DNA–ELISA for apoptosis detection in MiaPaCa-2
cells (⁄p <0.05), (D) Chemosensitisation to Gemcitabine by the analogs—ATQTHB and ATQTFB in MiaPaCa-2 cells.

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M. Yusufi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3101–3104
The anticancer potency of the synthesized analogs was evalu-
Further studies are in progress to delineate precise molecular mech-
anism relating to their anti-proliferative and pro-apoptotic effects.
ated in two pancreatic MiaPaCa-2 and BxPC-3 cancer cell lines.
The results obtained revealed differential sensitivity of the com-
pounds towards cell viability. ATQ and ATQTFB exhibited either
no significant effect or an effect similar to TQ at equimolar concen-
trations in BxPC-3 cell line that we investigated (Fig. 4B). In con-
trast, ATQTHB was promising amongst all; at equimolar
concentration of TQ, against MiaPaCa-2 cell line and it showed
>50% enhancement in the loss of cell viability compared to parental
TQ (Fig. 4A). Similar results were also noted in BxPC-3 cells
(Fig. 4B). This loss in cell viability, paralleled with apoptosis induc-
tion which was confirmed by Histone DNA ELISA in MiaPaCa-2
cells using ATQTFB and ATQTHB (Fig. 4C). Results revealed a trend
similar to cell viability results showing a significant increase in
apoptotic cells which was more pronounced in case of ATQTHB
treated cells than TQ at equivalent concentration (Fig. 4C). To
examine our hypothesis whether the Gemcitabine resistant Mia-
PaCa-2 cells when pre-treated with the synthesized ATQTHB or
ATQTFB analogs could be more sensitive to the cytotoxic effect
of Gemcitabine, we followed schedule of ATQTHB and ATQTFB
pre-treatment protocol wherein the effect of only ATQTHB
Acknowledgments
S.P. would like to acknowledge Mr. P. A. Inamdar, President,
MCES, Pune, for the encouragement. We are thankful to NMR Re-
search Centre, Indian Institute of Science, Bangalore for providing
NMR spectral data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
003.
References
1. Siegel, R.; Naishadham, D.; Jemal, A. CA Cancer J. Clin. 2012, 62, 10.
2. Banerjee, S.; Padhye, S.; Azmi, A.; Wang, Z.; Philip, P. A.; Kucuk, O.; Sarkar, F. H.;
Mohammad, R. M. Nutr. Cancer 2010, 62, 938.
3. Padhye, S.; Banerjee, S.; Ahmad, A.; Mohammed, R.; Sarkar, F. H. Cancer Ther.
2008, 6, 495.
4. Woo, C. C.; Kumar, A. P.; Sethi, G.; Tan, K. H. Biochem. Pharmacol. 2012, 83, 443.
5. Tageja, N.; Padhye, S.; Dandawate, P.; Al-katib, A.; Mohammed, R. J. Hematol.
Oncol. 2009, 2, 50.
6. Dandawate, P. R.; Vyas, A. C.; Padhye, S. B.; Singh, M. W.; Baruah, J. B. Mini-Rev.
Med. Chem. 2010, 10, 436.
7. Banerjee, S.; Azmi, A. S.; Padhye, S.; Singh, M. W.; Baruah, J. B.; Philip, P. A.;
Sarkar, F. H.; Mohammad, R. M. Pharm. Res. 2010, 27, 1146.
8. Effenberger, K.; Breyer, S.; Schobert, R. Chem. Biodivers. 2010, 7, 129.
9. Breyer, S.; Effenberger, K.; Schobert, R. Chem. Med. Chem. 2009, 4, 761.
10. Padhye, S.; Yang, H.; Jamadar, A.; Cui, Q. C.; Chavan, D.; Dominiak, K.;
McKinney, J.; Banerjee, S.; Dou, Q. P.; Sarkar, F. H. Pharm. Res. 2009, 26, 1874.
11. Padhye, S.; Ahmad, A.; Oswal, N.; Dandawate, P.; Rub, R. A.; Deshpande, J.;
Swamy, K. V.; Sarkar, F. H. Bioorg. Med. Chem. Lett. 2010, 20, 5818.
12. Moore, H. W.; Shelden, H. R. J. Org. Chem. 1968, 33, 4019.
13. Sadowski, J.; Gasteiger, J.; Klebe, G. J. Chem. Inf. Comput. Sci. 1994, 34, 1000.
14. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455.
(2.5
lM), Gemcitabine alone (0.5
l
M),33 and ATQTHB/ATQTFB
pre-treatment (2.5
l
M; 24 h) followed by Gemcitabine treatment
(72 hrs) on viability of MiaPaCa-2 cells evaluated by MTT
(Fig. 4D). Based on our data we conclude that sub-toxic dose of
ATQTFB did not exert any significant chemosensitizing effect on
the investigated cell lines. On the contrary we found treatment
of cells with Gemcitabine alone (for 72 h) caused >50% (p <0.05)
loss of MiaPaCa-2 cells viability (Fig. 4D). However, pre-treatment
of cells with ATQTHB for 24 h followed by treatment with the cyto-
toxic agent Gemcitabine for 72 h resulted in a significant loss of
viable cells (<80%; p <0.001) in the MiaPaCa-2 cell line indicating
that ATQTHB sensitizes resistant MiaPaCa-2 cells to the cytotoxic
effect of Gemcitabine.
