Asymmetrical meta-methoxylated diarylpentanoids: Rational design, synthesis and anti-cancer evaluation in-vitro

Article abstract of DOI:10.1016/j.ejmech.2018.08.039

In the present study, a series of forty-five asymmetrical meta-methoxylated diarylpentanoids have been synthesized, characterized and evaluated for their in-vitro anti-cancer potential. Among the forty-five analogs, three compounds (20, 33 and 42) have been identified as lead compounds due to their excellent inhibition against five human cancer cell lines including SW620, A549, EJ28, HT1080 and MCF-7. Structure-activity relationship study on cytotoxicity of tested compounds suggested that the presence of meta-oxygenated phenyl ring played a critical role in enhancing their cytotoxic effects. Compounds 33 and 42 in particular, exhibited strongest cytotoxicity against tested cell lines with the IC₅₀ values ranging from 1.1 to 4.3 μM. Subsequent colony formation assay on SW620 cell line showed that both compounds 33 and 42 possessed strong anti-proliferative activity. In addition, flow cytometry based experiments revealed that these compounds could trigger intracellular ROS production thus inducing G2/M-phase cell arrest and apoptosis. All these results suggested that poly meta-oxygenated diarylpentnoid is a promising scaffold which deserved further modification and investigation in the development of natural product-based anti-cancer drug.

Full text of DOI:10.1016/j.ejmech.2018.08.039

Accepted Manuscript
Asymmetrical meta-methoxylated diarylpentanoids: Rational design, synthesis and
anti-cancer evaluation in-vitro
Sze Wei Leong, Suet Lin Chia, Faridah Abas, Khatijah Yusoff
PII:
S0223-5234(18)30710-4
10.1016/j.ejmech.2018.08.039
EJMECH 10650
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 November 2017
Revised Date: 11 August 2018
Accepted Date: 14 August 2018
Please cite this article as: S.W. Leong, S.L. Chia, F. Abas, K. Yusoff, Asymmetrical meta-methoxylated
diarylpentanoids: Rational design, synthesis and anti-cancer evaluation in-vitro, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.039.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Asymmetrical meta-methoxylated diarylpentanoids: Rational design, synthesis and anti-cancer
evaluation in-vitro
a
a,b
b,c
a,d
Sze Wei Leong *, Suet Lin Chia , Faridah Abas , Khatijah Yusoff
*
a
Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia
b
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia
c
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400
UPM, Serdang, Selangor, Malaysia.
d
Malaysia Genome Institute (MGI), National Institute of Biotechnology Malaysia (NIBM), Jalan Bangi,
4
3000 Kajang, Selangor Darul Ehsan, Malaysia.
*Corresponding authors
Tel +603-89467765; Fax: +603-89467590
E-mail address: leongszewei@upm.edu.my (SW. Leong), kyusoff@upm.edu.my (K. Yusoff)

ACCEPTED MANUSCRIPT
Abstract
In the present study, a series of forty-five asymmetrical meta-methoxylated diarylpentanoids have been
synthesized, characterized and evaluated for their in-vitro anti-cancer potential. Among the forty-five
analogs, three compounds (20, 33 and 42) have been identified as lead compounds due to their excellent
inhibition against five human cancer cell lines including SW620, A549, EJ28, HT1080 and MCF-7.
Structure-activity relationship study on cytotoxicity of tested compounds suggested that the presence of
meta-oxygenated phenyl ring played a critical role in enhancing their cytotoxic effects. Compounds 33 and
4
1
4
2 in particular, exhibited strongest cytotoxicity against tested cell lines with the IC50 values ranging from
.1 to 4.3 µM. Subsequent colony formation assay on SW620 cell line showed that both compounds 33 and
2 possessed strong anti-proliferative activity. In addition, flow cytometry based experiments revealed that
these compounds could trigger intracellular ROS production thus inducing G2/M-phase cell arrest and
apoptosis. All these results suggested that poly meta-oxygenated diarylpentnoid is a promising scaffold
which deserved further modification and investigation in the development of natural product-based anti-
cancer drug.
Keywords:
Assymetrical diarylpentanoids; Cytotoxicity; Anti-proliferative; Cell cycle; Apoptosis; Intracellular ROS
1
. Introduction
Cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14
million new cases in 2012. Worryingly, this number is estimated to rise by about 70% over the next two
decades. According to the global cancer database (Globocan) in 2012, there are 32.6 million people who
were diagnosed with cancer and the number of cancer-related deaths has exceeded 8.2 million cases [1].
This disquieting situation is worsening by its effect on global economy as cancer has badly hit the economy
with an estimated cost of 1.16 trillion USD in 2010 [2]. Thus, considering the global human and economic
health, safer cancer therapies with better effectiveness are currently being pursued.
To date, cancer treatments are mostly relying on chemotherapy especially for late-stage or complex
cancers. Paclitaxel, cisplatin, and docetaxel are considered as the most prominent chemodrugs due to their
excellent killing effects on various cancer cell lines including breast, head and neck, prostate and lung
cancer cells. Paclitaxel and docetaxel belong to the taxane family that function by stabilizing the
microtubules thus thwarting cell mitosis while cisplatin is a platinum-based compound that prevents cell
mitosis by cross-linking DNA [3-6]. Unfortunately, the full potential of these drugs is far from being
realized due to their associated severe side effects such as extreme fatigue and leukopenia, which are
caused by their low selectivity towards cancer cells [7]. These unpleasant side effects not only reduce the
life quality of cancer patients, but may also threaten their survival. Thus, finding safer drugs with improved
selectivity has been the focus of current anticancer drug discovery.
Curcumin is a naturally occurring small molecule which is well-known for its medicinal potential.
Curcumin has been proven to exhibit remarkable antioxidant, anti-inflammatory and anticancer properties

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upon its strong superoxides and free radicals inhibition, pro-inflammatory cytokines and enzymes
suppression, and cancer cells apoptosis induction [8-12]. Together with its excellent safety profile and
extremely mild side effects, curcumin has been suggested to be the most potent candidate in the discovery
of alternative cancer therapeutic agents. However, the practical use of curcumin is derogated by its low
bioavailability due to its poor absorbability and stability which caused by the presence of unstable β-
diketone moiety [13]. Thus, structural optimization to remove the unstable β-diketone moiety of curcumin
is an inevitable effort to reveal its full therapeutic potential by developing a derivative molecule with
improved bioactivities and stability.
Diarylpentanoids, a 5-carbon spacer series formed by the synthetic modification of curcumin has gained
increasing attention due to their superior enhanced medicinal properties, particularly as anticancer agents
[
14-17]. They have been shown to exhibit remarkable anticancer properties as they significantly inhibited
the growth of various cancer cell lines including breast, lung, colon, pancreatic and prostate cancer cells,
through their anti-proliferative and anti-angiogenetic activities [18-22]. In addition, diarylpentanoids were
also found to induce cancer cell apoptosis by simultaneously inhibiting and activating the Bcl-2 proteins
and caspases, respectively [23-26]. Unlike curcumin, diarylpentanoids are chemically stable at
physiological pH and metabolically stable in rat liver microsomes, which prompted them to be the most
impactful candidates among the curcuminoids that deserve further intensive investigations with the goal of
developing novel safer chemodrugs [23, 27].
Previous research found that vanillin and adjacent methoxyphenyl moieties on both terminals of
diarylpentanoids are responsible for their apoptosis inducing ability [28-30]. However, our structure
activity relationship (SAR) study suggests that meta-methoxyphenyl group could be the most crucial
functional group due to its presence in majority of the reported structures. This idea is further supported by
their structure similarity with several known anti-tubulin agents including colchicine and combretastatin A-
4
, as well as their anti-tubulin enhancing effects on reported diarylpentanoids [31, 32]. Surprisingly, several
recent studies showed that the asymmetrical diarylpentanoids exhibited similar activity to their respective
symmetrical analogs suggesting that only a single substitution on the diarylpentanoid scaffold is sufficient
to prepare compounds active in inducing apoptosis [23, 26, 33]. On the basis of these observations, the aim
of the present study is to develop a series of rare asymmetrical meta-methoxyphenyl-containing
diarylpentanoids and evaluate for their oncolytic potential.
2
. Results and Discussion
2
.1 Chemistry
x
Scheme 1. General synthetic steps for compounds 1-45
x
Reagents and conditions: (a) morpholine, p-toluene-sulphonic acid, toluene, reflux (2h); (b) methoxylated
O, reflux (0.5h); (d) benzaldehyde, EtOH, 10M NaOH, RT (overnight).
benzaldehyde, 80˚C (8h); (c) H
2
The synthesis of meta-methoxyphenyl-containing diarylpentanoids is outlined in Scheme 1. As shown
in Scheme 1, compounds 1-45 were achieved through two different carbonyl-group addition reactions

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namely
Stork enamine alkylation and aldol condensation [34, 35]. Stork enamine alkylation was first
carried out to prepare key intermediate I by reacting cyclohexanone with morpholine under reflux
condition. In order to obtain maximum yield, Dean-Stark apparatus was employed to prevent hydrolysis of
desired enamine by removing water by product. Enamine I formed was then reacted with 2,3- or 3,4-
dimethoxybenzaldehyde to afford intermediate II. Upon purification, intermediate II was further reacted
with appropriate benzaldehydes under base-catalyzed aldol condensation conditions to give target
compounds 1-45. The formation of the final product was confirmed by detection of two singlets at chemical
shift of 7.5-8.0 ppm in the proton NMR spectra which represents the vinylic protons of bezylidenes. All
1
13
synthesized compounds were purified by column chromatography and characterized by H-NMR, C-
NMR, and high-resolution electron impact-mass spectrometry (HRMS). Prior to assays, the purity of
synthesized compounds were confirmed to be greater than 95% based on their respective HPLC profiles.
The synthesized compounds were listed in Table 1.
Table 1. Chemical structures of compounds 1-45
Compound
R
1
R
2
Compound
R
1
R
2
1
2
3
4
5
6
7
8
9
2-Cl
3-Cl
4-Cl
2-Br
3-Br
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
4-Br
2-OMe
3-OMe
4-OMe
3-OH
4-OH
2,3-Cl
2,4-Cl
3,4-Cl
3,5-Cl
2-OMe
3-OMe
4-OMe
3-OH
4-OH
2,3-Cl
2,4-Cl
3,4-Cl
3,5-Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
2,3-OMe
3,4-OMe
2,3-OMe
2,4-OMe
3,4-OMe
3,5-OMe
3,4,5-OMe
3-Cl-4-OH
4-OH-3-OMe
3,4-OH
2,4-OMe
3,4-OMe
3,5-OMe
3,4,5-OMe
3-Cl-4-OH
4-OH-3-OMe
3,4-OH
2
2
.2 Biological Studies
.2.1 In-vitro cytotoxic activity
The in-vitro cytotoxic activity of the synthesized compounds on five human cancer cell lines including
SW620, A549, EJ28, HT1080 and MCF-7 were determined by MTT assay as previously described [36].
The preliminary screening results are summarized and compiled in Figure 1.

