3-Aroylindoles display antitumor activity in?vitro and in?vivo: Effects of N1-substituents on biological activity

Article abstract of DOI:10.1016/j.ejmech.2016.11.033

A series of 3-aroylindole hydroxamic acids (10–17) were developed based on the concept of a structural combination of tubulin and histone deacetylase (HDAC) inhibitors. This was accomplished by introducing hydroxamic acid-containing moieties at the N1 pos

Full text of DOI:10.1016/j.ejmech.2016.11.033

European Journal of Medicinal Chemistry 125 (2017) 1268e1278
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
3-Aroylindoles display antitumor activity in vitro and in vivo: Effects
of N1-substituents on biological activity
Hsueh-Yun Lee a, Jiann-Fong Lee a, Sunil Kumar a, Yi-Wen Wu b, Wei-Chun HuangFu c,
Mei-Jung Lai d, Yu-Hsuan Li a, Hsiang-Ling Huang a, Fei-Chiao Kuo a, Che-Jen Hsiao e,
,
, **
Chun-Chun Cheng c, Chia-Ron Yang b *, Jing-Ping Liou a
a School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan
b School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
c The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
d Center for Translational Medicine, Taipei Medical University, Taiwan
e Department of Pharmacology, College of Medicine, Taipei Medical University, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-aroylindole hydroxamic acids (10e17) were developed based on the concept of a structural
combination of tubulin and histone deacetylase (HDAC) inhibitors. This was accomplished by introducing
hydroxamic acid-containing moieties at the N1 position of the tubulin assembly inhibitor, compound 9
(SCB01A, BPR0L075, phase II trial). Most of synthetic compounds produced in this way displayed com-
parable HDAC inhibitory activity, and four (10, 12e14) of them also inhibit tubulin assembly. Notably,
compound 12 possesses not only tubulin and HDAC inhibitory activity but also shows HDAC6 selectivity
over other HDAC isoforms. In addition, it exhibits remarkable inhibitory activity against the growth
cancer cells in vitro and in vivo (PC3 and RPMI-8226 cells). Notably, it suppresses the growth of multiple
myeloma xenografts without leading to the death of teated animals like reference compound. In sum,
this study provided potential compounds with safer profiles for cancer treatment.
Received 16 July 2016
Received in revised form
24 October 2016
Accepted 14 November 2016
Available online 15 November 2016
Keywords:
Anticancer agents
Histone deacetylase inhibitors
3-Aroylindoles
© 2016 Elsevier Masson SAS. All rights reserved.
Tubulin polymerization inhibition
1. Introduction
and azaindolylsulfonamides (8) [12] also exhibit potent HDAC
inhibitory activity and marked cytotoxicity. The potent anti-
To increase the structural diversity, the combination of specific
motif from different classes of bioactive molecules has become a
practical methodology in the field of medicinal chemistry. A liter-
ature survey indicated that histone deacetylase (HDAC) protein is
commonly used to validate this strategy of structural combination,
together with various biological targets. For example, Guerrant
et al. have developed dual HDAC/Topo II inhibitors [1], and Lin et al.
have synthesized a series of dual HDAC and HMG-coA reductase
inhibitors [2]. Histone deacetylase (HDAC) is an attractive target,
four HDAC inhibitors (SAHA [3], FK228 [4,5], PXD101 [6,7], and
LBH589 [8,9]) having been approved by the US FDA for the treat-
ment of cutaneous or refractory peripheral T-cell lymphoma or
multiple myeloma. In addition, many HDAC inhibitors such as MS-
275 (6) [10] are currently in clinical trials and our previously
developed 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles (7) [11]
proliferative activity of most HDAC inhibitors achieved by
addressing epigenetic disorders can be attributed to the charac-
teristic hydroxamic acid group [13,14]. The development of anti-
tubulin agents has been intensively progressed for several decades,
specifically in development of combretastatin analogous due to
their evident structural requirements such as Z-geometrical
conformation and 3,4,5-trimethoxyphenyl group [15]. Currently,
research attention was shifted to the strategy of structural combi-
nation. For instance, Zhang et al. combined natural antitubulin
agents or their analogous like colchicine and 4’-demethyl-4-
deoxypodophyllotoxin with HDAC specific moieties such as
hydroxamic acid and 2-aminobenzamide [16e18]. The strategy of
structural combination may open a new avenue and attracts our
attention to the utilization of our previously synthesized anti-
tubulin agents.
The previous study on the modification of 9 (SCB01A, BPR0L075)
revealed that the attachments such as furoyl, thiophenecarbonyl,
and benzoyl at N1 position still retained its antiproliferative activity
[19]. In addition, the presence of these groups cited above had no
effect on the mechanism of action of related derivatives, which
* Corresponding author.
** Corresponding author.
E-mail addresses: cryang@ntu.edu.tw (C.-R. Yang), jpl@tmu.edu.tw (J.-P. Liou).
http://dx.doi.org/10.1016/j.ejmech.2016.11.033
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
1269
revealed the tolerance of structural modification at N1 position of
compound 9. In order to explore the effect of N1 substitutions on
biological activity, we chose compound 9, a potent antitubulin
agent with concise structure and remarkable antiproliferative ac-
tivity in vitro and in vivo currently in clinical trials (phase II), for a
test of this precept [20,21]. A variety of hydroxamic acid-containing
moieties presented in Fig. 1 (in blue) were selected from surveys of
several potent HDAC inhibitors, which are expected to contribute
HDAC inhibitory activity. According to an earlier SAR study, the N1
position of compound 9 can accommodate substitutions without
significant influence on the antiproliferative activity. In an attempt
to develop dual functional compounds, introduction of hydroxamic
acid-containing moieties at the N1 position of compound 9 yielded
a series of 3-aroylindole hydroxamic acids (10e17) and the
structure-activity relationships (SAR), HDAC enzymatic assays, ef-
fects on tubulin assembly, and antitumor activity of the compounds
in vivo were explored (see Fig. 2).
Two 3-aroylindoles (9 and 18) were treated first with NaH and then
with bromobenzensulfonyl chloride, yielding compounds 19e21.