15. The PyMOL Molecular Graphics System, Schrödinger, LLC.
16. Mahadevan, D.; Periandy, S.; Ramalingam, S. Spectrochim. Acta, Part A 2011, 84,
86.
Over the years, the quest to explore and improve existing tra-
ditional phytochemicals for the treatment and prevention of can-
cer has driven the development of novel analogs with improved
anticancer therapeutic effecacy without toxicity to normal cells.
Several pre-clinical studies that have been reported reveal that
TQ exhibits multi-targeted pleiotropic effects associated with
chemoprevention and chemosensitisation of cancer. Previously,
we have demonstrated that TQ exhibited chemosensitisation ef-
fects in Gemcitabine and Oxaliplatin.2 The effect was attributed
to the inactivation of DNA binding activity of NF-kB, resulting
in the inactivation of multiple downstream survival molecules.
In the present study we have synthesized new TQ analogs that
reduce cell viability of pancreatic cancer cells by stimulating tu-
mor cells to undergo apoptosis. Of interest, compared to parental
compound TQ, one of the synthesized analog (ATQTHB) presents
a much superior and significant inhibition of viable cells at equi-
molar concentration of TQ. Although yet to be explored, we pro-
pose that our analogs sensitize tumor cells to Gemcitabine by
suppressing the pro-survival and pro-angiogenic molecule COX-
2.
17. Rozwadowski, Z.; Ambroziak, K.; Szypa, M.; Jagodzin´ ska, E.; Spychaj, S.; Schilf,
W.; Kamien´ ski, B. J. Mol. Struct. 2005, 734, 137.
18. Ding, X. Z.; Hennig, R.; Adrian, T. E. Mol. Cancer 2003, 2, 10.
19. Rioux, N.; Castonguay, A. Carcinogenesis 1998, 19(8), 1393.
20. Shureiqi, I.; Lippman, S. M. Cancer Res. 2001, 61, 6307.
21. Steele, V. E.; Holmes, C. A.; Hawk, E. T.; Kopelovich, L.; Lubet, R. A.; Crowell, J.
A.; Sigman, C. C.; Kelloff, G. J. Cancer Epidemiol. Biomarkers Prev. 1999, 8, 467.
22. Subbaramaiah, K.; Dannenberg, A. J. Trends Pharmacol. Sci. 2003, 24, 96.
23. Dannenberg, A. J.; Altorki, N. K.; Boyle, J. O.; Dang, C.; Howe, L. R.; Weksler, B.
B.; Subbaramaiah, K. Lancet Oncol. 2001, 2, 544.
24. Okami, J.; Yamamoto, H.; Fujiwara, Y.; Tsujie, M.; Kondo, M.; Noura, S.; Oshima,
S.; Nagano, H.; Dono, K.; Umeshita, K.; Ishikawa, O.; Sakon, M.; Matsuura, N.;
Nakamori, S.; Monden, M. Clin. Cancer Res. 1999, 5, 2018.
25. Kokawa, A.; Kondo, H.; Gotoda, T.; Ono, H.; Saito, D.; Nakadaira, S.; Kosuge, T.;
Yoshida, S. Cancer (Philadelphia) 2001, 91, 333.
26. Tucker, O. N.; Dannenberg, A. J.; Yang, E. K.; Zhang, F.; Teng, L.; Daly, J. M.;
Soslow, R. A.; Masferrer, J. L.; Woerner, B. M.; Koki, A. T.; Fahey, T. J. Cancer Res.
1999, 59, 987.
27. El-Rayes, B. F.; Ali, S.; Sarkar, F. H.; Philip, P. A. Mol. Cancer Ther. 2004, 3, 1421.
28. Arjona-Sánchez, A.; Ruiz-Rabelo, J.; Perea, M. D.; Vázquez, R.; Cruz, A.; Muñoz
Mdel, C.; Túnez, I.; Muntané, J.; Padillo, F. J. Pancreatology 2010, 10, 641.
29. Lipton, A.; Campbell-Baird, C.; Witters, L.; Harvey, H.; Ali, S. J. Clin.
Gastroenterol. 2010, 44, 286.
30. Dragovich, T.; Burris, H., 3rd.; Loehrer, P.; Von Hoff, D. D.; Chow, S.; Stratton, S.;
Green, S.; Obregon, Y.; Alvarez, I.; Gordon, M. Am. J. Clin. Oncol. 2008, 31, 157.
31. Chehl, N.; Chipitsyna, G.; Gong, Q.; Yeo, C. J.; Arafat, H. A. HPB (Oxford) 2009, 11,
373.
32. El Mezayen, R.; El Gazzar, M.; Nicolls, M. R.; Marecki, J. C.; Dreskin, S. C.;
Nomiyama, H. Immunol. Lett. 2006, 106, 72.
In conclusion, we have synthesized and structurally character-
ized novel analogs of thymoquinone which exhibit potent anti-pro-
liferative activities against pancreatic cancer cell lines which open
up the possibilities of optimizing application of these analogs for
translational research. Amongst these analogs, ATQTHB showed
superior sensitization of pancreatic cancer under in vitro system.
33. Li, Y.; VandenBoom, T. G.; Kong, D.; Wang, Z.; Ali, S.; Philip, P. A.; Sarkar, F. H.
Cancer Res. 2009, 69, 6704.