N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines: A novel class of antitumor agents targeting the colchicine site on tubulin

Article abstract of DOI:10.1016/j.ejmech.2013.06.041

Structural optimizations of the prior lead 1a led to the discovery of a series of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinoline derivatives as a novel class of tubulin polymerization inhibitors targeted at the colchicine binding site. The most active compound 6d showed extremely high cytotoxicity against a human tumor cell line panel (A549, KB, KBvin, and DU145) with GI₅₀ values ranging from 1.5 to 1.7 nM, significantly more potent than paclitaxel, especially against the drug-resistant KBvin cell line, in the same assays. Analogs 5f, 6b, 6c, and 6e were also quite potent, with a GI₅₀ range of 0.011-0.19 μM. In further studies, active compounds 6b-e and 5f significantly inhibited tubulin assembly, with IC 50 values of 0.92 -1.0 μM and strongly inhibited colchicine binding to tubulin, with inhibition rates of 75-99% (at 5 μM), comparable with or more potent than combretastatin A-4 (IC₅₀ 0.96 μM). Current studies included design, synthesis, and biological evaluations of 24 new compounds (series 3-6). Related SAR analysis, molecular modeling, and evaluation of essential drug-like properties, i.e. water solubility, log P, and in vitro metabolic stability, were also performed.

Full text of DOI:10.1016/j.ejmech.2013.06.041

European Journal of Medicinal Chemistry 67 (2013) 196e207
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
N-Aryl-6-methoxy-1,2,3,4-tetrahydroquinolines: A novel class of
antitumor agents targeting the colchicine site on tubulin
,
Xiao-Feng Wang a c, Sheng-Biao Wang a, Emika Ohkoshi b, Li-Ting Wang b, Ernest Hamel d,
,
,
*
Keduo Qian b, Susan L. Morris-Natschke b, Kuo-Hsiung Lee b e, Lan Xie a
a Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA
c Pharmacy Department, Urumqi General Hospital, Lanzhou Military Region, Urumqi 830000, China
d Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, MD 21702, USA
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural optimizations of the prior lead 1a led to the discovery of a series of N-aryl-6-methoxy-1,2,3,4-
tetrahydroquinoline derivatives as a novel class of tubulin polymerization inhibitors targeted at the
colchicine binding site. The most active compound 6d showed extremely high cytotoxicity against a
human tumor cell line panel (A549, KB, KBvin, and DU145) with GI50 values ranging from 1.5 to 1.7 nM,
significantly more potent than paclitaxel, especially against the drug-resistant KBvin cell line, in the same
Received 19 April 2013
Received in revised form
18 June 2013
Accepted 20 June 2013
Available online 29 June 2013
assays. Analogs 5f, 6b, 6c, and 6e were also quite potent, with a GI50 range of 0.011e0.19
studies, active compounds 6bee and 5f significantly inhibited tubulin assembly, with IC50 values of 0.92
e1.0 M and strongly inhibited colchicine binding to tubulin, with inhibition rates of 75e99% (at 5 M),
comparable with or more potent than combretastatin A-4 (IC50 0.96 M). Current studies included
mM. In further
Keywords:
m
m
N-Aryl-6-methoxy-1,2,3,4-
tetrahydroquinolines
Cytotoxicity
Tubulin polymerization inhibitors
Colchicine binding site
m
design, synthesis, and biological evaluations of 24 new compounds (series 3e6). Related SAR analysis,
molecular modeling, and evaluation of essential drug-like properties, i.e. water solubility, log P, and
in vitro metabolic stability, were also performed.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
some have exhibited high potency, lower toxicity, and greater
efficacy against cancers resistant to existing tubulin-targeting drugs
Microtubules, formed by a dynamic polymerization and depo-
lymerization of - and -tubulin heterodimers, play important roles
[3]. More attractively, some of these compounds exhibit a vascular
disrupting effect on vascular endothelial cells and have entered into
clinical development as vascular disrupting agents (VDAs) for cancer
treatment. Examples of this novel class of anticancer agents are
CA-4P and ZD6126 (Fig. 1) [4,5].
In our prior studies on new anticancer agents [6,7], we discov-
ered methyl 6-chloro-2-(N-(4-methoxyphenyl)-N-methyl)amino-
nicotinate (1a, Fig. 2) as a lead compound with potent cytotoxic
a
b
in cellular activities [1]. For many years, certain drugs that bind to
the taxol or vinca site on tubulin, such as paclitaxel and vinblastine,
have been widely used in the clinic to treat cancers [2]. However,
their narrow therapeutic windows and the emergence of drug
resistance have encouraged continued efforts to discover safer and
more effective agents capable of treating resistant cancer pheno-
types. Another natural product colchicine (Fig. 1) binds to a unique
site on tubulin, distinct from the above two binding sites, and can
effectively inhibit tubulin assembly. Even though colchicine is not
used clinically because of its high toxicity, other small molecules
bind at the colchicine site, e.g., N-deacetyl-N-(2-mercaptoacetyl)-
colchicine (DAMAecolchicine) and combretastatin A-4 (CA-4), and
activity (GI50 0.20e0.26 mM) against A549, KB, KBvin, and DU145
human tumor cell lines. Its biological target was identified as
tubulin, specifically the colchicine site. Previous related SAR studies
indicated that a para-methoxyphenyl moiety (B-ring) is an essen-
tial pharmacophore, that a tertiary amine linker can provide a
feasible binding conformation, and that the substituted pyridine
ring (A-ring) can be modified to improve potency. Based on these
known SAR findings, we pursued optimization of lead 1a, following
the strategies shown in Fig. 2, to identify additional tubulin
inhibitors with new scaffolds and high potency. Because the o-
* Corresponding author. Tel./fax: þ86 10 6931690.
E-mail address: lanxieshi@yahoo.com (L. Xie).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.041

X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
197
Fig. 1. Colchicine, DAMAecolchicine, and clinical trial candidates targeted at the colchicine site of tubulin.
chloropyridine moiety could potentially lead to metabolic toxicity
resulting from in vivo nucleophilic replacement [8], we initially
replaced the pyridine ring (A-ring) of 1a with a benzene ring and
also changed the identities and positions of substituents R1 and
X on the A-ring (series 3e4 compounds) to investigate how these
modifications impacted inhibitory activity against tumor cell
growth. Previously, we also postulated that the degree of torsional
angle between the two aryl rings (A- and B-rings) might be crucial
for activity; therefore, our next modification focused on the N-
linkage. By applying a conformation restriction strategy [9], we
connected a propyl group on the N-linker to the neighboring ben-
zene ring (B-ring) to form a six-membered fused tetrahydropyr-
idine ring (series 5 compounds). By constraining the flexible
conformation, we could better explore feasible binding conforma-
tions and possible ligandetarget interactions. We next postulated
that introducing a fused aromatic ring onto the A-ring might make
the molecular binding conformation of new series 6 compounds
more similar to that of colchicine and enhance affinity with the
binding site on tubulin, leading to better antitumor activity.
All newly synthesized compounds were evaluated by cellular
screening against a human tumor cell line (HTCL) panel, and
selected potent compounds were also assayed for tubulin inhibi-
tion. Molecular modeling was used to illustrate the interaction
between tubulin and the new ligand(s) as well as differences
among ligands. Furthermore, certain essential parameters related
to drug-like properties, such as water solubility, log P, and meta-
bolic stability, were determined for highly potent compounds.
Chemical synthesis, biological evaluation, molecular modeling, and
SAR analysis are presented in this paper.
reaction [10] of commercially available 4-methoxyaniline and
2,4-dichlorobenzoic acid in the presence of Cu/Cu2O in 2-
ethoxyethanol, following esterification with DMFeDMA (two
equivalents) in toluene at reflux for 24 h. Intermediates 2bed were
obtained by a BuchwaldeHartwig coupling reaction between 4-
methoxyaniline and methyl 5-substituted-3-bromo (or iodo) ben-
zoate in the presence of catalyst Pd(OAc)2 and Cs2CO3 either with X-
phos [11] under microwave (mw) irradiation at 120e150 ꢀC (Method
A for 2b and 2c) or with BINAP and traditional heating in toluene at
reflux under nitrogen protection (Method B for 2d). Subsequently,
secondary amines 2aed were treated with methyl iodide in the
presence of sodium hydride to afford corresponding tertiary diaryl-
amines 3a and 4aec. When using LiAlH4 in THF at 0 ꢀC for 1 h, the
ester group of 3a was reduced to a hydroxymethyl moiety, affording
3binnearlyquantitativeyield. Compound 3bwasfurthermethylated
with methyl iodide to give ether 3c. Alternatively, the ester group in
3a and 4aec was converted to an N-methylcarbamoyl group
by treatment with methylamine in MeOH under mw irradiation at
100e120 ꢀC to produce corresponding compounds 3d and 4def,
respectively, in high yields (79e89%). Methylation of 4f with methyl
iodide yielded compound 4g with a N,N-dimethylcarbamoyl group
on the A-ring. Ester compound 4c was hydrolyzed under basic con-
ditions to produce carboxylic acid compound 4h. Compound 4h was
then treated with 1-hydroxybenzotriazole (HOBt) in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) hydrochlo-
ride salt, followed by reaction with cyclopropylamine or cyclo-
pentylamine to produce corresponding N-cyclopropylamide and N-
cyclopentylamide compounds 4i and 4j, respectively [12]. By using
BuchwaldeHartwig coupling reactions and the same conditions,
compounds 5a,b and 6a,b were synthesized from intermediate 6-
methoxy-1,2,3,4-tetrahydroquinoline (7) [13] and an aryl halide,
such as phenyl, naphthyl, and quinolyl halides, in 68e75% yields.
