Natural products hybrids: 3,5,4′-Trimethoxystilbene-5,6,7-trimethoxyflavone chimeric analogs as potential cytotoxic agents against diverse human cancer cells

Article abstract of DOI:10.1016/j.ejmech.2018.10.062

Cancer still represents a major global health problem. All currently available anticancer agents have disadvantages like resistance or side effects. Therefore, introduction of novel anticancer agents is needed. Intrigued by the high success rate for natural products-based drug discovery, we designed and synthesized antiproliferative chemical entities as hybrids of two natural products; 3,5,4′-trimethoxystilbene and 5,6,7-trimethoxyflavone. To probe the spectrum of the synthesized compounds, in vitro evaluation was conducted against nine panels representing major cancer diseases. The results revealed the hybrid analogs 4f, 4h, 4k and 4q as promising broad-spectrum anticancer lead compounds eliciting high growth inhibition of several cell lines representing multiple cancers diseases. Evaluation of the promising lead compounds against normal human cell lines suggested a selective cytotoxic effect on cancer cells. Mechanistic investigation of the cytotoxic activity of compound 4f in human cervical cancer HeLa cells showed that it triggers cell death through induction of apoptosis. As a whole, this study presents the natural products hybrid analogs 4f, 4h, 4k and 4q as potential lead compounds for further development of novel anticancer therapeutics.

Full text of DOI:10.1016/j.ejmech.2018.10.062

Accepted Manuscript
Natural products hybrids: 3,5,4′-trimethoxystilbene-5,6,7-trimethoxyflavone chimeric
analogs as potential cytotoxic agents against diverse human cancer cells
Ahmed H.E. Hassan, Eunwoo Choi, Yoon Mi Yoon, Kun Won Lee, Sung Yeun Yoo,
Min Chang Cho, Ji Seul Yang, Hye In Kim, Joo Young Hong, Ji-Sun Shin, Kyung-
Sook Chung, Jeong-Hun Lee, Kyung-Tae Lee, Yong Sup Lee
PII:
S0223-5234(18)30939-5
DOI:
https://doi.org/10.1016/j.ejmech.2018.10.062
EJMECH 10848
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 August 2018
Revised Date: 25 October 2018
Accepted Date: 28 October 2018
Please cite this article as: A.H.E. Hassan, E. Choi, Y.M. Yoon, K.W. Lee, S.Y. Yoo, M.C. Cho, J.S.
Yang, H.I. Kim, J.Y. Hong, J.-S. Shin, K.-S. Chung, J.-H. Lee, K.-T. Lee, Y.S. Lee, Natural products
hybrids: 3,5,4′-trimethoxystilbene-5,6,7-trimethoxyflavone chimeric analogs as potential cytotoxic agents
against diverse human cancer cells, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.10.062.
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ACCEPTED MANUSCRIPT
Graphical Abstract:
%
Growth inhibition at 10 µM
RPMI8226 = 99.70
MDAMB435 = 93.64%
SKMEL5 = 91.66%
HS578T = 90.80%
MOLT4 = 89.58%
UO31 = 79.10%
H
CH O
O
O
N
OCH3
OCH3
3
O
CH O
3
CH O
OCH3
3
4
f
LOXIMVI = 75.56%
HOP92 = 75.26%
BT549 = 73.02%
K562 = 72.38%
Control
Compound 4f (10 µM)

ACCEPTED MANUSCRIPT
Natural products hybrids: 3,5,4'-Trimethoxystilbene-5,6,7-trimethoxyflavone
chimeric analogs as potential cytotoxic agents against diverse human cancer
cells
a,b
a,c
a,c
a,c
a,c
Ahmed H. E. Hassan , Eunwoo Choi , Yoon Mi Yoon , Kun Won Lee , Sung Yeun Yoo ,
a,c
a,c
a,c
c,e
e
Min Chang Cho , Ji Seul Yang , Hye In Kim , Joo Young Hong , Ji-Sun Shin , Kyung-Sook
e
c,e
c,e
a,c,d
Chung , Jeong-Hun Lee , Kyung-Tae Lee , Yong Sup Lee
*
a
Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Seoul 02447,
Republic of Korea
b
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, 26 Kyungheedae-ro, Seoul 02447, Republic of Korea
d
KHU-KIST Department of Converging Science and Technology, Kyung Hee University, 26 Kyungheedae-ro, Seoul 02447, Republic of Korea
e
Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul,
0
2447, Republic of Korea
*
Corresponding author: Prof. Dr. Yong Sup Lee (kyslee@khu.ac.kr)

ACCEPTED MANUSCRIPT
Abstract
Cancer still represents a major global health problem. All currently available anticancer agents
have disadvantages like resistance or side effects. Therefore, introduction of novel anticancer
agents is needed. Intrigued by the high success rate for natural products-based drug discovery,
we designed and synthesized antiproliferative chemical entities as hybrids of two natural
products; 3,5,4'-trimethoxystilbene and 5,6,7-trimethoxyflavone. To probe the spectrum of the
synthesized compounds, in vitro evaluation was conducted against nine panels representing
major cancer diseases. The results revealed the hybrid analogs 4f, 4h, 4k and 4q as promising
broad-spectrum anticancer lead compounds eliciting high growth inhibition of several cell lines
representing multiple cancers diseases. Evaluation of the promising lead compounds against
normal human cell lines suggested a selective cytotoxic effect on cancer cells. Mechanistic
investigation of the cytotoxic activity of compound 4f in human cervical cancer HeLa cells
showed that it triggers cell death through induction of apoptosis. As a whole, this study presents
the natural products hybrid analogs 4f, 4h, 4k and 4q as potential lead compounds for further
development of novel anticancer therapeutics.
Keywords:
3
,5,4'-Trimethoxystilbene; 5,6,7-Trimethoxyflavone; Chimeric molecules; Anticancer; Apoptosis.

ACCEPTED MANUSCRIPT
1
. Introduction
Ranked the second cause of death after cardiovascular diseases, cancer remains a major health
problem. A recent analysis of the intrinsic and extrinsic factors inducing cancers revealed that up
to 90% of cancers are caused by extrinsic factors like diet, lifestyle, pollution and infections [1].
Because many extrinsic predisposing factors for cancer incidence such as the modern life style,
popularity of certain foods and pollution are anticipated to accrue in future, more people might
be risked to cancer. The heterogeneity of cancers and evolving of resistance to the currently used
anticancer drugs render them ineffective overtime and necessitate the development of novel
anticancer agents to help in the fight against cancer.
Nature; the greatest invention machine, served as a plentiful source of drugs since old days [2]. A
recent survey of anticancer drugs approved over 34 years from 1981 to 2014 revealed that 113
new chemical entities out of the 136 approved small molecules anticancer drugs were either
natural products, derived from natural products or inspired by natural products [3]. Because of
the high success rate for natural products-based drug discovery and development, we initiated
our research to develop novel anticancer agents using the privileged scaffolds developed in the
R&D lab of nature as starting points.
3
,5,4'-Trimethoxystilbene (TMS; 1a; Fig. 1) is a natural product that has been isolated from
several plants [4-7]. It is a permethylated analog of resveratrol (RES; 1b); another natural
product that exists in a wide range of edible plants including vine grapes, legumes and berries [8].
Review of literature reports shows that RES (1b) has been the subject of several biological
evaluations, as well as, clinical trials as a potential anticancer agent impacting several pathways
that control the proliferation and survival of cells [9, 10]. However, the clinical benefit of RES

