Chem Biol Drug Des 2014

Research Article

Newly Designed and Synthesized Curcumin Analogs

with in vitro Cytotoxicity and Tubulin Polymerization
Activity
Iten M. Fawzy1, Khairia M. Youssef1, been focused on developing new chemotherapies with
Nasser S. M. Ismail2, Joachim Gullbo3 and minimal side-effects on mammalian cells. Natural products
Khaled A. M. Abouzid2,* have been found to be a source of novel and potent bio-
active compounds with minimal side-effects in vivo (1).
1
Pharmaceutical Chemistry Department, Faculty of
Pharmaceutical Sciences and Pharmaceutical Industries, Extensive research conducted within the past years revealed
Future University, Cairo 12311, Egypt that curcumin (1) [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
2
Pharmaceutical Chemistry Department, Faculty of heptadien-3,5-dione] (Figure S1) (2) extracted from the rhi-
Pharmacy, Ain Shams University, Cairo 11566, Egypt zome of the plant Curcuma longa modulates and interacts
3
Division of Clinical Pharmacology, Department of Medical
with a diverse range of molecular targets, and hence, it pos-
Sciences, Uppsala University Hospital, SE-751 85
Uppsala, Sweden sess antiproliferative activities against tumor cells in vitro, anti-
*Corresponding author: Khaled A. M. Abouzid, inflammatory, antibacterial, antiviral, antihepatotoxic,
[email protected] hypotensive, and anticholesterolemic activities (2–8). As can-
cer is a result of the dysregulation of multiple cell signaling
Novel curcumin analogs with 4-piperidone ring were pathways, curcumin’s multitargeting ability may be the key to
designed, synthesized, and evaluated for their its therapeutic potential against cancer. Reported structural
cytotoxic activities against five different cancer activity relationship studies performed on curcumin analogs
cell lines. 3,5-bis(4-Hydroxy-3-methoxybenzylidene)-4- highlighted the critical sites for developing novel analogs
oxo-N-phenylpiperidine-1-carbothioamide (XIIe) exhib- where 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore (2)
ited considerable cytotoxic activity with IC50 values in was found to be very important in activity of the compound
1–2.5 lM range. In silico and in vitro, studies were also as this group reacts with cellular constituents at a specific
performed to predict the binding affinity of the target
binding site (primary binding site A) (Figure S2) (9).
compounds to the b-chain of tubulin receptor (PDB
code 1SA1) and their abilities to affect microtubules
polymerization cycle. 3,5-bis(3-Iodo-5-methoxy-4-prop- This binding may be influenced by the nature of the
oxybenzylidene)-N-acetylpiperidin-4-one (VIIa) was group attached to the heterocyclic nitrogen atom where
found to exert 93.3% inhibition of tubulin and destabili- charged molecules were found to be unable to penetrate
zation of microtubules in vitro compared to vincristine cell membranes and exert a cytotoxic effect, and
while, 3,5-bis(3,4,5-trimethoxybenzylidene)-N-benzoylpi- hence, N-acylation was considered a route to render the
peridin-4-one (XIIc) showed high potency in a differ- nitrogen atom in a non-basic form (9,10). Recent
ent way where it exerted 94.8% stabilization of research also revealed that chalcones (3) (Figure S3),
microtubules in vitro compared to positive control curcumin, and their derivatives possess cytotoxic activity
paclitaxel. associated with tubulin inhibition and interference with
microtubule formation, which is essential in cellular pro-
Key words: curcumin analogs, cytotoxicity, molecular model- cesses such as mitosis and cell replication. It was
ing, tubulin polymerization assay found that curcumin exerted inhibition to tubulin inside
Plasmodium falciparum cells with IC50 = 5 lM (11). Also
Received 19 July 2014, revised 11 September 2014 and benzylidene curcumin derivative (4) (Figure S4) was found
accepted for publication 15 October 2014 to be more effective than curcumin in inhibiting tubulin
self-assembly (12), while chalcone derivative (Figure S5A)
exerted high tubulin inhibition activity compared to podo-
Cancer is a life-threatening disease and a leading cause of phyllotoxin (Figure S5B) (13).
death. Although a range of therapies based on chemother-
apy, surgery, and radiotherapy are available, yet they are According to all the previous findings in the literature,
of limited efficacy. Moreover, current anticancer regimens several modifications were applied to curcumin analogs
are associated with significant levels of toxicity and serious to develop novel curcumin analogs with cytotoxic activity
adverse side-effects. Hence, many research projects have

ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12464 1

Fawzy et al.

through targeting tubulin and inhibiting microtubules poly-

merization (13) where esterification and etherification of
the aromatic hydroxyl groups took place to increase the
hydrophobicity of the target compounds. Also, it was
noticed that methylene group in the curcumin structure is
unstable in acidic medium (14), so cyclization through the
use of 4-piperidone ring was applied. Besides, it was
found in the literature that oxygen atom in 4-piperidone
ring forms a bond with N-H terminal in protein sequence,
and so it was kept in the structure of target compounds.
N-terminal in 4-piperidone ring was acylated to render
targets non-basic, and hence, ease of penetration of cel-
lular membranes also some targets were synthesized as
thiourea derivatives as it was found in the literature that
the phenyl isothiocyanate group is responsible for thio-
carbomylation of thiol group of glutathione inside the cells
and hence leads to depletion of glutathione from cancer-
ous cells that leads to apoptosis (15,16). Conjugated
characteristics were kept in the structure of the target
compounds or elongated as it was found to be very
Scheme 1: Synthesis of 3,5-bis(4-Acyloxy-3-methoxybenzylidene)-
essential in activity (17). N-acylpiperidin-4-one (IVa-d). 1Reagents and conditions: i: acyl
chloride + triethylamine (5 °C), ii: 4-piperidone.HCl + conc. HCl, iii:
acyl chloride + triethylamine (5 °C). [R = -CH3].
Chemistry

Analogs IVa–d, VIa and VIIa,b, IXa and Xa–c, and XIIa–
e which represent four different series of compounds were
designed and synthesized with 1,5-diaryl-3-oxo-1,4-penta-
dienyl core moiety. Reported intermediates IIa–d of series
1, Va,b of series 2, and XIa,c,d of series 4 were prepared
according to reported procedures (18–25). The syntheses
of the target compounds IVa–d of series 1 follow two-step
procedure. First, condensation of the intermediates IIa–d
with 4-piperidone.HCl in acidic medium (26) yielded IIIa–d
that are pure enough to enter the final acylation step,
which was carried out using acetyl chloride or benzoyl
chloride (10) to give the corresponding target compounds
IVa–d as in (Scheme 1).

As for series 2, the target compounds VIa and VIIa,b

were synthesized by applying Claisen–Schmidt condensa-
tion reaction procedure (26) where the intermediates Va,b
were condensed with 4-piperidone.HCl in acidic medium.
VIa was obtained after neutralization and crystallization.
Then both VIa and the pure mixture VIb were acetylated
in a reaction catalyzed with triethylamine to obtain the final
compounds VIIa,b (Scheme 2).