The resulting compounds 19e21 were coupled with tert-butyl
acrylate under the conditions of Heck olefination to give the cor-
responding cinnamates (22e24). The ester groups in 22e24 were
hydrolyzed with TFA and the resulting products were allowed to
react with NH2OTHP in the presence of EDC$HCl and HOBt to yield
the corresponding O-protected N-hydroxamides which were
deprotected with 10% TFA to yield compounds 10, 11, and 15.
Scheme 2 shows the synthesis of compounds 12e14 and 16e17.
3-Aroyindoles (9, 18, and 25) was treated with NaH and then
reacted with substituted benzyl halides to afford compounds
26e29. The ester group of 26e27 was hydrolyzed with 1 N KOH to
yield the corresponding carboxylic acids which were then sub-
jected to amidation with NH2OTHP and deprotection with 10% TFA,
yielding the corresponding N-hydroxamic acids (12, 17). Alterna-
tively, the carboxylic acid obtained from compound 26 was reacted
with o-phenylenediamine, the core structure of compound 6, in the
presence of EDC$HCl and HOBt to afford compound 14. Compounds
28e29 underwent Heck olefination with tert-butyl acrylate and
following the subsequent ester hydrolysis, amide formation with
NH2OTHP and TFA deprotection, afforded the corresponding N-
hydroxamic acids (13, 16).
2. Results and discussion
2.1. Chemistry
Syntheses of compounds 10, 11, and 15 are shown in Scheme 1.
S
S
O
H
O
O
H
N
H
N
NHOH
NH
H
HN
O
O
O
1, SAHA
O
N
H
H
O
2, Romidepsin
O
NHOH
H
N
H
N
NHOH
S
O
O
O
HN
CH3
OCH3
OCH3
OCH3
O
4, PXD101
3, LBH589
CH3
N
H3CO
N
O
Linker
O
N
H
NH2
N
H
N
H
N
OH
O
NH
OH
N
O
O
6, MS-275
5, Tubastatin A
O
HO
N
Structural combination
N
H
O
N
N
OH
O2S
OCH3
O2S
N
H
O
OCH3
OCH3
7
H3CO
8
N
H
9, SCB01A, BPR0L075
Fig. 1. The design of 3-aroylindole hydroxamic acids.

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H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
Fig. 2. Structures of designed compounds (10e17).
OCH3
OCH3
OCH3
O
OCH3
OCH3
O
a
R1
N
R1
OCH3
O2S
N
H
R2
9, R1 = OMe
18, R1 = H
19, R1 = OMe; R2 = 3-Br
20, R1 = OMe; R2 = 4-Br
21, R1 = H; R2 = 3-Br
OCH3
OCH3
OCH3
OCH3
OCH3
O
O
b
c
R1
R1
OCH3
O
N
N
O
O2S
O2S
meta-
OH
OR2
N
H
para-
10, R1 = OMe; meta-
11, R1 = OMe; para-
15, R1 = H; meta-
22, R1 = OMe; meta-
23, R1 = OMe; para-
24, R1 = H; meta-
Scheme 1. Reagents and conditions: (a) NaH, bromobenzenesulfonyl chlorides, DMF, rt; (b) i. Pd(OAc)2, PPh3, NEt3, K2CO3, tert-butyl acrylate, DMF, 100 ꢀC; (c) i. CF3COOH, rt; ii.
EDC$HCl, HOBt, NH2OTHP, NMM, DMF, rt; iii CF3COOH/CH3OH, rt.
2.2. Biological evaluation
the N-hydroxylacrylamide moiety in 10 and 11, this moiety can be
seen to favor the meta position of the sulfonyl linker. However, this
is not observed in compound 13 which has a methylene linker para
to the N-hydroxylacrylamide moiety. Compounds 10, 13, and 17
exhibited comparable anti-HDAC activity in SAHA, whereas com-
pounds 16 and 15 showed slightly weaker potency than that of the
reference compound. These results indicate that the introduction of
a hydroxamic acid-containing moiety at N1 of compound 9 con-
tributes to the anti-HDAC activity of these compounds.
2.2.1. HeLa nuclear HDAC enzyme inhibition
To determine if the introduction of a hydroxamic acid moiety
confers anti-HDAC activity on this series of compounds, the inhi-
bition of HeLa nuclear HDAC activity by the synthesized com-
pounds was assessed (Table 1). The introduction of an o-
aminobenzamide unit at the N-1 position (14) has a negligible in-
fluence on HDAC enzymatic activity. Compared with the effect of

H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
1271
O
R
2
O
R
2
O
R
1
R
2
a
b or c
R
N
1
N
R
1
H
N
N
H
R
3
R
3
O
9, R = OMe; R = 3,4,5-OMe
1
2
12, R = OMe; R = 3,4,5-OMe; R = OH
26, R = OMe; R = 3,4,5-OMe; R = COOMe
1
2
3
1
2
3
18, R = H; R = 3,4,5-OMe
1
2
14, R = OMe; R = 3,4,5-OMe; R = 2-aminophenyl
27, R = OMe; R = 4-OMe; R = COOMe
1
2
3
1
2
3
25, R = 4-OMe; R = 4-OMe;
1
2
17, R = OMe; R = 4-OMe; R = OH
28, R = OMe; R = 3,4,5-OMe; R = Br
1
2
2
1
2
3
29, R = H; R = 3,4,5-OMe; R = Br
1
2
3
d
OCH
3
OCH
3
O
O
OCH
3
OCH
3
R
R
OCH
3
OCH
e
N
3
N
H
N
O
OH
O
O
13, R = OMe
16, R = H
30, R = OMe
31, R = H
Scheme 2. Reagents and conditions: (a) NaH, 4-bomobenzyl bromide or methyl 4-(chloromethyl)benzoate, DMF, rt; (b) i. 1 N LiOH, MeOH, reflux; ii. EDC$HCl, HOBt, NH2OTHP,
NMM, DMF, rt; iii CF3COOH/CH3OH, rt. (c) For 26: i. 1 N LiOH, MeOH, reflux; ii. o-phenylenediamine, HOBt, EDC$HCl, NMM, DMF; (d) For 28 and 29: i. Pd(OAc)2, PPh3, NEt3, K2CO3,
tert-butyl acrylate, DMF, 100 ꢀC; (e) i. CF3COOH, rt; ii. EDC$HCl, HOBt, NH2OTHP, NMM, DMF, rt; iii CF3COOH/CH3OH, rt.