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Figure 1. Inhibitory activities of synthesized compounds against various cancer cell lines at a testing concentration of
2
0 µM. The red line across the graph represents the threshold percentage for further IC50 determination
As depicted in Figure 1, all compounds displayed significant cytotoxic effect (above 50% inhibition)
against all tested cancer cell lines at a testing concentration of 20 µM suggesting that meta-methoxyphenyl-
containing diarylpentanoid is a promising anti-cancer scaffold. On the other hand, considering the
inhibitory selectivity of synthesized compounds on different cancer cell lines, this diarylpentanoid system
appeared to be a weaker A549 targeting structure as majority of the tested analogs showed lower A549
inhibition compared to their respective activity on other cell lines. In order to gain better insight into the
role of different structural functionalities on cytotoxic effect, IC50 determination and SAR study were
performed. The calculated cytotoxic IC50 values of the synthesized compounds on various cell lines are
presented in Table 2, in which curcumin and normal human dermal fibroblasts (NHDF) were served as
controls. Heatmap (Figure 2) was also generated using GraphPad Prism v7.0 (Graphpad Software, La
Jolla, California, USA) to provide an overview on the IC50 performance of the synthesized compounds
against the various cancer cells.
Table 2. Cytotoxicity of compounds 1-45 on SW620, A549, EJ28, HT1080, MCF-7 and NHDF cell lines
.
IC50 (µM)
Compound
R1
R2
SW620
14.0
10.3
9.1
11.1
12.1
8.8
11.1
>20
7.0
13.6
6.4
A549
17.9
>20
>20
>20
>20
>20
>20
>20
16.4
>20
>20
>20
>20
>20
EJ28
10.5
12.0
11.3
11.0
10.8
12.1
15.2
16.6
10.0
16.8
8.5
HT1080
13.1
11.7
10.3
11.1
10.8
12.2
13.7
14.1
9.9
16.3
8.5
>20
19.3
>20
MCF-7
9.8
11.3
12.0
10.3
9.7
NHDF
24.1
44.2
21.4
18.6
>50
Curcumin
-
-
1
2
3
4
5
6
7
8
9
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-OMe
3-OMe
4-OMe
3-OH
4-OH
2,3-Cl
2,4-Cl
11.7
13.8
12.0
5.9
18.6
8.8
>20
>20
>20
23.9
21.4
31.3
16.4
43.5
19.3
>50
1
1
1
1
0
1
2
3
>20
16.3
16.3
>20
18.1
>20
>50
49.2

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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
2,3-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-OMe
3,4-Cl
3,5-Cl
10.0
5.5
8.3
>20
6.4
3.8
2.5
16.8
8.6
6.3
7.1
5.1
5.9
5.3
4.2
5.2
7.8
3.7
6.6
1.9
7.7
10.8
10.3
4.0
2.5
>20
4.5
2.5
1.8
7.1
4.5
10.5
>20
12.5
11.4
>20
14.4
10.2
5.9
>20
>20
>20
>20
11.0
10.1
>20
12.8
16.0
>20
6.7
>20
4.3
>20
>20
>20
6.9
5.5
>20
8.5
12.1
7.5
8.4
>20
6.6
3.6
10.8
6.1
6.1
>20
6.4
4.5
2.3
14.3
5.9
4.7
6.8
4.9
4.8
8.4
5.2
4.3
8.1
4.6
7.2
1.7
16.8
8.8
10.5
3.1
3.0
17.1
3.0
2.6
1.1
7.6
4.6
4.2
9.0
4.3
5.0
>20
6.5
1.7
25.5
21.6
31.0
>50
14.9
13.1
7.9
39.5
14.3
17.9
21.6
11.9
11.2
21.8
12.4
13.1
18.1
11.6
30.9
6.8
2,3-OMe
2,4-OMe
3,4-OMe
3,5-OMe
3,4,5-OMe
3-Cl-4-OH
4-OH-3-OMe
3,4-OH
2-Cl
2.4
1.4
18.3
10.6
6.4
6.9
5.4
5.7
7.4
4.9
7.1
8.1
4.2
8.4
1.8
16.6
10.4
12.4
3.4
4.2
>20
3.5
16.4
11.6
4.7
10.4
6.0
6.3
9.5
5.3
6.9
8.0
4.6
7.8
2.9
17.0
14.3
16.2
4.4
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-OMe
3-OMe
4-OMe
3-OH
4-OH
2,3-Cl
2,4-Cl
3,4-Cl
3,5-Cl
26.1
18.5
>50
10.1
8.3
>50
9.7
6.5
3.8
2,4-OMe
3,4-OMe
3,5-OMe
3,4,5-OMe
3-Cl-4-OH
4-OH-3-OMe
3,4-OH
15.6
13.5
3.9
1.6
12.3
7.9
5.1
2.9
>20
10.3
>20
3.0
1.7
8.0
4.6
5.8
21.9
13.7
25.2
12.1
14.4
Figure 2. Overall IC50 distribution of compounds 1-45 against SW620, A549, EJ28, HT1080 and MCF-7
As shown in both Table 1 and heatmap (Figure 2), three compounds (20, 33 and 42) were found to
exhibit excellent cytotoxicity in which compounds 33 and 42 were identified as the most promising
candidates due to their lowest overall IC50 values. Meanwhile, six (19, 31, 37, 38, 40 and 41) and sixteen
(
8, 10, 15, 16, 18, 23, 24, 25, 26, 27, 28, 29, 30, 32, 43 and 44) other analogs have displayed strong and
moderate cytotoxic activity with overall IC50 values ranging from approximately 3.0 to 5.0 µM and 5.1 to
0 µM, respectively. These results suggest that meta-substitution of phenyl ring is important for cytotoxic
1
effect as all of the meta-substituted compounds possessed improved activity compared to their respective
ortho- and para-substituted analogs, regardless to the substituting functional groups. This trend is clearly
demonstrated in the comparisons of compounds 1-11 and 24-34 in which compounds 2, 5, 8, 10, 25, 28, 31
and 33 performed better in reducing cancer cells viability. On top of this, the enhanced killing effects of di-

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meta substituted diarylpentanoids including 15, 19, 20, 38, 41 and 42 further confirmed the positive
contribution of meta-substitution in anti-cancer activity.
In addition to substitution position, substituting functional group was also found to play a role in
altering the cytotoxicity of synthesized compounds. According to the IC50 values, halogen group appeared
to be essential for better cytotoxicity as brominated and chlorinated compounds including 1, 3, 4, 6, 24, 26,
2
7 and 29 shows higher inhibition than their respective methoxylated (7, 9, 30, 32) and hydroxylated (11
and 34) compounds. However, this trend is only applicable to ortho and para- substitutions. The trend was
completely reversed when only meta-substituted diarylpentanoids were being considered. As showed by
compound 25 and 28, halogenated analogs displayed much lower activity than their respective
methoxylated (31) and hydroxylated (33) analogs, of which compound 33 achieved the strongest
cytotoxicity with its lowest IC50 values ranging from 1.7 – 4.3 µM. Similar observations found in the
comparisons of compounds 2, 5, 8 and 10 further strengthen the reversed trend of meta-substituted
diarylpentanoids. All these results infer that meta-oxygenated phenyl ring is the most crucial functionality
that provides diarylpentanoid with excellent cytotoxic effects. Interestingly, compound 33 with only one
meta-hydroxy moiety was found to exhibit similar activity to poly-methoxylated analog 42, which implied
that meta-hydroxylation could be a better option in preparing potent anti-cancer diarylpentanoids. In the
view of their promising cytotoxicity, compounds 33 and 42 were further analyzed to evaluate and compare
their anti-cancer potential.
2
.2.2 Colony formation inhibition assay
Figure 3. Representative images of SW620 cells colonies after treatment with various concentrations of compounds 33
(A) and 42 (B) for 10 days
Colony formation assay on colorectal cancer cell line (SW620) was employed to evaluate the anti-
proliferative properties of compound 33 and 42. As showed in Figure 3A, no colonies were observed in the
cells treated with 2.5 and 5 µM of compounds 33. Meanwhile, only few colonies were formed in the cells
treated with 1.25 µM of the same compound. Similar observations were also made in cells treated with
compound 42 (Figure 3B). These results suggest that both compounds 33 and 42 are strong anti-
proliferative agents. Further quantification using ImegeJ software revealed that compound 42 performed
slightly better than compound 33 based on its relatively lower percentage of colony formation at the
concentration of 0.625 µM [37]. The quantified results were plotted into a bar graph as shown in Figure 4.