Similarly to series 3 and4, the ester group in 5a and5bwas converted
into an N-alkylcarbamoyl or carboxy group to provide corresponding
2. Chemistry
Thesynthesesoftargetcompounds3e6areoutlinedinSchemes1
and 2. Intermediate 2a was prepared by Ullmann condensation
R1
o-
OMe
Z
A
A
B
O
Cl
N
OMe
B
X
Y
N
o-
OMe
3
6
A
A Ring
Modification
Cl
N
N
fused A-ring
R1
O
R1
B
O
m-
OMe
1a
OMe
OMe
Linker
A
B
4
A
B
X
N
X
N
5
Fig. 2. Modification strategies and new target compounds 3e6 series.

198
X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
Scheme 1. a) i. K2CO3, Cu, Cu2O, 2-ethoxyethanol, N2, 130 ꢀC, 24 h, 67%; ii. DMFeDMA in toluene, reflux, 2 h, 85%; b) i. Cs2CO3, Pd(OAc)2, X-Phos, Celite, toluene/t-BuOH, N2, mw,
120e150 ꢀC, 30e60 min, 74% for 2b and 2c; c) Cs2CO3, Pd(OAc)2, BINAP, toluene, N2, reflux 12 h, 64% for 2d; d) MeI/NaH (60% oil suspension), 0 ꢀC, 1 h; e) LiAlH4/THF, 0 ꢀC, 1 h, 97%; f)
30% NH2Me/MeOH, 100e120 ꢀC, mw, 1e2 h for 3d, 4def; g) MeOH/THF, 3N NaOH aq, reflux, 5 h, 90% for 4h; h) EDCI, HOBt, cyclopropylamine or cyclopentylamine, CH2Cl2, rt, 24 h
for 4i and 4j.
compounds 5cef by using the same methods described above.
Differently from 6a and 6b, the preparation of 6c and 6d involved
coupling 7 with a 2-substituted 4-haloquinazoline underEtOH reflux
in the presence of NaHCO3, resulting in 79e87% yields. Using the
same method forpreparing5c, compound 6dwasconvertedto 6e. All
new series 3e6 compounds were identified by 1H NMR and MS
spectroscopic data, with purity determined by HPLC.
mouth), KBvin, a P-gp-expressing multidrug-resistant cell line
(vincristine-resistant KB) [14,15], and DU145 (prostate cancer) with
paclitaxel as a reference compound. The in vitro anticancer activity
(GI50) was determined using the established sulforhodamine B
(SRB) method [16]. The cytotoxicity data of all new compounds in
HTCL assays are listed in Tables 1 and 2. Subsequently, selected
compounds with high potency in cellular assays were further tested
in tubulin assays to determine biological target and binding site.
In series 3 compounds with a phenyl A-ring and substituent X
equal to m-chloro, two compounds 3a (R1 ¼ o-COOMe) and 3c
(R1 ¼ o-CH2OMe) showed promising potency with low micromolar
3. Results and discussion
3.1. Evaluation of cytotoxicity and tubulin inhibition activity in vitro
GI50 values of 1.68e5.24
mM, indicating that replacement of the
The 24 newly synthesized tertiary arylamines (series 3e6) were
evaluated in cellular cytotoxicity assays against a HTCL panel,
including A549 (lung carcinoma), KB (epidermoid carcinoma of the
pyridine A-ring in lead 1a is tolerated and R1 on the A-ring is
modifiable. When the COOMe group (R1) was moved to the meta-
position on the A-ring, the resulting compound 4a exhibited
X = Cl
X = Br
Scheme 2. a) Cs2CO3, Pd(OAc)2, X-Phos, Celite, N2, mw, 120 ꢀC, 30 min, 77% for 5a; b) Cs2CO3, Pd(OAc)2, BINAP, toluene, N2, reflux, 12 h, 68e75% for 5b and 6a,b; c) 30% NH2Me/
MeOH, 100 ꢀC, mw, 1 h for 5c, 5d, and 6e; d) MeOH/THF, 3 N NaOH aq, reflux, 5 h, 82% for 5e; e) EDCI, HOBt, cyclopropylamine, CH2Cl2, rt, 24 h, 90%; f) NaHCO3/EtOH, reflux, 3 h for
6c and 6d.

X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
199
Table 1
Cytotoxicity of series 3e5 compounds against human tumor cell lines (HTCL).
R1
R1
m-
COOMe
m-
OMe
OMe
A
o-
B
A
OMe
Cl
N
N
B
B
A
X
N
X
N
3-4
1a
5
X
R1
GI50
(
m
M)a
A549
KB
KBvin
DU145
1a
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
0.23 ꢁ 0.01
3.36 ꢁ 0.49
21.7 ꢁ 2.53
4.53 ꢁ 0.84
43.2 ꢁ 4.81
1.60 ꢁ 0.36
15.1 ꢁ 2.6
1.24 ꢁ 0.34
0.27 ꢁ 0.01
14.7 ꢁ 2.23
0.25 ꢁ 0.07
2.83 ꢁ 0.59
NAb
0.26 ꢁ 0.04
1.98 ꢁ 0.37
19.5 ꢁ 1.33
5.24 ꢁ 0.42
188 ꢁ 33.8
1.61 ꢁ 0.08
13.5 ꢁ 1.21
1.36 ꢁ 0.17
0.30 ꢁ 0.04
12.3 ꢁ 1.72
0.21 ꢁ 0.03
6.52 ꢁ 1.16
NA
0.20 ꢁ 0.03
1.89 ꢁ 0.17
18.1 ꢁ 2.24
2.85 ꢁ 0.62
61.0 ꢁ 5.13
1.57 ꢁ 0.10
13.1 ꢁ 1.41
1.20 ꢁ 0.39
0.23 ꢁ 0.04
12.6 ꢁ 1.81
0.14 ꢁ 0.01
2.38 ꢁ 0.37
NA
0.21 ꢁ 0.04
1.68 ꢁ 0.04
16.0 ꢁ 2.73
3.13 ꢁ 0.35
98.9 ꢁ 4.22
1.46 ꢁ 0.16
11.5 ꢁ 1.71
0.97 ꢁ 0.08
0.23 ꢁ 0.03
11.9 ꢁ 2.20
0.16 ꢁ 0.07
3.34 ꢁ 0.54
NA
Cl
Cl
Cl
Cl
o-COOMe
o-CH2OH
o-CH2OMe
o-CONHMe
m-COOMe
m-COOMe
m-COOMe
m-CONHMe
m-CONHMe
m-CONHMe
m-CONMe2
m-COOH
Cl
CF3
Br
Cl
CF3
Br
Br
Br
4g
4h
4i
4j
Br
Br
0.17 ꢁ 0.02
0.12 ꢁ 0.02
0.17 ꢁ 0.02
0.12 ꢁ 0.01
0.17 ꢁ 0.02
0.12 ꢁ 0.01
0.17 ꢁ 0.02
0.11 ꢁ 0.01
5a
5b
5c
5d
Cl
Br
Cl
Br
m-COOMe
m-COOMe
m-CONHMe
m-CONHMe
2.23 ꢁ 0.23
1.12 ꢁ 0.14
0.74 ꢁ 0.08
0.37 ꢁ 0.08
2.33 ꢁ 0.47
1.37 ꢁ 0.31
1.04 ꢁ 0.11
0.63 ꢁ 0.34
1.78 ꢁ 0.31
1.27 ꢁ 0.22
0.43 ꢁ 0.03
0.93 ꢁ 0.02
1.51 ꢁ 0.46
1.28 ꢁ 0.25
1.02 ꢁ 0.10
0.56 ꢁ 0.09
5f
Br
0.17 ꢁ 0.01
0.16 ꢁ 0.03
0.19 ꢁ 0.01
0.15 ꢁ 0.02
m-
Paclitaxelc
0.0076 ꢁ 0.0017
0.0064 ꢁ 0.0014
1.21 ꢁ 0.19
0.006 ꢁ 0.001
a
Concentration of compound that inhibits 50% human tumor cell growth, presented as mean ꢁ standard deviation (SD), performed at least in triplicate.
b
c
Not active; test compound (60
Positive control.
mM) did not reach 50% inhibition.
somewhat improved activity (GI50 1.46e1.61
m
M) compared with
potent than series 3 compounds (3a, 3d), we postulated that the
meta-substitution on the A-ring might favor an appropriate
torsional angle between the two aromatic rings, due to decreased
steric hindrance around the N-linker, which would be consistent
with our prior hypothesis. Moreover, the high potency of 4d, 4f, 4i,
and 4j demonstrated that a hydrophobic meta-N-alkylcarbamoyl
group with a suitable volume could enhance antitumor potency.
When the N-linker between the A- and B-rings was incorpo-
rated into a six-membered 1,2,3,4-tetrahydropyridine ring fused
with the B-ring, all five compounds 5aed, f exhibited high cyto-
3a (R1 ¼ o-COOMe, GI50 1.68e3.36
mM). This finding prompted us to
synthesize compounds 4bef, in which the meta-R1 was either
maintained as an ester (COOMe) or changed to a N-methyl-
carbamoyl (CONHMe) group, while substituent X was present as
either a chloro, trifluoromethyl or bromo group. Interestingly,
compounds 4a, 4c, 4d, and 4f where X is either chloride or bromide
were more potent than 3a. In addition, compounds 4d and 4f with a
meta-N-methylcarbamoyl group (R1) showed significantly
improved potency with a GI50 range of 0.14e0.30 mM, compared
with related meta-ester compounds 4a and 4c. In contrast, 4b and
4e with a trifluoromethyl (X) group were less active than corre-
sponding chloro- and bromo-compounds in the same assays. Next,
the cytotoxicity results for bromo-substituted compounds 4gej
revealed the impact of the m-alkanoyl group (R1). When the meta-
N-methylcarbamoyl (4f) or meta-ester (4c) was converted to a more
bulky N,N-dimethylcarbamoyl (4g) or more polar carboxy (4f)
group, potency decreased or was abolished completely, respec-
tively. But when R1 was an N-cyclopropylcarbamoyl or N-cyclo-
pentylcarbamoyl group, the corresponding compounds 4i and 4j
toxicity with GI50 values of 0.15e2.33 mM (Table 1). Thus, the ring-
restricted N-linker was favorable for cytotoxic activity. Conse-
quently, all series 6 compounds contain a 6-methoxy-1,2,3,4-
tetrahydroquinoline moiety connected to an aromatic (naphtha-
lene) or heteroaromatic (quinoline or quinazoline) bicyclic ring
system, in which the resonance system of the A-ring is extended
over an additional fused aromatic ring. As shown in Table 2, except
for 6a with a naphthyl A-ring system, compounds with either a
quinoline (6b) or quinazoline (6cee) moiety exhibited extremely
high cytotoxicity in the HTCL assays. Among them, 6d with a 2-
chloroquinazoline moiety (A-ring) was the most potent with GI50
values ranging from 1.5 to 1.7 nM. Thus, 6d was more potent than
paclitaxel in the same assays, especially against the drug-resistant
showed improved potency with GI50 values of 0.11e0.17 mM, more
potent than the other series 3 and 4 compounds, as well as 1a.
