Newly designed and synthesized curcumin analogs with in vitro cytotoxicity and tubulin polymerization activity

Article abstract of DOI:10.1111/cbdd.12464

Novel curcumin analogs with 4-piperidone ring were designed, synthesized, and evaluated for their cytotoxic activities against five different cancer cell lines. 3,5-bis(4-Hydroxy-3-methoxybenzylidene)-4-oxo-N-phenylpiperidine-1-carbothioamide (XIIe) exhib

Full text of DOI:10.1111/cbdd.12464

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
minimal side-effects on mammalian cells. Natural products
have been found to be a source of novel and potent bio-
active compounds with minimal side-effects in vivo (1).
Nasser S. M. Ismail2, Joachim Gullbo3 and
Khaled A. M. Abouzid2,*
1Pharmaceutical Chemistry Department, Faculty of
Pharmaceutical Sciences and Pharmaceutical Industries,
Future University, Cairo 12311, Egypt
Extensive research conducted within the past years revealed
that curcumin (1) [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadien-3,5-dione] (Figure S1) (2) extracted from the rhi-
zome of the plant Curcuma longa modulates and interacts
with a diverse range of molecular targets, and hence, it pos-
sess antiproliferative activities against tumor cells in vitro, anti-
2Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Ain Shams University, Cairo 11566, Egypt
3Division of Clinical Pharmacology, Department of Medical
Sciences, Uppsala University Hospital, SE-751 85
Uppsala, Sweden
inflammatory,
antibacterial,
antiviral,
antihepatotoxic,
*Corresponding author: Khaled A. M. Abouzid,
Khaled.abouzid@pharma.asu.edu.eg
hypotensive, and anticholesterolemic activities (2–8). As can-
cer is a result of the dysregulation of multiple cell signaling
pathways, curcumin’s multitargeting ability may be the key to
its therapeutic potential against cancer. Reported structural
activity relationship studies performed on curcumin analogs
highlighted the critical sites for developing novel analogs
where 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore (2)
was found to be very important in activity of the compound
as this group reacts with cellular constituents at a specific
binding site (primary binding site A) (Figure S2) (9).
Novel curcumin analogs with 4-piperidone ring were
designed, synthesized, and evaluated for their
cytotoxic activities against five different cancer
cell lines. 3,5-bis(4-Hydroxy-3-methoxybenzylidene)-4-
oxo-N-phenylpiperidine-1-carbothioamide (XIIe) exhib-
ited considerable cytotoxic activity with IC50 values in
1–2.5 lM range. In silico and in vitro, studies were also
performed to predict the binding affinity of the target
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-
oxybenzylidene)-N-acetylpiperidin-4-one (VIIa) was
found to exert 93.3% inhibition of tubulin and destabili-
zation of microtubules in vitro compared to vincristine
while, 3,5-bis(3,4,5-trimethoxybenzylidene)-N-benzoylpi-
peridin-4-one (XIIc) showed high potency in a differ-
ent way where it exerted 94.8% stabilization of
microtubules in vitro compared to positive control
paclitaxel.
This binding may be influenced by the nature of the
group attached to the heterocyclic nitrogen atom where
charged molecules were found to be unable to penetrate
cell membranes and exert
hence, N-acylation was considered a route to render the
nitrogen atom in non-basic form (9,10). Recent
a cytotoxic effect, and
a
research also revealed that chalcones (3) (Figure S3),
curcumin, and their derivatives possess cytotoxic activity
associated with tubulin inhibition and interference with
microtubule formation, which is essential in cellular pro-
cesses such as mitosis and cell replication. It was
found that curcumin exerted inhibition to tubulin inside
Plasmodium falciparum cells with IC50 = 5 lM (11). Also
benzylidene curcumin derivative (4) (Figure S4) was found
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-
phyllotoxin (Figure S5B) (13).
Key words: curcumin analogs, cytotoxicity, molecular model-
ing, tubulin polymerization assay
Received 19 July 2014, revised 11 September 2014 and
accepted for publication 15 October 2014
Cancer is a life-threatening disease and a leading cause of
death. Although a range of therapies based on chemother-
apy, surgery, and radiotherapy are available, yet they are
of limited efficacy. Moreover, current anticancer regimens
are associated with significant levels of toxicity and serious
adverse side-effects. Hence, many research projects have
According to all the previous findings in the literature,
several modifications were applied to curcumin analogs
to develop novel curcumin analogs with cytotoxic activity
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
essential in activity (17).
Scheme 1: Synthesis of 3,5-bis(4-Acyloxy-3-methoxybenzylidene)-
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).
Scheme 2: Synthesis of 3,5-bis(3-Methoxy-5-non-substituted/
To synthesize the final target compounds of series 3 (IXa
and Xa-c), thiation reaction was applied on the starting
materials Id,e in the presence of strong catalyst, then the
pure prepared mixture VIIIa,b entered the Claisen–
Schmidt condensation reaction with 4-piperidone.HCl to
obtain the target IXa and the mixture IXb which were pure
enough to be acetylated in the presence of triethylamine
yielding the final target compounds Xa,b. As for the target
Xc, it was prepared by reacting IXa with phenyl isothiocy-
anate in triethylamine under reflux (Scheme 3).
iodo-4-propoxybenzylidene/allylidene)
piperidin-4-one
and
3,5-bis(3-Methoxy-5-non-substituted/iodo-4-propoxybenzylidene/
allylidene)-N-acetylpiperidin-4-one (VIa and VIIa,b). 2Reagents
and conditions: i: Na metal/reflux + alkyl chloride in ethanol/
reflux, ii:4-piperidone.HCl + conc. HCl, iii: acyl chloride
triethylamine (5 °C). [R = -CH3].
+
Compounds XIIa–d of series 4 were prepared via reacting
the intermediates XIa–d with acetyl chloride or benzoyl
chloride in triethylamine to yield the corresponding final
2
Chem Biol Drug Des 2014

Curcumin Analogs
Synthesis of reported intermediates
compounds XIIa–d. Target XIIe of series 4 was prepared
through different pathway where 4-piperidone.HCl was
reacted with phenyl isothiocyanate in triethylamine under
reflux followed by condensation of the product with Ia in
the presence of acidic medium to obtain XIIe (Scheme 4).
