Effects of substituent pattern on the intracellular target of antiproliferative benzo[b]thiophenyl chromone derivatives

Article abstract of DOI:10.1016/j.ejmech.2021.113578

A new biological scaffold was produced by replacing the 6π-electron phenyl ring-B of a natural flavone skeleton with a 10π-electron benzothiophene (BT). Since aromatic rings are important for ligand protein interactions, this expansion of the π-electron system of ring-B might change the bioactivity profile. One of the resulting novel natural product-inspired compounds, 2-(benzo[b]thiophen-3-yl)-5-hydroxy-7-isopropoxy-6-methoxyflavone (6), effectively arrested the cell cycle at the G2/M phase and displayed significant antiproliferative effects with IC₅₀ values of 0.05–0.08 μM against multiple human tumor cell lines, including a multidrug resistant line. A structure-activity relationship study revealed that a 10π-electron system with high aromaticity, juxtaposed 4-oxo and 5-hydroxy groups, and 7-alkoxy groups were important for potent antimitotic activity. Interestingly, two BT-flavonols (3-hydroxyflavone), 16 and 20, with 3-hydroxy and 5-alkoxy groups, induced distinct biological profiles affecting the cell cycle at the G1/S phase by inhibition of DNA replication through an interaction with topoisomerase I.

Full text of DOI:10.1016/j.ejmech.2021.113578

Journal Pre-proof
Effects of substituent pattern on the intracellular target of antiproliferative
benzo[b]thiophenyl chromone derivatives
Yohei Saito, Yukako Taniguchi, Sachika Hirazawa, Yuta Miura, Hiroyuki Tsurimoto,
Tomoki Nakayoshi, Akifumi Oda, Ernest Hamel, Katsumi Yamashita, Masuo Goto,
Kyoko Nakagawa-Goto
PII:
S0223-5234(21)00427-X
DOI:
https://doi.org/10.1016/j.ejmech.2021.113578
EJMECH 113578
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2021
Revised Date: 9 May 2021
Accepted Date: 19 May 2021
Please cite this article as: Y. Saito, Y. Taniguchi, S. Hirazawa, Y. Miura, H. Tsurimoto, T. Nakayoshi,
A. Oda, E. Hamel, K. Yamashita, M. Goto, K. Nakagawa-Goto, Effects of substituent pattern on the
intracellular target of antiproliferative benzo[b]thiophenyl chromone derivatives, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2021.113578.
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Graphical Abstract

Effects of substituent pattern on the intracellular target of
antiproliferative benzo[b]thiophenyl chromone derivatives
a
a
a
a
a
Yohei Saito, Yukako Taniguchi, Sachika Hirazawa, Yuta Miura, Hiroyuki Tsurimoto,
b,1
b
a
d,*
c
Tomoki Nakayoshi, Akifumi Oda, Ernest Hamel, Katsumi Yamashita, Masuo Goto,
a,d,*
Kyoko Nakagawa-Goto
a
School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa
University, Kanazawa, 920-1192, Japan
b
Graduate School of Pharmacy, Meijo University, Tempaku-ku, Nagoya, 468-8503, Japan
cMolecular Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick,
Maryland, 21702, United States
d
Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North
Carolina, Chapel Hill, North Carolina 27599-7568, United States
Corresponding Authors
*
(K.N.G.) Phone: +81-76-264-6305, E-mail: kngoto@p.kanazawa-u.ac.jp
M.G.) Phone: +1-919-843-6325, E-mail: goto@med.unc.edu
(
1
(T.N) Current address: Graduate School of Information Sciences, Hiroshima City University, 3-
4
-1 Ozukahigasi, Asaminami-ku, Hiroshima, Hiroshima 731-3194, Japan
1

ABSTRACT
A new biological scaffold was produced by replacing the 6-election phenyl ring-B of a natural
flavone skeleton with a 10-electron benzothiophene (BT). Since aromatic rings are important
for ligand protein interactions, this expansion of the -electron system of ring-B might change
the bioactivity profile. One of the resulting novel natural product-inspired compounds, 2-
(benzo[b]thiophen-3-yl)-5-hydroxy-7-isopropoxy-6-methoxyflavone (6), effectively arrested the
cell cycle at the G2/M phase and displayed significant antiproliferative effects with IC values
5
0
of 0.05–0.08 M against multiple human tumor cell lines, including a multidrug resistant line. A
structure-activity relationship study revealed that a 10-electron system with high aromaticity,
juxtaposed 4-oxo and 5-hydroxy groups, and 7-alkoxy groups were important for potent
antimitotic activity. Interestingly, two BT-flavonols (3-hydroxyflavone), 16 and 20, with 3-
hydroxy and 5-alkoxy groups, induced distinct biological profiles affecting the cell cycle at the
G1/S phase by inhibition of DNA replication through an interaction with topoisomerase I.
Keywords: Benzothiophene, Flavone, Flavonol, Antiproliferative activity, Tubulin,
Topoisomerase I
2