To synthesize the final target compounds of series 3 (IXa Scheme 2: Synthesis of 3,5-bis(3-Methoxy-5-non-substituted/
iodo-4-propoxybenzylidene/allylidene) piperidin-4-one and
and Xa-c), thiation reaction was applied on the starting
3,5-bis(3-Methoxy-5-non-substituted/iodo-4-propoxybenzylidene/
materials Id,e in the presence of strong catalyst, then the allylidene)-N-acetylpiperidin-4-one (VIa and VIIa,b). 2Reagents
pure prepared mixture VIIIa,b entered the Claisen– and conditions: i: Na metal/reflux + alkyl chloride in ethanol/
Schmidt condensation reaction with 4-piperidone.HCl to reflux, ii:4-piperidone.HCl + conc. HCl, iii: acyl chloride +
obtain the target IXa and the mixture IXb which were pure triethylamine (5 °C). [R = -CH3].
enough to be acetylated in the presence of triethylamine
yielding the final target compounds Xa,b. As for the target Compounds XIIa–d of series 4 were prepared via reacting
Xc, it was prepared by reacting IXa with phenyl isothiocy- the intermediates XIa–d with acetyl chloride or benzoyl
anate in triethylamine under reflux (Scheme 3). chloride in triethylamine to yield the corresponding final

2 Chem Biol Drug Des 2014

 Curcumin Analogs

compounds XIIa–d. Target XIIe of series 4 was prepared Synthesis of reported intermediates
through different pathway where 4-piperidone.HCl was
reacted with phenyl isothiocyanate in triethylamine under 4-Acyloxy-3-methoxybenzaldehyde (IIa–d) General
reflux followed by condensation of the product with Ia in procedure
the presence of acidic medium to obtain XIIe (Scheme 4). 0.03 Moles of the appropriate acyl chloride and (0.06
moles, 6 mL) of triethylamine were dissolved in 10 mL
chloroform and added dropwise at 5 °C to solution of
Experimental (0.02 moles, 3 g) of vanillin ‘Ia’ in 5 mL chloroform then
stirred for 2 h at room temperature.
Materials and instrumentation
Starting materials and reagents were purchased from 4-Formyl-2-methoxyphenyl butyrate (IIa). Yield:
Sigma-Aldrich (Stockholm, Sweden). Melting points were 87%; melting point (104–105 °C) (18).
recorded on Stuart Scientific apparatus. 1H-NMR spectra
were recorded on a Varian Mercury VX-300 NMR spec- 4-Formyl-2-methoxyphenyl propionate (IIb). Yield:
trometer (International Equipment Trading Ltd, Vernon 66%; melting point (114–115 °C) (18, 19).
Hills, IL, USA). 13C-NMR spectra were recorded on a Var-
ian Mercury VX-300 NMR spectrometer. MS spectra Tricyclo[3.3.1.13,7]decane-1-carboxylic acid, 4-formyl-
mass were recorded on Shimadzu GCMS-QP 5050A gas 2-methoxyphenyl ester (IIc). Yield: 71%; melting
chromatograph mass spectrometer (70 eV). Elemental point (118–119 °C) (20).
analyses were performed at The Regional Center for
Mycology and Biotechnology Al-Azhar University, Cairo, 4-Formyl-2-methoxyphenyl heptanoate (IId). Yield:
Egypt. In vitro antiproliferative assay and tubulin polymeri- 75%; melting point (163–164 °C) (21).
zation assay were performed at Uppsala University Hospi-
tal, division of Clinical Pharmacology, department of
Medical Sciences, Uppsala, Sweden. One compound Methoxy-5-(unsubstituted/iodo)-4-
(XIIb) was tested for its cytotoxic activity by the American propoxybenzaldehyde/cinnmaldehyde (Va,b)
National Cancer Institute. General procedure
0.02 Moles of the appropriate available start Ib,c was ref-
luxed with (0.02 moles, 46 mg) sodium metal in (15 mL)
ethanol for one hour. The product was washed with
water and filtered and then alkylation reaction was per-
formed by the addition of chloroform solution of propyl
chloride (0.02 moles, 1.5 mL) to 0.02 moles of the acti-
vated sodium salt of Ib,c. The whole reaction mixture
was refluxed for further one hour to obtain the corre-
sponding intermediate Va,b.

Scheme 3: Synthesis of 3,5-bis(3-Substituted-4-mercaptoben

zylidene/allylidene) piperidin-4-one and 3,5-bis(3-Substituted-4-
mercaptobenzylidene/allylidene)-N-substituted piperidin-4-one (IXa Scheme 4: Synthesis of 3,5-bis(Substituted benzylidene)-N-
and Xa-c). 3Reagents and conditions: i: P2S5, CS2, conc. HCl, ii: substituted-piperidin-4-one (XIIa-e). 4Reagents and conditions:
4-piperidone.HCl, conc. HCl, iii: acyl chloride, triethylamine (5 °C), i: 4-piperidone.HCl, conc. HCl, ii: acyl chloride, triethylamine
iv; Phenylisothiocyanate, triethylamine (reflux). (5 °C), iii: N-phenylisothiocyanate-4-piperidone, conc. HCl.

Chem Biol Drug Des 2014 3

Fawzy et al.

1-Iodo-5-methoxy-4-propoxybenzaldehyde NMR (300 MHz, DMSO-d6): d 7.61 (d, 2H, J = 6.5 Hz, 2
(Va). Yield: 71%; melting point (148 °C) (22). [-CH=C]), 7.59 (d, 2H, J = 7 Hz, ArH), 7.56 (d, 2H,
J = 7 Hz, ArH), 7.31–7.36 (m, 2H, ArH), 5.05(q, 4H,-
3-Methoxy-4-propoxycinnamaldehyde (Vb). Yield: CH2NCH2), 3.85 (s, 6H, 2[OCH3]), 2.57 (t, 4H, J = 7.5 Hz,
36%; mp (164 °C) (23). 2[CO-CH2-CH2-CH3]), 2.29 (s, 3H, COCH3), 1.74–1.64 (m,
4H, J = 6 Hz, 2[CO-CH2-CH2-CH3]), 0.99 (t, 6H,
J = 7.5 Hz,2[CO-CH2-CH2-CH3]). Ms: (MW: 549.00): m/z
3,5-bis(Substituted benzylidene)-piperidin-4-one
549 (M+, 8.64%), 350.25 (100%). Anal. Calc. for
(XIa-d) General procedure C31H35NO8: C, 67.74; H, 6.42; N, 2.55; O, 23.29, Found:
Condensation of 0.02 moles of Ia,d–f dissolved in 6 mL C, 67.81; H, 6.48; N, 2.62.
chloroform with (0.01 mole, 1.5 g) of 4-piperidone.HCl
was carried out in presence of 2 mL conc. HCl, and the 3,5-bis(3-Methoxy-4-propyloyloxybenzylidene)-N-
mixture was stirred for 2 h at room temperature, then left benzoylpiperidin-4-one (IVb). The titled compound
for 2 days. The reaction mixture was neutralized and the was separated as yellow oily liquid, yield: 65.78%, boil-
chloroform layer was extracted with chloroform ing point: 199–204 °C. FT-IR (ύ max, cm 1): 3010 (Ar
(3 9 10 mL) and evaporated under vacuum. C-H), 2890 (C-H), 1750 (ketonic C=O), 1735 (ester C=O),
1700 (Ar C=C-), 1680 (C=C-) and 1679 (NH-C=O). 1H
3,5-bis(4-Hydroxy-3-methoxybenzylidene) piperidin-4- NMR (300 MHz, DMSO-d6): d 7.98 (d, J = 6 Hz, 2H,
one (XIa). Yield: 65%; melting point (254–256 °C) (24). ArH), 7.95 (d, 2H, 2[-CH=C]), 7.69 (t, 1H, ArH), 7.66 (t,
1H, ArH), 7.64 (t, 1H, ArH), 7.56–7.55 (m, 2H, ArH), 7.53–
3,5-bis(3,4,5-Trimethoxybenzylidene) piperidin-4-one 7.52 (m, 2H, ArH), 7.51–7.50 (m, 2H, ArH), 4.33 (d, 2H,
(XIc). Yield: 91%; melting point (251 °C) (24). J = 6 Hz, -CH2NCH2), 4.31 (d, 2H, J = 6 Hz, -CH2NCH2),
3.86 (s, 6H, 2[OCH3]), 1.36 (q, 4H, J = 4 Hz, 2[CO-CH2-]),
3,5-bis(3,4-Dihydroxybenzylidene) piperidin-4-one 1.33 (t, 6H, J = 6 Hz, 2[-CH3]). Ms: (MW: 583.00): m/z
(XId). Yield: 65%; melting point (>300 °C) (25). 582 (M+ 1, 0.42%), 75.85 (100%). Anal. Calc. for
C34H33NO8: C, 69.97; H, 5.70; N, 2.40; O, 21.93, Found:
Synthesis of unreported analogs C, 70.04; H, 5.74; N, 2.49.

