Antitubulin effects of aminobenzothiophene-substituted triethylated chromones

Article abstract of DOI:10.1016/j.bmcl.2017.04.055

In the course of our continuing studies on the 2-(benzo[b]thiophene-3′-yl)-6,8,8-triethyldesmosdumotin B (TEDB-TB) series, we designed and synthesized nine amino-TEDB-TB derivatives to improve pharmaceutical properties, identify structure activity relatio

Full text of DOI:10.1016/j.bmcl.2017.04.055

Accepted Manuscript
Antitubulin effects of aminobenzothiophene-substituted triethylated chromones
Yukiko Kobayashi, Yohei Saito, Masuo Goto, Kyoko Nakagawa-Goto
PII:
S0960-894X(17)30430-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.055
BMCL 24908
Reference:
Bioorganic & Medicinal Chemistry Letters
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17 March 2017
13 April 2017
17 April 2017
Please cite this article as: Kobayashi, Y., Saito, Y., Goto, M., Nakagawa-Goto, K., Antitubulin effects of
aminobenzothiophene-substituted triethylated chromones, Bioorganic & Medicinal Chemistry Letters (2017), doi:
http://dx.doi.org/10.1016/j.bmcl.2017.04.055
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Antitubulin effects of aminobenzothiophene-substituted triethylated chromones
Yukiko Kobayashia, Yohei Saitoa, Masuo Gotob*, Kyoko Nakagawa-Gotoa,b*
a School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences,
Kanazawa University, Kanazawa, 920-1192. Japan
b UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
27599-7568, United States
*Corresponding authors. K.N.G. Tel: +81-76-264-6305, e-mail: kngoto@p.kanazawa-u.ac.jp,
M.G. Tel: +1-919-843-6325, e-mail: goto@med.unc.edu
1

ABSTRACT
In the course of our continuing studies on the 2-(benzo[b]thiophene-3ʹ-yl)-6,8,8-
triethyldesmosdumotin B (TEDB-TB) series, we designed and synthesized nine amino-TEDB-
TB derivatives to improve pharmaceutical properties, identify structure activity relationships,
and discover novel antitubulin agents. Among all newly synthesized amino-TEDB-TBs, the 5′-
and 6′-amino derivatives, 6 and 7, exhibited significant antiproliferative activity against five
human tumor cell lines, including an MDR subline overexpressing P-gp. The IC50 values of
0.50–1.01 µM were 3–6 times better than those of previously reported hydroxy-TEDB-TBs.
Compounds 6 and 7 inhibited tubulin polymerization, induced both depolymerization of
interphase microtubules and multiple spindle formations, and caused cell arrest at prometaphase.
Among all compounds, compound 7 scored best pharmaceutically with LogP 2.11 and
biologically with greater antiproliferative activity and induction of cell cycle arrest at
prometaphase.
Keywords: aminobenzothiophene, triethylated chromones, antiproliferative activity, antitubulin
2

Desmosdumotin B (1, Figure 1)1 was characterized as a unique flavonoid with a
trimethylated A-ring structure. Our previous research revealed the following interesting
bioactivities: a) 1 showed selective inhibition of MDR-tumor cell growth showing high collateral
sensitivity (CS),2,3 b) a triethylated analogue, TEDB-MDR (2), enhanced the selectivity more
than 20-times,4-6 c) its benzothiophene analogue, TEDB-TB (3), dramatically changed the
bioactivity profile, and also demonstrated as a potent tubulin polymerization inhibitor binding to
the colchicine site.7 d) the hydroxylated analogues of TEDB-TB (4 and 5) efficiently induced
cell cycle arrest at the G2/M phase with formation of immature multipolar spindles.8 Despite the
potency of TEDB-TB as an antitumor agent, its low solubility prevented subsequent in vivo
investigation. In fact, the Clog P and the topological polar surface area (tPSA) values of 3 are
5.27 and 63.6 Å2, respectively.9 The insertion of an OH group into 3, as found in 4 and 5, led to
increased polarity as well as hydrogen bond formation with specific residues on tubulin; however,
the inhibitory effects of such analogues against tumor cell growth were less than that of the
parent 3. Thus, our subsequent study of TEDB-TB series described herein was performed to
optimize 3 toward in vivo screening.
R = H
R = OH
R = NH
2
3

