Functionalized curcumin analogs as potent modulators of the Wnt/β-catenin signaling pathway

DOI: 10.1016/j.ejmech.2013.10.073

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European Journal of Medicinal Chemistry p. 67 - 80 (2014)

Update date:2022-08-02

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Authors:

Leow, Pay-Chin

Bahety, Priti

Boon, Choon Pei

Lee, Chong Yew

Tan, Kheng Lin

Yang, Tianming

Ee, Pui-Lai Rachel

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Article abstract of DOI:10.1016/j.ejmech.2013.10.073

Osteosarcoma is a primary bone malignancy with aggressive metastatic potential and poor prognosis rates. In our earlier work we have investigated the therapeutic potential of curcumin as an anti-invasive agent in osteosarcoma by its ability to regulate the Wnt/β-catenin signaling pathway. However, the clinical use of curcumin is limited owing to its low potency and poor pharmacokinetic profile. In this study, an attempt was made to achieve more potent Wnt inhibitory activity in osteosarcoma cells by carrying out synthetic chemical modifications of curcumin. We synthesized a total of five series consisting of 43 curcumin analogs and screened in HEK293T cells for inhibition of β-catenin transcriptional activity. Six promising analogs, which were 6.5- to 60-fold more potent than curcumin in inhibiting Wnt activity, were further assessed for their anti-invasive activity and Wnt inhibitory mechanisms. Western blot analysis showed disruption of β-catenin protein nuclear translocation following treatment with analogs 2f, 3c and 4f. Using transwell assays, we also found that these compounds were more potent than 1a (curcumin) in impeding the invasion of osteosarcoma cells, possibly through suppressing MMP-9 activity. Structure-activity-relationship studies revealed that Wnt inhibitory effects could be enhanced by shortening and restraining the flexibility of the 7-carbon linker moiety connecting the terminal aromatic rings of curcumin and substituting both rings with appropriate substituents. Our results demonstrate that the synthesized curcumin analogs are more potent Wnt inhibitors in osteosarcoma cell lines as compared to parental curcumin and are good lead compounds for further development. Future in vivo tests with these compounds will define their therapeutic potentials as promising drug candidates for clinical treatment of osteosarcoma.

Full text of DOI:10.1016/j.ejmech.2013.10.073

European Journal of Medicinal Chemistry 71 (2014) 67e80

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Functionalized curcumin analogs as potent modulators

of the Wnt/ -catenin signaling pathway

Pay-Chin Leow, Priti Bahety, Choon Pei Boon, Chong Yew Lee, Kheng Lin Tan,

Tianming Yang, Pui-Lai Rachel Ee

Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore

a r t i c l e i n f o

a b s t r a c t

Article history:

Osteosarcoma is a primary bone malignancy with aggressive metastatic potential and poor prognosis

rates. In our earlier work we have investigated the therapeutic potential of curcumin as an anti-invasive

agent in osteosarcoma by its ability to regulate the Wnt/b-catenin signaling pathway. However, the

clinical use of curcumin is limited owing to its low potency and poor pharmacokinetic proﬁle. In this

study, an attempt was made to achieve more potent Wnt inhibitory activity in osteosarcoma cells by

carrying out synthetic chemical modiﬁcations of curcumin. We synthesized a total of ﬁve series con-

Received 26 June 2013

Received in revised form

28 October 2013

Accepted 29 October 2013

Available online 7 November 2013

sisting of 43 curcumin analogs and screened in HEK293T cells for inhibition of b-catenin transcriptional

activity. Six promising analogs, which were 6.5- to 60-fold more potent than curcumin in inhibiting Wnt

activity, were further assessed for their anti-invasive activity and Wnt inhibitory mechanisms. Western

Keywords:

Curcumin analog

Osteosarcoma

Wnt signaling

Invasion

blot analysis showed disruption of

b-catenin protein nuclear translocation following treatment with

analogs 2f, 3c and 4f. Using transwell assays, we also found that these compounds were more potent

than 1a (curcumin) in impeding the invasion of osteosarcoma cells, possibly through suppressing MMP-

9 activity. Structureeactivity-relationship studies revealed that Wnt inhibitory effects could be enhanced

by shortening and restraining the ﬂexibility of the 7-carbon linker moiety connecting the terminal ar-

omatic rings of curcumin and substituting both rings with appropriate substituents. Our results

demonstrate that the synthesized curcumin analogs are more potent Wnt inhibitors in osteosarcoma cell

lines as compared to parental curcumin and are good lead compounds for further development. Future

in vivo tests with these compounds will deﬁne their therapeutic potentials as promising drug candidates

for clinical treatment of osteosarcoma.

Drug discovery

1. Introduction

who suffer from long term toxicities following the standard

chemotherapy. These shortcomings have, thus, led to an increase in

Osteosarcoma is an aggressive primary malignancy of the bone

resulting from morphologically abnormal osteoblastic cells pro-

ducing defective immature bone or osteoid. It predominantly affects

children and adolescents and shows an aggressive tendency for

growth and metastasis, with as many as 20% of patients reported to

have developed pulmonary metastasis at the time of diagnosis [1].

Despite the use of various multi-modality regimens of intensive

chemotherapy and surgical resection since few decades, prognosis

of osteosarcoma patients with pulmonary metastasis is extremely

poor with 5-year survival rate of only 10e20% [2]. Moreover, effec-

tive therapeutic options are not available for patients with osteo-

sarcoma that relapses following multi-modal ﬁrst-line treatment or

the efforts invested in understanding the molecular signaling

pathways involved in the pathogenesis of the diseasewith the aim of

designing novel, safe and effective therapeutic strategies to help

improve clinical outcome among osteosarcoma patients.

With the exact mechanism behind the disease progression and

metastasis still remaining uncertain, reports on the contribution of

aberrant Wnt/b-catenin signaling, a key pathway in controlling

osteoblast development has come into the limelight in recent os-

teosarcoma research. In normal circumstances, the pathway is

initiated when Wnt ligands bind Frizzled (FZD) and low-density

lipoprotein receptor related protein 5/6 (LRP5/6) co-receptors and

trigger a cascade of processes, leading to the translocation of

catenin into the nucleus. In the nucleus, accumulated -catenin

partners with T-cell factor/lymphocyte enhancer factor (Tcf/lef)

family of transcription factors and activates downstream

target genes, mainly oncogenes, cell cycle regulators and matrix

* Corresponding author. Tel.: þ65 6516 2653; fax: þ65 6779 1554.

E-mail address: phaeplr@nus.edu.sg (P.-L.R. Ee).

http://dx.doi.org/10.1016/j.ejmech.2013.10.073

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Table 1

Structure of curcumin analogs (Series 1e5) and their physiochemical properties.

Compound no.

R₁(3⁰)

R₂(4⁰)

Molecular weight^a

No. of rotatable

bonds^b

No. of hydrogen

bond donors^b

No. of hydrogen

bond acceptors^b

Clog P^a

Series 1 analogs

1a (curcumin)

OCH₃

368.38

396.43

368.38

276.33

336.38

308.33

348.29

2.94

3.89

2.94

4.58

4.41

3.24

5.01

Series 2 analogs

OCH₃

326.34

354.40

326.34

234.39

294.34

266.29

306.25

2.64

3.59

2.64

4.28

4.11

2.94

4.71

Series 3 analogs

OCH₃

2⁰F

OCH₃

366.41

394.46

366.41

274.36

334.41

306.36

346.32

310.34

3.70

4.65

3.70

5.33

5.17

4.00

5.76

5.62

Series 4 analogs

OCH₃

2⁰F

OCH₃

352.38

380.43

352.38

260.33

320.38

292.33

332.29

296.31

3.14

4.09

3.14

4.77

4.61

3.44

5.20

5.06

Series 5 analogs

OCH₃

300.31

328.36

2.50

3.18

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Table 1 (continued )

Compound no.

R₁(3⁰)

R₂(4⁰)

Molecular weight^a

No. of rotatable

bonds^b

No. of hydrogen

bond donors^b

No. of hydrogen

bond acceptors^b

Clog P^a

Series 1 analogs

OCH₃

300.31

208.26

268.31

240.25

280.22

2.50

3.62

3.77

2.83

4.16

Clog P/molecular weight values were determined on ChemDraw Ultra 10.0, CambridgeSoft, Cambridge, MA.

No. of rotatable bonds/no. of hydrogen bond donors and acceptors values were determined on MOE, 2009. 10 Chemical Computing Group.

degrading enzymes [3e5]. In osteosarcoma, however, aberrant

Wnt/ -catenin activity resulting from -catenin mutations,

increased nuclear -catenin levels [6,7], over-expression of Wnt

2. Results and discussion

2.1. Rational drug design and synthesis

ligands [8,9], FZD receptors [9] and LRP5 co-receptor [9e11] have

been reported to correlate with metastasis and poorer survival

outcome in patients. Through our previous studies, we have

In this study, our aim was to synthesize more potent curcumin

analogs with lower EC₅₀for Wnt inhibition with limited cell cyto-

toxicity compared to the parental curcumin. To study the effect of

structural modiﬁcation on Wnt inhibition, we modiﬁed the lead

compound, curcumin 1-1, to produce ﬁve series of curcumin ana-

logs with diverse linkers joining the terminal phenyl rings as well

as different substituents at the phenyl rings. These compounds

were synthesized by base-catalyzed ClaiseneSchmidt condensa-

tion reactions of substituted aromatic aldehydes with the appro-

priate acetophenones. The structures of curcumin analogs and their

physicochemical properties are presented in Table 1. Series 1 con-

sists of curcumin-type compounds that retain the 7-carbon spacer

between the terminal phenyl rings known as the diarylheptanoids

while Series 2 represents diarylpentanoid analogs with a 5-carbon

spacer between the terminal phenyl rings. Series 3 (dibenzylidenee

cyclohexanones) and 4 (dibenzylideneecyclopentanones) consist

of diarylpentanoid-type compounds in which ﬂexibility of the 5-

carbon chain is constrained by incorporating it as part of a cyclo-

hexanone and cyclopentanone ring, respectively. Lastly, Series 5

compounds, known as chalcones, were considered as structures

equivalent to “half a curcuminoid” with loss of symmetry and a

shortened linker. The choice of these templates and the associated

substitutions at the terminal phenyl rings were prompted by the

following observations: Firstly, natural derivatives of curcumin

with different substitutions on the end rings were reported to

demonstrated that forced expression of

b-catenin signiﬁcantly

enhanced osteosarcoma cell invasiveness in vitro by causing aber-

rant activation of the pathway [12]. These lines of evidence present

Wnt/b-catenin signaling pathway as a potential therapeutic target

for treating osteosarcoma.

Over the past few decades, several potential anticancer com-

pounds have been derived from natural products and their con-

stituents, few of which are now either in advanced clinical

development or have already been approved for therapeutic use

[13]. Curcumin, a dietary phytochemical constituent extracted from

the plant turmeric, is one such well-known compound shown to

exhibit chemopreventive and chemotherapeutic properties against

various types of cancers with a good toxicity proﬁle [14]. The

mechanism by which curcumin acts is diverse, but at the molecular

level it has been shown to interact with multiple signaling path-

ways such as nuclear factor-kappa B (NF-

protein (AP)-1 [19,20], signal transducers and activators of tran-

scription (STAT) [18,21e27], Notch signaling [28,29] and Wnt/

kB) [15e18], activator

catenin signaling pathway [12,30e32]. However, clinical applica-

tions of curcumin are limited mainly due to its low potency, rapid

metabolism and low oral bioavailability [33e35]. Synthesizing

potent curcumin analogs or developing formulations for its selec-

tive delivery are some of the approaches being tried to improve its

pharmacological proﬁle and chemotherapeutic potentials [36e41].

In this study, we hypothesized that synthetic chemical modiﬁca-

tions of curcumin will yield compounds that combined improved

Wnt inhibitory potency with a suitable drug-like character, cir-

cumventing the limitations encountered with the parent analog. To

this end, we determined the effects of 43 curcumin analogs on the

suppress b-catenin/Tcf transcription. Hence, regioisomers of dia-

rylheptanoids (Series 1) were included to determine how changes

in substitution pattern would impact inhibitory activity [42]. Sec-

ondly, the deletion of the

b-ketone moiety in curcumin to give

diarylpentanoids (Series 2), dibenzylideneecyclohexanones (Series

3) and dibenzylideneecyclopentanones (Series 4) is known to give

rise to analogs with improved pharmacological and metabolic

proﬁles [39,43]. Finally, the substitution pattern on the terminal

phenyl rings were kept symmetrical (i.e. both rings share the same

substitution pattern) and largely restricted to groups present on

curcumin. Thus the 3⁰OCH₃e4⁰OH substitution pattern of curcumin

was modiﬁed by (i) excluding either one substituent to give 3⁰OCH₃

or 4⁰OH analogs (ii) removing both substituents, (iii) switching

their positions to give 3⁰OHe4⁰OCH₃substituted rings; (iv) intro-

ducing an additional OCH₃in place of OH. An exception was the

introduction of ﬂuorine which was made in view of the bioisosteric

relationship between F and H, as well as the anomalous properties

of ﬂuorine which have led to improved activities in many instances

[44]. It is noteworthy that all the curcumin analogs have physico-

chemical properties that comply with the Lipinski’s Rule of Five

Wnt/b-catenin signaling pathway and identiﬁed several promising

candidates that were effective in suppressing the activity of the

pathway in osteosarcoma. Results from cell invasion assays further

demonstrated the potency of these analogs as anti-invasive agents

with concomitant reduction in matrix metalloproteinase (MMP)-9

levels. In addition, our structureeactivity relationship studies

showed that Wnt inhibitory effects were markedly enhanced by

shortening and restraining the ﬂexibility of the 7-carbon linker

moiety connecting the terminal aromatic rings of curcumin. Our

results strongly suggest that curcumin analogs, especially those

with the dibenzylideneecyclohexanone and dibenzylidenee

cyclopentanone scaffolds, are promising templates for lead opti-

mization and may yield clinically useful drug candidates for the

treatment and prevention of osteosarcoma.