Because corresponding series 4 compounds (4a, 4d) were more

200
X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
Table 2
Cytotoxicity of 6 series against human tumor cell lines.
Fused A-ring
GI50
A549
(m
M)a
X
Y
Z
KB
15.6 ꢁ 1.3
KBvin
17.6 ꢁ 2.5
DU145
14.8 ꢁ 1.7
6a
6b
6c
6d
H
CH
CH
N
N
N
CH
N
N
N
N
17.8 ꢁ 2.6
Me
Me
Cl
0.045 ꢁ 0.008
0.018 ꢁ 0.001
0.0017 ꢁ 0.0003
0.027 ꢁ 0.005
0.0076 ꢁ 0.0017
0.091 ꢁ 0.009
0.011 ꢁ 0.001
0.0017 ꢁ 0.0008
0.025 ꢁ 0.002
0.0064 ꢁ 0.0014
0.055 ꢁ 0.010
0.018 ꢁ 0.004
0.0017 ꢁ 0.0003
0.029 ꢁ 0.005
1.21 ꢁ 0.19
0.038 ꢁ 0.008
0.015 ꢁ 0.005
0.0015 ꢁ 0.0003
0.023 ꢁ 0.003
0.006 ꢁ 0.001
6e
NHMe
Paclitaxelb
a
Concentration of compound that inhibits 50% human tumor cell growth.
Positive control.
b
KBvin cell line (GI50 0.0017
m
M versus 1.21
m
M, respectively).
comparable with and greater than those of 5f, respectively. Consis-
tent with the cellular assays, compound 6d was the most potent
Compounds 6b (2-methylquinoline), 6c (2-methylquinazoline), and
6e (2-methylaminoquinazoline) also showed high cytotoxicity with
a GI50 range of 11e91 nM, more potent by at least ten-fold than 1a
and series 3e5 compounds. Similarly to 6d, analogs 6b, 6c, and 6e
were also much more potent than paclitaxel against KBvin cell
growth, indicating that this compound type has great potential to
overcome resistance to paclitaxel.
compound with an IC50 value of 0.93
mM against tubulin assembly
and inhibitory rates of 99% and 95% at 5
m
M and 1 M, respectively,
m
for competitively inhibiting colchicine binding to tubulin. These data
are comparable or better than those for the reference compound CA-
4 (IC50 0.96 mM, 98% and 90% respectively). Thus, N-aryl-6-methoxy-
1,2,3,4-tetrahydroquinoline compounds, e.g., series 5e6, likely pre-
sent a novel class of highly potent tubulin polymerization inhibitors
targeted at the colchicine binding site. Meanwhile, we postulated
that a fused bicyclic aromatic A-ring might interact with the
colchicine site in some way, possibly resulting in increased affinity
for tubulin. On the other hand, the active 6 series compounds might
also interact with other unidentified target(s), because 6bee
displayed much higher potency than 5f in the cellular cytotoxicity
assays, but similar inhibitory activity against tubulin polymerization.
Based on these results in cellular assays, active compounds 4f, 4i,
5f, and 6bee (GI50 < 1 mM) were evaluated in tubulin inhibition
assays, in parallel with CA-4, a drug candidate in clinical trials, as a
reference. As shown in Table 3, while N-methyl linked compounds 4f
and 4i (open N-linker) showed good inhibitory potency against
tubulin assembly (IC50 1.6e2.2
1,2,3,4-tetrahydroquinoline compound 5f (ring-restricted N-linker)
exhibited improved potency in both tubulin assembly (IC50 1.0 M)
mM), the N-phenyl-6-methoxy-
m
and colchicine binding (75%) assays. Thus, the presence of the ring-
restricted N-linker probably increases compound affinity for tubulin.
Furthermore, 6bee with both a ring-restricted N-linker and a fused
bicyclic aromatic ring system (A-ring) also displayed high potency in
3.2. Molecular modeling
To better understand interactions between the newly synthe-
sized active inhibitors and tubulin, we investigated potential
binding modes of active compounds 4i, 5f, and 6d at the colchicine
site in the tubulin dimer by using the CDOCKER program in the
Discovery Studio 3.0 software with the tubulin crystal structure
(PDB: 1SA0) [17,18], as in our previous study. In the binding models
shown in Fig. 3A, both 4i (purple) and 5f (green) displayed low
energy binding conformations (ꢂ30.58 and ꢂ33.31 kcal/mol,
respectively) that superimposed well with each other and with
DAMAecolchicine (cyan), the ligand used in the tubulin crystal
structure. As seen in Fig. 3A, a hydrogen bond is present between
the tubulin assembly (IC50 0.92e1.00
mM) and inhibition of colchi-
cine binding (87e99% at 5
m
M and 61e95% at 1 M) assays,
m
Table 3
Inhibition of tubulin polymerizationa and colchicine binding to tubulin.b
Compound
Inhibition of tubulin
Inhibition of colchicine
binding (%)
inhibition ꢁ SD
assembly IC50
(m
M) ꢁ SD
At 5
m
M
At 1 mM
NDc
ND
ND
61 ꢁ 2.0
89 ꢁ 2.0
95 ꢁ 0.7
76 ꢁ 0.1
90 ꢁ 0.2
Val181 in the a-T5 loop of tubulin and the amide carbonyl group on
4f
4i
5f
6b
6c
6d
6e
CA4d
2.2 ꢁ 0.2
1.6 ꢁ 0.04
1.0 ꢁ 0.1
71 ꢁ 0.3
69 ꢁ 3.0
75 ꢁ 1.0
87 ꢁ 2.0
98 ꢁ 0.2
99 ꢁ 0.6
95 ꢁ 0.2
98 ꢁ 0.6
the A ring of 4i and 5f. This hydrogen bond is also observed
between Val181 and the carbonyl group on the C-ring of DAMAe
colchicine. In addition, the N-cyclopropyl of the amide group in
both 4i and 5f overlaps with the methoxy group on the seven-
membered ring (C-ring) of DAMAecolchicine and stretches into a
1.0 ꢁ 0.1
1.0 ꢁ 0.1
0.93 ꢁ 0.04
0.92 ꢁ 0.06
0.96 ꢁ 0.07
small lipophilic pocket around the amino acids in
a-T5, b-H8, b-S8
a
and -S9 of tubulin. In addition, the 4-methoxyphenyl moiety (B-
b
The tubulin assembly assay measured the extent of assembly of 10 mM tubulin
after 20 min at 30 ꢀC.
ring) in 4i and 5f, regardless of whether the N-linker is open or
ring-restricted, overlaps with the trimethoxyphenyl portion of
DAMAecolchicine. The 4-OCH3 group on the B-ring of 4i and 5f
b
Tubulin: 1
m
M. [3H]colchicine: 5
m
M. Inhibitor: 5
mM or 1
mM (compounds
inhibiting colchicine binding by more than 80% at 5
mM were further tested).
Incubation was performed for 10 min at 37 ꢀC.
c
forms a hydrogen bond with the Cys241 side chain in
b-H7 of
Not determined.
d
Reference compound is a drug candidate in phase II/III clinical trials.
tubulin, and a similar interaction is also observed with the 20-OCH3

X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
201
74.89ꢀ between the two aromatic rings in 4i, allowing the N-alkyl
amide group on the A-ring to interact with the binding site as
described above. When the N-linker is connected with additional
carbons into a six-membered ring, a similar torsional angle (70.52ꢀ)
was found, maintaining a favorable binding conformation for 5f
and all major interactions with the binding site as mentioned above
for 4i. Fig. 3B shows that 6d (binding energy: ꢂ34.39 kcal/mol) has
a similar binding orientation as those of 4i and 5f, as well as good
superimposition with DAMAecolchicine. The major interactions of
the B-ring and the cyclic linker with tubulin are maintained. The
torsional angle (69.07ꢀ) between the two rings in 6d is similar to
that of 5f, resulting in a superimposition between the quinazoline
ring of 6d and the aromatic C-ring of DAMAecolchicine. Although
we postulated that the fused aromatic ring introduced in 6d would
play a role similar to that of the C-ring of colchicine within the
binding site on tubulin, no interaction was obvious in the compu-
tational model. Because of the torsional angle between the two aryl
rings, the 2-chloro group on the quinazoline in 6d [as well as the m-
bromo group on the phenyl (A-ring) in 4i and 5f (Fig. 3A)] was
orientated similarly to the acetyl group on the B-ring in DAMAe
colchicine. Thus, the X substituent on the quinazoline (or other
aromatic ring system) might interact with tubulin in some way and
be modifiable.
3.3. Drug-like property evaluations
Next, we evaluated aqueous solubility and log P parameters of
4f, 4i, 5f, and 6bee as described previously [20]. As shown in
Table 4, compounds 6b, 6c, and 6e showed much better aqueous
solubility (3.21e7.67
mg/mL) than the other four compounds
(<1.0 g/mL). Consistently, log P values of 6b, 6c, and 6e (1.07e
m
3.98) were also lower (4f, 4i, 5f, 6d > 4). Subsequently, metabolic
stability of 6bee was further evaluated by an in vitro human liver
microsome incubation assay with propranolol (moderate meta-
bolism in vivo, t1/2 3e5 h) and terfenadine (fast metabolism in vivo,
t1/2 < 3 h) as positive controls. Compounds 6c (t1/2 25.19 min) and
6e (t1/2 25.97 min) were more stable than terfenadine (t1/2 21.14),
while 6b and 6d were even less stable with short metabolic half-
lives of 7.89 min and 10.59 min, respectively, indicating quick
metabolism. In general, a potent compound with low metabolic
stability is not usually recognized as a promising drug candidate.