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(1b) is not sufficiently confirmed [11]. In addition, it shows poor pharmacokinetics, as well as,
chemical instability that hinder further development [12, 13]. On the other side, TMS (1a); the
permethylated analog, is a more promising anticancer agent with an in vitro and in vivo proven
better anticancer activity, as well as, enhanced pharmacokinetic properties relative to RES [14-
1
6]. This stimulated researchers to investigate the cytotoxic activity of several TMS analogs
bearing various methoxylation patterns [17-19]. According to published reports, TMS (1a)
induces apoptosis and exhibits anticancer activity through multiple pathways including PI3K/Akt,
Wnt/β-catenin, MAPK pathway and decrement of expression of matrix metalloproteinase-2
(MMP-2) [20-22]. Despite the premises that TMS and stilbenoid analogs are promising
anticancer agents, the fact that such stilbenoid compounds are liable for cis/trans isomerization
in vivo raises doubts about the anticipated in vivo activity and complicates further development
of these compounds [23]. Although having much lower anticancer activity, isomerization is
nullified in the reported amide analog (2) [24].
Figure 1. Reported molecules served as starting points for the conducted study
Natural flavones belong to an important class of medicinally privileged scaffold with reported
diverse biological activities [25]. 5,6,7-Trimethoxyflavone (TMF; 3; Fig. 1) is one of these

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natural flavones that has been reported to be a constituent in the several plants [26-29]. At a
concentration of 25 µM. TMF produces considerable cytotoxic effects in Hep G2, but to a lesser
degree in Hep 3B and DU-145 cancer cells [30]. While the mechanism of the cytotoxic effects
of TMF is understudied, it is known that TMF suppress nuclear factor-kappa B (NF-κB) which is
a player in the development of cancer and inflammatory diseases [31-33]. In addition, in vitro
evaluation of TMF showed that it inhibits topoisomerase II [34]. Introduction of various
methoxylation patterns, hydroxy, nitro or amino moieties at B-ring of TMF provided compounds
with almost similar potency to TMF [30]. This might indicate that the simple substitution is not
sufficient to enhance the anticancer activity of TMF.
Combining the pharmacophoric features of two cytotoxic molecules into a single chemical entity
has been established as a powerful strategy to develop more promising novel cytotoxic
compounds that might elicit more than one mechanism of action [35, 36]. Accordingly, we
thought of this molecular hybridization strategy to develop chimeric molecules 4 (Fig. 2)
possessing the pharmacophoric features of both of the anticancer amide analog (2) of the natural
products TMS and of the natural product TMF (3) in attempts to identify promising anticancer
lead compounds. As shown in Fig. 2, the design involved replacement of one of the aromatic
phenyl rings of TMS analog (2) with TMF (3). The natural product TMS has two phenyl rings,
one has 4-methoxy substituent while the other has two methoxy substituents in 3,5-poistions.
Because of this variation in both of the number of methoxy substituents and their positions on the
phenyl rings, we wondered about the impact of such variations on the elicited biological activity
in the designed compounds. Accordingly, various monomethoxylation, 3,4-methylenedioxy and
3
,4,5-trimethoxy substitution patterns, as well as, the unsubstituted-phenyl moiety were planned
to be explored as a pharmacophoric feature inherited from TMS. In the design, the attachment of

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the amide moiety was established at 3-position of B-ring of TMF, meanwhile, the impact of the
two possible amide configuration on the activity was planned to be assessed. In addition, the
impact of switching the 5-methoxy substituent of the TMF moiety to 5-hydroxy was planned to
be assessed. The latter was done as we encountered some reports claiming an enhancement of the
cytotoxic activity of polymethoxylated flavones might happen upon selective demethylation of
the 5-methoxy group [37, 38]. In following sections, we report our promising results.
Figure 2. Design of TMS-TMF chimeric compounds
2
2
. Results and discussion
.1.Chemical Synthesis
As outlined in scheme 1, the targeted hybrid compounds (4a–u) were synthesized using
modifications of the previously reported procedures [39-41]. First, 3,4,5-trimethoxyphenol (5)
was acetylated using boron trifluoride diethyl etherate as a Lewis acid in the presence of a large
excess of acetic anhydride, followed by Fries rearrangement to yield acetophenone derivative (6).
Using sodium methoxide as a base and methanol as a solvent, compound 6 was condensed with
the appropriate benzaldehyde to afford the corresponding chalcone derivative (7) that was

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converted into a flavone derivative (8) in an intramolecular oxidative cyclization reaction using
iodine/dimethyl sulfoxide mixture. The methyl ester group of compound 8a was hydrolyzed into
carboxylic group by alkaline hydrolysis using potassium hydroxide in methanol to provide
carboxylic derivative 9a, while the nitro group of compound 8b was reduced into amino group
using tin(II) chloride to provide the amino derivative 9b. The carboxylic acid derivative (9a) was
advanced to the desired designed hybrid compounds 4a–f through amide coupling with the
appropriate amine derivative after activation of the carboxylic group by in situ prepared reagent
from trimethyl phosphite and iodine [40]. On the other side, the targeted hybrid compounds 4g–k
were accessed through reaction of the appropriate acid chloride with the amino derivative 9b in
the presence of potassium carbonate using acetone as a solvent. The desired 5-hydroxy
derivatives 4l–u, were prepared from the corresponding 5-methoxy derivatives 4a–k via a
regioselective 5-O-demethylation using boron trichloride in dichloromethane.
Scheme 1. Reagents and conditions: (a) Acetic anhydride, boron trifluoride diethyl etherate, reflux, overnight; (b)
Methyl 3-formylbenzoate (For 7a) or 3-nitrobenzaldehyde (For 7b), NaOMe, MeOH, rt, overnight; (c) I , DMSO,
2
7
0 °C, overnight; (d) For 9a: KOH, MeOH, reflux, overnight; For 9b: SnCl , EtOH, reflux, 2.5 h; (e) For 4a–f:
2
(
MeO)
3
P, I
2
, NEt
3
, appropriate amine, CH
2
Cl
2
, 0 °C to rt, 3 h; For 4g–k: K
2
3
CO , acetone, appropriate acid chloride,
rt;(f) BCl , CH Cl , 0 °C to rt, 3h.
3
2
2
2
.2. Biological evaluations

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2
.2.1. Sulforhodamine B (SRB) assay for the antiproliferative activity
While MTT assay, which depend on the metabolic activity of living cells, is popular, the
sulforhodamine B (SRB) assay, which measure the cellular proteins, has the advantages of better
measurements proportionality with cell numbers and high sensitivity independent on cell line [42,
4
3]. In fact, national cancer institute (NCI) adopts SRB assays for screening the antiproliferative
activity of candidate anticancer compounds. The synthesized hybrid compounds were assayed by
NCI against human cancer cell lines representing nine major cancer diseases. The screening was
performed at single dose of 10 µM to check the efficacy of tested compounds at this dose. The
antiproliferative activity of imatinib (Gleevec®; a FDA-approved anticancer tyrosine kinase
inhibitor drug marketed by Novartis pharma) against the used cell lines at the same single dose
of 10 µM was retrieved from NCI database (NSC 759854) and used as a reference standard for
comparison. The results were expressed as percent growth inhibition. The determined value
reflects a cytostatic activity of a compound if it is ≤ 100%. When the evaluated compounds
shows a cytocidal activity to cancer cells (lethality to cancer cells), the values exceeds 100%
indicating that the remaining population of cancer cells decreased relative to the initially
employed population. In the following sections, the interesting results are discussed.
2
.2.1.1. In vitro evaluation of antiproliferative activity in hematologic cancers
Among childhood cancers, leukemia comes to the fore accounting for one third. In addition, it
frequently afflicts elderly people too. Leukemia might be lymphoblastic or myeloid according
the type of leukemic progenitor cells. In addition, it might be also divided into acute or chronic
leukemia. In 2018, 60,300 new cases and 24,370 deaths because of leukemia are anticipated [44].
The designed hybrid compounds were evaluated against cell lines representing childhood and