3,5-bis(4-Acyloxy-3-methoxybenzylidene)-N- 3,5-bis(4-Adamantoyloxy-3-methoxybenzylidene)-N-
acylpiperidin-4-one (IVa–d) General procedure acetylpiperidin-4-one (IVc):. The product was crystal-
First, IIIa–d intermediates were prepared according to a lized from ethanol/benzene and separated as yellow crys-
reported condensation reaction (26) where 0.02 moles of tals, yield: 48.57%, melting point: 122–125 °C. FT-IR (ύ
IIa–d were dissolved in 5 mL chloroform and condensed max, cm 1): 3010 (Ar C-H), 2890 (C-H), 1750 (ketonic
with 0.01 mole, 1.5 g of 4-piperidone. HCl in the pres- C=O), 1735 (ester C=O), 1700 (Ar C=C-), 1680 (C=C-) and
ence of 2 mL conc. HCl and then stirred for 2 h at 1679 (NH-C=O). 1H NMR (300 MHz, DMSO-d6): d 7.59
room temperature, then left to stand for 2 days. The (d, 2H, 2[-CH=C]), 7.56 (d, 2H, ArH), 7.36–7.23 (m, 2H,
reaction mixture was neutralized with a suitable base as ArH), 7.29 (d, 2H, ArH), 3.91 (s, 2H, -CH2NCH2), 3.84 (s,
(10% sodium hydroxide, 10% sodium carbonate, 10% 6H, 2[OCH3]), 3.78 (s, 2H,-CH2NCH2), 2.29 (s, 3H,
ammonium hydroxide, or triethylamine). The above neu- COCH3), 2.06–1.66 (m, 30H, -CH2-, -CH-). Ms: (MW:
tralized solution was then extracted with CHCl3 733.00): m/z 733 (M+, 55.41%), 76 (100%). Anal. Calc.
(3 9 10 mL). The chloroform extract was evaporated for C45H51NO8: C, 73.65; H, 7.00; N, 1.91; O, 17.44,
under vacuum to afford IIIa–d as a sticky mass, which Found: C, 73.72; H, 6.98; N, 1.97.
is pure enough to the next step. Acylation of IIIa–d was
carried out according to a similar reported reaction (10) 3,5-bis(4-Heptanoyloxy-3-methoxybenzylidene)-N-
where 0.01 mole of the appropriate acyl chloride dis- acetylpiperidin-4-one (IVd):. The titled compound was
solved in chloroform (10 mL) was mixed with separated as orange oily liquid, yield: 37.5%, boiling
(0.02 moles, 2 mL) triethylamine and added dropwise to point: 177–178 °C. FT-IR (ύ max, cm 1): 3010 (Ar C-H),
0.006 mole of IIIa–d at 5 °C then stirred for 2 h at 2890 (C-H), 1750 (ketonic C=O), 1735 (ester C=O), 1700
room temperature. The reaction mixture was then re- (Ar C=C-), 1680 (C=C-), and 1679 (NH-C=O). 1H NMR
crystallized from appropriate solvent to afford target (300 MHz, DMSO-d6): d 7.61 (d, 2H, 2[-CH=C]), 7.59 (d,
compounds IVa–d. 2H, ArH), 7.57 (d, 2H, ArH), 7.33 (d, 2H, ArH), 3.87 (d,
2H, -CH2NCH2), 3.86 (s, 6H, 2[OCH3]), 3.84 (d, 2H,
3,5-bis(4-Butyryloxy-3-methoxybenzylidene)-N- -CH2NCH2), 2.59 (t, 4H, J = 6 Hz, 2[CO-CH2-CH2-CH2-
acetylpiperidin-4-one (IVa). The titled compound was CH2-CH2-CH3]), 2.3 (s, 3H, COCH3), 1.70–1.60 (m, 4H,
separated as dark brown oily liquid, yield: 66.24%, boil- J = 7.5 Hz, 2 CO-CH2-CH2-CH2-CH2-CH2-CH3]), 1.41–
ing point: 125–127 °C. FT-IR (ύ max, cm 1): 3010 (Ar C- 1.34 (m, 4H, 2 [CO-CH2-CH2-CH2-CH2-CH2-CH3]), 1.32–
H), 2890 (C-H), 1750 (ketonic C=O), 1735 (ester C=O), 1.24 (m, 8H, 4 [CO-CH2-CH2-CH2-CH2-CH2-CH3]), 1.06
1700 (Ar C=C-), 1680 (C=C-), and 1679 (NH-C=O). 1H (t, 6H, J = 7.5 Hz, 2[CO-CH2-CH2-CH2-CH2-CH2-CH3]).