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Table 2

Inhibition of Wnt/

cells.

which provide guidelines for drug-like proﬁles related to good oral

bioavailability (Table 1).

-catenin signaling pathway by curcumin analogs in HEK293T

Compound

Normalized luciferase activity (Fireﬂy/Renilla)^a

2.2. Evaluation of biological activities

Concentration of curcumin analogs

2.2.1. Inhibition of Wnt/

cells

b-catenin signaling activity in HEK293T

20 mM

Series 1 analogs

Owing to their low endogenous levels of

low -catenin/Tcf transcriptional activity, HEK293T cells were

chosen as a cell model for preliminary screening of Wnt inhibitory

activities [45]. Treatment with Wnt-3A CM stimulated Wnt/

b-catenin protein and

1a (curcumin)

99.4 ꢀ 3.6

98.2 ꢀ 3.3

82.5 ꢀ 7.9

62.8 ꢀ 12.4

1b^#

101.6 3.9

99.7 ꢀ 3.1

108.9 ꢀ 4.4

97.7 ꢀ 0.9

100.3 ꢀ 8.3

83.3 ꢀ 9.0

70.0 1.7

46.5 7.2

N.D^b

110.3 ꢀ 3.1

104.1 ꢀ 3.4

107.4 ꢀ 4.0

77.7 ꢀ 1.8

92.2 ꢀ 0.5

104.6 ꢀ 10.3 112.8 ꢀ 1.1

107.1 ꢀ 0.8

107.5 ꢀ 4.2

72.5 ꢀ 0.6

97.2 ꢀ 7.5

101.4 ꢀ 6.1

69.1 ꢀ 1.9

catenin signaling in HEK293T cells resulting in approximately 30-

fold increase in transcriptional activities. Table 2 shows the

normalized luciferase activity in the presence of curcumin analogs

89.2 ꢀ 19.2 93.1 ꢀ 20.0

Series 2 analogs

(1e20

Compared with control, Wnt-3A CM-induced

transcription (CRT) was inhibited by approximately 17.5% and

37.2% with 10 M and 20 M curcumin treatment, respectively.

Given that our lead compound, 1a (curcumin) was found to have

an EC₅₀value of 20.7 ꢀ 0.8 M (Table 3), a total of 16 other analogs

(Table 2, bold and indicated with #) that were capable of sup-

pressing the Wnt-3A-induced CRT by more than 50% at 20 M or

mM), expressed as % of DMSO with Wnt-3A activation.

118.4 ꢀ 4.1

107.1 ꢀ 1.2

82.5 4.1

93.0 ꢀ 2.4

100.4 ꢀ 5.7

62.3 2.4

110.9 ꢀ 10.5 110.4 ꢀ 23.0 90.4 ꢀ 13.0

-catenin response

107.6 ꢀ 0.1

38.8 4.1

82.7 ꢀ 9.0

113.9 ꢀ 3.4

51.3 6.6

108.6 ꢀ 0.2

103.4 ꢀ 1.1

2c^#

34.4 7.7

N.D^b

79.7 ꢀ 14.7 77.9 ꢀ 17.7

102.7 ꢀ 2.5

102.9 ꢀ 4.8

2f^#

52.0 5.0

37.8 3.6

103.0 ꢀ 13.0 100.0 ꢀ 12.5 110.1 ꢀ 6.2

110.9 ꢀ 9.7

Series 3 analogs

3a^#

86.1 0.8

97.7 3.1

37.3 1.9

85.8 5.7

93.3 ꢀ 11.2

51.9 5.9

94.5 ꢀ 3.2

52.2 1.8

96.1 ꢀ 9.9

70.3 ꢀ 5.4

28.9 5.6

64.3 7.7

27.7 1.5

49.6 6.1

60.7 ꢀ 17.7

26.9 3.5

21.3 3.8

62.8 3.5

24.7 3.7

40.6 9.2

72.7 ꢀ 5.8

26.3 4.3

N.D^b

3b^#

40.8 7.1

21.4 1.9

39.0 11.2

81.5 ꢀ 21.2

16.7 2.9

113.7 ꢀ 7.3

20.9 3.6

lower concentrations (i.e. more potent than curcumin), were

shortlisted for subsequent experiments. As shown in Table 3, Se-

ries 3 and Series 4 analogs were the most promising, with several

members such as 3c, f, h, 4c and f having EC₅₀values in the

submicromolar ranges. Speciﬁcally, the most potent analog from

3c^#

3d^#

3f^#

125.9 ꢀ 14.4 117.9 ꢀ 7.4

3h^#

21.9 1.5 14.5 0.6

these series, 3c (EC₅₀0.3 ꢀ 0.01

mM) and 4f (EC₅₀0.4 ꢀ 0.01

mM)

119.1 ꢀ 22.2 105.1 ꢀ 22.1 103.6 ꢀ 12.1

were approximately 60 and 51 times more potent than parental

curcumin in inhibiting Wnt-3A-induced CRT, respectively. Except

for 1b, 3a, b, 4a and b, all the other analogs had limited cytotox-

icity at their EC₅₀values (Table 3).

65.2 ꢀ 6.3

61.6 ꢀ 8.8

60.2 ꢀ 2.3

Series 4 analogs

4a^#

91.6 5.8

88.9 2.7

52.6 6.7

82.7 4.0

92.3 ꢀ 3.1

34.3 1.5

71.0 ꢀ 7.6

80.0 ꢀ 12.1

54.1 4.0

65.9 6.7

45.0 5.0

41.5 5.4

72.7 ꢀ 2.7

28.3 3.2

73.1 ꢀ 6.0

75.7 ꢀ 12.1

37.5 2.3

55.5 6.2

39.5 4.1

25.3 6.3

72.0 ꢀ 5.6

19.6 3.3

60.2 ꢀ 2.4

85.3 ꢀ 5.6

13.3 2.7

39.2 7.2

39.2 5.0

18.4 2.1

61.3 ꢀ 9.4

8.0 2.4

4b^#

4c^#

4d^#

2.2.2. Inhibition of

cells

b-catenin/Tcf transcriptional activity in U2OS

4f^#

59.5 ꢀ 6.7

The effects of selected curcumin analogs 2c, 3c, 4c, 2f, 3f and 4f

on the downstream -catenin/Tcf transcriptional activity were

further evaluated in U2OS osteosarcoma cells, which present with

activated Wnt/ -catenin signaling resulting in abnormal accumu-

lation of -catenin in the nucleus [6]. As shown in Fig. 1, 1a (cur-

cumin) signiﬁcantly inhibited -catenin/Tcf transcriptional activity

in U2OS cells by approximately 27.1% and 59.4% at 10 M and 20

respectively. In parallel to the results obtained in HEK293T cells,

analogs 3c, 4c, 2f, 3f and 4f effectively reduced -catenin/Tcf

transcriptional activity in U2OS cells by 25.7e52.7% at 1 M con-

centration, an effect comparable to 10 M of parental curcumin. The

least potent analog 2c was also capable of signiﬁcantly suppressing

the transcriptional activity by 49.4% at 5 M. In addition, there was

72.8 ꢀ 4.7

123.8 ꢀ 16.4 119.2 ꢀ 5.4

109.0 ꢀ 10.3 102.6 ꢀ 7.1

119.5 ꢀ 10.8 112.5 ꢀ 15.6 105.4 ꢀ 6.1

107.7 ꢀ 5.4

Series 5 analogs

5c^#

5d^#

112.0 ꢀ 8.3

95.0 ꢀ 6.5

95.7 3.9

101.5 5.7

106.8 ꢀ 4.3

92.7 ꢀ 1.7

115.3 ꢀ 13.9

86.6 ꢀ 7.7

53.9 6.9

90.6 14.1

107.8 ꢀ 6.4

89.6 ꢀ 8.7

86.9 ꢀ 9.7

60.3 ꢀ 4.3

42.0 4.7

69.0 8.8

111.2 ꢀ 10.5 72.9 ꢀ 7.0

70.9 ꢀ 0.4 68.3 ꢀ 7.0

90.2 ꢀ 15.7 59.1 ꢀ 6.8

69.6 ꢀ 6.7

41.6 ꢀ 5.5

25.7 2.8

18.9 5.4

117.1 ꢀ 14.8 111.5 ꢀ 13.3

# indicates analogs designated as ‘actives’ and shortlisted for EC₅₀determinations

with an estimated EC₅₀value of less than 20

mM compared to curcumin (EC₅₀

20.7 ꢀ 0.8).

Results are presented as the mean ꢀ S.E.M of normalized luciferase activity

no change in the transcriptional activities of the negative control

FOPglow plasmid in all instances, indicating the speciﬁcity of these

compounds for Wnt b-catenin/Tcf transcription.

(Fireﬂy/Renilla) from three independent experiments, expressed as % of DMSO

(vehicle control) under Wnt-3A induced condition.

Not determined (N.D). Tested at 1, 5 and 10 mM because of toxicity at higher

concentrations.

2.2.3. Effect of curcumin analogs on nuclear translocation of

catenin

Translocation and accumulation of nuclear

activation of -catenin/Tcf transcriptional activity [46]. As curcumin

disrupts the translocation of -catenin into the nucleus without

changing its total cellular levels [12], we evaluated the effect of

curcumin analogs on the cellular levels and localization of -cat-

enin protein. As shown in Fig. 2, we found that treatments with 2f,

3c and 4f analogs did not alter the amount of -catenin in the

cytoplasm, but caused a reduction in the nuclear fractions at 5 M,

suggesting a disruption in the translocation of nuclear -catenin. In

contrast, treatment with other analogs (2c, 3f and 4c) had no effects

on either the nuclear or cytoplasmic -catenin protein levels.

-catenin results in

2.2.4. U2OS cell invasion studies

In our previous work, we have shown that curcumin signiﬁ-

cantly reversed -catenin plasmid mediated increase in invasive

capacity of U2OS cells in a concentration-dependent manner [12].

To determine if curcumin analogs could exert similar, but more

potent anti-invasive effects, we performed Matrigel invasion assays

[47]. While curcumin treatment signiﬁcantly reduced the invasive

capacity of U2OS cells by 29.0 ꢀ 5.1% at 5

mM [12], treatment with

analog 2f, 3c and 4f drastically reduced U2OS cell invasiveness by

76.2 ꢀ 1.4%, 81.6 ꢀ 2.6%, and 72.8 ꢀ 1.0% at the same concentration,

respectively (Fig. 3B). We found that these analogs were also

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Table 3

EC₅₀values of selected curcumin analogs in HEK293T cells.

Series

Compound

R₁

R₂

EC₅₀of Wnt inhibition (

M)^a

Potency (fold)^b

Cell viability (%)^c

FOPglow (%)^c

Series 1

1a (curcumin)

OCH₃

2⁰F

OCH₃

20.7 ꢀ 0.8

8.3 ꢀ 0.2

1.8 ꢀ 0.3

3.2 ꢀ 0.2

2.6 ꢀ 0.2

15.9 ꢀ 0.4

0.3 ꢀ 0.0

2.0 ꢀ 0.3

0.8 ꢀ 0.0

0.9 ꢀ 0.1

5.9 ꢀ 0.5

17.1 ꢀ 1.8

0.5 ꢀ 0.0

3.1 ꢀ 0.4

0.4 ꢀ 0.0

3.9 ꢀ 0.5

11.0 ꢀ 1.0

1.0

2.5

11.2

6.5

7.9

1.3

59.7

10.1

25.8

22.8

3.5

1.2

40.4

6.7

42.4 ꢀ 2.3

41.6 ꢀ 0.1

86.6 ꢀ 1.5

99.2 ꢀ 6.9

57.6 ꢀ 12.4

62.1 ꢀ 8.6

91.6 ꢀ 7.6

100.0 ꢀ 11.9

105.9 ꢀ 10.1

87.0 ꢀ 1.8

38.4 ꢀ 7.8

40.0 ꢀ 9.5

86.9 ꢀ 4.5

83.5 ꢀ 3.3

81.4 ꢀ 1.8

87.2 ꢀ 6.9

73.1 ꢀ 12.4

89.3 ꢀ 3.6

117.6 ꢀ 17.3

105.1 ꢀ 16.0

100.4 ꢀ 11.1

87.6 ꢀ 7.1

OCH₃

Series 2

Series 3

OCH₃

103.6 ꢀ 6.4

93.8 ꢀ 8.3

90.7 ꢀ 7.1

85.1 ꢀ 9.2

3h^#

93.0 ꢀ 4.5

Series 4

Series 5

90.8 ꢀ 4.8

OCH₃

85.2 ꢀ 5.9

110.2 ꢀ 12.3

82.9 ꢀ 7.2

50.5

5.3

1.9

101.3 ꢀ 11.3

130.7 ꢀ 11.7

111.9 ꢀ 6.5

Ring substitutions at 2⁰position.

The concentration of curcumin analogs that inhibits 50% of TOPglow

independent experiments repeated in triplicate at least.

-catenin/Tcf transcriptional activity. EC₅₀values are presented as the mean ꢀ S.E.M from three

Potency fold: EC₅₀values of curcumin/EC₅₀values of other analogs.