1
. Introduction
Enzymes are required for the biosynthesis of all secondary metabolites (natural products)
produced by living organisms. With this innate affinity for enzymes, natural products might
unexpectedly bind to a protein/enzyme in a different species to produce unpredicted bioactivity.
Thus, we can exploit this innate affinity of natural products to discover and develop chemical
agents acting at desired protein targets.
Flavonoids are distributed abundantly in plants, including edible fruits and vegetables, as
secondary metabolites. They interact with various cellular targets involved in important signal
pathways in our bodies and generally act beneficially for human health with relatively low
toxicity [1]. Only a few cytotoxic flavonoids have been reported; examples are the highly
methoxylated
natural
flavones
centaureidin
[2]
and
5,3-dihydroxy-3,6,7,8,4-
pentamethoxyflavone (DH-PMF) [3], which displayed remarkable cytotoxicity (IC < 0.5 M)
5
0
against human tumor cell lines [4]. These flavones inhibit the assembly of tubulin by binding to
the colchicine site (CS).
The basic flavone skeleton has 15 carbons arranged in a C6-C3-C6 unit: two phenyl rings
(ring-A and ring-B) connected through a heterocyclic ring (ring-C) containing three carbons and
an oxygen atom. They are classified based on the oxidation stage of ring-C, e.g., flavone,
flavonol (3-hydroxyflavone), flavanone (2,3-dihydroflavone), etc., and the attachment of ring-B
(flavone vs. isoflavone). Except for isoflavones, a common flavone structure (2-phenyl-4-
chromone) has fused benzene and pyran-4-one rings (ring-A and -C, respectively) with a phenyl
group (ring-B) attached at C-2 (Figure 1).
3

Figure 1. Natural Flavone Skeleton and Proposed Artificial Flavone
Because the basic three-ring system is biosynthesized from 4-coumaroyl-CoA derived from
phenylalanine and three malonyl-CoAs, the ring-B in naturally occurring flavones is normally a
phenyl group, a 6-electron aromatic ring. Aromatic ring systems in bioactive compounds often
play an important role in binding to target proteins or enzymes [5-7]. They are rigid,
hydrophobic, structurally planar, and electron rich which can stabilize ligand-protein binding
through  stacking, cation-, and OH/interactions [8]. Bicyclic aromatic units, such as
benzothiophene (BT), benzofuran (BF), and naphthalene (Np), are 10-electron rather than 6-
electron systems; the electron distribution expands over an additional 4-orbitals. The higher
aromaticity might impact biological profiles, including absorption, distribution, metabolism, and
excretion (ADME) properties [9-12]. Thus, we postulated that a novel biological backbone
(Figure 1) could result from connecting a chromone to a bicyclic aromatic system rather than the
phenyl ring-B present in the natural flavone skeleton.
Among 10 aromatic systems, the BT skeleton is rarely found in secondary metabolites [13],
although it is often found in various clinical drugs [14]. The sulfur atom in BT contributes to
4

higher aromaticity through its 3d orbital, and its electronegativity is close to that of carbon. Other
0 systems, including BF, Np, and indole (X = NH), are biosynthesized through acetyl CoA as
1
well as shikimic acid and are abundant in natural products.
Previously, we found a dramatic change between the bioactivity profiles of
triethyldesmosdumotin B (TEDB) and TEDB-BT, which differ structurally only in ring-B:
phenyl in TEDB versus BT in TEDB-BT (Figure 2) [15-17]. TEDB is a derivative of
desmosdumotin B (DesB), an atypical flavonoid with a non-aromatic ring-A. It effectively
inhibited the growth of a multi-drug resistant (MDR) tumor cell line, while it was non-toxic
against the tested chemosensitive tumor cell lines. This unique biological phenomenon is called
collateral sensitivity [18]. However, TEDB-BT displayed potent cell growth inhibition against
multiple tumor cell lines and inhibited tubulin polymerization, potentially through the CS
[
[
17,19]. In addition, several BT derivatives were recently reported as potent antimitotic agents
20-27].
Figure 2. BT Effect on Desmosdumotin B
This notable effectiveness of BT strongly encouraged us to synthesize novel scaffolds,
specifically, the hybrids of chromones with bicyclic aromatic rings, including BT. Herein, we
5

discuss the syntheses, structure-activity relationship of BT-flavones and their mechanism of
action.
2
. Results and discussion
2
.1 Chemistry
All flavones were prepared by traditional flavone synthetic methods through Claisen-Schmidt
condensation for 1–13, 30 and 31 (Schemes 1 and 2) or Baker-Venkataraman rearrangement for
-substituted derivatives 14–27 (Scheme 3). Trihydroxyacetophenone 32 was the best candidate
3
as a starting material for di-substituted and tri-substituted BT-flavones (Scheme 1). The standard
methylation and isopropylation of the two phenolic hydroxy groups on 32 produced dimethoxy
3
3 and di-isopropoxy 34, respectively. The Claisen-Schmidt condensation of 33 and 34 with BT-
3
-carbaldehyde (35), followed by I catalyzed cyclization, yielded the related flavones 1 and 4,
2
respectively. With the latter compound, the isopropyl group at C-5 was lost during the
cyclization step to leave a hydroxy moiety. The treatment of 1 with BBr produced mono-
3
methoxy 2 and dihydroxy 3. In an Elbs persulfate oxidation, a hydroxy group was added to 34 to
give 38, which was then methylated to yield 39 [28]. A Claisen-Schmidt condensation of 39 with
BT-3-carboxaldehyde (35) followed by cyclization produced tri-substituted flavones 5 and 6.
Again, in the latter compound, the isopropyl group on the 5-OH of the starting material was
spontaneously removed during the cyclization. The remaining isopropyl group in 6 was
eliminated by using AlCl , and the 5-OH in the resulting 5,7-dihydroxy BT-flavone 7 was
3
selectively alkylated to produce 5-propoxy 8, 5-ethoxy 9, and 5-methoxy 10. Alternatively, the
treatment of 39 with benzofuran-3-carbaldehyde (36) [29] or 1-naphthaldehyde (37) produced the
related flavones 11 and 12, respectively. For comparison purposes, typical flavones 30 and 31
6