4-Acyloxy-3-methoxybenzaldehyde (IIa–d) General
procedure
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
(0.02 moles, 3 g) of vanillin ‘Ia’ in 5 mL chloroform then
stirred for 2 h at room temperature.
Experimental
Materials and instrumentation
Starting materials and reagents were purchased from
Sigma-Aldrich (Stockholm, Sweden). Melting points were
recorded on Stuart Scientific apparatus. 1H-NMR spectra
were recorded on a Varian Mercury VX-300 NMR spec-
trometer (International Equipment Trading Ltd, Vernon
Hills, IL, USA). 13C-NMR spectra were recorded on a Var-
ian Mercury VX-300 NMR spectrometer. MS spectra
mass were recorded on Shimadzu GCMS-QP 5050A gas
chromatograph mass spectrometer (70 eV). Elemental
analyses were performed at The Regional Center for
Mycology and Biotechnology Al-Azhar University, Cairo,
Egypt. In vitro antiproliferative assay and tubulin polymeri-
zation assay were performed at Uppsala University Hospi-
tal, division of Clinical Pharmacology, department of
Medical Sciences, Uppsala, Sweden. One compound
(XIIb) was tested for its cytotoxic activity by the American
National Cancer Institute.
4-Formyl-2-methoxyphenyl
butyrate
(IIa). Yield:
87%; melting point (104–105 °C) (18).
4-Formyl-2-methoxyphenyl propionate (IIb). Yield:
66%; melting point (114–115 °C) (18, 19).
Tricyclo[3.3.1.13,7]decane-1-carboxylic acid, 4-formyl-
2-methoxyphenyl ester (IIc). Yield: 71%; melting
point (118–119 °C) (20).
4-Formyl-2-methoxyphenyl heptanoate (IId). Yield:
75%; melting point (163–164 °C) (21).
Methoxy-5-(unsubstituted/iodo)-4-
propoxybenzaldehyde/cinnmaldehyde (Va,b)
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
and Xa-c). 3Reagents and conditions: i: P2S5, CS2, conc. HCl, ii:
4-piperidone.HCl, conc. HCl, iii: acyl chloride, triethylamine (5 °C),
iv; Phenylisothiocyanate, triethylamine (reflux).
Scheme 4: Synthesis of 3,5-bis(Substituted benzylidene)-N-
substituted-piperidin-4-one (XIIa-e). 4Reagents and conditions:
i: 4-piperidone.HCl, conc. HCl, ii: acyl chloride, triethylamine
(5 °C), iii: N-phenylisothiocyanate-4-piperidone, conc. HCl.
Chem Biol Drug Des 2014
3

Fawzy et al.
1-Iodo-5-methoxy-4-propoxybenzaldehyde
(Va). Yield: 71%; melting point (148 °C) (22).
NMR (300 MHz, DMSO-d6): d 7.61 (d, 2H, J = 6.5 Hz, 2
[-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,-
CH2NCH2), 3.85 (s, 6H, 2[OCH3]), 2.57 (t, 4H, J = 7.5 Hz,
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
549 (M+, 8.64%), 350.25 (100%). Anal. Calc. for
3-Methoxy-4-propoxycinnamaldehyde
36%; mp (164 °C) (23).
(Vb). Yield:
3,5-bis(Substituted benzylidene)-piperidin-4-one
(XIa-d) General procedure
C
31H35NO8: 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
chloroform with (0.01 mole, 1.5 g) of 4-piperidone.HCl
was carried out in presence of 2 mL conc. HCl, and the
mixture was stirred for 2 h at room temperature, then left
for 2 days. The reaction mixture was neutralized and the
chloroform layer was extracted with chloroform
(3 9 10 mL) and evaporated under vacuum.
C, 67.81; H, 6.48; N, 2.62.
3,5-bis(3-Methoxy-4-propyloyloxybenzylidene)-N-
benzoylpiperidin-4-one (IVb). The titled compound
was separated as yellow oily liquid, yield: 65.78%, boil-
ing point: 199–204 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar
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
NMR (300 MHz, DMSO-d6): d 7.98 (d, J = 6 Hz, 2H,
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–
7.52 (m, 2H, ArH), 7.51–7.50 (m, 2H, ArH), 4.33 (d, 2H,
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-]),
1.33 (t, 6H, J = 6 Hz, 2[-CH3]). Ms: (MW: 583.00): m/z
3,5-bis(4-Hydroxy-3-methoxybenzylidene) piperidin-4-
one (XIa). Yield: 65%; melting point (254–256 °C) (24).
3,5-bis(3,4,5-Trimethoxybenzylidene) piperidin-4-one
(XIc). Yield: 91%; melting point (251 °C) (24).
3,5-bis(3,4-Dihydroxybenzylidene)
piperidin-4-one
(XId). Yield: 65%; melting point (>300 °C) (25).
582 (M+À1
, 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-
acetylpiperidin-4-one (IVc):. The product was crystal-
lized from ethanol/benzene and separated as yellow crys-
tals, yield: 48.57%, melting point: 122–125 °C. FT-IR (ύ
max, cmÀ1): 3010 (Ar 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 NMR (300 MHz, DMSO-d6): d 7.59
(d, 2H, 2[-CH=C]), 7.56 (d, 2H, ArH), 7.36–7.23 (m, 2H,
ArH), 7.29 (d, 2H, ArH), 3.91 (s, 2H, -CH2NCH2), 3.84 (s,
6H, 2[OCH3]), 3.78 (s, 2H,-CH2NCH2), 2.29 (s, 3H,
COCH3), 2.06–1.66 (m, 30H, -CH2-, -CH-). Ms: (MW:
733.00): m/z 733 (M+, 55.41%), 76 (100%). Anal. Calc.