However, compounds targeting the colchicine site of tubulin are
proposed as VDAs to treat cancers. For this new type of anticancer
drug candidate, rapid metabolism might be desirable to decrease
drug toxicity in the body, as exemplified by ZD6126 (Fig. 1), which
was developed based on this hypothesis [21].
Fig. 3. (A) Predicted modes for 4i (purple stick) and 5f (green) binding with tubulin
(PDB code: 1SA0), and overlapping with DAMAecolchicine (cyan, the native ligand of
1SA0); (B) Superimposition of docked compound 6d (orange) with DAMAecolchicine
(cyan). Surrounding amino acid side chains are shown in gray stick format and labeled.
Hydrogen bonds are shown by green dashed lines, and the distance between ligands
ꢀ
and protein is less than 3 A. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
Table 4
Physicochemical parameters of selected compounds.
on the A-ring of DAMAecolchicine. Cys241 is a key amino acid that
interacts with most tubulin inhibitors bound at the colchicine site.
In the crystal structure, the C5 and C6 atoms of the B-ring of
DAMAecolchicine are involved in hydrophobic interactions with
Compound
At pH 7.4
Human liver microsome
t1/2 min
Water solubility
(mg/mL)
Log P
4f
4i
5f
6b
6c
6d
6e
0.81 ꢁ 0.01
0.16 ꢁ 0.03
0.26 ꢁ 0.05
3.21 ꢁ 0.60
7.67 ꢁ 0.40
0.45 ꢁ 0.06
7.21 ꢁ 0.06
4.02 ꢁ 0.11
4.33 ꢁ 0.12
4.75 ꢁ 0.09
3.98 ꢁ 0.02
3.65 ꢁ 0.03
4.13 ꢁ 0.05
1.07 ꢁ 0.21
NDc
ND
ND
7.89
25.19
10.59
25.97
40.82
21.14
the side chains of Alab250 and Leub255 in tubulin’s b-H8 region.
Although the NeCH3 linker in 4i is not in close proximity to
ꢀ
Ala
b250 and Leub255 (>4 A), the added carbons in the six-
membered tetrahydropyridine cyclic linker in 5f are closer to the
ꢀ
hydrophobic side chains of Ala
b
250 and Leu
b
255 (<4 A). The
additional van der Waal’s force between the cyclic linker and the
binding site would likely strengthen affinity of 5f for tubulin.
Therefore, our current modeling results might explain, at least
partially, the higher potency of 5f than 4f and 4i in the tubulin
assays (Table 3).
Propranolola
Terfenadineb
Data presented as mean from three separate experiments with or
without ꢁ standard deviation (SD).
a
Propranolol has moderate metabolic stability with t1/2 of 3e5 h in vivo.
Terfenadine has fast metabolic stability with t1/2 of <3 h in vivo.
Not determined.
b
Consistent with our earlier hypothesis [6,7], the steric hindrance
of the NeCH3 linker [19] results in a binding torsional angle of
c

202
X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
4. Conclusion
water 70e90/30e10]. For 6b, 6c and 6e, solvent B contained
0.025 mM ammonium acetate.
Modifications of prior lead 1a resulted in the synthesis and
biological evaluation of 24 new compounds (series 3e6). During this
process, active compound 6d showed extremely high cytotoxicity
against the screening HTCL panel with a GI50 value range of 1.5e
1.7 nM, more potent than paclitaxel in the same assays, especially
against resistant KBvin. Compound 6d also displayed significant
5.1.1. Methyl 4-chloro-2-(4-methoxyphenyl)aminobenzoate (2a)
A mixture of 2,4-dichlorobenzoic acid (1.91 g, 10 mmol), 4-
methoxyaniline (1.29 g, 10.5 mmol), copper powder (58 mg, 9%
mmol), cuprous oxide (58 mg, 4% mmol), anhydrous potassium car-
bonate (1.38 g, 10 mmol) and 2-ethoxyethanol (7.5 mL) was refluxed
under N2 protection for 24 h with stirring. The mixture was poured
into water and active carbon added. After filtration through Celite,
the filtrate was adjusted to pH 3e4 with aq HCl. The collected gray
solid was dried to obtain 1.85 g of 4-chloro-2-(4-methoxyphenyl)
aminobenzoic acid(0.83g, 3.0 mmol), whichwasmethylateddirectly
with DMFeDMA (0.79 mL, 6.0 mmol) in toluene (12 mL) under reflux
for 2 h. After removal of solvent in vacuo, the residue was purified by
flash column chromatography (gradient elution: EtOAc/petroleum
ether, 0e30%) to obtain 0.74 g of 2a in a 57% yield over two steps. It
was used next without further purification.
inhibitory activity in tubulin assembly (IC50 0.93
mM) and colchicine
binding (inhibition 99% at 5 M and 95% at 1
m
mM) assays, with
greater potency than CA-4. Similarly, analogs 6b, 6c, and 6e also
showed high potency in the cellular (GI50 11e91 nM) and tubulin
(IC50 0.92e1.0
mM) assays. Therefore, N-aryl-6-methoxy-1,2,3,4-
tetrahydroquinoline derivatives were identified as a novel class of
tubulin polymerization inhibitors targeted at the colchicine site.
Current SAR studies lead to the following conclusions: (1) a ring-
restricted N-linker is beneficial and provides a feasible torsional
angle (about 70e75ꢀ) between the two aryl ring systems (A and B
rings) for enhancing molecular affinity for tubulin; (2) the 6-
methoxy-1,2,3,4-tetrahydroquinoline moiety can serve as a phar-
macophore and the methoxy group on the B-ring is a necessary
binding point to tubulin; (3) isosteric replacement of the A-ring is
feasible and a fused aromatic ring, such as quinoline and quinazo-
line, improved molecular potency; (4) various m-substituents on the
A-ring are present in active compounds, and either halogen or N-
alkyl amide/amino might be beneficial. Molecular modeling results
illustrated some possible interactions of the active compounds with
the colchicine site of tubulin and supported our earlier hypothesis
that a feasible torsional angle between the two aryl rings might be
important for high potency. An evaluation of essential drug-like
properties, i.e. water solubility, log P, and metabolic stability
in vitro, also provided useful information. These results will help us
to develop drug candidates from this novel class of tubulin in-
hibitors targeted at the colchicine site in further studies.
5.1.2. Methyl 3-chloro-5-(4-methoxyphenylamino)benzoate (2b)
A
mixture of methyl 3-chloro-5-iodobenzoate (296 mg,
1.0 mmol), 4-methoxyaniline (148 mg, 1.2 mmol), Cs2CO3 (455 mg,
1.4 mmol), X-Phos (17 mg, 0.04 mmol), Pd(OAc)2 (11 mg,
0.05 mmol), Celite (228 mg), and t-BuOH (0.5 mL) in toluene (3 mL)
was heated at 120 ꢀC for 1 h under mw irradiation with stirring.
EtOAc (10 mL) was added to the mixture, and, after brief stirring,
insoluble solid was removed by filtration. After removal of organic
solvent in vacuo, the residue was purified by flash column chro-
matography (gradient elution: EtOAc/petroleum ether, 0e40%) to
obtain 2b as a yellow solid, 216 mg, 74% yield, which was used
directly in the next step without further purification.
5.1.3. Methyl 3-trifluoromethyl-5-(4-methoxyphenylamino)
benzoate (2c)
A
mixture of methyl 3-trifluoromethyl-5-bromobenzoate
5. Experimental section
(285 mg, 1.0 mmol), 4-methoxyaniline (147 mg, 1.2 mmol),
Cs2CO3 (455 mg, 1.4 mmol), X-Phos (17 mg, 0.04 mmol), Pd(OAc)2
(11 mg, 0.05 mmol), Celite (228 mg), and t-BuOH (0.5 mL) in
toluene (3.0 mL) was heated at 150 ꢀC for 30 min under mw irra-
diation. After work-up as for 2b, 240 mg of 2c was obtained in 74%
yield, yellow solid, which was directly used for the next step
without further purification.
5.1. Chemistry
Proton and carbon nuclear magnetic resonance (1H and 13C
NMR) spectra were measured on a JNM-ECA-400 (400 MHz)
spectrometer using tetramethylsilane (TMS) as internal standard.
The solvent used was CDCl3 unless otherwise indicated. Mass
spectra (MS) were measured on an API-150 mass spectrometer
with an electrospray ionization source from ABI, Inc. Melting points
were measured by a SGW X-4 Micro-Melting point detector
without correction. The mw reactions were performed on a mw
reactor from Biotage, Inc. Medium-pressure column chromatog-
raphy was performed using a CombiFlashÒ Companion system
from ISCO, Inc. Thin-layer chromatography (TLC) was performed on
silica gel GF254 plates. Silica gel GF254 and H (200e300 mesh)
from Qingdao Haiyang Chemical Company were used for TLC and
column chromatography, respectively. All commercial chemical
reagents were purchased from Beijing Chemical Works or Sigmae
Aldrich, Inc. Reagents NADPH, MgCl2, KH2PO4, K2HPO4, and refer-
ence compounds propranolol and terfenadine were purchased
from SigmaeAldrich. HPLC grade acetonitrile for LCeMS analysis
was purchased from VWR. Pooled human liver microsomes (lot no.
28831) were purchased from BD Biosciences (Woburn, MA).