Table 1
HDAC Inhibitory Activity (IC50) and Antiproliferative activity of Compounds 1, 9 and 10e17.
HeLa nuclear HDACs IC50 (nM)
IC50 SDa (nM)
A549
Compd
HCT116
PC3
10
11
12
13
14
15
16
17
1
135.45 4.23
>10,000
610.81 5.32
146.66 7.46
>10,000
172.22 8.22
177.55 13.61
144.69 4.62
120.06 5.55
e
72.52 5.22
43.91 0.81
70.34 1.71
484.57 3.94
1674.60 68.13
231.38 4.53
6684.85 50.68
5498.10 137.73
1896.28 19.16
1488.60 59.48
e
33.09 0.97
66.74 0.89
179.26 6.42
665.57 23.67
156.93 5.45
5774.80 299.39
2601.66 131.43
1841.38 50.50
873.26 50.85
e
119.56 7.15
205.68 3.38
677.67 7.04
208.38 5.41
4592.88 190.67
1898.46 96.21
939.90 34.26
476.86 20.09
9b
23
6
a
SD: standard deviation, all experiments were independently performed at least three times.
Data from ref. [20].
b
2.2.2. In vitro cell growth inhibition
retained cellular potency of compound 11 can probably be attrib-
uted to its inclusion of the structure of compound 9. A similar
phenomenon was observed with compound 12 which has better
cellular activity but poorer anti-HDAC activity than compound 1.
The impact of central core structure (9, shown in bold) on anti-
proliferative activity was assessed by altering the pattern of sub-
stitution shown in compounds 15e17. Removal of the 6-OMe group
led to dramatic losses of cytotoxicity in compounds 15 and 16,
when compared with 10 and 13, respectively. Replacement of the
30,40,5’-trimethoxybenzoyl group ring of antitubulin agents with 4’-
methoxybenzoyl (17) also resulted in a loss of cellular activity.
Despite all synthetic compounds have weaker antiproliferative
activity than compound 9, the core structure is essential for anti-
proliferative activity and it causes HDAC inhibitors to suppress the
The antiproliferative activities of the synthetic 3-aroylindole
hydroxamic acids (10e17), and the reference compounds SAHA
(1) and 9 were evaluated against three human cancer cell lines,
human non-small cell lung carcinoma A549 cells, human colorectal
HCT116 cells, and human prostate cancer PC3 cells (Table 1). Among
all the synthetic molecules, compound 10 showed the highest
antiproliferative activity. Although the IC50 values of compounds 10
and 1 against HeLa cellular HDAC are comparable,10 displayed 6- to
42-fold more potent antiproliferative activity than compound 1,
suppressing the growth of A549, HCT116, and PC3 cells with IC50
values of 72.52, 43.91, and 33.09 nM, respectively. Compound 11,
which has negligible anti-HDAC activity, showed 3- to 20-fold
improvement of antiproliferative activity as compared with 1. The

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H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
growth of cancer cells at submicromolar IC50 levels.
vehicle (1.0% carboxymethyl cellulose þ 0.5% Tween80 in D5W).
The result indicated that oral administration of compound 12 at
doses of 100 and 200 mg/kg led to the suppression of PC3 cancer
cell xenografts by p < 0.01 and factors of 24.8% and 68.5% (percent
tumor growth inhibition [%TGI] values), respectively. In addition,
the treatment with different doses of compound 12 had no signif-
icant effect on the body weight of tested mice (Fig. 5B).
2.2.3. In vitro tubulin polymerization assay
The effect of compounds 1, 10, and 12e17 on the assembly of
tubulin is shown in Fig. 3. To confirm their influence on microtubule
dynamics, paclitaxel, a tubulin stabilizer, and vincristine, a tubulin
assembly inhibitor, were selected as reference compounds in the
assay. The results indicate that compounds 12e14 dose-
dependently inhibit the polymerization of tubulin remarkably
and this explains the inconsistency of the anti-HDAC activity with
the antiproliferative activity of compounds 12 and 14 (Table 1).
Aside from HDAC inhibitory activity, compounds 12 and 14 may
suppress the growth of cancer cells by inhibiting the polymeriza-
tion of tubulin. In addition, the loss of cellular activity after altering
the substitution pattern of the core structure (15 vs 10, 16 vs 11, and
17 vs 12) may be attributed to the disappearance of tubulin depo-
lymerization activity. In summary, this series of 3-aroylindole
hydroxamic acids could possibly serve as potent anticancer
agents with dual tubulin and HDAC inhibitory activity.
2.2.7. Growth inhibition of multiple myeloma RPMI-8226
xenografts in vivo
Recently, the use of LBH589 in combination with bortezomib
and dexamethasone has been approved by US FDA in 2015 for the
treatment of multiple myeloma [8,9]. In addition, the combination
therapy using ACY1215, an HDAC6 inhibitor, with bortezomib is
currently undergoing clinical trials for treatment of multiple
myeloma [22]. As a result, compound 12 was also tested for its
ability to inhibit the growth of multiple myeloma xenografts
in vivo, using bortezomib (Velcade®) (1 mg/kg, ip, once a week) as a
reference compound. The result showed that compound 12 dose-
dependently suppressed the growth of multiple myeloma
(Fig. 6A). The intraperitoneal administration of compound 12 at
doses of 50 and 100 mg/kg daily led to the suppression of RPMI-
8226 cancer cell xenografts by TGI values of 35.8% (p < 0.05) and
58.2% (P < 0.01) (percent tumor growth inhibition [%TGI] values),
respectively. It is noteworthy that compound 12 did not influence
the change of weight of test animals, whereas the treatment with
bortezomib caused three deaths of test animals (Fig. 6B).
2.2.4. HDAC isoform inhibition
Table 2 shows the selective inhibitory activity of the tested
compounds (10e13 and 15e17) and the reference compound (1,
SAHA) against HDAC1, 2, and 6. When compared with other iso-
enzymes, compound 15 tends to suppress the activity of HDAC1. It
inhibits HDAC1 with an IC50 value of 138.76 nM, and is thus 16.7-
and 5.7-fold more selective over HDAC2 and 6, respectively. The
profile of compound 13 is similar to that of compound 1. Notably,
compound 12 with the same 4-(N-hydroxyaminocarbonyl)benzyl
moiety as compound 5 shows highly selective activity on HDAC6
with selectivity ratios of 8, 21, and 14 over HDAC1, 2, and 8,
respectively. Comparison of 10 and 15 reveals that the removal of
the 6-OMe group possibly contributes to a slight enhancement of
HDAC1 and HDAC2 inhibitory activity, as well as a slight decrease
against HDAC6. This result is also seen in the comparison of 12 with
17. The conversion of the 30,40,5’-trimethoxybenzoyl motif (12) into
4’-methoxybenzoyl one (17) led to the loss of HDAC6 selectivity
over that of HDAC1 or HDAC2.