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Figure 4. Relative colony formation of SW620 cells after 10 days of treatments with selected compounds at various
concentrations. Blue and red bars represent the effects of compounds 33 and 42 on SW620 cell colonies, respectively.
Since anti-proliferative activity may be due to cell cycle arrest and/or cell apoptosis, we further carried
out both analyses using flow cytometry to examine the possible anti-proliferative mechanism of compound
3
3 and 42.
2
.2.3 Cell cycle analysis
Cell cycle analyses were performed using a NovoCyte flow cytometer (ACEA Biosciences, Inc., San
Diego, CA, USA). Figure 5A illustrates the representative cell cycle histograms of treated and untreated
SW620 cells while Figure 5B represents the line graph plotted based on the data obtained from the flow
cytometry experiments.
Figure 5. Representative profiles (A) and quantified data (B) of cell cycle analysis on SW620 after 24 h of treatment
with various concentrations of compounds 33 and 42. Green and blue peaks in 5A represent G1 and G2 checkpoints,
respectively, while blue and red lines in 5B represent percentage of cells arrested in G0/G1 and G2/M phases,
respectively.

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As depicted in Figure 5A and Figure 5B, 1.25 µM of compound 33 and 42 were found to slightly
increase the percentage of cells in G2/M phase from 19% to 24% and 20%, respectively. The percentages
of G2/M-phase cells were further increased to 49% and 50%, respectively when 2.5 µM of same
compounds were used. In contrast, the number of cells in G0/G1 phase was found to be inversely
proportional to the concentration of compounds 33 and 42 as the increase in their concentrations reduced
the percentage of G0/G1-arrested cells. All these results indicate that both compounds 33 and 42 could
induce G2/M arrest in colorectal cancer cell line SW620.
2
.2.4 Cell apoptosis study
Flow cytometry-based apoptosis analysis was carried out on SW620 cell line using Annexin V and 7-
AAD double staining method (BD Biosciences, SanJose, CA, USA). Figure 6A and Figure 6B represent
the dot plot diagrams resulted from the apoptosis assay using compound 33 and 42, respectively, while the
data obtained were summarized and compiled into bar graph as shown in Figure 6C.
Figure 6. (A) Apoptosis profiles of SW620 treated with compound 33 at 24, 48 and 72h. (B) Apoptosis profiles of
SW620 treated with compound 42 at 24, 48 and 72h. (C) Comparison of total apoptotic cells induced by compound 33
and 42 at 24, 48 and 72h.
As shown in Figure 6A, the treatment with 1.25 µM of compound 33 was found to barely shift the dots
distribution to the right. This indicates that low dosage of compound 33 could only induce mild elevation of
apoptotic cells number. However, when higher dosage of the same compound was used, the green dots that
represent florescent cells were shifted further right reflecting an increment in the number of apoptotic cells.
This dose-dependent response was also observed in cells treated with compound 42 (Figure 6B), whereby
the increase in treatment concentrations of compound 42 has induced higher levels of cell apoptosis.
Although the dot plot patterns obtained for both compounds seemed to be identical, further bar chart

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comparison (Figure 6C) shows that compound 42 possessed a slightly stronger apoptosis inducing ability
based on the higher number of apoptotic cells.
2
.2.5 Intracellular reactive oxygen species (ROS) detection assay
Previous studies reported that the cell cycle arrest and cell apoptosis in response to drug treatment could
be caused by the stimulation of intracellular ROS [38]. Therefore, we further explored the ability of
compounds 33 and 42 in triggering the production of intracellular ROS using 2',7'-dichlorofluorescin-
diacetate (DCFDA) method. DCFDA is a non-fluorescent ROS-sensitive chemical which can be easily
absorbed by cells. Upon the exposure to ROS, the uptaken DCFDA will be converted into its respective
highly fluorescent derivative namely 2',7'-dichlorodihydrofluorescein (DCF), and thereby causes the cells
to be fluorescent. On the basis of this, the production degree of triggered ROS at different treatment
concentrations can be distinguished by measuring the fluorescence intensity of treated cells. Figure 7A and
Figure 7B represent the flow cytometry-based ROS profiles of cells treated with compounds 33 and 42,
respectively, while the peaks observed are corresponding to the cells with different fluorescence intensities.
Figure 7. Representative flow cytometry-based ROS profiles of SW620 cells after treated with compounds 33 (A) and
4
2 (B) for 12 h.
As shown in Figure 7, the peak responsible for the detection of untreated cells is located at the most left
region while the peak that corresponds to hydrogen peroxide treated cells is observed at the most right
region. The shift from left to right indicates an increment in cellular fluorescence intensity. Interestingly,
the fluorescence intensities of the treated cells were found to be directly proportional to the concentrations
of compounds 33 and 42, implying that both compounds were able to trigger the production of intracellular
ROS in dose-dependent manners. Similar to previous analyses, compound 42 appeared to be a slightly
better candidate based on it barely stronger intracellular ROS inducing ability.
3
. Conclusion
In summary, 45 analogs of asymmetrical meta-methoxyphenyl-containing diarylpentanoid have been
synthesized, characterized and evaluated for their anti-cancer potential against five human cancer cell lines
including SW620, A549, EJ28, HT1080 and MCF-7. Compounds 33 and 42 appeared to be the most active
analogs based on the determined IC50 values. The overall result suggested that meta-oxygenated phenyl
ring is crucial for cytotoxicity. Subsequent colony formation assay showed that compounds 33 and 42

ACCEPTED MANUSCRIPT
possess strong anti-proliferative activity. Meanwhile, further flow cytometry experiments proved that both
compounds could induce G2/M phase arrest and apoptosis in cancer cells via intracellular ROS stimulation.
These results suggest that both compounds 33 and 42 are potentially to be served as antimitotic agents.
Although the overall result shows that compound 42 is slightly better than compound 33, the anti-cancer
potential of meta-hydroxyphenyl should not be underestimated as compounds with single meta-
hydroxylation (33) have displayed comparable activities to that of di-meta-methoxylated analogs (41 and
4
2). In conclusion, we believe that the asymmetrical diarylpentanoid system with poly meta-oxygenated
functional groups is a promising class of natural product-based derivatives, which deserve further
investigation in the development of small molecule-based anti-cancer drug.
4
. Experimental Section
4
.1 Chemistry
Starting materials and chemical reagents were purchased from Sigma-Aldrich and Merck, and were
used without purification. Solvents were purchased from common commercial suppliers, were dried and
distilled before use. The chemical reactions were routinely checked on 0.20 mm Merck TLC plate silica gel
6
0 F254 in every reaction step. Purification procedures were conducted using column chromatography on
Merck silica gel 60 (mesh 70-230). Melting points were determined using Fisher-Johns melting point
apparatus and were uncorrected. High-resolution electron ionization-mass spectrometry (HREI-MS) was
determined using a DFS high resolution GC/MS (Thermo Scientific, San Jose, CA, USA). Nuclear
Magnetic Resonance Spectra were recorded on Varian 500 MHz NMR Spectrometer.
4
.1.1 General procedure for synthesis of I and II
Catalytic amount of p-toluenesulphonic acid was added into a mixture of cyclohexanone (20 mmol) and
morpholine (20 mmol) in 30 mL of toluene kept in 100 mL SNRB at room temperature. The mixture was
refluxed on a Dean & Stark apparatus for 2 h to obtain intermediate I. Upon completion, 20 mmol of 2,3-
or 3,4-dimethoxybenzaldehyde in 20 mL of toluene was added dropwise into the reaction solution and
heated at 80˚C for 8 h. Distilled water (10 ml) was then added and further refluxed for 30 min. The
resulting reaction mixture was extracted thrice with 3M HCl and once with 20 mL water. The toluene layer
was then dry over anhydrous magnesium sulphate and concentrated in vacuo followed by purification using
column chromatography to give pure 2-benzylidenecyclohexanone analog, the crucial intermediate II.
4
.1.2 General procedure for synthesis of compound 1-45
-Benzylidenecyclohexanone II (5 mmol) and appropriate aromatic aldehydes (5 mmol) were dissolved
2
in 30 mL of absolute ethanol in a 50 mL SNRB. Catalytic amount of 6M NaOH solution was then added
and stirred overnight. Subsequently, the resulting mixture was neutralized with 3M HCl followed by
extraction with ethyl acetate. The organic layer was then collected and dried over anhydrous magnesium
sulfate followed by evaporation with rotatory evaporator to give the crude of desired compounds. The pure
asymmetrical diarylpentanoids were finally obtained through column chromatography purification.
2
7
-(2-Chlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (1). Yellow viscous oil; yield:
1
6.21%; m.p.: -ºC; Mass calculated: 368.8533; Mass found: 368.8547. H NMR (500 MHz, CDCl ) δ: 1.74
3
(
dt, J=12.36, 6.33 Hz, 2 H) 2.74 - 2.78 (m, 2 H) 2.79 - 2.83 (m, 2 H) 3.83 (s, 3 H) 3.88 (s, 3 H) 6.93 (d,
J=7.94 Hz, 2 H) 7.04 - 7.09 (m, 1 H) 7.25 - 7.29 (m, 2 H) 7.31 - 7.35 (m, 1 H) 7.41 - 7.45 (m, 1 H) 7.89 (s,
1
3
1
1
H) 7.95 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.2, 28.4, 28.7, 55.8, 61.2, 112.8, 122.1, 123.5, 126.2,
3
29.5, 129.7, 130.3, 130.5, 133.0, 133.6, 134.5, 135.0, 137.3, 138.0, 148.3, 152.8, 190.6