Clog P
tPSA
5.267
63.6
4.759
83.83
4.19
89.62
Figure 1. Summary of Desmosdumotin B Analogues
The polar amino group (NH2) is a classic bioisostere of the hydroxy group (OH). The
replacement of a hydroxy group by an amino group often results in a significant change in
molecular properties, not only the lipid-aqueous solubility and pKa of the compound, but also the
chemical reactivity. With Clog P and tPSA values of 4.19 and 89.6 Å2, respectively, amino
derivatives of TEDB-TB, such as 6 and 7 (Scheme 1), could have better drug-like properties than
3. Furthermore, various functional groups can be attached to an amine to produce alkylamines
and amides, such as 8–14, which might provide additional proton acceptor for hydrogen bonding
with the biological target.
4

Scheme 1. Syntheses of new TEDB-TB analogues. Reagents and conditions: (a) 16 or 17, 50% KOH,
EtOH, rt, overnight, 30% for 18, 65% for 19; (b) I2, cH2SO4 (cat.), DMSO, 90°C, 34% for 20, 21% for
21, 35% for 22, 9% for 23, 3% for 24; (c) BBr3, CH2Cl2, –10°C to 0°C, 70% for 6, 95% for 7; (d)
Ac2O, CH2Cl2, rt for 8 and 12, 98% for 8, 100% for 12; propionyl chloride, NaHCO3, EtOAc, H2O, rt
for 9 and 13, 94% for 9, 100% for 13; Boc2O, K2CO3, THF, H2O, 40°C for 10 and 14, 100% for 10,
77% for 14; Me2SO4, K2CO3, acetone, rt for 11, 41%.
The desired compounds were synthesized via our established method starting from
triethylated acetophloroglucinol 15.4 Claisen-Schmidt condensation of 15 with
aminobenzothiophene aldehydes 16 and 17, which were protected by t-butoxycarbonyl (BOC),
under basic conditions generated chalcones 18 and 19 in 30% and 65% yields, respectively. The
treatment of 5′-aminobenzothiophenylchalcone 18 with catalytic iodine and conc. H2SO4 in
DMSO afforded the related flavones 20 and 21, in which the BOC group was also removed.
Demethylation of 20 with BBr3 provided the desired 5′-NH2 derivative 6 together with NBOC
derivative 10 in 70% and 25% yields, respectively. The reaction temperature and time had to be
carefully controlled; otherwise, decomposition occurred. In the same manner, 6′-
aminobenzothiophenylchalcone 19 was converted to the related flavones 22 and 23. In this
reaction, a trace amount of 3-iodo derivative 24 was also isolated. BOC protected flavone 22 was
treated carefully with BBr3 to obtain 6′-NH2-TEDB-TB (7) in 95% yield. Amino-TEDB-TBs 6
and 7 were acylated with Ac2O and propionated with propionyl chloride to obtain acetylamides 8
and 12, as well as propylamides 9 and 13, respectively. tert-Butyl carbamates 10 and 14 were
also prepared by the treatment of 6 and 7 with Boc2O in the presence of K2CO3. Furthermore,
secondary amino-TEDB-TB 11 was prepared by adding a methyl group, the smallest alkyl group,
to primary amino-TEDB-TB 6 to determine its biological effect.
5

Scheme 2 shows the preparation of 5- and 6-NHBoc benzo[b]thiophene aldehyde, 16 and 17,
which were obtained from 3- and 4-nitrobenzenethiol (2510 and 2611) by known methods. The
introduction of a propynyl group to thiols 25 and 26, followed by oxidation of the resulting 27
and 28 with hydrogen peroxide provided the related propargyl sulfoxides, 29 and 30,
respectively.12 The tandem [2,3]/[3,3] thio-Claisen rearrangement followed by cyclization of 1-
nitro-4-(prop-2-yn-1-ylsulfinyl)benzene (30) under acidic conditions generated (5-
nitrobenzo[b]thiophen-3-yl)methanol (32). The nitro group was reduced to an amine and
protected with BOC to produce 36.13 The cyclization of 3-nitro 29 produced 31 and 33 in 48%
and 30% yields, respectively, depending on the cyclization position. (6-Nitrobenzo[b]thiophen-3-
yl)methanol (33) was converted to 37 in the same manner as 32 to 36. Alcohols 36 and 37 were
oxidized with 2-iodoxybenzoic acid to obtain aldehydes 16 and 17, respectively. (4-
Nitrobenzo[b]thiophen-3-yl)methanol (31) was also converted to the related 4-
aminobenzothiophene-3-carbaldehyde. However, the bulky NHBOC group at C-4 prevented the
subsequent condensation with 15, and 4-amino-TEDB-TB derivatives were not produced.
6