Normalized FOPglow transcriptional activities and cell viability of the cells on treatment with analogs at the concentrations of EC₅₀

effective in suppressing U2OS cell invasion by between 34.8 ꢀ 2.2%

to 71.0 ꢀ 5.9% at a 1 M concentration. The anti-invasive effects of

2.3. Structure activity relationship (SAR)

the analogs were not a result of cell toxicity as no signiﬁcant growth

inhibition was observed at these concentrations (Supplementary

data, Fig. S1). In addition, western blot analysis also showed

down-regulation in expression of MMP-9 protein at same con-

centrations (Fig. 3C). Altogether, our results suggest that analogs 2f,

3c and 4f were effective in inhibiting U2OS cell invasiveness at

much lower concentrations than curcumin, possibly through

down-regulating Wnt target gene, MMP-9.

To have a better understanding of the structural features impor-

tant for Wnt inhibitory effects, we examined the SAR of the curcumin

analogs that were identiﬁed to be more potent than parental curcu-

min in inhibiting Wnt/b-catenin signaling activities in U2OS cells. To

study the effect of carbon spacers on SAR, analogs were synthesized

with different number of rotatable bonds: Series 3 and 4 (two rotat-

able bonds), chalcones (three rotatable bonds), Series 2 (four rotat-

able bonds) and Series

(ﬁve rotatable bonds). Series 3

120

110

100

DMSO

10 20

(μM)

1a (curcumin)

Curcumin analogs

Fig. 1. Effect of curcumin analogs on b-catenin/Tcf transcriptional activity in U2OS cell line. U2OS cells were co-transfected with reporter genes harboring Tcf-4 binding sites

(TOPglow) or a mutant Tcf-4 binding site (FOPglow) and pCMV Renilla gene. 20 h post-transfection, U2OS cell were treated with curcumin analogs. 24 h post-treatment ﬁreﬂy

luciferase activity was determined and normalized against values for the corresponding Renilla luciferase activity. Results were expressed as the means ꢀ S.E.M of normalized ratios

of ﬁreﬂy luciferase and renilla luciferase measurements of three independent experiments. Reporter activity in compound-treated cells is expressed as the percentage of DMSO-

treated samples. *p < 0.05, **p < 0.01.

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Curcumin analogs (5 µM)

2f 3f 4f

Curcumin analogs (5 µM)

2c 3c 4c

DMSO

nuclear β-catenin

lamin A/C

cytoplasmic β-catenin

α-tubulin

Cytoplasmic

Nuclear

120

100

CyNtoupcllCaesaymNrtouipccllCaesaymNrtouipccllCaesaymNrtouipccllCaesaymNrtouipccllCaesaymNrtouipccllCaesaymNrtouipccllaesamr ic

DMSO 2c 3c 4c 2f 3f 4f

Curcumin analogs (5 μM)

Fig. 2. Effect of curcumin analogs on the cellular and nuclear accumulation of

24 h, followed by collection of protein from the cytoplasmic and nuclear fractions.

respectively. (B) Quantity One software (Bio-Rad, Hercules, CA) was used for quantifying the bands and the results were then expressed relative to the lamin A/C and

-catenin. (A) U2OS cells were pre-treated with curcumin analogs at the stated concentrations for

-tubulin and lamin A/C were used for cytoplasmic and nuclear protein loading controls,

-tubulin

-catenin protein expression in compound-treated cells was expressed as the percentage of

loading controls. All western blot experiments were repeated at least in triplicates.

DMSO-treated samples. *p < 0.05, **p < 0.01.

(dibenzylideneecyclohexanones) and

(dibenzylideneecyclo-

different series. Overall, the inﬂuence of the nature of ring sub-

stituent on the Wnt inhibitory activities may be broadly ranked in

the sequence: 3⁰-OHe4⁰-OCH₃z 4⁰-OH (most active) >> 3⁰-He4⁰-

H > 3⁰-OCH₃4⁰-OH > 3⁰-OCH₃e4⁰-OCH₃>> 3⁰-OCH₃z 4⁰-F z 3⁰-

4⁰-F (least active).

pentanones) analogs, both with conformationally restricted and

bulkier tethers between the terminal phenyl rings, exhibited much

higher Wnt inhibitory potency than others in which the terminal

ringswerelinkedbylongerandmoreﬂexiblecarbonspacers(Series1,

2, 5).

The poor activity of the 3⁰OCH₃analogs in all the series sug-

gested the need to restrain the steric dimensions of the groups at

this position. It also reﬂected a preference for groups that were not

electron withdrawing at this position. It was notable that 3⁰OCH₃

was electron withdrawing unlike 4⁰OCH₃which was electron

The importance of maintaining 3⁰-OCH₃e4⁰-OH substituents at

the terminal rings of Series 1e5 compounds was investigated by

various permutations centered on the OH/OCH₃groups. For Series

1 analogs, only the dimethoxy analog 1b was found to be more

potent than curcumin. These results suggested a limited tolerance

for the type of groups that can be introduced into phenyl rings of

the Series 1 template. A similar trend was observed in Series 2 and

5 where no more than 2 different substitution patterns were

permissible, with the preferred substituents being 3⁰-OHe4⁰-OCH₃

and not dimethoxy as noted for Series 1. On the other hand, the

Wnt inhibitory activities of Series 3 and 4 were less inﬂuenced by

the type of substituents on the terminal phenyl rings. In addition,

all non-ﬂuorinated cyclic analogs from both series, except those

with 3⁰-methoxy ring substituent (3e and 4e) were found to be

more potent than curcumin. Interestingly, compound 3h, which

was mono-ﬂuorinated at the 2⁰position, was found to be active,

with luciferase activity reduced to approximately 45.7% and 15% at

donating, as seen from their Hammett sigma values

(sp

OCH3 ꢁ0.27; m OCH3 ¼ 0.12). Inhibitory activity may be deter-

mined by interplay of the size and electrostatic nature of groups at

this position. Thus, the poor activity of 3⁰F analogs would suggest

that the strong electron withdrawing effect of F has more than

offset its small steric dimensions while the moderate activities of

analogs with unsubstituted phenyl rings (3d, 4d) may be attributed

to their limited size requirements. From these results, it is tempting

to suggest that Wnt inhibitory activity is favored by the absence of

electron withdrawing groups on the terminal phenyl rings of Series

3 and 4. Support for this view was seen from the exceptionally good

activities of the 4⁰OH analogs (3f, 4f) and the 3⁰OHe4⁰OCH₃analogs

(3c, 4c). As to why switching OH/OCH₃to 3⁰OCH₃e4⁰OH resulted in

analogs (3a, 4a) with a modest decline in activity, may be attributed

to the location of the bulkier OCH₃at the 3⁰position. Taken

together, some general SAR trends were deduced from the results

mM and 10 mM concentrations, respectively. This trend was

however not seen for compound 4h in Series 4 or among other

ﬂuorinated analogs (4⁰-F or 3⁰, 4⁰-diF) represented across the

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Curcumin analogs (1 µM)

DMSO

100

(μM)

DMSO

Curcumin analogs

DMSO

(µM)

Fig. 3. Effect of curcumin analogs on U2OS cell invasion. (A) U2OS cells were pre-treated with curcumin analogs for 24 h before they were seeded into Matrigel-coated inserts. Cells

that invaded to the lower surface of the insert were captured with a light microscope at 200ꢂ magniﬁcation after staining with crystal violet dye. Images were representations from

three independent experiments. (B) Ten random ﬁelds were counted for the number of invaded U2OS cells. Cell invasion data was presented as means ꢀ S.E.M of three independent

experiments. Cell invasion in compound-treated cells was expressed as the percentage of DMSO-treated samples, *p < 0.05, **p < 0.01. (C) U2OS cells were treated with indicated

concentrations of curcumin analogs or DMSO (vehicle control) for 24 h before proteins were collected for western blot. The blots shown were representative of three independent

experiments. a-tubulin was used as a loading control.

obtained and were summarized in Fig. 4. These requirements may

reﬂect the involvement of these groups in key interactions with

target proteins and would require further conﬁrmation with addi-

tional analogs.

drugs for further development as chemotherapeutic agents for the

treatment and prevention of osteosarcoma.

4. Experimental protocols

3. Conclusion

4.1. Chemistry

Our study identiﬁed three new curcumin analogs that were

more potent than parent curcumin as effective Wnt inhibitors and

anti-invasive agents in human osteosarcoma. SAR studies revealed

that the Wnt inhibitory effects could be markedly enhanced by

introducing conformational restriction in the central linker and

appropriate ring substituents such as a strong electron donating

group at the 4⁰ring position. These SAR trends observed may reﬂect

the involvement of these groups in key interactions with the target

proteins. Our results strongly suggest that curcumin analogs

especially those with the dibenzylideneecyclohexanones and

dibenzylideneecyclopentanones templates are promising scaffolds

for future structural optimization and are promising candidate

4.1.1. General experimental details for synthesis

Reagents (synthetic grade or better) were obtained from

SigmaeAldrich Chemical Company Inc (Singapore) and used

without further puriﬁcation. Melting points were determined in

open capillary tubes on a Gallenkamp melting point apparatus and

reported as uncorrected values. Mass spectra were captured on an

LCQ Finnigan MAT equipped with an Atmospheric Chemical Ioni-

zation (APCI) probe and m/z ratios for the molecular ions (M þ 1)^þ

were reported. Chemical shifts of ¹H NMR and ¹³C NMR spectra,

obtained on a Bruker Spectrospin 300 Ultrashield spectrometer at

300 MHz and 75 MHz respectively, were analyzed using MestRec-

C 4.9.9.6 (Mestrelab Research SL, Spain) and reported in

d (ppm)

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

H₃C

CH₃

B₂0₃

OCH

B₂O₃

+ BO₂

+ H₂0

H₃C

CH₃

Deletion of the beta-diketone

moiety to obtain mono-carbonyl analogues

Electron donating ring substitutions

(b, c)

Ring substitutions with

(d)

limited size requirements

preferred at 3' position.

n-butylamine

(d)

(a)

Reducing the flexibility of

the intermediate linker

Fig. 4. Structural features of curcumin analogs important for enhanced Wnt inhibitory

activity. (a) Reducing the ﬂexibility of the intermediate linker joining the terminal

phenyl rings improved Wnt inhibitory activity. Incorporating the linker as part of a ring

structure such as dibenzylideneecyclohexanone (Series 3) and dibenzylideneecyclo-

pentanone (Series 4) spacers are favored. (b) Flexibility of the intermediate side chain

inﬂuenced the substitution on the terminal phenyl rings. Only limited ring substitution

patterns were tolerated in analogs with ﬂexible linkers (Series 1, 2, 5), but a wider

range of substitution patterns were permitted in analogs that had less ﬂexible linkers

(Series 3, 4). (c) Electron donating ring substitutions are more favorable. (d) Ring

substitutions with limited size requirements are preferred at the 3⁰position.

HCl

relative to tetramethylsilane (TMS) as an internal standard. Silica

60 F254 sheets (Merck, Darmstadt, Germany) and silica gel 60

(0.040e0.063) (Merck, Darmstadt, Germany) were used for Thin

Layer Chromatography (TLC) and ﬂash chromatography respec-

tively. Purity of the ﬁnal compounds were veriﬁed either by

combustion analysis (C, H) on a Perkin Elmer PRE-2400 Elemental

Analyzer or by High Pressure Liquid Chromatography (HPLC) using

two different solvent systems. Spectroscopic data, melting points,

yields and purities of individual compounds were reported.

Scheme 1. General method for the synthesis of Series 1 curcumin analogs.

used instead. The resultant mixturewas stirredat room temperature

for 20 min before 20% (w/v) NaOH (3.0 mL, 15 mmol) was added

dropwise. After the reaction has completed, the residue was poured

into saturated NH₄Cl solution and ﬁltered. The precipitate was

washed with brine and cold ethanol, dried with anhydrous NaSO₄,

evaporated in vacuo to give either a solid or liquid residue and pu-

riﬁed by column chromatography on silica gel using hexane:ethyl

acetate as eluting solvents. Further puriﬁcation by re-crystallization

from ethyl acetate or ethanol yielded yellow crystals.

4.1.2. General procedure for the synthesis of Series 1 curcumin

analogs

Curcumin analogs from Series 1 were prepared according to

Pedersen method with slight modiﬁcations [48] (Scheme 1). A

mixture of boric anhydride (0.35 g, 5 mmol), suspended in ethyl

acetate (EtOAc), and acetylacetone (1.00 g, 10 mmol) was ﬁrst

stirred for 3 h at 70 ^ꢃC. After removing the solvent, the resultant

white residue was washed with hexane. Following this,

substituted aldehyde (20 mmol), tributyl borate (4.60 g, 20 mmol)

and 20 mL EtOAc were added and stirred at room temperature for

a further 30 min. Butylamine (73 mg, 1 mmol) dissolved in EtOAc

(5 mL) was then added dropwise over 15 min and the mixture was

stirred at 70 ^ꢃC for another 24 h. Next, the reaction mixture was

heating for 30 min at 60 ^ꢃC after adjusting to pH5 by adding 1 N

HCl. EtOAc (3 ꢂ 50 mL) was then used to extract the crude product

from the water layer. The organic layer was washed with brine,

dried with anhydrous NaSO₄and evaporated in vacuo to give

either a solid or liquid residue and puriﬁed by column chroma-

tography on silica gel using hexane:ethyl acetate as eluting sol-

vents. Further puriﬁcation by re-crystallization from ethyl acetate

yielded yellow crystals.