ACCEPTED MANUSCRIPT
2
6
-(3-Chlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (2). Yellow viscous oil; yield:
1
5.81%; m.p.:- ; Mass calculated: 368.8533; Mass found: 368.8541. H NMR (500 MHz, CDCl ) δ: 1.8 (dt,
3
J=12.67, 6.18 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 - 3.8 (m, 3 H) 3.9 (s, 3 H) 6.9 (d, J=7.94
1
3
Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.3 - 7.4 (m, 3 H) 7.4 (s, 1 H) 7.7 (s, 1 H) 7.9 (s, 1 H). C NMR (126 MHz,
CDCl ) δ: 23.0, 28.4, 28.6, 55.8, 61.1, 112.8, 122.1, 123.5, 128.4, 128.5, 129.6, 129.9, 130.2, 132.8, 134.3,
3
1
35.3, 137.2, 137.4, 137.8, 148.3, 152.8, 190.1
2
-(4-Chlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (3). Yellow solid; yield: 85.17%;
1
m.p.: 94-96ºC; Mass calculated: 368.8533; Mass found: 368.8541. H NMR (500 MHz, CDCl
3
) δ: 1.8
(
7
2
quin, J=6.26 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=7.94 Hz, 2 H)
.0 - 7.1 (m, 1 H) 7.3 - 7.4 (m, 4 H) 7.7 (s, 1 H) 7.9 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.1, 28.4,
3
8.6, 55.8, 61.1, 112.8, 122.1, 123.5, 128.6, 130.3, 131.6, 132.7, 134.4, 135.6, 137.2, 148.3, 152.8, 190.1
1
3
2
7
-(2-Bromobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (4). Yellow viscous oil; yield:
1
4.22%; m.p.: -; Mass calculated: 413.3043; Mass found: 413.3068. H NMR (500 MHz, CDCl ) δ: 1.7 (dt,
3
J=12.36, 6.33 Hz, 2 H) 2.7 - 2.8 (m, 2 H) 2.8 - 2.8 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 - 7.0 (m, 2 H) 7.0 -
1
3
7
.1 (m, 1 H) 7.2 - 7.2 (m, 1 H) 7.3 - 7.4 (m, 2 H) 7.6 (d, J=7.94 Hz, 1 H) 7.8 (s, 1 H) 8.0 (s, 1 H). C NMR
(
126 MHz, CDCl ) δ: 23.2, 28.3, 28.7, 55.8, 61.2, 112.8, 122.1, 123.5, 125.1, 126.8, 129.6, 130.3, 130.5,
3
1
32.9, 133.0, 135.9, 136.4, 137.2, 137.7, 152.8, 190.0
2
-(3-Bromobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (5). Yellow solid; yield: 54.28%;
1
m.p.: 81-83ºC; Mass calculated: 413.3043; Mass found: 413.3057. H NMR (500 MHz, CDCl ) δ: 1.8
3
(
quin, J=6.26 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=7.93 Hz, 2 H)
7
7
1
.0 - 7.1 (m, 1 H) 7.2 - 7.3 (m, 1 H) 7.4 (d, J=7.94 Hz, 1 H) 7.5 (d, J=7.94 Hz, 1 H) 7.6 (s, 1 H) 7.7 (s, 1 H)
13
.9 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.0, 28.4, 28.6, 55.8, 61.2, 112.8, 122.1, 122.4, 123.5, 128.9,
3
29.9, 130.2, 131.3, 132.8, 132.8, 135.2, 137.2, 137.5, 138.1, 148.3, 152.8, 190.0
2
-(4-Bromobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (6). Yellow solid; yield: 77.71%;
1
m.p.: 101-102ºC; Mass calculated: 413.3043; Mass found: 413.3048. H NMR (500 MHz, CDCl
3
) δ: 1.8
(
7
quin, J=6.26 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.8 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=7.93 Hz, 2 H)
.0 - 7.1 (m, 1 H) 7.3 (d, J=8.55 Hz, 2 H) 7.5 (d, J=8.55 Hz, 2 H) 7.7 (s, 1 H) 7.9 (s, 1 H). C NMR (126
1
3
MHz, CDCl ) δ: 23.1, 28.4, 28.7, 55.8, 61.1, 112.8, 122.1, 122.8, 123.5, 130.3, 131.6, 131.8, 132.7, 134.9,
3
1
35.6, 136.9, 137.2, 148.3, 152.8, 190.1
2
4
-(2,3-Dimethoxybenzylidene)-6-(2-methoxybenzylidene)cyclohexanone (7). Yellow viscous oil; yield:
1
3.21%; m.p.: - ; Mass calculated: 364.4343; Mass found: 364.4353. H NMR (500 MHz, CDCl ) δ: 1.7
3
(
dt, J=12.67, 6.18 Hz, 2 H) 2.8 (t, J=6.41 Hz, 2 H) 2.8 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 3.9 - 3.9 (m, 3
H) 6.9 - 6.9 (m, 3 H) 7.0 (t, J=7.63 Hz, 1 H) 7.0 - 7.1 (m, 1 H) 7.3 - 7.4 (m, 2 H) 7.9 (s, 1 H) 8.0 (s, 1 H).
13
C NMR (126 MHz, CDCl ) δ: 23.4, 28.7, 28.8, 55.5, 55.8, 61.1, 110.6, 112.6, 119.9, 122.2, 123.4, 125.0,
3
1
30.1, 130.3, 132.2, 132.7, 136.4, 137.7, 148.3, 152.8, 158.4, 190.4
2
3
-(2,3-Dimethoxybenzylidene)-6-(3-methoxybenzylidene)cyclohexanone (8). Yellow viscous oil; yield:
1
8.77%; m.p.: - ; Mass calculated: 364.4343; Mass found: 364.4361. H NMR (500 MHz, CDCl ) δ: 1.8
3
(
6
dt, J=12.36, 6.33 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 -
1
3
.9 (m, 3 H) 7.0 (s, 1 H) 7.0 - 7.1 (m, 2 H) 7.3 (t, J=7.94 Hz, 1 H) 7.8 (s, 1 H) 7.9 (s, 1 H). C NMR (126
) δ: 23.1, 28.5, 28.7, 55.3, 55.8, 61.1, 112.7, 114.2, 115.7, 122.1, 122.8, 123.5, 129.3, 130.4,
32.5, 136.6, 136.9, 137.3, 137.4, 148.3, 152.8, 159.4, 190.3
MHz, CDCl
1
3

ACCEPTED MANUSCRIPT
2
4
-(2,3-Dimethoxybenzylidene)-6-(4-methoxybenzylidene)cyclohexanone (9). Yellow solid; yield:
1
3.15%; m.p.: 114-115ºC; Mass calculated: 364.4343; Mass found: 364.4352. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.26 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 -
13
7
.0 (m, 4 H) 7.0 - 7.1 (m, 1 H) 7.5 (d, J=8.55 Hz, 2 H) 7.8 (s, 1 H) 7.9 (s, 1 H). C NMR (126 MHz,
CDCl ) δ: 23.2, 28.4, 28.8, 55.3, 55.8, 61.1, 112.6, 113.9, 122.1, 123.4, 128.7, 130.5, 132.0, 132.3, 134.2,
3
1
37.1, 137.6, 148.2, 152.8, 160.0, 190.2
2
2
-(2,3-Dimethoxybenzylidene)-6-(3-hydroxybenzylidene)cyclohexanone (10). Yellow solid; yield:
1
2.34%; m.p.: 115-117ºC; Mass calculated: 350.4077; Mass found: 350.4091. H NMR (500 MHz, CDCl
3
)
δ: 1.7 - 1.8 (m, 2 H) 2.7 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.4 (br. s., 1 H) 6.9 (dd,
J=8.24, 2.14 Hz, 1 H) 6.9 (d, J=7.93 Hz, 2 H) 7.0 - 7.0 (m, 2 H) 7.0 - 7.1 (m, 1 H) 7.2 - 7.3 (m, 1 H) 7.8 (s,
1
3
1
1
3
H) 7.9 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.0, 28.4, 28.6, 55.9, 61.2, 110.0, 112.9, 116.1, 117.4,
22.1, 122.5, 123.5, 129.5, 130.2, 132.9, 136.4, 137.3, 137.5, 148.3, 152.8, 156.0, 191.0
2
3
-(2,3-Dimethoxybenzylidene)-6-(4-hydroxybenzylidene)cyclohexanone (11). Yellow solid; yield:
3.82%; m.p.: 161-163ºC; Mass calculated: 350.4077; Mass found: 350.4084. H NMR (500 MHz,
1
acetone) δ: 1.8 (quin, J=6.36 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (d,
J=8.67 Hz, 2 H) 7.0 - 7.0 (m, 1 H) 7.0 - 7.1 (m, 2 H) 7.5 (d, J=8.67 Hz, 2 H) 7.7 (s, 1 H) 7.9 (s, 1 H) 8.8 (s,
1
3
1
1
H). C NMR (126 MHz, acetone) δ: 23.0, 28.1, 28.5, 55.3, 60.0, 113.2, 115.5, 121.8, 123.5, 130.2, 130.8,
32.5, 133.7, 136.5, 137.3, 148.4, 153.0, 158.3, 188.4
2
-(2,3-Dichlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (12). Yellow viscous oil;
1
yield: 44.57%; m.p.:- ; Mass calculated: 403.2984; Mass found: 403.2993. H NMR (500 MHz, CDCl ) δ:
3
1
.7 (dt, J=12.67, 6.18 Hz, 2 H) 2.7 - 2.7 (m, 2 H) 2.8 - 2.8 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=7.93
1
3
Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.2 - 7.2 (m, 2 H) 7.4 - 7.5 (m, 1 H) 7.8 (s, 1 H) 8.0 (s, 1 H). C NMR (126
MHz, CDCl ) δ: 23.2, 28.4, 28.7, 55.9, 61.2, 112.9, 122.1, 123.5, 126.6, 126.7, 128.6, 130.0, 130.2, 132.3,
32.9, 133.3, 136.8, 137.1, 138.6, 148.3, 152.8, 189.9
3
1
2
5
1
-(2,4-Dichlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (13). Yellow solid; yield:
1
3.14%; m.p.: 96-96ºC; Mass calculated: 403.2984; Mass found: 403.2992. H NMR (500 MHz, CDCl
3
) δ:
.7 (dt, J=12.57, 6.14 Hz, 2 H) 2.7 - 2.7 (m, 2 H) 2.8 - 2.8 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=8.09
1
3
Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.2 (s, 2 H) 7.4 (s, 1 H) 7.8 (s, 1 H) 7.9 (s, 1 H). C NMR (126 MHz, CDCl ) δ:
3
2
1
3.2, 28.5, 28.6, 55.8, 61.1, 112.9, 122.1, 123.5, 126.6, 129.6, 130.2, 131.2, 132.3, 133.0, 133.2, 134.6,
35.7, 137.0, 138.5, 148.3, 152.8, 189.8
2
5
-(3,4-Dichlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (14). Yellow solid; yield:
1
8.35%; m.p.: 105-106ºC; Mass calculated: 403.2984; Mass found: 403.2989. H NMR (500 MHz, CDCl )
3
δ: 1.8 (dt, J=12.36, 6.33 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.8 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (dd,
J=8.24, 3.97 Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.2 - 7.3 (m, 1 H) 7.5 (d, J=8.55 Hz, 1 H) 7.5 (s, 1 H) 7.6 (s, 1 H)
1
3
7
1
.9 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.0, 28.4, 28.6, 55.8, 61.2, 112.9, 122.1, 123.5, 129.5, 130.1,
3
30.4, 131.7, 132.5, 132.6, 133.0, 134.1, 136.0, 137.0, 137.8, 148.3, 152.8, 189.8
2
2
-(3,5-Dichlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (15). Yellow solid; yield:
1
7.76%; m.p.: 136-138ºC; Mass calculated: 403.2984; Mass found: 403.2997. H NMR (500 MHz, CDCl
3
)
δ: 1.8 (dt, J=12.28, 6.29 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.8 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 (dd,
13
J=8.09, 3.47 Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.3 - 7.3 (m, 3 H) 7.6 (s, 1 H) 7.9 (s, 1 H). C NMR (126 MHz,
CDCl ) δ: 22.9, 28.4, 28.5, 55.8, 61.1, 112.9, 122.0, 123.5, 128.2, 128.2, 133.2, 133.7, 134.9, 135.6, 135.7,
3
1
36.7, 136.9, 138.5, 138.9, 148.3, 152.8, 189.7