Purities of target compounds were determined by using an Agilent
HPLC-1200 with UV detector and an Agilent Eclipse XDB-C18
5.1.4. Methyl 3-bromo-5-(4-methoxyphenylamino)benzoate (2d)
A mixture of methyl 3,5-dibromobenzoate (294 mg, 1.0 mmol),
4-methoxyaniline (146 mg, 1.2 mmol), Cs2CO3 (455 mg, 1.4 mmol),
BINAP (31 mg, 0.05 mmol), and Pd(OAc)2 (11 mg, 0.05 mmol) in
toluene (5e10 mL) was refluxed for 12 h under nitrogen protection.
After the mixture was cooled to rt, EtOAc (10 mL) was added. After
stirring, the insoluble material was removed by filtration. The sol-
vent was removed in vacuo, and the residue was purified by flash
column chromatography (gradient elution: EtOAc/petroleum ether,
0e40%) to produce 215 mg of 2d in 64% yield as a brown oil.
5.1.5. General procedure for methylation of aniline NH with methyl
iodide to prepare 3a and 4aec
To a solution of a diarylamine (2) and MeI (molar ratio: 1/2e3)
in anhydrous DMF (ca. 3 mL) was slowly added NaH (2e3 equiv,
60% oil suspension) at 0 ꢀC with stirring over about 1 h. When the
reaction was completed as monitored by TLC, the mixture was
poured into ice-water and extracted with EtOAc three times. The
combined organic phase was washed with water and brine suc-
cessively and dried over anhydrous Na2SO4 overnight. After
removal of solvent in vacuo, the crude product was purified by flash
column chromatography (gradient elution: EtOAc/petroleum ether,
column (150 mm ꢃ 4.6 mm, 5
m
m), flow rate 0.8 mL/min, UV
detection at 254 nm, and injection volume of 15
m
L. Mobile elution
was conducted with a mixture of solvents A and B [Condition
1: acetonitrile (ACN)/water 60e80/40e20; Condition 2: MeOH/

X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
203
0e50%) to give the corresponding methylated tertiary amine
compounds.
dd, J ¼ 8.4 & 2.0 Hz, ArH-5), 7.45 (1H, d, J ¼ 8.4, ArH-6). MS m/z (%)
292 (M þ 1, 22), 294 (M þ 3, 7), 228 (M ꢂ 63, 100).
5.1.5.1. Methyl 4-chloro-2-(N-(4-methoxyphenyl)-N-methylamino)
benzoate (3a). Starting with 2a (670 mg, 2.3 mmol), methyl iodide
(0.28 mL, 4.6 mmol) and NaH (184 mg, 4.6 mmol) to produce
662 mg of 3a in 82% yield, yellow solid, mp 52e53 ꢀC; 1H NMR
5.1.8. General procedure for preparing N-methylamides 3d, 4def
from esters under mw irradiation
A solution of ester compound (0.17 mmole0.40 mmol) in 3 mL
30% methylamine in MeOH was heated to 100e120 ꢀC under mw
irradiation for 1ꢂ2 h. Then the mixture was poured into ice-water,
neutralized with aq HCl (2 N), and extracted with EtOAc three
times. The combined organic phase was washed with water and
brine, successively, and dried over anhydrous Na2SO4 overnight.
After removal of solvent in vacuo, the crude product was purified by
flash column chromatography (gradient elution: EtOAc/petroleum
ether, 0e70%) to give the corresponding target compound.
d
ppm 3.27 (3H, s, NCH3), 3.51 (3H, s, OCH3), 3.76 (3H, s, OCH3), 6.80
(4H, m, ArH-20,30,50,60), 7.02 (1H, dd, J ¼ 8.4 and 2.0 Hz, ArH-5), 7.15
(1H, d, J ¼ 2.0 Hz, ArH-3), 7.55 (1H, d, J ¼ 8.0, ArH-6); MS m/z (%) 306
(M þ 1, 100), 308 (M þ 3, 26); HPLC purity 98.3%.
5.1.5.2. Methyl 3-chloro-5-(N-(4-methoxyphenyl)-N-methylamino)
benzoate (4a). Starting with 2b (322 mg, 1.1 mmol), methyl iodide
(0.14 mL, 2.2 mmol) and NaH (88 mg, 2.2 mmol) to produce 328 mgof
4a in 98%yield, yellow solid, mp 58e60 ꢀC; 1H NMR
d
ppm 3.27 (3H, s,
5.1.8.1. N-Methyl-4-chloro-2-(N-(4-methoxy)phenyl-N-methyl-
amino)benzamide (3d). Starting with 3a (115 mg, 0.38 mmol) at
120 ꢀC for 2 h to produce 92 mg of 3d, 79% yield, yellow oil; 1H NMR
NCH3), 3.84 (3H, s, OCH3), 3.87 (3H, s, OCH3), 7.04 (1H, t, J ¼ 2.0 Hz,
ArH-2), 6.94 (2H, d, J ¼ 8.8 Hz, ArH-20,60), 7.10 (2H, d, J ¼ 8.8 Hz, ArH-
30,50), 7.26 (1H, m, ArH-4), 7.35 (1H, t, J ¼ 2.0 Hz, ArH-6). MS m/z (%)
306 (M þ 1, 39), 308 (M þ 3, 13), 291 (100); HPLC purity 99.3%.
d
ppm 2.89 (3H, d, J ¼ 5.2 Hz, NCH3), 3.12 (3H, s, NCH3), 3.78 (3H, s,
OCH3), 6.82 (4H, m, ArH on the B-ring), 7.02 (1H, d, J ¼ 2.0 Hz, ArH-
3), 7.26 (1H, m, ArH-5), 8.14 (1H, d, J ¼ 8.0, ArH-6). MS m/z (%) 305
(M þ 1, 12), 307 (M þ 3, 4), 274 (M ꢂ 31, 100).
5.1.5.3. Methyl 3-trifluoromethyl-5-(N-(4-methoxyphenyl)-N-meth-
ylamino)benzoate (4b). Starting with 2c (188 mg, 0.58 mmol),
methyl iodide (0.07 mL, 1.2 mmol), and NaH (48 mg, 1.2 mmol) to
5.1.8.2. 3-Chloro-5-(N-(4-methoxyphenyl)-N-methylamino)-N-
methylbenzamide (4d). Starting with 4a (61 mg, 0.20 mmol) at
100 ꢀC for 1 h to produce 52 mg of 4d, 85% yield, white solid, mp
produce 176 mg of 4c in 89% yield, yellow oil; 1H NMR
d ppm 3.32
(3H, s, NCH3), 3.85 (3H, s, OCH3), 3.90 (3H, s, OCH3), 6.95 (2H, d,
J ¼ 8.8 Hz, ArH-20,60), 7.04 (1H, s, ArH-2), 7.12 (2H, d, J ¼ 8.8 Hz, ArH-
30,50), 7.51 (1H, s, ArH-4), 7.61 (1H, s, ArH-6). MS m/z (%) 340 (M þ 1,
55), 325 (M ꢂ 14, 100); HPLC purity 98.7%.
144e145 ꢀC; 1H NMR
d
ppm 2.96 (3H, d, J ¼ 4.8 Hz, NCH3), 3.26 (3H,
s, NCH3), 3.83 (3H, s, OCH3), 5.96 (1H, s, CONH), 6.73 (1H, s, ArH-2),
6.93 (2H, d, J ¼ 8.8 Hz, ArH-20,60), 6.96 (1H, s, ArH-4), 6.99 (1H, s,
ArH-6), 7.10 (2H, d, J ¼ 8.8 Hz, ArH-30,50). MS m/z (%) 305 (M þ 1,
100), 307 (M þ 3, 31); HPLC purity 99.3%.
5.1.5.4. Methyl
3-bromo-(N-(4-methoxyphenyl)-N-methylamino)
benzoate (4c). Starting with 2d (128 mg, 0.38 mmol), methyl iodide
(0.07 mL, 1.1 mmol) and sodium hydride (44 mg, 1.1 mmol) to
5.1. 8. 3. 5-(N-(4-Methoxyphenyl)-N-methylamino)-3-
trifluoromethyl-N-methylbenzamide (4e). Starting with 4b (102 mg,
0.30 mmol) at 100 ꢀC for 1 h to produce 90 mg of 4e in 89% yield,
produce 115 mg of 4c in 90% yield, yellow oil; 1H NMR
d ppm 3.27
(3H, s, NCH3), 3.84 (3H, s, OCH3), 3.87 (3H, s, OCH3), 6.94 (3H, m,
ArH-2,20,60), 7.10 (2H, d, J ¼ 8.8 Hz, ArH-30,50), 7.29 (1H, m, ArH-4),
7.50 (1H, s, ArH-6). MS m/z (%) 350 (M þ 1, 34), 352 (M þ 3, 29), 337
(M ꢂ 14, 100); HPLC purity 95.0%.
yellow solid, mp 115e116 ꢀC; 1H NMR
d
ppm 2.99 (3H, d, J ¼ 4.8 Hz,
NCH3), 3.31 (3H, s, NCH3), 3.84 (3H, s, OCH3), 6.05 (1H, s, CONH),
6.94 (2H, d, J ¼ 8.8 Hz, ArH-20,60), 6.97 (1H, s, ArH-2), 7.11 (2H, d,
J ¼ 8.8 Hz, ArH-30,50), 7.20 (1H, s, ArH-4), 7.26 (1H, s, ArH-6). MS m/z
(%) 339 (M þ 1, 100); HPLC purity 96.3%.