Scheme 2. Syntheses of aminobenzo[b]thiophene-3-carbaldehydes. Reagents and conditions: (a)
propargyl bromide, NaOH, TBAB, H2O/benzene, 0°C, 97% for 27, 99% for 28; (b) H2O2, HCOOH, 0°C
to rt, 94% for 29, 75% for 30; (c)1,4-dioxane, reflux then TsOH, H2O, reflux, 48% for 31 and 30% for 33,
78% for 32; (d) SnCl2, EtOH, 80°C, 78% for 34, 100% for 35; (e) Boc2O, K2CO3, THF/H2O, rt, 93% for
36, 90% for 37; (f) IBX, DMSO, rt, 95% for 16, 100% for 17.
All synthesized compounds were evaluated for antiproliferative activity against five human
tumor cell lines, MDA-MB-231 (triple-negative breast cancer), MCF-7 (estrogen receptor-
positive and HER2-negative breast adenocarcinoma), A549 (lung carcinoma), KB (originally
isolated from epidermoid carcinoma of the nasopharynx), and KB subline KB-VIN showing
MDR phenotype with overexpression of P-gp (Table 1). Unsubstituted aminobenzothiophene
derivatives 6 and 7 displayed the best antiproliferative activity among all compounds, with
higher potency than the related hydroxy derivatives, 4 and 5. Unfortunately, acylation or
alkylation of the amino substituent reduced the activity. The substituent group at C-6′ had a
significant effect on the activity; an increase in size induced a reduction in activity (e.g., 13 and
14 with N-propionyl and NHBOC, respectively) suggesting that the biological binding pocket
has limited space available around the ligand’s C-6′ position. In contrast, the C-5′ position was
comparatively tolerant. Even compound 10 with a NHBOC group displayed significant
antiproliferative activity against all tested tumor cell lines, except MDA-MB-231. Compound 10
was even more active than N-propionyl derivative 9. Interestingly, this phenomenon was totally
different in the case of 7-methoxy flavones, 20–24. NBOC derivatives 20 and 22 showed potent
activity (IC50 5.02–9.15 µM), while NH2 derivatives 21 and 23 exhibited only weak inhibition of
tested tumor cell growth. It should be noted that all active compounds overcame the development
of MDR, since the compounds showed comparable or even greater potency against KB-VIN (P-
7

gp overexpressing KB subline) compared with KB. Especially, 6′-acetamide 12 was three times
more potent against KB-VIN than KB.
Table 1. Antiproliferative effects of amino-TEDB-TBs against human tumor cell lines.
Cell lines/IC50 (µM) a
MDA-MB-231 b
MCF-7
A549
KB
KB-VIN
5′-substituted target compounds
2.21
0.72
6.44
1.01
2.46
0.79
2.46
0.85
1.76
0.63
4
6
19.39
26.51
32.50
24.36
30.03
32.24
9.67
8.33
15.97
9.16
29.11
23.47
7.49
12.08
18.34
8.16
8
9
10
11
20.18
18.59
18.04
5.35
6′-substituted target compounds
1.55
0.83
7.94
>40
>40
1.81
0.97
26.34
>40
1.66
0.73
9.71
31.70
>40
2.98
0.57
21.90
>40
1.51
0.50
7.13
>40
>40
5
7
12
13
14
>40
>40
7-methoxy flavone intermediates
34.37
9.15
39.22
6.52
22.77
23.32
8.18
30.21
6.01
16.03
25.92
6.80
25.06
5.62
8.06
21.28
7.20
26.31
5.13
9.89
16.93
6.07
25.55
5.02
13.22
21
20
23
22
24
Chalcone intermediates
7.48
6.07
9.63
7.01
7.42
11.46
5.10
5.04
6.85
5.88
5.77
5.90
5.45
5.63
2035
18
19
PXL (nM)
aAntiproliferative activity as IC50 values, the concentration of compound that caused 50%
reduction relative to unreacted cells using the sulforhodamine B assay, calculated from at least
three independent experiments for each cell line. Paclitaxel (PXL) was used as an experimental
control.
bTriple-negative breast cancer (MDA-MB-231), breast adenocarcinoma (MCF-7), lung
carcinoma (A549), originally isolated as human epidermoid carcinoma of the nasopharynx (KB),
multidrug-resistant KB subline overexpressing P-glycoprotein (KB-VIN).
8