with a phenyl ring-B were also synthesized from 39 and benzaldehyde. Claisen-Schmidt
condensation of 39 with 35 followed by acidic treatment produced 2,3-dihydroflavone 28 as a
racemate. The removal of the isopropyl group on the 5-OH generated 29, which is equivalent to
6
, except for the oxidation state of the C2-C3 bond (single bond in 29, double bond in 6).
Mono-substituted flavone 13 was prepared from resacetophenone (40) through the selective
isopropylation of a phenolic hydroxy group, followed by Claisen-Schmidt condensation with 35
and cyclization with catalytic I in dimethyl sulfoxide (DMSO) (Scheme 2).
2
Scheme 1. Preparations of Di-and Tri-substituted BT-Flavones 1–12 and 28–31
Reagents and conditions: a) Me SO , K CO , DMF, 80 °C; b) 50% KOH aq., EtOH, rt for BT-CHO (35),
2
4
2
3
Np-CHO (37), or PhCHO, Ba(OH) , DMF, 90 °C for BF-CHO (36); c) I (cat.), DMSO, 180 °C; d) BBr ,
2
2
3
CH Cl , 0 to 45 °C; e) iPrBr, K CO , DMF, 80 °C, 3.5 h; f) K S O , NaOH, Py, rt; g) AlCl , CH Cl , 0 °C
2
2
2
3
2
2
8
3
2
2
to rt; h) RI (R = Et for 9, R = Pr for 8) or Me SO for R = Me, K CO , acetone, reflux; i) NaOAc, EtOH,
2
4
2
3
H O, 80 °C.
2
7

Scheme 2. Preparation of Mono-substituted BT-Flavone 13
Reagents and conditions: a) iPrBr, K CO , DMF, 80 °C, 3.5 h; b) BT-CHO (35), 50% KOH aq., EtOH, rt;
2
3
c) I (cat.), DMSO, 180 °C.
2
3
-Substituted BT-flavones were prepared by Fougerousse’s method [30] through a Baker-
Venkataraman rearrangement (Scheme 3). The hydroxy group on 39 was condensed with the
carboxylic acid of 42, derived from 35, to yield ester 43. Then, the  position on the acetyl group
was selectively brominated by using phenyltrimethylammonium tribromide to produce 44, which
was treated with potassium benzoate to generate 45. A Baker-Venkataraman rearrangement of 45
was performed with sodium hydride in THF, and the resulting 1,3-diketone 46 was cyclized
under acidic conditions to form 3-benzoylated BT-flavone 14, which was treated with TiCl to
4
obtain its de-isopropyl analog 15. Basic hydrolysis of 14 with NaOH produced the related 3-
hydroxy BT-flavone 16. Further treatment of 16 with Me SO produced 17, which was reacted
2
4
with aluminum trichloride for selective removal of the isopropyl group to give 18. Hydrolysis of
5 produced 3,5-dihydroxy BT-flavonol 19, a 3-hydroxy analog of 6. Methylation of the 5-OH
1
of 15, followed by hydrolysis of the benzoate, generated 20. Subsequently, the 3-OH of 20 was
methylated to obtain 3,5,6-trimethoxy 21. The same sequence of reactions using 6-hydroxy-
2
,3,4-trimethoxyacetophenone [31], 2-hydroxy-4,5-methylenedioxyacetophenone [32], or 2-
hydroxy-4,5-dimethoxyacetophenone [33] provided benzoates 22, 24, and 26, respectively,
which were then hydrolyzed to produce 23, 25, and 27, respectively.
8

Scheme 3. Preparations of 3-Substituted BT-Flavones 14–27
Reagents and conditions: a) Jones Reagent, acetone; b) EDCI, DMAP, CH Cl , rt; c) PhMe N Br , THF,
+
-
2
2
3
3
rt; d) PhCOOK, CH CN, reflux; e) NaH, THF, 65 °C; f) 2.5% H SO /AcOH, 60 °C; g) 5% NaOH, EtOH,
3
2
4
6
0 °C; h) Me SO , K CO , DMF, 80 °C; i) AlCl , CH Cl , 0 °C to rt; k) TiCl , CH Cl , 0 °C to rt.
2 4 2 3 3 2 2 4 2 2
2
.2. Biological Evaluation
.2.1. Antiproliferative effects and SAR analysis
The newly synthesized flavones and flavonols were evaluated for antiproliferative activity
2
against five human tumor cell lines: A549 (lung carcinoma), two breast cancer cell lines (MDA-
MD-231 and MCF-7), KB (cervical cancer cell line derived from HeLa cells), and the KB-
subline KB-VIN showing the MDR phenotype with overexpression of the drug transporter P-
glycoprotein (P-gp) (Tables 1 and 2). Paclitaxel, a cytotoxic, antitubulin P-gp substrate, was used
as an experimental control. The antiproliferative effects of the compounds were measured with a
sulforhodamine B (SRB) assay, and IC values were calculated from at least three independent
5
0
9

experiments, each performed in duplicate. For comparison, IC values obtained with a typical
5
0
flavone, chrysin (5,7-dihydroxyflavone), were also obtained.
Among all compounds, five (three 2-(benzo[b]thiophen-3-yl)-5-hydroxy-6-methoxy-7-
alkoxyflavones [R = alkoxy, R = OMe, R = OH, Ar = BT (6, 8, and 9)]; the naphthyl
1
2
3
derivative 12; and the flavanone 29) showed potent antiproliferative activity (IC50 0.05–0.12
M) against all tested tumor cell lines, including the MDR cell line, KB-VIN (highlighted in