ACCEPTED MANUSCRIPT
2
,6-Bis(2,3-dimethoxybenzylidene)cyclohexanone (16). Yellow solid; yield: 69.57%; m.p.: 116-118ºC;
1
Mass calculated: 394.4602; Mass found: 394.4627. H NMR (500 MHz, CDCl ) δ: 1.7 (quin, J=6.26 Hz, 2
H) 2.8 (t, J=6.41 Hz, 4 H) 3.8 (s, 6 H) 3.9 (s, 6 H) 6.9 - 7.0 (m, 4 H) 7.0 - 7.1 (m, 2 H) 7.9 (s, 2 H).
3
13
C
NMR (126 MHz, CDCl ) δ: 23.3, 28.7, 55.8, 61.1, 112.7, 122.1, 123.4, 130.4, 132.4, 137.5, 148.3, 152.8,
3
1
90.3
2
4
-(2,3-Dimethoxybenzylidene)-6-(2,4-dimethoxybenzylidene)cyclohexanone (17). Yellow solid; yield:
1
3.84%; m.p.: 125-127ºC; Mass calculated: 394.4602; Mass found: 394.4609. H NMR (500 MHz, CDCl )
3
δ: 1.7 (dt, J=12.28, 6.29 Hz, 2 H) 2.7 - 2.8 (m, 2 H) 2.8 - 2.9 (m, 2 H) 3.8 (s, 3 H) 3.8 (d, J=1.73 Hz, 6 H)
.9 (s, 3 H) 6.5 (d, J=2.89 Hz, 1 H) 6.5 - 6.5 (m, 1 H) 6.9 - 6.9 (m, 2 H) 7.0 - 7.1 (m, 1 H) 7.3 (d, J=8.67
3
13
Hz, 1 H) 7.9 (s, 1 H) 8.0 (s, 1 H). C NMR (126 MHz, CDCl
3
) δ: 23.5, 28.7, 29.0, 55.4, 55.5, 55.8, 61.1,
9
1
8.2, 104.1, 112.5, 118.0, 122.2, 123.4, 130.6, 131.2, 131.8, 132.5, 134.6, 137.8, 148.2, 152.8, 160.0,
61.6, 190.3
2
6
-(2,3-Dimethoxybenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (18). Yellow solid; yield:
1
0.48%; m.p.: 105-107ºC; Mass calculated: 394.4602; Mass found: 394.4618. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.18 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 3.9 (s,
13
3
H) 6.9 - 6.9 (m, 3 H) 7.0 (s, 1 H) 7.0 - 7.1 (m, 1 H) 7.1 (d, J=8.78 Hz, 1 H) 7.8 (s, 1 H) 7.9 (s, 1 H).
C
NMR (126 MHz, CDCl ) δ: 23.2, 28.4, 28.8, 55.8, 55.9, 55.9, 61.1, 110.8, 112.6, 113.7, 122.1, 123.4,
3
1
24.0, 129.0, 130.5, 132.1, 134.5, 137.3, 137.5, 148.2, 148.6, 149.6, 152.8, 190.1
2
4
1
-(2,3-Dimethoxybenzylidene)-6-(3,5-dimethoxybenzylidene)cyclohexanone (19). Yellow solid; yield:
1
3.32%; m.p.: 85-87ºC; Mass calculated: 394.4602; Mass found: 394.4618. H NMR (500 MHz, CDCl ) δ:
3
.7 (dt, J=12.28, 6.29 Hz, 2 H) 2.7 - 2.8 (m, 2 H) 2.9 - 2.9 (m, 2 H) 3.8 (s, 9 H) 3.9 - 3.9 (m, 3 H) 6.4 (t,
J=2.31 Hz, 1 H) 6.6 (d, J=2.31 Hz, 2 H) 6.9 (d, J=8.09 Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.7 (s, 1 H) 7.9 (s, 1 H).
13
3
C NMR (126 MHz, CDCl ) δ: 23.1, 28.5, 28.7, 55.4, 55.8, 61.1, 100.7, 108.3, 112.7, 122.1, 123.4, 130.3,
1
32.5, 136.7, 136.9, 137.4, 137.8, 144.1, 148.3, 152.8, 160.5, 190.3
2
-(2,3-Dimethoxybenzylidene)-6-(3,4,5-trimethoxybenzylidene)cyclohexanone (20). Yellow solid;
1
yield: 41.98%; m.p.: 141-143ºC; Mass calculated: 424.4862; Mass found: 424.4871. H NMR (500 MHz,
CDCl ) δ: 1.8 (quin, J=6.18 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 12 H) 6.7 (s, 2
3
1
3
H) 6.9 (d, J=7.68 Hz, 2 H) 7.0 - 7.1 (m, 1 H) 7.7 - 7.7 (m, 1 H) 7.9 (s, 1 H). C NMR (126 MHz, CDCl ) δ:
3
2
1
3.2, 28.4, 28.7, 55.8, 56.2, 61.0, 61.1, 107.8, 112.7, 122.1, 123.5, 130.4, 131.5, 132.4, 135.6, 137.2, 137.4,
48.3, 152.9, 153.0, 190.1
2
-(3-Chloro-4-hydroxybenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (21). Yellow solid;
1
yield: 67.88%; m.p.: 98-100ºC; Mass calculated: 384.8527; Mass found: 384.8539. H NMR (500 MHz,
acetone) δ: 1.7 - 1.8 (m, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 7.0 - 7.0 (m, 1 H)
13
7
.0 - 7.1 (m, 3 H) 7.4 (dd, J=8.38, 2.02 Hz, 1 H) 7.6 (d, J=2.31 Hz, 1 H) 7.6 (s, 1 H) 7.9 (s, 1 H). C NMR
(
126 MHz, acetone) δ: 22.9, 28.1, 28.5, 55.3, 60.1, 110.0, 113.3, 116.7, 120.4, 121.8, 123.5, 128.9, 130.1,
1
30.7, 131.2, 131.9, 134.8, 135.2, 137.2, 148.4, 153.0, 188.4
2
-(2,3-Dimethoxybenzylidene)-6-(4-hydroxy-3-methoxybenzylidene)cyclohexanone (22). Yellow solid;
1
yield: 64.58%; m.p.: 121-123ºC; Mass calculated: 380.4337; Mass found: 380.4349. H NMR (500 MHz,
acetone) δ: 1.8 (quin, J=6.36 Hz, 2 H) 2.8 - 2.8 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 3.9 (s, 3
H) 6.9 - 6.9 (m, 1 H) 7.0 - 7.0 (m, 1 H) 7.0 - 7.1 (m, 3 H) 7.2 (s, 1 H) 7.7 (s, 1 H) 7.9 (s, 1 H) 8.1 (s, 1 H).
13
C NMR (126 MHz, acetone) δ: 23.1, 28.1, 28.5, 55.3, 55.4, 60.1, 113.2, 114.2, 115.1, 121.8, 123.5, 124.5,
1
27.9, 130.2, 130.8, 133.9, 135.1, 136.8, 137.4, 147.3, 147.7, 153.0, 188.4