for C45H51NO8: C, 73.65; H, 7.00; N, 1.91; O, 17.44,
Found: C, 73.72; H, 6.98; N, 1.97.
acylpiperidin-4-one (IVa–d) General procedure
First, IIIa–d intermediates were prepared according to a
reported condensation reaction (26) where 0.02 moles of
IIa–d were dissolved in 5 mL chloroform and condensed
with 0.01 mole, 1.5 g of 4-piperidone. HCl in the pres-
ence of 2 mL conc. HCl and then stirred for 2 h at
room temperature, then left to stand for 2 days. The
reaction mixture was neutralized with a suitable base as
(10% sodium hydroxide, 10% sodium carbonate, 10%
ammonium hydroxide, or triethylamine). The above neu-
tralized solution was then extracted with CHCl3
(3 9 10 mL). The chloroform extract was evaporated
under vacuum to afford IIIa–d as a sticky mass, which
is pure enough to the next step. Acylation of IIIa–d was
carried out according to a similar reported reaction (10)
where 0.01 mole of the appropriate acyl chloride dis-
solved in chloroform (10 mL) was mixed with
(0.02 moles, 2 mL) triethylamine and added dropwise to
0.006 mole of IIIa–d at 5 °C then stirred for 2 h at
room temperature. The reaction mixture was then re-
crystallized from appropriate solvent to afford target
compounds IVa–d.
3,5-bis(4-Heptanoyloxy-3-methoxybenzylidene)-N-
acetylpiperidin-4-one (IVd):. The titled compound was
separated as orange oily liquid, yield: 37.5%, boiling
point: 177–178 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar 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 NMR
(300 MHz, DMSO-d6): d 7.61 (d, 2H, 2[-CH=C]), 7.59 (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,
-CH2NCH2), 2.59 (t, 4H, J = 6 Hz, 2[CO-CH2-CH2-CH2-
CH2-CH2-CH3]), 2.3 (s, 3H, COCH3), 1.70–1.60 (m, 4H,
J = 7.5 Hz, 2 CO-CH2-CH2-CH2-CH2-CH2-CH3]), 1.41–
1.34 (m, 4H, 2 [CO-CH2-CH2-CH2-CH2-CH2-CH3]), 1.32–
1.24 (m, 8H, 4 [CO-CH2-CH2-CH2-CH2-CH2-CH3]), 1.06
(t, 6H, J = 7.5 Hz, 2[CO-CH2-CH2-CH2-CH2-CH2-CH3]).
3,5-bis(4-Butyryloxy-3-methoxybenzylidene)-N-
acetylpiperidin-4-one (IVa). The titled compound was
separated as dark brown oily liquid, yield: 66.24%, boil-
ing point: 125–127 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar 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
4
Chem Biol Drug Des 2014

Curcumin Analogs
Ms: (MW: 633.00): m/z 633 (M+, 3.24%), 151.6 (100%).
Anal. Calc. for C37H47NO8: C, 70.12; H, 7.47; N, 2.21;
O, 20.20, Found: C, 70.22; H, 7.53; N, 2.28.
separated as reddish brown powder, yield: 72.34%, melt-
ing point: 114–116 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar C-
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-
CH]), 7.24 (d, 2H, ArH), 7.17 (d, 2H, ArH), 6.91 (d, 2H, 2
[=CH-C]), 6.74 (q, 2H, J = 8 Hz, ArH), 3.83 (t, 4H, 2[OCH2-
CH2-CH3]), 2.58 (q, 4H, J = 6 Hz, -CH2NCH2), 2.5 (s, 6H, 2
[OCH3]), 2.30–2.10 (m, 4H, J = 6 Hz, 2[OCH2-CH2-CH3]),
1.88 (s, 3H, COCH3), 1.01 (t, 6H, 2[OCH2-CH2-CH3]). Ms:
(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,
Found: C, 72.75; H, 7.27; N, 2.69.
3,5-bis[3-Methoxy-5-(non-substittuted/iodo)-4-
propoxybenzylidene/allylidene] piperidin-4-one
(VIa,b) General procedure
VIa,b were prepared by condensation reaction procedure
applied in the preparation of compounds IIIa–d. Compound
VIb gave sticky mass pure enough to enter next step.
3,5-bis(3-Iodo-5-methoxy-4-propoxybenzylidene)
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, cmÀ1): 3450
(NH), 3010 (Ar C-H), 2890 (C-H), 1750 (ketonic C=O),
1700 (Ar C=C-), and 1680 (C=C-). 1H NMR (300 MHz,
DMSO-d6): d 9.7 (s, 1H, NH), 7.88 (d, 2H, 2[-CH=C]),
7.47 (s, 2H, ArH), 7.42 (d, 2H, ArH), 3.89 (s, 6H, 2
[OCH3]), 3.38 (t, 4H, J = 6 Hz, 2[OCH2-CH2-CH3]), 3.06
(q, 4H, J = 8 Hz, -CH2NCH2), 2.08 (s, 1H, NH), 1.79–
1.75 (m, 4H, 2[OCH2-CH2-CH3]), 1.21 (t, 6H, 2[OCH2-
CH2-CH3]). Ms: (MW: 703.00): m/z 703 (M+, 0.04%),
277.55 (100%). Anal. Calc. for C27H31I2NO5: C, 46.11;
H, 4.44; I, 36.09; N, 1.99; O, 11.37, Found: C, 46.18;
H, 4.47; N, 2.06.
3,5-bis(3-Substituted-4-mercaptobenzylidene/
allylidene) piperidin-4-one (IXa,b) General
procedure
An equimolar mixture of ‘4-hydroxy-3-methoxy cinnamal-
dehyde’ Ic (0.01 mole, 1.78 g)/‘3, 4 dihydroxybenzalde-
hyde’ Id (0.01 mole, 1.38 g) and phosphorus
pentasulfide (0.01 mole, 1.78 g) were mixed and stirred
together at room temperature with the addition of
(12 mL) of carbon disulfide dropwise and (6 mL) of conc.