4.1.4. General procedure for the synthesis of Series 2, 3 and 4

hydroxylated curcumin analogs

For compounds with phenolic hydroxyl substituent, additional

protection and de-protection of the phenolic hydroxyl groups on

the benzaldehyde were required and the method by Liang et al. was

Series 2

Series 3

4.1.3. General procedure for the synthesis of Series 2, 3, 4 and 5

alkoxylated curcumin

Series 4

Series 5

The method by Liang et al. was followed [49] (Scheme 2). To a

solution of substituted benzaldehyde (30 mmol) in methanol

(20 mL) was added appropriatelysubstituted ketone (15 mmol) such

as acetone (Series 2), cyclohexanone (Series 3) and cyclopentanone

(Series 4). For the synthesis of Series 5 analogs, 15 mmol of

substituted benzaldehyde and 15 mmol of substituted ketone were

CH₃

Scheme 2. General synthesis of curcumin analogs from Series 2, 3, 4 and 5.

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

with water, followed by extracting with ethyl acetate (50 mL ꢂ 3)

and the combined organic phasewas washed with brine (50 mL ꢂ 3),

dried over anhydrous NaSO₄, evaporated in vacuo and puriﬁed by

column chromatography and/or re-crystallization as described

earlier.

CHO

(i)

4.1.6. Characterization of synthesized compounds

(ii)

4.1.6.1. Compound 1a (curcumin). IUPAC name: 5-hydroxy-1,7-bis

(4-hydroxy-3-methoxy phenyl) hepta-1,4,6-trien-3-one. Yellowe

orange crystals. C₂₁H₂₀O₆. Melting point: 183 ^ꢃC. ¹H NMR (300 MHz,

CDCl₃)

: 3.95 (6H, s, OCH₃ꢂ2), 5.80 (1H, s, COCH]C), 6.48 (2H, d,

J ¼ 16 Hz, CHCO ꢂ2), 6.93 (2H, d, J ¼ 8 Hz, AreH), 7.05 (2H, s, AreH),

7.12 (2H, d, J ¼ 8 Hz, AreH), 7.59 (2H, d, J ¼ 16 Hz, AreCH]C ꢂ2).

¹³C NMR (75 MHz, CDCl₃)

d: 55.928, 101.089, 109.626, 114.800,

(iii)

121.769, 122.828, 127.677, 140.491, 140.491, 146.755, 147.823,

151.915, 183.229. MS (APCI) m/z: 369.0 (M þ 1)^þ. Anal calcd for

C₁₇H₁₄O₅: C, 68.45; H, 4.73; Found: C, 68.03; H, 4.78.

4.1.6.2. Compound 1b. IUPAC name: 5-hydroxy-1,7-bis (3,4-

dimethoxy phenyl) hepta-1,4,6-trien-3-one. Orange powder.

C₂₃H₂₄O₆. Yield: 39.0%. Melting point: 130e131 ^ꢃC. ¹H NMR

(300 MHz, CDCl₃)

: 3.92 (6H, s, OCH₃ꢂ2), 3.93 (6H, s, OCH₃ꢂ2),

Scheme 3. Protection and deprotection of phenolic hydroxyl groups for the synthesis

of compound 2f. Reagents and conditions: (i) pyridinium p-toluenesulfonate, RT, 4 h

(ii) 20% NaOH, RT, 3 h. (iii) 4 M HCl, RT, 4 h.

5.82 (1H, s, COCH]C), 6.50 (2H, d, J ¼ 16 Hz, CHCO ꢂ2), 6.88 (2H, d,

J ¼ 8 Hz, AreH), 7.10 (4H, m, AreH), 7.61 (2H, d, J ¼ 16 Hz, AreCH]

C ꢂ2). ¹³C NMR (75 MHz, CDCl₃)

d: 55.843, 55.912, 76.642, 77.066,

77.490, 101.274, 109.754, 111.096, 121.967, 122.578, 128.002,

140.334, 149.173, 150.992, 183.204. MS (APCI) m/z: 396.9 (M þ 1)^þ.

HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 9.394. P_HPLC/%: 97.50. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 2.931. P_HPLC/%: 100.00.

followed [49] (Scheme 3). A solution of 3,4-dihydro-a-pyran

(44 mmol) in dichloromethane (40 mL) was added dropwise to a

well stirred suspension of hydroxyl benzaldehyde (28.8 mmol) and

pyridinium p-toluenesulfonate (0.32 mmol) in dichloromethane

(80 mL) and stirred at room temperature for 4 h. The reaction

mixture was then washed with 1 M NaCO₃solution (60 mL ꢂ 3) and

brine (60 mL ꢂ 3), dried with anhydrous NaSO₄, evaporated in

vacuo and puriﬁed by column chromatography on silica gel using

hexane:ethyl acetate as eluting solvents to yield 4-(tetrahy-

dropyran-2-yloxy) as a pale yellow oil. This puriﬁed protected de-

rivative was condensed with substituted ketone at a ratio of 2:1

using similar procedures as described earlier for alkoxylated cur-

cumin analogs. At the end of the reaction, the protecting groups

were removed by acidifying with 4 M HCl, followed by stirring the

mixture for 4 h at room temperature. The reaction mixture was

then diluted with water, followed by extracting with ethyl acetate

(50 mL ꢂ 3) and the combined organic phase was washed with

brine (50 mL ꢂ 3), dried over anhydrous NaSO₄, evaporated in vacuo

and puriﬁed by column chromatography and/or re-crystallization

as described earlier.

4.1.6.3. Compound 1c. IUPAC name: 5-hydroxy-1,7-bis (3-hydroxy-

4-methoxy phenyl) hepta-1,4,6-trien-3-one. Orange powder.

₂₁H₂₀O₆. Yield: 35.0%. Melting point: 192 ^ꢃC. ¹H NMR (300 MHz,

CDCl₃)

: 3.92 (6H, s, OCH₃ꢂ2), 5.82 (1H, s, COCH]C), 6.48 (2H, d,

J ¼ 16 Hz, CHCO ꢂ2), 6.87 (2H, d, J ¼ 8 Hz, AreH), 7.04 (2H, d,

J ¼ 8 Hz, AreH),7.15 (2H, s, AreH), 7.53 (2H, d, J ¼ 16 Hz, AreCH]

C ꢂ2). MS (APCI) m/z: 369.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R

(min): 2.450. P_HPLC/%: 95.97. HPLC (ACN:H₂O ¼ 80:20) t_R(min):

1.849. P_HPLC/%: 95.48.

4.1.6.4. Compound 1d. IUPAC name: 5-hydroxy-1,7-diphenyl

hepta-1,4,6-trien-3-one. Yellow powder. C₁₉H₁₆O₂. Yield: 50.0%.

Melting point: 138e140 ^ꢃC. ¹H NMR (400 MHz, CDCl₃)

d: 7.68 (2H,d,

J ¼ 16 Hz), 7.57e7.55 (4H, m),7.40e7.39 (6H, m), 6.64 (2H,d,

J ¼ 15.6 Hz), 5.86 (1H, s). ¹³C NMR (100 MHz, CDCl₃)

d: 101.752,

124.194, 128.237, 128.878, 130.200, 134.551, 140.264, 183.092. MS

(APCI) m/z: 277.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

9.281. P_HPLC/%: 99.86. HPLC (ACN:H₂O ¼ 70:30) t_R(min): 12.003.

P_HPLC/%: 98.50.

4.1.5. General procedure for the synthesis of Series 5 hydroxylated

chalcones

For chalcones with phenolic hydroxyl substituent, additional

protection and de-protection of the phenolic hydroxyl groups on

both the acetophenone and substituted aromatic aldehyde were

required. The benzaldehyde (15 mmol) and aromatic ketone

(15 mmol), were reacted separately each with pyridinium p-tolue-

nesulfonate (1 mmol) and 3,4-dihydro-2H-pyran (40 mmol) in

dichloromethane (40 mL) and stirred at room temperature for 4 h.

The reaction mixture was then washed with 1 M NaCO₃solution

(60 mL ꢂ 3) and brine (60 mL ꢂ 3), dried with anhydrous NaSO₄,

evaporated in vacuo to yield the crude tetrahydropyranyl ether as a

pale yellow oil and used without puriﬁcation. This crude protected

aromatic aldehyde and ketones was then reacted at a ratio of 1:1

using similar procedures as described earlier for alkoxylated Series 5

analogs. At the end of the reaction, the protecting groups were

removed byacidifying with 4 M HCl, followed bystirring the mixture

for 4 h at room temperature. The reaction mixture was then diluted

4.1.6.5. Compound 1e. IUPAC name: 5-hydroxy-1,7-bis (3-methoxy

phenyl) hepta-1,4,6-trien-3-one. Brownish-orange powder.

C₂₃H₂₄O₄. Yield: 40.0%. Melting point: 59e60 ^ꢃC. ¹H NMR

(300 MHz, CDCl₃)

: 3.85 (6H, s, OCH₃ꢂ2), 5.86 (1H, s, COCH]C),

6.62 (2H, d, J ¼ 16 Hz, eCHCOe ꢂ2), 6.88 (2H, d, J ¼ 2 Hz, AreH),

6.95 (2H, d, J ¼ 10 Hz, AreH), 7.08 (2H, s, AreH),7.30 (2H, s, AreH),

7.633 (2H, d, J ¼ 16 Hz, AreCH]C ꢂ2). ¹³C NMR (75 MHz, CDCl₃)

55.289, 101.794, 113.037, 115.908, 120.794, 124.328, 129.882,

136.329, 140.543, 159.889, 183.230. MS (APCI) m/z: 337.0 (M þ 1)^þ.

HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 1.784. P_HPLC/%: 95.81. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 1.608. P_HPLC/%: 97.30.

4.1.6.6. Compound 1f. IUPAC name: 5-hydroxy-1,7-bis (4-hydroxy

phenyl) hepta-1,4,6-trien-3-one. Orangeered crystals. C₁₉H₁₆O₆.

Yield: 49.0%. Melting point: 229e230 ^ꢃC. ¹H NMR (400 MHz,

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

DMSO-d₆)

: 10.10 (2H, s),7.57e7.52 (6H,m), 6.82 (4H, d, J ¼ 8.8 Hz),

129.759, 135.958, 143.043, 159.751, 188.641. MS (APCI) m/z: 295.0

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 6.637. P_HPLC/%:

96.48. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 3.040. P_HPLC/%: 96.71.

6.68 (2H, d, J ¼ 15.6 Hz), 6.04 (1H, s). ¹³C NMR (100 MHz, DMSO-d₆)

d: 100.93, 115.92, 120.891, 125.979, 130.464, 140.522, 159.893,

183.325. MS (APCI) m/z: 369.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20)

t_R(min): 5.055. P_HPLC/%: 96.85. HPLC (ACN:H₂O ¼ 80:20) t_R(min):

1.670. P_HPLC/%: 99.14.

4.1.6.13. Compound 2f. IUPAC name: 1,5-bis (4-hydroxy phenyl)

penta-1,4-dien-3-one. Dark yellow powder.

C₁₇H₁₄O₃. Yield:

24.26%. Melting point: 236e239 ^ꢃC. ¹H NMR (400 MHz, DMSO-d₆)

4.1.6.7. Compound 1g. IUPAC name: 1,7-bis (3,4-diﬂuoro phenyl)-

: 10.09 (2H,s), 7.68e7.60 (6H, m),7.09 (2H,d, J ¼ 16 Hz), 6.83 (4H,d,

5-hydroxy hepta-1,4,6-trien-3-one. Yellow powder.

Yield: 94.0%. Melting point: 229e230 ^ꢃC. ¹H NMR (300 MHz,

DMSO-d₆)

: 5.82 (1H, s, COCH]C), 6.53 (2H, J ¼ 16 Hz, eCHCOe

ꢂ2), 7.14e7.41 (m, 7H, AreH þ AreCH]C ꢂ2), 7.57 (2H, d, 8 Hz, Are

H). ¹³C NMR (75 MHz, DMSO-d₆)

: 76.5764, 77.0000, 77.2022,

₁₉H₁₂F₄O.

J ¼ 8.8 Hz). ¹³C NMR (100 MHz, DMSO-d₆)

d: 115.836, 122.656,

125.821, 130.443, 142.400, 159.821, 188.047. MS (APCI) m/z: 267.2

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 2.738. P_HPLC/%:

99.42. HPLC (ACN:H₂O ¼ 70:30) t_R(min): 1.793. P_HPLC/%: 99.67.

77.4236, 78.3575, 102.1870, 116.0418, 117.7460, 117.9771, 124.8419,

132.1689, 138.4753, 182.7933. MS (APCI) m/z: 309.2 (M þ 1)^þ. HPLC

(MeOH:H₂O ¼ 80:20) t_R(min): 14.846. P_HPLC/%: 99.85. HPLC

(ACN:H₂O ¼ 70:30) t_R(min): 2.198. P_HPLC/%: 98.68.

4.1.6.14. Compound 2g. IUPAC name: 1,5-bis (3,4-diﬂuoro phenyl)

penta-1,4-dien-3-one. Yellow crystals. C₁₇H₁₀F₄O. Yield: 90.0%.

Melting point: 132e133 ^ꢃC. ¹H NMR (300 MHz, DMSO-d₆)

d: 6.955

(2H, d, J ¼ 7.95, AreH), 7.199 (2H, t, J ¼ 8.9 Hz, AreH), 7.332 (2H, d,

J ¼ 16 Hz, eCOCH] ꢂ2), 7.443 (2H, m, AreH), 7.637 (2H, d,

J ¼ 16 Hz, AreCH]C ꢂ2). MS (APCI) m/z: 306.9 (M þ 1)^þ. HPLC

4.1.6.8. Compound 2a. IUPAC name: 1,5-bis (4-hydroxy-3-methoxy

phenyl) penta-1,4-dien-3-one. Greenish-yellow powder. C₁₉H₁₈O₅.