5.1.6. 5-Chloro-2-hydroxymethyl-N-(4-methoxy)phenyl-N-
methylaniline (3b)
A solution of 3a (427 mg, 1.40 mmol) in THF (4 mL) was added
dropwise to LiAlH4 (108 mg, 2.89 mmol, excess) in anhydrous THF
(5 mL) at 0 ꢀC with stirring, which was continued at the same
temperature for another 1 h. After the reaction was complete, as
monitored by TLC, to the mixture was added 0.11 mL of water,
0.33 mL of 15% aq NaOH, and 0.33 mL of water, successively, with
stirring for another 10 min at rt. Then the mixture was filtered
through Celite, solvent was removed in vacuo, and the residue was
purified by flash column chromatography (gradient elution: EtOAc/
petroleum ether 0e40%) to produce 377 mg of 3b in 97% yield,
5.1.8.4. 3-Bromo-5-(N-(4-methoxyphenyl)-N-methylamino)-N-
methylbenzamide (4f). Starting with 4c (100 mg, 0.29 mmol) at
100 ꢀC for 1 h to produce 82 mg of 4f in 82% yield, white solid, mp
155e156 ꢀC; 1H NMR
d
ppm 2.96 (3H, d, J ¼ 4.4 Hz, NCH3), 3.26 (3H,
s, NCH3), 3.83 (3H, s, OCH3), 5.98 (1H, s, CONH), 6.89 (1H, s, ArH-2),
6.93 (2H, d, J ¼ 8.8 Hz, ArH-20,60), 7.03 (1H, s, ArH-4), 7.08 (1H, s,
ArH-6), 7.10 (2H, d, J ¼ 8.8 Hz, ArH-30,50). MS m/z (%) 349 (M þ 1,
100), 351 (M þ 3, 95); HPLC purity 97.9%.
5.1.9. 3-Bromo-5-(N-(4-methoxyphenyl)-N-methylamino)-N,N-
dimethylbenzamide (4g)
brown oil; 1H NMR
d ppm 3.19 (3H, s, NCH3), 3.76 (3H, s, OCH3), 4.48
(2H, s, OCH2), 6.68 (2H, d, J ¼ 9.2 Hz, ArH-20,60), 6.79 (2H, d,
J ¼ 9.2 Hz, ArH-30,50), 7.12 (1H, d, J ¼ 2.0 Hz, ArH-3), 7.19 (1H, dd,
J ¼ 8.0 & 2.0 Hz, ArH-5), 7.38 (1H, d, J ¼ 8.0, ArH-6). MS m/z (%) 278
(M þ 1, 100), 280 (M þ 3, 36).
Prepared in the same manner as 3c. Starting with 4f (70 mg,
0.20 mmol), methyl iodide (0.02 mL, 0.32 mmol) and NaH (16 mg,
0.40 mmol) to produce 65 mg of 4g, 90% yield, brown solid, mp 80e
81 ꢀC; 1H NMR
d ppm 2.94 (3H, s, NCH3), 3.05 (3H, s, NCH3), 3.23
(3H, s, NCH3), 3.83 (3H, s, OCH3), 6.61 (1H, dd, J ¼ 1.2 and 1.2 Hz,
ArH-4) 6.82 (1H, m, ArH-2), 6.83 (1H, m, ArH-6), 6.92 (2H, d,
J ¼ 8.8 Hz, ArH-20,60), 7.10 (2H, d, J ¼ 8.8 Hz, ArH-30,50). MS m/z (%)
363 (M þ 1, 100), 365 (M þ 3, 95); HPLC purity 98.4%.
5.1.7. 5-Chloro-2-(methoxymethyl)-N-(4-methoxy)phenyl-N-
methylaniline (3c)
Prepared in the same manner as for 3a, starting with 3b
(227 mg, 0.82 mmol), methyl iodide (0.10 mL, 1.6 mmol), and NaH
(66 mg, 1.6 mmol) to produce 228 mg of 3c in 95% yield, yellow oil;
5.1.10. 3-Bromo-5-(N-(4-methoxyphenyl)-N-methylamino)benzoic
acid (4h)
To a solution of 4c (76 mg, 0.20 mmol) in THF/MeOH (2/2 mL)
was added aq NaOH (3 N, 2 mL), and the mixture was heated at
1H NMR
d ppm 3.17 (3H, s, NCH3), 3.32 (3H, s, OCH3), 3.76 (3H, s,
OCH3), 4.26 (2H, s, OCH2), 6.60 (2H, d, J ¼ 9.2 Hz, ArH-20,60), 6.79
(2H, d, J ¼ 9.2 Hz, ArH-30,50), 7.11 (1H, d, J ¼ 2.0 Hz, ArH-3), 7.20 (1H,

204
X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
reflux for 5 h. After removal of solvent in vacuo, the mixture was
poured into ice-water, and the insoluble solid (pH 12) was removed
by filtration. The filtrate was acidified to pH 2, until there was no
further precipitation. The solid was collected by filtration, washed
with water until neutral, and dried to obtain 178 mg of 4h in 90%
5.1.15. N-(3-Chloro-5-(N-methyl)carbamoyl)phenyl-6-methoxy-
1,2,3,4-tetrahydroquinoline (5c)
Prepared in the same manner as 3d. Starting with 5a (100 mg,
0.30 mmol) at 100 ꢀC for 1 h to produce 91 mg of 5c, 92% yield, pale
yellow solid, mp 179e180 ꢀC; 1H NMR
d
ppm 1.96 (2H, m, 30-CH2),
yield, yellow solid, mp 242e244 ꢀC; 1H NMR (DMSO-d6)
d
ppm 3.17
2.75 (2H, t, J ¼ 6.0 Hz, 40-CH2), 2.98 (3H, d, J ¼ 4.8 Hz, NCH3), 3.61
(2H, t, J ¼ 6.0 Hz, 20-CH2), 3.78 (3H, s, OCH3), 6.05 (1H, bs, NH), 6.68
(2H, m, ArH-50,70), 6.97 (1H, d, J ¼ 8.4 Hz, ArH-80), 7.17 (1H, d,
J ¼ 1.6 Hz, ArH-6), 7.20 (1H, d, J ¼ 1.6 Hz, ArH-4), 7.40 (1H, s, ArH-2).
MS m/z (%) 331 (M þ 1, 100), 333 (M þ 3, 32); HPLC purity 99.0%.
(3H, s, NCH3), 3.77 (3H, s, OCH3), 7.46 (1H, m, ArH-4), 6.97 (2H, d,
J ¼ 8.8 Hz, ArH-20,60), 7.11 (2H, d, J ¼ 8.8 Hz, ArH-30,50), 7.18 (1H, d,
J ¼ 1.2 Hz, ArH-6), 7.31 (1H, s, ArH-2). MS m/z (%) 336 (M þ 1, 100),
338 (M þ 3, 97).
5.1.11. 3-Bromo-5-(N-(4-methoxyphenyl)-N-methylamino)-N-
cyclopropylbenzamide (4i)
5.1.16. N-(3-Bromo-5-(N-methyl)carbamoyl)phenyl-6-methoxy-
1,2,3,4-tetrahydroquinoline (5d)
A mixture of 4h (105 mg, 0.31 mmol), EDCI (90 mg, 0.47 mmol),
and HOBt (64 mg, 0.47 mmol) in CH2Cl2 (5 mL) was stirred at rt for
30 min, and then cyclopropylamine (27 mg, 0.47 mmol) was added
dropwise at 0 ꢀC with stirring over 15 min, followed by warming to
rt for an additional 24 h. The mixture was added to water (20 mL)
and extracted with CH2Cl2 three times. The combined organic
phase was washed with water and brine, successively, and dried
over anhydrous Na2SO4 overnight. After removal of solvent in
vacuo, crude product was purified by flash column chromatography
(gradient elution: EtOAc/petroleum ether, 0e50%) to give 95 mg of
Prepared in the same manner as 3d. Starting with 5b (64 mg,
0.17 mmol) at 100 ꢀC for 1 h to produce 57 mg of 5d, 92% yield,
white solid, mp 161e162 ꢀC; 1H NMR
d
ppm 1.96 (2H, m, 30-CH2),
2.75 (2H, t, J ¼ 6.0 Hz, 40-CH2), 2.99 (3H, d, J ¼ 4.8 Hz, NCH3), 3.60
(2H, t, J ¼ 6.0 Hz, 20-CH2), 3.78 (3H, s, OCH3), 6.02 (1H, bs, NH), 6.68
(2H, m, ArH-50,70), 6.96 (1H, d, J ¼ 8.4 Hz, ArH-80), 7.32 (1H, s, ArH-
4), 7.36 (1H, s, ArH-6), 7.44 (1H, s, ArH-2). MS m/z (%) 375 (M þ 1,
100), 377 (M þ 3, 97); HPLC purity 98.2%.
5.1.17. N-(3-Bromo-5-carboxy)phenyl-6-methoxy-1,2,3,4-
tetrahydroquinoline (5e)
pure 4i in 82% yield, white solid, mp 184e186 ꢀC; 1H NMR
d ppm
0.59 (2H, m, CH2), 0.85 (2H, m, CH2), 2.84 (1H, m, CH), 3.26 (3H, s,
NCH3), 3.83 (3H, s, OCH3), 6.09 (1H, br, NH), 6.87 (1H, dd, J ¼ 1.6 &
2.0 Hz, ArH-2), 6.93 (2H, d, J ¼ 8.8 Hz, ArH-20,60), 7.05 (2H, m, ArH-
4,6), 7.09 (2H, d, J ¼ 8.8 Hz, ArH-30,50). MS m/z (%) 375 (M þ 1, 69),
377 (M þ 3, 79), 320 (M － 54, 100); HPLC purity 99.1%.
