Exploring pyrimidine-substituted curcumin analogues: Design, synthesis and effects on EGFR signaling

Article abstract of DOI:10.1016/j.bmc.2013.06.053

Epidermal growth factor receptor (EGFR) is an effective molecular target of anti-cancer therapies. Curcumin inhibits cancer cell growth in vitro by suppressing gene expression of EGFR and reduces tumor growth in various animal models. To overcome instable and insoluble properties of curcumin as therapeutics, we designed and synthesized six novel pyrimidine-substituted curcumin analogues with or without a hydroxyl group originally present in curcumin. The cell viability tests indicated that IC₅₀ of the analogues containing hydroxyl group were 3 to 8-fold lower than those of the analogues without hydroxyl group in two colon cancer cell lines tested. Western blot analysis indicates the analogues containing hydroxyl group inhibited expression and tyrosine phosphorylation of EGFR. Further protein analyses showed that the analogues had anti-cellular proliferation, pro-apoptosis, and cell cycle arrest properties associated with suppressed EGFR expression. These results indicate that the hydroxyl groups in curcumin and the analogues were critical for observed biological activities.

Full text of DOI:10.1016/j.bmc.2013.06.053

Bioorganic & Medicinal Chemistry 21 (2013) 5012–5020
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploring pyrimidine-substituted curcumin analogues: Design,
synthesis and effects on EGFR signaling q
⇑
Peiju Qiu , Lingling Xu , Lei Gao , Meng Zhang, Shixi Wang, Sheng Tong, Yue Sun, Lijuan Zhang ,
Tao Jiang
⇑
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Epidermal growth factor receptor (EGFR) is an effective molecular target of anti-cancer therapies. Curcu-
min inhibits cancer cell growth in vitro by suppressing gene expression of EGFR and reduces tumor
growth in various animal models. To overcome instable and insoluble properties of curcumin as thera-
peutics, we designed and synthesized six novel pyrimidine-substituted curcumin analogues with or with-
out a hydroxyl group originally present in curcumin. The cell viability tests indicated that IC50 of the
analogues containing hydroxyl group were 3 to 8-fold lower than those of the analogues without hydro-
xyl group in two colon cancer cell lines tested. Western blot analysis indicates the analogues containing
hydroxyl group inhibited expression and tyrosine phosphorylation of EGFR. Further protein analyses
showed that the analogues had anti-cellular proliferation, pro-apoptosis, and cell cycle arrest properties
associated with suppressed EGFR expression. These results indicate that the hydroxyl groups in curcumin
and the analogues were critical for observed biological activities.
Received 9 May 2013
Revised 21 June 2013
Accepted 22 June 2013
Available online 2 July 2013
Keywords:
Apoptosis
Cell cycle
Colon cancer
Curcumin analogues
EGFR
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Curcumin, a compound that is responsible for tumeric thera-
peutic properties in traditional medicine, has been shown to inhi-
Epithelial growth factor receptor family with its most prominent
member of EGFR represents valid targets for anti-cancer therapy.
Anti-EGFRs MoAbs (cetuximab, panitumumab, and trastuzumab)
and TKIs (gefitinib, erlotinib, and lapatinib) have now been ap-
proved for the treatment of advanced colorectal cancer, squamous
cell carcinoma of the head and neck, advanced non small cell lung
cancer, as well as pancreatic and breast cancer because majority
of human cancers are originated from accumulated genetic muta-
tions of epithelia cells and many types of human cancers are asso-
ciated with over-expressed EGFR.1 However, the benefits of costly
TKIs and anti-EGFRs MoAbs treatment to most cancer patients are
very limited due to complications of drug-resistant and side effects.
The median survival rates are only a few months more than those
patients treated with conventional therapies. Hence there is a great
need in developing inexpensive and more effective anti-EGFR
drugs.
bit human colon cancer cell growth by suppressing gene
expression of EGFR through reducing the trans-activation activity
of Egr-1.2 Curcumin also inhibits a variety of tumor growth in var-
ious animal models such as mammary adenocarcinoma, stomach,
duodenal in addition to colon cancers.3–6 Therefore, understanding
the relationship between structure and biological activities of cur-
cumin will be important for developing novel anti-EGFR drugs.
Curcumin (Fig. 1) consists of a phenolic ring, alternating double
bond and b-dicarbonyl group, which form a conjugated
p bond
skeleton,7 wherein the b-dicarbonyl group and substituted phenol
rings are the pharmacophore.8–12 Curcumin can exist in several
tautomeric forms, including a 1,3-diketo form and 2 equiv enolate
forms. The enolate form is more energetically stable in the solid
phase and in solution.13 Curcumin has poor water solubility due
to its rigid and planarity structure, which affects its absorption
from digestive system into blood circulation.14 Moreover, the b-
dicarbonyl group in curcumin is unstable and easily decomposed,
which leads to an even lower blood concentration and further re-
duces pharmacological potency of curcumin.14 Indeed, turmeric
containing ꢀ5% curcumin is pharmacological safe even when con-
sumed up to 100 g per day in diet.15,16 Thus, we have designed
and synthesized six novel pyrimidine-substituted curcumin ana-
logues with or without hydroxyl group in which the plane conju-
gate structure of curcumin was retained while the chemical
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding authors. Tel.: +86 532 82033054.
E-mail addresses: lijuanzhang@ouc.edu.cn (L. Zhang), jiangtao@ouc.edu.cn
(T. Jiang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.053

P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
5013
between structure and biological activity of curcumin analogues
towards EGFR regulations.
2. Results and discussion
2.1. Chemical synthesis of six novel primidine substituted
curcumin analogues
To retain the plane conjugate structure and to improve the
chemical stability and solubility in water and organic solvent of
curcumin, we used the following scheme to synthesize the curcu-
min analogues (Scheme 1).
Reagents and conditions: (i) 4,6-dimethyl-2-hydroxyl-pyrimi-
dine hydrochloride, HCl, EtOH, PhMe, 110 °C, 36 h, 65–70%. (ii)
POCl3, DIPEA, 110 °C, 12 h, 40–45%. (iii) Appropriate amine, EtOH,
90 °C, 12 h, 52–69%.