(MeOH:H₂O

80:20) t_R(min): 2.728. P_HPLC/%: 99.28. HPLC

Yield: 33.0%. Melting point: 106 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

(ACN:H₂O ¼ 70:30) t_R(min): 1.793. P_HPLC/%: 99.67.

3.96 (6H, s, OCH₃ꢂ2), 6.90e6.95 (4H, m, AreH þ COCH] ꢂ2),

7.09e7.17 (m, 4H, AreH), 7.68 (2H, d, J ¼ 16 Hz, AreCH] ꢂ2). ¹³

4.1.6.15. Compound 3a. IUPAC name: 2,6-bis (4-hydroxy-3-

methoxy benzylidene) cyclohexanone. Yellow solid. C₂₂H₂₂O₅.

Yield: 30.0%. Melting point: 177e178 ^ꢃC. ¹H NMR (300 MHz, DMSO,

NMR (75 MHz, DMSO-d₆)

d: 55.689, 111.350, 115.632, 122.965,

123.334, 126.306, 142.754, 149.942, 188.015. MS (APCI) m/z: 326.9

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 1.794. P_HPLC/%: 95.81.

HPLC (ACN:H₂O ¼ 70:30) t_R(min): 1.608. P_HPLC/%: 97.40.

ppm):

J ¼ 9 Hz), 6.84e6.87 (d, 2H, J ¼ 9 Hz), 3.82 (s, 6H), 2.90 (s, 4H), 1.71e

1.75 (m, 2H); ¹³C NMR (75 MHz, DMSO, ppm):

189.53, 148.87,

d 9.53 (s, 2H), 7.57 (s, 2H), 7.12 (s, 2H), 7.02e7.05 (d, 2H,

4.1.6.9. Compound 2b. IUPAC name: 1,5-bis (3,4-dimethoxy

phenyl) penta-1,4-dien-3-one. Orangeeyellow solid. C₂₁H₂₂O₅.

148.48, 137.19, 134.55, 127.97, 125.27, 116.59, 115.85, 56.69, 29.01,

23.63. MS (APCI) m/z: 367.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R

(min): 3.351. P_HPLC/%: 97.88. HPLC (ACN:H₂O ¼ 80:20) t_R(min):

3.209. P_HPLC/%: 99.31.

Yield: 52.0%. Melting point: 93e95 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

3.93 (6H, s, OCH₃ꢂ2), 3.95 (6H, s, OCH₃ꢂ2), 6.89 (2H, d, J ¼ 8.1 Hz,

AreH), 6.96 (d, J ¼ 16 Hz, COCH] ꢂ2), 7.14e7.21 (m, 4H, AreH),

7.69 (d, J ¼ 16 Hz, AreCH] ꢂ2). ¹³C NMR (75 MHz, CDCl₃)

: 55.845,

4.1.6.16. Compound 3b. IUPAC name: 2,6-bis (3,4-dimethoxy ben-

55.892, 109.840, 111.035, 123.033, 123.530, 127.763, 142.984,

zylidene) cyclohexanone. Yellow crystals. C₂₄H₂₆O₅. Yield: 47.7%.

149.156, 151.252, 188.629. MS (APCI) m/z: 355.0 (M þ 1)^þ. HPLC

Melting point: 149e151 ^ꢃC. ¹H NMR (400 MHz, DMSO, ppm):

d 7.59

(MeOH:H₂O

80:20) t_R(min): 3.129. P_HPLC/%: 95.49. HPLC

(s, 1H), 7.14 (m, 4H), 7.06e6.99 (m, 2H), 3.80 (S, 12H), 2.91 (t, 4H,

(ACN:H₂O ¼ 70:30) t_R(min): 2.762. P_HPLC/%: 94.59.

J ¼ 5.3 Hz), 1.73 (M, 2H); ¹³C NMR (101 MHz, DMSO, ppm):

d 188.60,

149.47, 148.40, 135.79, 134.26, 128.09, 123.72, 114.01, 111.51, 55.47,

4.1.6.10. Compound 2c. IUPAC name: 1,5-bis (3-hydroxy-4-

methoxy phenyl) penta-1,4-dien-3-one. Yellow powder. C₁₉H₁₈O₅.

Yield: 33.27%. Melting point: 189e190 ^ꢃC. ¹H NMR (300 MHz,

27.82, 22.44. MS (APCI) m/z: 395.1 (M

(MeOH:H₂O ¼ 80:20) t_R(min): 5.691. P_HPLC/%: 100.00. HPLC

1)^þ. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 2.750. P_HPLC/%: 97.80.

DMSO-d₆)

: 3.77 (s, 6H, OCH₃ꢂ2), 6.93 (d, 2H, J ¼ 8 Hz, AreH), 7.03

(d, J ¼ 16 Hz, COCH] ꢂ2), 7.14e7.17 (m, 4H, AreH), 7.57 (d,

4.1.6.17. Compound 3c. IUPAC name: 2,6-bis (3-hydroxy-4-

J ¼ 16 Hz, 2H, AreCH] ꢂ2). ¹³C NMR (75 MHz, DMSO-d₆)

: 56.0,

methoxy benzylidene) cyclohexanone. Brownish-yellow powder.

112.592, 114.706, 122.818, 123.919, 128.057, 143.757, 147.087,

₂₂H₂₂O₅. Yield: 31.09%. Melting point: 189 ^ꢃC. ¹H NMR (300 MHz,

150.906, 189.454. MS (APCI) m/z: 327.0 (M

(MeOH:H₂O 80:20) t_R(min): 3.129. P_HPLC/%: 95.49. HPLC

(ACN:H₂O ¼ 70:30) t_R(min): 2.762. P_HPLC/%: 94.59.

1)^þ. HPLC

DMSO-d₆, ppm) d: 9.18 (br s, 2H), 7.48 (s, 2H), 6.99e7.01 (m, 6H),

3.81 (s, 6H), 2.86 (s, 4H), 1.72 (t, 2H, J ¼ 5 Hz); ¹³C NMR (75 MHz,

DMSO-d₆, ppm) d:188.51, 148.48, 146.17, 135.69, 133.96, 129.17,

122.88, 117.06, 111.91, 55.50, 27.85, 22.24. MS (APCI) m/z: 367.3

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 4.810. P_HPLC/%:

100.00. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 1.747. P_HPLC/%: 99.83.

4.1.6.11. Compound 2d. IUPAC name: 1,5-diphenyl penta-1,4-dien-

3-one. Yellow crystals. C₁₇H₁₄O. Yield: 28.3%. Melting point: 113 ^ꢃC.

¹H NMR (400 MHz, CDCl₃)

: 7.75 (2H,d, J ¼ 16 Hz), 7.63e7.61

(4H,m), 7.42e7.41 (6H,m), 7.09 (2H,d, J ¼ 17.3 Hz). ¹³C NMR

4.1.6.18. Compound 3d. IUPAC name: 2,6-dibenzylidene cyclohex-

anone. Yellow crystals. C₂₀H₁₈O. Yield: 59.5%. Melting point: 116e

(100 MHz, CDCl₃) d: 125.327, 128.310, 128.872, 130.411, 134.964,

143.206, 188.805. MS (APCI) m/z: 235.3 (M þ 1)^þ. P_HPLC/%: 95.37.

HPLC (ACN:H₂O ¼ 80:20) t_R(min): 3.075. P_HPLC/%: 99.72.

120 ^ꢃC. ¹H NMR (400 MHz, DMSO, ppm):

7.63 (s, 2H), 7.53 (d, 4H,

J ¼ 7 Hz), 7.46 (dd, 4H, J ¼ 10 Hz), 7.42e7.36 (m, 2H), 2.93e2.84 (m,

4H), 1.75e1.65 (m, 2H); ¹³C NMR (101 MHz, DMSO, ppm):

188.94,

4.1.6.12. Compound 2e. IUPAC name: 1,5-bis (3-methoxyphenyl)

136.25, 135.65, 135.25, 130.23, 128.75, 128.49, 27.76, 22.33. MS

(APCI) m/z: 275.1 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

12.149. P_HPLC/%: 98.71. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 5.549.

P_HPLC/%: 98.60.

penta-1,4-dien-3-one. Yellow solid. C₁₉H₁₈O₃. Yield: 45.7%. Melting

point: 50e51 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

d: 3.847 (6H, s, CH₃e

O ꢂ2), 6.959 (2H, d, J ¼ 3.15 Hz, AreH), 7.056 (4H, m, AreH), 7.208

(2H, d, J ¼ 16 Hz, -COCH] ꢂ2), 7.326 (2H, t, J ¼ 8 Hz, AreH), 7.699

(2H, d, J ¼ 16 Hz, AreCH]C ꢂ2). ¹³C NMR (75 MHz, CDCl₃)

4.1.6.19. Compound 3e. IUPAC name: 2,6-bis (3-methoxy benzyli-

dene) cyclohexanone. Yellow crystals. C₂₂H₂₂O₃. Yield: 41.1%.

55.100, 113.127, 116.158, 120.915, 113.127, 116.158, 120.915, 125.451,

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

Melting point: 59e60 ^ꢃC. ¹H NMR (400 MHz, DMSO, ppm):

7.61 (s,

OCH₃ꢂ2), 6.89 (2H, d, J ¼ 8 Hz, AreH), 7.16 (2H, d, J ¼ 8 Hz, AreH),

2H), 7.45e7.26 (m, 2H), 7.15e7.00 (m, 4H), 6.99e6.88 (m, 2H), 3.77

7.24 (2H, s, AreH), 7.35 (2H, s, CH]C ꢂ2), 9.69 (2H, br s, OH ꢂ2). ¹³

(s, 6H), 2.84 (t, 4H, J ¼ 5.2 Hz), 1.66 (m, 2H); ¹³C NMR (101 MHz,

NMR (75 MHz, DMSO-d₆)

d: 26.914, 56.605, 115.545, 116.922,

DMSO, ppm):

188.75, 159.11, 136.59, 136.37,135.64, 129.43, 122.43,

125.793, 128.179, 133.841, 135.768, 148.727, 149.537, 195.480. MS

(APCI) m/z: 353.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

2.464. P_HPLC/%: 98.66. HPLC (ACN:H₂O ¼ 70:30) t_R(min): 1.953.

P_HPLC/%: 100.00.

115.48, 114.45, 54.99, 27.78, 22.28. MS (APCI) m/z: 335.0 (M þ 1)^þ.

HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 5.049. P_HPLC/%: 95.43. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 4.033. P_HPLC/%: 96.79.

4.1.6.20. Compound 3f. IUPAC name: 2,6-bis (4-hydroxy benzyli-

dene) cyclohexanone. Brownish-yellow powder. C₂₀H₁₈O₃. Yield:

25.44%. Melting point: 270e271 ^ꢃC. ¹H NMR (400 MHz, DMSO,

4.1.6.26. Compound 4b. IUPAC name: 2,5-bis (3,4-dimethoxy

benzylidene) cyclopentanone. Yellow powder. C₂₃H₂₄O₅. Yield:

44.0%. Melting point: 196e199 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

ppm):

7.54 (s, 2H), 7.41 (d, 4H, J ¼ 8.7 Hz), 6.88e6.78 (m, 4H), 2.85

3.10 (4H, s, CH₂eCH₂), 3.93 (12H, s, OCH₃ꢂ4), 6.93 (2H, d, J ¼ 8 Hz,

(t, 4H, J ¼ 5.34 Hz), 1.71 (m, 2H); ¹³C NMR (101 MHz, DMSO, ppm):

AreH), 7.12e7.27 (m, 4H, AreH), 7.53 (2H, s, CH]C ꢂ2). ¹³C NMR

188.70, 158.44, 135.92, 133.48, 132.59, 126.59, 115.67, 28.09, 22.67.

(75 MHz, DMSO-d₆) d: 26.387, 55.822, 55.899, 111.112, 113.409,

MS (APCI) m/z: 307.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

7.542. P_HPLC/%: 98.28. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 3.075.

P_HPLC/%: 97.68.

124.534, 128.946, 133.622, 135.319, 148.856, 150.223, 195.946. MS

(APCI) m/z: 381.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

7.734. P_HPLC/%: 96.91. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 2.377.

P_HPLC/%: 96.60.

4.1.6.21. Compound 3g. IUPAC name: 2,6-bis (3,4-diﬂuoro benzyli-

dene) cyclohexanone. Yellow crystals. C₂₀H₁₄F₄O Yield: 12.5%.

4.1.6.27. Compound 4c. IUPAC name: 2,5-bis (3-hydroxy-4-

Melting point: 105e106 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

d: 1.79e1.87

methoxy benzylidene) cyclopentanone. Dark yellow powder.

(m, 2H, eCeCH₂e), 2.89 (4H, t, J ¼ 5.7 Hz, eCeCH₂e ꢂ2), 7.18e7.31

(m, 6H, AreH), 7.67 (2H, s, AreCH]C ꢂ2). MS (APCI) m/z: 347.0

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 6.923. P_HPLC/%:

92.43. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 5.013. P_HPLC/%: 96.79.