Table 1). These compounds were dramatically more potent as compared to the related flavone 31
with a phenyl ring-B (IC = 8.95–29.1 M). These results demonstrated a key effect with BT or
5
0
Np as ring-B.
In contrast, compound 3 (BT-chrysin) did not show improved potency as compared to natural
chrysin. However, while most natural flavones exhibit only moderate to weak cytotoxicity, a few
highly functionalized flavones were exceptions. For instance, 5,3′-dihydroxy-3,6,7,8,4′-
pentamethoxyflavone, first isolated in 1986 from Gutierrezia microcephala [34], showed potent
antimitotic antiproliferative activity with IC values of 0.08 and 1.49 M against KB and A549
5
0
cells, respectively [35]. Lewin’s group vigorously pursued further research on cytotoxic
flavonoids and concluded that a 5-hydroxy-6,7,8-trimethoxy arrangement on ring-A and 3-
hydroxy/3-amino-4-methoxy groups on ring-B were the most favorable substitution patterns for
increased potency [36]. However, our study suggests the importance of the aromaticity of ring-B,
with a 10 electron system significantly influencing antiproliferative activity. The order of
potency in our assays was 6  12 > 11, which correlated with the order of ring-B aromaticity, BT

Np > BF. Because the electronegativities of sulfur (BT), carbon (Np), and oxygen (BF) atoms
are 2.5, 2.5 and 3.5, respectively, the lone pair delocalization is less effective on the oxygen than
on the sulfur and carbon atoms, which reduces the aromaticity of BF. In addition, the 3d orbital
1
0

of the sulfur atom efficiently contributes to resonance. As noted above, flavone 31 with the
natural 6-electron ring-B (phenyl) was substantially less potent as compared to the compounds
with a 10-electron ring-B.
Table 1. Antiproliferative Activities of Synthesized Flavones 1–13 and 28–31.
a
b
Compounds
Human cancer cell lines /IC (μM)
50
MDA-
MB-231
>40
R1
R2
R3
Ar
A549
MCF-7
KB
KB-VIN
1
2
3
4
5
6
7
8
9
OMe
OMe
OH
OMe
10.8
36.3
20.5
8.41
7.23
0.05
23.3
0.07
0.07
0.44
6.83
0.19
0.08
31.1
10.1
28.0
5.40
0.06
0.0057
>40
>40
36.8
>40
13.3
>40
>40
29.9
8.34
11.8
0.08
37.3
0.08
0.07
0.50
18.8
0.25
0.12
40.2
29.1
33.0
16.8
0.09
0.00917
H
OH
24.7
8.53
7.48
0.08
9.84
0.07
0.07
0.70
6.77
0.52
0.08
32.2
18.0
26.9
8.23
0.10
24.9
8.74
5.84
0.07
20.6
0.06
0.06
0.57
5.34
0.31
0.10
35.3
22.4
29.6
4.84
0.06
19.3
5.88
6.06
0.06
18.5
0.05
0.05
0.37
4.31
0.09
0.09
33.0
8.95
11.5
1.91
0.06
2.105
OiPr
OiPr
OiPr
OH
OPr
OEt
OMe
BT
OMe
OH
1
0
3
1
2
0
1
1
1
1
3
3
H
H
BF
Np
OH
OiPr
OH
OiPr
OMe
Ph
c
Ch OH
H
2
2
8
9
OiPr
OH
OiPr
OMe
BT
Paclitaxel
0.0633 0.00579
a
A549 (lung carcinoma), MDA-MB-231 (triple-negative breast cancer), MCF-7 (estrogen receptor-
positive & HER2-negative breast cancer), KB (cervical cancer cell line derived from HeLa cells.
Antiproliferative activity as IC values for each cell line, the concentration of compound that caused 50%
b
50
reduction relative to untreated cells determined by the SRB assay. Each experiment was performed three
c
times, and standard deviations were within 10% of the means (see Supplemental Table S1). Chrysin
Among the flavones with no substituent at C-3 (Table 1), the combination of 5-hydroxy (R =
3
OH), 6-methoxy (R = OCH ) and 7-alkoxy (R = O-alkyl) substitution in ring-A was crucial for
2
3
1
1
1

potent antiproliferative activity. The size of the 7-alkoxy group also influenced activity. A
comparison of the results obtained with 6 and 810 showed that compound 10 with a 7-methoxy
group was clearly less potent (IC50 0.370.70 µM) than 6 (isopropoxy), 8 (propoxy), and 9
(
ethoxy) with larger alkoxy groups (IC 0.050.08 µM). In addition, compound 7, which differs
5
0
from 6 and 810 only by a 7-hydroxy instead of a 7-alkoxy group, dramatically lost activity
IC > 10 µM). The 5-hydroxy moiety (R = OH), which could form a hydrogen bond with the
(
5
0
3
ketone at C-4, was another key for better activity. Compound 5 with a 5-isopropoxy group
clearly showed decreased cell growth inhibition as compared to 6 with a 5-hydroxy group.
Flavanone 29 and flavone 6 exhibited significant potency with similar IC values. Since the two
5
0
compounds have identical structures except for the saturated C2-C3 bond in ring-C of the former
compound, a double bond between C-2 and C-3 is not crucial. The comparable results for
flavanone 28 and the analogous flavone 5 were consistent with this conclusion.
Notably, most derivatives displayed similar antiproliferative effects against KB-VIN, a P-gp-
overexpressing MDR tumor cell line, and the chemosensitive tumor cell lines. This finding
indicates that these compounds are not P-gp substrates and could be effective against MDR
tumors. Moreover, compounds 1, 11, and 31 exhibited collateral sensitivity [18], showing two- to
three-fold greater antiproliferative activity against KB-VIN than the parental non-MDR tumor
cell line, KB.
Among the synthesized flavonols (14–20), compounds 15 and 18–20 exhibited the highest
activity (IC 0.241.05 µM) (Table 2), although they were generally tenfold less potent than
5
0
flavones 6, 8, and 9 and compounds 12 and 29. At C-3 (R ), flavonols 19 and 20 have a free OH,
4
and flavonols 15 and 18 have benzoate and methoxy groups, respectively, indicating some
tolerance in the substitution at this position. At C-7 (R ) and C-6 (R ), all four active flavonols
1
2
1
2