HCl then afterward continue stirring with gentle heating
at (40–50 °C) for 1 h. The product was then washed
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
was carried out to prepare the new intermediates IXa,b,
whereas compound IXb gave sticky mass pure enough
to enter next step.
3,5-bis(3-Methoxy-5-non-substituted/iodo-4-
propoxybenzylidene/allylidene)-N-acetylpiperidin-
4-one (VIIa,b) General procedure
0.03 Mole of acetyl chloride dissolved in chloroform (4 mL)
was mixed with (0.06 moles, 6 mL) triethylamine and
added dropwise to 0.02 mole of VIa,b at 5 °C then stirred
for 1 h at room temperature. The reaction mixture was
then recrystallized from appropriate solvent to afford target
compounds VIIa,b.
3,5-bis[(4-Mercapto-3-methoxyphenyl)
allylidene]
piperidin-4-one (IXa). The titled compound was sepa-
rated as violet powder, yield: 16.9%, melting point:
234–246 °C. FT-IR (ύ max, cmÀ1): 3450 (NH), 3010 (Ar
C-H), 2890 (C-H), 1750 (ketonic C=O), 1700 (Ar C=C-),
and 1680 (C=C-). 1H NMR (300 MHz, DMSO-d6): d
7.25 (d, 2H, 2[-CH=C]), 7.23 (d, 2H, ArH), 7.18 (d, 2H,
2[CH-CH=C]), 7.16 (d, 2H, ArH), 6.74 (m, 4H, =CH-C,
ArH), 3.81–3.80 (s, 6H, 2[OCH3]), 3.21 (s, 2H, 2[SH]),
3.02 (q, 4H, -CH2NCH2), 1.91 (s, 1H, NH). Ms: (MW:
451.00): m/z 451 (M+, 9.38%), 51 (100%). Anal. Calc.
for C25H25NO3S2: C, 66.49; H, 5.58; N, 3.10; O,
10.63; S, 14.20, Found: C, 66.52; H, 5.64; N, 3.08; S,
13.14.
3,5-bis(3-Iodo-5-methoxy-4-propoxybenzylidene)-N-
acetylpiperidin-4-one
(VIIa). Recrystallization
was
applied for the above titled product from ethanol/benzene
and separated as brown crystals, yield: 46.41%, melting
point: 102–107 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar C-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 7.99 (d, 2H, 2[-CH=C]), 7.87 (s, 2H, ArH), 7.60 (d,
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,
6H, 2[OCH2-CH2-CH3]). Ms: (MW: 745.00): m/z 747
3,5-bis(3-Substituted-4-mercapto-benzylidene/
allylidene)-N-substituted piperidin-4-one (Xa–c).
General procedure
Compounds Xa,b were obtained via acetylation reaction
according to a similar reported reaction (10). (0.03 moles,
1 mL) of acetyl chloride dissolved in chloroform (5 mL)
mixed with (0.06 moles, 6 mL) triethylamine and added
dropwise to 0.002 moles of IXa,b at 5 °C then stirred
for 2 h at room temperature. The reaction mixture was
(M++2
29H33I2NO6: C, 46.73; H, 4.46; I, 34.05; N, 1.88; O,
,
0.06%), 50.90 (100%). Anal. Calc. for
C
12.88, Found: C, 46.79; H, 4.52; N, 1.93.
3,5-bis[(3-Methoxy-4-propoxyphenyl)
allylidene]-N-
acetylpiperidin-4-one (VIIb). Recrystallization from eth-
anol/benzene was applied for the above titled product and
Chem Biol Drug Des 2014
5

Fawzy et al.
then recrystallized from appropriate solvent to afford tar-
get compounds Xa,b. The target compound Xc was
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.
Dropwise addition of (1–3 mL) of triethylamine was
applied along the reflux. The product Xc was then
washed with acetone, filtered, and crystallized from
toluene.
5.15; N, 4.77; O, 8.18; S, 16.39, Found: C, 65.58; H,
5.19; N, 4.82; S, 16.42.
3,5-bis(Substituted benzylidene)-N-acylpiperidin-4-
one (XIIa-d) General procedure
The target compounds XIIa–d were prepared by acylation
reaction of 0.01 mole of acetyl chloride (1 mL)/0.01 mole
of benzoyl chloride (2 mL) dissolved in chloroform (6 mL)
mixed with (0.02 moles, 2 mL) triethylamine and added
dropwise to 0.006 mole of the previously prepared XIa–d
at 5 °C then stirred for 2 h at room temperature. Recrystal-
lization from the appropriate solvent was then carried out.
3, 5-bis ([4-Mercapto-3-methoxyphenyl] allylidene)-N-
acetylpiperidin-4-one (Xa). The above titled compound
was crystallized from ethanol/benzene and separated as
brown powder, yield: 81.21%, melting point: 240–
247 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar C-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 8.96
(s, 2H, 2[SH]), 7.24 (d, 2H, J = 6 Hz, 2[-CH=C]), 7.19–
7.14 (m, 4H, ArH), 7.17 (d, 2H, J = 6 Hz, 2[CH=CH-CH]),
6.90–6.60 (dd, 4H,2[=CH-C]), 3.79 (q, 4H, -CH2NCH2),
3.30 (s, 6H, 2[OCH3]), 2.43 (s, 2H, 2[SH]), 2.30 (s, 3H,
COCH3). Ms: (MW: 493.00): m/z 494 (M++1, 2.86%),
49.90 (100%). Anal. Calc. for C27H27NO4S2: C, 65.69;
H, 5.51; N, 2.84; O, 12.96; S, 12.99, Found: C, 65.73;
H, 5.57; N, 2.93; S, 13.05.