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adult leukemia of acute, chronic lymphoblastic and myeloid types. The results are shown in
Table 1.
Table 1. % Growth inhibition of cell lines representing hematologic cancers by compounds 4a–u and imatinib at 10
µM Conc.
1
2
Comp.
R
R
X-Y
CCRFCEM
35.13
48.31
59.46
50.73
36.58
68.74
54.27
83.98
56.13
72.49
111.94
-0.19
HL60(TB)
12.70
10.10
31.29
21.16
30.28
64.01
39.49
42.34
14.76
55.20
65.43
7.30
K562
29.56
51.17
59.69
50.44
25.79
72.38
56.90
63.61
62.03
79.44
82.60
2.93
MOLT4
43.20
38.74
51.49
36.78
35.20
89.58
59.35
71.17
53.31
76.69
97.10
0.26
RPMI8226
59.43
73.69
83.05
87.45
79.10
99.70
73.38
93.25
83.18
81.23
153.74
16.50
45.41
53.57
7.48
SR
4
4
a
MeO
MeO
MeO
MeO
MeO
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
35.94
32.43
58.14
18.36
52.37
70.19
68.40
ND
b
2-MeO
3-MeO
4-MeO
4
c
4
d
4
e
2 2
3,4-CH O
4
f
MeO 3,4,5-TriMeO
4
4
g
MeO
MeO
MeO
MeO
MeO
OH
H
h
2-MeO
3-MeO
4-MeO
4
i
ND
4
j
71.43
88.49
4.29
4
k
3,4-CH
H
2 2
O
4
l
4
m
OH
2-MeO
3-MeO
4-MeO
39.80
34.70
16.00
2.77
9.41
22.15
16.93
8.23
27.94
24.21
-2.73
-0.50
27.18
6.77
21.51
47.66
19.21
24.15
45.62
16.50
54.82
20.83
18.98
14.60
4
n
o
p
q
OH
17.49
-29.67
5.71
4
OH
4
4
OH
3,4-CH
2
O
2
5.51
20.24
72.98
21.63
74.17
25.21
20.63
12.60
OH
3,4,5-TriMeO
2-MeO
41.89
5.53
22.01
0.60
45.48
15.48
69.21
11.93
15.04
ND
4
r
OH
4
s
OH
3-MeO
50.52
11.95
11.68
6.60
19.48
15.39
14.02
-6.80
49.26
19.11
15.77
18.00
4
t
OH
4-MeO
4
u
OH
2 2
3,4-CH O
Imatinib
All data are reported as the average of duplicates.
As presented in Table 1, imatinib; the reference drug used for comparison, elicited no to low
antiproliferative activity (≤ 18.00%) at the employed 10 µM dose. As illustrated in Fig. 3, most
of the synthesized and evaluated hybrid compounds were much more effective relative to the
reference imatinib. In general, compounds 4a–k possessing TMF moiety showed higher
activities relative to compounds 4l–u possessing 6,7-dimethoxy-5-hydroxyflavone moiety.
Among the used cell lines, RPMI8226; a plasma cell myeloma, was the most responsive,
showing very high to average inhibition by all TMF derivatives. Among them, compounds 4k

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was lethal to RPMI8226 cell line (153.74% growth inhibition, Table 1). In addition, compounds
4
f, 4h, 4d, 4i, 4c, and 4j in decreasing order respectively elicited an excellent inhibition within
the range of 99.70.74~81.23%. Furthermore, compounds 4e, 4b, 4g and 4a in decreasing order
respectively elicited an inhibition range of 79.10~59.43%. This indicates that the leukemic
RPMI8226 cell line is highly susceptible to this class of compounds. On the other hand, only
three compounds (4s, 4q and 4n) among the 6,7-dimethoxy-5-hydroxyflavone derivatives
produced highly significant inhibition values of 74.17, 72.98 and 53.57%. Among the rest of
evaluated leukemic cell lines, the growth of the chronic myelogenous leukemia K562 cell line
and the childhood T acute lymphoblastic leukemia CCRFCEM cell line were also significantly
inhibited by compounds possessing TMF moiety, but lesser than RPMI8226 cell line. As
illustrated in Fig. 3, compound 4g–k and 4r–u, whose B-ring of the flavone moiety was attached
to the linker’s nitrogen atom, were generally more active against the employed leukemic cell
lines relative to the corresponding compounds 4a–f and 4m–p; whose B-ring of the flavone
moiety was attached to the linker’s carbonyl group. In addition, the results revealed different
profiles for the compounds bearing identical methoxylation pattern on the aromatic phenyl
moiety retained from TMS on the activity, depending on the configuration of the amide linker.
This indicates that the amide linker plays a crucial role than simply linking the two-
pharmacophoric TMS and the flavone moieties. Thus, among TMF derivatives whose B-ring of
the flavone moiety was linked to the carbonyl group of the linker, derivative 4f bearing 3,4,5-
trimethoxyphenyl moiety possessed a high efficacy showing an inhibition range of 99.70~64.01%
for all evaluated leukemic cell lines. Meanwhile, derivatives 4e and 4a bearing 3,4-
methylenedioxyphenyl and unsubstituted-phenyl moieties respectively were the least effective
relative to other derivatives (Table 1). Among TMF derivatives 4b–d bearing

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monomethoxyphenyl moieties, compound 4c having 3-methoxyphenyl moiety was the most
active showing an inhibition range of 83.05~59.46% against five out the six evaluated leukemic
cell lines. In contrast, when the configuration of the amide linker was reversed, derivative 4k
bearing 3,4-methylenedioxyphenyl moiety became the most active among TMF derivatives
whose B-ring of the flavone moiety is linked to the linker’s nitrogen atom (Fig. 3). Among TMF
derivatives 4g–j possessing the latter amide configuration and bearing monomethoxyphenyl
moieties, compound 4j bearing 4-methoxyphenyl moiety was the most active eliciting inhibition
8
1.23~55.20% against all of the evaluated six leukemic cell lines (Fig. 3).
1
1
60
10
CCRFCEM
HL60(TB)
K562
MOLT4
RPMI8226
SR
6
1
0
0
-40
Figure 3. Percentage inhibition of growth inhibition of leukemic cells elicited by the synthesized TMS-TMF hybrid
compounds and imatinib at a single dose of 10 µM.
2
.2.1.2. In vitro evaluation of antiproliferative activity in non-small cell lung cancers
Lung adenocarcinoma, squamous cell lung carcinoma and large-cell lung carcinoma, collectively
known as non-small cell lung cancers (NSCLC) are less sensitive to chemotherapeutic agents
than the small lung cancers. Accordingly, NCI cancer cells panel incorporates several cell lines
representing this difficult-to-treat class of lung cancers. The synthesized hybrid compounds were
evaluated against these cells and the results are presented in Table 2.

