Antimitotic and vascular disrupting agents: 2-Hydroxy-3,4,5- trimethoxybenzophenones

Article abstract of DOI:10.1016/j.ejmech.2014.02.061

2-Hydroxy-3,4,5-trimethoxybenzophenones (8-16) manifest pseudo-ring formation involving intramolecular hydrogen bonding of the 2-OH and the carbonyl group. Among the synthetic products described in this report, (3-hydroxy-4-methoxyphenyl)(2-hydroxy-3,4,5-trimethoxyphenyl)-methanone (14) and (3-amino-4-methoxyphenyl)(2-hydroxy-3,4,5-trimethoxy-phenyl)methanone (16) exhibit significant antiproliferative activity against KB cells with IC ₅₀ values of 11.1 and 11.3 nM, respectively. These two compounds also displayed tubulin affinity comparable to that of combretastatin A-4. In studies with human umbilical vein endothelial cells, compounds 14 and 16 revealed concentration-dependent vascular-disrupting properties. The results support the rationale of the pseudo-ring concept and suggest further investigation of A-ring modification in these benzophenones.

Full text of DOI:10.1016/j.ejmech.2014.02.061

European Journal of Medicinal Chemistry 77 (2014) 306e314
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimitotic and vascular disrupting agents: 2-Hydroxy-3,4,5-
trimethoxybenzophenones
Chih-Yi Chang a, Hsun-Yueh Chuang a, Hsueh-Yun Lee a, Teng-Kuang Yeh e,
,
d
,
a,c,
**
Ching-Chuan Kuo e, Chi-Yen Chang b, Jang-Yang Chang b , Jing-Ping Liou
*
a School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan, ROC
b National Institute of Cancer Research, National Health Research Institutes, Tainan 70456, Taiwan, ROC
c School of Pharmacy, National Defense Medical Center, 161 Minchuan East Road, Section 6, Taipei 114, Taiwan, ROC
d Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC
e Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 350, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Hydroxy-3,4,5-trimethoxybenzophenones (8e16) manifest pseudo-ring formation involving intra-
molecular hydrogen bonding of the 2-OH and the carbonyl group. Among the synthetic products
described in this report, (3-hydroxy-4-methoxyphenyl)(2-hydroxy-3,4,5-trimethoxyphenyl)-methanone
(14) and (3-amino-4-methoxyphenyl)(2-hydroxy-3,4,5-trimethoxy-phenyl)methanone (16) exhibit sig-
nificant antiproliferative activity against KB cells with IC50 values of 11.1 and 11.3 nM, respectively. These
two compounds also displayed tubulin affinity comparable to that of combretastatin A-4. In studies with
human umbilical vein endothelial cells, compounds 14 and 16 revealed concentration-dependent
vascular-disrupting properties. The results support the rationale of the pseudo-ring concept and sug-
gest further investigation of A-ring modification in these benzophenones.
Received 28 November 2013
Received in revised form
26 February 2014
Accepted 28 February 2014
Available online 1 March 2014
Keywords:
Vascular-disrupting agents
Antiproliferative
CA-4
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
selectively cytotoxic VDAs [4,5]. A variety of CA-4 derivatives such
as combretastatin A-4 phosphate (CA-4P, fosbretabulin, 2) and AVE-
Tumor vasculature associated with abnormal properties such as
poor perfusion, hyperpermeability and disorganization is a basis for
tumor proliferation and survival and targeting tumor vasculature
has become an important approach in the development of anti-
cancer therapy [1,2]. Among antivascular agents, vascular-
disrupting agents (VDAs) with the capability of destroying estab-
lished tumor vasculature causing widespread tumor necrosis and
regression are particularly different from other anti-angiogenic
strategies and have recently shown potential in clinical trials [3].
Tubulin-depolymerizing agents structurally related to com-
bretastatin A-4 (CA-4, 1) have exhibited promising results for
cancer treatment. Recent reports have demonstrated that tubulin-
binding agents not only inhibit microtubule polymerization at
mitosis with consequent apoptosis in cancer cells but also over-
come the problem of multi-drug resistance (MDR) in cancer
chemotherapy. Moreover, they reveal the novel characteristic of
8062 (ombrabulin, 3) (Fig. 1) are currently in clinical trials [6]. The
clinical data however indicated unexpected systemic toxicity
including cardiotoxicity, neurotoxicity, dose-limiting toxicities, and
other undesirable pharmaceutical properties which impeded the
development of microtubule-targeting agents for the treatment of
cancer [7]. In the development of small molecules as anti-
proliferative agents based on the optimization of CA-4, benzophe-
none-contained
analogs
including
phenstatin
(4),
hydroxyphenstatin (5), and compound 6 exhibit potent cytotox-
icity. Furthermore, they displayed considerable pharmacological
features and convenient synthetic access in which construction of
Z-stilbene type of CA-4 derivatives was avoided [8e11].
Numerous studies have demonstrated that the trimethox-
ybenzene A ring of the combretastatins is pivotal for cytotoxic ac-
tivity. Recently different trimethoxyindoles have been developed as
surrogates of this trimethoxybenzene A-ring. For instance, in our
previous
studies
[12e14],
N-aryl-,
N-benzyl-4,5,6-
trimethoxyindoles, and N-aryl-5,6,7-trimethoxyindoles were
found to be potent antiproliferative compounds and in addition to
N1 substitution, C2 and C3 replacements of trimethoxyindoles
* Corresponding author. National Institute of Cancer Research, National Health
Research Institutes, Tainan 70456, Taiwan, ROC.
** Corresponding author. School of Pharmacy, College of Pharmacy, Taipei Medical
University, Taipei 11031, Taiwan, ROC.
were
described
[15,16].
Furthermore,
2-amino-3,4,5-
trimethoxybenzophenones forming
a
pseudo-ring exhibited
E-mail addresses: jychang@nhri.org.tw (J.-Y. Chang), jpl@tmu.edu.tw (J.-P. Liou).
http://dx.doi.org/10.1016/j.ejmech.2014.02.061
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
307
achieved by treating compound 18 with hexamethylenetetramine
and trifluoroacetic acid under Duff conditions [22] and then with
tetrabutylammonium tribromide in CH2Cl2 [23]. The hydroxyl
groups of 19 and 20 were protected by the reaction with tert-
butyldimethylsilyl chloride (TBDMS-Cl) and methyl chloromethyl
ether to afford compounds 21 and 22, respectively.