3,5-bis(4-Hydroxy-3-methoxybenzylidene)-N-
acetylpiperidin-4-one (XIIa). The titled compound was
subjected to recrystallization from ethanol/benzene and
separated as dark yellow powder, yield: 86.81%, melting
point: 94–102 °C. FT-IR (ύ max, cmÀ1): 3500 (Ar-OH),
3010 (Ar C-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 9.97 (s, 2H, 2[OH]),7.61–7.58
(dd, 2H, 2[-CH=C]), 7.57 (d, 2H, ArH), 7.35 (d, 2H, ArH),
6.97 (d, 2H, J = 6 Hz, ArH), 3.875 (s, 2H, 2[OH]), 3.02 (q,
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
(100%). Anal. Calc. for C23H23NO6: C, 67.47; H, 5.66; N,
3.42; O, 23.45, Found: C, 67.56; H, 5.69; N, 3.53.
3,5-bis(3, 4 Dimercaptobenzylidene)-N-acetylpiperidin-
4-one (Xb). The above titled compound was crystallized
from ethanol/benzene and separated as dark blue pow-
der, yield: 93.02%, melting point: 238–243 °C. FT-IR
(ύ max, cmÀ1): 3010 (Ar C-H), 2890 (C-H), 1750 (keton-
ic C=O), 1700 (Ar C=C-), 1680 (C=C-), and 1679 (NH-
C=O). 1H NMR (300 MHz, DMSO-d6): d 9.70 (s, 4H, 4
[SH]), 6.82 (d, 2H, 2[-CH=C]), 6.80–6.72 (m, 2H, ArH),
6.69 (d, 2H, ArH), 5.53 (d, 2H, ArH), 3.45 (q, 4H,
J = 6 Hz, -CH2NCH2), 2.85 (s, 4H, 2[SH]), 1.10 (s, 3H,
3,5-bis(3,4-Dioxymethylene-6-methylbenzylidene)-N-
acetylpiperidin-4-one (XIIb). 0.02 moles of Ie was dis-
solved in 6 ml chloroform and condensed with (0.01 mole,
1.5 g) of 4-piperidone.HCl in the presence of 2 mL conc.
HCl and stirred for 2 h at room temperature, then left for
2 days. The reaction mixture was neutralized and the chlo-
roform layer was extracted and evaporated to give XIb
pure enough intermediate to be acetylated according to
the general procedure explained in 6.3.3 to yield the final
target XIIb, which was crystallized from ethanol/benzene.
COCH3). 13C-NMR (300 MHz, DMSO-d6):
d 186.89
(1C, C=O), 161.89 (1C, N-C=O), 146.00 (2C, -CH=),
145.47 (2C, C-CO), 131.13 (4C, SH), 116.002 (4C, ArC),
114.71 (4C, ArC), 45.61 (2C, -CH2NCH2), 38.64 (1C,
CH3). Ms: (MW: 445.00): m/z 445 (M+, 0.40%), 67.90
(100%). Anal. Calc. for C21H19NO2S4: C, 56.60; H,
4.30; N, 3.14; O, 7.18; S, 28.78, Found: C, 56.68; H,
4.32; N, 3.19; S, 28.86.
The above titled product was separated as yellow powder,
yield: 42.55%, melting point: 126–129 °C. FT-IR (ύ
max, cmÀ1): 3010 (Ar C-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 7.29 (s, 2H, 2[-CH=C]),
6.91 (d, 2H, ArH), 6.11 (s, 2H, ArH), 6.11 (s, 2H, OCH2O),
3.29 (s, 4H, -CH2NCH2), 2.56 (s, 6H, 2[Ar-CH3]), 2.50 (s,
3H, COCH3). Ms: (MW: 433.00): m/z 433 (M+, 21.68%),
76 (100%). Anal. Calc. for C25H23NO6: C, 69.27; H,
5.35; N, 3.23; O, 22.15, Found: C, 69.33; H, 5.39; N,
3.31.
3,5-bis[(4-Mercapto-3-methoxyphenyl)allylidene]-4-
oxo-N-phenylpiperidine-1-carbothioamide (Xc). The
above titled compound was crystallized from acetone/tolu-
ene and separated as brown powder, yield: 24.6%, melt-
ing point: 216–219 °C. FT-IR (ύ max, cmÀ1): 3450 (NH),
3010 (Ar C-H), 2890 (C-H), 1750 (ketonic C=O), 1700 (Ar
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,
ArH), 7.28 (t, 2H, ArH), 6.80–6.62 (m, 11H, =CH-CH=C,
ArH, =CH-C, ArH), 5.31–5.29 (m, 4H, -CH2NCH2), 5.14 (s,
1H, NH), 3.75–3.67 (s, 6H, 2[OCH3]), 2.75–.72 (s, 2H, 2
[SH]). Ms: (MW: 586.00): m/z 580 (M+À6, 0.74%), 85.80
(100%). Anal. Calc. for C32H30N2O3S3: C, 65.50; H,
3,5-bis(3, 4, 5-Trimethoxybenzylidene)-N-benzoylpi-
peridin-4-one (XIIc). The above titled compound was
crystallized from ethanol/benzene and separated as yel-
lowish brown powder, yield: 89.34%, melting point:
115–122 °C. FT-IR (ύ max, cmÀ1): 3010 (Ar C-H), 2890
(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
7.94 (d, 2H, ArH), 7.61 (t, 1H, ArH), 7.48 (t, 2H, J = 6 Hz,
ArH), 7.27 (d, 2H, J = 6 Hz, 2[-CH=C]), 6.69 (d, 4H, ArH),
3.06 (q, 4H, J = 6 Hz, -CH2NCH2), 3.86–3.47 (m, 18H, 6
[OCH3]). Ms: (MW: 559.00): m/z 559 (M+, 0.33%), 103.95
(100%). Anal. Calc. for C32H33NO8: C, 68.68; H, 5.94;
N, 2.50; O, 22.87, Found: C, 68.77; H, 5.98; N, 2.61.