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Table 2. % Growth inhibition of cell lines representing non-small cell lung cancers by compounds 4a–u and
imatinib at 10 µM Conc.
1
2
Comp.
R
R
X-Y
A549
16.66
31.15
37.21
22.37
25.51
56.48
31.13
51.79
44.06
35.29
48.72
39.70
18.54
26.63
18.74
25.13
59.46
22.14
35.36
15.12
13.52
3.80
HOP62
13.71
30.65
40.50
31.02
26.63
62.50
21.60
42.20
30.25
27.54
45.65
91.11
0.21
HOP92
29.18
62.45
58.14
52.69
21.94
75.26
52.36
100.39
51.80
93.33
106.11
94.72
15.77
33.59
76.39
26.99
43.85
5.45
H226
12.73
37.25
36.69
18.47
21.19
41.45
18.63
41.61
28.02
30.06
34.30
26.76
23.93
41.48
8.07
H23
7.79
H322M
17.66
18.93
19.91
24.49
22.56
30.51
16.23
56.40
27.12
44.94
56.46
33.58
1.56
H460
11.22
41.14
39.89
33.87
26.91
69.96
21.30
ND
H522
21.89
56.55
53.37
39.05
26.87
65.28
50.71
55.74
58.37
55.55
63.27
8.74
4
4
a
MeO
MeO
MeO
MeO
MeO
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
b
2-MeO
3-MeO
4-MeO
34.05
36.87
28.67
10.27
51.21
27.12
37.60
33.94
42.54
47.07
13.10
13.93
25.42
8.74
4
c
4
d
4
e
2 2
3,4-CH O
4
f
MeO 3,4,5-TriMeO
4
4
g
MeO
MeO
MeO
MeO
MeO
OH
H
h
2-MeO
3-MeO
4-MeO
4
i
ND
4
j
57.87
72.81
66.31
-3.83
31.39
7.30
4
k
3,4-CH
H
2 2
O
4
l
4
m
OH
2-MeO
3-MeO
4-MeO
26.65
59.84
20.56
23.95
58.77
40.98
56.86
34.41
41.48
ND
4
n
o
p
q
OH
37.53
27.78
39.65
88.33
16.51
37.09
30.20
47.86
ND
12.86
0.54
4
OH
4
4
OH
3,4-CH
2
O
2
21.48
92.31
12.98
36.33
41.43
55.40
10.60
27.32
65.14
1.58
19.03
25.62
8.64
45.73
46.22
-2.21
45.62
17.35
16.72
3.20
OH
3,4,5-TriMeO
2-MeO
4
r
OH
4
s
OH
3-MeO
31.22
42.34
38.50
43.70
32.65
15.66
13.39
17.10
30.95
8.16
4
t
OH
4-MeO
4
u
OH
3,4-CH
2
O
2
15.72
ND
Imatinib
All data are reported as the average of duplicates.
At the used 10 µM dose, imatinib; the standard drug used for comparison, elicited 43.70%
growth inhibition against HOP92 cell line, while the determined growth inhibition percent of the
rest of NSCLC cell lines was low (≤17.10%, Table 2). In comparison, several compounds among
the synthesized and evaluated hybrid compounds were much more active relative to imatinib
against the employed NSCLC cell lines as illustrated in Fig. 4. Depending on methoxylation
pattern, configuration of amide linker and the switch between TMF and 6,7-dimethoxy-5-
hydroxyflavone moieties, distinct profiles could be observed for the synthesized hybrid
compounds. Comparing compounds 4a and 4g possessing unsubstituted-phenyl moiety as a
pharmacophore inherited from TMS hybridized with TMF moiety, compound 4g whose TMF

ACCEPTED MANUSCRIPT
moiety was attached to linker’s nitrogen atom was more active than compound 4a whose TMF
moiety was attached to the linker’s carbonyl group (Fig. 4). In contrast to the decrement of
activity that has been revealed in leukemic cell lines upon replacing TMF moiety of compound
4
a with 6,7-dimethoxy-5-hydroxyflavone moiety to afford compound 4l, the activity profile was
reversed in NSCLC cell lines as the compound 4l showed much improved activity eliciting high
inhibition of growth of lung adenocarcinoma HOP92 and HOP62 cell lines, as well as large cell
lung carcinoma H460 cell line by 94.72, 91.11 and 66.31% respectively. This might indicate that
the pathway(s) impacted by compound 4l in NSCLC cells is almost inactive in leukemic cells.
This activity pattern, which is the reverse to that observed in leukemic cell lines upon replacing
TMF moiety with 6,7-dimethoxy-5-hydroxyflavone moiety, was also noted for compounds
bearing the 3,4,5-trimethoxyphenyl moiety. As Fig. 4 shows, compound 4f bearing the 3,4,5-
trimethoxyphenyl was the most active among derivatives possessing TMF moiety attached to the
carbonyl group of the amide linker. It elicited 75.26~51.21% inhibition against six cell lines out
of the eight cell lines representing NSCLC. However, the corresponding compound 4q, which
possess 6,7-dimethoxy-5-hydroxyflavone moiety instead of TMF moiety, elicited a remarkable
higher activity against some NSCLC cells lines. Thus, the growth of lung squamous carcinoma
cell line H226 and lung adenocarcinoma cell line HOP62 were inhibited by 92.31 and 88.33%
respectively. Again, this pattern of activity, which was absent in leukemic cells, might indicate
the presence of a pathway(s) impacted by 6,7-dimethoxy-5-hydroxyflavone derivatives 4q and 4l
in NSCLC, but absent in leukemic cells. Nevertheless, 6,7-dimethoxy-5-hydroxyflavone
derivative 4m–p and 4r–u bearing monomethoxyphenyl or 3,4-methylenedioxyphenyl moieties
were less active relative to the corresponding TMF derivatives 4b–e and 4r–u. As Fig. 4
illustrates, compounds 4r–u whose TMF moiety was attached to the linker’s nitrogen atom

ACCEPTED MANUSCRIPT
possessed higher activity in general relative to compounds 4b–e whose configuration of amide
linker was the reverse. This emphasize amide linker as a crucial player in elicited activity rather
than simply linking the two-combined pharamcophoric moieties. Among the evaluated NSCLC
cell lines, the lung adenocarcinoma cell line HOP92 was the most sensitive to the synthesized
hybrid compounds (Table 2). Its growth was inhibited by more than 100% by two compounds
(4k and 4h), within the range of 94.72~75.26% by four compounds (4l, 4j, 4o and 4f) and within
the range of 62.45~51.80% by five compounds (4b, 4c, 4d, 4g and 4i) which collectively
accounts for eleven compounds out of the evaluated twenty-one compounds. Also, the lung
adenocarcinoma cell line H522 was highly responsive to this class of compounds eliciting
growth inhibition within the range of 65.28~50.71% by eleven compounds out of the evaluated
twenty-one compounds (Table 2).
1
1
20
00
A549
HOP62
HOP92
H226
H23
H322M
H460
H522
8
6
4
2
0
0
0
0
0
-20
Figure 4. Percentage inhibition of growth inhibition of non-small cell lung cancer cells elicited by the synthesized
TMS-TMF hybrid compounds and imatinib at a single dose of 10 µM.
2
.2.1.3. In vitro evaluation of antiproliferative activity in colorectal cancers
Colorectal cancers’ global incidence is anticipated to increase to more than 2.2 million cases and
1
.1 million deaths by 2030 [45]. As of 2016, colorectal cancers are among the four most