Prepared in the same manner as 4h. Starting with 5b (226 mg,
0.60 mmol) to produce 178 mg of 5e in 82% yield, yellow solid, mp
176e178 ꢀC;1H NMR
d
ppm 1.97 (2H, m, 30-CH2), 2.76 (2H, t, J ¼ 6.0 Hz,
40-CH2), 3.61 (2H, t, J ¼ 6.0 Hz, 20-CH2), 3.79 (3H, s, OCH3), 6.68 (2H, m,
ArH-50,70), 6.98 (2H, d, J ¼ 8.8 Hz, ArH-80), 7.48 (1H, t, J ¼ 2.0 Hz, ArH-
6), 7.70 (1H, dd, J ¼ 2.0 and 1.6 Hz, ArH-4), 7.48 (1H, dd, J ¼ 2.0 and
1.6 Hz, ArH-2). MS m/z (%) 362 (M þ 1, 100), 364 (M þ 3, 72).
5.1.12. 3-Bromo-5-(N-(4-methoxyphenyl)-N-methylamino)-N-
cyclopentylbenzamide (4j)
As for 4i. Starting with 4h (168 mg, 0.50 mmol), EDCI (144 mg,
0.75 mmol), HOBt (101 mg, 0.75 mmol), and cyclopentylamine
(64 mg, 0.75 mmol) to produce 161 mg of 4j in 80% yield, white
5.1.18. N-(3-Bromo-5-(N-cyclopropyl)carbamoyl)phenyl-6-
methoxy-1,2,3,4-tetrahydroquinoline (5f)
Prepared in the same manner as 4i. Starting with 5e (109 mg,
0.30 mmol), EDCI (115 mg, 0.60 mmol), HOBt (81 mg, 0.60 mmol),
and cyclopropylamine (56 mg, 1.0 mmol) to produce 108 mg of 5f in
solid, mp 173e174 ꢀC; 1H NMR
d ppm1.46 (2H, m, CH2), 1.69 (4H, m,
2ꢃ CH2), 2.08 (2H, m, CH2), 3.26 (3H, s, NCH3), 3.83 (3H, s, OCH3),
4.33(1H, m, CH), 5.90 (1H, br, NH), 6.86 (1H, dd, J ¼ 2.0 and 2.0 Hz,
ArH-2), 6.93 (2H, d, J ¼ 10.0 Hz, ArH-20,60), 7.04 (1H, s, ArH-4), 7.10
(3H, m, ArH-30,50,6). MS m/z (%) 403 (M þ 1, 100), 405 (M þ 3, 91).
90% yield, white solid, mp 163e164 ꢀC; 1H NMR
d ppm 0.61 (2H, m,
CH2), 0.86 (2H, m, CH2), 1.95 (2H, m, 30-CH2), 2.75 (2H, t, J ¼ 6.4 Hz,
40-CH2), 2.86 (1H, m, CH), 3.60 (2H, t, J ¼ 6.0 Hz, 20-CH2), 3.78 (3H, s,
OCH3), 6.12 (1H, br, NH), 6.66 (2H, m, ArH-50,70), 6.95 (1H, d,
J ¼ 8.4 Hz, ArH-80), 7.26 (1H, t, J ¼ 2.0 Hz, ArH-4), 7.35 (1H, d,
5.1.13. N-(3-Chloro-5-carbomethoxy)phenyl-6-methoxy-1,2,3,4-
tetrahydroquinoline (5a)
J ¼ 2.0 Hz, ArH-2), 7.43 (1H, t, J ¼ 2.0 Hz, ArH-6); 13C NMR
d ppm
Prepared in the same manner as 2b. Starting with methyl 3-
chloro-5-iodobenzoate (297 mg, 1.0 mmol), 6-methoxy-1,2,3,4-
tetrahydroquinoline (7) (163 mg, 1.0 mmol) at 120 ꢀC for 30 min
6.91 (CH2), 23.38 (CH), 23.39 (CH2), 27.71 (CH2), 49.78 (CH2), 55.71
(OCH3), 112.59 (CH), 114.35 (CH), 117.61 (CH), 120.62 (CH), 121.48
(CH), 123.02 (C), 125.54 (CH), 130 40 (C), 135.66 (C), 137.15 (C),
150.75 (C), 154.51 (C), 167.95 (C), 195.50 (CO). MS m/z (%) 401
(M þ 1, 100), 403 (M þ 3, 90); HPLC purity 98.4%.
to produce 255 mg of 5a in 77% yield, yellow oil; 1H NMR
d ppm 1.96
(2H, m, 30-CH2), 2.76 (2H, t, J ¼ 6.4 Hz, 40-CH2), 3.61 (2H, t, J ¼ 6.4 Hz,
20-CH2), 3.78 (3H, s, OCH3), 3.89 (3H, s, OCH3), 6.64 (1H, d,
J ¼ 3.2 Hz, ArH-50), 6.67 (1H, dd, J ¼ 8.8 & 3.2 Hz, ArH-70), 6.96 (1H,
d, J ¼ 8.8 Hz, ArH-80), 7.28 (1H, m, ArH-6), 7.51 (1H, m, ArH-4), 7.66
(1H, m, ArH-2). MS m/z (%) 332 (M þ 1, 100), 334 (M þ 3, 30); HPLC
purity 97.2%.
5.1.19. 6-Methoxy-N-(naphthalen-1-yl)-1,2,3,4-
tetrahydroquinoline (6a)
Prepared in the same manner as 2d. Starting with 1-
bromonaphthalene (206 mg, 1.0 mmol) and 7 (179 mg, 1.1 mmol)
for 12 h to produce 196 mg of 6a in 68% yield, pale yellow solid, mp
5.1.14. N-(3-Bromo-5-carbomethoxy)phenyl-6-methoxy-1,2,3,4-
tetrahydroquinoline (5b)
Prepared in the same manner as 2d. Starting with methyl 3,5-
dibromobenzoate (294 mg, 1.0 mmol), 7 (179 mg, 1.1 mmol) for
12 h to produce 255 mg of 5b in 68% yield, yellow oil; 1H NMR
93e95 ꢀC; 1H NMR
d ppm 2.15 (2H, br, 3-CH2), 2.98 (2H, br, 4-CH2),
3.63 (2H, t, J ¼ 5.6 Hz, 2-CH2), 3.72 (3H, s, 6-OCH3), 6.06 (1H, d,
J ¼ 8.8 Hz, ArH-8), 6.41 (1H, dd, J ¼ 8.8 & 2.8 Hz, ArH-7), 6.68 (1H, d,
J ¼ 2.8 Hz, ArH-5), 6.97 (1H, d, J ¼ 7.2 Hz, ArH-20), 7.46 (3H, m, ArH-
30,60,70), 7.74 (1H, d, J ¼ 8.0 Hz, ArH-40), 7.89 (1H, d, J ¼ 8.0 Hz, ArH-
50), 8.02 (1H, d, J ¼ 8.0 Hz, ArH-80). MS m/z (%) 290 (M þ 1, 100), 274
(M ꢂ 15, 100); HPLC purity 98.2%.
d
ppm 1.96 (2H, m, 30-CH2), 2.76 (2H, t, J ¼ 6.4 Hz, 40-CH2), 3.61 (2H,
t, J ¼ 6.4 Hz, 20-CH2), 3.78 (3H, s, OCH3), 3.89 (3H, s, OCH3), 6.65 (1H,
dd, J ¼ 9.2 and 2.8 Hz, ArH-70), 6.68 (1H, d, J ¼ 2.8 Hz, ArH-50), 6.95
(1H, d, J ¼ 9.2 Hz, ArH-80), 7.43 (1H, t, J ¼ 2.0 Hz, ArH-4), 7.66 (1H, t,
J ¼ 2.0 Hz, ArH-6), 7.66 (1H, m, ArH-2). MS m/z (%) 376 (M þ 1, 100),
378 (M þ 3, 91).
5.1.20. 6-Methoxy-20-methyl-3,4-dihydro-2H-1,40-biquinoline (6b)
Prepared in the same manner as 2d. Starting with 4-chloro-2-
methylquinoline (178 mg, 1.0 mmol) and 7 (180 mg, 1.1 mmol) for

X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
205
12 h to produce 228 mg of 6b in75% yield, yellow solid, mp 120e
121 ꢀC; 1H NMR ppm 2.03 (2H, m, 3-CH2), 2.65 (3H, s, 20-CH3), 2.94
5.2. Antiproliferative activity assay
d
(2H, t, J ¼ 6.4 Hz, 4-CH2), 3.71 (2H, t, J ¼ 5.6 Hz, 2-CH2), 3.77 (3H, s,
6-OCH3), 6.46 (1H, d, J ¼ 9.2 Hz, ArH-8), 6.53 (1H, dd, J ¼ 9.2 &
2.8 Hz, ArH-7), 6.71 (1H, d, J ¼ 2.8 Hz, ArH-5), 6.97 (1H, s, ArH-30),
7.38 (1H, m, ArH-60), 7.64 (1H, m, ArH-70), 7.90 (1H, d, J ¼ 8.4 Hz,
Target compounds were assayed by the SRB method for cyto-
toxic activity using a HTCL assay according to procedures described
previously [22e24]. The panel of cell lines included human lung
carcinoma (A549), epidermoid carcinoma of the nasopharynx (KB),
P-gp-expressing epidermoid carcinoma of the nasopharynx
(KBvin), and prostate cancer (DU145). The cytotoxic effects of each
compound were expressed as GI50 values, which represent the
molar drug concentrations required to cause 50% tumor cell growth
inhibition.
ArH-80), 8.01 (1H, d, J ¼ 8.4 Hz, ArH-50); 13C NMR
d ppm 22.41 (CH2),
25.59 (CH3), 27.67 (CH2), 51.39 (CH2), 55.68 (OCH3), 112.78 (CH),
114.34 (CH), 116.44 (CH), 120.25 (CH), 123.58 (C), 124.31 (CH),
124.96 (CH), 127.55 (C), 129.27 (CH), 129.50 (CH), 138.14 (C), 149.92
(C), 153.93 (C), 154.75 (C), 159.78 (C). MS m/z (%) 305 (M þ 1, 100);
HPLC purity 99.3%.
5.3. Tubulin assays
5.1.21. 6-Methoxy-N-(20-methylquinazol-40-yl)-1,2,3,4-
tetrahydroquinoline (6c)
Tubulin assembly was measured by turbidimetry at 350 nm as
described previously [25]. Assay mixtures contained 1.0 mg/mL
(10 mM) tubulin and varying compound concentrations were pre-
A mixture of 4-chloro-2-methylquinazoline (89 mg, 0.5 mmol),
7 (98 mg, 0.5 mmol), and NaHCO3 (126 mg, 1.5 mmol) in absolute
anhydrous EtOH (5 mL) was refluxed for 3 h. The mixture was
poured into ice-water, acidified to pH 3 with aq HCl (2 N), and
extracted with EtOAc three times. The combined organic phases
were washed with water and brine, successively, and dried over
anhydrous Na2SO4 overnight. After removal of solvent in vacuo, the
crude product was purified by flash column chromatography
(gradient elution: EtOAc/petroleum ether, 0e50%) to obtain 121 mg
incubated for 15 min at 30 ꢀC without guanosine 50-triphosphate
(GTP). The samples were placed on ice, and 0.4 mM GTP was added.