4,6-Dimethyl-2-hydroxyl-pyrimidine hydrochloride was syn-
thesized in one-pot as previously reported.17
Compounds 4a–f were synthesized following the convergent
synthetic approach depicted in Scheme 1. The compounds 2a–b
were prepared from the appropriate aromatic aldehydes with
4,6-dimethyl-2-hydroxyl-pyrimidine hydrochloride according to
the procedure we reported previously.21
Figure 1. Chemical structure of curcumin.
Subsequently, the compounds 3a–b were synthesized by reflux-
ing a solution of 2a–b with N,N-diethylaniline in an excess of POCl3
overnight in the yields of 40–45%. In the last step, target com-
pounds 4a–f could be prepared by refluxing an ethanol solution
of the appropriate amine with compounds 3a–b in good yields
(52–69%). Comparing with curcumin, the target compounds 4a–f
can be dissolved in polar solvent such as ethanol and in water with
acid as well.
stability and solubility of the analogues were greatly improved
(Fig. 2).17
Colorectal cancer is one of the leading causes of cancer death in
the developed Western countries18 and increases in an alarming
rate in Asian.19 EGFR has been reported to be over-expressed from
25% to 82% in colorectal cancers.20 Therefore, we used two colon
cancer cell lines as a model system to understand the relationships
Figure 2. Chemical structures of 4a–f.

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P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
OH
O
N
N
R1
R2
R1
R2
R1
R2
H
ii
i
R3
R3
R3
1a: R1=OCH3; R2=OCH3; R3=H
2a: R1=OCH3; R2=OCH3; R3=H
1b: R1=OCH3; R2=OCH3; R3=OCH3
2b: R1=OCH3; R2=OCH3; R3=OCH3
R
N
HN
N
Cl
N
N
R1
R2
R1
R2
R1
R2
R1
R2
iii
R3
R3
R3
R3
3a: R1=OCH3; R2=OCH3; R3=H
4a: R1=OCH3; R2=OCH3; R3=H;R=CH2CH3
3b: R1=OCH3; R2=OCH3; R3=OCH3
4b: R1=OCH3; R2=OCH3; R3=H;R=CH2CH2OH
4c: R1=OCH3; R2=OCH3; R3=H;R=CH2CH2CH3
4d: R1=OCH3; R2=OCH3; R3=H;R=CH2(CH2)2OH
4e: R1=OCH3; R2=OCH3; R3=OCH3;R=CH2CH2CH3
4f : R1=OCH3; R2=OCH3; R3=OCH3;R=CH2(CH2)2OH
Scheme 1. Synthesis of compounds 4a–f.
HT29_4e
HT29_4f
HT29_4a
HT29_4b
HCT116_4b
HT29_4c
HT29_4d
HCT116_4e
HCT116_4f
HCT116_4a
HCT116_4c
HCT116_4d
125
100
75
50
25
0
125
100
75
50
25
0
2.5
7.5
12.5
17.5
2.5
7.5
12.5
17.5
2.5
7.5
12.5
17.5
Concentration(µM)
Concentration(µM)
Concentration(µM)
Figure 3. Growth inhibitory effect of 4a–f on HCT116 and HT29 human colon cancer cells. HCT116 and HT29 were seeded in 96-well plates, respectively. After 24 h, cells
were treated with serial concentrations of curcumin analogues. After 48 h of treatment, cell viability was measured by resazurin assay as described under Materials and
Methods. Data are expressed as the mean standard deviation.
2.2. Curcumin analogues with hydroxyl group were more
effective than their counterparts in inhibiting colon cancer cell
growth
inhibition on the cell growth and the analogues with hydroxyl
group showed much stronger grow inhibition effects than their
corresponding curcumin analogues without hydroxyl group
(Fig. 3). For example, three curcumin analogues with hydroxyl
We compared the inhibitory effects of the six analogues on two
colon cancer cell lines using resazurin assay. Three curcumin ana-
logues with hydroxyl group were 4b, 4d, and 4f and their corre-
sponding curcumin analogues without hydroxyl group were 4a,
4c, and 4e, respectively. In both HCT116 and HT29 cells, all five
curcumin analogues except 4e in HT29 showed dose-dependent
group (4b, 4d, and 4f) showed IC50 from 6.2 to 12.8 lM in both
cancer cells, while IC50 of the three analogues without hydroxyl
group (4a, 4c, and 4e) were 3 to 8-fold higher (from 21.9 to
>100 lM) (Table 1).
2.3. Analogue 4b suppressed EGFR expression and
phosphorylation
Table 1
Curcumin is a component of turmeric. Turmeric is a spice derived
from a member of ginger family. Traditional medicine uses turmeric
for biliary disorders, anorexia, cough, diabetic wounds, hepatic dis-
orders, rheumatism, and sinusitis.22 Modern research indicates cur-
cumin is responsible for many of tumeric therapeutic properties. It is
subsequently demonstrated that curcumin inhibits human colon
cancer cell growth by suppressing gene expression of EGFR.2
Data in Figure 3 and Table 1 showed that the curcumin ana-
logues inhibited the growth of two human colon cells. Since EGFR
might be the most important growth signaling pathway in the two
colon cancer cell lines, we then tested if the observed cytotoxicity
Compound
IC50 SDa
(lM)
HT29
HCT116
4a
4b
4c
4d
4e
4f
38.5 25.4
7.1 0.4
>100
12.8 1.9
>100
46.4 23.6
6.2 1.2
28.0 12.7
9.9 6.9
21.9 7.0
7.4 1.8
12.4 0.9
a
IC50, compound concentration required to inhibit cell proliferation by 50%.

P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
5015
was due to suppressed EGFR expression and phosphorylation by
the analogues.
tional in several major cancers. One function of Rb is to prevent
excessive cell growth by inhibiting cell cycle progression.