ACCEPTED MANUSCRIPT
common cancers in both men and women [46]. Furthermore, it was found that patients who had a
colorectal cancer are prone to develop a second primary colorectal cancer unrelated to the first
primary cancer [47]. Accordingly, the panel of cancer cell lines used for assay of the synthesized
hybrid compounds by NCI included several cell lines representing colorectal adenocarcinoma.
The outcome of the assay is summarized in Table 3.
Table 3. % Growth inhibition of cell lines representing colorectal cancers by compounds 4a–u and imatinib at 10
µM Conc.
1
2
Comp.
R
R
X-Y
COLO205
2.76
HCC2998
1.29
HCT116
18.31
31.88
39.28
36.60
27.32
51.40
32.28
63.27
46.44
52.19
64.53
53.74
14.48
30.73
29.53
43.01
55.23
11.68
44.48
30.20
21.03
18.60
HCT15
20.75
48.38
45.33
52.88
28.24
59.30
34.82
58.46
46.65
54.76
67.89
7.23
HT29
11.47
38.65
49.17
32.94
17.21
68.95
22.94
75.39
53.63
53.26
70.59
45.85
10.83
15.64
12.32
23.69
37.19
2.63
KM12
24.34
34.80
46.30
45.19
32.67
54.24
35.58
61.62
53.93
53.71
48.72
42.23
0.81
SW620
-0.72
12.22
10.40
6.52
4
4
a
MeO
MeO
MeO
MeO
MeO
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
b
2-MeO
3-MeO
4-MeO
14.81
31.68
30.60
24.39
55.70
15.64
ND
13.61
12.23
26.31
2.03
4
c
4
d
4
e
3,4-CH
2
O
2
-4.89
33.02
8.49
4
f
MeO 3,4,5-TriMeO
25.88
12.19
32.21
19.52
29.67
46.90
10.27
-2.45
-3.54
-1.16
-4.53
34.05
-9.63
11.82
19.36
9.76
4
4
g
MeO
MeO
MeO
MeO
MeO
OH
H
h
2-MeO
3-MeO
4-MeO
36.65
22.76
11.98
36.02
5.67
4
i
ND
4
j
26.91
31.88
7.73
4
k
3,4-CH
H
2 2
O
4
l
4
m
OH
2-MeO
3-MeO
4-MeO
-7.01
24.29
2.39
12.96
25.70
-7.36
6.45
-0.74
18.36
-1.58
15.53
26.74
10.54
18.98
9.52
4
n
o
p
q
OH
35.48
-3.03
29.39
46.09
5.13
4
OH
4
4
OH
3,4-CH
2
O
2
5.22
OH
3,4,5-TriMeO
2-MeO
32.32
-7.03
5.46
40.12
3.51
4
r
OH
4
s
OH
3-MeO
58.35
7.47
27.91
1.12
43.73
-0.05
1.48
4
t
OH
4-MeO
-7.69
2.46
4
u
OH
3,4-CH
2
O
2
0.04
15.57
47.10
-1.42
-10.00
Imatinib
-1.10
-22.00
11.50
-6.70
All data are reported as the average of duplicates.
While imatinib inhibited the growth of HT29 colorectal cell line by 47.10%, it was ineffective at
the 10 µm dose against four colorectal cell lines (COLO205, HCC2998, KM12 and SW620) and
elicited only 18.60 and 11.50 against two cell lines (HCT116 and HCT15). As illustrated in Fig.
5
, most of the evaluated compounds showed much better activity profiles relative to imatinib. In

ACCEPTED MANUSCRIPT
general the percent growth inhibition induced by TMS analogs hybridized with TMF moiety
(compounds 4a–k) were higher than those triggered by corresponding TMS analogs hybridized
with 6,7-dimethoxy-5-hydroxyflavone moiety (compounds 4l–u). Considering the configuration
of the amide linker, compounds 4g–k, whose TMF moiety was attached to the linker’s nitrogen
atom, were more active relative to corresponding compounds whose TMF moiety is attached to
the linker’s carbonyl group. Among the latter, TMS-TMF hybrid compounds 4f possessing 3,4,5-
trimethoxyphenyl moiety was prominently active eliciting 68.95~51.40% growth inhibition
against five out of seven cell lines representing colorectal cancers (Table 3). On the other side,
among compounds 4g–k, compounds 4h possessing 2-methoxyphenyl moiety was prominently
active compound eliciting 75.39~58.46% growth inhibition against four out of the six evaluated
cell lines. In addition, compound 4j bearing 4-methoxyphenyl moiety elicited 54.76~52.19%
growth inhibition against four out of the seven tested cell lines. Furthermore, compound 4k
having 3,4-methylenedioxyphenyl moiety was highly active eliciting growth inhibition against
three cell lines within the range of 70.59~64.53% while the corresponding compound 4e was
much less active. These, collectively, emphasize the influence of the amide linker configuration
on the elicited activity. Replacement of TMF moiety of compound 4a possessing unsubstituted-
phenyl moiety as pharmacophore from TMS, by 6,7-dimethoxy-5-hydroxyflavone moiety in the
corresponding compound 4l resulted in some enhancement of activity. However, similar
replacement of TMF in compounds 4b–k possessing methoxylated-phenyl moieties, by 6,7-
dimethoxy-5-hydroxyflavone moiety in the corresponding compound 4m–u resulted in less
effective compounds. This might indicate difference between molecular targets in colorectal cells
influenced by compounds 4a and 4l bearing unsubstituted-phenyl moiety and those molecular
target influenced by compounds 4b–k and 4m–u bearing methoxylated-phenyl moieties.

ACCEPTED MANUSCRIPT
Regarding the susceptibility of the employed colorectal cell lines to this class of compounds,
HCC2998 and SW620 cell lines were the least responsive cell lines showing inhibition percent
lower than 50% for all of the evaluated hybrid compounds 4a–u. Meanwhile, HCT116 and
HCT15 cell lines showed 67.89~51.40% growth inhibition by six compounds; and HT29 showed
7
5.39~53.26% growth inhibition by five compounds. Accordingly, HCT116, HCT15 and HT29
cell lines were more responsive than other employed colorectal cell lines to the evaluated hybrid
compounds.
9
7
5
3
1
0
0
0
0
0
COLO205
HCC2998
HCT116
HCT15
HT29
KM12
SW620
-10
-30
Figure 5. Percentage inhibition of growth inhibition of colon cancer cells elicited by the synthesized TMS-TMF
hybrid compounds and imatinib at a single dose of 10 µM.
2
.2.1.4. In vitro evaluation of antiproliferative activity in CNS cancers
Mortalities because of CNS cancers are the highest among other cancers mortalities in men and
women before ages of 40 and 20 respectively [48]. Treatment of gliomas; the most common
CNS cancers afflicting adults, is often hampered by molecular heterogeneity and limitations of
drug’s crossing of BBB [49]. Accordingly, the synthesized hybrid compounds were evaluated by
NCI against six cell lines representing gliomas and the results are summarized in Table 4.
Table 4. % Growth inhibition of cell lines representing CNS cancers by compounds 4a–u and imatinib at 10 µM
Conc.

ACCEPTED MANUSCRIPT
1
2
Comp.
R
R
X-Y
SF268
16.73
29.33
36.92
27.86
12.46
32.85
27.72
45.54
42.65
41.57
51.88
63.01
12.92
22.91
45.38
41.28
38.12
8.58
SF295
4.34
SF539
19.84
30.51
22.50
27.66
14.65
47.44
32.24
36.21
33.71
33.67
43.04
80.58
6.01
SNB19
4.37
SNB75
7.02
U251
9.89
4
4
a
MeO
MeO
MeO
MeO
MeO
H
CONH
CONH
CONH
CONH
CONH
b
2-MeO
3-MeO
4-MeO
38.21
29.20
33.62
2.37
17.52
17.51
8.54
30.05
10.24
19.73
-1.27
25.33
22.56
16.80
32.60
32.38
32.60
78.23
13.01
1.94
11.03
37.90
13.86
9.36
4
c
4
d
4
e
3,4-CH
2
O
2
5.13
4
f
MeO 3,4,5-TriMeO CONH
55.01
22.22
40.83
42.74
40.40
46.09
30.00
10.77
17.64
-3.77
7.93
43.73
8.59
55.08
16.34
61.04
36.22
27.67
67.48
63.13
-0.16
23.94
26.78
44.19
55.17
33.08
28.37
28.93
27.05
10.60
4
4
g
MeO
MeO
MeO
MeO
MeO
OH
H
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
CONH
h
2-MeO
3-MeO
4-MeO
63.79
29.56
35.41
54.43
42.33
6.18
4
i
4
j
4
k
3,4-CH
H
2 2
O
4
l
4
m
OH
2-MeO
3-MeO
4-MeO
4
n
o
p
q
OH
20.74
43.91
11.19
48.34
9.52
16.39
38.43
27.85
50.05
25.37
16.64
34.11
33.83
-12.20
4
OH
105.24
15.42
67.75
16.45
13.10
32.53
47.64
0.60
4
4
OH
2 2
3,4-CH O
OH
3,4,5-TriMeO CONH
38.50
6.16
4
r
OH
2-MeO
3-MeO
4-MeO
NHCO
NHCO
NHCO
NHCO
4
s
OH
29.79
13.90
32.54
-3.80
33.14
20.63
18.96
15.10
22.93
15.31
77.40
24.50
4
t
OH
4
u
OH
2 2
3,4-CH O
Imatinib
All data are reported as the average of duplicates.
At the used 10 µM dose, imatinib showed low growth inhibition against three cell lines (24.50,
5.10 and 10.60% for SF539, SF295 and U251 respectively) and was ineffective against another
1
three cell lines (SNB19, SF268 and SNB75). As illustrated in Fig. 6, most of the evaluated
compounds showed much better activity relative to imatinib. As illustrated in Fig. 6, compounds
4
g–k, whose TMF moiety was attached to the nitrogen atom of the linker, were more active
relative to corresponding compounds 4a–e whose TMF moiety was attached to the carbonyl
group of the linker. As table 4 shows, compound 4g possessing an unsubstituted-phenyl moiety
elicited better activity, albeit low, relative to the corresponding compound 4a. In addition,
reversing the configuration of the amide linker of compounds 4b/4c possessing 2-/3-
methoxyphenyl moiety provided the corresponding compounds 4h and 4i eliciting more