C-CO), 139.58 (1C, ArC-NH), 139.34 (2C, ArC), 125.16
(2C, ArC-CH), 124.71 (2C, ArC), 124.46 (1C, ArC), 115.99
(2C, ArC), 115.64 (2C, ArC), 115.41 (2C, ArC), 55.86
(2C, OCH3), 55.58 (2C, -CH2NCH2). Ms: (MW: 502.00): m/z
502 (M+, 0.57%), 161.10 (100%). Anal. Calc. for
C
28H26N2O5S: C, 66.91; H, 5.21; N, 5.57; O, 15.92; S,
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
ethanol/benzene and separated as reddish brown powder,
yield: 56.15%, melting point: 160–162 °C. FT-IR (ύ
max, cmÀ1): 3500 (Ar-OH), 3010 (Ar C-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 8.15
(d, 2H, ArH), 7.93 (d, 2H, J = 6 Hz, 2[-CH=C]), 7.77 (t,
1H, ArH), 7.65 (t, 2H, ArH), 7.49 (d, 2H, ArH), 7.33 (d, 2H,
ArH), 6.90 (d, 2H, ArH), 5.10–4.90 (s, 4H, 4[OH]), 2.63 (d,
Biology
Cell lines
The in vitro analysis were carried out in a panel of can-
cer cell lines, including A2780 (ECACC Salisbury, UK),
ACHN, Hct-116 and PC-3 (all American Type Culture
ꢀ
Collection, LGC Standards, Boras, Sweden), and U937-
GTB (kind gift from Kennet Nilsson, Department of
pathology, Uppsala University). The different cell lines
were selected as representatives of various kinds of can-
cer types, including ovarian cancer (A2780), renal adeno-
carcinoma (ACHN), prostate cancer (PC-3), colorectal
cancer (Hct-116), and a leukemic monocyte lymphoma
(U937-GTB). Cell growth medium RPMI 1640 (Sigma-
Aldrich), supplemented with 10% heat-inactivated fetal
bovine serum (FCS; Sigma-Aldrich), 2 mmol/L L-gluta-
mine, 100 lg/mL streptomycin, and 100 U/mL penicillin
(Sigma-Aldrich), was used.
4H, -CH2NCH2). 13C-NMR (300 MHz, DMSO-d6):
d
180.91 (1C, C=O), 168.42 (1C, N-C=O), 147.23 (2C, ArC-
OH), 145.31 (2C, ArC-OH), 134.43 (2C, -CH=), 132.31
(2C, C-CO), 131.52 (1C, ArC-C=O), 129.60 (1C, ArC),
129.27 (2C, ArC-CH=), 128.90 (2C, ArC), 128.06 (2C,
ArC), 125.36 (2C, ArC), 117.89 (2C, ArC), 115.75 (2C,
ArC), 45.30 (2C, -CH2NCH2). Ms: (MW: 443.00): m/z 443
(M+, 0.36%), 50.10 (100%). Anal. Calc. for C26H21NO6:
C, 70.42; H, 4.77; N, 3.16; O, 21.65, Found: C, 70.51;
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
for different types of cancer as (leukemia, non-small cell
lung cancer, colon cancer, CNS cancer, melanoma, ovar-
ian cancer, prostate cancer, renal cancer, and breast can-
cer).
Synthesis of target 3,5-bis(4-Hydroxy-3-
methoxybenzylidene)-4-oxo-N-phenylpiperidine-1-
carbothioamide (XIIe)
The final target compound XIIe was obtained through a
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
stirring while dropwise addition of (1–3 mL) of triethylamine
along the reflux. The product ‘4-oxo-N-phenylpiperidine-1-
carbothioamide’ was then washed with acetone and fil-
tered. (1 mole, 1.8 g) of that product was then reacted
with (2 moles, 2.4 g) of the available vanillin Ia through
condensation procedure mentioned previously under the
preparation of IIIa–d. The product XIIe was then washed
again with acetone and petroleum ether and subjected to
recrystallization from methanol/benzene.
Cytotoxic study
A semi-automated fluorometric microculture cytotoxicity
assay (FMCA) was used to assess drug sensitivity (27,28).
The method was based on measurement of fluorescence
generated from hydrolysis of fluorescein diacetate (FDA) to
fluorescein by cells with intact plasma membranes. Using
the pipetting robot BioMek 2000 (Beckman Coulter, Fuller-
ton, CA, USA), 384-well microplates (NUNC) were prepared
with 5 lL drug solution in 10 times the final drug concen-
tration. The plates were then stored at À70 °C until further
use. Tumor cells from cell lines (5000 cells/well) were
seeded in the drug-prepared 384-well plates using the pip-
etting robot Precision 2000 (Bio-Tek Instruments, Winooski,
VT, USA). Three columns without drugs served as controls
and one column with medium only served as a blank. The
plates were incubated at 37 °C for 72 h and were then
analyzed using the FMCA. Cell survival, expressed as sur-
vival index (SI), is defined as fluorescence in test wells
divided by fluorescence of control wells, with blank values
subtracted, 9100. Quality criteria for a successful assay
included a mean coefficient of variation of <30% in the con-
trol and a fluorescence signal in control wells of more than
The titled compound was separated as orange powder,
yield: 18.05%, melting point: 236–245 °C. FT-IR (ύ max,
cmÀ1): 3500 (Ar-OH), 3450 (NH), 3010 (Ar C-H), 2890
(C-H), 1750 (ketonic C=O), 1700 (Ar C=C-), 1680 (C=C-),
and 1500 (C=S). 1H NMR (300 MHz, DMSO-d6): d 9.85–
9.84 (s, 2H, 2[OH]), 9.47 (s, 1H, NH), 7.81 (s, 2H,2[-CH=C]),
7.13 (d, 4H, ArH), 7.01–6.92 (m, 7H, ArH), 4.51 (s, 4H,
-CH2NCH2), 4.39 (s, 1H, NH), 3.84–3.81 (s, 6H, 2[OCH3]),
3.17 (s, 2H, 2[OH]). 13C-NMR (300 MHz, DMSO-d6): d
181.94 (1C, C=O), 181.84 (1C, C=S), 150.00 (2C, ArC-O),
149.18 (2C, -CH=), 147.69 (2C, ArC-OH), 147.60 (2C,
Chem Biol Drug Des 2014
7

Fawzy et al.
five times the blank. From the mean SI% curves, the half
maximal inhibitory concentration (IC50) was determined
using nonlinear regression analysis in Prism 5 Software
Package (Graph Pad, San Diego, CA, USA).