3. Conclusion
The modification of compound 9 with various hydroxamic acid-
containing groups provided a series of 3-aroylindole hydroxamic
acids (10e17). Five of the eight compounds showed HDAC enzy-
matic activity comparable to that of compound 1. An in vitro anti-
proliferation assay showed that the presence of the core structure
(9) plays a crucial role in the cytotoxicity regardless of the HDAC
enzymatic potency. In addition to the HDAC inhibitory activity,
compounds 10 and 12e14 also possess tubulin depolymerization
ability. Compound 12 displayed 8-fold selectivity for HDAC6 over
HDAC1 and 21-fold over HDAC2, and suppressed the growth of
tumors in human prostate PC3 xenograft models with a p < 0.01 by
TGI factors of 24.8% and 68.5% after oral administration with 100
and 200 mg/kg, respectively. The intraperitoneal administration of
compound 12 at doses of 50 and 100 mg/kg also suppresses RPMI-
8226 cancer cell xenografts by TGI of 35.8% and 58.2%, respectively.
Taken together, the combination of compound 9 with hydroxamic
acid-containing moieties results in a series of novel 3-aroylindoles
as antitumor molecules with safer profiles.
2.2.5. Western Blot analysis
The influence of compounds 10, 12, 13, 15, 16, 17, and the refer-
ence compound 1 on the expression of a-tubulin and histone H3
were assessed by the Western Blot analysis shown in Fig. 4. The
results indicated that all tested compounds, with the exception of
10 and 15, are able to increase the acetylation level of a-tubulin and
histone H3 in dose-dependent manner, behavior comparable to
that of compound 1. Compound 10 showed a slight effect on the
acetylation of a-tubulin, and compound 15 had negligible impact
on the acetylation of histone H3. The expression of MPM2 (mitotic
protein monoclonal-2) bands shows the cell cycle progression after
treatment with tested compounds. The distinct bands appearing
after treatment with compounds 10 and 12 indicated these two
compounds probably cause cell cycle arrest at the M-phase, and
this can be attributed to the tubulin polymerization inhibition of
the compounds. Compounds 15e17 showed slight to negligible
expression of MPM2 bands, which is consistent with the results
shown in Fig. 3.
4. Experimental section
4.1. Chemistry
Nuclear magnetic resonance (1H and 13C NMR) spectra were
recorded with
500 MHz) or a Bruker Fourier 300 (operating at 300 MHz), with
chemical shifts in parts per million ( ) downfield from TMS, the
a Bruker DRX-500 spectrometer (operating at
d
internal standard. High-resolution mass spectra (HRMS) were
recorded with a JEOL (JMS-700) electron impact (EI) mass spec-
trometer. The purities of the final compounds were determined
using an Agilent 1100 series HPLC system using a C-18 column
2.2.6. Growth inhibition of human prostate cancer xenografts
in vivo
Compound 12 was further tested for its in vivo efficacy using a
PC3 xenograft nude mouse model (Fig. 5A). Once a tumor was
approximately 150 mm3 in size after subcutaneous injection with
1 ꢁ 107 PC3 cells per mouse, the mice were randomized into vehicle
control and treatment groups, with control mice receiving only the
(Agilent ZORBAX Eclipse XDB-C18 5
mm, 4.6 mm ꢁ 150 mm) and
were found to be ꢂ 95%. Flash column chromatography was con-
ducted using silica gel (Merck Kieselgel 60, No. 9385, 230e400 mesh
ASTM). All reactions were conducted under an atmosphere of dry N2.

H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
1273
Fig. 3. In vitro tubulin polymerization assay. Microtubule assembly was assessed using the CytoDYNAMIX Screen kit BK006P, (Cytoskeleton Inc., Denver, CO). Purified porcine
tubulin proteins (>99% purity) were suspended in G-PEM buffer containing 80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP (pH 6.9), and 15% glycerol in the absence or
presence of indicated compounds at 4 ꢀC. The mixture was immediately transferred to pre-warmed 96-well plates, and absorbance was measured at 340 nm every 1 min for 30 min
using a 37 ꢀC plate reader (SpectraMAX Plus, Molecular Devices Inc., Sunnyvale, CA).
4.1.1. (E)-N-Hydroxy-3-(3-(6-methoxy-3-(3,4,5-
organic layer was extracted with saturated aqueous NaHCO3
(50 mL ꢁ 2). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure to give a brown
residue which was purified by silica gel chromatography (EtOAc/n-
hexane ¼ 1:2) to give the acrylic acid product. The resulting product
trimethoxybenzoyl)-1H-indol-1-ylsulfonyl)phenyl)- acrylamide (10)
A mixture of compound 22 (0.5 g, 0.82 mmol) and trifluoroacetic
acid (10 mL) was stirred at room temperature for 16 h. The reaction
was quenched with H2O and extracted with EtOAc (25 mL ꢁ 3). The

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H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
Table 2
HDAC inhibition activity and isoform selectivity of tested compounds.
Compd
IC50 (nM)a
HDAC1
selectivity ratio
HDAC1/HDAC6
HDAC2
HDAC6
HDAC2/HDAC6
10
11
12
13
15
16
17
1
467.82 7.80
>10,000
3215.90 31.73
>10,000
275.35 14.47
>10,000
1.7
e
11.68
e
573.43 40.21
129.57 8.48
138.76 19.46
141.12 4.57
157.62 10.17
101.98 8.73
1408.14 63.24
287.59 4.37
2319.76 87.21
385.51 18.98
209.35 3.15
332.65 15.52
64.50 2.44
70.49 5.64
791.60 48.78
138.13 3.88
108.48 5.84
72.34 1.89
8.89
1.84
0.18
1.02
1.45
1.41
21.83
4.08
2.93
2.79
1.93
4.60
a
SD: standard deviation. All experiments were independently performed at least three times.