ACCEPTED MANUSCRIPT
enhanced activity. Thus, compound 4h elicited 63.79 and 61.04% growth inhibition against
SNB19 and U251 cell lines respectively. When the TMF moiety of compound 4a was replaced
by 6,7-dimethoxy-5-hydroxyflavone moiety, an abrupt increase of activity against multiple
glioma cell lines was elicited by the corresponding compound 4l (Fig. 6). Thus, compound 4l
inhibited SF539, SNB75, U251 and SF268 cell lines by 80.58, 78.23, 63.13 and 63.01%
respectively. In contrast, replacement of the TMF moiety of compounds 4b, 4c, 4h, 4i or 4j by
6
,7-dimethoxy-5-hydroxyflavone moiety to afford the corresponding compound 4m, 4n, 4r, 4s
or 4t resulted in reduction of the activity. However, such replacement in compounds 4d or 4e
having 4-methoxyphenyl moiety or 3,4-methylenedioxyphenyl to afford compounds 4o or 4p
resulted in enhancement of activity against several cell lines representing gliomas. Noticeably,
compound 4o exerted a cytocidal effect on SNB75 glioma cell line inhibiting its growth by
1
05.24%. Despite the activity of compound 4f, which have TMF and 3,4,5-trimethoxyphenyl
moieties was prominent in leukemia, NSCLC and colorectal cancers, it was less prominent
against the assessed CNS cancers. Thus, compound 4f showed inhibition by more than 50%
against only two cell lines out the six cell lines displayed in Table 4. Replacement of TMF
moiety in compound 4f by 6,7-dimethoxy-5-hydroxyflavone moiety afforded the corresponding
compound 4q showing approximately the same level of activity against most of the glioma cell
lines evaluated. Among the evaluated glioma cell lines, SNB75 and SF539 cell lines showed the
highest growth inhibition by two compounds for each within the ranges of 105.24~78.23% and
8
0.58~77.40% respectively. Meanwhile, U251 was responsive to the highest number of tested
compounds eliciting growth inhibition by five evaluated compounds within the range of
7.48~55.08%.
6

ACCEPTED MANUSCRIPT
1
1
20
00
SF268
SF295
SF539
SNB19
SNB75
U251
8
6
4
2
0
0
0
0
0
-20
Figure 6. Percentage inhibition of growth inhibition of CNS cancer cells elicited by the synthesized TMS-TMF
hybrid compounds and imatinib at a single dose of 10 µM.
2
.2.1.5. In vitro evaluation of antiproliferative activity in melanoma
Among skin cancers, melanoma is the most dangerous, In 2018, the number of patients
anticipated to be afflicted with melanoma in US only is 87,290 new cases [48]. The fact that in
melanomas there is intratumor and intertumor heterogenicity undermine the therapeutic benefits
of currently used anticancer agents [50, 51]. Accordingly, the synthesized hybrid compounds
were evaluated by NCI against eight cell lines representing melanomas. The results are
summarized in Table 5.
Table 5. % Growth inhibition of cell lines representing melanoma by compounds 4a–u and imatinib at 10 µM Conc.
1
2
Comp
R
R
X-Y
LOXIMVI
33.03
52.09
50.15
75.43
81.49
75.56
38.30
64.61
42.57
58.14
60.51
11.89
MALME3M
12.51
22.85
15.15
21.42
1.22
MDAMB435
SKMEL2
15.39
14.39
24.30
15.31
-2.38
SKMEL28
SKMEL5
35.29
58.35
36.13
59.06
47.23
91.66
36.79
74.96
39.36
49.84
83.60
28.29
UACC257
UACC62
29.84
46.55
37.89
35.23
19.13
49.52
33.20
56.86
41.10
44.57
50.01
61.09
4
4
a
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
OH
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
8.87
8.31
5.48
b
2-MeO
3-MeO
4-MeO
22.39
18.83
23.77
4.94
24.55
26.58
28.68
20.39
36.45
26.83
43.96
27.04
31.00
44.87
12.57
-6.14
7.62
4
c
4
d
-2.76
5.96
4
e
2 2
3,4-CH O
4
f
3,4,5-TriMeO
H
43.58
13.16
34.50
12.78
46.62
23.57
35.47
93.64
19.27
56.45
23.41
30.44
46.70
41.72
58.33
23.18
68.85
38.11
38.71
37.64
35.04
47.21
0.33
4
4
g
h
2-MeO
3-MeO
4-MeO
38.53
3.66
4
4
i
j
-4.69
22.71
15.72
4
k
2 2
3,4-CH O
4
l
H

ACCEPTED MANUSCRIPT
4
m
OH
OH
OH
OH
OH
OH
OH
OH
OH
2-MeO
3-MeO
4-MeO
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
16.66
36.76
20.25
11.79
67.01
4.40
5.55
12.33
19.38
11.90
49.05
-12.70
29.49
0.28
2.77
22.61
-5.05
20.87
34.63
1.26
14.05
2.64
5.62
14.23
-4.12
10.23
22.57
-1.90
22.50
9.40
3.77
26.82
8.61
1.19
8.74
26.52
44.42
18.93
18.57
50.65
30.17
39.09
33.74
41.40
7.70
4
n
o
p
q
4
16.62
10.11
30.39
0.56
20.81
21.82
33.92
31.90
25.86
13.56
17.18
2.80
4
4
3,4-CH
2
O
2
16.04
60.83
13.42
48.93
16.24
25.85
22.30
3,4,5-TriMeO
2-MeO
4
r
4
s
3-MeO
57.56
6.47
33.62
5.59
21.56
16.65
6.66
4
t
4-MeO
4
u
3,4-CH
2
O
2
5.74
7.67
5.39
15.22
-9.40
Imatinib
11.60
-13.70
7.40
ND
All data are reported as the average of duplicates.
At the used 10 µM dose, imatinib elicited 22.30, 11.60, 7.70 and 7.40% growth inhibition against
SKMEL5, LOXIMVI, UACC62 and MDAMB435 cell lines respectively, while it was almost
ineffective against MALME3M, SKMEL28 and UACC257 cell lines (Table 5). Relative to
imatinib, several compounds among the synthesized and evaluated hybrid compounds were
much more active against the employed melanoma cell lines as illustrated in Fig. 7. The results
indicated that compound 4f, which is a TMS-TMF hybrid compound having 3,4,5-
trimethoxyphenyl moiety and an amide linker configuration in which the TMF moiety was
attached to linker’s carbonyl group, possessed an excellent activity against MDAMB435 and
SKMEL5 cell lines evident by measured 93.64 and 91.66% growth inhibition against these two
cell lines respectively. In addition, it elicited good inhibitory activity against LOXIMVI and
SKMEL2 cell lines showing 75.56 and 58.33% growth inhibition respectively. The growth of the
other three melanoma cell lines was also inhibited by compound 4f, albeit with a lower inhibition
percent. Thus, compound 4f elicited the highest antiproliferative activity among all evaluated
compounds against melanoma cell lines. When the same amide linker configuration was retained
but the TMF moiety of compound 4f was replaced with 6,7-dimethoxy-5-hydroxyflavone moiety
to afford the corresponding compound 4q, the elicited antiproliferative activity was reduced (Fig.
7
). Also, replacing the 3,4,5-trimethoxyphenyl moiety of compound 4f with unsubstituted-phenyl,