The synthesis of 2-hydroxy-3,4,5-trimethoxybenzoyl de-
rivatives is described in Schemes 2 and 3. Compound 23aeb was
synthesized by reacting compound 21 with the appropriate
substituted Grignard reagent followed by oxidation with pyr-
idinium dichromate (PDC). Removal of the TBMDS group was
accomplished with tetrabutylammonium fluoride (TBAF) affording
compounds 8 and 14. Compound 22 was sequentially treated with
n-BuLi, various benzaldehydes, and PDC to afford the correspond-
ing TBDMS-protected benzophenones 24a, 24b, 24e, and 24f. Sur-
prisingly, compound 10 was obtained simultaneously during the
PDC oxidation. Reduction of the nitro group of compounds 24c and
24f by iron powder yielded compounds 24d and 24g. Finally, the
deprotection of MOM group under acidic conditions provided the
desired compounds 9e16. In compounds such as 8e16, NMR sig-
nals corresponding to C2eOH are observed at downfield regions (
11e12 ppm). This is attributed to the formation of the intra-
d
Fig. 1. Potential antitubulin agents.
molecular hydrogen bond shown in Fig. 2.
marked antitubulin and antiproliferation activity [17]. These
interesting results revealed that an extra ring fused with C1eC2
bond of 3,4,5-trimethoxybenzene is an acceptable form of A-ring
modification. In an attempt to maintain both fused ring charac-
teristics and the potency of the benzophenone structure, we have
2.2. Biological evaluation
2.2.1. In vitro cell growth inhibitory activity
Reference compounds, all synthesized compounds (8e16), and
CA4 and colchicine were evaluated for cytotoxic activity against
three human cancer cell lines, cervical carcinoma KB cells, stomach
carcinoma MKN45 cells and colorectal carcinoma HT29 cells
(Table 1).
designed and synthesized
a
series of 2-hydroxy-3,4,5-
trimethoxybenzophenones on the basis of pseudo-ring approach
[18e20] and the relevant biological assays are reported in this pa-
per (Fig. 2).
HT29 cells are not sensitive to the compounds tested, with the
exception of compound 16. Comparison of compounds 8, 9 and 10
revealed a substitution effect at C4 of the B-ring. Compound 9 with
an N-methylamino group, showed the most potent activity against
KB and MKN45 cells with IC50 values of 30.9 and 43.5 nM,
respectively. Conversion of the 4-NHMe to 4-OMe (8) resulted in a
5-fold decrease of potency. Addition of an extra methyl group, as in
compound 10, led to a 10-fold decrease in cytotoxicity. In the B-ring
modification, additional eF, eNH2, and eOH substituents accom-
panied by a 4-OMe were found to improve biological activity and
the effect of eF, eNH2, and eOH at the C2 or C3 position was
therefore evaluated. Compounds with additional substitution at C2
or C3, with the exception of compound 16, showed considerable
loss of inhibitory activity with MKN45 cells. The comparison of 8,11,
and 12 demonstrated that a 2-NH2 group was associated with a 2-
fold increase of inhibitory activity against KB cells but a 2-OH
substituent has no influence on the potency. Compound 15
bearing a nitro group displayed a dramatic loss of cytotoxicity
indicating the electron-withdrawing NO2 group is disfavored. A
similar phenomenon is observed in compound 13 with a fluorine
atom at C3 position. Notably, the addition of a 3-OH (14) or a 3-NH2
(16) led to a 13-fold improvement of activity, compared with 8,
against KB cells with IC50 values of 11.1 and 11.3 nM, respectively.
Comparison of compounds 11 and 12 with compounds 14 and 16
revealed that the 3-OH and 3-NH2 substituents respectively pro-
vided a 13- and 6-fold enhancement of antiproliferative activity
against KB cells. Since 30-hydroxy-40-methoxyphenyl (14) and 30-
amino-40-methoxyphenyl substitution (16) produces the same B-
ring as in CA-4 and AVE-8062, this result indicates that such
pseudo-ring methodology, which maintains the conformation of A-
ring/Z-double bond part of CA-4 is feasible. In general, this group of
compounds displays no cytotoxicity against HT29 cells and the
introduction of additional groups at C2 or C3 is detrimental to
2. Results and discussion
2.1. Chemistry
The synthetic strategy for compounds 21 and 22 is shown in
Scheme 1. Compound 18 was synthesized by a BaeyereVilliger re-
action in the presence of H2O2 [21]. Preparation of 19 and 20 was
Ring expansion
from bond C1-C2
OMe
MeO
MeO
2
N
N
MeO
1
R
R
R
MeO
OMe
MeO
MeO
MeO
Pseudo-ring
H
O
O
MeO
MeO
R
OMe
8: 4'-OCH3
13: 3'-F, 4'-OCH3
9: 4'-NHCH3
14: 3'-OH, 4'-OCH3
15: 3'-NO2,4'-OCH3
16: 3'-NH2, 4'-OCH3
10: 4'-N(CH3)2
11: 2'-OH, 4'-OCH3
12: 2'-NH2, 4'-OCH3
Fig. 2. Ring-opening strategy for novel anticancer agents.

308
C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
Scheme 1. Synthetic approaches to compounds 21 and 22. Reagents and conditions: (a) 30% H2O2, H2SO4, MeOH, rt; (b) hexamethylenetetramine, TFA, reflux; (c) tetrabuty-
lammonium tribromide, DCM, rt; (d) TBDMSCl, DIPEA, DCM, 0 ꢀC to r.t; (e) MOMCl, DIPEA, DCM, 0 ꢀC to r.t.
Scheme 2. Synthetic approaches to compounds 8 and 14. Reagents and conditions: (a) substituted phenyl magnesium bromide, THF, 0 ꢀC to r.t; (b) PDC, DCM, r.t; (c) TBAF, THF, 0 ꢀ
C
to rt.
MKN45 inhibitory activity, but these changes, together with
retention of the B-rings in CA-4 and AVE-8062 leads to remarkable
improvement of antiproliferative activity. Compounds 14 and 16
were further evaluated for antiproliferative activity against a vari-
ety of resistant cell lines and the results are shown in Table 2.
Despite the high level of expression of drug-resistant efflux protein
(MDR/P-gp or MRP) in KB-Vin10, KB-S15, and KB-7D cells, com-
pounds 14 and 16 show similar efficacy in parental cells and in
these resistant cell lines.