4 Chem Biol Drug Des 2014

 Curcumin Analogs

Ms: (MW: 633.00): m/z 633 (M+, 3.24%), 151.6 (100%). separated as reddish brown powder, yield: 72.34%, melt-
Anal. Calc. for C37H47NO8: C, 70.12; H, 7.47; N, 2.21; ing point: 114–116 °C. FT-IR (ύ max, cm 1): 3010 (Ar C-
O, 20.20, Found: C, 70.22; H, 7.53; N, 2.28. H), 2890 (C-H), 1750 (ketonic C=O), 1700 (Ar C=C-), 1680
(C=C-), and 1679 (NH-C=O). 1H NMR (300 MHz, DMSO-
d6): d 7.59 (d, 2H,2[-CH=C]), 7.36–7.27 (m, 2H, 2[CH=CH-
3,5-bis[3-Methoxy-5-(non-substittuted/iodo)-4- CH]), 7.24 (d, 2H, ArH), 7.17 (d, 2H, ArH), 6.91 (d, 2H, 2
propoxybenzylidene/allylidene] piperidin-4-one [=CH-C]), 6.74 (q, 2H, J = 8 Hz, ArH), 3.83 (t, 4H, 2[OCH2-
(VIa,b) General procedure CH2-CH3]), 2.58 (q, 4H, J = 6 Hz, -CH2NCH2), 2.5 (s, 6H, 2
VIa,b were prepared by condensation reaction procedure [OCH3]), 2.30–2.10 (m, 4H, J = 6 Hz, 2[OCH2-CH2-CH3]),
applied in the preparation of compounds IIIa–d. Compound 1.88 (s, 3H, COCH3), 1.01 (t, 6H, 2[OCH2-CH2-CH3]). Ms:
VIb gave sticky mass pure enough to enter next step. (MW: 545.00): m/z 550 (M++5, 0.16%), 76 (100%). Anal.
Calc. for C33H39NO6: C, 72.64; H, 7.20; N, 2.57; O, 17.59,
3,5-bis(3-Iodo-5-methoxy-4-propoxybenzylidene) Found: C, 72.75; H, 7.27; N, 2.69.
piperidin-4-one (VIa). The above titled compound was
separated as yellow buff powder, yield: 41.42%, melt-
ing point: 149–155 °C. FT-IR (ύ max, cm 1): 3450 3,5-bis(3-Substituted-4-mercaptobenzylidene/
(NH), 3010 (Ar C-H), 2890 (C-H), 1750 (ketonic C=O), allylidene) piperidin-4-one (IXa,b) General
1700 (Ar C=C-), and 1680 (C=C-). 1H NMR (300 MHz, procedure
DMSO-d6): d 9.7 (s, 1H, NH), 7.88 (d, 2H, 2[-CH=C]), An equimolar mixture of ‘4-hydroxy-3-methoxy cinnamal-
7.47 (s, 2H, ArH), 7.42 (d, 2H, ArH), 3.89 (s, 6H, 2 dehyde’ Ic (0.01 mole, 1.78 g)/‘3, 4 dihydroxybenzalde-
[OCH3]), 3.38 (t, 4H, J = 6 Hz, 2[OCH2-CH2-CH3]), 3.06 hyde’ Id (0.01 mole, 1.38 g) and phosphorus
(q, 4H, J = 8 Hz, -CH2NCH2), 2.08 (s, 1H, NH), 1.79– pentasulfide (0.01 mole, 1.78 g) were mixed and stirred
1.75 (m, 4H, 2[OCH2-CH2-CH3]), 1.21 (t, 6H, 2[OCH2- together at room temperature with the addition of
CH2-CH3]). Ms: (MW: 703.00): m/z 703 (M+, 0.04%), (12 mL) of carbon disulfide dropwise and (6 mL) of conc.
277.55 (100%). Anal. Calc. for C27H31I2NO5: C, 46.11; HCl then afterward continue stirring with gentle heating
H, 4.44; I, 36.09; N, 1.99; O, 11.37, Found: C, 46.18; at (40–50 °C) for 1 h. The product was then washed
H, 4.47; N, 2.06. with water and filtered to afford a sticky mass VIIIa,b
pure enough for next step. The same condensation
reaction applied in the preparation of compounds IIIa–d
3,5-bis(3-Methoxy-5-non-substituted/iodo-4- was carried out to prepare the new intermediates IXa,b,
propoxybenzylidene/allylidene)-N-acetylpiperidin- whereas compound IXb gave sticky mass pure enough
4-one (VIIa,b) General procedure to enter next step.
0.03 Mole of acetyl chloride dissolved in chloroform (4 mL)
was mixed with (0.06 moles, 6 mL) triethylamine and 3,5-bis[(4-Mercapto-3-methoxyphenyl) allylidene]
added dropwise to 0.02 mole of VIa,b at 5 °C then stirred piperidin-4-one (IXa). The titled compound was sepa-
for 1 h at room temperature. The reaction mixture was rated as violet powder, yield: 16.9%, melting point:
then recrystallized from appropriate solvent to afford target 234–246 °C. FT-IR (ύ max, cm 1): 3450 (NH), 3010 (Ar
compounds VIIa,b. C-H), 2890 (C-H), 1750 (ketonic C=O), 1700 (Ar C=C-),
and 1680 (C=C-). 1H NMR (300 MHz, DMSO-d6): d
3,5-bis(3-Iodo-5-methoxy-4-propoxybenzylidene)-N- 7.25 (d, 2H, 2[-CH=C]), 7.23 (d, 2H, ArH), 7.18 (d, 2H,
acetylpiperidin-4-one (VIIa). Recrystallization was 2[CH-CH=C]), 7.16 (d, 2H, ArH), 6.74 (m, 4H, =CH-C,
applied for the above titled product from ethanol/benzene ArH), 3.81–3.80 (s, 6H, 2[OCH3]), 3.21 (s, 2H, 2[SH]),
and separated as brown crystals, yield: 46.41%, melting 3.02 (q, 4H, -CH2NCH2), 1.91 (s, 1H, NH). Ms: (MW:
point: 102–107 °C. FT-IR (ύ max, cm 1): 3010 (Ar C-H), 451.00): m/z 451 (M+, 9.38%), 51 (100%). Anal. Calc.
2890 (C-H), 1750 (ketonic C=O), 1700 (Ar C=C-), 1680 for C25H25NO3S2: C, 66.49; H, 5.58; N, 3.10; O,
(C=C-), and 1679 (NH-C=O). 1H NMR (300 MHz, DMSO- 10.63; S, 14.20, Found: C, 66.52; H, 5.64; N, 3.08; S,
d6): d 7.99 (d, 2H, 2[-CH=C]), 7.87 (s, 2H, ArH), 7.60 (d, 13.14.
2H, ArH), 3.84 (t, 4H, 2[OCH2-CH2-CH3]), 3.05 (q, 4H,
J = 6 Hz, -CH2NCH2), 2.36 (s, 6H, 2[OCH3]), 2.29 (s, 3H,
COCH3), 2.09–1.91 (m, 4H, 2[OCH2-CH2-CH3]), 1.18 (t, 3,5-bis(3-Substituted-4-mercapto-benzylidene/
6H, 2[OCH2-CH2-CH3]). Ms: (MW: 745.00): m/z 747 allylidene)-N-substituted piperidin-4-one (Xa–c).
(M++2, 0.06%), 50.90 (100%). Anal. Calc. for General procedure
C29H33I2NO6: C, 46.73; H, 4.46; I, 34.05; N, 1.88; O, Compounds Xa,b were obtained via acetylation reaction
12.88, Found: C, 46.79; H, 4.52; N, 1.93. according to a similar reported reaction (10). (0.03 moles,
1 mL) of acetyl chloride dissolved in chloroform (5 mL)
3,5-bis[(3-Methoxy-4-propoxyphenyl) allylidene]-N- mixed with (0.06 moles, 6 mL) triethylamine and added
acetylpiperidin-4-one (VIIb). Recrystallization from eth- dropwise to 0.002 moles of IXa,b at 5 °C then stirred
anol/benzene was applied for the above titled product and for 2 h at room temperature. The reaction mixture was

Chem Biol Drug Des 2014 5

Fawzy et al.