ACCEPTED MANUSCRIPT
other methoxylated-phenyl or 3,4-methylenedioxyphenyl moiety while retaining the same amide
linker configuration, resulted in the less active compounds 4a–e. Replacement of the TMF
moiety of compounds 4b–e with 6,7-dimethoxy-5-hydroxyflavone moiety to afford the
corresponding compounds 4m–p resulted in further reduction of the elicited antiproliferative
activity against the employed melanoma cell lines. In contrast, replacement of the TMF moiety
of compound 4a possessing unsubstituted-phenyl moiety with 6,7-dimethoxy-5-hydroxyflavone
moiety to provide compound 4l resulted in some enhancement of the antiproliferative activity
(
Fig. 7). This different behavior might indicate that the pathways contributing to the triggered
growth inhibition are different in the case of compounds having unsubstituted-phenyl moiety
from the cases of compounds having methoxylated-phenyl moieties. When the amide linker
configuration of compounds 4a–e was reversed to provide the corresponding compounds 4g–k,
the activity was enhanced (Fig. 7). Among the latter compounds, the 2-methoxyphenyl and the
3
,4-methylenedioxyphenyl derivatives 4h and 4k elicited prominent antiproliferative activity
(
Fig. 7). Replacing the TMF moiety of compounds 4h–k with 6,7-dimethoxy-5-hydroxyflavone
moiety resulted in compounds 4r–u with lower activity which is a similar behavior to that noted
above for other tested compounds. The overall results showed that LOXIMVI and SKMEL5
were the most susceptible cell lines among the employed melanoma cell lines to the tested
compounds while MALME3M, SKMEL28 and UACC257 were the least affected cell lines by
these compounds

ACCEPTED MANUSCRIPT
LOXIMVI
MALME3M
MDAMB435
SKMEL2
SKMEL28
SKMEL5
UACC257
UACC62
1
00
8
6
4
2
0
0
0
0
0
-20
Figure 7. Percentage inhibition of growth inhibition of melanoma cells elicited by the synthesized TMS-TMF
hybrid compounds and imatinib at a single dose of 10 µM.
2
.2.1.6. In vitro evaluation of antiproliferative activity in ovarian cancers
Because the symptoms of ovarian cancers are more noticeable when the disease progress,
most women diagnosed with ovarian cancers are in advanced stages which are difficult to cure
[
52]. Accordingly, development of novel therapeutics for ovarian cancers is extremely needed.
The synthesized hybrid compounds were evaluated by NCI against a panel of cell lines
representing ovarian cancers. The results are summarized in Table 6.
Table 6. % Growth inhibition of cell lines representing ovarian cancers by compounds 4a–u and imatinib at 10 µM
Conc.
1
2
Comp.
R
R
X-Y
IGROV1
19.39
27.52
46.73
38.66
39.82
48.21
21.64
57.11
42.56
46.09
52.97
14.61
OVCAR3
12.34
47.51
33.39
54.26
12.66
59.16
15.81
48.65
24.65
33.19
46.95
29.87
OVCAR4
1.91
OVCAR5
16.21
18.65
9.93
OVCAR8
34.85
42.67
52.96
46.12
45.94
69.12
39.05
61.29
47.74
42.12
53.70
84.60
ADRRES
18.25
46.70
35.17
47.21
35.10
48.93
25.46
41.75
34.53
39.83
42.41
20.86
4
4
a
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
OH
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
b
2-MeO
3-MeO
4-MeO
19.41
37.43
15.33
3.72
4
c
4
d
16.77
16.44
13.97
-0.09
18.80
10.44
4.43
4
e
2 2
3,4-CH O
4
f
3,4,5-TriMeO
H
25.15
18.28
40.95
37.74
38.04
41.87
120.24
4
4
g
h
2-MeO
3-MeO
4-MeO
4
4
i
j
4
k
3,4-CH
2
O
2
6.14
4
l
H
5.59

ACCEPTED MANUSCRIPT
4
m
OH
OH
OH
OH
OH
OH
OH
OH
OH
2-MeO
3-MeO
4-MeO
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
-5.97
12.00
4.86
-0.89
10.55
49.39
10.51
76.24
-14.59
34.15
-0.49
3.74
48.03
2.54
8.10
8.50
35.16
62.49
19.37
69.42
76.91
17.48
50.66
35.51
37.76
8.40
19.83
25.82
14.68
29.41
50.62
5.26
4
n
o
p
q
4
2.51
4
4
3,4-CH
2
O
2
18.38
29.60
3.04
99.93
41.43
-5.24
33.44
4.66
11.13
22.62
5.27
3,4,5-TriMeO
2-MeO
4
r
4
s
3-MeO
25.67
2.38
9.51
43.69
13.29
14.79
-0.50
4
t
4-MeO
0.04
4
u
3,4-CH
2
O
2
5.56
-4.70
25.76
-0.10
13.65
-22.80
Imatinib
-18.60
-10.30
All data are reported as the average of duplicates.
At the used 10 µM dose, imatinib was almost ineffective against the employed ovarian cancer
cell lines. As shown in table 6, it did not inhibit the growth of all employed cell lines except
for very weak inhibition of OVCAR8 by only 8.40%. In contrast, almost all of the synthesized
and evaluated compounds possessed much better activities as illustrated in Fig. 8. The
antiproliferative activity of both compounds 4a and 4g, which represent the two possible
configurations of amide linked TMS-TMF hybrids possessing unsubstituted-phenyl moiety, was
relatively weak against the employed ovarian cancer cell lines. However, replacement of TMF
moiety with 6,7-dimethoxy-5-hydroxyflavone moiety while retaining the unsubstituted-phenyl
moiety afforded compound 4l showing a strikingly enhanced activity against two cell lines, as
illustrated in Fig. 8. Thus, compound 4l was lethal to OVCAR4 cell line (120.24% growth
inhibition) and highly active against OVCAR8 cell line (84.60% growth inhibition).
Comparing the activity of TMF derivatives 4b–e and 4h–k having reversed amide linker
configuration-relationship reveals some enhancement of activity, albeit not high, for compounds
whose TMF moiety was attached to the linker’s nitrogen atom. In general, compounds 4f and 4q
sharing the presence of 3,4,5-trimethoxyphenyl moiety were the most active among compounds
having methoxylated-phenyl moieties. The activity of compound 4q which possesses 6,7-
dimethoxy-5-hydroxyflavone moiety was higher than compounds 4f possessing TMF moiety. In

ACCEPTED MANUSCRIPT
contrast to the noticed enhancement of activity upon replacing TMF moiety with 6,7-dimethoxy-
5
-hydroxyflavone moiety in derivatives 4l and 4q, compounds 4m, 4n 4o, 4r, 4t and 4u having
the 6,7-dimethoxy-5-hydroxyflavone moiety were in general less active than the corresponding
compounds having the TMF moiety. Among the employed ovarian cancer cell lines, OVCAR8
was the most responsive to the evaluated compounds eliciting more than 50% inhibition by nine
compounds out of the evaluated twenty-one compounds. Meanwhile, OVCAR5 was the least
inhibited ovarian cancer cell line showing inhibition not more than 50% by all of the evaluated
compounds.
IGROV1
OVCAR3
OVCAR4
OVCAR5
OVCAR8
ADRRES
1
1
20
00
8
6
4
2
0
0
0
0
0
-20
Figure 8. Percentage inhibition of growth inhibition of ovarian cancer cells elicited by the synthesized TMS-TMF
hybrid compounds and imatinib at a single dose of 10 µM.
2
.2.1.7. In vitro evaluation of antiproliferative activity in renal cancers
The global number of new renal cancer cases surpassed 350,000 in 2013 resulting in more than
1
40,000 deaths [53]. The rate of renal cancers incidence are increasing with the highest rates in
the developed western countries [54]. The hybrid compounds were assessed by NCI against a
panel of renal cancer cells and the results are presented in Table 7.