As shown in Figure 4, after incubation with analogue 4a or 4b
As shown in Figure 5C, 4a at 20 lM and 4b at 5, 10, and 20 lM
for 72 h in both colon cancer cells, 4a at 20
lM concentration did
concentrations induced significant p21WAF-1/CIP-1 expression (20.0,
not significantly decrease the levels of P-EGFR(Tyr1068) or EGFR
in both of HCT116 and HT29 cells. In contrast, 4b significantly de-
creased the levels of both phosphorylated P-EGFR(Tyr1068) and
EGFR in a concentration-dependent manner.
21.4, 28.6, and 25.7 fold-increases, respectively). Analogue 4a at
20 lM did not induce significant changes in all other proteins
tested. In contrast, 4b inhibited expression of Cyclin D1, Cyclin
E1, and Rb in a concentration-dependent manner even though 4b
did not induce significant changes in CDK2 and CDK4 proteins at
all concentrations tested.
2.4. Analogue 4b induced cell cycle arrest in a concentration-
dependent manner
We concluded from the data in Figure 5 that the curcumin ana-
logues, especially 4b, caused cell cycle arrest in both HCT116 and
HT29 human colon cancer cells by inducing significant p21WAF-1/
It was reported that curcumin inhibits HT29 colon cancer cell
growth by suppressing gene expression of EGFR.2 We found that
the curcumin analogues inhibited EGFR expression and phosphor-
ylation in both HT29 and HCT116 cells (Fig. 4). It was reported in
literature that HCT116 cells treated with curcumin are largely
accumulated in G2/M phase.23 We thus tested how the curcumin
analogues affect cell cycles in both HCT116 and HT29 cells. As
shown in Figure 5A (HCT116 cells), 4a at the concentration of
CIP-1
expression. Analogue 4b also inhibited CyclinD1, CyclinE1,
and Rb expression in a concentration-dependent manner.
2.5. Effects of the curcumin analogues on proteins involved in
apoptosis
Studies indicated that intracellular caspases are one of the ma-
jor signaling pathways involved in apoptotic cell death. Caspases
are a family of structurally related cysteine proteases. For example,
caspases-2, -3, -6, -7 and -9 can cleave poly (ADP ribose) polymer-
ase (PARP).24 Curcumin was reported to activate caspases 3, 8, and
9 in the colon cancer cell lines SW480 and SW620.25
We first examined the expression levels of activated caspase 3
and cleaved PARP because PARP is a substrate of caspase 3 after
treating the HCT116 colon cancer cells with or without the ana-
20 lM and 4b at the concentrations of 5, 10, and 20 lM increased
G2/M cell population to 1.03-, 1.54-, 1.78-, and 2.05-fold of that of
the control cells, respectively. In HT29 cells (Fig. 5B), 4a at the con-
centration of 20
20 M increased G2/M cell population to 1.10-, 1.86-, 1.94-, and
2.40-fold of that of the control cells, respectively. Interestingly,
4b at 5 M concentration induced G0/G1 cell cycle arrest in both
lM and 4b at the concentrations of 5, 10, and
l
l
HCT116 and HT29 cells.
It was reported that curcumin induced G2/M cell cycle arrest is
accompanied by increased p21WAF-1/CIP-1 expression.5 p21WAF-1/CIP-1
is a potent cyclin-dependent kinase inhibitor (CKI). p21WAF-1/CIP-1
binds to and inhibits the activity of cyclin-CDK2 or cyclin-CDK1
complexes, and thus functions as a regulator of cell cycle progres-
logues. As shown in Figure 6A, 4a at 20 lM concentration did not
cause any noticeable change in both caspase 3 and PARP. In con-
trast, 4b caused activated caspase 3 and cleaved PARP in a concen-
tration-dependent manner.
Heat shock proteins (HSP) are a class of functionally related pro-
teins involved in the folding and unfolding of other proteins. Their
expression is increased when cells are exposed to elevated temper-
atures or other stress including harmful chemicals. It has been
established that HSP70 directly inhibits apoptosis whereas HSP90
stabilizes various growth factor receptors including EGFR. Hence
inhibition of HSP90 may induce apoptosis.26 We found that 4a at
sion. Since 4b at 5 lM concentration induced both G0/G1 and G2/
M cell cycle arrests (Fig. 5A and B), we assessed cell cycle signaling
checkpoint proteins, including p21WAF-1/CIP-1, CDK2, CDK4, Cyclin
D1, Cyclin E1, and Rb systematically in HCT116 cells after treating
the cells with or without the analogues. Rb stands for retinoblas-
toma protein, which is a tumor suppressor protein that is dysfunc-
20 lM concentration had no effect on the expression of HSP-70
and HSP-90, while 4b decreased the levels of HSP90 and HSP70
expressions in a concentration-dependent manner.
Concentration
(A)
(μM)
Bcl-2 is a family of evolutionarily related proteins. These pro-
teins govern mitochondrial outer membrane permeabilization
and can be either pro-apoptotic, such as Bax, or anti-apoptotic such
as Bcl-2.27 We measured the levels of Bcl-2 and Bax expression in
HCT116 colon cancer cells after treating the cells with different
concentrations of 4a and 4b for 72 h. Compound 4a decreased
slightly the level of anti-apoptotic Bcl-2 expression but increased
the level of pro-apoptotic protein Bax expression (Fig. 6B). Com-
pound 4b had a stronger effect on the expression of Bcl-2 and
Bax and the effect was concentration-dependent.
P-EGFR(Tyr1068)
1.0
1.0
0.9
0.7
0.8
0.5
0.4
0.3
0.3
EGFR
0.5
β-tubulin
Concentration
(B)
(μM)
P-EGFR(Tyr1068)
A quantification of BAX and Bcl-2 expression indicated that 4a
at 20 lM concentration increased the ratio of Bax/Bcl-2 by 1.57-
fold compared to the untreated control, whereas 4b increased
the ratio of Bax/Bcl-2 by 2.1-, 17-, and 19-fold at 5, 10, and
1.0
1.0
1.0
1.4
0.6
1.4
0.6
0.1
0.6
EGFR
0.4
20 lM concentrations, respectively. Therefore, the curcumin ana-
β-tubulin
logues worked on multiple protein targets to promote apoptosis
in the human colon cell line HTC116, which was consistent with
the multiple functions of EGFR signaling.