then recrystallized from appropriate solvent to afford tar- 5.15; N, 4.77; O, 8.18; S, 16.39, Found: C, 65.58; H,
get compounds Xa,b. The target compound Xc was 5.19; N, 4.82; S, 16.42.
obtained by reacting a solution of (0.25 g) IXa in chloro-
form (5 mL) with (3 mL) phenyl isothiocyanate in equimo-
lar quantity and then refluxed for 2 h with stirring. 3,5-bis(Substituted benzylidene)-N-acylpiperidin-4-
Dropwise addition of (1–3 mL) of triethylamine was one (XIIa-d) General procedure
applied along the reflux. The product Xc was then The target compounds XIIa–d were prepared by acylation
washed with acetone, filtered, and crystallized from reaction of 0.01 mole of acetyl chloride (1 mL)/0.01 mole
toluene. of benzoyl chloride (2 mL) dissolved in chloroform (6 mL)
mixed with (0.02 moles, 2 mL) triethylamine and added
3, 5-bis ([4-Mercapto-3-methoxyphenyl] allylidene)-N- dropwise to 0.006 mole of the previously prepared XIa–d
acetylpiperidin-4-one (Xa). The above titled compound at 5 °C then stirred for 2 h at room temperature. Recrystal-
was crystallized from ethanol/benzene and separated as lization from the appropriate solvent was then carried out.
brown powder, yield: 81.21%, melting point: 240–
247 °C. FT-IR (ύ max, cm 1): 3010 (Ar C-H), 2890 (C-H), 3,5-bis(4-Hydroxy-3-methoxybenzylidene)-N-
1750 (ketonic C=O), 1700 (Ar C=C-), 1680 (C=C-), and acetylpiperidin-4-one (XIIa). The titled compound was
1679 (NH-C=O). 1H NMR (300 MHz, DMSO-d6): d 8.96 subjected to recrystallization from ethanol/benzene and
(s, 2H, 2[SH]), 7.24 (d, 2H, J = 6 Hz, 2[-CH=C]), 7.19– separated as dark yellow powder, yield: 86.81%, melting
7.14 (m, 4H, ArH), 7.17 (d, 2H, J = 6 Hz, 2[CH=CH-CH]), point: 94–102 °C. FT-IR (ύ max, cm 1): 3500 (Ar-OH),
6.90–6.60 (dd, 4H,2[=CH-C]), 3.79 (q, 4H, -CH2NCH2), 3010 (Ar C-H), 2890 (C-H), 1750 (ketonic C=O), 1700 (Ar
3.30 (s, 6H, 2[OCH3]), 2.43 (s, 2H, 2[SH]), 2.30 (s, 3H, C=C-), 1680 (C=C-), and 1679 (NH-C=O). 1H NMR
COCH3). Ms: (MW: 493.00): m/z 494 (M++1, 2.86%), (300 MHz, DMSO-d6): d 9.97 (s, 2H, 2[OH]),7.61–7.58
49.90 (100%). Anal. Calc. for C27H27NO4S2: C, 65.69; (dd, 2H, 2[-CH=C]), 7.57 (d, 2H, ArH), 7.35 (d, 2H, ArH),
H, 5.51; N, 2.84; O, 12.96; S, 12.99, Found: C, 65.73; 6.97 (d, 2H, J = 6 Hz, ArH), 3.875 (s, 2H, 2[OH]), 3.02 (q,
H, 5.57; N, 2.93; S, 13.05. 4H, -CH2NCH2), 2.30 (s, 6H, 2[OCH3]), 1.89 (s, 3H,
COCH3). Ms: (MW: 409.00): m/z 409 (M+, 40.39%), 350.20
3,5-bis(3, 4 Dimercaptobenzylidene)-N-acetylpiperidin- (100%). Anal. Calc. for C23H23NO6: C, 67.47; H, 5.66; N,
4-one (Xb). The above titled compound was crystallized 3.42; O, 23.45, Found: C, 67.56; H, 5.69; N, 3.53.
from ethanol/benzene and separated as dark blue pow-
der, yield: 93.02%, melting point: 238–243 °C. FT-IR 3,5-bis(3,4-Dioxymethylene-6-methylbenzylidene)-N-
(ύ max, cm 1): 3010 (Ar C-H), 2890 (C-H), 1750 (keton- acetylpiperidin-4-one (XIIb). 0.02 moles of Ie was dis-
ic C=O), 1700 (Ar C=C-), 1680 (C=C-), and 1679 (NH- solved in 6 ml chloroform and condensed with (0.01 mole,
C=O). 1H NMR (300 MHz, DMSO-d6): d 9.70 (s, 4H, 4 1.5 g) of 4-piperidone.HCl in the presence of 2 mL conc.
[SH]), 6.82 (d, 2H, 2[-CH=C]), 6.80–6.72 (m, 2H, ArH), HCl and stirred for 2 h at room temperature, then left for
6.69 (d, 2H, ArH), 5.53 (d, 2H, ArH), 3.45 (q, 4H, 2 days. The reaction mixture was neutralized and the chlo-
J = 6 Hz, -CH2NCH2), 2.85 (s, 4H, 2[SH]), 1.10 (s, 3H, roform layer was extracted and evaporated to give XIb
COCH3). 13C-NMR (300 MHz, DMSO-d6): d 186.89 pure enough intermediate to be acetylated according to
(1C, C=O), 161.89 (1C, N-C=O), 146.00 (2C, -CH=), the general procedure explained in 6.3.3 to yield the final
145.47 (2C, C-CO), 131.13 (4C, SH), 116.002 (4C, ArC), target XIIb, which was crystallized from ethanol/benzene.
114.71 (4C, ArC), 45.61 (2C, -CH2NCH2), 38.64 (1C,
CH3). Ms: (MW: 445.00): m/z 445 (M+, 0.40%), 67.90 The above titled product was separated as yellow powder,
(100%). Anal. Calc. for C21H19NO2S4: C, 56.60; H, yield: 42.55%, melting point: 126–129 °C. FT-IR (ύ
4.30; N, 3.14; O, 7.18; S, 28.78, Found: C, 56.68; H, max, cm 1): 3010 (Ar C-H), 2890 (C-H), 1750 (ketonic
4.32; N, 3.19; S, 28.86. C=O), 1700 (Ar C=C-), 1680 (C=C-), and 1679 (NH-C=O).
1
H NMR (300 MHz, DMSO-d6): d 7.29 (s, 2H, 2[-CH=C]),
3,5-bis[(4-Mercapto-3-methoxyphenyl)allylidene]-4- 6.91 (d, 2H, ArH), 6.11 (s, 2H, ArH), 6.11 (s, 2H, OCH2O),
oxo-N-phenylpiperidine-1-carbothioamide (Xc). The 3.29 (s, 4H, -CH2NCH2), 2.56 (s, 6H, 2[Ar-CH3]), 2.50 (s,
above titled compound was crystallized from acetone/tolu- 3H, COCH3). Ms: (MW: 433.00): m/z 433 (M+, 21.68%),
ene and separated as brown powder, yield: 24.6%, melt- 76 (100%). Anal. Calc. for C25H23NO6: C, 69.27; H,
ing point: 216–219 °C. FT-IR (ύ max, cm 1): 3450 (NH), 5.35; N, 3.23; O, 22.15, Found: C, 69.33; H, 5.39; N,
3010 (Ar C-H), 2890 (C-H), 1750 (ketonic C=O), 1700 (Ar 3.31.
C=C-), 1680 (C=C-), and 1500 (C=S). 1H NMR (300 MHz,
DMSO-d6): d 7.51 (d, 2H, 2[-CH=C]), 7.32–7.30 (m, 2H, 3,5-bis(3, 4, 5-Trimethoxybenzylidene)-N-benzoylpi-
ArH), 7.28 (t, 2H, ArH), 6.80–6.62 (m, 11H, =CH-CH=C, peridin-4-one (XIIc). The above titled compound was
ArH, =CH-C, ArH), 5.31–5.29 (m, 4H, -CH2NCH2), 5.14 (s, crystallized from ethanol/benzene and separated as yel-
1H, NH), 3.75–3.67 (s, 6H, 2[OCH3]), 2.75–.72 (s, 2H, 2 lowish brown powder, yield: 89.34%, melting point:
[SH]). Ms: (MW: 586.00): m/z 580 (M+ 6, 0.74%), 85.80 115–122 °C. FT-IR (ύ max, cm 1): 3010 (Ar C-H), 2890
(100%). Anal. Calc. for C32H30N2O3S3: C, 65.50; H, (C-H), 1750 (ketonic C=O), 1700 (Ar C=C-), 1680 (C=C-),