ACCEPTED MANUSCRIPT
2
7
-(3,4-Dihydroxybenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (23). Yellow solid; yield:
8.24%; m.p.: 122-124ºC; Mass calculated: 366.4071; Mass found: 366.4083. H NMR (500 MHz,
1
acetone) δ: 1.8 (quin, J=6.21 Hz, 2 H) 2.9 - 2.9 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 - 6.9
m, 1 H) 6.9 - 7.0 (m, 1 H) 7.0 - 7.0 (m, 1 H) 7.1 (d, J=1.73 Hz, 1 H) 7.1 (d, J=4.05 Hz, 2 H) 7.5 - 7.6 (m, 1
(
1
3
H) 7.6 - 7.7 (m, 1 H) 8.2 (br. s., 1 H) 8.3 (br. s., 1 H). C NMR (126 MHz, acetone) δ: 22.4, 28.3, 28.3,
5
1
5.2, 55.2, 110.0, 111.5, 114.1, 115.3, 117.4, 123.6, 123.8, 128.2, 128.9, 133.8, 134.6, 135.7, 136.2, 144.9,
49.2, 150.1, 188.3
2
6
-(2-Chlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (24). Yellow solid; yield:
1
6.83%; m.p.: 107-109ºC; Mass calculated: 368.8533; Mass found: 368.8544. H NMR (500 MHz, CDCl
3
)
δ: 1.79 (quin, J=6.31 Hz, 2 H) 2.72 - 2.78 (m, 2 H) 2.92 - 2.99 (m, 2 H) 3.91 (s, 3 H) 3.92 (s, 3 H) 6.91 (d,
J=8.23 Hz, 1 H) 7.02 (s, 1 H) 7.09 - 7.13 (m, 1 H) 7.25 - 7.28 (m, 2 H) 7.31 - 7.34 (m, 1 H) 7.42 - 7.45 (m,
1
3
1
1
3
H) 7.78 (s, 1 H) 7.88 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.1, 28.1, 28.7, 55.9, 55.9, 110.8, 113.7,
24.1, 126.2, 126.9, 128.8, 129.4, 129.7, 130.5, 133.2, 134.2, 134.6, 134.9, 137.8, 148.6, 149.7, 189.8
2
5
1
-(3-Chlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (25). Yellow solid; yield:
1
3.77%; m.p.: 96-97ºC; Mass calculated: 368.8533; Mass found: 368.8547. H NMR (500 MHz, CDCl ) δ:
3
.81 (quin, J=6.31 Hz, 2 H) 2.86 - 2.92 (m, 2 H) 2.92 - 2.99 (m, 2 H) 3.91 (s, 3 H) 3.92 (s, 3 H) 6.91 (d,
J=8.23 Hz, 1 H) 7.02 (s, 1 H) 7.12 (dd, J=8.23, 1.65 Hz, 1 H) 7.29 - 7.35 (m, 3 H) 7.42 (s, 1 H) 7.70 (s, 1
13
H) 7.76 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 22.9, 28.3, 28.5, 55.9, 55.9, 110.9, 113.7, 124.1, 128.4,
3
1
28.4, 128.8, 129.6, 129.8, 134.1, 134.3, 134.8, 137.4, 137.6, 137.8, 148.7, 149.8, 189.9
2
8
-(4-Chlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (26). Yellow solid; yield:
1
1.31%; m.p.: 137-139ºC; Mass calculated: 368.8533; Mass found: 368.8547. H NMR (500 MHz, CDCl )
3
δ: 1.81 (quin, J=6.31 Hz, 2 H) 2.86 - 2.90 (m, 2 H) 2.92 - 2.98 (m, 2 H) 3.91 (s, 3 H) 3.92 (s, 3 H) 6.91 (d,
J=8.23 Hz, 1 H) 7.02 (d, J=1.65 Hz, 1 H) 7.11 (d, J=8.23 Hz, 1 H) 7.35 - 7.40 (m, 4 H) 7.72 (s, 1 H) 7.75
1
3
(
s, 1 H). C NMR (126 MHz, CDCl
3
) δ: 22.9, 28.3, 28.5, 55.9, 56.0, 110.9, 113.7, 124.0, 128.6, 128.8,
1
31.5, 134.1, 134.4, 134.5, 135.1, 136.7, 137.5, 148.6, 149.7, 189.9
2
6
-(2-Bromobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (27). Yellow solid; yield:
1
4.71%; m.p.: 111-112ºC; Mass calculated: 413.3043; Mass found: 413.3055. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.21 Hz, 2 H) 2.7 - 2.8 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=8.09 Hz,
1
1
1
H) 7.0 (s, 1 H) 7.1 (d, J=8.09 Hz, 1 H) 7.2 - 7.2 (m, 1 H) 7.3 - 7.3 (m, 2 H) 7.6 (d, J=8.09 Hz, 1 H) 7.8 (s,
13
H) 7.8 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.1, 28.0, 28.7, 55.9, 110.9, 113.8, 124.1, 125.0, 126.8,
3
28.9, 129.5, 130.5, 131.5, 132.9, 134.2, 135.5, 136.5, 137.8, 148.7, 149.8, 189.8
2
5
-(3-Bromobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (28). Yellow solid; yield:
1
6.99%; m.p.: 183-185 ºC; Mass calculated: 413.3043; Mass found: 413.3048. H NMR (500 MHz, CDCl )
3
δ: 1.8 (dt, J=12.28, 6.29 Hz, 2 H) 2.9 - 2.9 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d,
J=8.09 Hz, 1 H) 7.0 (s, 1 H) 7.1 (d, J=8.67 Hz, 1 H) 7.2 - 7.3 (m, 1 H) 7.3 - 7.4 (m, 1 H) 7.4 - 7.5 (m, 1 H)
1
3
7
1
1
.6 (s, 1 H) 7.7 (s, 1 H) 7.8 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 22.9, 28.3, 28.5, 55.9, 55.9, 110.9,
3
13.7, 122.4, 124.1, 128.8, 128.8, 129.9, 131.3, 132.7, 134.1, 134.7, 137.4, 137.6, 138.1, 148.6, 149.8,
89.8
2
7
-(4-Bromobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (29). Yellow solid; yield:
1
8.29%; m.p.: 130-131ºC; Mass calculated: 413.3043; Mass found: 413.3061. H NMR (500 MHz, CDCl
3
)
δ: 1.8 (quin, J=6.36 Hz, 2 H) 2.8 - 2.9 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=8.09 Hz,
H) 7.0 (s, 1 H) 7.1 (d, J=8.09 Hz, 1 H) 7.3 (d, J=8.09 Hz, 2 H) 7.5 (d, J=8.09 Hz, 2 H) 7.7 (s, 1 H) 7.7 (s,
1