4 were evaluated in the tubulin inhibition assay and for colchicine
binding activity (Table 3). Compounds 14 and 16 exhibited com-
parable antitubulin activity to that of colchicine but with IC50 values
of 4.1 and 3.7 mM, respectively, are slightly less potent than CA-4. In
the [3H]colchicine binding assay, compound 16 has a similar affinity
as CA-4 for the colchicine binding site. The interaction between
compound 14 and tubulin is slightly weaker than is shown by
compound 16, and this is consistent with the results of the tubulin
inhibition assay. Compounds 14 and 16 inhibited polymerization of
pure MAP-rich tubulins in a concentration-dependent manner
(Fig. 3.) and were identified as competitive inhibitors targeting the
colchicine binding site in tubulin (Fig. 4.). Taken together, the
marked cytotoxicity of compounds 14 and 16 is associated with the
inhibition of microtubules, and supports the pharmacological
mechanism of pseudo-ring products.
2.2.2. Inhibition of tubulin polymerization and colchicine binding
activity
To investigate whether the pseudo-ring approach affects the
original interaction mode with the microtubule system, com-
pounds 14 and 16 and the reference compounds colchicine and CA-
Scheme 3. Synthetic approaches to compounds 9e16. Reagents and conditions: (a) n-BuLi, THF, ꢁ78 ꢀC to 0 ꢀC; (b) substituted benzaldehyde, THF, 0 ꢀC; (c) PDC, DCM, r.t; (d) Fe,
NH4Cl, IPA-H2O, reflux; (e) 1 N HCl, MeOH, rt.

C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
309
Table 1
Table 3
Antiproliferative activity of compounds 8e16 and reference compounds.
Inhibition of tubulin polymerization and colchicine binding inhibition by com-
pounds 14, 16, and reference compounds.
Compd
Cell type (IC50 nM ꢄ SDa)
Compound
Tubulina IC50 ꢄ SD (
m
M)
Colchicine bindingb (%)
KB
MKN45
HT29
1
m
M
5 mM
8
9
10
11
12
13
14
15
145.6 ꢄ 48
30.9 ꢄ 3.8
517.3 ꢄ 59.3
146.3 ꢄ 5.9
70.3 ꢄ 10.2
183 ꢄ 40.6
11.1 ꢄ 0.8
3667 ꢄ 190
11.3 ꢄ 1.1
2.5 ꢄ 0.8
235 ꢄ 0.8
43.5 ꢄ 1.2
412 ꢄ 76.6
484 ꢄ 128
447 ꢄ 11.3
1130 ꢄ 107
493 ꢄ 35.1
＞5000
＞5000
206 ꢄ 30.7
1093 ꢄ 109
＞5000
14
16
4.1
3.7
4.9
1.9
79.3 ꢄ 1.2
81.1 ꢄ 2.1
32.2 ꢄ 3.3
86.2 ꢄ 0.6
88.2 ꢄ 1.4
91.7 ꢄ 1.5
72.4 ꢄ 0.6
93.5 ꢄ 0.3
colchicine
CA4
3301 ꢄ 116
＞5000
a
Inhibition of tubulin polymerization.
Inhibition of [3H]colchicine binding. Tubulin was at 1
2500
b
m
M; [3H]colchicine was at
1312 ꢄ 560
51.8 ꢄ 15.6
87.2 ꢄ 27.8
12.8 ꢄ 1.3
5
m
M.
16
CA4
colchicine
13.1 ꢄ 2.3
3.2 ꢄ 1.5
12.4 ꢄ 2.5
14.9 ꢄ 2.5
studies of HUVECs tube formation assay, compounds 14 and 16
have been recognized as vascular disrupting agents with
concentration-dependent suppression activity.
a
SD: standard deviation. All experiments were independently performed at least
three times.
In summary, this paper not only widens the exploration of A-
ring modification but also identifies a series of 2-hydroxy-3,4,5-
trimethoxybenzophenones as novel promising anti-mitotic and
anti-vascular agents.
2.2.3. In vitro vascular disrupting effect
Recent reports indicate that antitubulin agents also exhibit a
vascular disrupting effect, causing destruction of tumor vascula-
ture. Compounds 14 and 16 were explored for the VDA property
using a human umbilical vein endothelial cells (HUVECs) culture
assay. HUVECs on Matrigel were allowed to form capillary tubes in
the presence of VEGF (20 ng/mL) and then were exposed to
different concentrations of selected compounds. The results shown
in Fig. 5A indicated that compounds 14 and 16 both reveal a
concentration-dependent inhibition of the formation of capillary
tubes. In addition, compounds 14 and 16 with the concentration
value of 500 nM entirely destroyed established tubes without
altering cell viability (Fig. 5B.).
4. Experimental section
4.1. Chemistry
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra
were obtained with Bruker Fourier 300 and DRX-500 spectrome-
ters, with chemical shift reported in parts per million (ppm, d)
downfield from TMS as an internal standard. High-resolution mass
spectra (HRMS) were recorded with a FINNIGAN MAT 95S Mass
Spectrometer. Purity of the final compounds was determined using
a Hitachi 2000 series HPLC system using C-18 column (Agilent
3. Conclusion
ZORBAX Eclipse XDB-C18 5
m
m, 4.6 mm ꢂ 150 mm) with the sol-
This paper studies the introduction of C2eOH of the A-ring to
form intramolecular H-bond-containing benzophenone de-
rivatives. The existence of a pseudo-ring structure is supported by a
distinct downfield signal in the NMR spectra of the compounds
vent system (elution conditions: mobile phase A consisting of
acetonitrile; mobile phase B consisting of water containing 0.1%
formic acid þ 10 mmol NH4OAc) and was found to be ꢃ95%. Flash
column chromatography was done using silica gel (Merck Kieselgel
60, no. 9385, 230ꢁ400 mesh ASTM). All reactions were carried out
under an atmosphere of dry nitrogen.
derived from trimethoxyindole.