Figure 4. Effects of the curcumin analogues on EGFR and P-EGFR (Tyr1068) in
human colon cancer cells HCT116 and HT29. Both cells were seeded in 15 cm dishes
for 24 h, and then cells were treated with 4a at 20 lM or 4b at 5, 10 and 20 lM,
respectively. After 72 h of incubation, cells were harvested for Western blotting
analysis. The numbers underneath the blots represent band intensities (normalized
to the loading controls, means of three independent experiments) measured by the
Image J software. The standard deviations were all within 15% of the means (data
not shown). b-Tubulin was used as equal loading controls. The experiments were
repeated for three times.
3. Conclusions
In summary, we describe the synthesis of six novel pyrimidine-
substituted curcumin analogues with or without hydroxyl group in
which the critical plane conjugate structure of curcumin was

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P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
(A)
4b
4b
4a
4b
control
20µM
5µM
20µM
10µM
DNA Content
DNA Content
DNA Content
DNA Content
DNA Content
(B)
4b
4b
control
4a
4b
20µM
10µM
20µM
5µM
DNA Content
DNA Content
DNA Content
DNA Content
4b 5µM
DNA Content
control
4a 20µM
4b 10µM
4b 20µM
Figure 5. Cell cycle-related analysis of human colon cancer cells after treating with the curcumin analogues. HCT-116(A) and HT29 (B) cells after treating with the curcumin
analogues 4a or 4b. The HCT116 and HT29 cells were seeded in 6-well plates (4 Â 104/ml, 2 ml/well) for 24 h, and then the cells were treated with 4a at 20
lM or 4b at 5, 10
and 20 lM concentrations. After 24 h, cells were harvested and subject to cell cycle analyses as described in Section 4. All results shown are mean SD, and are representative
of three independent assays. Statistical analyses were conducted among control and treated groups in G0/G1, S, and G2/M phases. (C) Effects of 4a and 4b on cell cycle related
proteins in HCT116 human colon cancer cells.
retained while the chemical stability and solubility were improved.
The cell viability tests indicated that IC50 of the three curcumin
analogues containing hydroxyl group was 3 to 8-fold lower than
that of the three analogues without hydroxyl group in two colon
cancer cell lines tested. Western blot analysis indicates the ana-
logues containing hydroxyl group inhibited expression and tyro-
sine phosphorylation of EGFR. Cell cycle and further cell cycle
and apoptotic check point protein analyses showed that the ana-
logues had expected anti-cellular proliferation, pro-apoptosis,
and cell cycle arresting properties associated with suppressed
EGFR expression and activation. These results suggest that the hy-
droxyl group in curcumin and the analogues (Fig. 7) might be crit-
ical for EGFR regulating activities.
4. Experimental section
4.1. Cell culture
Human colon cancer cells HCT116 and HT29 were obtained
from Shanghai Cell Bank of Chinese Academy of Science (China)
and were maintained in McCoy’s 5A (Sigma–Aldrich) supple-
mented with 5% heat inactivated FBS (Hyclone), 100 U/mL of pen-
icillin, and 0.1 mg/mL streptomycin (Sigma–Aldrich) at 37 °C with
5% CO2 and 95% air. Cells were kept subconfluent, and media were
changed every other day. All cells used were within 3–30 passages.
DMSO was used to dissolve the six curcumin analogues, and the fi-
nal concentration of DMSO in all culture media was 0.1%. PI/RNase

P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
5017
d = 151.1, 149.6, 138.8, 128.6, 122.7, 112.3, 110.4, 56.1 ppm; MS
(ESI) m/z 421.2 [M+H]+.
(C)
Concentration
(μM)
4.3.2. (E,E)-4,6-Bis(3,4,5-trimethoxystyryl)-2-hydroxyl-
pyrimidine (2b)
p21 Cip1/Waf1
1.0
20.0
21.4
28.6
25.7
The titled compound 2b was obtained from 4,6-dimethyl-2-hy-
droxyl-pyrimidine hydrochloride (2.18 g, 15 mmol), 3,4,5-trimeth-
oxybenzaldehyde (8.83 g, 45 mmol), conc HCl (3.75 mL 45 mmol),
anhydrous EtOH (150 mL) and toluene (50 mL) according to the
procedure described above as brown powder (4.68 g, 65%):
Rf = 0.20 (CHCl3/MeOH 9:1); mp: 222–224 °C, 1H NMR (600 MHz,
[D6]DMSO): d = 11.71 (s, 1H), 7.79 (d, J = 16.5 Hz, 2H), 6.99 (m,
7H); 3.85 (s, 12H); 3.71 ppm (s, 6H); 13C NMR (150 MHz, [D6-
]DMSO): d = 153.7, 139.6, 139.0, 131.4, 107.6, 105.7, 67.2, 60.7,
56.5 ppm; MS (ESI) m/z 481.2 [M+H]+.
General method for the synthesis of compounds (3a–b). A
mixture of compound 2a–b (3.0 mmol) and POCl3 (25 mL) was
stirring in a round-bottomed flask (50 mL), then DIPEA (1 mL,
5.8 mmol) was added and the mixture was refluxed at 100 °C
for 2 h. After completion of the reaction, the POCl3 was evapo-
rated under reduce pressure and the residue was poured into cold
saturated NaHCO3 solution. The precipitate was collected by fil-
tration and washed with water to give crude product. The crude
product was purified through column chromatography on silica
gel (eluent dichloromethane/methanol) to give compounds 3a–b
as yellow powder.
CDK4
1.0
1.0
1.1
0.9
1.2
0.7
1.0
1.1
0.7
0.4
0.8
0.8
Cyclin D1
0.8
1.2
0.03
CDK2
1.0
1.0
1.0
0.9
Cyclin E1
0.8
1.1
0.3
0.2
0.04
0.2
Rb
β-actin
Fig. 5 (continued)
staining buffer was purchased from BD Pharmingen. All other
chemicals are of analytical grade.