Fig. 4. Effect of a-tubulin acetylation and histone H3 acetylation in cultured human prostate PC3 cells by compounds 10, 12, 13, 15, 16, 17, and 1 using Western blot analysis.
(0.3 g, 0.54 mmol) was dissolved in DMF (4 mL), then EDC$HCl
(0.16 g, 0.81 mmol), HOBt (0.09 g, 0.65 mmol), NMM (0.15 mL,
1.23 mmol), and NH2OTP (0.08 g, 0.64 mmol) were added and the
mixture was stirred at room temperature for 2 h. The reaction was
quenched with water and extracted with EtOAc (100 mL ꢁ 3). The
organic layer was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The resulting residue was purified by flash
column chromatography (CH2Cl2/CH3OH/aqueous NH3 ¼ 20:1:1) to
give a white solid. To the resulting product was added 10% tri-
fluoroacetic acid in MeOH (10 mL) and the mixture was stirred at
room temperature for 16 h. The reaction was quenched with H2O
and extracted with EtOAc (25 mL ꢁ 3). The organic layer was

H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
1275
Fig. 5. (A) Anticancer activity of compound 12 in a xenograft model of human prostate PC3 cells. Tumor growth of PC3 xenograft nude mice treated with or without compound 12
(100 and 200 mg/kg) was tracked by the mean tumor volume (mm3) S.E and calculated as % tumor growth inhibition (%TGI). Tumor volume was determined using caliper
measurements and was calculated as the product of 1/2 ꢁ length ꢁ width2. (B) Body weight (g) of the mice. *, p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control
group.
extracted with saturated aqueous NaHCO3 (50 mL ꢁ 2). The com-
bined organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure to give a brown residue
which was purified by column chromatography (EtOAc: n-
hexane ¼ 1: 2) to afford compound 10 (43%). mp 151e152 ꢀC; 1H
stirred at room temperature for 16 h. The reaction mixture was
quenched with H2O and extracted with EtOAc (25 mL ꢁ 3), and then
the organic layer was washed with saturated aqueous NaHCO3
(50 mL ꢁ 2). The combined organic layers were dried over anhy-
drous MgSO4 and concentrated under reduced pressure to give a
brown residue, which was purified by silica gel chromatography
NMR (300 MHz, Acetone-d6) d 10.51 (s, 1H), 8.10e8.24 (m, 3H), 7.98
(d, J ¼ 7.8 Hz, 1H), 7.70 (d, J ¼ 7.8 Hz, 1H), 7.58e7.64 (m, 2H), 7.23 (s,
2H), 7.08 (dd, J ¼ 2.1, 8.7 Hz, 1H), 6.72 (d, J ¼ 15.6 Hz, 1H), 3.96 (s,
9H), 3.88 (s, 3H). HRMS (ESI) for C28H26N2O9S (M þ Hþ) calcd
566.1359, found 566.1436. HPLC purity of 94.10% (retention
time ¼ 34.48).
(EtOAc/n-hexane
¼
1:2) to give compound 12 (66%). mp
8.27 (d, J ¼ 9.0 Hz,
181e182 ꢀC; 1H NMR (300 MHz, acetone-d6)
d
1H), 8.06 (s, 1H), 7.82 (d, J ¼ 8.4 Hz, 2H), 7.45 (d, J ¼ 8.4 Hz, 2H), 7.15
(s, 2H), 7.10 (d, J ¼ 2.1 Hz, 1H), 6.96 (dd, J ¼ 2.1, 8.7 Hz, 1H), 5.64 (s,
2H), 3.89 (s, 6H), 3.82 (s, 6H). HRMS (ESI) for C27H26N2O7 (Mþ) calcd
490.1740, found 490.1817. HPLC purity of 95.00% (retention
time ¼ 25.21).
4.1.2. (E)-N-Hydroxy-3-(4-(6-methoxy-3-(3,4,5-
trimethoxybenzoyl)-1H-indol-1-ylsulfonyl)phenyl)- acrylamide (11)
This compound was obtained from in 55% overall yield com-
pound 20 in a manner similar to that described for the preparation
of 10 from 19: mp 225e226 ꢀC; 1H NMR (500 MHz, Acetone-d6)
4.1.4. (E)-N-Hydroxy-3-(4-((6-methoxy-3-(3,4,5-
trimethoxybenzoyl)-1H-indol-1-yl)methyl)-phenyl)- acrylamide
(13)
d
8.22e8.26 (m, 3H), 8.13 (d, J ¼ 9.0 Hz, 1H), 7.96 (d, J ¼ 8.4 Hz, 2H),
This compound was obtained in 55% overall yield from com-
pound 28 in a manner similar to that described for the preparation
of 10 from 19: mp 186e187 ꢀC; 1H NMR (300 MHz, acetone-d6)
7.69 (d, J ¼ 16.2 Hz, 1H), 7.63 (d, J ¼ 2.1 Hz, 1H), 7.24 (s, 2H), 7.10 (dd,
J ¼ 2.4, 9.0 Hz, 1H), 7.69 (d, J ¼ 16.2 Hz, 1H), 3.97 (s, 9H), 3.89 (s, 3H).
HRMS (ESI) for C28H26N2O9S (Mþ) calcd 566.1359, found: 566.1407.
HPLC purity of 97.33% (retention time ¼ 35.93).
d
8.28 (d, J ¼ 8.4 Hz, 1H), 8.05 (s, 1H), 7.50e7.63 (m, 3H), 7.41 (d,
J ¼ 8.4 Hz, 2H), 7.13e7.20 (m, 3H), 6.97 (dd, 2.4, 8.7 Hz, 1H), 5.60 (s,
2H), 3.90 (s, 6H), 3.85 (s, 3H), 3.83 (s, 3H). HRMS (ESI) for
C
29H28N2O7 (M þ) calcd 516.1897, found: 516.1962. HPLC purity of
4.1.3. N-Hydroxy-4-((6-methoxy-3-(3,4,5-trimethoxybenzoyl)-1H-
indol-1-yl)methyl)benzamide (12)
98.06% (retention time ¼ 26.67).