₂₁H₂₀O₅. Yield: 27.19%. Melting point: 224e225 ^ꢃC. ¹H NMR

(300 MHz, DMSO-d₆)

d: 3.02 (4H, s, CH₂eCH₂), 3.82 (6H, s,

OCH₃ꢂ2), 7.02 (2H, d, J ¼ 8 Hz, AreH), 7.12e7.14 (m, 4H, AreH), 7.28

(2H, s, CH]C ꢂ2), 9.28 (2H, s, OH ꢂ2). ¹³C NMR (75 MHz, DMSO-d₆)

: 25.846, 55.534, 112.095, 116.836, 123.566, 128.334, 132.410,

4.1.6.22. Compounds 3h. IUPAC name: 2,6-bis (2-ﬂuoro benzyli-

135.260, 146.481, 149.148, 194.795. MS (APCI) m/z: 353.0 (M þ 1)^þ.

HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 6.566. P_HPLC/%: 99.28. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 3.083. P_HPLC/%: 99.79.

dene) cyclohexanone. Yellow powder.

₂₀H₁₆F₂O. Yield: 98%.

: 1.7821 (2H,

Melting point: 95e96 ^ꢃC. ¹H NMR (300 MHz, CHCl₃)

m, eCH₂e), 2.8086 (4H, t, J ¼ 5.67 Hz ]CeCH₂e ꢂ2), 7.1324 (4H, m,

AreH), 7.3441 (4H, m, AreH), 7.8224 (2H, s, eCH]Ce ꢂ2). ¹³C NMR

4.1.6.28. Compound 4d. IUPAC name: 2,5-dibenzylidene cyclo-

(75 MHz, DMSO-d₆)

: 22.9383, 28.5130, 76.5764, 77.0000, 77.4236,

pentanone. Yellow powder. C₁₉H₁₆O. Yield: 63.9%. Melting point:

115.5604, 115.8589, 123.6673, 123.7154, 123.7636, 123.9455,

129.7041, 129.7522, 130.2722, 130.3781, 130.6669, 130.7054,

138.1961, 159.1660, 162.4877, 189.5330. MS (APCI) m/z: 311.0

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 5.535. P_HPLC/%:

97.00. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 4.435. P_HPLC/%: 95.99.

194e195 ^ꢃC. ¹H NMR (400 MHz, CDCl₃)

: 7.61e7.59 (6H, m), 7.46e

7.36 (6H, m), 3.11 (4H, s). ¹³C NMR (100 MHz, CDCl₃)

: 26.434,

128.675, 129.286, 130.652, 133.722, 135.717, 137.212, 196.220. MS

(APCI) m/z: 381.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

22.011. P_HPLC/%: 97.12. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 4.199.

P_HPLC/%: 99.28.

4.1.6.23. Compound 3i. IUPAC name: 2,6-bis (3-ﬂuoro benzylidene)

cyclohexanone. Yellow powder. C₂₀H₁₆F₂O. Yield: 99%. Melting

4.1.6.29. Compound 4e. IUPAC name: 2,5-bis (3-methoxy benzyli-

point: 84 ^ꢃC. ¹H NMR (300 MHz, CHCl₃)

d: 1.8000 (2H, m, eCH₂e),

dene) cyclopentanone. Yellow crystals. C₂₁H₂₀O₃. Yield: 56.8%.

2.9128 (4H, t, J ¼ 5.28 Hz ]CeCH₂e ꢂ2), 7.2148 (8H, m, AreH ꢂ2),

Melting point: 144e147 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

d: 3.05 (4H, s,

7.7270 (2H, s, eCH]Ce ꢂ2). ¹³C NMR (75 MHz, DMSO-d₆)

CH₂eCH₂), 3.82 (6H, s, MeO ꢂ2), 6.91 (dd, J ¼ 2 Hz, J ¼ 10 Hz, 2H,

22.6206, 28.2241, 76.5764, 77.0000, 77.4236, 115.2909, 115.5701,

116.4452, 116.7351, 126.1321, 126.1706, 129.7619, 129.8678,

135.6254, 135.6542, 135.8577, 137.8206, 137.9265, 160.8509,

164.1148, 189.6774. MS (APCI) m/z: 311.0 (M þ 1) ^þ. HPLC

AreH), 7.08 (s, 2H, AreH), 7.16 (d, J ¼ 8 Hz, 2H, AreH), 7.30e7.35 (m,

2H, AreH), 7.52 (s, 2H, CH]C ꢂ2). ¹³C NMR (75 MHz, DMSO-d₆)

26.387, 55.154, 114.968, 115.888, 123.179, 129.592, 133.647, 136.978,

137.405, 159.579, 196.103. MS (APCI) m/z: 321.0 (M þ 1)^þ. HPLC

(MeOH:H₂O ¼ 80:20) t_R(min): 13.048. P_HPLC/%: 98.85. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 4.022. P_HPLC/%: 99.09.

(MeOH:H₂O

80:20) t_R(min): 6.683. P_HPLC/%: 99.41. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 5.609. P_HPLC/%: 97.23.

4.1.6.24. Compound 3j. IUPAC name: 2,6-bis (4-ﬂuoro benzylidene)

4.1.6.30. Compound 4f. IUPAC name: 2,5-bis (4-hydroxy benzyli-

cyclohexanone. Yellow powder. C₂₀H₁₆F₂O. Yield: 99%. Melting

dene) cyclopentanone. Greenish-yellow powder. C₁₉H₁₆O₃. Yield:

point: 151 ^ꢃC. ¹H NMR (300 MHz, CHCl₃)

d: 1.8055 (2H, m, eCH₂e),

31.60%. Melting point: 314 ^ꢃC. ¹H NMR (300 MHz, DMSO-d₆)

d: 3.02

2.8952 (4H, t, J ¼ 5.28 Hz ]CeCH₂e ꢂ2), 7.0943 (4H, m, AreH),

(4H, s, CH₂eCH₂), 6.87 (4H, d, J ¼ 8 Hz, AreH^3,5ꢂ2), 7.33 (s, 2H,

7.4458 (4H, m, AreH), 7.7458 (2H, s, eCH]Ce ꢂ2). ¹³C NMR

CH]C ꢂ2), 7.54 (4H, d, J ¼ 8 Hz, AreH^2,6ꢂ2), 10.1 (br s, 2H, OH ꢂ2).

(75 MHz, DMSO-d₆)

: 22.8709, 28.3108, 115.3583, 115.6375,

¹³C NMR (75 MHz, DMSO-d₆)

d: 26.063, 116.110, 126.800, 132.912,

132.1689, 132.2844, 135.6928, 135.8468, 160.9857, 164.2978,

190.0048. MS (APCI) m/z: 310.9 (M

1)^þ. HPLC

(MeOH:H₂O 80:20) t_R(min): 5.699. P_HPLC/%: 96.45. HPLC

134.787, 159.078, 195.280. MS (APCI) m/z: 293.0 (M þ 1)^þ. HPLC

(MeOH:H₂O

80:20) t_R(min): 3.854. P_HPLC/%: 98.60. HPLC

(ACN:H₂O ¼ 70:30) t_R(min): 1.784. P_HPLC/%: 99.66.

(ACN:H₂O ¼ 80:20) t_R(min): 5.316. P_HPLC/%: 95.44.

4.1.6.31. Compound 4g. IUPAC name: 2,5-bis (3,4-diﬂuoro benzyli-

4.1.6.25. Compound 4a. IUPAC name: 2,5-bis (4-hydroxy-3-

dene) cyclopentanone. Yellow crystals. C₁₉H₁₂F₄O. Yield: 38.5%.

methoxy benzylidene) cyclopentanone. Dark yellow powder.

Melting point: 239e240 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

d: 3.10 (4H, s,

₂₁H₂₀O₅. Yield: 27.0%. Melting point: 198e202 ^ꢃC. ¹H NMR

CH₂eCH₂), 7.19e7.45 (m, 6H, AreH), 7.50 (s, 2H, CH]C ꢂ2). MS

(300 MHz, DMSO-d₆)

d: 3.06 (4H, s, eCH₂eCH₂), 3.84 (6H, s,

(APCI) m/z: 332.9 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

20.240. P_HPLC/%: 96.92. HPLC (ACN:H₂O ¼ 70:30) t_R(min): 13.712.

P_HPLC/%: 98.41.

: 3.94 (s, 3H, OCH₃), 3.98 (s, 3H, OCH₃), 6.86 (d, J ¼ 8 Hz, 1H, AreH),

6.93 (d, J ¼ 8 Hz,1H, AreH), 7.13 (dd, J ¼ 1.8 Hz, J ¼ 10 Hz,1H, AreH),

7.27e7.28 (m, 1H, AreH), 7.39 (d, J ¼ 16 Hz, 1H, COCH]), 7.61e7.63

(m, 2H, AreH), 7.72 (d, J ¼ 16 Hz, 1H, ArCH]). ¹³C NMR (75 MHz,

4.1.6.32. Compound 4h. IUPAC name: 2,5-bis (2-ﬂuoro benzyli-

dene) cyclopentanone. Yellow powder. C₁₉H₁₄F₂O. Yield: 99%.

DMSO-d₆) d: 56.197, 56.288, 111.821, 112.542, 114.804, 115.151,

Melting point: 210 ^ꢃC. ¹H NMR (300 MHz, CHCl₃)

d: 3.0484 (4H, s,

119.781, 122.406, 122.779, 128.088, 131.317, 144.265, 146.882,

147.027, 150.793, 152.766, 188.593. MS (APCI) m/z: 301.0 (M þ 1)^þ.

HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 3.366. P_HPLC/%: 99.40. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 2.934. P_HPLC/%: 99.91.

CH₂eCH₂), 7.7.167 (4H, m, AreH), 7.3667 (2H, m, AreH),7.5776 (2H,

m, AreH),7.8111 (2H, s, AreCH]C ꢂ2). ¹³C NMR (75 MHz, DMSO-

d₆)

d: 26.0771, 76.1527, 76.5764, 77.0000, 115.3775, 115.6664,

123.3496, 123.5132, 123.6095, 123.6577, 125.2655, 125.3329,

129.7041, 130.5802, 130.6958, 138.5041, 159.6281, 195.1076. MS

(APCI) m/z: 297.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min):

6.816. P_HPLC/%: 99.89. HPLC (ACN:H₂O:80 ¼ 20) t_R(min): 4.057.

P_HPLC/%: 94.18.

4.1.6.38. Compound 5d. IUPAC name: 1,3-diphenyl prop-2-en-1-

one. Light yellow crystals. C₁₅H₁₂O. Yield: 66.1%. Melting point:

53e55 ^ꢃC. ¹H NMR (400 MHz, CDCl₃)

: 8.04e8.02 (2H, m), 7.82 (1H,

d, J ¼ 15.6 Hz), 7.66e7.64 (2H, m), 7.61e7.49 (4H, m), 7.43e7.41 (3H,

m). ¹³C NMR (100 MHz, CDCl₃)

: 190.51, 144.79, 138.20, 134.88,

4.1.6.33. Compound 4i. IUPAC name: 2,5-bis (3-ﬂuoro benzylidene)

132.73, 130.49, 128.92, 128.58, 128.47, 128.41, 122.11. MS (APCI) m/z:

209.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 8.273. P_HPLC/%:

100.00. HPLC (ACN:H₂O ¼ 70:30) t_R(min): 4.465. P_HPLC/%: 99.27.

cyclopentanone. Yellow powder. C₁₉H₁₄F₂O. Yield: 99%. Melting

point: 191e192 ^ꢃC. ¹H NMR (300 MHz, CHCl₃)

d: 2.9969 (4H, s, CH₂e

CH₂), 7.0071 (2H, m, AreH), 7.2633 (6H, m, AreH), 7.4257 (2H, s,

AreCH]C ꢂ2). ¹³C NMR (75 MHz, DMSO-d₆)

d: 26.3370, 76.5764,

4.1.6.39. Compound 5e. IUPAC name: 1,3-bis (3-methoxy phenyl)

77.0000, 77.4236, 100.6658, 116.1863, 116.4751, 116.5907, 116.8795,

126.7001, 126.7386, 130.1662, 130.2818, 132.7273, 132.7658,

137.6954, 137.8109, 138.0420, 139.6595, 161.1494, 164.4229,

prop-2-en-1-one. Yellow liquid. C₁₇H₁₆O₃. Yield: 15.8%. Liquid state.

¹H NMR (300 MHz, CDCl₃)

d: 3.85 (s, 3H, OMe), 3.88 (s, 3H, OMe),

1)^þ. HPLC

6.97 (d, J ¼ 8 Hz, 1H, AreH), 7.12e7.61 (m, 8H, AreH þ COCH]),

195.9068. MS (APCI) m/z: 297.0 (M

7.77 (d, J ¼ 16 Hz, 1H, AreCH]). ¹³C NMR (75 MHz, CDCl₃)

(MeOH:H₂O 80:20) t_R(min): 5.848. P_HPLC/%: 97.32. HPLC

121.980, 128.358, 55.038, 55.156, 112.704, 113.263, 116.053, 118.978,

120.818, 120.857, 122.060, 129.343, 129.696, 135.993, 144.437,

159.650, 159.700, 189.78. MS (APCI) m/z: 241.2 (M þ 1)^þ. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 4.303. P_HPLC/%: 93.77.

4.1.6.34. Compound 4j. IUPAC name: 2,5-bis (4-ﬂuoro benzylidene)

(MeOH:H₂O

80:20) t_R(min): 6.073. P_HPLC/%: 98.13. HPLC

cyclopentanone. Yellow powder. C₁₉H₁₄F₂O. Yield: 99%. Melting

point: 239e240 ^ꢃC. ¹H NMR (300 MHz, CHCl₃)

d: 2.7885 (4H, s, CH₂e

(ACN:H₂O ¼ 70:30) t_R(min): 2.867. P_HPLC/%: 97.85.