A series of 2-hydroxy-3,4,5-
trimethoxybenzophenones were synthesized (8e16) and were
found generally to exhibit superior activity against KB cells than
against MKN45 and HT29 cells. In addition, C3eNH2 and C3eOH
substituents on the B-ring contributed to an improvement of
cytotoxicity. This result is similar to those obtained with CA-4 and
AVE-8062, and supports the pseudo-ring rationale. Among the
synthetic compounds, compounds 14 and 16 showed marked ac-
tivity against KB cells with IC50 values of 11.1 and 11.3 nM,
respectively. These two compounds were identified as inhibitors of
tubulin polymerization which bind at the colchicine binding site. In
4.1.1. 2,3,4-Trimethoxyphenol (18)
A solution of 2,3,4-trimethoxybenzaldehyde (20 g, 100.9 mmol)
and sulfuric acid (2 mL) in MeOH (200 mL) was stirred at 0 ꢀC. To
the previous solution, 30% H2O2 (13.6 mL, 131.2 mmol) was added
dropwise at 0 ꢀC and then was stirred at room temperature for
30 min. The solution was evaporated and extracted with EtOAc. The
crude product was purified through chromatography to provide a
transparent oil (14.8 g, 79%; EtOAc/hexane, Rf ¼ 0.25). 1H NMR
(300 MHz, CDCl3):
d 3.79 (s, 3H), 3.88 (s, 3H), 3.94 (s, 3H), 5.54 (s,
Table 2
1H), 6.54 (d, 1H, J ¼ 9.0 Hz), 6.62 (d, 1H, J ¼ 9.0 Hz).
Growth inhibition of compounds 14, 16 and reference compounds against drug-
resistant cell lines.
Compd
(IC50 nM ꢄ SDa)
4.1.2. 2-Hydroxy-3,4,5-trimethoxybenzaldehyde (19)
Parental cells Multidrug resistant cells
A solution of 2,3,4-trimethoxyphenol (14.7 g, 80 mmol) and
hexamethylenetetramine (11.4 g, 80 mmol) in trifluoroacetic acid
(80 mL) was heated under reflux for 4 h. The stirred solution was
cooled to room temperature and ice was added to the cooled
mixture. Subsequently, the mixture was extracted with EtOAc and
the organic layer was evaporated. The product was purified through
chromatography to provide a solid (6.9 g, 41%; EtOAc/hexane,
KB
KB-VIN10
(P-gp170/MDR[) (P-gp170/MDR[)
KB-S15
KB-7D (MRP[)
14
16
11.1 ꢄ 0.8
13.8 ꢄ 3.3
8.0 ꢄ 4.1
10 ꢄ 1.0
7.4 ꢄ 3.3
11.7 ꢄ 2.3
7.5 ꢄ 4.3
11.3 ꢄ 1.1
0.9 ꢄ 0.4
5.1 ꢄ 2.3
Vincristine
Paclitaxel
VP-16
96.4 ꢄ 8.5
2.1 ꢄ 0.6
1.5 ꢄ 0.6
14,800 ꢄ 890
135 ꢄ 7.8
3500 ꢄ 400
8.4 ꢄ 0.3
1500 ꢄ 500 26,400 ꢄ 2800
46,100 ꢄ 5400
Rf ¼ 0.2). 1H NMR (500 MHz, CDCl3):
d 3.81 (s, 3H), 3.89 (s, 3H), 3.99
(s, 3H), 6.73 (s, 1H), 9.72 (s, 1H), 10.93 (s, 1H).
a
SD: standard deviation. All experiments were independently performed at least
three times.

310
C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
Fig. 3. Compounds 14 and 16 depolymerize microtubule in vitro by binding to the colchicine-binding site. Effect of compounds 14 and 16 on in vitro tubulin polymerization. MAP-
rich tubulins were incubated at 37 ꢀC in the absence [dimethyl sulfoxide (DMSO) control] or presence of drugs (colchicine or serial concentrations of compounds 14 and 16).
Absorbance at 350 nm was measured every 30 s for 30 min and is presented as the polymerized microtubule increases.
4.1.3. 6-Bromo-2,3,4-trimethoxyphenol (20)
(0.4 mL, 4.6 mmol) at 0 ꢀC. Subsequently, the resulting mixture was
stirred at room temperature for 4 h. The solvent was evaporated
and the crude product was purified through chromatography to
afford the product (0.93 g, 80%; EtOAc/hexane ¼ 1:6, Rf ¼ 0.6). 1H
To a solution of 2,3,4-trimethoxyphenol (2 g, 10.9 mmol) in
CH2Cl2 (60 mL)-MeOH (40 mL) was added tetrabutylammonium
tribromide (5.6 g, 11.4 mmol) in portions at room temperature.
Subsequently, the resulting mixture was stirred at room tempera-
ture for 2 h. The solvent was evaporated and the crude product was
purified through chromatography to afford the product (2.3 g, 81%;
NMR (500 MHz, CDCl3): d 3.63 (s, 3H), 3.80 (s, 3H), 3.85 (s, 3H), 3.88
(s, 3H), 5.09 (s, 2H), 6.82 (s, 1H).
EtOAc/hexane ¼ 1:6, Rf ¼ 0.3). 1H NMR (300 MHz, CDCl3):
d 3.80 (s,
3H), 3.87 (s, 3H), 3.96 (s, 3H), 5.65 (brs, 1H), 6.77 (s, 1H).
4.1.6. General procedure A for synthesis of 2-(tert-
butyldimethylsilyloxy)-3,4,5-trimethoxy- benzophenone (23a and
23b)
4.1.4. 2-((Tert-Butyldimethylsilyl)oxy)-3,4,5-
trimethoxybenzaldehyde (21)
A solution of the substituted benzene magnesium bromide was
prepared by achieving the reaction of substituted bromobenzene
(1.2 equiv), magnesium (1.3 equiv) and a catalytic amount of iodine
in THF (0.5 M) under reflux condition for 4 h. The solution was
cooled to room temperature and then was added to a solution of 2-
((tert-butyldimethylsilyl)oxy)-3,4,5-trimethoxybenzaldehyde
(1 equiv) in THF (0.5 M) at 0 ꢀC. The solution was stirred at 0 ꢀC for
1 h and then was quenched with water. The mixture was extracted
with EtOAc and the organic layer was collected. The crude product
was purified through chromatography (EtOAc/hexane) to afford the
substituted benzhydrol. To a solution of the substituted benzhydrol
To
a solution of 2-hydroxy-3,4,5-trimethoxybenzaldehyde
(6.1 g, 28.8 mmol) and N,N-diisopropylethylamine (15.4 mL,
86.3 mmol) in CH2Cl2 (56 mL) was added dropwise tert-butyldi-
methylsilyl chloride (5.3 g, 34.5 mmol) at 0 ꢀC. Subsequently, the
resulting mixture was stirred at room temperature for 6 h. The
solvent was evaporated and the crude product was purified
through chromatography to afford the product (7.59 g, 81%; EtOAc/
hexane ¼ 95:5, Rf ¼ 0.2). 1H NMR (300 MHz, CDCl3):
d 0.20 (s, 6H),
1.00 (s, 9H), 3.81 (s, 3H), 3.85 (s, 3H), 3.97 (s, 3H), 7.07 (s, 1H), 10.33
(s, 1H).