ACCEPTED MANUSCRIPT
Table 7. % Growth inhibition of cell lines representing renal cancers by compounds 4a–u and imatinib at 10 µM
Conc.
1
2
Comp
R
R
X-Y
786-0
7.46
ACHN
8.22
CAKI1
4.59
RXF393
24.94
29.26
52.11
59.88
29.04
46.78
25.64
40.97
28.94
46.43
53.51
43.46
21.77
25.82
8.01
SN12C
27.34
34.60
37.67
34.99
30.76
54.08
23.81
58.95
38.69
50.03
57.91
25.16
5.99
TK10
8.98
UO31
35.62
69.58
68.44
56.15
40.99
79.10
48.96
84.75
66.69
73.51
75.77
40.34
30.03
41.69
6.60
4
4
a
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
OH
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
b
2-MeO
3-MeO
4-MeO
39.19
31.10
29.33
7.44
43.80
37.49
33.37
10.18
43.33
38.36
57.02
42.08
53.03
52.24
41.96
9.27
43.00
25.27
63.66
27.89
59.37
12.77
34.88
18.06
42.56
22.93
51.13
6.75
10.41
21.20
15.72
16.13
30.59
8.72
4
c
4
d
4
e
2 2
3,4-CH O
4
f
3,4,5-TriMeO
H
68.25
21.85
54.66
47.22
45.04
80.49
68.53
9.66
4
4
g
h
2-MeO
3-MeO
4-MeO
46.56
17.21
23.32
38.37
39.23
14.08
6.27
4
i
4
j
4
k
3,4-CH
H
2 2
O
4
l
4
m
OH
2-MeO
3-MeO
4-MeO
4
n
o
p
q
OH
14.23
56.97
35.66
33.68
11.35
26.66
24.53
29.90
5.00
33.80
16.39
24.75
83.67
5.14
32.38
6.41
18.96
15.71
31.30
37.00
5.29
4
OH
-5.09
1.40
4
4
OH
3,4-CH
2
O
2
23.07
37.47
9.48
18.53
25.01
-12.13
1.37
17.88
65.67
15.19
52.86
4.63
OH
3,4,5-TriMeO
2-MeO
25.17
-0.86
16.71
18.42
15.32
-10.00
4
r
OH
4
s
OH
3-MeO
35.47
23.74
30.49
-1.00
23.76
20.04
34.41
-0.20
34.94
25.67
9.48
4
t
OH
4-MeO
4.27
4
u
OH
3,4-CH
2
O
2
32.62
5.70
15.84
7.60
Imatinib
6.00
All data are reported as the average of duplicates.
While the standard imatinib at the employed 10 µM dose elicited low activity (≤ 7.60%, Table 7),
most of the evaluated compounds showed much better activity against the employed seven cell
lines representing renal cancers (Fig. 9). In general, TMF derivatives 4g–k in which the amide
linker was configured so that the nitrogen atom of the linker was attached to the TMF moiety
were more active than the corresponding compounds 4a–e with the opposite linker configuration.
As Table 7 shows, derivative 4h and 4k having 2-methoxyphenyl and 3,4-methylenedioxyphenyl
moieties inhibited four cell lines by 84.75~54.66% and five cell lines by 75.77~52.24%
respectively. In comparison, the corresponding derivatives 4b and 4e having linker configuration,
in which the carbonyl group was attached to the TMF moiety were less effective as only one cell

ACCEPTED MANUSCRIPT
line was inhibited by more than 50% by compound 4b while none showed more than 50%
inhibition by compound 4e. Replacement of TMF moiety of the relatively weak compound 4a
which has unsubstituted-phenyl moiety, with 6,7-dimethoxy-5-hydroxyflavone moiety resulted
in compound 4l with more enhanced activity. Thus, while compound 4a showed inhibition of the
growth of 786-0 and CAKI1 cell lines by 7.46 and 4.59% respectively, compound 4l showed
inhibition of these two cell lines by 64.53 and 51.13 respectively. As noticed before in the
assessments of antiproliferative activity, this enhancement of activity by replacing 6,7-
dimethoxy-5-hydroxyflavone for TMF moiety in compounds with unsubstituted-phenyl moiety
was reversed in compounds with monomethoxyphenyl moieties. Thus, compounds 4m–o elicited
much weaker activity relative to the corresponding compounds 4b–d. In addition, replacing the
TMF moiety of the highly active 3,4,5-trimethoxyphenyl derivative 4f with 6,7-dimethoxy-5-
hydroxyflavone moiety afforded the corresponding compound 4q which was less active except
against ACHN cell line (Table 7). This difference in the elicited activity upon replacing TMF
with 6,7-dimethoxy-5-hydroxyflavone moiety might arise from presence of different active
pathways in the different cells. Among active compounds possessing TMF moiety compounds 4f,
4
h, 4j and 4k possessed prominent activity against several cell lines (Fig. 9). Also, the results
showed that UO31 cell line as the most responsive among evaluated cell lines to the set of
evaluated compounds showing growth inhibition within the range of 84.75~52.86% by ten
compounds out of the evaluated twenty-one compounds. On the other hand, TK10 cells were the
least affected cell line showing a maximum inhibition of 46.56% by the best inhibiting
compound to this cell line.

ACCEPTED MANUSCRIPT
7
86-0
ACHN
CAKI1
RXF393
SN12C
TK10
UO31
8
6
4
2
0
0
0
0
0
-20
Figure 9. Percentage inhibition of growth inhibition of renal cancer cells elicited by the synthesized TMS-TMF
hybrid compounds and imatinib at a single dose of 10 µM.
2
.2.1.8. In vitro evaluation of antiproliferative activity in prostate cancers
Prostate cancer is ranked the second cancer in men. Annually, there is 1.6 million new cases and
66,000 deaths because of prostate cancer [55]. In contrast to several androgen dependent
3
prostate cancers, the androgen independent PC3 and DU145 cell lines show an aggressive and
invasive behavior [56, 57]. Accordingly, the synthesized hybrid compounds were evaluated at 10
µM concentration against these two cell lines and the results are summarized in Table 8.
Table 8. % Growth inhibition of cell lines representing prostate cancers by compounds 4a–u and imatinib at 10 µM
Conc.
1
2
1
2
Comp
R
R
X-Y
PC3
28.98
43.63
42.88
38.78
26.46
47.57
46.86
70.04
48.76
68.08
111.96
DU145
12.22
29.65
28.25
26.33
18.67
45.69
14.52
55.06
32.65
43.54
47.07
Comp
4l
R
R
X-Y
PC3
DU145
34.41
5.49
4
4
a
MeO
MeO
MeO
MeO
MeO
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
NHCO
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCO
NHCO
NHCO
28.99
25.15
39.29
13.80
27.73
50.19
10.33
59.64
19.59
22.55
10.60
b
2-MeO
3-MeO
4-MeO
4m
4n
2-MeO
3-MeO
4-MeO
4
c
19.45
3.06
4
d
4o
4
e
3,4-CH
2
O
2
4p
3,4-CH
2
O
2
19.58
42.75
-2.87
36.69
5.40
4
f
MeO 3,4,5-TriMeO
4q
3,4,5-TriMeO
2-MeO
4
4
g
MeO
MeO
MeO
MeO
MeO
H
4r
h
2-MeO
3-MeO
4-MeO
4s
3-MeO
4
i
4t
4-MeO
4
j
4u
3,4-CH
2
O
2
2.12
4
k
3,4-CH
2
O
2
Imatinib
14.40
All data are reported as the average of duplicates.