CH₂), 6.8292 (8H, m, AreH), 7.3077 (2H, s, AreCH]C ꢂ2). ¹³C NMR

4.1.6.40. Compound 5f. IUPAC name: 1,3-bis (4-hydroxy phenyl)

(75 MHz, DMSO-d₆)

d: 26.3274, 76.5764, 77.0000, 77.4236,

prop-2-en-1-one. Pale yellow powder. C₁₅H₁₂O₃. Yield: 12.05%.

115.8108, 116.0996, 131.9956, 132.0437, 132.5444, 132.6599,

132.7177, 136.6556, 161.4864, 164.8177, 196.0416. MS (APCI) m/z:

297.0 (M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 7.108. P_HPLC/%:

99.57. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 4.387. P_HPLC/%: 95.02.

Melting point: 190e195 ^ꢃC. ¹H NMR (300 MHz, DMSO-d₆)

d: 6.80e

6.88 (m, 4H, AreH), 7.56e7.62 (m, 4H, AreH þ eCH]CHe), 7.94 (d,

J ¼ 8 Hz, 2H, AreH). MS (APCI) m/z: 241.2 (M þ 1)^þ. HPLC

(MeOH:H₂O

80:20) t_R(min): 3.326. P_HPLC/%: 99.69. HPLC

(ACN:H₂O ¼ 70:30) t_R(min): 2.941. P_HPLC/%: 99.69.

4.1.6.35. Compound 5a. IUPAC name: 1,3-bis (4-hydroxy-3-

methoxy phenyl) prop-2-en-1-one. Yellow powder. C₁₇H₁₆O₅.

Yield: 34.07%. Melting point: 108e110 ^ꢃC. ¹H NMR (400 MHz,

4.1.6.41. Compound 5g. IUPAC name: 1,3-bis (3,4-diﬂuoro phenyl)

prop-2-en-1-one. White crystals. C₁₅H₈F₄O. Yield: 14.3%. Melting

DMSO-d₆) d: 10.01 (1H,s), 9.68 (1H,s), 7.79e7.71 (2H, m), 7.63e7.59

point: 132e134 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

: 7.18e7.39 (m, 4H,

AreH þ COCH]), 7.45e7.51 (m, 1H, AreH), 7.73 (d, J ¼ 16 Hz, 1H,

AreCH]), 7.79e7.90 (m, 2H, AreH). ¹³C NMR (75 MHz, CDCl₃)

(2H, m), 7.47 (1H, d, J ¼ 1.6 Hz), 7.28e7.25 (1H, m), 6.91 (1H, d,

J ¼ 8.4 Hz), 6.83 (1H, d, J ¼ 8.4 Hz), 3.86 (6H, s). ¹³C NMR (100 MHz,

DMSO-d₆)

d: 55.637, 55.776, 111.519, 111.722, 114.848, 115.540,

55100.711, 116.482, 116.715, 117.508, 117.742, 117.894, 117.978,

118.127, 121.670, 121.694, 125.335, 125.403, 131.783, 131.841,

134.884, 143.229, 148.946, 152.249, 187.095. MS (APCI) m/z: 280.9

(M þ 1)^þ. HPLC (MeOH:H₂O ¼ 80:20) t_R(min): 4.523. P_HPLC/%:

95.64. HPLC (ACN:H₂O ¼ 80:20) t_R(min): 3.812. P_HPLC/%: 96.05.

118.643, 123.480, 126.445, 129.786, 143.527, 147.711, 149.334,

151.593, 186.957. MS (APCI) m/z: 301.0 (M

1)^þ. HPLC

(MeOH:H₂O 80:20) t_R(min): 4.252. P_HPLC/%: 97.30. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 3.234. P_HPLC/%: 95.49.

4.1.6.36. Compound 5b. IUPAC name: 1,3-bis (3,4-dimethoxy

phenyl) prop-2-en-1-one. Light yellow solid. C₁₉H₂₀O₅. Yield:

69.0%. Melting point: 110 ^ꢃC. ¹H NMR (400 MHz, CDCl₃) : 7.92e7.89

(1H, m), 7.81 (1H, d, J ¼ 15.6 Hz), 7.67 (1H, d, J ¼ 15.2 Hz), 7.59 (1H,

d, J ¼ 1.6 Hz), 7.51 (1H, d, J ¼ 1.6 Hz),7.40e7.37 (1H, m), 7.09 (1H, d,

J ¼ 8.4 Hz),7.01 (1H, d, J ¼ 18.4 Hz), 3.86e3.85 (9H, m), 3.81 (3H, s).

4.2. Biology

4.2.1. Cell lines and plasmids

All cell culture reagents were procured from Sigma Chemical Co.

(St Louis, MO). The U2OS human osteosarcoma cell line was bought

from American Tissue Culture Collection (Rockville, MD) and grown

and maintained in McCoy’s 5A medium. The human embryonic

kidney cells (HEK293T) was a gift from Dr Yi Yan Yang (Institute of

Biotechnology and Nanotechnology, Singapore). These cells were

cultured and maintained in Dulbecco’s Modiﬁed Eagle’s Medium

(DMEM). Both L-cells and L-cells stably transfected with a Wnt-3A

expression factor (L-Wnt-3A cells) were kindly provided by Pro-

fessor Victor Nurcombe (Institute of Medical Biology, A*STAR,

Singapore) and were maintained in DMEM supplemented with

¹³C NMR (100 MHz, CDCl₃)

d: 187.35, 153.06, 151.09, 148.98, 148.78,

143.53, 130.75, 127.63, 123.50, 123.25, 119.57, 111.57, 110.99, 110.78,

110.73, 55.72, 55.56. MS (APCI) m/z: 329.0 (M þ 1)^þ. HPLC

(MeOH:H₂O

80:20) t_R(min): 7.043. P_HPLC/%: 99.34. HPLC

(ACN:H₂O ¼ 80:20) t_R(min): 3.742. P_HPLC/%: 99.12.

4.1.6.37. Compound 5c. IUPAC name: 1,3-bis (3-hydroxy-4-

methoxy phenyl) prop-2-en-1-one. Yellow powder.

C₁₇H₁₆O₅.

Yield: 34.1%. Melting point: 144e147 ^ꢃC. ¹H NMR (300 MHz, CDCl₃)

400 mg/mL of G418. All culture media were supplemented with 10%

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 100

mg/mL

We used 30

mg Matrigel (BD Bioscience, San Jose, CA) precoated 8

streptomycin and 10 U/mL of penicillin G. The cells were grown in a

humidiﬁed atmosphere at 37 ^ꢃC containing 5% CO₂. Wnt-3A

conditioned medium (Wnt-3A CM) was prepared from Wnt-3A

secreting L-Wnt-3A cells according to ATCC instructions. TOPglow

and FOPglow reporter genes were purchased from Upstate

Biotechnology (Lake Placid, NY). The pCMV-RL renilla control vec-

tor was purchased from Promega (Madison, WI). The synthesized

curcumin analogs were dissolved in dimethyl sulfoxide (DMSO)

with the absolute concentration of DMSO not exceeding 0.1% in all

the experiments.

pore size polyethylene terephthalate (PET) membrane inserts (BD

Bioscience, San Jose, CA) for the assay. U2OS cells were treated with

different concentrations of curcumin analogs for 24 h. The viability

of the cells was conﬁrmed by trypan blue exclusion assay beforethey

were seeded onto the upper wells of the precoated membrane in-

serts at a density of 1.5 ꢂ10⁵/200

mL in serum-free medium. McCoy’s

5A medium with 15% FBS was added as a chemoattractant in the

lower compartment while control wells contained serum-free me-

dium in the lower chamber. Pre-treated U2OS cells were allowed to

invade the matrigel coated inserts for 48 h, following which the non-

invasive cells were removed with the help of a cotton swab. Cells in

thelowersurface of themembranewerethen ﬁxed with70%ethanol

and stained with 0.2% w/v crystal violet. Ten random microscopic

ﬁelds (200ꢂ magniﬁcations) were counted for invaded cells using

Nikon Eclipse TE2000U microscope (Melville, NY). All experiments

were performed at least in triplicates.

4.2.2. Dual luciferase reporter assay

HEK293T cells (1.4 ꢂ 10⁶) grown to 40% conﬂuency in 60 mm

culture dish were transiently co-transfected with either 3 mg TOP-

glowor 3 mg FOPglowand 0.012 mgpCMV-RL renilla control plasmids

for normalization of transfection efﬁciency using Lipofectamine

2000 (Invitrogen, Carlsbad, CA), following manufacturer’s in-

structions. On the other hand, U2OS cells (1.0 ꢂ 10⁵/well) were

grown in 24-well plates to 90e95% conﬂuency and then transiently

4.2.5. Western blot analysis

The expression levels of

examined pre- and post-24 h treatment with curcumin analogs

using western blot analysis as previously described [12]. -Catenin

b-catenin and MMP-9 protein was

co-transfected with either 0.3

0.012 g pCMV-RL renilla plasmids. Twenty hours post transfection,

both the cell types were co-incubated with Wnt-3A CM and curcu-

min analogs at various concentrations (0.01e20 M) for 24 h. For

mg TOPglow or 0.3 mg FOPglow, and

and MMP-9 primary antibodies were obtained from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA), whereas those for lamin A/C

HEK293T cells, they were re-seeded into 96-well plates at a density

of 18 ꢂ 10³cells/well before treatment. Luciferase assays were per-

formed with the Dual Luciferase Reporter assay system (Promega,

and a-tubulin were purchased from Sigma Chemical Co. (St Louis,

MO) and BD Bioscience (San Jose, CA), respectively. Anti-mouse IgG

horseradish peroxidase secondary antibody was from Bio-Rad

(Hercules, CA). Quantity One software (Bio-Rad, Hercules, CA)

was used for quantifying the bands and the results were then

Madison, WI) as per manufacturer’s instructions. Brieﬂy, 20

L cell

L Dual-Glo^ÒLuciferase Reagent

L of Dual-Glo^ÒStop & Glo^ÒRe-

lysate was incubated with 100

followed by incubation with 100

expressed relative to the lamin A/C and a-tubulin loading controls.

agent and the luminescence was measured at both the steps with a

luminometer (Tecan, MTX Lab Systems Inc., Vienna, VA). All the

readings were performed in triplicates and expressed as

mean ꢀ S.E.M. of the normalized ratios of Fireﬂy to Renilla luciferase

and as the percentage of DMSO-treated samples. The EC₅₀values for

HEK293T cells were obtained from the sigmoidal curve by plotting

the percentage normalized luciferase activity against the concen-

tration of curcumin analogs using GraphPad Prism version 4.00,

GraphPad Software (San Diego, CA).

All western blot experiments were repeated at least in triplicates.

4.2.5.1. Statistical analysis. The two-tailed Student’s t-test (SPSS,

Chicago, IL) was used to analyze statistical signiﬁcance for treat-

ment groups. p-value of less than 0.05 was used for statistical

comparison between the treated and the control samples.

Acknowledgments

We thank Dr Yi-Yan Yang and Professor Victor Nurcombe for

providing the HEK293T cells and L Wnt-3A cells respectively. We

also thank A/Prof Mei Lin Go for her helpful comments and

suggestions. This work was made possible by the Ministry of

Education Academic Research Fund (AcRF) R148000150112 to

PLRE and National University of Singapore Graduate Scholarships

to PCL, KLT and PB.

4.2.3. MTS assay

MTS cell cytotoxicity assay was used to evaluate the cytotoxic

proﬁle of the curcumin analogs. HEK293T and U2OS cells were

seeded into 96-well plates at a density of 18 ꢂ 10³cells/well and

10 ꢂ 10³cells/well, respectively. On the subsequent day, HEK293T

cells were treated with curcumin analogs at their respective EC₅₀

concentrations for Wnt inhibition for 24 h while U2OS cells were

treated with curcumin analogs at 1 and 5

treatment, cell viability was analyzed by adding 20

M for 24 h. After

Appendix A. Supplementary data

L of CellTiter

96 AQ_ueousOne Solution Reagent (Promega, Madison, WI).

Following an incubation period of 4 h, the absorbance of the formed

formazan crystals was measured at l_maxof 490 nm using a Tecan

Spectra Fluor spectrophotometer (MTX Lab Systems Inc Vienna,

VA). The percent cell viability after treatment with curcumin ana-

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2013.10.073.

References

logs was calculated using the following formula:

[1] C.A. Arndt, W.M. Crist, Common musculoskeletal tumors of childhood and

adolescence, N. Engl. J. Med. 341 (1999) 342e352.

[2] P.A. Meyers, G. Heller, J.H. Healey, A. Huvos, A. Applewhite, M. Sun,

M. LaQuaglia, Osteogenic sarcoma with clinically detectable metastasis at

initial presentation, J. Clin. Oncol. 11 (1993) 449e453.

[3] H.C. Crawford, B.M. Fingleton, L.A. Rudolph-Owen, K.J. Goss, B. Rubinfeld,

P. Polakis, L.M. Matrisian, The metalloproteinase matrilysin is a target of beta-

catenin transactivation in intestinal tumors, Oncogene 18 (1999) 2883e2891.

[4] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin,

B. Vogelstein, K.W. Kinzler, Identi ﬁ cation of c-MYC as a target of the APC

pathway, Science 281 (1998) 1509e1512.

viability ¼ (A_Analoguesꢁ A_Blank)/(A_Control

ꢁ A_Blank) ꢂ 100%,

(DMSO)

where A_Analogues¼ absorbance of wells with cells treated with

curcumin analogs, A_Blank¼ absorbance of wells with media and

A_{Control (DMSO)}¼ absorbance of wells with DMSO treated cells. Each

concentration of curcumin analog was performed in triplicate on

three separate occasions.