A mixture of compound 26 (1.0 g, 2.04 mmol) dissolved in
MeOH (20 mL), and 1 N LiOH (5 mL) was heated under reflux for
3 h. To the reaction was added H2O (50 mL) followed by acidifica-
tion with 3 N HCl and extraction with EtOAc (50 mL ꢁ 3). The
organic layers were collected and purified by column chromatog-
raphy to afford a white solid (0.87 g). The resulting product (0.6 g,
1.26 mmol) was dissolved in DMF (4 mL), and EDC$HCl (0.32 g,
1.62 mmol), HOBt (0.18 g, 1.3 mmol), NMM (0.15 mL, 1.23 mmol)
and NH2OTP (0.16 g, 1.28 mmol) were added. After stirring at room
temperature for 2 h, the reaction was quenched with H2O and
extracted with EtOAc (50 mL ꢁ 3). The combined organic layer was
dried over anhydrous MgSO4 and concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(CH2Cl2/CH3OH/aqueous NH3 ¼ 20:1:1) to give a white solid. To the
solid was added 10% TFA in MeOH (10 mL) and the mixture was
4.1.5. N-(2-Amino-phenyl)-4-[6-methoxy-3-(3,4,5-trimethoxy-
benzoyl)-indol-1-ylmethyl]-benzamide (14)
Compound 26 (1.0 g, 2.04 mmol) was dissolved in MeOH
(20 mL), 1 N LiOH (5 mL) was added and the mixture was heated
under reflux for 3 h. To the reaction was added H2O (50 mL) and this
was followed by acidification with 3 N HCl and extraction with
EtOAc (50 mL ꢁ 3). The organic layers were collected and purified
by column chromatography to afford a white solid (0.87 g). The
resulting product (0.4 g, 0.8 mmol) was dissolved in DMF (4 mL),
and HOBt (0.2 g, 1.5 mmol), EDC$HCl (0.17 g, 0.8 mmol), NMM
(0.25 mL), and o-phenylenediamine (0.12 g, 1.1 mmol) were added.
After stirring at room temperature for 2 h, H2O (30 mL) was added
to the reaction mixture and the resulting brown precipitate was
collected by filtration and purified by column chromatography (n-

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H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
Fig. 6. (A) Anticancer activity of compound 12 in a xenograft model of multiple myeloma RPMI-8226 cells. Tumor growth of RPMI-822 xenograft nude mice treated with or without
compound 12 (50 and 100 mg/kg) was tracked by the mean tumor volume (mm3) S.E and calculated as % tumor growth inhibition (%TGI). Tumor volume was determined using
caliper measurements and was calculated as the product of 1/2 ꢁ length ꢁ width2. (B) Body weight (g) of the mice. *, p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the
control group. TR: treatment-related death.
hexane/EtOAc ¼ 1:4) to afford compound 14 (70%) as a brown solid.
mp 150e151 ꢀC; 1H NMR (300 MHz, DMSO-d6)
9.58 (s,1H), 8.25 (s,
4.1.6. (E)-N-Hydroxy-3-(3-(3-(3,4,5-trimethoxybenzoyl)-1H-indol-
1-ylsulfonyl)phenyl)-acrylamide (15)
d
1H), 8.12 (d, J ¼ 8.7 Hz,1H), 7.91 (s, J ¼ 8.1 Hz, 2H), 7.46 (d, J ¼ 8.1 Hz,
2H), 7.08e7.15 (m, 4H), 6.89e6.98 (m, 2H), 6.75 (d, J ¼ 7.2 Hz, 1H),
6.56 (t, J ¼ 7.8 Hz, 1H), 5.59 (s, 2H), 3.86 (s, 6H), 3.79 (s, 3H), 3.76 (s,
3H). HRMS (ESI) for C33H31N3O6 (Mþ) calcd 565.2213, found
565.2264. HPLC purity of 99.20% (retention time ¼ 34.75).
This compound was obtained in 40% overall yield from com-
pound 21 in a manner similar to that described for the preparation
of 10 from 19: mp 172e173 ꢀC; 1H NMR (300 MHz, DMSO-d6)
d 8.40
(s, 2H), 8.13e8.19 (m, 2H), 8.08 (d, J ¼ 8.1 Hz, 2H), 7.94 (d, J ¼ 7.8 Hz,
1H), 7.65 (t, J ¼ 7.8 Hz, 1H), 7.4e7.53 (m, 3H), 7.19 (s, 2H), 6.57 (d,

H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
1277
J ¼ 15.9 Hz, 2H), 3.86 (s, 6H), 3.80 (s, 3H). HRMS (ESI) for
27H24N2O8S (Mþ) calcd 536.1253, found 536.1332. HPLC purity of
100.0% (retention time ¼ 33.24).
5 h. The reaction mixture was cooled to ambient temperature and
filtered through a pad of Celite. The filtrate was concentrated under
reduced pressure and the resulting brown residue was purified by
flash column chromatography (n-hexane: EtOAc ¼ 2: 1) to afford
C
4.1.7. N-Hydroxy-4-[3-(3,4,5-trimethoxy-benzoyl)-indol-1-
ylmethyl]-benzamide (16)
compound 22 as a yellow oil. 1H NMR (500 MHz, CDCl3)
d 8.11 (d,
J ¼ 9.0 Hz, 1H), 8.02 (s, 1H), 7.93 (s, 1H), 7.86 (d, J ¼ 8.0 Hz, 1H),
7.60e7.72 (m, 2H), 7.45e7.53 (m, 2H), 7.37 (d, J ¼ 7.0 Hz, 1H), 7.13 (s,
2H), 7.02 (dd, J ¼ 2.5, 9.0 Hz, 1H), 6.38 (d, J ¼ 16.0 Hz, 1H), 3.97 (s,
3H), 3.92 (m, 9H), 1.33 (s, 9H).
This compound was obtained in 48% overall yield from com-
pound 29 in a manner similar to that described for the preparation
of 10 from 19: 1H NMR (500 MHz, acetone-d6)
d 10.35 (s, 1H),
8.37e8.41 (m, 1H), 8.18 (s, 1H), 7.51e7.58 (m, 4H), 7.37 (d, J ¼ 7.5 Hz,
2H), 7.27e7.30 (m, 2H), 7.14 (s, 2H), 6.53 (d, J ¼ 16.0 Hz, 1H), 5.63 (s,
2H), 3.88 (s, 6H), 3.80 (s, 3H). HRMS (ESI) for C28H27N2O6 (MþHþ)
calcd 487.1869, found: 487.1858. HPLC purity of 98.23% (retention
time ¼ 30.04).