ꢀ
in CH2Cl2 (0.2 M), pyridinium dichromate (1.5 equiv) and 4A mo-
4.1.5. 1-Bromo-3,4,5-trimethoxy-2-(methoxymethoxy)benzene (22)
lecular sieves (0.3 g) was added at room temperature. The resulting
mixture was stirred at room temperature for 12 h. The mixture was
diluted with EtOAc and filtered through a pad of Celite. The organic
layer was collected and the crude product was purified by flash
To
a solution of 6-bromo-2,3,4-trimethoxyphenol (1 g,
3.8 mmol) and N,N-diisopropylethylamine (2 mL, 10.2 mmol) in
CH2Cl2 (8 mL) was added dropwise methyl chloromethyl ether
Fig. 4. The binding site of compounds 14 and 16 on tubulin was examined by using a competition-binding scintillation proximity assay, as described under Materials and Methods.
The 100% binding represents 3H-labeled ligand binding of the control group without tested compounds. The double-reciprocal plot indicates that compounds 14 and 16 are
competitive inhibitors for the colchicine-binding site on tubulin. Each data point represents the mean ꢄ S.D.

C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
311
Fig. 5. Investigation of the vascular disrupting activity of compounds 14 and 16. HUVECs were plated on Matrigel and allowed to form capillary tubes in the presence of VEGF
(20 ng/mL) followed by exposure to different concentrations of test compound. Cultures were photographed, and the number of capillary tube networks was determined by
counting under a microscope (original magnification of 100ꢂ). Data reflect the mean number of capillary tube networks to vehicle control group (DMSO) ꢄ the standard deviation
(SD) from three separate experiments. In the meantime, cell viability was also determined in parallel.
chromatography (EtOAc/hexane) to yield the substituted
benzophenone.
23a (200 mg, 0.47 mmol) according to general procedure B (147 mg,
98%). 1H NMR (500 MHz, CDCl3):
3.68 (s, 3H), 3.84 (s, 3H), 3.92
d
(s, 3H), 4.00 (s, 3H), 6.85 (s, 1H), 6.95 (d, 2H, J ¼ 8.5 Hz), 7.65 (d, 2H,
J ¼ 9.0 Hz), 12.09 (s, 1H). 13C NMR (75 MHz, CDCl3):
d 55.2, 56.3,
4.1.6.1. (2-((Tert-Butyldimethylsilyl)oxy)-3,4,5-trimethoxyphenyl)(4-
methoxyphenyl)methanone (23a). The title compound was ob-
tained from 2-((tert-butyldimethylsilyl)oxy)-3,4,5-trimethoxy-
benzaldehyde (1.7 g, 5.20 mmol) according to general procedure A
60.8, 61.0, 110.2, 113.4, 113.6, 130.2, 131.2, 141.3, 144.5, 149.1, 153.1,
162.6,198.7. HRMS-ESI calcd. for C17H19O6 [M þ H]þ 319.1182, found
319.1181.
(1.17 g, 52%). 1H NMR (300 MHz, CDCl3):
d 0.03 (s, 6H), 0.64 (s, 9H),
3.81 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 3.93 (s, 3H), 6.66 (s, 1H), 6.88e
6.92 (m, 2H), 7.79e7.83 (m, 2H).
4.1.7.2. (2-Hydroxy-3,4,5-trimethoxyphenyl)(3-hydroxy-4-
methoxyphenyl)methanone (14). The title compound was obtained
from compound 23b (2.12 g, 3.77 mmol) according to general
4.1.6.2. (2-(Tert-butyldimethylsilyloxy)-3,4,5-trimethoxyphenyl)(3-
procedure B (1.06 g, 84%). 1H NMR (300 MHz, CDCl3):
d 3.71 (s, 3H),
(tert-butyldimethylsilyloxy)-4-methoxyphenyl)methanone
The title compound was obtained from 2-((tert-butyldimethylsilyl)
(23b).
3.95 (s, 6H), 3.97 (s, 3H), 4.04 (s, 3H), 5.71 (brs, 1H), 6.90 (s, 1H), 6.93
(d, 1H, J ¼ 8.4 Hz), 7.23e7.30 (m, 2H), 12.10 (s, 1H). 13C NMR
oxy)-3,4,5-trimethoxy- benzaldehyde (2 g, 6.13 mmol) according to
(75 MHz, CDCl3): d 56.1, 56.7, 61.1, 61.3, 109.9, 110.6, 113.8, 115.6,
general procedure A (2.22 g, 64%). 1H NMR (300 MHz, CDCl3):
d
0.64
122.5, 131.3, 141.5, 144.7, 145.5, 149.5, 150.0, 153.4, 199.0. HRMS-ESI
(s, 9H), 0.96 (s, 9H), 3.80 (s, 3H), 3.84 (s, 6H), 3.91 (s, 3H), 6.63
(s, 1H), 6.83 (d, 1H, J ¼ 8.4 Hz), 7.34 (d, 1H, J ¼ 2.1 Hz), 7.46 (dd, 1H,
J ¼ 2.1, 8.4 Hz).
calcd. for C17H19O7 [M þ H]þ 335.1131, found 335.1138.
4.1.8. General procedure C for synthesis of 2-(methoxymethoxy)-
3,4,5-trimethoxy benzophenone (24aec, 24eef and 10)
4.1.7. General procedure B for cleavage of tert-butyldimethylsilyl
group to provide 8 and 14
A stirred solution of 1-bromo-3,4,5-trimethoxy-2-(methox-
ymethoxy)benzene (1.2 equiv) in THF (0.13 M) was cooled to ꢁ78 ꢀC
and n-BuLi (1.2 equiv, 1.6 M in hexane) was added dropwise. The
resulting solution was stirred at ꢁ78 ꢀC for 1 h. The previous so-
lution was added slowly to a solution of the substituted benzal-
dehyde (1 equiv) in THF (0.3 M) at 0 ꢀC. The solution was stirred at
TBAF solution (2 equiv) was added to a stirred solution of
TBDMS-protected 2-hydroxybenzophenone (1 equiv) in THF
(0.2 M) at 0 ꢀC and the resulting mixture was stirred at room
temperature for 1 h. The reaction solution was quenched with
water. The crude product was extracted with EtOAc and purified by
flash chromatography (EtOAc/hexane).