4.2.4. Cell invasion assay

Matrigel invasion assay was used to determine the effect of cur-

cumin analogs on U2OS cell invasion as previously described [12].

[5] O. Tetsu, F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon

carcinoma cells, Nature 398 (1999) 422e426.

P.-C. Leow et al. / European Journal of Medicinal Chemistry 71 (2014) 67e80

factor kappa B and Notch 1 in Raji cells, Basic Clin. Pharmacol. Toxicol. 101

[6] R.C. Haydon, A. Deyrup, A. Ishikawa, R. Heck, W. Jiang, L. Zhou, T. Feng, D. King,

H. Cheng, B. Breyer, T. Peabody, M.A. Simon, A.G. Montag, T.C. He, Cytoplasmic

and/or nuclear accumulation of the beta-catenin protein is a frequent event in

human osteosarcoma, Int. J. Cancer 102 (2002) 338e342.

(2007) 427e433.

[29] Z. Wang, Y. Zhang, S. Banerjee, Y. Li, F.H. Sarkar, Notch-1 down-regulation by

curcumin is associated with the inhibition of cell growth and the induction of

apoptosis in pancreatic cancer cells, Cancer 106 (2006) 2503e2513.

[30] A.S. Jaiswal, B.P. Marlow, N. Gupta, S. Narayan, Beta-catenin-mediated trans-

activation and cell-cell adhesion pathways are important in curcumin

(diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells,

Oncogene 21 (2002) 8414e8427.

[31] M. Kakarala, D.E. Brenner, H. Korkaya, C. Cheng, K. Tazi, C. Ginestier, S. Liu,

G. Dontu, M.S. Wicha, Targeting breast stem cells with the cancer preventive

compounds curcumin and piperine, Breast Cancer Res. Treat. 122 (2010) 777e

785.

[32] C.H. Park, E.R. Hahm, S. Park, H.K. Kim, C.H. Yang, The inhibitory mechanism of

curcumin and its derivative against beta-catenin/Tcf signaling, FEBS Lett. 579

(2005) 2965e2971.

[33] G. Garcea, D.P. Berry, D.J. Jones, R. Singh, A.R. Dennison, P.B. Farmer,

R.A. Sharma, W.P. Steward, A.J. Gescher, Consumption of the putative che-

mopreventive agent curcumin by cancer patients: assessment of curcumin

levels in the colorectum and their pharmacodynamic consequences, Cancer

Epidemiol. Biomarkers Prev. 14 (2005) 120e125.

[34] R.A. Sharma, S.A. Euden, S.L. Platton, D.N. Cooke, A. Shafayat, H.R. Hewitt,

T.H. Marczylo, B. Morgan, D. Hemingway, S.M. Plummer, M. Pirmohamed,

A.J. Gescher, W.P. Steward, Phase I clinical trial of oral curcumin: bio-

markers of systemic activity and compliance, Clin. Cancer Res. 10 (2004)

6847e6854.

[35] A.L. Cheng, C.H. Hsu, J.K. Lin, M.M. Hsu, Y.F. Ho, T.S. Shen, J.Y. Ko, J.T. Lin,

B.R. Lin, W. Ming-Shiang, H.S. Yu, S.H. Jee, G.S. Chen, T.M. Chen, C.A. Chen,

M.K. Lai, Y.S. Pu, M.H. Pan, Y.J. Wang, C.C. Tsai, C.Y. Hsieh, Phase I clinical trial

of curcumin, a chemopreventive agent, in patients with high-risk or pre-

malignant lesions, Anticancer Res. 21 (2001) 2895e2900.

[36] M.M. Yallapu, M. Jaggi, S.C. Chauhan, Curcumin nanoformulations: a future

nanomedicine for cancer, Drug Discov. Today 17 (2012) 71e80.

[37] A. Vyas, P. Dandawate, S. Padhye, A. Ahmad, F. Sarkar, Perspectives on new

synthetic curcumin analogs and their potential anticancer properties, Curr.

Pharm. Des. 19 (2013) 2047e2069.

[7] K. Iwao, Y. Miyoshi, G. Nawa, H. Yoshikawa, T. Ochi, Y. Nakamura, Frequent

beta-catenin abnormalities in bone and soft-tissue tumors, Jpn. J. Cancer Res.

90 (1999) 205e209.

[8] K. Chen, S. Fallen, H.O. Abaan, M. Hayran, C. Gonzalez, F. Wodajo,

T. MacDonald, J.A. Toretsky, A. Uren, Wnt10b induces chemotaxis of osteo-

sarcoma and correlates with reduced survival, Pediatr. Blood Cancer 51 (2008)

349e355.

[9] B.H. Hoang, T. Kubo, J.H. Healey, R. Sowers, B. Mazza, R. Yang, A.G. Huvos,

P.A. Meyers, R. Gorlick, Expression of LDL receptor-related protein 5 (LRP5) as

a novel marker for disease progression in high-grade osteosarcoma, Int. J.

Cancer 109 (2004) 106e111.

[10] Y. Guo, X. Zi, Z. Koontz, A. Kim, J. Xie, R. Gorlick, R.F. Holcombe, B.H. Hoang,

Blocking Wnt/LRP5 signaling by a soluble receptor modulates the epithelial to

mesenchymal transition and suppresses met and metalloproteinases in os-

teosarcoma Saos-2 cells, J. Orthop. Res. 25 (2007) 964e971.

[11] Y. Guo, E.M. Rubin, J. Xie, X. Zi, B.H. Hoang, Dominant negative LRP5 decreases

tumorigenicity and metastasis of osteosarcoma in an animal model, Clin.

Orthop. Relat. Res. 466 (2008) 2039e2045.

[12] P.C. Leow, Q. Tian, Z.Y. Ong, Z. Yang, P.L. Ee, Antitumor activity of natural

compounds, curcumin and PKF118-310, as Wnt/beta-catenin antagonists

against human osteosarcoma cells, Invest New Drugs 28 (2010) 766e782.

[13] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the

last 25 years, J. Nat. Prod. 70 (2007) 461e477.

[14] Y.J. Surh, Cancer chemoprevention with dietary phytochemicals, Nat. Rev.

Cancer 3 (2003) 768e780.

[15] C. Sharma, J. Kaur, S. Shishodia, B.B. Aggarwal, R. Ralhan, Curcumin down

regulates smokeless tobacco-induced NF-kappaB activation and COX-2

expression in human oral premalignant and cancer cells, Toxicology 228

(2006) 1e15.

[16] S. Singh, B.B. Aggarwal, Activation of transcription factor NF-kappa B is sup-

pressed by curcumin (diferuloylmethane) [corrected], J. Biol. Chem. 270

(1995) 24995e25000.

[17] A.P. Zambre, V.M. Kulkarni, S. Padhye, S.K. Sandur, B.B. Aggarwal, Novel cur-

cumin analogs targeting TNF-induced NF-kappaB activation and proliferation

in human leukemic KBM-5 cells, Bioorg. Med. Chem. 14 (2006) 7196e7204.

[18] N. Dhillon, B.B. Aggarwal, R.A. Newman, R.A. Wolff, A.B. Kunnumakkara,

J.L. Abbruzzese, C.S. Ng, V. Badmaev, R. Kurzrock, Phase II trial of curcumin in pa-

tients with advanced pancreatic cancer, Clin. Cancer Res. 14 (2008) 4491e4499.

[19] S. Balasubramanian, R.L. Eckert, Curcumin suppresses AP1 transcription

factor-dependent differentiation and activates apoptosis in human epidermal

keratinocytes, J. Biol. Chem. 282 (2007) 6707e6715.

[20] Y.J. Surh, S.S. Han, Y.S. Keum, H.J. Seo, S.S. Lee, Inhibitory effects of curcumin

and capsaicin on phorbol ester-induced activation of eukaryotic transcription

factors, NF-kappaB and AP-1, Biofactors 12 (2000) 107e112.

[21] A.C. Bharti, N. Donato, B.B. Aggarwal, Curcumin (diferuloylmethane) inhibits

constitutive and IL-6-inducible STAT3 phosphorylation in human multiple

myeloma cells, J. Immunol. 171 (2003) 3863e3871.

[38] C. Zhao, Z. Liu, G. Liang, Promising curcumin-based drug design: mono-

carbonyl analogues of curcumin (MACs), Curr. Pharm. Des. 19 (2013) 2114e

2135.

[39] G. Liang, L. Shao, Y. Wang, C. Zhao, Y. Chu, J. Xiao, Y. Zhao, X. Li, S. Yang,

Exploration and synthesis of curcumin analogues with improved structural

stability both in vitro and in vivo as cytotoxic agents, Bioorg. Med. Chem. 17

(2009) 2623e2631.

[40] H. Ohtsu, Z. Xiao, J. Ishida, M. Nagai, H.K. Wang, H. Itokawa, C.Y. Su, C. Shih,

T. Chiang, E. Chang, Y. Lee, M.Y. Tsai, C. Chang, K.H. Lee, Antitumor agents. 217.

Curcumin analogues as novel androgen receptor antagonists with potential as

anti-prostate cancer agents, J. Med. Chem. 45 (2002) 5037e5042.

[41] J.R. Fuchs, B. Pandit, D. Bhasin, J.P. Etter, N. Regan, D. Abdelhamid, C. Li, J. Lin,

P.-K. Li, Structureeactivity relationship studies of curcumin analogues, Bioorg.

Med. Chem. Lett. 19 (2009) 2065e2069.

[42] M.J. Ryu, M. Cho, J.Y. Song, Y.S. Yun, I.W. Choi, D.E. Kim, B.S. Park, S. Oh,

Natural derivatives of curcumin attenuate the Wnt/beta-catenin pathway

through down-regulation of the transcriptional coactivator p300, Biochem.

Biophys. Res. Commun. 377 (2008) 1304e1308.

[22] S. Bhattacharyya, D. Mandal, B. Saha, G.S. Sen, T. Das, G. Sa, Curcumin prevents

tumor-induced T cell apoptosis through Stat-5a-mediated Bcl-2 induction,

J. Biol. Chem. 282 (2007) 15954e15964.

[23] R. Blasius, S. Reuter, E. Henry, M. Dicato, M. Diederich, Curcumin regulates

signal transducer and activator of transcription (STAT) expression in K562

cells, Biochem. Pharmacol. 72 (2006) 1547e1554.

[43] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavail-

ability of curcumin: problems and promises, Mol. Pharmacol. 4 (2007)

807e818.

[44] K.L. Kirk, Selective ﬂ uorination in drug design and development: an overview

of biochemical rationales, Curr. Top. Med. Chem. 6 (2006) 1447e1456.

[45] X. Li, T. Ohtsuki, T. Koyano, T. Kowithayakorn, M. Ishibashi, New Wnt/beta-

catenin signaling inhibitors isolated from Eleutherine palmifolia, Chem.

Asian J. 4 (2009) 540e547.

[46] P. Polakis, Wnt signaling and cancer, Genes Dev. 14 (2000) 1837e1851.

[47] C.R. Dass, E.T. Ek, K.G. Contreras, P.F. Choong, A novel orthotopic murine

model provides insights into cellular and molecular characteristics

contributing to human osteosarcoma, Clin. Exp. Metastasis 23 (2006)

367e380.

[48] X. Qiu, Z. Liu, W.Y. Shao, X. Liu, D.P. Jing, Y.J. Yu, L.K. An, S.L. Huang, X.Z. Bu,

Z.S. Huang, L.Q. Gu, Synthesis and evaluation of curcumin analogues as potential

thioredoxin reductase inhibitors, Bioorg. Med. Chem. 16 (2008) 8035e8041.

[49] G. Liang, S. Yang, L. Jiang, Y. Zhao, L. Shao, J. Xiao, F. Ye, Y. Li, X. Li, Synthesis

and anti-bacterial properties of mono-carbonyl analogues of curcumin, Chem.

Pharm. Bull (Tokyo) 56 (2008) 162e167.

[24] H.Y. Kim, E.J. Park, E.H. Joe, I. Jou, Curcumin suppresses Janus kinase-STAT

in ﬂ ammatory signaling through activation of Src homology

domain-

containing tyrosine phosphatase

2 in brain microglia, J. Immunol. 171

(2003) 6072e6079.

[25] C. Natarajan, J.J. Bright, Curcumin inhibits experimental allergic encephalo-

myelitis by blocking IL-12 signaling through Janus kinase-STAT pathway in T

lymphocytes, J. Immunol. 168 (2002) 6506e6513.

[26] J. Rajasingh, H.P. Raikwar, G. Muthian, C. Johnson, J.J. Bright, Curcumin induces

growth-arrest and apoptosis in association with the inhibition of constitu-

tively active JAK-STAT pathway in T cell leukemia, Biochem. Biophys. Res.

Commun. 340 (2006) 359e368.

[27] M. Saydmohammed, D. Joseph, V. Syed, Curcumin suppresses constitutive

activation of STAT-3 by up-regulating protein inhibitor of activated STAT-3

(PIAS-3) in ovarian and endometrial cancer cells, J. Cell Biochem. 110 (2010)

447e456.

[28] Y. Chen, W. Shu, W. Chen, Q. Wu, H. Liu, G. Cui, Curcumin, both histone

deacetylase and p300/CBP-speci ﬁ c inhibitor, represses the activity of nuclear

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