have isopropoxy and methoxy substituents, respectively, while at C-5 (R ), three compounds (15,
3
1
8, 19) have a hydroxy, and flavonol 20 has a methoxy group. Compounds 24–27, with no
substituent on C-5, were clearly less active, suggesting the importance of a hydroxy/alkoxy
group at C-5. Comparison of the data for 20 and 23 indicated the same SAR effect found with
flavones 6 and 10. The compound with the larger 7-isopropoxy group (20) was more potent than
the compound with the smaller 7-methoxy moiety (23).
a
Table 2. Antiproliferative Activities of Synthesized Flavonols 14–27.
Compounds
Cell lines/IC (μM)
50
MDA-
MB-231
R1
OiPr
OMe
R2
R3
R4
A549
MCF-7
KB
KB-VIN
1
1
1
1
1
1
2
2
2
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
4
5
6
7
OiPr
3.87
0.64
5.08
17.8
0.36
0.93
0.58
5.31
5.23
6.04
4.47
8.10
5.20
6.76
6.62
0.75
6.62
27.4
0.71
2.58
1.00
8.97
7.78
11.5
7.31
10.1
8.81
18.3
6.42
0.81
6.19
21.6
0.45
7.43
1.05
7.72
6.46
7.92
6.70
>40
5.64
0.56
5.96
19.0
0.25
0.87
0.65
5.37
5.29
8.06
5.32
>40
6.38
0.55
6.02
14.7
0.24
0.64
0.68
4.83
4.59
6.97
4.70
>40
OBz
OH
OH
OiPr
OMe
OMe
OH
OH
OMe
OBz
OH
OMe
OBz
OH
-OCH O-
2
H
OBz
OH
8.04
23.1
6.25
18.6
6.52
13.1
2.105
OMe OMe
Paclitaxel
0.0057 0.00917
0.00633 0.00579
a
Each experiment was performed three times, and standard deviations were within 10% of the means (see
Supplemental Table S2).
1
3

It is noteworthy that compound 6 showed 45-fold lower antiproliferative activity against
human umbilical vein endothelial cells (HUVECs), with an IC value of 2.26 M, indicating
5
0
that 6 selectively inhibited the growth of tumor cells. Next, the biological effects and targets of
the new synthetic flavones were examined to clarify the different SAR observations.
2
.2.2. Identification of cell cycle phases affected by compound modifications
To determine the cellular target of the active compounds, the initial mechanism of action (MOA)
studies were performed using flavone 6. Because TEDB-BT, the BT modified atypical flavonoid,
induces cell cycle arrest at G2/M by inhibiting tubulin polymerization [17,19], the effects of 6 on
cell cycle progression and tubulin polymerization were analyzed by flow cytometry and
immunocytochemistry (Figure 3). Compound 6 induced cell cycle arrest at G2/M in a dose-
dependent manner. The immunostaining of 6-treated cells with antibodies to α-tubulin and
Ser10-phosphorylated histone H3 (p-H3), a mitotic marker of condensed chromosomes, as well
as with 4ʹ,6-diamidino-2-phenylindole (DAPI) for DNA, clearly demonstrated that
depolymerization of microtubules and abnormal centrosomal amplification with immature
spindles had occurred in p-H3-positive cells. These results confirmed that compound 6 induced
cell cycle arrest at prometaphase by targeting tubulin. The phenotype of cells treated with an
equitoxic concentration of combretastatin A-4 (CA-4), a CS tubulin polymerization inhibitor,
differed from that of 6-treated cells in that no p-H3-positive chromosome condensation occurred,
suggesting that 6 and CA-4 may target tubulin by slightly different mechanisms.
1
4

Figure 3. Dose-dependent Effects of 6 on Cell Cycle Progression, Microtubules, and Spindle
Formation. PC-3 cells were treated with 6 for 24 h at the indicated compound concentrations.
DMSO was used as a vehicle control. Cell cycle distributions of treated cells were analyzed by
flow cytometry after staining with PI. A CS tubulin polymerization inhibitor combretastatin A-4
(CA-4) was used at 200 nM as a control for accumulation of G2/M phase cells. Treated cells
were triple-stained with antibodies to α-tubulin (green), Ser10-phosphorylated histone H3 (p-H3,
red), a marker of mitotic chromosome condensation, and DAPI (blue) for DNA. Stained cells
were observed with a confocal fluorescence microscope. The represented merged image was a
projection of 16-24 optical sections. Multipolar immature spindles (red arrows), abnormal
amplification of spindle poles without spindle elongation (pink arrows). Bar, 0.025 mm.
1
5