0
ꢀC for 1 h and then was quenched with water. The mixture was
extracted with EtOAc and the organic layer was collected. The crude
product was purified through chromatography to afford (EtOAc/
hexane) the substituted benzhydrol. To a solution of the substituted
benzhydrol (0.1 M) in CH2Cl2, pyridinium dichromate (2 equiv) and
4.1.7.1. (2-Hydroxy-3,4,5-trimethoxyphenyl)(4-methoxyphenyl)
methanone (8). The title compound was obtained from compound

312
C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
ꢀ
4A molecular sieves (0.3 g) was added at room temperature. The
resulting mixture was stirred at room temperature for 6 h. The
mixture was diluted with EtOAc and filtered through a pad of Celite.
The organic layer was collected and the crude product was purified
by flash chromatography (EtOAc/hexane) to yield the substituted
benzophenone.
by flash chromatography (EtOAc/hexane) to afford the desired
compound.
4.1.9.1. (2-Amino-4-methoxyphenyl)(3,4,5-trimethoxy-2-(methox-
ymethoxy)phenyl)methanone (24d). The title compound was ob-
tained from compound 24c (600 mg, 1.5 mmol) according to
general procedure D (400 mg, 72%). 1H NMR (300 MHz, CDCl3):
d
3.08 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H), 3.97 (s, 3H), 3.99 (s, 3H), 4.93
4.1.8.1. (4-(Methylamino)phenyl)(3,4,5-trimethoxy-2-(methox-
ymethoxy)phenyl)methanone (24a). The title compound was ob-
tained from 4-(methylamino)benzaldehyde (600 mg, 4.44 mmol)
according to general procedure C (330 mg, 21%). 1H NMR (500 MHz,
(s, 2H), 6.66e6.71 (m, 2H), 7.04 (s, 1H), 7.69e7.74 (m, 1H).
4.1.9.2. (3-Amino-4-methoxyphenyl)(3,4,5-trimethoxy-2-(methox-
ymethoxy)phenyl)methanone (24g). The title compound was ob-
tained from compound 24f (710 mg, 1.8 mmol) according to general
CDCl3):
d 3.09 (s, 3H), 3.27 (s, 3H), 3.74 (s, 3H), 3.87 (s, 3H), 4.81
(s, 2H). 6.60 (s, 1H), 7.19 (d, 2H, J ¼ 8.5 Hz), 7.83 (d, 2H, J ¼ 8.5 Hz),
procedure D (516 mg, 78%). 1H NMR (300 MHz, CDCl3):
d 3.23
8.60 (s, 1H).
(s, 3H), 3.80 (s, 3H), 3.90e3.95 (m, 12H), 4.93 (s, 2H), 6.61 (s, 1H),
6.78 (d, 1H, J ¼ 8.4 Hz), 7.22 (dd, 1H, J ¼ 2.1, 8.4 Hz), 7.31 (d, 1H,
J ¼ 1.8 Hz).
4.1.8.2. (4-Methoxy-2-(methoxymethoxy)phenyl)(3,4,5-trimethoxy-
2-(methoxymethoxy)phenyl)methanone (24b). The title compound
was obtained from 4-methoxy-2-(methoxymethoxy)benzaldehyde
(500 mg, 2.55 mmol) according to general procedure C (510 mg,
4.1.10. General procedure E for cleavage of methoxymethoxy group
to provide 9e15
47%). 1H NMR (300 MHz, CDCl3):
d3.26 (s, 3H) 3.30 (s, 3H) 3.79
2 N HCl (5 mL) was added at room temperature to a stirred
solution of MOM-protected 2-hydroxybenzophenone (1 equiv) in
MeOH (5 mL) and the resulting mixture was stirred for 4 h. The
solution was evaporated in vacuo and diluted with water. The
diluted solution was extracted with EtOAc. The organic layer was
collected and the crude product was purified by flash chromatog-
(s, 3H) 3.82 (s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 4.83 (s, 2H), 4.98 (s, 2H),
6.56 (dd,1H, J ¼ 2.4, 8.7 Hz), 6.66 (d,1H, J ¼ 2.4 Hz), 6.73 (s,1H), 7.59
(d, 1H, J ¼ 8.7 Hz).
4.1.8.3. (4-Methoxy-2-nitrophenyl)(3,4,5-trimethoxy-2-(methox-
ymethoxy)phenyl)methanone (24c). The title compound was ob-
tained from 4-methoxy-2-nitrobenzaldehyde (800 mg, 4.42 mmol)
according to general procedure C (760 mg, 42%). 1H NMR (300 MHz,
raphy
(EtOAc/hexane)
to
afford
the
substituted
2-
hydroxybenzophenone.
CDCl3):
d 3.23 (s, 3H), 3.82 (s, 3H), 3.83 (s, 3H), 3.90 (s, 3H), 3.94
4.1.10.1. (2-Hydroxy-3,4,5-trimethoxyphenyl)(4-(methylamino)
phenyl)methanone (9). The title compound was obtained from
compound 24a (365 mg, 1.0 mmol) according to general procedure
(s, 3H), 4.74 (s, 1H), 7.03 (s, 1H), 7.13 (dd, 1H, J ¼ 2.4, 8.4 Hz), 7.41e
7.47 (m, 2H).
E (301 mg, 83%). 1H NMR (500 MHz, CDCl3):
d
2.93 (d, 3H, J ¼ 4.5 Hz,
4.1.8.4. (3-Fluoro-4-methoxyphenyl)(3,4,5-trimethoxy-2-(methox-
ymethoxy)phenyl)methanone (24e). The title compound was ob-
tained from 3-fluoro-4-methoxybenzaldehyde (200 mg, 1.3 mmol)
according to general procedure C (130 mg, 22%). 1H NMR (300 MHz,
NCH3), 3.76 (s, 3H), 3.97 (s, 3H), 4.05 (s, 3H), 4.33 (brs, 1H), 6.63
(d, 2H, J ¼ 9.0 Hz), 6.96 (s, 1H), 7.63 (d, 2H, J ¼ 8.5 Hz), 12.13 (s, 1H).