Results and Discussion
Pharmacology
Antiproliferative activity
Compound XIIb was selected and tested by the NCI on
60 different cell lines using concentration of 1–5 M and
both mean growth percent and growth percent of the can-
cer cell lines were calculated.
The newly synthesized compounds IVa,c and d, VIa and
VII a,b, IXa and Xa–c, and XIIa–e were screened for their
antiproliferative activities against: ovarian cancer (A2780),
renal adenocarcinoma (ACHN), prostate cancer (PC-3),
colorectal cancer (Hct-116), and a leukemic monocyte
lymphoma (U937-GTB) utilizing the fluorometric microcul-
ture cytotoxicity assay FMCA method.
Tubulin polymerization assay
All compounds have been tested at 10 lM. The sub-
stances’ effect on tubulin polymerization were investigated
using the Tubulin Polymerization Assay Kit (porcine tubulin
and fluorescence based), which utilize fluorescent reporter
enhancement (Cytoskeleton Inc. Denver, CO, USA). Fluo-
rescence was measured using a FLUOstar OPTIMA instru-
From the observed results (Table S1), it was noticed that
most of the synthesized compounds revealed mild to
moderate cytotoxic properties. However, compounds XIIe
and IVc were found to be extremely potent, as both
exhibited considerable antiproliferative activity against the
five cancer cell lines used in the assay with (IC50, concen-
tration required to produce 50% inhibition of cell growth
compared to control experimental) = 1–2.5 lM and 11.4–
23.2 lM, respectively. Compound XIId showed moderate
cytotoxicity with IC50 = 18.9–52.7 lM, while rest of com-
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-
cer cell lines by The American National Cancer Institute
showing mild cytotoxic activity among them, with mean
value 101.15 and range of 35.20 of the growth percents
of the cancer cell lines. The assay was inapplicable on
compound IVb due to its oily nature and poor solubility.
ment. Paclitaxel was used as
a positive control for
microtubule-stabilizing agent and vincristine was used as a
second positive control for microtubule destabilization. The
positive controls were obtained from the Swedish Phar-
macy and diluted with PBS to a final concentration of
3 lM.
Molecular modeling
All molecular modeling studies were performed using Ac-
celrys Discovery Studio 2.5 operating system (Accelrys
Inc., San Diego, CA, USA), at the Faculty of Pharmacy,
Ain Shams University; Cairo, Egypt. Molecules were built
within DS, and conformational models for each com-
pound were generated automatically. This emphasizes
representative coverage over a 20 Kcal/mol energy range
above the estimated global energy minimum, and the
best quality generation technique was chosen. Docking
study involved the following steps: the docking analysis
was carried out on b-chain of tubulin protein. The 3D
protein structure of tubulin cocrystallized with podophyllo-
toxin (code; 1SA1) was downloaded from the Protein
Data Bank of the Research Collaboration for Structural
Bioinformatics (RCSB) Web site [www.rcsb.org]. The col-
chicine binding pocket of the b-chain of tubulin was
docked with podophyllotoxin and the test analogs IVa–d,
VIa and VIIa,b, IXa and Xa-c, and XIIa-e, after deleting
the water structure and a, c chains of tubulin protein,
cleaning the protein, adding the missing hydrogens and
side chains, and energy minimization according to DS
protocol. The binding pocket of the complexed
compound (podophyllotoxin) with the connected amino
acid molecules at sphere of radius = 14 Å was identified
and then docked with test analogs using CDocker-
CHARMm-based technique. After that the docking scores
(-CDOCKER interaction energy) of the best-fitted confor-
mation of each of the docked molecules as well as the
total number of hydrogen bonds and Pi-bonds with the
amino acids at the colchicine binding pocket were
recorded.
Tubulin polymerization assay
The effect of 10 lM of the compounds IVa,c and d, VIa
and VIIa,b, IXa and Xa–c, and XIIa–e on tubulin poly-
merization were investigated using the Tubulin Polymeriza-
tion Assay Kit (porcine tubulin- and fluorescence based),
which utilize fluorescent reporter enhancement (Cytoskele-
ton Inc.). The results are summarized in (Tables S2 and
S3) where inhibition % of the newly synthesized com-
pounds were calculated using (equation 1) ‘Inhibition % =
[((Test sample) – (Vehicle control))/((Control ligand) – (Vehi-
cle control))] 9 100’, and stabilization % were also calcu-
lated using (equation 2) ‘Stabilization
% = {1-[((Test
sample) – (Vehicle control))/((Control ligand) – (Vehicle con-
trol))] 9 100}’.
The in vitro assay revealed that only few compounds inhib-
ited microtubule assembly. Compound VIIa displayed the
highest activity in destabilizing microtubules compared to
the positive control drug, ‘vincristine’ as a microtubule de-
stabilizing agent (Figure S6).
On the other hand, compound XIIc was found to be the
most potent one in stabilizing microtubules compared to
the other positive control drug, ‘paclitaxel’ as an example
of microtubule-stabilizing agent (Figure S7).
8
Chem Biol Drug Des 2014

Curcumin Analogs
Compound XIIc acted by different mechanism where sta-
bilizing microtubules causes chromosomes to become
unable to achieve a metaphase spindle configuration,
which blocks progression of mitosis, also prolonged acti-
vation of the mitotic checkpoint triggers apoptosis or
reversion to the G-phase of the cell cycle without cell
division.
All these findings proved that the in silico study results cor-
related with the in vitro tubulin polymerization assay. A vali-
dation of the ideal pose was also performed by alignment
of the X-ray bioactive conformers of (VIIa and XIIc), with
the best-fitted pose of podophyllotoxin. The alignment
showed good coincidence between them with RMSD =
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-
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-
ing the code 1SA1 obtained from the protein data bank
without change in its conformation to investigate the
detailed intermolecular interactions between the ligand and
the target protein then to validate this study, it was
matched with another peer article (27) where the study of
the binding mode of Podophyllotoxin and chalcone deriva-
tives-structures which mimic our compounds- was carried
out with the same procedure.
attain a pose, and the docking operation did not work out.