To ascertain whether compounds with various ring-B types affected cell cycle and mitotic
inhibition differently, cells treated with natural chrysin and four additional synthetic flavones (8,
1
1, 12, 31) were also analyzed by flow cytometry and immunocytochemistry. The natural
compound and all four synthetic analogues showed the same effect as 6 on mitotic progression
Figure 4), although they exhibited different IC50 values in the antiproliferative assays. Four
(
compounds, 31, 12, 11, and 6, have the same substituted chromone connected to a different ring-
B, Ph, Np, BF, and BT, respectively. From an overall evaluation of bioactivity based on IC and
5
0
induction of cell cycle arrest at G2/M, the effects of ring-B were conjectured to follow the order,
Ph (31) << BF (11) < Np (12)  BT (6), which correlated well with the order of ring aromaticity.
Figure 4. Effect of Ring-B on Induction of G2/M Arrest.
MDA-MB-231 cells were treated with compound for 24 h at the indicated concentrations.
DMSO was used as a vehicle control. Treated cells were subjected to analysis of the effects on
cell cycle progression by flow cytometry and immunocytochemistry. Cell cycle distributions of
treated cells were analyzed by flow cytometry after staining with PI. Treated cells were triple-
stained with antibodies to α-tubulin (green), Ser10-phosphorylated histone H3 (p-H3, red), a
marker of mitotic chromosome condensation, and DAPI (blue) for DNA. Stained cells were
1
6

observed with a confocal fluorescence microscope. The represented merged image was a
projection of 18-24 optical sections. Normal bipolar spindles (white arrow), multipolar immature
spindles (red arrows), amplification of spindle poles without spindle elongation (pink arrows),
cells with nuclear fragmentation with apoptotic microtubules (yellow arrows), fragmented and
dispersed chromatin (yellow arrowhead). Bar, 0.025 mm.
The effects of ring-A substituents in 46, 8, and 9 on mitotic arrest were also analyzed by flow
cytometry (Figure 5). A 5-hydroxy group was critical (see 5 vs 6) and a 6-methoxy group
improved the induction of cell cycle arrest at G2/M (see 4 vs 6).
Figure 5. Effect of Ring-A Modification on Induction of G2/M Arrest.
MDA-MB-231 cells were treated with compound for 24 h at the indicated concentrations.
DMSO or 200 nM CA-4 (10× IC50) was used as a vehicle control or tubulin polymerization
inhibitor arresting cells at G2/M, respectively. Cell cycle distributions of treated cells were
analyzed by flow cytometry after staining with PI.
1
7

The MOA studies were also conducted on the four bioactive flavonols 16, 18, 19, and 20. Flow
cytometric analysis of cells treated with flavonols 18 and 19, which, like 6, have a 5-OH,
displayed accumulation of cells in the G2/M phase after 24 h (Figure 6), similar to that found
with CA-4, regardless of their 3-alkoxy or -hydroxy group. In contrast, flavonols 20 and 16,
which, in addition to a 3-OH, possess a 5-alkoxy rather than hydroxy group, showed a different
effect on the cell cycle. Both compounds impacted cell cycle progression at the G1/S phase
(Figure 7), not the G2/M phase. Thus, a 5-OH is likely crucial for the compound to target tubulin
and arrest cells at G2/M. Furthermore, compounds with both 3-hydroxy and 5-alkoxy groups
affected the G1/S phase, instead of the G2/M phase. These results suggested that the 3-OH and
5
-OH are key factors in determining the target of the flavonols.
Figure 6. Effects of Flavonols 18 and 19 on Induction of G2/M Arrest.
MDA-MB-231 cells were treated with compound 18 or 19 for 24 h at 1-fold (1× IC50) or 3-fold
(
3× IC50) the IC values. DMSO or 20 nM CA-4 (1× IC50) was used as a vehicle control or
50
tubulin polymerization inhibitor arresting cells at G2/M, respectively. Cell cycle distributions of
treated cells were analyzed by flow cytometry after staining with PI.
1
8

Figure 7. Effects of Flavonols 16 and 20 on Accumulation of Cells in S-phase.
MDA-MB-231 cells were treated with compound 16, 20, or reference compound 5-FU or CPT
for 24 or 48 h at the indicated concentrations. DMSO was used as a vehicle control. Cell cycle
distributions of treated cells were analyzed by flow cytometry after staining with PI.
The S phase in the cell cycle is involved in DNA replication. Camptothecin (CPT) and 5-
fluorouracil (5-FU) are well-known agents that damage DNA through different mechanisms of
action, which can be distinguished by analyzing the distribution of cells in the cell cycle. Based
on the flow cytometric analysis, compound 16 and 5-FU act by a similar mechanism, showing
cell accumulation at the S phase, while 16 is 3.8-fold more potent than 5-FU (Figure 7).
Meanwhile, the cell cycle distributions with 20 and CPT were similar (Figure 7); thus, the likely
molecular target of 20 is topoisomerase (Topo) I, which is the target of CPT. When DNA is
damaged, its repair pathway is activated, which leads to the induction of phosphorylated histone
1
9

H2AX (γH2AX). To confirm the DNA damage, the cells treated with flavonols 16, 18, 19, and
0 were stained with antibodies to α-tubulin and γH2AX as well as DAPI for DNA (Figure 8).
2
Among the four compounds, the 3-hydroxy analogues 16 and 20 clearly induced DNA damage
as did the control 5-FU. Considering the immunostaining and flow cytometry results, it is
postulated that 16 acts similarly to 5-FU, by impairing DNA replication through depletion of
newly synthesized nucleotides [37]. In contrast, compounds 18 and 19 did not show significant
induction of γH2AX positive cells, while they did inhibit tubulin polymerization with abnormal
centrosomal amplification; thus, like 6, these two compounds target tubulin.
2
0