Induction of cell cycle arrest in G2/M phase is one of the typical effects of MTAs on tumor
cells. Our previous studies of TEDB-TBs7,9 suggested that aminobenzothiophene TEDB-TBs
would also target the tubulin. Thus, the effects of the newly synthesized compounds, 6 and 7, on
cell cycle progression were investigated by flow cytometry (Figure 2). Compound 6 strongly
induced cell cycle arrest at G2/M at the 10-fold IC50 concentration (7.2 µM) with accumulation
of sub-G1 cells, while compound 7 showed a similar effect at the IC50 concentration (0.83 µM).
These observations suggested that, compared with 6, compound 7 was more selective to tubulin.
Compound 6 may be bifunctional possessing both antitubulin effect and apoptosis induction
independently.
Figure 2. Induction of cell cycle arrest at G2/M. MDA-MB-231 cells were seeded in a 12-well plate (8
× 104 cells/well) 24 h prior to treatment with compounds 6 and 7 for 24 h at the same (1× IC50) or 10-
fold (10× IC50) concentrations of their IC50. Harvested and fixed cells were stained with propidium
iodide (PI) subjected to flow cytometry. DMSO or 0.2 µM CA-4 was used for negative control or
tubulin polymerization inhibitor arresting cells at G2/M, respectively.
To study other effects of these compounds on microtubules and spindle formation, as well as
to determine their point of impact on the cell cycle, compound-treated MDA-MB-231 cells were
9

labeled with antibodies to α-tubulin and serine 10-phosphorylated histone H3 (p-H3), and 4ʹ,6-
diamidino-2-phenylindole (DAPI) was used for DNA (Figure 3). Most of the cells treated with 6
at 0.72 µM (1 × IC50) looked normal with normal microtubules in interphase cells, as well as
bipolar spindle formation in metaphase cells. At an increased dose of 7.2 µM (10 × IC50) clearly
depolymerized microtubules and multiple spindles were formed abnormally in the p-H3-positive
prometaphase cells. Similar effects were seen in the cells treated with 7 at 0.83 µM (1 × IC50),
suggesting that compound 7 is more selective than 6 against tubulin. The phenotype of
microtubule depolymerization in interphase was similar to that of combretastatin A-4 (CA-4).
However, induction of multiple spindle formations in prometaphase was distinctive to 6 and 7,
suggesting that these compounds might target tubulin via a different mode of binding.
10

Figure 3. Effects of compounds on microtubules and spindle formation. MDA-MB-231 cells
were seeded in a 8-well chamber slide (2.4 × 104 cells/well) 24 h prior to treatment with
compounds for 24 h at the same (1× IC50) or 10-fold (10× IC50) concentrations of their IC50.
DMSO or 0.2 µM CA-4 was used for negative control or tubulin polymerization inhibitor,
respectively. Fixed cells were labeled with antibodies to α-tubulin (green) and serine 10-
phosphorylated histone H3 (p-H3, red) as a chromosome condensation marker, DAPI was used
for DNA (blue). Confocal images were stacked and merged. Cells expressing polymerized
interphase microtubules or bipolar spindles are indicated with white arrow or white arrowheads,
respectively. Typical cells with multiple spindles and condensed chromosomes are shown by
yellow arrowheads. Bar, 25 µm.
11

Herein, we designed and synthesized nine 5′- and 6′-amino-TEDB-TBs with improvement of
pharmaceutical properties, and evaluated their antiproliferative activities against human tumor
cell lines including MDR subline. All of compounds were effective against MDR subline.
Compounds 6 and 7 showed improved antiproliferative activity by arresting cell cycle at G2/M.
They induced depolymerization of microtubules and multiple spindle formations in interphase
and prometaphase cells, respectively. These observations suggest that compounds 6 and 7
directly target tubulin. Among our novel aminobenzothiophene chromones, compound 7 scored
best pharmaceutically, i.e., improved LogP, as well as biologically, i.e., enhanced
antiproliferative activity and induction of cell cycle arrest at prometaphase.
Acknowledgments
We wish to thank Microscopy Service Laboratory (UNC-CH) for their expertise in the confocal
microscope. In addition, we appreciate critical comments, suggestions, and editing on the
manuscript by Dr. Susan L. Morris-Natschke (UNC-CH). This study was supported by Grant-in-
Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT
KAKENHI, Japan), awarded to K.N.G. (Grant Numbers 25293024 & 25670054) as well as by
Terumo Life Science Foundation awarded to K.N.G. This work was also supported in part by the
Eshelman Institute for Innovation, Chapel Hill, North Carolina awarded to M.G.
Supplementary data
Supplementary data associated with this article can be found, in the online version,
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Graphic Abstract
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