Reaction mixtures were transferred to 0 ꢀC cuvettes, and turbidity
development was followed for 20 min at 30 ꢀC following a rapid
temperature jump. Compound concentrations that inhibited
increase in turbidity by 50% relative to a control sample were
determined.
of pure 6c in79% yield, yellow solid, mp 134e136 ꢀC; 1H NMR
d
ppm
Inhibition of the binding of [3H]colchicine to tubulin was
2.11 (2H, m, 30-CH2), 2.73 (3H, s, CH3), 2.88 (2H, t, J ¼ 6.8 Hz, 40-CH2),
3.79 (3H, s, OCH3), 4.05 (2H, t, J ¼ 6.8 Hz, 20-CH2), 6.53 (1H, dd,
J ¼ 8.8 & 2.8 Hz, ArH-70), 6.63 (1H, d, J ¼ 8.8 Hz, ArH-80), 6.78 (1H, d,
J ¼ 2.8 Hz, ArH-50), 7.12 (1H, m, ArH-6), 7.32 (1H, dd, J ¼ 8.4 &1.2 Hz,
ArH-5), 7.62 (1H, m, ArH-7), 7.79 (1H, d, J ¼ 8.0 Hz, ArH-8); 13C NMR
measured as described previously [26]. Incubation of 1.0 mM
tubulin with 5.0 m mM or 1.0 mM inhibitor
M [3H]colchicine and 5.0
was for 10 min at 37 ꢀC, when about 40e60% of maximum
colchicine binding occurs in control samples.
d
ppm 24.42 (CH2), 26.65 (CH3), 27.39 (CH2), 47.49 (CH2), 55.62
5.4. Aqueous solubility
(OCH3), 112.00 (CH), 113.70 (CH), 115.68 (C), 122.28 (CH), 124.40
(CH), 126.29 (CH), 128.00 (CH), 132.35 (CH), 132.45 (C), 136.22 (C),
152.43 (C), 156.07 (C), 161.95 (C), 163.99 (C). MS m/z (%) 306 (M þ 1,
100); HPLC purity 98.8%.
Determination. Solubility was measured at pH 7.4 by using an
HPLCꢂUV method. Test compounds were initially dissolved in
DMSO at a concentration of 1.0 mg/mL. Ten microliters of this stock
solution were added to pH 7.4 phosphate buffer (1.0 mL) with the
final DMSO concentration being 1%. The mixture was stirred for 4 h
at rt and then centrifuged at 3000 rpm for 10 min. The saturated
supernatants were transferred to other vials for analysis by HPLCe
UV. Each sample was performed in triplicate. For quantification, a
model 1200 HPLCeUV (Agilent) system was used with an Agilent
5.1.22. N-(20-Chloroquinazol-40-yl)-6-methoxy-1,2,3,4-
tetrahydroquinoline (6d)
Similar procedure to that of 6c. Starting with 2,4-
dichloroquinazoline (200 mg, 1.0 mmol), 7 (163 mg, 1.0 mmol),
and NaHCO3 (252 mg, 3.0 mmol) in absolute anhydrous EtOH
(8 mL) for 3 h to produce 282 mg of 6d in 87% yield, yellow solid, mp
Eclipse XDB-C18 column (150 mm ꢃ 4.6 mm, 5
with 50%e80% ACN in water. The flow rate was 0.8 mL/min, and
injection volume was 20 L. Aqueous concentration was deter-
mm) and elution was
136e138 ꢀC; 1H NMR
d
ppm 2.12 (2H, m, 30-CH2), 2.86 (2H, t,
J ¼ 6.8 Hz, 40-CH2), 3.81 (3H, s, OCH3), 4.07 (2H, t, J ¼ 6.8 Hz, 20-CH2),
6.55 (1H, dd, J ¼ 8.8 and 2.8 Hz, ArH-70), 6.70 (1H, d, J ¼ 8.8 Hz, ArH-
80), 6.81 (1H, d, J ¼ 2.8 Hz, ArH-50), 7.13 (1H, m, ArH-6), 7.32 (1H, dd,
J ¼ 8.8 and 1.2 Hz, ArH-5), 7.63 (1H, m, ArH-7), 7.78 (1H, dd,
m
mined by comparison of the peak area of the saturated solution
with a standard curve plotted peak area versus known concentra-
tions, which were prepared by solutions of test compound in ACN
J ¼ 8.8 Hz and 1.2 Hz, ArH-8); 13C NMR
d
ppm 23.67 (CH2), 26.84
at 50, 12.5, 3.13, 0.78, and 0.20 mg/mL.
(CH2), 48.86 (CH2), 55.78 (OCH3), 112.82 (CH), 113.14 (C), 114.06
(CH), 123.21 (CH), 123.99 (CH), 126.68 (CH ꢃ2), 132.44 (C), 135.44
(CH), 135.86 (C), 145.79 (C), 152.50 (C), 159.10 (C), 160.89 (C). MS m/z
(%) 326 (M þ 1, 100), 328 (M þ 3, 31); HPLC purity 98.4%.
5.5. Log P measurement
1e2 mg of test compound was dissolved in 1.0e2.0 mL of n-
octane to obtain a 1.0 mg/mL solution. Next, the same volume of
water as n-octane was added to each vial. The mixture was stirred
at rt for 24 h, and then was left without stirring overnight. The
aqueous and organic phases of each mixture were transferred to
separate vials for HPLC analysis. The instrument and conditions
were the same as those for water solubility determinations. The log
P was calculated by the peak area ratio in n-octane and in water.
5.1.23. 6-Methoxy-N-(20-(N-methyl)aminoquinazol-40-yl)-1,2,3,4-
tetrahydroquinoline (6e)
Prepared in the same manner as 3d. Staring with 6d (65 mg,
0.20 mmol) in 3 mL of methylamine (30%) was heated to 100 ꢀC for
1 h to produce 42 mg of 6e in 66% yield, yellow solid, mp 139e
140 ꢀC; 1H NMR
d
ppm 2.07 (2H, m, 30-CH2), 2.85 (2H, t, J ¼ 6.8 Hz,
40-CH2), 3.10 (3H, d, J ¼ 5.2 Hz, NCH3), 3.79 (3H, s, OCH3), 3.94 (2H, t,
J ¼ 6.8 Hz, 20-CH2), 6.53 (1H, dd, J ¼ 9.2 & 2.8 Hz, ArH-70), 6.67 (1H,
d, J ¼ 9.2 Hz, ArH-80), 6.76 (1H, d, J ¼ 2.8 Hz, ArH-50), 6.83 (1H, m,
ArH-6), 7.29 (1H, d, J ¼ 8.0 Hz, ArH-5), 7.46 (1H, m, ArH-7), 7.52 (1H,
d, J ¼ 8.0 Hz, ArH-8). MS m/z (%) 321 (M þ 1, 100); HPLC purity
98.6%.
5.6. Microsomal stability assay
Stock solutions of test compounds (1 mg/mL) were prepared by
dissolving the pure compound in DMSO, and the solutions were
stored at 4 ꢀC. Before assay, the stock solution was diluted with ACN

206
X.-F. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 196e207
to 0.1 mM. For measurement of metabolic stability, all compounds
were brought to a final concentration of 1 M with 0.1 M potassium
within the above defined sphere were allowed to move freely
during the minimization, while the outer atoms were frozen. The
implicit solvent model of Generalized Born with Molecular Volume
(GBMV) was also used to calculate the binding energies.
m
phosphate buffer at pH 7.4, which contained 0.1 mg/mL human
liver microsomes and 5 mM MgCl2. The incubation volumes were
300
by adding 60
quenched by adding 600
15, 30, and 60 min time points. Samples at the 0 min time point
were prepared by adding 600 L of ice-cold ACN first, followed by
60 L of NADPH. All samples were prepared in duplicate. After
quenching, all samples were centrifuged at 12,000 rpm for 5 min at
ꢀC. The supernatant was collected, and 20
L of the supernatant
m
L, and reaction temperature was 37 ꢀC. Reactions were started
mL of NADPH (final concentration, 1.0 mM) and
Acknowledgments
mL of ice-cold ACN to stop the reaction at 5,
This investigation was supported by Grants 81120108022 and
30930106 from the Natural Science Foundation of China (NSFC)
awarded to Dr. Lan Xie and NIH Grant CA17625-32 from the
National Cancer Institute awarded to Dr. K. H. Lee. This study was
also supported in part by the Taiwan Department of Health, China
Medical University Hospital Cancer Research Center of Excellence
(DOH100-TD-C-111-005).
m
m
0
m
was directly injected into a Shimadzu LCeMS 2010 system with an
electrospray ionization source for further analysis. The following
controls were also used: (1) positive control incubation containing
liver microsomes, NADPH, reference compound propranolol or
terfenadine; (2) negative control incubation omitting NADPH; (3)
baseline control containing only liver microsomes and NADPH. The
peak heights of test compounds at different time points were
converted to percentage of compound remaining, and the peak
height values at initial time (0 min) served as 100% values. The
slope of the linear regression from log percentage remaining versus
incubation time relationships (ꢂk) was used to calculate the in vitro
half-life (t1/2) by the formula of in vitro t1/2 ¼ 0.693/k, regarded as
first-order kinetics. Conversion to in vitro CLint (in units of mL/min/
mg of protein) was calculated by the formula CLint ¼ [0.693/(in vitro
t1/2)][(mL incubation)/(mg of microsomes)]. The HPLCeMS analysis
was carried out on a Shimadzu LCeMS 2010 with an electrospray
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.06.
041. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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mm,150 mm ꢃ 2.1 mm)
was used for HPLC with a gradient elution at a flow rate of 0.2 mL/
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