Figure 8. Induction of DNA Damage by Compounds.
MDA-MB-231 cells were treated with compounds 16, 18, 19, or 20 for 24 h at the IC50
concentrations. DMSO or 5-FU was used as a vehicle control or positive control, respectively.
Treated cells were triple-stained with antibodies to α-tubulin (green), γ-H2AX (red), a marker of
DNA double-strand breaks (DSBs), and DAPI (blue) for DNA. Stained cells were observed by
confocal fluorescence microscopy. The merged images shown were projections of 16-20 optical
sections. Bar, 0.025 mm.
2
1

2
2
.3. Molecular MOA
.3.1. Effect of 3-OH flavones on tubulin polymerization and colchicine binding to tubulin
Antitubulin agents binding to the CS, including CA-4, disrupt cell cycle progression at the
G2/M phase. Selected antiproliferative flavones were evaluated in a cell-free system to evaluate
a direct inhibitory effect on tubulin assembly (ITA), as measured by turbidimetry, and for
3
inhibition of [ H]colchicine binding to tubulin, i.e., whether compounds could compete with
colchicine. The 50% effective concentration for inhibiting tubulin assembly (IC ) and percent
5
0
inhibition of colchicine binding to tubulin (ICB) of flavones 6, 8, and 9, flavonols 16 and 18–20,
and flavanone 29 were determined (Table 3). Typical turbidimetry experiments are shown in
Supplemental Materials, Fig. S76, for compounds 18 (Panel A), 8 (Panel B) and 20 (Panel C). As
expected, flavone 6 strongly inhibited tubulin assembly with an IC -ITA value of 1.3 μM; it also
5
0
inhibited colchicine binding by 61%. Flavanone 29 showed similar inhibitory effects to those of
3
2
6
, indicating that both sp and sp carbons at C2 and C3 were tolerated for inhibition of tubulin.
The size of the 7-alkoxy group influenced the inhibition of tubulin assembly as well as apparent
binding to the CS. While compound 8 with a 7-propoxy group exhibited an IC -ITA value of 1.9
5
0
μM, it was less effective than 6 with a 7-isopropoxy group at inhibiting colchicine binding to
tubulin. 7-Ethoxy derivative 9 did not significantly inhibit tubulin assembly in the cell-free
system, while its in vitro antiproliferative activities were almost identical to those of 6 and 9.
Like flavone 6, 5-hydroxy-3-methoxy flavonol 18 mildly inhibited colchicine binding to tubulin,
but it showed a potent inhibitory effect on tubulin assembly. As predicted from the results of
flow cytometry and immunocytochemical analysis, 5-alkoxy flavonols 16 and 20 did not inhibit
tubulin assembly. Interestingly, 5-hydroxy flavonol 19 also did not inhibit tubulin assembly in
this assay, although the compound arrested cell progression at the G2/M phase. These results
2
2

supported the postulate that 6, 8, 18, and 29 directly inhibit tubulin polymerization in a slightly
different manner from colchicine binding to tubulin because these compounds only partially
competed with colchicine for binding to tubulin.
a
b
Table 3. Inhibition of Tubulin Assembly and Colchicine Binding.
c
Compound
IC (M) ± SD
ICB (%)  SD
61 ± 3
37 ± 3
34 ± 3
0
5
0
d
6
8
9
1.3 ± 0
1.9 ± 0.3
>20
16
18
19
20
29
>20
1.1 ± 0.1
>20
59 ± 4
32 ± 3
0
>20
1.4 ± 0.1
76 ± 2
98 ± 0.7
CA-4
0.65 ± 0.09
a
The tubulin assembly assay measured the extent of assembly of 10 M tubulin after 20 min at
3
0 C.
b
3
Tubulin: 0.5 M, [ H]colchicine: 5 M, inhibitor: 5 M. Incubation was for 10 min at 37 C.
c
Inhibition of colchicine binding.
d
SD of 0 indicates same IC was obtained in all assays performed with compound 6. Actual
5
0
reproducibility of the assay from evaluation of multiple compounds over 30 years is in the range
of 15-20%.
2
.3.2. Effect of 3-OiPr flavones on Topo I.
One way for a compound to affect the cellular S phase it to inhibit Topo. Topos are enzymes
involved in the overwinding or unwinding of DNA and are generally classified into two types, I
Topo I) and II (Topo II). Flavonol 20 was evaluated for Topo I inhibitory activity in a cell-free
(
system using a relaxation assay by simultaneous incubation of supercoiled plasmid DNA as a
substrate with purified Topo I in the presence of compound (Figure 9). The assay was performed
under a condition where the Topo I could nick supercoiled DNA at almost 100%. In this assay,
2
3

compound 20 protected supercoiled DNA from the effects of Topo I in a dose dependent manner,
and its inhibitory effect was comparable to that of CPT.
Figure 9. Effects of 20 on Topoisomerase I-mediated DNA Relaxation.
Supercoiled plasmid DNA was treated with Topo I in the presence of vehicle (DMSO),
compound 20 or CPT for 30 min at 37 °C. DNA was separated by 0.8% agarose gel
electrophoresis followed by staining with ethidium bromide.
2
1
2
3
.4. Summary of SAR findings (Figure 10)
) A ring-B 10π electron aromatic system was essential for potent antiproliferative activity.
) The ring-B aromaticity influenced the activity order: BT  Np > BF >> Ph
) A 3-hydroxy group (R = OH) was crucial for cell cycle arrest at G2/M, while a 5-hydroxy
4
group (R = OH) was important for arrest at G1/S.
3
For tubulin inhibitory effect (green box in Figure 10),
i) No substituent at C-3 was preferred rather than hydroxy, alkoxy, or benzoate.
ii) A 6-methoxy group was important.
iii) A certain size of 7-alkoxy was necessary.
For the arrest of cell cycle at G1/S (orange box in Figure 10),
2
4