4.2. General methods for chemical synthesis
4.3.3. (E,E)-4,6-Bis(3,4-dimethoxystyryl)-2-chloro-pyrimidine
(3a)
All starting materials and solvents were obtained from com-
mercial sources and used without further purification. Thin-layer
chromatography (TLC) was performed on precoated E. Merck sil-
ica-gel 60 F254 plates. Column chromatography was performed
on silica gel (200–300 mesh). Melting points were determined on
a Mitamura–Riken micro-hot stage and were not corrected. 1H
NMR and 13C NMR spectra were obtained on a Jeol JNM-ECP 600
spectrometer with tetramethylsilane (Me4Si) as the internal stan-
dard, and chemical shifts were recorded in d values. Mass spectra
were recorded on a Q-TOF Global mass spectrometer.
The title compound 3a was obtained from 2a (1.26 g, 3.0 mmol),
POCl3 (25 mL) and DIPEA (1 mL, 5.8 mmol) according to the proce-
dure described above as yellow powder (591 mg, 45%): Rf = 0.90
(CH2Cl2/MeOH 20:1); mp: 108–110 °C; 1H NMR (600 MHz, CDCl3):
d = 7.85 (d, J = 15.9, 2H), 7.16–7.15 (dd, J = 8.22, 1.44, 2H), 7.12–
7.11 (d, J = 2.22, 2H), 7.05 (s, 2H), 6.90 (s, 1H), 6.88 (s, 1H), 6.86
(d, J = 15.96, 2H), 3.92 (s, 6H), 3.91 ppm (s, 6H); 13C NMR
(150 MHz, CDCl3): d = 165.8, 161.5, 150.8, 149.3, 138.6, 128.4,
122.5, 122.2, 113.8, 111.2, 109.6, 56.0 ppm; MS (ESI) m/z 439.1
[M+H]+.
4.3. General procedure for the synthesis of compounds 4a–f
4.3.4. (E,E)-4,6-Bis(3,4,5-trimethoxystyryl)-2-chloro-pyrimidine
(3b)
4,6-Dimethyl-2-hydroxyl-pyrimidine hydrochloride (1 equiv)
and appropriate substituted benzaldehydes (3 equiv) were dis-
solved in anhydrous ethyl alcohol and toluene in a round-bot-
tomed flask (500 mL). Then concentrated hydrochloric acid
(3 equiv) was added and the mixture was refluxed at 100 °C for
36 h. After completion of the reaction, the solvent was evaporated
under reduce pressure. The saturated NaHCO3 solution (250 mL)
was added into the brown oil residue for desalting. After stirring
at room temperature for 5 h, the resulting precipitate was filtered
and washed with water and ether to obtain crude product and then
purified by chromatography on silica gel (eluent, dichloromethane/
methanol) to give 2a–b as brown powder.
The title compound 3b was obtained from 2b (1.44 g,
3.0 mmol), POCl3 (25 mL) and DIPEA (1 mL, 5.8 mmol) according
to the procedure described above as yellow powder (672 mg,
45%): Rf = 0.80 (CH2Cl2/MeOH 20:1); mp: 104–106 °C; 1H NMR
(600 MHz, CDCl3): d = 7.85–7.82 (d, J = 15.84, 2H), 7.19 (s, 1H),
6.93–6.91 (d, J = 15.42, 2H), 6.83 (s, 4H), 3.91 (s, 12H), 3.90 ppm
(s, 6H); 13C NMR (150 MHz, CDCl3): d = 165.64, 161.54, 153.57,
139.91, 138.81, 130.89, 123.82, 114.24, 105.11, 56.24 ppm; MS
(ESI) m/z 499.1 [M+H]+.
General method for the synthesis of compounds (4a–f). A mix-
ture of compound 3a–b (1 mmol) and appropriate amine
(10 mmol) in anhydrous EtOH (50 mL) was stirred at 90 °C for
12 h and monitored with TLC. When the reaction was finished,
the solvent was evaporated under reduce pressure and the residue
was purified by column chromatography (silica gel dichlorometh-
ane/methanol) to yield the desired compounds.
4.3.1. (E,E)-4,6-Bis(3,4-dimethoxystyryl)-2-hydroxyl-pyrimidine
(2a)
The titled compound 2a was obtained from 4,6-dimethyl-2-hy-
droxyl-pyrimidine hydrochloride (2.18 g, 15 mmol), 3,4-dime-
thoxybenzaldehyde (7.48 g, 45 mmol), conc HCl (3.75 mL
45 mmol), anhydrous EtOH (150 mL) and toluene (50 mL) accord-
ing to the procedure described above as brown powder (4.41 g,
70%): Rf = 0.20 (CHCl3/MeOH 9:1); mp: 255–257 °C; 1H NMR
(600 MHz, [D6]DMSO): d = 11.55 (s, 1H), 7.80–7.77 (d,
J = 17.28 Hz, 2H), 7.29 (s, 2H), 7.20 (d, J = 7.74 Hz, 2H), 7.04–7.02
(d, J = 7.92 Hz, 2H), 6.94–6.92 (d, J = 15.42 Hz, 2H), 6.85 (s, 1H),
3.84 (s, 6H), 3.81 ppm (s, 6H); 13C NMR (150 MHz, [D6]DMSO):
4.3.5. 4,6-Bis((E)-3,4-dimethoxystyryl)-N-ethylpyrimidin-2-
amine (4a)
The title compound 4a was obtained from 3a (0.44 g 1 mmol),
ethylamine (3 mL) and anhydrous EtOH (50 mL) according to the
procedure described above as yellow powder (232 mg, 52%):
Rf = 0.50 (CH2Cl2/MeOH 20:1); mp: 74–76 °C; 1H NMR (600 MHz,
CDCl3): d = 7.71 (d, J = 15.9, 2H), 7.14 (d, J = 6.6, 4H), 6.88 (d,

5018
P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
(A)
Concentration
(μM)
Full length PARP
Cleaved PARP
1.0
0.9
0.9
3.5
2.5
3.8
Cleaved caspase 3
HSP-90
1.0
1.0
2.7
30.5
38.1
0.7
0.9
0.7
0.8
0.8
HSP-70
1.0
0.7
0.8
0.3
β-actin
(B)
Concentration
(μM)
Bcl-2
1
0.7
1.1
0.7
1.5
0.1
1.7
0.1
1.9
Bax
1.0
β-actin
20
16
12
8
4
0
Control20
μM5
μM10
μM
20μM
Figure 6. Effects of the curcumin analogues on apoptosis-related proteins in HCT116 human colon cancer cells after treating with the curcumin analogues. HCT116 cells were
seeded in 15 cm dishes for 24 h, and then cells were treated with 4a at 20 M or 4b at 5, 10 and 20 M. After 72 h of incubation, cells were harvested for Western blotting
l
l
analysis as described in Section 4. The numbers underneath of the blots represent band intensity (normalized to b-actin, means of three independent experiments) measured
by Image J software. The standard deviations were all within 15% of the means (data not shown). b-Actin was served as an equal loading control. The experiments were
repeated three times.