6 Chem Biol Drug Des 2014

 Curcumin Analogs

and 1679 (NH-C=O). 1H NMR (300 MHz, DMSO-d6): d C-CO), 139.58 (1C, ArC-NH), 139.34 (2C, ArC), 125.16
7.94 (d, 2H, ArH), 7.61 (t, 1H, ArH), 7.48 (t, 2H, J = 6 Hz, (2C, ArC-CH), 124.71 (2C, ArC), 124.46 (1C, ArC), 115.99
ArH), 7.27 (d, 2H, J = 6 Hz, 2[-CH=C]), 6.69 (d, 4H, ArH), (2C, ArC), 115.64 (2C, ArC), 115.41 (2C, ArC), 55.86
3.06 (q, 4H, J = 6 Hz, -CH2NCH2), 3.86–3.47 (m, 18H, 6 (2C, OCH3), 55.58 (2C, -CH2NCH2). Ms: (MW: 502.00): m/z
[OCH3]). Ms: (MW: 559.00): m/z 559 (M+, 0.33%), 103.95 502 (M+, 0.57%), 161.10 (100%). Anal. Calc. for
(100%). Anal. Calc. for C32H33NO8: C, 68.68; H, 5.94; C28H26N2O5S: C, 66.91; H, 5.21; N, 5.57; O, 15.92; S,
N, 2.50; O, 22.87, Found: C, 68.77; H, 5.98; N, 2.61. 6.38, Found: C, 66.98; H, 5.27; N, 5.62; S, 6.43.

3,5-bis(3, 4-Dihydroxybenzylidene)-N-benzoylpiperidin-
4-one (XIId). The above compound was crystallized from Biology
ethanol/benzene and separated as reddish brown powder,
yield: 56.15%, melting point: 160–162 °C. FT-IR (ύ Cell lines
max, cm 1): 3500 (Ar-OH), 3010 (Ar C-H), 2890 (C-H), The in vitro analysis were carried out in a panel of can-
1750 (ketonic C=O), 1700 (Ar C=C-), 1680 (C=C-), and cer cell lines, including A2780 (ECACC Salisbury, UK),
1679 (NH-C=O). 1H NMR (300 MHz, DMSO-d6): d 8.15 ACHN, Hct-116 and PC-3 (all American Type Culture
(d, 2H, ArH), 7.93 (d, 2H, J = 6 Hz, 2[-CH=C]), 7.77 (t, Collection, LGC Standards, Bor
as, Sweden), and U937-
1H, ArH), 7.65 (t, 2H, ArH), 7.49 (d, 2H, ArH), 7.33 (d, 2H, GTB (kind gift from Kennet Nilsson, Department of
ArH), 6.90 (d, 2H, ArH), 5.10–4.90 (s, 4H, 4[OH]), 2.63 (d, pathology, Uppsala University). The different cell lines
4H, -CH2NCH2). 13C-NMR (300 MHz, DMSO-d6): d were selected as representatives of various kinds of can-
180.91 (1C, C=O), 168.42 (1C, N-C=O), 147.23 (2C, ArC- cer types, including ovarian cancer (A2780), renal adeno-
OH), 145.31 (2C, ArC-OH), 134.43 (2C, -CH=), 132.31 carcinoma (ACHN), prostate cancer (PC-3), colorectal
(2C, C-CO), 131.52 (1C, ArC-C=O), 129.60 (1C, ArC), cancer (Hct-116), and a leukemic monocyte lymphoma
129.27 (2C, ArC-CH=), 128.90 (2C, ArC), 128.06 (2C, (U937-GTB). Cell growth medium RPMI 1640 (Sigma-
ArC), 125.36 (2C, ArC), 117.89 (2C, ArC), 115.75 (2C, Aldrich), supplemented with 10% heat-inactivated fetal
ArC), 45.30 (2C, -CH2NCH2). Ms: (MW: 443.00): m/z 443 bovine serum (FCS; Sigma-Aldrich), 2 mmol/L L-gluta-
(M+, 0.36%), 50.10 (100%). Anal. Calc. for C26H21NO6: mine, 100 lg/mL streptomycin, and 100 U/mL penicillin
C, 70.42; H, 4.77; N, 3.16; O, 21.65, Found: C, 70.51; (Sigma-Aldrich), was used.
H, 4.82; N, 3.25.
Target XIIb was the only compound tested by the Ameri-
can National Cancer Institute (NCI) on 60 different cell lines
Synthesis of target 3,5-bis(4-Hydroxy-3- for different types of cancer as (leukemia, non-small cell
methoxybenzylidene)-4-oxo-N-phenylpiperidine-1- lung cancer, colon cancer, CNS cancer, melanoma, ovar-
carbothioamide (XIIe) ian cancer, prostate cancer, renal cancer, and breast can-
The final target compound XIIe was obtained through a cer).
reverse pathway by mixing 4-piperidone.HCl (1.5 g) dis-
solved in 4 mL chloroform with phenyl isothiocyanate
(9 mL) in equimolar amount and then refluxing for 2 h with Cytotoxic study
stirring while dropwise addition of (1–3 mL) of triethylamine A semi-automated fluorometric microculture cytotoxicity
along the reflux. The product ‘4-oxo-N-phenylpiperidine-1- assay (FMCA) was used to assess drug sensitivity (27,28).
carbothioamide’ was then washed with acetone and fil- The method was based on measurement of fluorescence
tered. (1 mole, 1.8 g) of that product was then reacted generated from hydrolysis of fluorescein diacetate (FDA) to
with (2 moles, 2.4 g) of the available vanillin Ia through fluorescein by cells with intact plasma membranes. Using
condensation procedure mentioned previously under the the pipetting robot BioMek 2000 (Beckman Coulter, Fuller-
preparation of IIIa–d. The product XIIe was then washed ton, CA, USA), 384-well microplates (NUNC) were prepared
again with acetone and petroleum ether and subjected to with 5 lL drug solution in 10 times the final drug concen-
recrystallization from methanol/benzene. tration. The plates were then stored at 70 °C until further
use. Tumor cells from cell lines (5000 cells/well) were
The titled compound was separated as orange powder, seeded in the drug-prepared 384-well plates using the pip-
yield: 18.05%, melting point: 236–245 °C. FT-IR (ύ max, etting robot Precision 2000 (Bio-Tek Instruments, Winooski,
cm 1): 3500 (Ar-OH), 3450 (NH), 3010 (Ar C-H), 2890 VT, USA). Three columns without drugs served as controls
(C-H), 1750 (ketonic C=O), 1700 (Ar C=C-), 1680 (C=C-), and one column with medium only served as a blank. The
and 1500 (C=S). 1H NMR (300 MHz, DMSO-d6): d 9.85– plates were incubated at 37 °C for 72 h and were then
9.84 (s, 2H, 2[OH]), 9.47 (s, 1H, NH), 7.81 (s, 2H,2[-CH=C]), analyzed using the FMCA. Cell survival, expressed as sur-
7.13 (d, 4H, ArH), 7.01–6.92 (m, 7H, ArH), 4.51 (s, 4H, vival index (SI), is defined as fluorescence in test wells
-CH2NCH2), 4.39 (s, 1H, NH), 3.84–3.81 (s, 6H, 2[OCH3]), divided by fluorescence of control wells, with blank values
3.17 (s, 2H, 2[OH]). 13C-NMR (300 MHz, DMSO-d6): d subtracted, 9100. Quality criteria for a successful assay
181.94 (1C, C=O), 181.84 (1C, C=S), 150.00 (2C, ArC-O), included a mean coefficient of variation of <30% in the con-
149.18 (2C, -CH=), 147.69 (2C, ArC-OH), 147.60 (2C, trol and a fluorescence signal in control wells of more than