13C NMR (75 MHz, CDCl3):
d 30.0, 56.6, 61.1, 61.3, 110.6, 111.1, 114.4,
126.0, 132.1, 141.6, 144.5, 148.7, 152.7, 152.8, 198.2. HRMS-ESI calcd.
CDCl3):
d 3.19 (s, 3H), 3.82 (s, 3H), 3.94e3.95 (m, 9H), 4.90 (s, 2H),
for C17H20NO5 [M þ H]þ 318.1341, found 318.1340.
6.63 (s, 1H), 6.94e7.00 (m, 1H), 7.59e7.66 (m, 2H).
4.1.10.2. (2-Hydroxy-3,4,5-trimethoxyphenyl)(2-hydroxy-4-
methoxyphenyl)methanone (11). The title compound was obtained
from compound 24b (423 mg, 1.0 mmol) according to general
4.1.8.5. (4-Methoxy-3-nitrophenyl)(3,4,5-trimethoxy-2-(methox-
ymethoxy)phenyl)methanone (24f). The title compound was ob-
tained from 4-methoxy-3-nitrobenzaldehyde (603 mg, 1.3 mmol)
according to general procedure C (1 g, 75%). 1H NMR (300 MHz,
procedure E (272 mg, 81%). 1H NMR (500 MHz, CDCl3):
d 3.77
(s, 3H), 3.84 (s, 3H), 3.94 (s, 3H), 4.01 (s, 3H), 6.44 (dd, 1H, J ¼ 2.0,
CDCl3):
d 3.17 (s, 3H), 3.84 (s, 3H), 3.95 (s, 3H), 3.97 (s, 3H), 4.04 (s,
9.0 Hz), 6.50 (d, 1H, J ¼ 2.0 Hz), 6.79 (s, 1H), 7.54 (d, 1H, J ¼ 9.0 Hz),
9.50 (s, 1H), 11.64 (s, 1H). 13C NMR (75 MHz, CDCl3):
d 55.0, 56.0,
3H), 4.89 (s, 2H), 6.68 (s, 1H), 7.14 (d, 1H, J ¼ 8.7 Hz), 8.08 (d, 1H,
J ¼ 2.1, 8.7 Hz), 8.32 (d, 1H, J ¼ 2.1 Hz).
60.6, 100.9, 106.7, 108.9, 113.0, 115.6, 134.3, 141.0, 145.0, 147.2, 148.1,
164.7, 165.5, 198.7. HRMS-ESI calcd. for C17H19O7 [M þ H]þ 335.1131,
found 335.1143.
4.1. 8. 6. (4-(Dimethylamino)phenyl)(2-hydroxy-3, 4, 5-
trimethoxyphenyl)methanone (10). The title compound was ob-
tained from 4-(dimethylamino)benzaldehyde (370 mg, 2.5 mmol)
according to general procedure C (380 mg, 46%). 1H NMR (500 MHz,
4.1.10.3. (2-Amino-4-methoxyphenyl)(2-hydroxy-3,4,5-
trimethoxyphenyl)methanone (12). The title compound was ob-
tained from compound 24d (320 mg, 0.79 mmol) according to
general procedure E (221 mg, 84%). 1H NMR (500 MHz, CDCl3):
CDCl3):
d 3.03 (s, 6H), 3.79 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 6.61
(d, 2H, J ¼ 11.0 Hz), 6.63 (s, 1H), 7.28 (d, 2H, J ¼ 11.5 Hz). 13C NMR
(75 MHz, CDCl3):
d 39.9, 56.0, 60.7, 61.0, 106.9, 110.4, 125.3, 127.6,
d
3.88 (s, 3H), 3.90 (s, 3H), 3.98 (s, 3H), 4.03 (s, 3H), 6.29 (s, 1H), 6.65
132.6, 140.5, 144.4, 145.0, 147.6, 153.5, 194.0. HRMS-ESI calcd. for
(dd, 1H, J ¼ 2.0, 9.5 Hz), 6.67e6.68 (m, 1H), 7.14 (s, 1H), 7.89 (d, 1H,
C
18H22NO5 [M þ H]þ 332.1498, found 332.1499.
J ¼ 9.5 Hz). 13C NMR (75 MHz, CDCl3):
d
55.1, 56.2, 60.8, 61.1, 88.8,
106.5, 109.1, 111.9, 119.5, 123.9, 140.9, 141.7, 144.1, 146.7, 159.1, 161.4,
161.8. HRMS-ESI calcd. for C17H20NO6 [M þ H]þ 334.1291, found
334.1190.
4.1.9. General procedure D for reduction of nitro group to provide
24d and 24g
To a solution of nitro compound (1 equiv) in isopropanol and
water (5:1, 0.1 M) iron powder (2 equiv) and ammonium chloride
(2 equiv) was added. The mixture was heated to reflux for 2 h and
then diluted with EtOAc. The mixture was filtered off with celite
and the organic layer was collected. The crude product was purified
4.1.10.4. (3-Fluoro-4-methoxyphenyl)(2-hydroxy-3,4,5-
trimethoxyphenyl)methanone (13). The title compound was ob-
tained from compound 24e (140 mg, 0.37 mmol) according to
general procedure E (110 mg, 88%). 1H NMR (300 MHz, CDCl3):

C.-Y. Chang et al. / European Journal of Medicinal Chemistry 77 (2014) 306e314
313
d
3.74 (s, 3H), 3.96 (s, 3H), 3.98 (s, 3H), 4.06 (s, 3H), 6.85 (s, 1H),
7.02e7.09 (m, 1H), 7.47e7.49 (m, 1H), 7.50e7.52 (m, 1H), 11.96 (s,
1H). 13C NMR (75 MHz, CDCl3):
56.3, 56.6, 61.1, 61.3, 110.1, 112.4,
protein (MAP)-rich tubulin (from bovine brain, Cytoskeleton, Inc.)
was dissolved in reaction buffer [100 mM PIPES (pH 6.9), 2 mM
MgCl2, and 1 mM GTP] in preparing the 4 mg/mL tubulin solution.
d
112.5, 113.5, 117.0, 117.3, 126.4, 126.5, 130.6, 130.7, 141.6, 144.8, 153.5,
197.6. HRMS-ESI calcd. for C17H18FO6 [M þ H]þ 337.1087, found
337.1100.