4.1.13. 4-[6-Methoxy-3-(3,4,5-trimethoxy-benzoyl)-indol-1-
ylmethyl]-benzoic acid methyl ester (26)
NaH (0.15 g, 3.8 mmol) was added to a solution of compound 9
(0.9 g, 2.6 mmol) in DMF (5 mL) and the mixture was stirred for
20 min. Methyl (4-chloromethyl)benzoate (0.52 g, 2.8 mmol) was
added to the resulting mixture which was then stirred for 16 h. The
reaction was quenched with H2O (50 mL) and extracted with EtOAc
(50 mL ꢁ 3). The organic layers were collected and dried in vacuum
to afford compound 26 (1.19 g, 94%) as an oily product. 1H NMR
4.1.8. N-Hydroxy-4-((6-methoxy-3-(4-methoxybenzoyl)-1H-indol-
1-yl)methyl)benzamide (17)
This compound was obtained in 65% overall yield from com-
pound 25 in a manner similar to that described for the preparation
of 12 from 9: mp 120e121 ꢀC; 1H NMR (300 MHz, acetone-d6)
d
8.26
(500 MHz, CDCl3)
d
7.94 (d, J ¼ 8.5 Hz, 2H), 7.60 (d, J ¼ 9.0 Hz, 1H),
(d, J ¼ 8.4 Hz, 1H), 7.99 (s, 1H), 7.87e7.92 (m, 2H), 7.81 (d, J ¼ 8.4 Hz,
2H), 7.39 (d, J ¼ 8.4 Hz, 2H), 7.04e7.08 (m, 3H), 6.94 (dd, J ¼ 2.4,
8.7 Hz, 1H), 5.64 (s, 2H), 3.91 (s, 3H), 3.80 (s, 3H). MS (EI): m/z
7.19 (d, J ¼ 8.0 Hz, 2H), 7.12e7.15 (m, 3H), 6.86 (dd, J ¼ 2.0, 8.5 Hz,
1H), 6.68 (s, 1H), 5.83 (s, 2H), 5.29 (s, 1H), 3.93 (s, 3H), 3.89 (s, 6H),
3.87 (s, 3H), 3.81 (s, 3H).
(%):431.1602 (100); HRMS (ESI) for
C
25H22N2O5 (Mþ) calcd
430.1602, found 430.1529. HPLC purity of 97.11% (retention
time ¼ 30.45).
4.1.14. (1-(4-Bromobenzyl)-6-methoxy-1H-indol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (28)
NaH (0.15 g, 3.8 mmol) was added to a solution of compound 9
(0.9 g, 2.6 mmol) in DMF (5 mL) and the mixture was stirred for
20 min. Methyl 4-bromobenzyl bromide (0.8 g, 3.2 mmol) was
added to the resulting mixture which was then stirred for 16 h. The
reaction was quenched with H2O (50 mL) and extracted with EtOAc
(50 mL ꢁ 3). The organic layers were collected and dried in vacuum
to afford compound 28 as an oily product. 1H NMR (300 MHz,
4.1.9. [1-(3-Bromo-benzenesulfonyl)-6-methoxy-1H-indol-3-yl]-
(3,4,5-trimethoxy-phenyl)- methanone (19)
3-Bromobenzenesulfonyl chloride (4.12 g, 16.11 mmol) was
added to a mixture of 9 (5.0 g, 10.65 mmol) and 60% of NaH (0.64 g,
16.11 mmol) in anhydrous DMF (50 mL) and the mixture was stirred
at room temperature for 3 h. The reaction was quenched with H2O,
and extracted with EtOAc (100 mL ꢁ 3). The combined organic layer
was dried over anhydrous MgSO4 and dried in vacuo to give a yel-
low residue which was purified by flash column chromatography
(n-hexane: EtOAc ¼ 4: 1) to afford compound 19 as a white powder.
CDCl3)
d
8.27 (d, J ¼ 6.0 Hz, 1H), 7.40e7.51 (m, 3H), 6.97e7.10 (m,
5H), 6.74 (d, J ¼ 3.0 Hz, 1H), 5.25 (s, 2H), 3.91 (s, 3H), 3.84 (s, 6H),
3.83 (s, 3H).
1H NMR (300 MHz, CDCl3)
(s, 1H), 7.84 (d, J ¼ 9.0 Hz, 1H), 7.73 (d, J ¼ 9.0 Hz, 1H), 7.50 (d,
J ¼ 0.9 Hz,1H), 7.39 (t, J ¼ 9.0 Hz,1H), 7.13 (s, 2H), 7.02e7.05 (m,1H),
3.92e3.97 (m, 12H).
d
8.13 (d, J ¼ 9.0 Hz, 1H), 8.06 (s, 1H), 7.90
4.2. Biology
4.2.1. HeLa nuclear HDAC enzyme inhibition
The HeLa nuclear extract HDAC activity was measured by using
the HDAC Fluorescent Activity Assay Kit (BioVision, CA) according
to manufacturer's instructions. Briefly, the HDAC fluorometric
substrate and assay buffer were added to HeLa nuclear extracts in a
96-well format and incubated at 37 ꢀC for 30 min. The reaction was
stopped by adding lysine developer, and the mixture was incubated
for another 30 min at 37 ꢀC. Additional negative controls included
incubation without the nuclear extract, without the substrate, or
4.1.10. [1-(4-Bromo-benzenesulfonyl)-6-methoxy-1H-indol-3-yl]-
(3,4,5-trimethoxy-phenyl)- methanone (20)
This compound was obtained yield from compound 9 in a
manner similar to that described for the preparation of 19: 1H NMR
(500 MHz, CDCl3)
d
8.11 (d, J ¼ 10.0 Hz, 1H), 7.89 (s, 1H), 7.75 (dd,
J ¼ 1.5, 7.0 Hz, 2H), 7.61 (dd, J ¼ 1.5, 7.0 Hz, 2H), 7.49 (d, J ¼ 2.5 Hz,
1H), 7.12 (s, 2H), 7.02 (dd, J ¼ 2.5, 8.5 Hz, 1H), 3.96 (s, 3H), 3.91 (s,
6H), 3. 93 (s, 3H).
without both. TSA at 1
mM was used as the positive control. A
fluorescence plate reader with excitation at 355 nm and emission at
460 nm was used to quantify HDAC activity.