Structure–Activity Relationship (SAR)
Based on the in vitro studies, it was concluded that acyla-
tion of heterocyclic nitrogen of 4-piperidone ring is very
essential for enhancing cytotoxic activity. This was sup-
ported by the structure–activity relationship study per-
formed previously on curcumin analogs (9), which stated
the importance of such acylation to render nitrogen atom
in non-basic form to be able to penetrate cell membrane
and hence exerting efficient cytotoxic effect. Also, intro-
duction of thiourea moiety produced potent cytotoxic com-
pounds which coincides with reports describing that
phenylisothiocyanate group is responsible for thiocarbomy-
lation of thiol group of glutathione inside cancerous cells
which leads to its depletion and hence apoptosis (15,16).
Additionally, introduction of iodo group at the aromatic
rings could enhance the tubulin destabilization activity of
compounds as the size of iodine atom just perfectly fitted
into the binding pocket of tubulin; this was concluded from
the in silico docking study performed to iodine containing
compound compared with other halide containing com-
pounds and validated by the results of the in vitro assay.
Interactive docking using CDOCKER protocol was then
applied between the designed analogs IVa–d, VIa and
VIIa,b, IXa and Xa–c, and XIIa–e, and the binding site of
the prepared b-chain of tubulin protein. Each tested mole-
cule gave 10 possible docked poses. The ideal pose of
each molecule was selected according to the similarity of
its binding mode in the binding site to that of podophyllo-
toxin. The corresponding CDOCKER interaction energy
(kcal/mole) was considered in our study to prioritize their
virtual affinity to the binding site, in comparison to the ideal
pose of the podophyllotoxin, curcumin, and other reported
analogs.
Results revealed that these compounds have the ability
to interact with the deep two hydrophobic centers of col-
chicine binding site region in the b-chain of tubulin similar
to podophyllotoxin. One hydrophobic center is sur-
rounded by Met 259, Ala 316, and Lys 352 (occupied by
the benzodioxole fragment in podophyllotoxin), and the
other one is surrounded by Leu 242, Ala 250, and Leu
255 (occupied by the trimethoxyphenyl moiety) (29) (Fig-
ure S8).
Conclusion
Esterification/etherification or sulfurization of vanillin and its
derivatives yielded products that could be condensed suc-
cessfully with 4-piperidone ring, where its heterocyclic
N-atom was then acylated, benzoylated, or reacted with
phenylisothiocyanate to give the targeted compounds IVa–
d, VIa and VIIa,b, IXa and Xa–c, and XIIa–e in high yield.
Compound VIIa, which showed the highest destabilizing
activity on microtubules in silico as well as in the in vitro
tubulin polymerization assay, interacted in the same bind-
ing mode as podophyllotoxin forming an extra hydrogen
bond with Lys 352 (Figure S9).
The target compounds that were tested in vitro for their
cytotoxicity on five different cancer cell lines and for their
activity on tubulin polymerization proved to possess high to
moderate cytotoxic activity. Also, in silico study results were
consistent with that obtained from in vitro tubulin polymeri-
zation assay. Based on the previous results, it was con-
cluded that the thiourea derivative XIIe was the most potent
cytotoxic compound with IC50 values in 1–2.5 lM range,
while it showed moderate effect on tubulin polymerization.
Also compound XIIc with the highest stabilizing activity on
microtubules interacted similarly in the same binding mode
as podophyllotoxin (Figure S10), where it was ranked
within the compounds which showed high interaction
energy in silico while in vitro assay revealed its high stabil-
ization activity mechanism on microtubules.
Compound IVc displayed lower cytotoxicity with IC50 val-
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
it showed failure of docking operation in the in silico study,
whereas compound VIIa destabilized microtubules with
93.3% of inhibition of tubulin and showed cytotoxicity with
IC50 = 36–102.6 lM. On the other hand, compound XIIc
stabilized microtubules with 94.8% and showed cytotoxic-
ity with IC50 = 54.4–187.4 lM. Hence, we concluded that
the synthesized curcumin analogs provide a nucleus for
further optimization, which could lead to the development
of new active chemical entities against cancer via tubulin
targeting.
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Selvakumar P., Sharma R.K., Dimmock J.R. (2010)
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nylcarbonyl]-4-piperidone hydrochloride: a lead tumor-
specific cytotoxin which induces apoptosis and auto-
phagy. Biorg Med Chem Lett;20:912–917.
10. Das U., Alcorn J., Shrivastav A., Sharma R.K., De
Clercq E., Balzarini J., Dimmock J.R. (2007) Design,
synthesis and cytotoxic properties of novel 1-[4-(2-al-
kylaminoethoxy) phenylcarbonyl]-3,5-bis(arylidene)-4-
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bules. PLoS ONE;8:e57302.
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Acknowledgments
The authors are grateful to Professor J.Gullbo; Department
of Medical Sciences, Uppsala University Hospital, Uppsala,
Sweden, for his great collaboration in this study also would
like to express sincere thanks to Future University in Egypt
for funding this research project. Finally, thanks are due to
the department of pharmaceutical chemistry, faculty of
pharmacy, Ain Shams University for providing CADD Lab
facility, the Regional Center for Mycology and Biotechnol-
ogy Al-Azhar University for performing elemental analysis
and American National Cancer Institute for performing
cytotoxic activity test on a selected compound.
€
ꢁ
13. Dyrager C., Wickstrom M., Friden-Saxin M., Friberg A.,
ꢁ
ꢁ
Dahlen K., Wallen E.A., Gullbo J., Grøtli M., Luthman
K. (2011) Inhibitors and promoters of tubulin polymeri-
zation: synthesis and biological evaluation of chalcones
and related dienones as potential anticancer agents.
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2659–2665.
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Supporting Information
Table S3. Stabilization % of target compounds towards
tubulin with paclitaxel as control ligand.
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Curcumin structure (2).
Figure S2. Primary binding site shown on curcumin ana-
log (9).
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