Chem Biol Drug Des 2014 7

Fawzy et al.

five times the blank. From the mean SI% curves, the half Results and Discussion
maximal inhibitory concentration (IC50) was determined
using nonlinear regression analysis in Prism 5 Software Pharmacology
Package (Graph Pad, San Diego, CA, USA).
Antiproliferative activity
Compound XIIb was selected and tested by the NCI on The newly synthesized compounds IVa,c and d, VIa and
60 different cell lines using concentration of 1–5 M and VII a,b, IXa and Xa–c, and XIIa–e were screened for their
both mean growth percent and growth percent of the can- antiproliferative activities against: ovarian cancer (A2780),
cer cell lines were calculated. renal adenocarcinoma (ACHN), prostate cancer (PC-3),
colorectal cancer (Hct-116), and a leukemic monocyte
lymphoma (U937-GTB) utilizing the fluorometric microcul-
Tubulin polymerization assay ture cytotoxicity assay FMCA method.
All compounds have been tested at 10 lM. The sub-
stances’ effect on tubulin polymerization were investigated From the observed results (Table S1), it was noticed that
using the Tubulin Polymerization Assay Kit (porcine tubulin most of the synthesized compounds revealed mild to
and fluorescence based), which utilize fluorescent reporter moderate cytotoxic properties. However, compounds XIIe
enhancement (Cytoskeleton Inc. Denver, CO, USA). Fluo- and IVc were found to be extremely potent, as both
rescence was measured using a FLUOstar OPTIMA instru- exhibited considerable antiproliferative activity against the
ment. Paclitaxel was used as a positive control for five cancer cell lines used in the assay with (IC50, concen-
microtubule-stabilizing agent and vincristine was used as a tration required to produce 50% inhibition of cell growth
second positive control for microtubule destabilization. The compared to control experimental) = 1–2.5 lM and 11.4–
positive controls were obtained from the Swedish Phar- 23.2 lM, respectively. Compound XIId showed moderate
macy and diluted with PBS to a final concentration of cytotoxicity with IC50 = 18.9–52.7 lM, while rest of com-
3 lM. pounds exhibited mild to moderate activity according to
the type of cancer cell line with IC50 ranging from 36 to
269 lM. Compound XIIb was tested on 60 different can-
Molecular modeling cer cell lines by The American National Cancer Institute
All molecular modeling studies were performed using Ac- showing mild cytotoxic activity among them, with mean
celrys Discovery Studio 2.5 operating system (Accelrys value 101.15 and range of 35.20 of the growth percents
Inc., San Diego, CA, USA), at the Faculty of Pharmacy, of the cancer cell lines. The assay was inapplicable on
Ain Shams University; Cairo, Egypt. Molecules were built compound IVb due to its oily nature and poor solubility.
within DS, and conformational models for each com-
pound were generated automatically. This emphasizes
representative coverage over a 20 Kcal/mol energy range Tubulin polymerization assay
above the estimated global energy minimum, and the The effect of 10 lM of the compounds IVa,c and d, VIa
best quality generation technique was chosen. Docking and VIIa,b, IXa and Xa–c, and XIIa–e on tubulin poly-
study involved the following steps: the docking analysis merization were investigated using the Tubulin Polymeriza-
was carried out on b-chain of tubulin protein. The 3D tion Assay Kit (porcine tubulin- and fluorescence based),
protein structure of tubulin cocrystallized with podophyllo- which utilize fluorescent reporter enhancement (Cytoskele-
toxin (code; 1SA1) was downloaded from the Protein ton Inc.). The results are summarized in (Tables S2 and
Data Bank of the Research Collaboration for Structural S3) where inhibition % of the newly synthesized com-
Bioinformatics (RCSB) Web site [www.rcsb.org]. The col- pounds were calculated using (equation 1) ‘Inhibition % =
chicine binding pocket of the b-chain of tubulin was [((Test sample) – (Vehicle control))/((Control ligand) – (Vehi-
docked with podophyllotoxin and the test analogs IVa–d, cle control))] 9 100’, and stabilization % were also calcu-
VIa and VIIa,b, IXa and Xa-c, and XIIa-e, after deleting lated using (equation 2) ‘Stabilization % = {1-[((Test
the water structure and a, c chains of tubulin protein, sample) – (Vehicle control))/((Control ligand) – (Vehicle con-
cleaning the protein, adding the missing hydrogens and trol))] 9 100}’.
side chains, and energy minimization according to DS
protocol. The binding pocket of the complexed The in vitro assay revealed that only few compounds inhib-
compound (podophyllotoxin) with the connected amino ited microtubule assembly. Compound VIIa displayed the
acid molecules at sphere of radius = 14 Å was identified highest activity in destabilizing microtubules compared to
and then docked with test analogs using CDocker- the positive control drug, ‘vincristine’ as a microtubule de-
CHARMm-based technique. After that the docking scores stabilizing agent (Figure S6).
(-CDOCKER interaction energy) of the best-fitted confor-
mation of each of the docked molecules as well as the On the other hand, compound XIIc was found to be the
total number of hydrogen bonds and Pi-bonds with the most potent one in stabilizing microtubules compared to
amino acids at the colchicine binding pocket were the other positive control drug, ‘paclitaxel’ as an example
recorded. of microtubule-stabilizing agent (Figure S7).

8 Chem Biol Drug Des 2014

 Curcumin Analogs

Compound XIIc acted by different mechanism where sta- All these findings proved that the in silico study results cor-
bilizing microtubules causes chromosomes to become related with the in vitro tubulin polymerization assay. A vali-
unable to achieve a metaphase spindle configuration, dation of the ideal pose was also performed by alignment
which blocks progression of mitosis, also prolonged acti- of the X-ray bioactive conformers of (VIIa and XIIc), with
vation of the mitotic checkpoint triggers apoptosis or the best-fitted pose of podophyllotoxin. The alignment
reversion to the G-phase of the cell cycle without cell showed good coincidence between them with RMSD =
division. 0.401 Å, indicating the validity of the selected poses for
both analogs (Figures S11 and S12).