ACCEPTED MANUSCRIPT
1
3
1
1
H). C NMR (126 MHz, CDCl ) δ: 22.9, 28.3, 28.5, 55.9, 110.8, 113.7, 122.7, 124.0, 128.5, 128.8,
3
31.6, 131.7, 134.1, 134.9, 135.2, 136.8, 137.5, 148.6, 149.7, 189.9
2
3
-(3,4-Dimethoxybenzylidene)-6-(2-methoxybenzylidene)cyclohexanone (30). Yellow solid; yield:
1
3.09%; m.p.: 125-126ºC; Mass calculated: 364.4343; Mass found: 364.4355. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.18 Hz, 2 H) 2.8 - 2.9 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 -
.9 (m, 2 H) 7.0 (t, J=7.41 Hz, 1 H) 7.0 (d, J=2.20 Hz, 1 H) 7.1 (dd, J=8.23, 1.65 Hz, 1 H) 7.3 - 7.3 (m, 2
6
13
H) 7.8 (t, J=2.47 Hz, 1 H) 8.0 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.2, 28.4, 28.8, 55.5, 55.9, 55.9,
3
1
1
10.0, 110.6, 110.8, 113.7, 119.9, 123.9, 129.1, 130.0, 130.3, 132.3, 134.6, 136.3, 137.0, 148.6, 149.5,
58.3, 190.2
2
4
-(3,4-Dimethoxybenzylidene)-6-(3-methoxybenzylidene)cyclohexanone (31). Yellow viscous oil; yield:
1
4.03%; m.p.: -; Mass calculated: 364.4343; Mass found: 364.4350. H NMR (500 MHz, CDCl
3
) δ: 1.8
(
7
quin, J=6.18 Hz, 2 H) 2.9 - 3.0 (m, 4 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 - 6.9 (m, 2 H) 7.0 (s, 1 H)
.0 (d, J=1.65 Hz, 1 H) 7.1 (d, J=7.68 Hz, 1 H) 7.1 (d, J=8.23 Hz, 1 H) 7.3 - 7.3 (m, 1 H) 7.8 (s, 2 H). C
13
NMR (126 MHz, CDCl ) δ: 23.0, 28.4, 28.6, 55.3, 55.9, 55.9, 110.9, 113.7, 114.1, 115.7, 122.8, 124.0,
3
1
28.9, 129.3, 134.4, 136.4, 136.5, 137.2, 137.4, 148.6, 149.7, 159.4, 190.2
2
5
-(3,4-Dimethoxybenzylidene)-6-(4-methoxybenzylidene)cyclohexanone (32). Yellow solid; yield:
1
1.36%; m.p.: 142-144ºC; Mass calculated: 364.4343; Mass found: 364.4353. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.31 Hz, 2 H) 2.9 - 3.0 (m, 4 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=8.23 Hz, 1 H)
.9 - 6.9 (m, 2 H) 7.0 (d, J=2.20 Hz, 1 H) 7.1 (dd, J=8.78, 1.65 Hz, 1 H) 7.4 - 7.5 (m, 2 H) 7.8 (d, J=8.23
6
1
3
Hz, 2 H). C NMR (126 MHz, CDCl ) δ: 23.0, 28.5, 28.5, 55.3, 55.9, 55.9, 110.8, 113.6, 113.9, 123.9,
3
1
28.7, 129.0, 132.2, 134.2, 134.6, 136.6, 136.7, 148.6, 149.5, 159.9, 190.1
2
3
-(3,4-Dimethoxybenzylidene)-6-(3-hydroxybenzylidene)cyclohexanone (33). Yellow solid; yield:
1
1.84%; m.p.: 129-131ºC; Mass calculated: 350.4077; Mass found: 350.4085. H NMR (500 MHz, CDCl
3
)
δ: 1.8 (dt, J=12.49, 6.11 Hz, 2 H) 2.9 - 3.0 (m, 4 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.5 (br. s., 1 H) 6.9 (dd, J=8.23,
.74 Hz, 1 H) 6.9 (d, J=8.23 Hz, 1 H) 7.0 - 7.0 (m, 3 H) 7.1 (d, J=8.78 Hz, 1 H) 7.2 - 7.3 (m, 1 H) 7.7 - 7.8
2
13
(
m, 2 H). C NMR (126 MHz, CDCl
3
) δ: 22.9, 28.4, 28.5, 55.9, 110.0, 110.9, 113.8, 116.0, 117.4, 122.4,
1
24.2, 128.8, 129.6, 134.3,136.4, 137.3, 137.9, 148.6, 149.8, 156.0, 190.9
2
5
-(3,4-Dimethoxybenzylidene)-6-(4-hydroxybenzylidene)cyclohexanone (34). Yellow solid; yield:
0.19%; m.p.: 146-148ºC; Mass calculated: 350.4077; Mass found: 350.4083. H NMR (500 MHz,
1
acetone) δ: 1.8 (dt, J=12.49, 6.11 Hz, 2 H) 2.9 - 3.0 (m, 2 H) 3.0 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 -
.9 (m, 2 H) 7.0 - 7.0 (m, 1 H) 7.1 - 7.1 (m, 2 H) 7.4 (d, J=8.78 Hz, 2 H) 7.6 (d, J=2.20 Hz, 2 H) 8.8 - 8.9
6
13
(
m, 1 H). C NMR (126 MHz, acetone) δ: 22.9, 28.3, 28.3, 55.2, 55.2, 111.4, 114.1, 115.4, 123.8, 127.5,
1
28.8, 132.4, 133.8, 134.6, 135.8, 135.9, 149.1, 150.1, 158.2, 188.3
2
4
-(2,3-Dichlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (35). Yellow solid; yield:
1
0.25%; m.p.: 121-122ºC; Mass calculated: 403.2984; Mass found: 403.2996. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.18 Hz, 2 H) 2.7 - 2.7 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=8.23 Hz,
1
7
1
H) 7.0 (d, J=1.65 Hz, 1 H) 7.1 - 7.1 (m, 1 H) 7.2 - 7.2 (m, 2 H) 7.4 (dd, J=6.59, 2.74 Hz, 1 H) 7.8 (s, 1 H)
.8 (s, 1 H). C NMR (126 MHz, CDCl ) δ: 23.0, 28.0, 28.7, 55.9, 55.9, 110.0, ) 110.9, 113.7, 124.2,
3
26.7, 128.6, 128.7, 130.0, 132.9, 133.5, 134.0, 136.7, 136.9, 138.2, 138.6, 148.7, 189.6
13
2
4
-(2,4-Dichlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (36). Yellow solid; yield:
1
8.41%; m.p.: 143-145ºC; Mass calculated: 403.2984; Mass found: 403.2991. H NMR (500 MHz, CDCl )
3
δ: 1.8 (dt, J=12.28, 6.29 Hz, 2 H) 2.7 - 2.7 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d,

ACCEPTED MANUSCRIPT
J=8.09 Hz, 1 H) 7.0 (d, J=1.73 Hz, 1 H) 7.1 - 7.1 (m, 1 H) 7.2 (s, 2 H) 7.4 (s, 1 H) 7.8 (s, 1 H) 7.8 (s, 1 H).
13
C NMR (126 MHz, CDCl ) δ: 23.0, 28.1, 28.7, 55.9, 110.8, 113.7, 124.2, 126.6, 129.6, 131.2, 132.0,
3
1
33.1, 133.9, 134.6, 135.7, 137.0, 138.1, 138.5, 148.6, 149.8, 189.6
2
5
2-(3,4-Dichlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (37). Yellow solid; yield:
1
1.83%; m.p.: 115-117ºC; Mass calculated: 403.2984; Mass found: 403.2991. H NMR (500 MHz, CDCl )
3
δ: 1.8 (quin, J=6.31 Hz, 2 H) 2.8 - 2.9 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d, J=8.23 Hz,
H) 7.0 (d, J=1.65 Hz, 1 H) 7.1 (d, J=8.23 Hz, 1 H) 7.2 - 7.3 (m, 1 H) 7.5 (d, J=8.23 Hz, 1 H) 7.5 (d,
1
13
J=1.65 Hz, 1 H) 7.6 (s, 1 H) 7.8 (s, 1 H). C NMR (126 MHz, CDCl
3
) δ: 22.8, 28.3, 28.5, 55.9, 55.9,
1
1
10.9, 113.7, 124.1, 128.7, 129.5, 130.3, 131.6, 132.4, 132.6, 133.7, 133.9, 136.0, 137.7, 137.8, 148.7,
49.8, 189.6
2
1
-(3,5-Dichlorobenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (38). Yellow solid; yield:
1
8.05%; m.p.: 106-108ºC; Mass calculated: 403.2984; Mass found: 403.2994. H NMR (500 MHz, CDCl
3
)
δ: 1.77 - 1.84 (m, 2 H) 2.84 (t, J=6.36 Hz, 2 H) 2.94 (t, J=6.65 Hz, 2 H) 3.89 (s, 3 H) 3.90 (s, 3 H) 6.89 (d,
13
J=8.09 Hz, 1 H) 7.00 (s, 1 H) 7.10 (d, J=8.67 Hz, 1 H) 7.21 - 7.32 (m, 3 H) 7.59 (s, 1 H) 7.74 (s, 1 H).
C
NMR (126 MHz, CDCl ) δ: 22.8, 28.2, 28.5, 55.9, 110.9, 113.7, 124.2, 125.0, 126.6, 128.2, 128.6, 133.3,
3
1
33.8, 134.9, 138.1, 138.4, 138.9, 148.7, 149.9, 189.5
2
2
-(2,4-Dimethoxybenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (39). Yellow solid; yield:
1
7.22%; m.p.: 145-147ºC; Mass calculated: 394.4602; Mass found: 394.4627. H NMR (500 MHz, CDCl )
3
δ: 1.78 (dt, J=12.72, 6.36 Hz, 3 H) 2.81 - 2.85 (m, 2 H) 2.89 - 2.95 (m, 2 H) 3.83 (s, 2 H) 3.84 (s, 3 H) 3.90
(
7
s, 3 H) 3.91 (s, 3 H) 6.47 (d, J=2.89 Hz, 1 H) 6.49 (dd, J=8.38, 2.60 Hz, 1 H) 6.89 (d, J=8.67 Hz, 1 H)
.00 (d, J=1.73 Hz, 1 H) 7.09 (d, J=8.67 Hz, 1 H) 7.28 (d, J=8.67 Hz, 1 H) 7.73 (s, 1 H) 7.98 (s, 1 H). C
13
NMR (126 MHz, CDCl ) δ: 22.7, 28.6, 28.7, 55.4, 55.5, 55.9, 98.2, 104.1, 110.8, 113.6, 118.0, 123.8,
3
1
29.1, 131.2, 132.1, 134.2, 134.6, 134.8, 136.6, 149.4, 159.9, 161.6, 190.2
2
,6-Bis(3,4-dimethoxybenzylidene)cyclohexanone (40). Yellow solid; yield: 66.33%; m.p.: 135-137ºC;
1
Mass calculated: 394.4602; Mass found: 394.4624. H NMR (500 MHz, CDCl
3
) δ: 1.82 (quin, J=6.31 Hz, 2
H) 2.92 - 2.97 (m, 4 H) 3.91 (s, 6 H) 3.92 (s, 6 H) 6.90 (d, J=8.23 Hz, 2 H) 7.02 (d, J=1.65 Hz, 2 H) 7.11
13
(
dd, J=8.23, 1.65 Hz, 2 H) 7.75 (s, 2 H). C NMR (126 MHz, CDCl ) δ: 23.0, 28.5, 55.9, 110.8, 113.7,
3
1
23.9, 129.0, 134.5, 136.8, 148.6, 149.6, 190.0
2
2
1
6
-(3,4-Dimethoxybenzylidene)-6-(3,5-dimethoxybenzylidene)cyclohexanone (41). Yellow solid; yield:
1
9.33%; m.p.: 90-91ºC; Mass calculated: 394.4602; Mass found: 394.4609. H NMR (500 MHz, CDCl
3
) δ:
.78 (dt, J=12.28, 6.29 Hz, 2 H) 2.91 (dt, J=11.85, 6.21 Hz, 4 H) 3.79 (s, 6 H) 3.89 (s, 3 H) 3.90 (s, 3 H)
.44 (s, 1 H) 6.58 (s, 2 H) 6.89 (d, J=8.09 Hz, 1 H) 7.00 (s, 1 H) 7.10 (d, J=8.09 Hz, 1 H) 7.69 (s, 1 H) 7.73
1
3
(
s, 1 H). C NMR (126 MHz, CDCl ) δ: 22.9, 28.4, 28.6, 55.4, 55.9, 55.9, 100.6, 108.2, 110.8, 113.7,
3
1
24.0, 128.9, 136.5, 136.7, 137.3, 137.8, 148.6, 149.7, 160.5, 190.1
2
-(3,4-Dimethoxybenzylidene)-6-(3,4,5-trimethoxybenzylidene)cyclohexanone (42). Yellow solid;
1
yield: 17.94%; m.p.: 148-149ºC; Mass calculated: 424.4862; Mass found: 424.4874. H NMR (500 MHz,
CDCl ) δ: 1.8 (quin, J=6.31 Hz, 2 H) 2.9 - 3.0 (m, 4 H) 3.9 (s, 9 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.7 (s, 2 H) 6.9
3
13
(
d, J=8.23 Hz, 1 H) 7.0 (s, 1 H) 7.1 (dd, J=8.51, 1.92 Hz, 1 H) 7.7 (s, 1 H) 7.8 (s, 1 H). C NMR (126
MHz, CDCl ) δ: 23.0, 28.4, 28.5, 55.9, 56.2, 61.0, 107.8, 110.9, 113.7, 124.0, 128.9, 134.3, 135.5, 136.8,
3
1
37.2, 153.0, 190.0
2
-(3-Chloro-4-hydroxybenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (43). Yellow solid;
1
yield: 35.54%.; m.p.: 132-134ºC; Mass calculated: 384.8527; Mass found: 384.8541. H NMR (500 MHz,