i) A 5-methoxy group was suitable.
ii) A 7-isopropoxy group was better than a 7-methoxy moiety.
Figure 10. Structure-Activity Relationships of Antiproliferative Flavones.
2.5. Computer Modeling
The theoretical docking form of flavone 6 at the CS in the tubulin dimer was examined by
using 1SAO in the Protein Data Bank as a structure of the α/β-tubulin dimer. The modeling
indicated that the BT ring-B inserts on the β-tubulin side, and the chromone ring is located in the
gap between α- and β-tubulin with adequate room for a bulky isopropyl group (Figure 11).
Hydrogen bonds were observed between αSer178 and the 4-oxo group (2.78 Å) as well as
between αThr179 and the 5-hydroxy group (3.10 Å). Herein, the formation of a hydrogen bond
was defined when the distance between the heavy atoms of the hydrogen-bond donor and
2
5

acceptor was within 3.9 Å, which is an empirically proposed rule [38, 39]. In the docking
simulation, the single-bond rotation is allowed for the calculation of the docking poses and
docking scores. That is, note that the hydrogen atoms were not fixed at specific positions. Thus,
only the distance between the heavy atoms of the hydrogen-bond donor and acceptor was
adopted as the criterion for the formation of a hydrogen bond. The bicyclic ring-B system fit well
in the cavity, which might favor hydrophobic contacts. Since no aromatic amino acids were
observed around the BT bicycle, the compound is likely stabilized by hydrophobic interactions
with the alkyl groups of aliphatic amino acids, such as Leu and Ile.
In addition, the possible docking pose of flavonol 20 at the CPT binding site in Topo I was
studied by using 1T81 in the Protein Data Bank as a structure of Topo I. In the model (Figure
1
2), both BT and chromone rings are located almost parallel to the nucleobase of the DNA.
Hydrogen bonding was observed between the 3-hydroxy group of 20 and the guanidino group of
Arg364 (2.78 Å). The amino residues, including Thr718, Leu721, and Asn722, close to the
chromone moiety could prohibit the insertion of a bulky 5-alkoxy group, such as isopropoxy.
This observation may explain why the antiproliferative activity of flavonol 16 was 10-fold
weaker than that of 20 (Table 2). In addition, a pocket around C-7 is formed by Ala351, Asn352,
Ile427, Met428, Leu429, Asn430, Pro431, and Lys436, which creates an appropriate
hydrophobic pocket for a 7-isopropoxy group as found in the four most potent flavonols.
2
.6. In vitro metabolic stability
A compound’s metabolic stability is an important factor in drug discovery and development.
Flavone 6 was investigated for microsomal stability using human liver microsomes with the
reference compound propranolol, which has a moderate half-life in vivo of 3–6 h. Compound 6
2
6

showed better metabolic stability (t1/2 288.75 min) than propranolol (t1/2 177.69 min) (Figure 13,
Table 4). Its estimated half-life in vivo was 4.8 to 9.8 h.
Figure 11. Models for 6 Binding to Tubulin. (A) Docking model of 6 (gray skeleton with
oxygen in red and sulfur in yellow) with CS in α- (white) and β-tubulin (red) heterodimer of the
tubulin crystal structure (PDB: 1SA0). (B) Docking model of 6 into the CS. (a) Hydrogen bonds
calculated to be less than 3.9 Å between amino acid and compounds are represented by dashed
lines. Superimposition of docked compound 6 shows hydrogen bonds between αSer178 (gray
skeleton with oxygen in red and nitrogen in blue) and a ketone at C-4 as well as αThr179 and a
5
-hydroxy group. (b), (c) The residues near the ligand within a radius of 4 Å are indicated. b)
View of ligand from upper side. (c) View of ligand from ring plane side. No aromatic amino acid
is observed around bicyclic BT ring-B, indicating the compound can be stabilized by
hydrophobic interactions with alkyl functions of aliphatic amino acids.
2
7

Figure 12. Predicted Docking Models for Compound 20 to Topo I. Docking simulation of
compound 20 to Topo I (PDB: 1T8I) was carried out by the calculation program GOLD. The
docking score was 59.57. (A) The docking was set so that compound 20 entered within a radius
of 10 Å from the coordinates (22.517, −3.374, 27.845). Both BT and chromone rings are located
almost parallel to the nucleobase of DNA. (B) Amino residues around chromone moiety on
compound 20. The surface of amino residues is shown as mesh. The presence of Thr718,
Leu721, and Asn722 might prohibit the insertion of a bulky group such as isopropyl at C-5. A
hydrophobic pocket formed by Ala351, Asn352, Ile427, Met428, Leu429, Asn430, Pro431, and
Lys436 around C-7 is an appropriate pocket for a 7-isopropoxy group. (C) Nucleic acid bases
around compound 20. The surface of nucleic acid bases is shown as mesh. The planar chromone
and benzothiophene units are in the narrow pocket formed by nucleic acid bases. The bulky 3-
alkoxy group might disturb insertion. (D) Hydrogen bonding between 3-OH on compound 20
and guanidino group on Arg364.
2
8