Molecular modeling Compound IVc having adamantyl bulky group failed to

The molecular docking study was carried out using Dis- attain a pose, and the docking operation did not work out.
covery Studio 2.5 software. The study was started by
determining the binding mode of bioactive conformation of
podophyllotoxin cocrystallized with b-chain of tubulin hav- Structure–Activity Relationship (SAR)
ing the code 1SA1 obtained from the protein data bank
without change in its conformation to investigate the Based on the in vitro studies, it was concluded that acyla-
detailed intermolecular interactions between the ligand and tion of heterocyclic nitrogen of 4-piperidone ring is very
the target protein then to validate this study, it was essential for enhancing cytotoxic activity. This was sup-
matched with another peer article (27) where the study of ported by the structure–activity relationship study per-
the binding mode of Podophyllotoxin and chalcone deriva- formed previously on curcumin analogs (9), which stated
tives-structures which mimic our compounds- was carried the importance of such acylation to render nitrogen atom
out with the same procedure. in non-basic form to be able to penetrate cell membrane
and hence exerting efficient cytotoxic effect. Also, intro-
Interactive docking using CDOCKER protocol was then duction of thiourea moiety produced potent cytotoxic com-
applied between the designed analogs IVa–d, VIa and pounds which coincides with reports describing that
VIIa,b, IXa and Xa–c, and XIIa–e, and the binding site of phenylisothiocyanate group is responsible for thiocarbomy-
the prepared b-chain of tubulin protein. Each tested mole- lation of thiol group of glutathione inside cancerous cells
cule gave 10 possible docked poses. The ideal pose of which leads to its depletion and hence apoptosis (15,16).
each molecule was selected according to the similarity of Additionally, introduction of iodo group at the aromatic
its binding mode in the binding site to that of podophyllo- rings could enhance the tubulin destabilization activity of
toxin. The corresponding CDOCKER interaction energy compounds as the size of iodine atom just perfectly fitted
(kcal/mole) was considered in our study to prioritize their into the binding pocket of tubulin; this was concluded from
virtual affinity to the binding site, in comparison to the ideal the in silico docking study performed to iodine containing
pose of the podophyllotoxin, curcumin, and other reported compound compared with other halide containing com-
analogs. pounds and validated by the results of the in vitro assay.

Results revealed that these compounds have the ability

to interact with the deep two hydrophobic centers of col- Conclusion
chicine binding site region in the b-chain of tubulin similar
to podophyllotoxin. One hydrophobic center is sur- Esterification/etherification or sulfurization of vanillin and its
rounded by Met 259, Ala 316, and Lys 352 (occupied by derivatives yielded products that could be condensed suc-
the benzodioxole fragment in podophyllotoxin), and the cessfully with 4-piperidone ring, where its heterocyclic
other one is surrounded by Leu 242, Ala 250, and Leu N-atom was then acylated, benzoylated, or reacted with
255 (occupied by the trimethoxyphenyl moiety) (29) (Fig- phenylisothiocyanate to give the targeted compounds IVa–
ure S8). d, VIa and VIIa,b, IXa and Xa–c, and XIIa–e in high yield.

Compound VIIa, which showed the highest destabilizing The target compounds that were tested in vitro for their
activity on microtubules in silico as well as in the in vitro cytotoxicity on five different cancer cell lines and for their
tubulin polymerization assay, interacted in the same bind- activity on tubulin polymerization proved to possess high to
ing mode as podophyllotoxin forming an extra hydrogen moderate cytotoxic activity. Also, in silico study results were
bond with Lys 352 (Figure S9). consistent with that obtained from in vitro tubulin polymeri-
zation assay. Based on the previous results, it was con-
Also compound XIIc with the highest stabilizing activity on cluded that the thiourea derivative XIIe was the most potent
microtubules interacted similarly in the same binding mode cytotoxic compound with IC50 values in 1–2.5 lM range,
as podophyllotoxin (Figure S10), where it was ranked while it showed moderate effect on tubulin polymerization.
within the compounds which showed high interaction
energy in silico while in vitro assay revealed its high stabil- Compound IVc displayed lower cytotoxicity with IC50 val-
ization activity mechanism on microtubules. ues in 11.4–23.2 lM range and 79.8% stabilization of

Chem Biol Drug Des 2014 9

Fawzy et al.

microtubules in the tubulin polymerization assay, although 9. Das U., Sakagami H., Chu Q., Wang Q., Kawase M.,
it showed failure of docking operation in the in silico study, Selvakumar P., Sharma R.K., Dimmock J.R. (2010)
whereas compound VIIa destabilized microtubules with 3,5-Bis(benzylidene)-1-[4-2-(morpholin-4-yl)ethoxyphe-
93.3% of inhibition of tubulin and showed cytotoxicity with nylcarbonyl]-4-piperidone hydrochloride: a lead tumor-
IC50 = 36–102.6 lM. On the other hand, compound XIIc specific cytotoxin which induces apoptosis and auto-
stabilized microtubules with 94.8% and showed cytotoxic- phagy. Biorg Med Chem Lett;20:912–917.
ity with IC50 = 54.4–187.4 lM. Hence, we concluded that 10. Das U., Alcorn J., Shrivastav A., Sharma R.K., De
the synthesized curcumin analogs provide a nucleus for Clercq E., Balzarini J., Dimmock J.R. (2007) Design,
further optimization, which could lead to the development synthesis and cytotoxic properties of novel 1-[4-(2-al-
of new active chemical entities against cancer via tubulin kylaminoethoxy) phenylcarbonyl]-3,5-bis(arylidene)-4-
targeting. piperidones and related compounds. Eur J Med
Chem;42:71–80.
11. Chakrabarti R., Rawat P.S., Cooke B.M., Coppel R.L.,
Acknowledgments Patankar S. (2013) Cellular effects of curcumin on
Plasmodium falciparum include disruption of microtu-
The authors are grateful to Professor J.Gullbo; Department bules. PLoS ONE;8:e57302.
of Medical Sciences, Uppsala University Hospital, Uppsala, 12. Chakraborti S., Das L., Kapoor N., Das A., Dwivedi V.,
Sweden, for his great collaboration in this study also would Poddar A., Chakraborti G., Janik M., Basu G., Panda
like to express sincere thanks to Future University in Egypt D., Chakrabarti P., Surolia A., Bhattacharyya B. (2011)
for funding this research project. Finally, thanks are due to Curcumin recognizes a unique binding site of tubulin. J
the department of pharmaceutical chemistry, faculty of Med Chem;54:6183–6196.
pharmacy, Ain Shams University for providing CADD Lab 13. Dyrager C., Wickstro €m M., Fride
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 Curcumin Analogs

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Design and biological evaluation of novel tubulin inhibi-
tors as antimitotic agents using a pharmacophore Table S1. Results of the cytotoxicity assay of the new
binding model with tubulin. J Med Chem;49:5664. curcumin analogs expressed as IC50 in lM.

Table S2. Inhibition % of target compounds towards tubu-

Supporting Information lin with vincristine as control ligand.

Additional Supporting Information may be found in the Table S3. Stabilization % of target compounds towards
online version of this article: tubulin with paclitaxel as control ligand.

Figure S1. Curcumin structure (2).

Figure S2. Primary binding site shown on curcumin ana-

log (9).

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