ACCEPTED MANUSCRIPT
acetone) δ: 1.8 (quin, J=6.31 Hz, 2 H) 2.9 - 3.0 (m, 2 H) 3.0 - 3.0 (m, 2 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 7.0 - 7.0
(
m, 1 H) 7.1 (d, J=8.78 Hz, 1 H) 7.1 - 7.2 (m, 2 H) 7.4 (dd, J=8.78, 2.20 Hz, 1 H) 7.6 (d, J=2.20 Hz, 1 H)
1
3
7
1
1
.6 (s, 1 H) 7.6 - 7.7 (m, 1 H) 9.3 (br. s., 1 H). C NMR (126 MHz, acetone) δ: 22.8, 28.2, 28.3, 55.2, 55.2,
11.4, 114.1, 116.7, 123.9, 128.7, 128.9, 130.6, 130.7, 131.8, 131.9, 134.2, 134.4, 135.3, 136.2, 149.1,
50.2, 188.2
2
-(3,4-Dimethoxybenzylidene)-6-(4-hydroxy-3-methoxybenzylidene)cyclohexanone (44). Yellow solid;
1
yield: 41.09%; m.p.: 89-91ºC; Mass calculated: 380.4337; Mass found: 380.4349. H NMR (500 MHz,
acetone) δ: 1.8 (dt, J=12.57, 6.14 Hz, 2 H) 2.9 - 3.0 (m, 4 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 3.9 (s, 3 H) 6.9 (d,
13
J=8.09 Hz, 1 H) 7.0 - 7.0 (m, 1 H) 7.1 (d, J=8.67 Hz, 1 H) 7.1 - 7.2 (m, 3 H) 7.6 (s, 2 H) 8.1 (s, 1 H).
C
NMR (126 MHz, acetone) δ: 22.9, 28.3, 28.3, 55.2, 55.2, 55.4, 111.5, 114.1, 114.1, 115.1, 123.8, 124.3,
1
28.0, 128.9, 134.0, 134.6, 135.7, 136.2, 147.3, 147.6, 149.2, 150.1, 188.3
2
2
-(3,4-Dihydroxybenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone (45). Yellow solid; yield:
5.52%; m.p.: 108-110ºC; Mass calculated: 366.4071; Mass found: 366.4082. H NMR (500 MHz,
1
acetone) δ: 1.8 (quin, J=6.21 Hz, 2 H) 2.8 - 2.9 (m, 2 H) 2.9 - 3.0 (m, 2 H) 3.8 (s, 3 H) 3.9 (s, 3 H) 6.9 - 6.9
1
3
(
m, 1 H) 7.0 - 7.0 (m, 2 H) 7.0 - 7.1 (m, 1 H) 7.1 - 7.1 (m, 2 H) 7.6 (s, 1 H) 7.9 (s, 1 H). C NMR (126
MHz, acetone) δ: 23.0, 28.1, 28.6, 55.3, 60.1, 110.0, 113.2, 115.3, 117.4, 121.8, 123.5, 123.8, 128.1, 130.2,
1
4
4
30.8, 133.7, 136.8, 137.3, 144.9, 146.4, 153.0, 188.4
.2 Biology
.2.1 Cells culture
A series of human cancer cell lines including colorectal (SW620), lung (A549), bladder (EJ28),
fibroblast (HT1080) and breast (MCF-7) cancer cells were grown in Dulbecco's modified Eagle's medium
DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37˚C under 5% CO2
(
while NHDF cells was cultured in DMEM with 5% FBS under the same condition. The medium was
changed every two days. After reaching 80-90% confluence, cells were treated with 0.25% trypsin-EDTA
for further passage.
4
.2.2 MTT assay
Cells were seeded at a density of 1500-5000 cells per well in a 96 well plate depending on the doubling
time and cell size. After 24 h of incubation at 37˚C and 5% CO atmosphere, the culture media was
2
replaced with 100 µL of fresh media followed by the addition of test compounds (100 µL) in DMSO-
containing culture media. The final concentration of DMSO in the well is 0.1%. The plate was further
incubated for 72 h under the same condition. Upon completion, culture medium was removed and 100 µL
of MTT (Thiazoyl blue tertrazolium bromide) solution (5 mg/mL in culture media) was added. After 4 h
incubation at 37˚C, the MTT solution was replaced with 100 µL of DMSO. The absorbance was measured
in microtiter plate reader at 570 nm.
4
.2.3 Colony formation assay
Cells were grown in a 6 well plate (1000 cells/well) in 2 mL of DMEM at 37˚C and 5% CO2
atmosphere overnight. After incubation, different concentrations of selected compound were added to the
cells for ten days. The culture medium was replaced by the fresh drug containing medium every three days
to keep cells growing for ten days. Upon completion, cells were washed with PBS and fixed with 4%
paraformaldehyde for 15 min followed by cell staining with 0.5% (w/v) Crystal violet for 10 min. The plate

ACCEPTED MANUSCRIPT
was then washed with tap water and dried at room temperature. Upon drying, the plate image was
photographed [39].
4
.2.4 Cell cycle analysis
5
Cells (2 × 10 cells/well) was seeded in a 6-well plate and allowed to adhere overnight. The seeded cells
were then treated with selected compounds at different concentrations for 24 h. After treatment, the cells
were harvested and washed with PBS followed by fixation with 70% ethanol for overnight at 4˚C. The
fixed cells were then centrifuged (1200 rpm for 5 min) and the pellet was washed twice with PBS followed
by addition of DNAse-free RNAse A solution (1 mg/ml). After 15 min of incubation, propidium iodide
staining buffer (PI; 10 mg/ml) was added and the resulting mixture was further incubated for 20 min at 4˚C
in the dark. Lastly, samples were analyzed for propidium iodide-DNA fluorescence from 10000 events by
flow cytometry [40].
4
.2.5 Cell apoptosis analysis
Cells were seeded in a 6 well plate (2 × 10 cells/well) and allowed to adhere overnight. The seeded
5
cells were then treated with the selected compounds at different concentrations for 24, 48 and 72 h. After
treatment, the cells were harvested and washed with PBS followed by the addition of 1× binding buffer
(
500 µl) and Annexin V-FITC (5 µl). The cell containing mixture was then incubated in dark at 4˚C for 20
min. Prior to analysis on a flow cytometer, 5 µl of PI was added [41].
4
.2.6 Measurement of reactive oxygen species (ROS) levels
5
Cells were seeded in a 6 well plate (2 × 10 cells/well) and allowed to adhere overnight. The seeded
cells were then treated with 10 µM of DCFDA in culturing media for 1 h. Upon completion, the DCFDA
solution was aspirated and the cells were washed with PBS for twice. Then, the cells were treated with
selected compounds at different concentrations for 12 h. After treatments, resultant cells were harvested,
washed and resuspended in PBS. Intensity of the formed 2’,7’-dichlorofluorescein as a result of Carboxy-
DCFDA hydrolysis, was finally analysed with flow-cytometer at an excitation and emission wavelength of
4
88 and 525 nm respectively [41].
Acknowledgments
The authors thank the Ministry of Higher Education (MOHE) of Malaysia and for financial the support
under the project FRGS/1/2014/ST01/UPM/02/1 (5538011). SW. Leong is supported by the university
Putra Malaysia under the post-doctoral programme.
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•
•
A series of asymmetrical meta-methoxylated diarylpentanoids were synthesized as anti-cancer
agents.
Poly-meta-oxygenated analogs 33 and 42 showed potent cytotoxicity against SW620, A549, EJ28,
HT1080 and MCF-7 human cancer cell lines.
•
•
Analogs 33 and 42 showed anti-proliferative activity.
Analogs 33 and 42 could induce intracellular ROS-mediated G2/M cell arrest and cell apoptosis.