4.1.11. (1-(3-Bromophenylsulfonyl)-1H-indol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (21)
4.2.2. In vitro cell growth inhibitory activity
This compound was obtained from compound 18 in a manner
4.2.2.1. Cell culture. All human cancer cells were maintained in
RPMI 1640 medium containing 100 units/mL penicillin G sodium,
similar to that described for the preparation of 19 from 9: 1H NMR
(300 MHz, CDCl3)
7.80e8.02 (m, 2H), 7.85e7.89 (m, 1H), 7.70e7.74 (m, 1H), 7.33e7.47
d
8.23e8.28 (m, 1H), 8.05 (t, J ¼ 1.8 Hz, 1H),
100
25
m
g/mL streptomycin sulfate, 0.25
mg/mL amphotericin B, and
mg/mL gentamicin. The medium was supplemented with 10%
(m, 3H), 7.13 (s, 2H), 3.97 (s, 3H), 3.92 (s, 6H).
fetal bovine serum. The cells were cultured in a humidified incu-
bator at 37 ꢀC, in an atmosphere of 5% CO2 and 95% air.
4.1.12. (E)-tert-Butyl-3-(3-(6-methoxy-3-(3,4,5-
trimethoxybenzoyl)-1H-indol-1-ylsulfonyl)) phenyl acrylate (22)
A mixture of compound 19 (6.7 g, 11.95 mmol), Pd(OAc)2 (1.47 g,
6.58 mmol), PPh3 (1.57 g, 5.98 mmol), K2CO3 (1.65 g, 11.95 mmol),
triethylamine (1.7 mL, 11.95 mmol), tert-butyl acrylate (2.6 mL,
17.93 mmol), and anhydrous DMF (5 mL) was stirred at 100 ꢀC for
4.2.2.2. The sulforhodamine B (SRB) assay. Cell proliferation was
determined with the SRB assay. Human cancer A549 (non-small
cell lung cancer), HCT116 (human colon carcinoma), and PC-3
(prostate) cells were seeded in 96-well plates in medium with 5%
FBS. Cells were fixed with 10% trichloroacetic acid (TCA) to

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H.-Y. Lee et al. / European Journal of Medicinal Chemistry 125 (2017) 1268e1278
represent the cell population at the time of compound addition (T0)
after 24 h. Then additional incubation of DMSO or test compound
for 48 h, cells were fixed with 10% TCA and SRB at 0.4% (w/v) in 1%
acetic acid. Unbound SRB was washed out by 1% acetic acid and SRB
bound cells were solubilized with 10 mM Trizma base to determine
the absorbance at 515 nm. Using the following absorbance mea-
surements, time zero (T0), control growth (C), and cell growth in the
presence of the compound (Tx), the percentage growth was calcu-
lated at each of the compound concentration levels. Growth inhi-
bition of 50% (GI50) was calculated from [(Ti-Tz)/(C-Tz)] x 100 ¼ 50.
GI50 (inhibition rate of cell proliferation) was calculated as the test
drug concentration that reduced cell proliferation by 50%.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.11.033.
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agents, Chem. Med. Chem. 1 (2006) 1106e1118.
4.2.5. In vitro tubulin polymerization assay
Microtubule assembly was assessed using the CytoDYNAMIX
Screen kit (BK006P, Cytoskeleton Inc., Denver, CO). Purified porcine
tubulin proteins (>99% purity) were suspended in G-PEM buffer
containing 80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA,1 mM GTP (pH
6.9), and 15% glycerol in the absence or presence of indicated
compounds at 4 ꢀC. The mixture was immediately transferred to
pre-warmed 96-well plates, and absorbance was measured at
340 nm every 1 min for 30 min using a plate reader at 37 ꢀC
(SpectraMAX Plus, Molecular Devices Inc., Sunnyvale, CA).
4.2.6. Antitumor activity in vivo
Male nude athymic mice (male, 5e6 weeks) were subcutane-
ously injected with 1 ꢁ 107 prostate cancer PC3 cells and multiple
myeloma RPMI-8226 cells per mouse. When tumor volumes
reached approximately 150e350 mm3, mice were randomized by
tumor size as follows: In the prostate cancer PC3 xenograft model:
vehicle (5% DMSO þ 5% Cremophor in D5W), 100 mg/kg compound
12, 200 mg/kg compound 12 by oral administration daily. In the
multiple myeloma RPMI-8226 xenograft model: vehicle (5%
DMSO þ 5% Cremophor in D5W), 50 mg/kg compound 12, 100 mg/
kg compound 12 by intraperitoneal injection daily. Tumor volumes
were calculated using caliper measurements twice per week using
the formula volume (mm3) ¼ (length ꢁ width2)/2. %TGI (Tumor
Growth Inhibition) as determined by the formula: {1-[(Tt/T0)/(Ct/
C0)]/1-[C0/Ct]}*100. Tt: tumor volume of treated at time t. T0: tumor
volume of treated at time 0. Ct: tumor volume of control at time t.
C0: tumor volume of control at time 0. Body weights were
measured daily during the first week and twice per week.
[20] J.P. Liou, Y.L. Chang, F.M. Kuo, C.W. Chang, H.Y. Tseng, C.C. Wang, Y.N. Yang,
J.Y. Chang, S.J. Lee, H.P. Hsieh, Concise synthesis and structure-activity re-
lationships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroy-
lindoles, as novel classes of potent antitubulin agents, J. Med. Chem. 47 (2004)
4247e4257.
Acknowledgments
[21] C.C. Kuo, H.P. Hsieh, W.Y. Pan, C.P. Chen, J.P. Liou, S.J. Lee, Y.L. Chang, L.T. Chen,
C.T. Chen, J.Y. Chang, BPR0L075, a novel synthetic indole compound with
antimitotic activity in human cancer cells, exerts effective antitumoral activity
in vivo, Cancer Res. 64 (2004) 4621e4628.
This research were supported by the National Science Council of
the Republic of China (grant no. MOST 103-2113-M-038 -001
-MY3).
[22] https://clinicaltrials.gov/ct2/home.