The tubulin solution (240 mg of MAP-rich tubulin per well) was
placed in a 96-well microtiter plate in the presence of test com-
pounds or 2% (v/v) DMSO as a vehicle control. The increase in
absorbance at 350 nm was measured in a PowerWave X Microplate
Reader (BIO-TEK Instruments, Winooski, VT) at 37 ꢀC and recorded
every 30 s for 30 min. The area under the curve (AUC) was used to
determine the concentration that inhibited tubulin polymerization
4.1.10.5. (2-Hydroxy-3,4,5-trimethoxyphenyl)(4-methoxy-3-
nitrophenyl)methanone (15). The title compound was obtained
from compound 24f (150 mg, 0.37 mmol) according to general
procedure E (116 mg, 86%). 1H NMR (500 MHz, CDCl3):
d
3.74 (s, 3H),
by 50% (IC50). The AUCs of the untreated control and 10 mM
3.96 (s, 3H), 4.07 (s, 3H), 4.08 (s, 3H), 6.77 (s, 1H), 7.23 (d, 1H,
colchicine were set to 100 and 0% polymerization, respectively, and
the IC50 was calculated by nonlinear regression in at least three
experiments.
J ¼ 9.0 Hz), 7.96 (d, 1H, J ¼ 2.0, 9.0 Hz), 8.25 (d, 1H, J ¼ 3.0 Hz). 13
C
NMR (75 MHz, CDCl3):
d 56.6, 56.9, 61.1, 61.3, 109.4, 113.0, 113.5,
127.1, 130.0, 135.0, 139.0, 141.6, 145.1, 150.0, 153.6, 155.5, 196.3.
HRMS-ESI calcd. for C17H18NO8 [M þ H]þ 364.1032, found 364.1056.
4.2.3. Tubulin competition binding scintillation proximity assay
[26e28]
4.1.10.6. (3-Amino-4-methoxyphenyl)(2-hydroxy-3,4,5-
trimethoxyphenyl)methanone (16). The title compound was
obtained from compound 24g (300 mg, 0.8 mmol) according to
general procedure E (226 mg, 85%). 1H NMR (500 MHz, CDCl3):
This assay was performed in a 96-well plate. In brief, 0.08
[3H]colchicine was mixed with the test compound and 0.5
special long-chain biotin-labeled tubulin (0.5 g) then incubated in
100 L of reaction buffer [80 mM PIPES (pH 6.8), 1 mM EGTA, 10%
glycerol, 1 mM MgCl2, and 1 mM GTP] for 2 h at 37 ꢀC. Then 80
g of
streptavidin-labeled SPA beads was added to each reaction mixture.
The radioactive counts were then directly measured with a scin-
tillation counter and the inhibition constant (Ki) was calculated
using the ChengePrusoff equation [29].
mM
g of
m
m
m
d
3.72 (s, 3H), 3.92 (s, 3H), 3.95 (s, 3H), 4.04 (s, 3H), 6.83 (d, 1H,
m
J ¼ 8.5 Hz), 6.95 (s, 1H), 7.08e7.09 (m, 2H), 12.16 (s, 1H). 13C NMR
(75 MHz, CDCl3):
d 55.5, 56.5, 60.9, 61.1, 109.0, 110.7, 113.9, 114.9,
120.7, 130.7, 136.4, 141.3, 144.4, 149.1, 150.3, 153.1, 199.4. HRMS-ESI
calcd. for C17H20NO6 [M þ H]þ 334.1291, found 364.1298.
4.2. Biology
4.2.4. Capillary disruption assays [30]
Capillary disruption assays were conducted in a 96-well plate
format using human umbilical vein endothelial cells (HUVECs)
plated at a density of 2 ꢂ 104 cells/well in 20% FBS M199 medium
containing 20 ng/mL VEGF on a Matrigel layer (BD Biosciences).
Capillaries were allowed to form over a 4 h period before the
addition of test compound or vehicle control. Images were acquired
immediately following addition of the compound and 4 h after
exposure to the test compound. Tube formation was quantified by
measuring the network number of capillary structures manually by
counting under a microscope (original magnification of 100ꢂ).
Reagents for cell culture were obtained from Gibco-BRL Life
Technologies (Gaithersburg, MD). Microtubule-associated protein
(MAP)-rich tubulin was purchased from Cytoskeleton, Inc. (Denver,
CO). [3H]Colchicine (specific activity, 60e87 Ci/mmol) was
purchased from PerkinElmer Life Sciences (Boston, MA).
4.2.1. Cell growth inhibitory assay
Human cancer cell lines (KB, MKN45, and HT29) used in this
study were procured from American Type Culture Collection
(Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium,
minimal essential medium, or RPMI 1640 medium. Resistant cell
lines KB-Vin10, KB-7D, and KB-S15 were maintained in medium
Acknowledgments
containing additional 10 nM vincristine, 7
paclitaxel, respectively. All cell cultures were supplemented with
10% fetal bovine serum, 2 M glutamine, 100 units/mL penicillin,
and 100 g/mL streptomycin and incubated in a humidified at-
mM VP16, and 50 nM
This research were supported by the National Science Council of
the Republic of China (grant no. NSC 100-2628-M-038-001-MY3,
and NSC 101-2325-B-038-002).
m
m
mosphere (95% air and 5% CO2) at 37 ꢀC. KB-Vin10 and KB-S15 cell
lines were resistant to vincristine and paclitaxel, respectively, and
both overexpressed the MDR drug efflux protein. KB-7D cells were
resistant to VP16 and overexpressed MRP. All resistant cell lines
were incubated in drug-free medium for 3 days before being har-
vested for the growth inhibition assay. In vitro growth inhibition
was assessed with the methylene blue assay [24]. Exponentially
growing cells were seeded into 24-well culture plates at a density of
8000e20,000 cells per mL per well (depending on the doubling
time of the cell line) and allowed to adhere overnight. Cells were
incubated with various concentrations of drugs for 72 h. Then the
A595 of the resulting solution from 1% N-lauroylsarcosine extrac-
tion was measured. Fifty percent growth inhibition (IC50) was
calculated on the basis of the A595 of untreated cells (taken as
100%). The values shown are the means and standard errors of at
least three independent experiments performed in duplicate.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.061.
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