J = 8.82, 2H), 6.84 (s, 1H), 6.81 (s, 1H), 6.67 (s, 1H), 5.32 (t, J = 6.6,
1H), 3.95 (s, 6H), 3.91 (s, 6H), 3.58 (m, 2H), 1.29 ppm (t, J = 8.34,
3H); 13C NMR (150 MHz, CDCl3): d = 163.70, 162.50, 150.10,
149.22, 135.45, 129.28, 125.03, 121.60, 111.16, 109.39, 106.45,
56.02, 36.45, 15.24 ppm; HRMS m/z [M+H]+ calculated for
136.1, 129.0, 124.4, 121.7, 111.2, 109.4, 107.0, 64.7, 56.0,
45.1 ppm; HRMS m/z [M+H]+ calcd for C26H30N3O5: 464.2180,
found 464.2187.
4.3.7. 4,6-Bis((E)-3,4-dimethoxystyryl)-N-propylpyrimidin-2-
amine (4c)
C26H30N3O4: 448.2231, found 448.2225.
The title compound 4c was obtained from 3a (0.44 g 1 mmol),
propylamine (3 mL) and anhydrous EtOH (50 mL) according to
the procedure described above as yellow powder (258 mg, 56%):
Rf = 0.40 (CH2Cl2/MeOH 20:1); mp: 67–69 °C; 1H NMR (600 MHz,
CDCl3): d = 7.71 (d, J = 15.4, 2H), 7.13 (d, J = 4.38, 4H), 6.87 (d,
J = 8.82, 2H), 6.83 (d, J = 15.4, 2H), 6.66 (s, 1H), 5.09 (t, J = 5.49,
1H), 4.12 (q, J = 7.14, 2H), 3.94 (s, 6H), 3.90 (s, 6H), 3.52 (q,
J = 6.87, 2H), 1.16 (m, 2H), 1.25 (t, J = 7.14, 3H), 1.03 ppm (t,
J = 7.41, 3H); 13C NMR (150 MHz, CDCl3): d = 163.7, 162.7, 150.0,
149.2, 135.3, 129.3, 125.1, 121.5, 111.2, 109.4, 106.3, 56.0, 43.4,
23.1, 11.7 ppm; HRMS m/z [M+H]+ calcd for C27H32N3O4:
462.2387, found 461.2382.
4.3.6. 2-(4,6-Bis((E)-3,4-dimethoxystyryl)pyrimidin-2-
ylamino)ethanol (4b)
The title compound 4b was obtained from 3a (0.44 g 1 mmol),
2-aminoethanol (3 mL) and anhydrous EtOH (50 mL) according to
the procedure described above as yellow powder (315 mg, 68%):
Rf = 0.45 (CH2Cl2/MeOH 20:1); mp: 145–147 °C; 1H NMR
(600 MHz, CDCl3): d = 7.66 (d, J = 15.9, 2H), 7.15 (d, J = 2.16, 1H),
7.13 (d, J = 2.22, 1H), 7.12 (d, J = 1.68, 2H), 6.88 (d, J = 8.22, 2H),
6.82 (s, 1H), 6.79 (s, 1H), 6.71 (s, 1H), 5.53 (t, J = 5.79, 1H), 3.94
(s, 6H), 3.92 (s, 6H), 3.91 (t, J = 4.98, 2H), 3.70 ppm (q, J = 5.46,
2H); 13C NMR (150 MHz, CDCl3): d = 163.9, 163.0, 150.3, 149.2,

P. Qiu et al. / Bioorg. Med. Chem. 21 (2013) 5012–5020
5019
4.4. Cellular viability assay
For cellular viability assay, a 96-wells plate (Nest) was seeded
with 2000 cells/well in complete medium. After 24 h, the medium
was removed and 200
amount of drugs was added to each well. The cells were incubated
for 48 h, followed by adding 20 L of resazurin (2 mg/mL dissolved
lL complete medium containing different
l
in water, catalog no. R7017-5G, Sigma) to the media for 16 h. The
fluorescent signal was monitored using 544 nm excitation wave-
length and 595 nm emission wavelength by Spectramax M5 plate
reader (Molecular Devices). The relative fluorescence unit (RFU)
generated from the assay was proportional to the number of living
cells in each well.28 The IC50 value of each drug was calculated by
the Logit approach.
4.5. Cell cycle analyses
HCT116 and HT29 cells were seeded in 6-well plates
(4 Â 104 cells/ml, 2 ml/well). After 24 h incubation for attach-
ment, cells were treated with or without different amount of
4a or 4b in 2 ml of complete media. After another 24 h, media
was collected and combined with adherent cells that were de-
tached by brief trypsinization (0.25% trypsin-EDTA; Sigma–Al-
drich). Cell pellets were washed with 1 ml of ice-cold PBS two
times and then resuspended in 1 ml of 70% ethanol in 4 °C over-
night. After centrifugation (1600g, 1 min), the supernatant was
removed and cells were incubated with 0.5 ml of PI/RNase stain-
ing buffer for 15 min at room temperature. Single-cell suspension
was generated by gentle pipetting. Cell cycle was analyzed using
a FC500 MCL/MPL cell analyzer and data were processed using
MultiCycle software.29
Figure 7. Enolate form of curcumin might be critical for EGFR regulating activities.
4.3.8. 3-(4,6-Bis((E)-3,4-dimethoxystyryl)pyrimidin-2-
ylamino)propan-1-ol (4d)
The title compound 4d was obtained from 3a (0.44 g 1 mmol),
3-amino-1-propanol (3 mL) and anhydrous EtOH (50 mL) accord-
ing to the procedure described above as yellow powder (253 mg,
53%): Rf = 0.34 (CH2Cl2/MeOH 20:1); mp: 142–144 °C; 1H NMR
(600 MHz, CDCl3): d = 7.64 (d, J = 15.9, 2H), 7.14 (d, J = 1.62, 1H),
7.12 (s, 1H), 7.11 (s, 2H), 6.87 (d, J = 8.28, 2H), 6.81 (d, J = 15.9,
2H), 6.7 (s, 1H), 5.56 (t, J = 6.6, 1H), 3.93 (s, 6H), 3.90 (s, 6H),
3.72(q, J = 6.33, 2H), 3.68 (t, J = 5.52, 2H), 1.79 ppm (m, 2H); 13C
NMR (150 MHz, CDCl3): d = 163.9, 163.0, 150.3, 149.2, 136.1,
128.9, 124.3, 121.7, 111.1, 109.4, 106.2, 58.4, 55.9, 37.2,
33.3 ppm; HRMS m/z [M+H]+ calcd for C27H32N3O5: 478.2336,
found 478.2330.
4.6. Whole cell lysate preparation
Cells were seeded in cell culture dishes at a density of 8 Â 104 -
cells/ml and 20 ml per dish in McCoy’s 5A medium containing 5%
FBS for 24 h. Different amount of 4a or 4b were then added directly
to the medium of the cultured cells and incubated for another 72 h.
Cells were rinsed with PBS and total cell lysates were prepared by
adding 200 lL of whole cell lysis buffer containing 20 mM Tris–
HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton,
2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM
Na3VO4, 1 lg/ml leupeptin (pH 7.5) to each dish and collected with
cell scrapers into microcentrifuge tubes. Cell suspensions were
then subjected to sonication (5s, three times). After further incuba-
tion for 30 min, supernatants were collected by centrifugation at
10,000g for 10 min.30 Protein concentrations were determined by
BCA protein assay kit (Beyotime Institute of Biotechnology, China),
following manufacturer’s instruction.
4.3.9. 4,6-Bis((E)-3,4,5-trimethoxystyryl)-N-propylpyrimidin-2-
amine (4e)
The title compound 4e was obtained from 3b (0.50 g 1 mmol),
propylamine (3 mL) and anhydrous EtOH (50 mL) according to
the procedure described above as yellow powder (339 mg, 65%):
Rf = 0.35 (CH2Cl2/MeOH 20:1); Mp:102–104 °C; 1H NMR (CDCl3,
600 M) d 7.69 (2H, d, J = 15.9), 6.87 (2H, d, J = 15.9), 6.81 (4H, s),
6.70 (1H, s), 5.11–5.10 (1H, t, J = 5.22), 3.91–3.88 (18H, s), 3.51–
3.50 (2H, q, J = 6.6), 1.62–1.67 (2H, m), 1.05–1.01 (3H, t, J = 7.41)
ppm; 13C NMR (CDCl3, 150 M) d 163.5, 162.7, 153.4, 139.0, 135.5,
131.8, 126.4, 106.5, 104.6, 61.0, 56.2, 43.4, 23.1, 11.7 ppm; HRMS
+
(ESI) m/z calcd for C29H36N3O6 522.2599, found 522.2584.
4.7. Preparation of cell membrane fraction
4.3.10. 3-(4,6-Bis((E)-3,4,5-trimethoxystyryl)pyrimidin-2-
ylamino)propan-1-ol (4f)
Trypsinized cells were washed with ice-cold PBS and then incu-
bated in the presence of buffer (10 mM Tris–HCl, pH 7.5, 1 mM
EDTA, 0.1 mM DTT) containing protease inhibitor cocktail (1 mM
Na3VO4, 1 mM leupeptin, 1 mM PMSF). The cell suspension was
sonicated 3s on ice four times followed by centrifugation at
100,000g for 1 h. The pellets were solubilized with lysis buffer
(20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1% Triton,
2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate) sup-
plemented with protease inhibitor cocktail (1 mM Na3VO4, 1 mM
leupeptin, 1 mM PMSF) on ice for 30 min after thorough mixing.
After centrifugation at 10,000g for 30 min, the cell membrane frac-
tion present in supernatant was collected.29
The title compound 4f was obtained from 3b (0.50 g 1 mmol), 3-
amino-1-propanol (3 mL) and anhydrous EtOH (50 mL) according
to the procedure described above as yellow powder (376 mg,
70%): Rf = 0.30 (CH2Cl2/MeOH 20:1); mp: 102–104 °C; 1H NMR
(600 MHz, CDCl3): d = 7.64 (d, J = 15.9, 2H), 6.88 (d, J = 15.36, 2H),
6.81 (s, 4H), 6.74 (s, 1H), 5.30–5.28 (t, J = 6.87, 1H), 3.91 (s, 12H),
3.89 (s, 6H), 3.76–3.73 (q, J = 6.22, 2H), 3.69–3.68 (t, J = 5.22, 2H),
1.83–1.79 ppm (m, 2H); 13C NMR (150 MHz, [D6]DMSO):
d = 163.18, 153.51, 139.35, 136.22, 131.59, 125.95, 106.82,
104.78, 61.08, 58.42, 56.25, 37.24, 33.42 ppm; HRMS m/z [M+H]+
calcd for C29H36N3O7: 538.2548, found 538.2537.

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5. Liu, E.; Wu, J.; Cao, W.; Zhang, J.; Liu, W.; Jiang, X.; Zhang, X. J. Neurooncol. 2007,
4.8. Western blotting assay
85(3), 263.
6. Shehzad, A.; Lee, J.; Lee, Y. S. Biofactors 2013.
For immunoblot analyses, equal amounts of proteins (20–
300 lg, depending on the proteins of interest) were subjected to
7. Pascal, L.; Eynde, J. J. V.; Van Haverbeke, Y.; Dubois, P.; Michel, A.; Rant, U.;
Zojer, E.; Leising, G.; Van Dorn, L. O.; Gruhn, N. E. J. Phys. Chem. B 2002, 106(25),
6442.
12% SDS–polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The membranes were incubated in
blocking buffer (Tris–buffered saline-0.05% Tween-20 (TBST) con-
taining 5% nonfat milk) at room temperature for 1 h, then incu-
bated with appropriate antibodies according to the protocols of
the manufacturers and washed with TBST. After incubation with
appropriate secondary antibodies, the membranes were washed
three times with Tris–buffered saline containing Tween-20 and
then visualized using enhanced chemiluminescence. Antibodies
for PARP, BAX, Bcl-2, HSP-70, HSP-90, p21WAF-1/CIP-1, P-EGFR
(Tyr1068), Rb, cyclinD1, cleaved caspase-3, caspase-8, CDK-2,
CDK-4 and b-actin were obtained from Cell Signaling Technology,
Inc. Antibodies for EGFR, cyclin E1, b-tubulin were purchased from
Immunoway, Inc.
8. Masuda, T.; Hidaka, K.; Shinohara, A.; Maekawa, T.; Takeda, Y.; Yamaguchi, H. J.
Agric. Food. Chem. 1999, 47(1), 71.
9. Mishra, S.; Narain, U.; Mishra, R.; Misra, K. Bioorg. Med. Chem. 2005, 13(5), 1477.
10. Kumar, S.; Narain, U.; Tripathi, S.; Misra, K. Bioconjugate Chem. 2001, 12(4), 464.
11. Ferrari, E.; Lazzari, S.; Marverti, G.; Pignedoli, F.; Spagnolo, F.; Saladini, M.
Bioorg. Med. Chem. 2009, 17(8), 3043.
12. Ishida, J.; Ohtsu, H.; Tachibana, Y.; Nakanishi, Y.; Bastow, K. F.; Nagai, M.;
Wang, H. K.; Itokawa, H.; Lee, K. H. Bioorg. Med. Chem. 2002, 10(11), 3481.
13. Kolev, T. M.; Velcheva, E. A.; Stamboliyska, B. A.; Spiteller, M. Int. J. Quantum
Chem. 2005, 102(6), 1069.
14. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Mol. Pharm.
2007, 4(6), 807.
15. Bradford, P. G. Biofactors 2013.
16. Chainani-Wu, N. J. Altern. Complement. Med. 2003, 9(1), 161.
17. Vlad, G.; Horvath, I. T. J. Org. Chem. 2002, 67(18), 6550.
18. Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Thun, M. J. CA Cancer J. Clin. 2009,
59(4), 225.
19. Sung, J. J.; Lau, J. Y.; Goh, K. L.; Leung, W. K. Lancet Oncol. 2005, 6(11), 871.
20. Normanno, N.; De Luca, A.; Bianco, C.; Strizzi, L.; Mancino, M.; Maiello, M. R.;
Carotenuto, A.; De Feo, G.; Caponigro, F.; Salomon, D. S. Gene 2006, 366(1), 2.
21. Gao, L.; Liu, Q.; Ren, S.; Wan, S.; Jiang, T.; Wong, I. L. K.; Chow, L. M. C.; Wang, S.
J. Carbohydr. Chem. 2012, 31(8), 620.
Acknowledgments
This work was supported by Natural Science Foundation of Chi-
na (Grant Nos. 21171154 and 91129706), Special Fund for Marine
Scientific Research in the Public Interest (01005024), and Program
for Science and Technology Development of Shandong Province
(Grant No. 2012GSF11912).
22. Gupta, S. C.; Kismali, G.; Aggarwal, B. B. Biofactors 2013.
23. Lu, J. J.; Cai, Y. J.; Ding, J. Mol. Cell. Biochem. 2011, 354(1–2), 247.
24. Fernandes-Alnemri, T.; Litwack, G.; Alnemri, E. S. J. Biol. Chem. 1994, 269(49),
30761.
25. Rashmi, R.; Santhosh, K. T.; Karunagaran, D. FEBS Lett. 2003, 538(1–3), 19.
26. Nollen, E. A.; Morimoto, R. I. J. Cell Sci. 2002, 115(Pt 14), 2809.
27. Murphy, K. M.; Ranganathan, V.; Farnsworth, M. L.; Kavallaris, M.; Lock, R. B.
Cell Death Differ. 2000, 7(1), 102.
28. Pan, J.; Qian, Y.; Zhou, X.; Lu, H.; Ramacciotti, E.; Zhang, L. J. Biol. Chem. 2010,
285(30), 22966.
29. Qiu, P.; Guan, H.; Dong, P.; Guo, S.; Zheng, J.; Li, S.; Chen, Y.; Ho, C. T.; Pan, M.
H.; McClements, D. J.; Xiao, H. Mol. Nutr. Food Res. 2011, 55(10), 1523.
30. Qiu, P.; Dong, P.; Guan, H.; Li, S.; Ho, C. T.; Pan, M. H.; McClements, D. J.; Xiao, H.
Mol. Nutr. Food Res. 2010, 54(Suppl. 2), S244.
References and notes
1. Nedergaard, M. K.; Hedegaard, C. J.; Poulsen, H. S. Biodrugs 2012, 26(2), 83.
2. Chen, A.; Xu, J.; Johnson, A. C. Oncogene 2006, 25(2), 278.
3. Sharma, R. A.; Gescher, A. J.; Steward, W. P. Eur. J. Cancer 2005, 41(13), 1955.
4. Agrawal, D. K.; Mishra, P. K. Med. Res. Rev. 2010, 30(5), 818.